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## **Meet the editor**

Dr Rakesh Sharma, Ph.D Biochemistry and Ph.D Magnetic Resonance Spectroscopy, is a professor teaching nanotechnology at Amity University. Since 2004 he does research in Biomedical Engineering, National High Magnetic Field Lab. Florida and Center of Nanobiotechnology at Florida State University, Tallahassee. Dr Sharma is affiliated with the department of Medicine, Columbia

University, New York since 2000. His research interest is development of enzyme assays.

Contents

**Preface IX** 

Rakesh Sharma

Chapter 3 **Pharmacomodulation of** 

Chapter 5 **Inhibition of Nitric Oxide** 

Rakesh Sharma

Rakesh Sharma

Chapter 4 **Non-Enzymatic** 

Chapter 1 **Enzyme Inhibition: Mechanisms and Scope 3** 

Chapter 2 **Cytochrome P450 Enzyme Inhibitors from Nature 39**  Simone Badal, Mario Shields and Rupika Delgoda

**Broad Spectrum Matrix Metalloproteinase** 

**and the Possibilities of Its Modulation 85**  Iva Boušová, Lenka Srbová and Jaroslav Dršata

**Synthase Gene Expression:** *In vivo* **Imaging** 

Chapter 6 **Transcriptional Bursting in the Tryptophan Operon of** 

Emanuel Salazar-Cavazos and Moisés Santillán

**and Regeneration: Enzyme Inhibition by** 

**Nitroimidazole and Human Liver Regeneration 195** 

Chapter 7 **Mechanisms of Hepatocellular Dysfunction** 

**Approaches of Nitric Oxide with Multimodal Imaging 115** 

*E. coli* **and Its Effect on the System Stochastic Dynamics 179** 

Erika Bourguet, William Hornebeck,

**Glycation of Aminotransferases** 

**Inhibitors Towards Regulation of Gelatinases 57** 

Janos Sapi, Alain Jean-Paul Alix and Gautier Moroy

**Section 2 Applications of Enzyme Inhibition 37** 

**Section 1 Basic Concepts 1** 

## Contents

#### **Preface XI**


X Contents

#### Chapter 8 **Reversible Inhibition of Tyrosine Protein Phosphatases by Redox Reactions 253**  Daniela Cosentino-Gomes and José Roberto Meyer-Fernandes


## Preface

Enzyme is a protein molecule exhibiting specific activity and binding affinity with its substrate molecule to complete enzyme reaction or biocatalytic reaction. Substrate analogues can inhibit the enzyme reaction and act as enzyme inhibitor. Enzyme inhibition (**Enz-ai-m ie-ni-hi-bi-son**) means reducing or blocking an enzyme action on specific location of enzyme active site by specific substrate or analogue so called enzyme inhibitor. In modern times, most of the pharmaceutical as well as nutriceutical compounds are marketed as enzyme inhibitors and such inhibitors exhibit their specific action in enzyme inhibition inside cells, bacteria, virus, animal plants and human body. The action of enzyme inhibitors in drug discovery has become a fundamental approach to pharmacology at any pharmaceutical industry, university research lab or drug research center. The present issue has been compiled from various data sources with aim of incorporating a wide range of basic concepts and applied enzyme inhibition evaluation methods in drug discovery. It is aimed at those who are embarking on drug discovery research projects, immobilized enzyme solid state devices as well as relatively experienced pharmacologists, biochemists and pharmacy scientists who might wish to develop their experiments further to the advanced level. While it is not possible to detail and include every possible technique related with enzyme inhibition evaluation in drug design by using specific inhibitors at specific metabolic mechanism(s), the present issue attempts to provide working tips with examples and analysis relevant to a wide range of more commonly available enzyme inhibition techniques.

The methods and concepts described in this book are aimed at giving the reader a glimpse of some existing enzyme inhibition studies and also methods with context of each enzyme inhibition method applied for, as well as providing some basis of familiarizing oneself with these biochemical methods. While enzyme inhibition has been used as major approach in drug design in the research and industry over last two decades, it was only later part of 20th century that it has become a major part of so many applications in biochemical engineering, biomedical engineering of miniatured clinical chemistry devices in microbiological, bacteriological, immunological, hormonal testing, nanotechnology, physiological monitoring in health science, plant science and environment research work. This is, at least in part, due to the continued development of new solid state polymer platform, pure enzymes and specific

#### XII Preface

inhibitors available, better understanding of enzyme inhibition mechanisms and precise detection methods, new awareness of drug discovery and design, with scanning and monitoring accessories. Thanks to the continued joint efforts of governmental, industrial and academic institutions globally to promote the need of new generations of drugs or enzyme inhibitors and new mechanisms of enzyme inhibition. Regardless of the enzymes or drugs and their brands that are used, one should always be able to understand and justify the use of right inhibitor or drug action on enzyme of right choice to drug design for specific study. With this aim, different approaches of enzyme inhibition methods are presented in separate chapters on the use of different enzyme inhibitors. For learners, basic concepts, mechanistic issues, limitations in drug testing, skepticism in enzyme inhibition approach and drug variability due to non-specific analogues of enzyme inhibitors or substrates is presented with a working enzyme inhibition protocols for drug design and analysis of their inhibitory action.

Preface XI

examples of inhibitors including pharmacomodulation of galardin®, a powerful broad spectrum MMPI, hydrazide and sulfonylhydrazide-type functions as potential Zinc Binding Compounds for gelatinase enzyme inhibition, double-headed elastase-MMP inhibitor (d-hEMI) able to block elastase and MMP activities, with display of structural-functional models in the last sections. Authors speculate to develop hybrid nanoprobes build from MMPI and fluorescent nanocrystal quantum dots (QDs). Design and chemical synthesis of derivatives of galardin®, selective inhibitors of MMP-2, tagging with QDs. In hope of photo- and chemical stability of QDs, it is possible that apporoach will enable long-term spatiotemporal tracking of the process of inhibition of MMP-2 enzymes to offer better understanding of physiological process of invasion of melanoma. In chapter 4, authors introduce the readers with nonenzymatic glycation of aminotransferases and modulation of these enzymes as model protein. Further authors emphasized the significance of advanced glycation end-products with introduction of structure, targeting with aim to evaluate potential antiglycation activity of two mitochondrial antioxidants, α-phenyl *N*-*tert*-butyl nitrone (PBN) and *Ntert*-butyl hydroxylamine (NtBHA). In next section, authors established the effect of natural compound on fructose induced AST glycation, basic techniques to determine primary amino groups, enzyme molecular charge of AST modulated by fructose by electrophoresis. Authors attempted to search the compounds with antioxidant and potential antiglycating activities in an illustrated description with a perspective of their use as remedies against diabetic complications. In chapter 5, author introduces the readers with basic mechanism of reduced nitric oxide synthase gene expression and applications of nitric oxide synthase (NOS) inhibition and approaches of nitric oxide (NO) content by using less known multimodal imaging. Nitric oxide imaging techniques utilize mapping NO in tissue using NO specific imaging contrast agents sensitive to fluorescence, magnetic resonance and electron spin resonance. A handful account is presented on NOS expression inhibitors, possibility of dithiacarbamates, paramagnetic complexes for bioimaging of NO. For learners, MRI protocol is given as example. In the light of recent developments in multimodal bioimaging of NO/NOS expression by bioluminescence, fluorescence techniques, a handful information is given in cells, animals, plants and humans body in different diseases including endothelial cell injury, apoptosis, renal, liver, lung, muscle, brain, inflammation, bones, retina with emphasis on multimodal techniques of calcium, ion channels, iron bound complexes. Author speculated the future possibility of NO/NOS bioimaging by combined radioimaging techniques and it remains to see if real-time imaging becomes routine modality. In chapter 6, transcriptional bursting in E.coli tryptophan operon is introduced to explain stochastic dynamics of the tryptophan synthase enzyme feedback inhibition regulatory mechanism at various levels of Trp operon genes. Authors have described in chapter various components of trp operon structure, model development, parameter estimation, and numerical methods with results on stochastic stimulations to evidence the least response time with inhibition-less strain. Authors further investigated that the repressor-operator interaction stimulates transcriptional bursting which shows several dynamic effects on transcriptional bursting possibly by a feedback enzyme-inhibition regulatory mechanism. In chapter 7, a hepatocellular

In chapter 1, the authors have introduced the basic concepts on enzyme, enzyme reaction, inhibitors and types of inhibition with a handful established applications in drug discovery, immobilized enzyme engineering, and biosensing. Major three basic types of enzyme inhibition kinetics is highlighted for beginners. Examples of substrate analogues and their enzyme inhibition behaviour are illustrated with color schemes. Application of immobilized enzyme on chips for environment monitoring and biosensor development is quite intriguing for engineers, scientists and industrialists. In chapter 2, authors overviewed isolates from *Pepermia amplexicaulis* and *Spathelia sorbifolia* plants have examined for CYP inhibitions to exhibit antiprotozoal, chemopreventive and anti-cancer activity. Other plant sources of tea and fruits of *Rhytidophyllum tomentosa*, *Psidium guajava, Symphytium officinale, Momordica charantia*  showed inhibition properties of these teas against a panel of CYP450 enzymes in order to assess the potential for drug interactions with co-medicated pharmaceuticals. The chapter highlights the potential of a few natural products emanating from the Caribbean: chromene amides isolated from *Amyris plumieri*, quassinoids isolated from *Picrasma excelsa*, anhydrosorbifolin isolated from *Spathelia sorbifolia* and 5-Hydroxy-2,7 dimethyl-8-(3-methyl-but-2-enyl)-2-(4-methyl-penta-1,3-dienyl)-chroman-6-carboxylic acid isolated from *Peperomia amplexicaulis.* New information is presented on bioactive screens against CYP enzymes in the presence of five aqueous infusions of popularly used herbs; *Rhytidophyllum tomentosa*, *Psidium guajava, Symphytium officinale, Momordica charantia* and *Picrasma excelsa. Authors emphasized search of* CYPs 1A1 and 1B1 activity inhibitors as chemoprotectors such as CA1 and quassin for CYP 1A1; and anhydrosorbifolin and 5-Hydroxy-2,7-dimethyl-8-(3-methyl-but-2-enyl)-2-(4-methylpenta-1,3-dienyl)-chroman-6-carboxylic acid for CYP 1B1 in search of safer herbal remedies in co-adiministration of medicines in cancer prevention. In chapter 3, authors described pharmacomodulation in metalloprotease enzymes by inhibitors. Authors aim to achieve better metallloprotease (MMP) enzyme inhibitors with good MMP-2 selectivities to increase hydrophobicity and rigidity with the dehydro and didehydro analogues and synthesized (analogue 2a-d and 3a-h). Authors displayed sevral examples of inhibitors including pharmacomodulation of galardin®, a powerful broad spectrum MMPI, hydrazide and sulfonylhydrazide-type functions as potential Zinc Binding Compounds for gelatinase enzyme inhibition, double-headed elastase-MMP inhibitor (d-hEMI) able to block elastase and MMP activities, with display of structural-functional models in the last sections. Authors speculate to develop hybrid nanoprobes build from MMPI and fluorescent nanocrystal quantum dots (QDs). Design and chemical synthesis of derivatives of galardin®, selective inhibitors of MMP-2, tagging with QDs. In hope of photo- and chemical stability of QDs, it is possible that apporoach will enable long-term spatiotemporal tracking of the process of inhibition of MMP-2 enzymes to offer better understanding of physiological process of invasion of melanoma. In chapter 4, authors introduce the readers with nonenzymatic glycation of aminotransferases and modulation of these enzymes as model protein. Further authors emphasized the significance of advanced glycation end-products with introduction of structure, targeting with aim to evaluate potential antiglycation activity of two mitochondrial antioxidants, α-phenyl *N*-*tert*-butyl nitrone (PBN) and *Ntert*-butyl hydroxylamine (NtBHA). In next section, authors established the effect of natural compound on fructose induced AST glycation, basic techniques to determine primary amino groups, enzyme molecular charge of AST modulated by fructose by electrophoresis. Authors attempted to search the compounds with antioxidant and potential antiglycating activities in an illustrated description with a perspective of their use as remedies against diabetic complications. In chapter 5, author introduces the readers with basic mechanism of reduced nitric oxide synthase gene expression and applications of nitric oxide synthase (NOS) inhibition and approaches of nitric oxide (NO) content by using less known multimodal imaging. Nitric oxide imaging techniques utilize mapping NO in tissue using NO specific imaging contrast agents sensitive to fluorescence, magnetic resonance and electron spin resonance. A handful account is presented on NOS expression inhibitors, possibility of dithiacarbamates, paramagnetic complexes for bioimaging of NO. For learners, MRI protocol is given as example. In the light of recent developments in multimodal bioimaging of NO/NOS expression by bioluminescence, fluorescence techniques, a handful information is given in cells, animals, plants and humans body in different diseases including endothelial cell injury, apoptosis, renal, liver, lung, muscle, brain, inflammation, bones, retina with emphasis on multimodal techniques of calcium, ion channels, iron bound complexes. Author speculated the future possibility of NO/NOS bioimaging by combined radioimaging techniques and it remains to see if real-time imaging becomes routine modality. In chapter 6, transcriptional bursting in E.coli tryptophan operon is introduced to explain stochastic dynamics of the tryptophan synthase enzyme feedback inhibition regulatory mechanism at various levels of Trp operon genes. Authors have described in chapter various components of trp operon structure, model development, parameter estimation, and numerical methods with results on stochastic stimulations to evidence the least response time with inhibition-less strain. Authors further investigated that the repressor-operator interaction stimulates transcriptional bursting which shows several dynamic effects on transcriptional bursting possibly by a feedback enzyme-inhibition regulatory mechanism. In chapter 7, a hepatocellular

X Preface

their inhibitory action.

inhibitors available, better understanding of enzyme inhibition mechanisms and precise detection methods, new awareness of drug discovery and design, with scanning and monitoring accessories. Thanks to the continued joint efforts of governmental, industrial and academic institutions globally to promote the need of new generations of drugs or enzyme inhibitors and new mechanisms of enzyme inhibition. Regardless of the enzymes or drugs and their brands that are used, one should always be able to understand and justify the use of right inhibitor or drug action on enzyme of right choice to drug design for specific study. With this aim, different approaches of enzyme inhibition methods are presented in separate chapters on the use of different enzyme inhibitors. For learners, basic concepts, mechanistic issues, limitations in drug testing, skepticism in enzyme inhibition approach and drug variability due to non-specific analogues of enzyme inhibitors or substrates is presented with a working enzyme inhibition protocols for drug design and analysis of

In chapter 1, the authors have introduced the basic concepts on enzyme, enzyme reaction, inhibitors and types of inhibition with a handful established applications in drug discovery, immobilized enzyme engineering, and biosensing. Major three basic types of enzyme inhibition kinetics is highlighted for beginners. Examples of substrate analogues and their enzyme inhibition behaviour are illustrated with color schemes. Application of immobilized enzyme on chips for environment monitoring and biosensor development is quite intriguing for engineers, scientists and industrialists. In chapter 2, authors overviewed isolates from *Pepermia amplexicaulis* and *Spathelia sorbifolia* plants have examined for CYP inhibitions to exhibit antiprotozoal, chemopreventive and anti-cancer activity. Other plant sources of tea and fruits of *Rhytidophyllum tomentosa*, *Psidium guajava, Symphytium officinale, Momordica charantia*  showed inhibition properties of these teas against a panel of CYP450 enzymes in order to assess the potential for drug interactions with co-medicated pharmaceuticals. The chapter highlights the potential of a few natural products emanating from the Caribbean: chromene amides isolated from *Amyris plumieri*, quassinoids isolated from *Picrasma excelsa*, anhydrosorbifolin isolated from *Spathelia sorbifolia* and 5-Hydroxy-2,7 dimethyl-8-(3-methyl-but-2-enyl)-2-(4-methyl-penta-1,3-dienyl)-chroman-6-carboxylic acid isolated from *Peperomia amplexicaulis.* New information is presented on bioactive screens against CYP enzymes in the presence of five aqueous infusions of popularly used herbs; *Rhytidophyllum tomentosa*, *Psidium guajava, Symphytium officinale, Momordica charantia* and *Picrasma excelsa. Authors emphasized search of* CYPs 1A1 and 1B1 activity inhibitors as chemoprotectors such as CA1 and quassin for CYP 1A1; and anhydrosorbifolin and 5-Hydroxy-2,7-dimethyl-8-(3-methyl-but-2-enyl)-2-(4-methylpenta-1,3-dienyl)-chroman-6-carboxylic acid for CYP 1B1 in search of safer herbal remedies in co-adiministration of medicines in cancer prevention. In chapter 3, authors described pharmacomodulation in metalloprotease enzymes by inhibitors. Authors aim to achieve better metallloprotease (MMP) enzyme inhibitors with good MMP-2 selectivities to increase hydrophobicity and rigidity with the dehydro and didehydro analogues and synthesized (analogue 2a-d and 3a-h). Authors displayed sevral

dysfunction criteria with possible mechanism of hepatocellular dysfunction is proposed to evaluate liver degeneration due to amoebic infection and hepatic recovery by nitroimidazole administration. The main focus is the evaluation of regulatory enzyme inhibition effects on major energy metabolism. Liver regeneration is an excitement to reverse the process of enzyme inhibition in defense. Author introduces enzymes in liver abscess, programmed cell death, hepatocellular criteria of hypoxia, loss of metabolic integrity with low NADH/ATP, parenchymal lysosomal enzyme inhibition illustrated by regulatory behavior changes of glucokinase, phiosphofructokinase, pyruvate kinase, phosphodiesterase, respiratory burst, superoxide dismutase, cytochrome oxidase, adenylate cyclase, inhibition of drug metabolizing microsomal enzymes, phagocytosis, and DNA synthesis. A model is proposed on proteolysis in isolated lysosomes to establish the mechanism of degradation and proteolysis inhibition. Further, role of lysosomal enzymes is proposed for liver regeneration and recovery after nitroimidazole treatment. The emerging state of art is presented on role of enzyme inhibition in liver transplantation and tissue engineering. Still, art of liver regeneration is not free from several challenges such as lack of ideal stimulator of hepatic recovery. In attempt to stimulate the liver regeneration by nitroimidazole, it was explored that nitroimidazole is becoming a multi-organ therapeutic drug for tumor, tuberculosis, myocardial infarction, hypoxia and diagnostic tool in imaging, chemosensor, and tissue engineering in addition to hepatic recovery. In chapter 8, authors describe reversal inhibition of tyrosine protein phosphatases and explore it by redox reactions. Investigators describe a structural mechanistic action of ROS in the tyrosine phosphatase enzymatic activity to demonstrate how it interacts with their target molecules; the reversible regulation of this enzyme by oxidants and antioxidants; and the major consequences of this tightly controlled mechanism on cell signalling. Authors describe tyrosine phosphatase family, catalytic domain of tyrosine phosphatase, sources of redox radical, reactive nitrogen, antioxidant mechanisms, redox inhibition, and mechanism of cellular signalling. Authors further emphasize an urgent need of a mechanism(s) available on specific tyrosine phosphatase action to identify the sources of reactive radical species formation. In chapter 9, a novel technique 'response surface methodology' is proposed using lipase inhibition by Novozyme 435-catalyse process to produce fatty acid methyl ester from waste frying oil. The issue of preserving lipase in the reaction is addressed without any loss of enzyme activity due to methanol in the reactor. The chapter describes high yield lipase catalysed process, optimization conditions, properties of feedstock, economic biodiesel production with lipase reutilization, and kinetics of tert-butanol system. Authors highlighted several issues such as short reaction time, flexible enzyme process, and utilization of low cost raw materials as environment-friendly chemical catalysis in economic enzyme process. In chapter 10, author introduces urease inhibition, chemical reactions of urease organic, metal inhibitors, and urease as virulence factor for the urinary tract infections, gastrointestinal infection by inhibition of urease. Author describes urease inhibition mechanism, kinetics, action of inhibition, potential inhibitors, and structure activity relations. Author speculates the potentials of Preface XIII

inhibitors in biomedical science with more applications in pharmaceutical, agriculture,

In last three decades, science of enzyme inhibition has grown by leaps and bounds in the field of biochemistry, medicine and pharmacology. With tremendous developments in technology, now enzymes are valuable research tools as biomarkers, biosensors, detection miniature biodevices, point-of-care pocket monitors in emergency care. Use of enzyme inhibitors in valuable drug testing has rocked the business in pharmaceutical industries and biotechnology industries in search of suitable and effective drug. Present time, science has grown mainly in three directions.

First, precision and accuracy in measurement of enzyme reaction has made possible to measure and localize the site of enzyme inhibition at the level of **picoscale** to down side of 10-12 meters. For example, enzyme reaction of caspases and metalloproteases can be detected and visualized by **vision®** at the site of tumor under powerful microscope. In future, it is speculated that **picotechnology** will replace nanotechnology in terms of more detectable applications at picoscale instead of nanoscale with better understanding of enzyme-substrate or inhibitor complexes. Further, solid phase enzyme devices or immobilized enzyme fixed on polymeric base have emerged as detection tools in agriculture, environment, healthcare, and

Second, enzyme inhibitors do behave very precise at the exact location of enzyme active site in the 'lock-key' configuration. The 3D structure and conformation of active site permits very powerful enzyme inhibitors or substrate analogues to bind with enzyme molecule to make desirable product and accomplish the goal as pharmaceutical drug. Many drug classes and variants have been explored and developed for almost each and every disease, infection and immunity. Better sources of structural-functional relationship enzyme-inhibitor and database available have changed the scenario. Now it is a fashion of new generation medication available based on new drug discovery almost every year. It is all possible due to new developments in drug testing or enzyme inhibitor development in pre-clinical and

Third, enzyme inhibitors play a major role in enzyme engineering and many other related fields. Design of portable devices using enzyme reaction as detection mechanism need a polymer base with layer of enzyme molecules fixed on its surface and placed in device at the point-of-detection. Such miniature detection devices are becoming popular in detection of microbial, bacterial contamination, pollutes, organic, chemical toxicity, allergens, harmful gases, paints, vapors, bioconversion recovery, molecular biology products, biopharmaceutical products, hormones, agroproducts, nutraceutical products, and list is growing in use at clinical chemistry, microbiology,

toxicology, pathology labs and quick diagnostic tools in hospitals.

biotechnology industries.

clinical use.

environmental research such as sensors, adsorbent and commercial devices.

inhibitors in biomedical science with more applications in pharmaceutical, agriculture, environmental research such as sensors, adsorbent and commercial devices.

XII Preface

dysfunction criteria with possible mechanism of hepatocellular dysfunction is proposed to evaluate liver degeneration due to amoebic infection and hepatic recovery by nitroimidazole administration. The main focus is the evaluation of regulatory enzyme inhibition effects on major energy metabolism. Liver regeneration is an excitement to reverse the process of enzyme inhibition in defense. Author introduces enzymes in liver abscess, programmed cell death, hepatocellular criteria of hypoxia, loss of metabolic integrity with low NADH/ATP, parenchymal lysosomal enzyme inhibition illustrated by regulatory behavior changes of glucokinase, phiosphofructokinase, pyruvate kinase, phosphodiesterase, respiratory burst, superoxide dismutase, cytochrome oxidase, adenylate cyclase, inhibition of drug metabolizing microsomal enzymes, phagocytosis, and DNA synthesis. A model is proposed on proteolysis in isolated lysosomes to establish the mechanism of degradation and proteolysis inhibition. Further, role of lysosomal enzymes is proposed for liver regeneration and recovery after nitroimidazole treatment. The emerging state of art is presented on role of enzyme inhibition in liver transplantation and tissue engineering. Still, art of liver regeneration is not free from several challenges such as lack of ideal stimulator of hepatic recovery. In attempt to stimulate the liver regeneration by nitroimidazole, it was explored that nitroimidazole is becoming a multi-organ therapeutic drug for tumor, tuberculosis, myocardial infarction, hypoxia and diagnostic tool in imaging, chemosensor, and tissue engineering in addition to hepatic recovery. In chapter 8, authors describe reversal inhibition of tyrosine protein phosphatases and explore it by redox reactions. Investigators describe a structural mechanistic action of ROS in the tyrosine phosphatase enzymatic activity to demonstrate how it interacts with their target molecules; the reversible regulation of this enzyme by oxidants and antioxidants; and the major consequences of this tightly controlled mechanism on cell signalling. Authors describe tyrosine phosphatase family, catalytic domain of tyrosine phosphatase, sources of redox radical, reactive nitrogen, antioxidant mechanisms, redox inhibition, and mechanism of cellular signalling. Authors further emphasize an urgent need of a mechanism(s) available on specific tyrosine phosphatase action to identify the sources of reactive radical species formation. In chapter 9, a novel technique 'response surface methodology' is proposed using lipase inhibition by Novozyme 435-catalyse process to produce fatty acid methyl ester from waste frying oil. The issue of preserving lipase in the reaction is addressed without any loss of enzyme activity due to methanol in the reactor. The chapter describes high yield lipase catalysed process, optimization conditions, properties of feedstock, economic biodiesel production with lipase reutilization, and kinetics of tert-butanol system. Authors highlighted several issues such as short reaction time, flexible enzyme process, and utilization of low cost raw materials as environment-friendly chemical catalysis in economic enzyme process. In chapter 10, author introduces urease inhibition, chemical reactions of urease organic, metal inhibitors, and urease as virulence factor for the urinary tract infections, gastrointestinal infection by inhibition of urease. Author describes urease inhibition mechanism, kinetics, action of inhibition, potential inhibitors, and structure activity relations. Author speculates the potentials of

In last three decades, science of enzyme inhibition has grown by leaps and bounds in the field of biochemistry, medicine and pharmacology. With tremendous developments in technology, now enzymes are valuable research tools as biomarkers, biosensors, detection miniature biodevices, point-of-care pocket monitors in emergency care. Use of enzyme inhibitors in valuable drug testing has rocked the business in pharmaceutical industries and biotechnology industries in search of suitable and effective drug. Present time, science has grown mainly in three directions.

First, precision and accuracy in measurement of enzyme reaction has made possible to measure and localize the site of enzyme inhibition at the level of **picoscale** to down side of 10-12 meters. For example, enzyme reaction of caspases and metalloproteases can be detected and visualized by **vision®** at the site of tumor under powerful microscope. In future, it is speculated that **picotechnology** will replace nanotechnology in terms of more detectable applications at picoscale instead of nanoscale with better understanding of enzyme-substrate or inhibitor complexes. Further, solid phase enzyme devices or immobilized enzyme fixed on polymeric base have emerged as detection tools in agriculture, environment, healthcare, and biotechnology industries.

Second, enzyme inhibitors do behave very precise at the exact location of enzyme active site in the 'lock-key' configuration. The 3D structure and conformation of active site permits very powerful enzyme inhibitors or substrate analogues to bind with enzyme molecule to make desirable product and accomplish the goal as pharmaceutical drug. Many drug classes and variants have been explored and developed for almost each and every disease, infection and immunity. Better sources of structural-functional relationship enzyme-inhibitor and database available have changed the scenario. Now it is a fashion of new generation medication available based on new drug discovery almost every year. It is all possible due to new developments in drug testing or enzyme inhibitor development in pre-clinical and clinical use.

Third, enzyme inhibitors play a major role in enzyme engineering and many other related fields. Design of portable devices using enzyme reaction as detection mechanism need a polymer base with layer of enzyme molecules fixed on its surface and placed in device at the point-of-detection. Such miniature detection devices are becoming popular in detection of microbial, bacterial contamination, pollutes, organic, chemical toxicity, allergens, harmful gases, paints, vapors, bioconversion recovery, molecular biology products, biopharmaceutical products, hormones, agroproducts, nutraceutical products, and list is growing in use at clinical chemistry, microbiology, toxicology, pathology labs and quick diagnostic tools in hospitals.

#### XVI Preface

I acknowledge Anja Filipovic for the guidance in book production, the continuous efforts made by Miss Nisha Keval to assist me in whole editorial work, in shaping the chapters at various points of time in presentable form, and Professor P.V.Pannirselvam from Brazil, Federal University Riogrande Norte, Natal, Brazil in chapter 1.

Finally, I thank all authors and co-authors for their chapter contributions and for selecting the right topics suitable for this book.

I hope that readers will enjoy reading the book and that it will serve the purpose of basic concepts on enzyme inhibition with advanced applications in the field of applied enzyme inhibition in pharmaceutical, biotech industries and academic research.

> **Rakesh Sharma, Ph.D**  MS-Ph.D, ABR II Professor (Nanotechnology) Amity University, India

 Research Professor, Center of Nano-Biotechnology, Florida State University, Tallahassee, FL, USA

## **Section 1**

**Basic Concepts** 

**1** 

*1,2USA 3India* 

Rakesh Sharma1,2,3

*3Amity University, NOIDA, UP* 

**Enzyme Inhibition: Mechanisms and Scope** 

*1Center of Nanomagnetics Biotechnology, Florida State University, Tallahassee, FL* 

Enzyme is a protein molecule acting as catalyst in enzyme reaction. Enzyme inhibition is a science of enzyme-substrate reaction influenced by the presence of any organic chemical or inorganic metal or biosynthetic compound due to their covalent or non-covalent interactions with enzyme active site. It is well known that all these inhibitors follow same rule to interplay in enzyme reaction. Present chapter introduces beginners with basic tenets of classic presumptions of enzyme inhibition, types of enzyme inhibitors, different models of enzyme inhibition with established examples cited in literature, and scientific basis of emerging immobilized enzyme technology in different applications. In the end, limitations of using classic presumptions and variants of enzyme inhibition are highlighted with new challenges to achieve best results. Present time, best approach is 'customize new technology with detailed analysis to make it highly efficient' in both drug discovery and enzyme biosensor industry. However, other applications are described in following chapters on

The enzyme inhibitors are low molecular weight chemical compounds. They can reduce or completely inhibit the enzyme catalytic activity either reversibly or permanently (irreversibly). Inhibitor can modify one amino acid, or several side chain(s) required in enzyme catalytic activity. To protect enzyme catalytic site from any change, ligand binds with critical side chain in enzyme. Safely, chemical modification can be done to test inhibitor

In drug discovery, several drug analogues are chosen and/or designed to inhibit specific enzymes. However, detoxification or reduced toxic effect of many antitoxins is also accomplished mainly due to their enzyme inhibitory action. Therefore, studying the aforementioned enzyme kinetics and structure-function relationship is vital to understand the kinetics of enzyme inhibition that in turn is fundamental to the modern design of pharmaceuticals in industries [Sami et al. 2011]. Enzyme inhibition kinetics behavior and inhibitor structure-function relationship with enzyme active site clarify the mechanisms of

**1. Introduction** 

pesticides, herbicides.

for any drug value.

**2. What are enzyme inhibitors?** 

*2Innovations and Solutions Inc. USA, Tallahassee, FL* 

## **Enzyme Inhibition: Mechanisms and Scope**

#### Rakesh Sharma1,2,3

*1Center of Nanomagnetics Biotechnology, Florida State University, Tallahassee, FL 2Innovations and Solutions Inc. USA, Tallahassee, FL 3Amity University, NOIDA, UP 1,2USA 3India* 

#### **1. Introduction**

Enzyme is a protein molecule acting as catalyst in enzyme reaction. Enzyme inhibition is a science of enzyme-substrate reaction influenced by the presence of any organic chemical or inorganic metal or biosynthetic compound due to their covalent or non-covalent interactions with enzyme active site. It is well known that all these inhibitors follow same rule to interplay in enzyme reaction. Present chapter introduces beginners with basic tenets of classic presumptions of enzyme inhibition, types of enzyme inhibitors, different models of enzyme inhibition with established examples cited in literature, and scientific basis of emerging immobilized enzyme technology in different applications. In the end, limitations of using classic presumptions and variants of enzyme inhibition are highlighted with new challenges to achieve best results. Present time, best approach is 'customize new technology with detailed analysis to make it highly efficient' in both drug discovery and enzyme biosensor industry. However, other applications are described in following chapters on pesticides, herbicides.

#### **2. What are enzyme inhibitors?**

The enzyme inhibitors are low molecular weight chemical compounds. They can reduce or completely inhibit the enzyme catalytic activity either reversibly or permanently (irreversibly). Inhibitor can modify one amino acid, or several side chain(s) required in enzyme catalytic activity. To protect enzyme catalytic site from any change, ligand binds with critical side chain in enzyme. Safely, chemical modification can be done to test inhibitor for any drug value.

In drug discovery, several drug analogues are chosen and/or designed to inhibit specific enzymes. However, detoxification or reduced toxic effect of many antitoxins is also accomplished mainly due to their enzyme inhibitory action. Therefore, studying the aforementioned enzyme kinetics and structure-function relationship is vital to understand the kinetics of enzyme inhibition that in turn is fundamental to the modern design of pharmaceuticals in industries [Sami et al. 2011]. Enzyme inhibition kinetics behavior and inhibitor structure-function relationship with enzyme active site clarify the mechanisms of

Enzyme Inhibition: Mechanisms and Scope 5

These inhibitors may act in reversible or irreversible manner. Non-specific irreversible noncompetitive inhibitors include all protein denaturating factors (physical and chemical denaturation factors). The specific inhibitors attack a specific component of the holoenzyme system. The action depends on increased amount of substrate or by other means of physiological conditions, toxins. Specific inhibitors can be described in several forms including; *1) coenzyme inhibitors*: e.g., cyanide, hydrazine and hydroxylamine that inhibit pyridoxal phosphate, and, dicumarol that is a competitive antagonist for vitamin K; *2) inhibitors of specific ion cofactor*: e.g., fluoride that chelates Mg2+ of enolase enzyme; *3) prosthetic group inhibitors*: e.g., cyanide that inhibits the heme prosthetic group of cytochrome oxidase; and, *4) apoenzyme inhibitors* that attack the apoenzyme component of the holoenzyme; 5) *physiological modulators* of reaction pH and temperature that denature the

The apoenzyme inhibitors are of two types; i) *Reversible inhibitors;* their inhibitory action is reversible because they make reversible association with the enzyme, and, ii) *Irreversible inhibitors;* because they make inactivating irreversible covalent modification of an essential residue of the enzyme. Apoenzyme inhibitors show effect on Km and Vmax. The reversible apoenzyme inhibitors are also called metabolic antagonists. They are of three subtypes; *a) competitive*, *b) uncompetitive* and *c) non-competitive* or mixed type. For example: enzyme

Discovery of useful new enzyme inhibitors used to be done by trial and error through screening a huge library of compounds against a target enzyme at allosteric catalytic site. This approach is still in use for compounds with combinatorial chemistry and highthroughput screening technology as described in following description based on recent concepts [El-Metwally et al. 2010]. However, rational drug design as an alternative approach uses the three-dimensional structure of an enzyme's active site or transition-state conformation to predict which molecules might be ideal inhibitors as given an example of urease in chapter 11 in this book. 3D-structure shortens the long screening list towards a right set of novel inhibitor which kinetically characterizes and allows specific structural changes in amino acids of catalytic site chain to optimize inhibitor-enzyme binding. Alternatively, molecular docking and molecular mechanics are computer-based methods that predict the affinity of an inhibitor for an enzyme. In following description, a glimpse of these mechanisms is given on different types of inhibitors based on recent classic book [El-Metwally et al. 2010]. Readers are requested to read other classic details from advanced text

The irreversible apoenzyme inhibitors have no structural relationship to the substrate and bind covalently. They also bind stable non-covalently with the active site of the enzyme or destroy an essential functional group of active site. So, irreversible inhibitors are used to identify functional groups of the enzyme active sites at which location they bind. Although inhibitors have limited therapeutic applications because they are usually act as poisons. A subset of irreversible inhibitors called *suicide irreversible inhibitors,* are relatively inactive compounds. They get activated upon binding with the active site of a specific enzyme. After such binding, the suicide irreversible inhibitor is activated by the first few intermediary

enzyme catalytic site.

inhibitors are used in drug design.

books [Dixon and Webb, 1979].

**3. Irreversible inhibition** 

enzyme inhibition action and physiological regulation of metabolic enzymes as evidenced in following chapters in this book. Some notable classic examples are: drug and toxin action and/or drug design for therapeutic uses e.g., iodoacetamide deactivates cys amino acid in enzyme side chain; methotrexate in cancer chemotherapy through semi-selectively inhibit DNA synthesis of malignant cells; aspirin inhibits the synthesis of the proinflammatory prostaglandins; sulfa drugs inhibit the folic acid synthesis essential for growth of pathogenic bacteria and so many other drugs. Many life-threatening poisons, e.g., cyanide, carbon monoxide and polychlorinated biphenols are all enzyme inhibitors.

Conceptually, enzyme inhibitors are classified into two types: non-specific inhibitors and specific inhibitors.

The enzyme inhibition reactions follow a set of rules as mentioned in following rules. Presently, computer based enzyme kinetics data analysis softwares are developed using following basic presumptions.


Other factors are also significant in determining enzyme inhibition reaction as described in each individual inhibitor in following sections. For basic principles of enzyme units (apoenzyme, holoenzyme, co-factor, co-enzyme) in enzyme catalysis, active energy loss, Michaelis-Menton Equations, LeChatelier's principle, Lineweaber-Burk and semi-log plots, apparent and actual plots, readers are requested to read text books [Schnell et al. 2003, Nelson, et al. 2008, Jakobowski 2010a, Strayer et al. 2011]. Our focus is enzyme inhibition mechanisms with examples in following description. For multisubstrate enzymes, pingpong mechanism, allosteric mechanisms, and diffusion kinetics, readers are requested to read original papers [Pryciak 2008, Bashor 2008, Jakobowski 2010b]

enzyme inhibition action and physiological regulation of metabolic enzymes as evidenced in following chapters in this book. Some notable classic examples are: drug and toxin action and/or drug design for therapeutic uses e.g., iodoacetamide deactivates cys amino acid in enzyme side chain; methotrexate in cancer chemotherapy through semi-selectively inhibit DNA synthesis of malignant cells; aspirin inhibits the synthesis of the proinflammatory prostaglandins; sulfa drugs inhibit the folic acid synthesis essential for growth of pathogenic bacteria and so many other drugs. Many life-threatening poisons, e.g., cyanide, carbon

Conceptually, enzyme inhibitors are classified into two types: non-specific inhibitors and

The enzyme inhibition reactions follow a set of rules as mentioned in following rules. Presently, computer based enzyme kinetics data analysis softwares are developed using

2. Enzyme binds with substrate at active site in the form of a lock-key 3D arrangement for

3. Inhibitor active groups compete with substrate active groups and/or active groups at enzyme allosteric catalytic site in a synergistic manner or first cum first preference (competition) to make enzyme-inhibitor-substrate/enzyme-substrate/enzyme-inhibitor

4. Enzyme-inhibitor-substrate complex formation depends on active free energy loss and

5. Enzyme and substrate or inhibitors react with each other as active masses and reaction

6. Kinetic nature of inhibitor or substrate binding with enzyme is expressed as kinetic

7. Enzyme reaction(s) are highly depend on physiological conditions such as pH, temperature, concentration of reactants, reaction period to determine the rate of reaction. 8. Substrate and inhibitor molecules arrange over enzyme active site on specific sub unit(s) in 3D manner. As a result enzyme-substrate-inhibitor exhibit binding rates depend on allosteric sites or subunit-subunit homotropic or heterotropic interactions. 9. Intermolecular forces between enzyme subunits, substrate or inhibitor active group interactions, physical properties of binding nature: electrophilic, hydrophilic, nucleophilic and metalloprotein nature; hydrogen bonding affect the overall enzyme reaction rates and

mode of inhibition (3D orientation of inhibitor molecule on enzyme active site).

Other factors are also significant in determining enzyme inhibition reaction as described in each individual inhibitor in following sections. For basic principles of enzyme units (apoenzyme, holoenzyme, co-factor, co-enzyme) in enzyme catalysis, active energy loss, Michaelis-Menton Equations, LeChatelier's principle, Lineweaber-Burk and semi-log plots, apparent and actual plots, readers are requested to read text books [Schnell et al. 2003, Nelson, et al. 2008, Jakobowski 2010a, Strayer et al. 2011]. Our focus is enzyme inhibition mechanisms with examples in following description. For multisubstrate enzymes, pingpong mechanism, allosteric mechanisms, and diffusion kinetics, readers are requested to

progresses in kinetic manner of forward or backward reaction.

read original papers [Pryciak 2008, Bashor 2008, Jakobowski 2010b]

1. Enzyme interacts with substrate in 1:1 ratio at active site to catalyze the reaction.

monoxide and polychlorinated biphenols are all enzyme inhibitors.

specific inhibitors.

induced fit.

complexes.

following basic presumptions.

thermodynamic principles.

constants of a catalytic reaction.

These inhibitors may act in reversible or irreversible manner. Non-specific irreversible noncompetitive inhibitors include all protein denaturating factors (physical and chemical denaturation factors). The specific inhibitors attack a specific component of the holoenzyme system. The action depends on increased amount of substrate or by other means of physiological conditions, toxins. Specific inhibitors can be described in several forms including; *1) coenzyme inhibitors*: e.g., cyanide, hydrazine and hydroxylamine that inhibit pyridoxal phosphate, and, dicumarol that is a competitive antagonist for vitamin K; *2) inhibitors of specific ion cofactor*: e.g., fluoride that chelates Mg2+ of enolase enzyme; *3) prosthetic group inhibitors*: e.g., cyanide that inhibits the heme prosthetic group of cytochrome oxidase; and, *4) apoenzyme inhibitors* that attack the apoenzyme component of the holoenzyme; 5) *physiological modulators* of reaction pH and temperature that denature the enzyme catalytic site.

The apoenzyme inhibitors are of two types; i) *Reversible inhibitors;* their inhibitory action is reversible because they make reversible association with the enzyme, and, ii) *Irreversible inhibitors;* because they make inactivating irreversible covalent modification of an essential residue of the enzyme. Apoenzyme inhibitors show effect on Km and Vmax. The reversible apoenzyme inhibitors are also called metabolic antagonists. They are of three subtypes; *a) competitive*, *b) uncompetitive* and *c) non-competitive* or mixed type. For example: enzyme inhibitors are used in drug design.

Discovery of useful new enzyme inhibitors used to be done by trial and error through screening a huge library of compounds against a target enzyme at allosteric catalytic site. This approach is still in use for compounds with combinatorial chemistry and highthroughput screening technology as described in following description based on recent concepts [El-Metwally et al. 2010]. However, rational drug design as an alternative approach uses the three-dimensional structure of an enzyme's active site or transition-state conformation to predict which molecules might be ideal inhibitors as given an example of urease in chapter 11 in this book. 3D-structure shortens the long screening list towards a right set of novel inhibitor which kinetically characterizes and allows specific structural changes in amino acids of catalytic site chain to optimize inhibitor-enzyme binding. Alternatively, molecular docking and molecular mechanics are computer-based methods that predict the affinity of an inhibitor for an enzyme. In following description, a glimpse of these mechanisms is given on different types of inhibitors based on recent classic book [El-Metwally et al. 2010]. Readers are requested to read other classic details from advanced text books [Dixon and Webb, 1979].

#### **3. Irreversible inhibition**

The irreversible apoenzyme inhibitors have no structural relationship to the substrate and bind covalently. They also bind stable non-covalently with the active site of the enzyme or destroy an essential functional group of active site. So, irreversible inhibitors are used to identify functional groups of the enzyme active sites at which location they bind. Although inhibitors have limited therapeutic applications because they are usually act as poisons. A subset of irreversible inhibitors called *suicide irreversible inhibitors,* are relatively inactive compounds. They get activated upon binding with the active site of a specific enzyme. After such binding, the suicide irreversible inhibitor is activated by the first few intermediary

Enzyme Inhibition: Mechanisms and Scope 7

 Thrombin inhibition is common in saliva of leeches and other blood-sucking organisms. They contain the anticoagulant hirudin that irreversibly inhibits thrombin, and, to regain thrombin action synthesis of new thrombin molecules is required. This made it unsafe as an anticoagulation drug. However, based on hirudin structure, rational drug design synthesized 20-amino acids peptide known as bivalirudin that is safe for longterm use because of its reversible effects on thrombin; despite its high binding affinity

 Ornithine decarboxylase by difluoromethylornithine is used to treat African trypanosomiasis (sleeping sickness). The enzyme initially decarboxylates difluoromethylornithine instead of ornithine and releases a fluorine atom, leaving the rest of the molecule as a highly electrophilic conjugated imine. The later reacts with either a cysteine or lysine residue in the active site to irreversibly inactivate the enzyme. Inhibition of thymidylate synthase by fluoro-dUMP. Imidazole antimycotic drugs are examples of such group that inhibit several subtypes of cytochrome P450 [Sharma, 1990]. The mechanisms of toxicities and antidotes of irreversible inhibitors are of medical pathological importance. Because of the irreversible inactivation of the enzyme, irreversible inhibition is of long duration in the biological system because reversal of their action requires synthesis of new enzyme molecules at the enzyme gene-

 Inhibition of acetylcholine esterase (ACE) by diisopropylfluorophosphate (DPFP), the ancestor of current organophosphorus nerve gases (e.g., Sarin and Tabun) and other organophosphorus toxins (e.g., the insecticides Malathion and Parathion and chlorpyrifos). ACE hydrolyzes the acetylcholine into acetate and choline to terminate the transmission of the neural signal form the neuromuscular excitatory acetylcholine presynaptic cell to somatic neuromuscular junction (see Figure 2). DPFP as a potent neurotoxin inhibits ACE and acetylcholine hydrolysis. Failure of hydrolysis leads to persistent acetylcholine excitatory state and improper vital function particularly respiratory muscles that may lead to suffocation; with a lethal dose of less than 100 mg. DPFP inhibits other enzymes with the reactive serine residue at the active site, e.g., serine proteases such as trypsin and chymotrypsin, but the inhibition is not as lethal as that of acetylcholine esterase. Similar to DPFP, malaoxon the toxic reactive derivative from Malathion (after its metabolism by the liver) binds initially reversibly and then irreversibly (after dealkylation of the inhibitor) to the active site serine and inactivates ACE and other enzymes. Lethal doses of oral Malathion are estimated at 1 g/kg of body

 Inhibition of ACE by these poisons leads to accumulation of acetylcholine that overstimulates the autonomic nervous system (including heart, blood vessels, and glands), thereby accounting for the poisoning symptoms of vomiting, abdominal cramps, nausea, salivation, and sweating. Acetylcholine is also a neurotransmitter for the somatic motor nervous system, where its accumulation resulted in poisoning symptom of involuntary muscle twitching (muscle fasciculation), convulsions, respiratory failure and coma. Intoxication of Malathion is treated by the antidote drug Oxime that reactivates the acetylcholine esterase and by intravenous injection of the anticholinergic (antimuscarinic) drug atropine to antagonize the action of the excessive amounts of

decreasing their dependence on it.

and specificity for thrombin.

transcription-translation level.

weight for humans.

acetylcholine [El-Metwally et al. 2010].

used as injection drug to rapidly destroy cocaine in the blood of addicted individuals to

steps of the biochemical reaction - like the normal substrate. However, it does not release any product because of its irreversible binding at the enzyme active site. Inhibitors make use of the normal enzyme reaction mechanism to get activated and subsequently inactivate the enzyme. Due to this very nature, suicide irreversible inhibitors are also called *mechanismbased inactivators* or *transition state analog inhibitors*. Thus, inhibitor exploits the transition state stabilizing effect of the enzyme, resulting in a better binding affinity (lower Ki) than substrate-based designs. An example of such a transition state inhibitor is active form of the antiviral drug oseltamivir (Tamiflu; see Figure 1); this drug mimics the planar nature of the ring oxonium ion in the reaction of the viral enzyme neuraminidase [El-Metwally et al. 2010]. After drug activation in the liver, the drug replaces sialic acid as the normal substrate found on the surface proteins of normal host cells. It prevents the release of new viral particles from infected cells. It has been used to treat and prevent Influenza virus A and Influenza virus B infections. Most of such inhibitors are classified as tight-binding competitive inhibitors in other references of enzymes. However, their reaction kinetics is essentially irreversible.

Fig. 1. The transition state analog oseltamivir - the viral neuraminidase inhibitor.

The present art of drug discovery and design of new drugs is based on suicidal irreversible inhibitors. Chemicals are synthesized based on knowledge of 3D conformation of substrateactive site binding at specific binding rates in presence of co-factors, co-enzyme (enzyme reaction mechanisms) to inhibit at specific enzyme active site with minimal side-effects due to its non-specific binding nature. Transition state analogs are extremely potent and specific inhibitors of enzymes because they have higher affinity and stronger binding to the active site of the target enzyme than the natural substrates or products. However, exact design of drugs that precisely mimic the transition state is a challenge because of unstable structure of transition state in the free-state. Prodrugs undergo initial reaction(s) to form an overall electrostatic and three-dimensional intermediate transition state complex form with close similarity to that of the substrate. These prodrugs serve as guideline for drug development to form transition state suitable for stable modification; or, using the transition state analog to design a complementary catalytic antibody; called *Abzyme.* Example: Abzymes are used in catalytic antibodies and ribozymes in catalytic ribosomes [El-Metwally et al. 2010].

 Abzymes are antibodies generated against analogs of the transition state complex of a specific chemical. The arrangement of amino acid side chains at the abzyme variable regions is similar to the active site of the enzyme in the transition state and work as artificial enzymes. For example, an abzyme was developed against analogs of the transition state complex of cocaine esterase, the enzyme that degrades cocaine in the body [El-Metwally et al. 2010]. Thus, this abzyme has similar esterase activity that is

steps of the biochemical reaction - like the normal substrate. However, it does not release any product because of its irreversible binding at the enzyme active site. Inhibitors make use of the normal enzyme reaction mechanism to get activated and subsequently inactivate the enzyme. Due to this very nature, suicide irreversible inhibitors are also called *mechanismbased inactivators* or *transition state analog inhibitors*. Thus, inhibitor exploits the transition state stabilizing effect of the enzyme, resulting in a better binding affinity (lower Ki) than substrate-based designs. An example of such a transition state inhibitor is active form of the antiviral drug oseltamivir (Tamiflu; see Figure 1); this drug mimics the planar nature of the ring oxonium ion in the reaction of the viral enzyme neuraminidase [El-Metwally et al. 2010]. After drug activation in the liver, the drug replaces sialic acid as the normal substrate found on the surface proteins of normal host cells. It prevents the release of new viral particles from infected cells. It has been used to treat and prevent Influenza virus A and Influenza virus B infections. Most of such inhibitors are classified as tight-binding competitive inhibitors in other references of enzymes. However, their reaction kinetics is

**O**

**H2N**

Fig. 1. The transition state analog oseltamivir - the viral neuraminidase inhibitor.

in catalytic antibodies and ribozymes in catalytic ribosomes [El-Metwally et al. 2010].

 Abzymes are antibodies generated against analogs of the transition state complex of a specific chemical. The arrangement of amino acid side chains at the abzyme variable regions is similar to the active site of the enzyme in the transition state and work as artificial enzymes. For example, an abzyme was developed against analogs of the transition state complex of cocaine esterase, the enzyme that degrades cocaine in the body [El-Metwally et al. 2010]. Thus, this abzyme has similar esterase activity that is

The present art of drug discovery and design of new drugs is based on suicidal irreversible inhibitors. Chemicals are synthesized based on knowledge of 3D conformation of substrateactive site binding at specific binding rates in presence of co-factors, co-enzyme (enzyme reaction mechanisms) to inhibit at specific enzyme active site with minimal side-effects due to its non-specific binding nature. Transition state analogs are extremely potent and specific inhibitors of enzymes because they have higher affinity and stronger binding to the active site of the target enzyme than the natural substrates or products. However, exact design of drugs that precisely mimic the transition state is a challenge because of unstable structure of transition state in the free-state. Prodrugs undergo initial reaction(s) to form an overall electrostatic and three-dimensional intermediate transition state complex form with close similarity to that of the substrate. These prodrugs serve as guideline for drug development to form transition state suitable for stable modification; or, using the transition state analog to design a complementary catalytic antibody; called *Abzyme.* Example: Abzymes are used

**HN**

**O**

**O**

essentially irreversible.

used as injection drug to rapidly destroy cocaine in the blood of addicted individuals to decreasing their dependence on it.


Enzyme Inhibition: Mechanisms and Scope 9

**N H**

**N H N H C**

**N**

*Xanthine oxidase (Mo=S)*

*Xanthine oxidase (Mo=S); inactive complex*

**HN**

**O**

**N H**

*Uric acid*

**O**

**N H** **O**

**H N**

**H2O + H<sup>+</sup> 3H+ + 2e***to O2 to give H2O2 (Oxidase), or, to NAD+ to give NADH.H+ (Dehydrogenase)*

**HN**

**HN**

**O**

**HN**

**O**

**<sup>N</sup> <sup>N</sup>**

 *The antibiotic penicillin* is another transition state analog suicidal inhibitor that binds irreversibly covalently to serine at the active site of the bacterial enzyme glycopeptide transpeptidase. The enzyme is a serine protease required for synthesis of the bacterial cell wall and is essential for bacterial growth and survival. It normally cleaves the

**H2N O Aciclovir**

Fig. 5. Aciclovir; the prodrug for the suicidal irreversible inhibition of the viral DNA

**N**

**OH**

**O**

*Xanthine oxidase (Mo=S)*

*Xanthine oxidase (Mo=S)*

**H2O + H<sup>+</sup> 3H+ + 2e***to O2 to give H2O2 (Oxidase), or, to NAD+ to give NADH.H+ (Dehydrogenase)*

**H2O + H<sup>+</sup> 3H+ + 2e***to O2 to give H2O2 (Oxidase), or, to NAD+ to give NADH.H+ (Dehydrogenase)*

**N H**

**N H**

*Oxypurinol*

Fig. 4. The suicidal irreversible mechanism-based inhibition of the enzyme xanthine oxidase

 Guanosine analogue antiviral drug aciclovir - acycloguanosine (2-amino-9-((2 hydroxyethoxy)methyl)-1H-purin-6(9H)-one), as one of the most commonly-used antiviral drugs, it is primarily used for the treatment of herpes simplex and herpes zoster (shingles) viral infections. Aciclovir (see Figure 5) started a new era in antiviral therapy, as it is extremely selective and low in cytotoxicity. Aciclovir as a prodrug differs from previous nucleoside analogues in that it contains only a partial nucleoside structure: the sugar ring is replaced by an open-chain structure. It is selectively converted into acyclo-guanosine monophosphate (acyclo-GMP) by viral thymidine kinase, which is far more effective (3000 times) in phosphorylation than cellular thymidine kinase. Subsequently, the monophosphate form is further phosphorylated into the active triphosphate form, acyclo-guanosine triphosphate (acyclo-GTP), by cellular kinases. Acyclo-GTP is a very potent inhibitor of viral DNA polymerase; it has approximately 100 times greater affinity for viral than cellular polymerase. As a substrate, acyclo-GTP is incorporated into viral DNA, resulting in chain termination. Acyclo-GTP is fairly rapidly metabolized within the cell, possibly by cellular

**O**

*Xanthine*

**O**

**HN**

**HN**

**O**

**O**

**<sup>N</sup> <sup>N</sup> H N H C**

**<sup>N</sup> <sup>N</sup> H**

*Hypoxanthine*

**N**

*Allopurinol*

by allopurinol.

phosphatases.

polymerase.

Fig. 2. Organophosphorus compounds and the suicidal irreversible mechanism-based inhibition of the enzyme acetylcholine esterase by diisopropylfluorophosphate. Malathion and parathion are organophosphorus insecticides. The nerve gases Tabun and Sarin are other organophosphorus compounds.

Another example of irreversible inhibition is iodoacetate inhibition of the glycolytic glyceraldehyde-3-phosphate dehydrogenase (GPD). Iodoacetate is a sulfhydryl compound that covalently alkylates and blocks the sulfhydryl group at the active site of the enzyme. Iodoacetate also inhibits other enzymes with -SH at the active site (Figure 3).

Fig. 3. The suicidal irreversible mechanism-based inhibition of the enzyme glyceraldehyde-3-phosphate dehydrogenase by iodoacetate.

 Allopurinol - the anti-gout drug - is a suicidal irreversible mechanism-based inhibitor of the enzyme xanthine oxidase that works as oxidase or dehydrogenase. The enzyme commits suicide by initial activating allopurinol into a transition state analog oxypurinol - that bind very tightly to molybdenum-sulfide (Mo-S) complex at the active site (Figure 4). This enzyme accounts for the human dietary requirement for the trace mineral molybdenum. The molybdenum-sulfide (Mo-S) complex binds the substrates and transfers the electrons required for the oxidation reactions.

**P O**

**F**

**CH H3C**

**C O CH2 CH3**

**O CH2 CH3**

**H2O**

**NO2**

**O CH H3C CH3**

**CH H3C CH3**

Iodoacetate also inhibits other enzymes with -SH at the active site (Figure 3).

and transfers the electrons required for the oxidation reactions.

**CH2 COOH** *Iodoacetate*

Fig. 3. The suicidal irreversible mechanism-based inhibition of the enzyme glyceraldehyde-

 Allopurinol - the anti-gout drug - is a suicidal irreversible mechanism-based inhibitor of the enzyme xanthine oxidase that works as oxidase or dehydrogenase. The enzyme commits suicide by initial activating allopurinol into a transition state analog oxypurinol - that bind very tightly to molybdenum-sulfide (Mo-S) complex at the active site (Figure 4). This enzyme accounts for the human dietary requirement for the trace mineral molybdenum. The molybdenum-sulfide (Mo-S) complex binds the substrates

Fig. 2. Organophosphorus compounds and the suicidal irreversible mechanism-based inhibition of the enzyme acetylcholine esterase by diisopropylfluorophosphate. Malathion and parathion are organophosphorus insecticides. The nerve gases Tabun and Sarin are

**H3C**

*Diisopropylfluorophosphate*

**H3C COOH**

**HO** *Acetylcholine esterase*

*DPFP*

Another example of irreversible inhibition is iodoacetate inhibition of the glycolytic glyceraldehyde-3-phosphate dehydrogenase (GPD). Iodoacetate is a sulfhydryl compound that covalently alkylates and blocks the sulfhydryl group at the active site of the enzyme.

**IH**

**O CH**

**CH3**

**H3C CH2**

**CH2**

*Choline*

*Sarin*

*Tabun*

**F**

*Acetate*

**CH3 P O**

> **CH2 N+ CH3 CH3**

**O CH**

**O P O**

**CN**

**CH3**

**CH3**

**CH3**

**N CH3**

**HF O**

**I** GPD **Cysteine CH2 S CH2 COOH**

**CH3**

ACE **Serine CH2 P O**

**CH H3C CH3**

> *Inhibited acetylcholine esterase*

**CH H3C CH3**

*Inhibited glyceraldehyde-3-phosphate dehydrogenase*

**CH3**

*Acetylcholine*

**O P <sup>H</sup> <sup>O</sup> <sup>S</sup> 3C**

**O P <sup>O</sup> <sup>S</sup> CH2**

**H3C**

ACE **Serine CH2 OH F P O**

**H3C CH2**

other organophosphorus compounds.

GPD **Cysteine CH2 SH**

3-phosphate dehydrogenase by iodoacetate.

*Active glyceraldehyde-3-phosphate dehydrogenase*

*Active acetylcholine esterase*

**C O O**

**CH2**

**H3C H3C**

> **CH2 N+ CH3 CH3**

> > **CH3**

*Malathion*

*Parathion*

**C**

**O**

**O**

**S CH H2C**

**S**

Fig. 4. The suicidal irreversible mechanism-based inhibition of the enzyme xanthine oxidase by allopurinol.

 Guanosine analogue antiviral drug aciclovir - acycloguanosine (2-amino-9-((2 hydroxyethoxy)methyl)-1H-purin-6(9H)-one), as one of the most commonly-used antiviral drugs, it is primarily used for the treatment of herpes simplex and herpes zoster (shingles) viral infections. Aciclovir (see Figure 5) started a new era in antiviral therapy, as it is extremely selective and low in cytotoxicity. Aciclovir as a prodrug differs from previous nucleoside analogues in that it contains only a partial nucleoside structure: the sugar ring is replaced by an open-chain structure. It is selectively converted into acyclo-guanosine monophosphate (acyclo-GMP) by viral thymidine kinase, which is far more effective (3000 times) in phosphorylation than cellular thymidine kinase. Subsequently, the monophosphate form is further phosphorylated into the active triphosphate form, acyclo-guanosine triphosphate (acyclo-GTP), by cellular kinases. Acyclo-GTP is a very potent inhibitor of viral DNA polymerase; it has approximately 100 times greater affinity for viral than cellular polymerase. As a substrate, acyclo-GTP is incorporated into viral DNA, resulting in chain termination. Acyclo-GTP is fairly rapidly metabolized within the cell, possibly by cellular phosphatases.

Fig. 5. Aciclovir; the prodrug for the suicidal irreversible inhibition of the viral DNA polymerase.

 *The antibiotic penicillin* is another transition state analog suicidal inhibitor that binds irreversibly covalently to serine at the active site of the bacterial enzyme glycopeptide transpeptidase. The enzyme is a serine protease required for synthesis of the bacterial cell wall and is essential for bacterial growth and survival. It normally cleaves the

Enzyme Inhibition: Mechanisms and Scope 11

reduces protein synthesis and a single molecule of ricin is enough to kill a cell.

Reversible inhibitors may be competitive, noncompetitive, or uncompetitive inhibitors relative to a particular substrate. Products of enzymatic reactions are reversible inhibitors of the enzymes. A decrease in the rate of an enzyme caused by the accumulation of its own product plays an important role in the balance and most economic usage of metabolic pathways. It prevents one enzyme in a sequence of reactions from generating a new product more than the capacity of the next enzyme in that sequence, e.g., inhibition of hexokinase by

With the reduction in the inhibitor concentration, the enzyme activity is regenerated due to the non-covalent association and the reversible equilibrium with the enzyme. The equilibrium constant for the dissociation of enzyme inhibitor complexes is known as Ki that equals [E][I]/[EI] [Cheng et al. 1973]. The inhibition efffect of Ki on the reaction kinetics is reflected on the normal Km and or Vmax observed in Lineweaver-Burk plots; in a pattern dependent on the type of the inhibitor [Nelson et al. 2008]. The inhibitor is removable by

The competitive inhibitor is structurally related to the substrate and binds reversibly at the active site of enzyme and occupies it in a mutually exclusive manner with the substrate. Therefore, the competitive inhibitor competes with the substrate for the active site. The binding is mutually exclusive because of their free competition. According to the law of mass action, relatively higher inhibitor concentration prevents the substrate binding. Since the reaction rate is directly proportional to [ES], reduction in ES formation for EI formation lowers the rate. Increasing substrate towards a saturating concentration alleviates competitive inhibition. In the time enzyme-substrate complex releases the free enzyme and a product, the enzyme-inhibitor complex does release neither free enzyme nor a product.

**4. Reversible inhibition** 

accumulating glucose 6-phosphate.

 Competitive reversible inhibition. Uncompetitive reversible inhibition.

**4.1 Competitive reversible inhibition** 

several ways. *The three common types of reversible inhibitions are:*

Mixed reversible inhibition (or non-competitive inhibition).

potent enzyme inhibitor, in this case preventing the RNA polymerase II enzyme from transcribing DNA. The algal toxin microcystin is also a peptide and is an inhibitor of protein phosphatases. This toxin can contaminate water supplies after algal blooms and is a known carcinogen that can also cause acute liver hemorrhage and death at higher doses. Proteins can also be natural poisons or antinutrients, such as the trypsin inhibitors that are found in some legumes, potato, and tomato. Several invertebrate and vertebrate venoms contain protein and peptide enzyme inhibitors for, e.g., plasmin, renin and angiotensin converting enzymes. Inhibitory enzymes are enzymes that irreversibly inhibit other enzymes by chemically modifying them. In the broad sense, they include all proteases and lysosomal enzymes. Some of them are toxic plant products, e.g., ricin, a glycosidase that is an extremely potent protein toxin found in castor oil beans. It inactivates ribosomes by cleavage the eukaryotic 28S rRNA and

peptide bond between two D-alanine residues in a polypeptide. Penicillin structure contains a strained peptide bond within the β-lactam ring that resembles the transition state of the normal cleavage reaction, and thus penicillin binds very readily to the enzyme active site. The partial reaction to cleave the imitating penicillin peptide bond activates penicillin to bind irreversibly covalently to the active site serine (Figure 6).

Fig. 6. The suicidal irreversible mechanism-based inhibition of the bacterial enzyme glycopeptide transpeptidase by the antibiotic penicillin.


Kinetically, the irreversible inhibitors decrease the concentration of active enzyme and in turn decrease the maximum possible concentration of ES complex with ultimate reduction in the reaction rate of the inactivated individual enzyme molecules. The remaining unmodified enzyme molecules are normally functional considering their turnover number and Km. For example: Natural poisons act as Enzyme inhibitors and Inhibitory enzymes

In nature, animals and plants are rich in poisons as secondary metabolites, peptides and proteins that can act as enzyme inhibitors. Natural toxins are small organic molecules and act as natural inhibitors for enzymes in metabolic pathways and non-catalytic proteins.

 Neurotoxins are natural inhibitors, toxic but valuable for therapeutic uses at lower doses. For example, glycoalkaloids from Solanaceae family plants (potato, tomato and eggplant) act as acetylcholinesterase inhibitors to increase the acetylcholine neurotransmitter, muscular paralysis and then death. Many natural toxins are secondary metabolites. These neurotoxins also include peptides and proteins. An example of a toxic peptide is alpha-amanitin, found in death cap mushroom and acts

**HC C N CH**

*Strained peptide bond*

glycopeptide transpeptidase by the antibiotic penicillin.

**O**

Fig. 6. The suicidal irreversible mechanism-based inhibition of the bacterial enzyme

the prostaglandin precursor that is a physiologic substrate for the enzyme.

**NH C R O**

**HO** *- Serine-Glycopeptide Transpeptidase; Free and active*

preparations.

peptide bond between two D-alanine residues in a polypeptide. Penicillin structure contains a strained peptide bond within the β-lactam ring that resembles the transition state of the normal cleavage reaction, and thus penicillin binds very readily to the enzyme active site. The partial reaction to cleave the imitating penicillin peptide bond activates penicillin to bind irreversibly covalently to the active site serine (Figure 6).

> **C H**

 *Aspirin (acetylsalicylic acid)* provides an example of a pharmacologic drug that exerts its effect through the covalent acetylation of an active site serine in the enzyme cyclooxygenase (prostaglandin endoperoxide synthase). Aspirin resembles a portion of

 *Heavy metal toxicity* is caused by tight binding of a metal such as mercury, lead, aluminum, or iron, to a functional group at the active site of an enzyme. At high concentration of the toxin, heavy metals are relatively nonspecific for the enzymes they inhibit and inhibit a large number of enzymes. For example, it is impossible to specify which particular enzyme is implicated in mercury toxicity that binds reactive -SH groups at the active sites. Lead developmental and neurologic toxicity is caused by its ability to replace the normal functional metal in target enzymes; particularly Ca2+ in important enzymes, e.g., Ca2+-calmodulin and protein kinase C. Because of their irreversible effect, heavy metals are routinely use as fixatives in histological

Kinetically, the irreversible inhibitors decrease the concentration of active enzyme and in turn decrease the maximum possible concentration of ES complex with ultimate reduction in the reaction rate of the inactivated individual enzyme molecules. The remaining unmodified enzyme molecules are normally functional considering their turnover number and Km. For example: Natural poisons act as Enzyme inhibitors and Inhibitory enzymes

In nature, animals and plants are rich in poisons as secondary metabolites, peptides and proteins that can act as enzyme inhibitors. Natural toxins are small organic molecules and act as natural inhibitors for enzymes in metabolic pathways and non-catalytic proteins.

 Neurotoxins are natural inhibitors, toxic but valuable for therapeutic uses at lower doses. For example, glycoalkaloids from Solanaceae family plants (potato, tomato and eggplant) act as acetylcholinesterase inhibitors to increase the acetylcholine neurotransmitter, muscular paralysis and then death. Many natural toxins are secondary metabolites. These neurotoxins also include peptides and proteins. An example of a toxic peptide is alpha-amanitin, found in death cap mushroom and acts

**C S Penicillin**

**CH3 CH3 COO-**

**HC C HN CH**

**O**

**NH C R O**

> **C H**

**C S CH3**

**CH3 COO-**

**O -** *Serine-Glycopeptide Transpeptidase; Covalently bound and inactive*

potent enzyme inhibitor, in this case preventing the RNA polymerase II enzyme from transcribing DNA. The algal toxin microcystin is also a peptide and is an inhibitor of protein phosphatases. This toxin can contaminate water supplies after algal blooms and is a known carcinogen that can also cause acute liver hemorrhage and death at higher doses. Proteins can also be natural poisons or antinutrients, such as the trypsin inhibitors that are found in some legumes, potato, and tomato. Several invertebrate and vertebrate venoms contain protein and peptide enzyme inhibitors for, e.g., plasmin, renin and angiotensin converting enzymes. Inhibitory enzymes are enzymes that irreversibly inhibit other enzymes by chemically modifying them. In the broad sense, they include all proteases and lysosomal enzymes. Some of them are toxic plant products, e.g., ricin, a glycosidase that is an extremely potent protein toxin found in castor oil beans. It inactivates ribosomes by cleavage the eukaryotic 28S rRNA and reduces protein synthesis and a single molecule of ricin is enough to kill a cell.

#### **4. Reversible inhibition**

Reversible inhibitors may be competitive, noncompetitive, or uncompetitive inhibitors relative to a particular substrate. Products of enzymatic reactions are reversible inhibitors of the enzymes. A decrease in the rate of an enzyme caused by the accumulation of its own product plays an important role in the balance and most economic usage of metabolic pathways. It prevents one enzyme in a sequence of reactions from generating a new product more than the capacity of the next enzyme in that sequence, e.g., inhibition of hexokinase by accumulating glucose 6-phosphate.

With the reduction in the inhibitor concentration, the enzyme activity is regenerated due to the non-covalent association and the reversible equilibrium with the enzyme. The equilibrium constant for the dissociation of enzyme inhibitor complexes is known as Ki that equals [E][I]/[EI] [Cheng et al. 1973]. The inhibition efffect of Ki on the reaction kinetics is reflected on the normal Km and or Vmax observed in Lineweaver-Burk plots; in a pattern dependent on the type of the inhibitor [Nelson et al. 2008]. The inhibitor is removable by several ways. *The three common types of reversible inhibitions are:*


#### **4.1 Competitive reversible inhibition**

The competitive inhibitor is structurally related to the substrate and binds reversibly at the active site of enzyme and occupies it in a mutually exclusive manner with the substrate. Therefore, the competitive inhibitor competes with the substrate for the active site. The binding is mutually exclusive because of their free competition. According to the law of mass action, relatively higher inhibitor concentration prevents the substrate binding. Since the reaction rate is directly proportional to [ES], reduction in ES formation for EI formation lowers the rate. Increasing substrate towards a saturating concentration alleviates competitive inhibition. In the time enzyme-substrate complex releases the free enzyme and a product, the enzyme-inhibitor complex does release neither free enzyme nor a product.

Enzyme Inhibition: Mechanisms and Scope 13

CH2 COO-

COO-COO-

*Oxalate*

COO-

COO-

CH2 CH2

COO-

*Glutamate*

replication (Figure 9).

N

O

N N

NH2

N

HN

H2N

reductase.

N

H

N H

N

CH2 N CH3

*Methotrexate*

them competitive inhibitors to the enzyme (Figure 10).

**N**

**N N**

*Pteridine ring*

**HN**

during the bacterial folate synthesis.

**H2N**

**O**

CH

H2N

C O

CH2 COO-

*Oxaloacetate*

COO-

*Malonate*


CH2 NH NH CH2 CH2 COO- CH - OOC

Fig. 9. The substrate and methotrexate as a competitive inhibitor for dihydrofolate

*Sulfanilamides* - the simplest form of Sulfa drugs - were among earliest antibacterial chemotherapeutic drugs classified as enzyme inhibitors. They are competitive inhibitors of the bacterial folic acid synthesizing enzyme system from *p*-aminobenzoic acid. Bacterial cannot absorb pre-made folate that is necessary to be synthesized *de novo*. Structural similarity of sulfanilamide (and other sulfas derived from it) to *p*-aminobenzoic acid made

C O

C O

Fig. 8. The substrate and different competitive inhibitors of succinate dehydrogenase (SD).

*Methotrexate* - competitive inhibitor of dihydrofolate reductase (DHFR) is another example. The drug is used as anticancer antimetabolite chemotherapy particularly for pediatric leukemia. It hinders the availability of tetrahydrofolate as a carrier for one-carbon moieties important for anabolic pathways -particularly synthesis of purine nucleotides for DNA

*Succinate*

NH CH2 CH2 COO- CH

*Dihydrofolate Tetrahydrofolate*

**CH2 NH NH CH2 CH2 COO- CH - OOC**

**NH2**

**C O**

*p-Aminobenzoic acid Glutamate*

*Folate*

Fig. 10. The *p*-aminobenzoic acid substrate and sulfanilamide as a competitive inhibitor

**H2N COOH**

**H2N SO2** *Sulfanilamide*

CH2 COO- **+**

*SD*

CH2 COO-COO- **+** *SD-FAD SD-FADH2*

*Succinate dehydrogenase* CH

CH

N H

H

H R

N N

H2N <sup>H</sup>

HN

NADPH.H+ NADP<sup>+</sup> *DHFR*

O

COO-*Fumarate* COO-

Reversible inhibition is of short duration in the biological system because it depends on substrate availability and/or rate of the catabolic clearance of the inhibitor (Figure 7).

Fig. 7. The equation and the effect of the competitive inhibitor on the double reciprocal plot of the substrate-reaction rate relationship.

*Kinetically,* the inhibitor (I) binds the free enzyme reversibly to form enzyme inhibitor complex (EI) that is catalytically inactive and cannot bind the substrate. The competitive inhibitor reduces the availability of free enzyme for the substrate binding. Thus, the Km of the normal reaction is increased to a new Km (aKm) as a function of the inhibitor concentration (expressed in the "a" factor - *a*pparent Km in presence of the inhibitors), where the substrate concentration at Vo = ½ Vmax is equal to aKm. The "a" can be calculated from the change in the slope of the line at a given inhibitor concentration;

$$a = 1 + \frac{\text{[I]}}{\text{K}\_{\text{I}}}, \text{ where, } \text{K}\_{\text{I}} = \frac{\text{[E]}\{\text{I}\}}{\text{[EI]}} \tag{1}$$

Therefore, competitive inhibitors do not affect the turnover number (active site catalysis per unit time) or the efficiency of the enzyme because once enzyme is free, enzyme behaves normally. The Michaelis-Menten equation for competitive inhibitors becomes

$$V\_o = \frac{V\_{\text{max}}\,\text{[S]}}{aK\_m + \text{[S]}} \tag{2}$$

 Consequently, the double reciprocal form of the equation is also modified so as the line slope becomes *<sup>m</sup> max aK V* and the intercept with y-Axis stays at *<sup>1</sup> Vmax* but the intercept with the x-axis at *m <sup>1</sup> aK* will differ according to the concentration of the competitive inhibitor.

The later property is characteristic for competitive inhibitors.

*Examples* include the classical competitive inhibitory effect of *malonic acid* on succinate dehydrogenase (SD) of the Krebs' cycle that reversibly dehydrogenates succinate into fumarate. Other less potent competitive inhibitors of succinate dehydrogenase include; oxalate, glutamate and oxaloacetate. The common molecular geometric feature of these compounds is the presence of two negatively charged -COOH groups suggesting that the active site of the flavoprotein SD has specifically positioned two positively charged binding groups (Figure 8).

Reversible inhibition is of short duration in the biological system because it depends on

*Increases in*

Fig. 7. The equation and the effect of the competitive inhibitor on the double reciprocal plot

*Kinetically,* the inhibitor (I) binds the free enzyme reversibly to form enzyme inhibitor complex (EI) that is catalytically inactive and cannot bind the substrate. The competitive inhibitor reduces the availability of free enzyme for the substrate binding. Thus, the Km of the normal reaction is increased to a new Km (aKm) as a function of the inhibitor concentration (expressed in the "a" factor - *a*pparent Km in presence of the inhibitors), where the substrate concentration at Vo = ½ Vmax is equal to aKm. The "a" can be calculated from the

*[I] [E][I] a = 1+ , where, K = <sup>K</sup> [EI]*

Therefore, competitive inhibitors do not affect the turnover number (active site catalysis per unit time) or the efficiency of the enzyme because once enzyme is free, enzyme behaves

> *max <sup>o</sup> m V [S] V = aK + [S]*

and the intercept with y-Axis stays at *<sup>1</sup>*

Consequently, the double reciprocal form of the equation is also modified so as the line

*Examples* include the classical competitive inhibitory effect of *malonic acid* on succinate dehydrogenase (SD) of the Krebs' cycle that reversibly dehydrogenates succinate into fumarate. Other less potent competitive inhibitors of succinate dehydrogenase include; oxalate, glutamate and oxaloacetate. The common molecular geometric feature of these compounds is the presence of two negatively charged -COOH groups suggesting that the active site of the flavoprotein SD has specifically positioned two positively charged binding

*I*

*Vmax*

**[S] 1**

*1*

*Increases in inhibitor concentration*

(1)

but the intercept with

(2)

*Vmax*

will differ according to the concentration of the competitive inhibitor.

*aKm 1*

**1 Vo**

substrate availability and/or rate of the catabolic clearance of the inhibitor (Figure 7).

E + S ES E + P *K1 K2*

E + I EI + S No product

of the substrate-reaction rate relationship.

change in the slope of the line at a given inhibitor concentration;

The later property is characteristic for competitive inhibitors.

*I*

normally. The Michaelis-Menten equation for competitive inhibitors becomes

*K-1*

*Ki*

slope becomes *<sup>m</sup>*

groups (Figure 8).

the x-axis at

*max aK V*

*m <sup>1</sup> aK*

Fig. 8. The substrate and different competitive inhibitors of succinate dehydrogenase (SD).

*Methotrexate* - competitive inhibitor of dihydrofolate reductase (DHFR) is another example. The drug is used as anticancer antimetabolite chemotherapy particularly for pediatric leukemia. It hinders the availability of tetrahydrofolate as a carrier for one-carbon moieties important for anabolic pathways -particularly synthesis of purine nucleotides for DNA replication (Figure 9).

Fig. 9. The substrate and methotrexate as a competitive inhibitor for dihydrofolate reductase.

*Sulfanilamides* - the simplest form of Sulfa drugs - were among earliest antibacterial chemotherapeutic drugs classified as enzyme inhibitors. They are competitive inhibitors of the bacterial folic acid synthesizing enzyme system from *p*-aminobenzoic acid. Bacterial cannot absorb pre-made folate that is necessary to be synthesized *de novo*. Structural similarity of sulfanilamide (and other sulfas derived from it) to *p*-aminobenzoic acid made them competitive inhibitors to the enzyme (Figure 10).

Fig. 10. The *p*-aminobenzoic acid substrate and sulfanilamide as a competitive inhibitor during the bacterial folate synthesis.

Enzyme Inhibition: Mechanisms and Scope 15

Uncompetitive inhibitor has no structural similarity to the substrate. It may bind the free enzyme or enzyme substrate complex that exposes the inhibitor binding site (ESI). Its binding, although away from the active site, causes structural distortion of the active and allosteric sites of the complexed enzyme that inactivates the catalysis. This leads to a decrease in both Km and Vmax. Increasing substrate towards a saturating concentration does not reverse this type of inhibition and reversal requires special treatment, e.g., dialysis. This type of inhibition is also encountered in multi-substrate enzymes, where the inhibitor competes with one substrate (S2) to which it has some structural similarity and is uncompetitive for the other (S1). The reaction without the inhibitor would be; **E + S1 ES1 + S2 ES1S2 E + Ps** and with uncompetitive inhibitor becomes; **E + S1 ES1 + I ES1I** (prevents S2 binding)  **no product**. It is a rare

*Kinetically,* uncompetitive inhibition modifies the Michaelis-Menten equation by (a') factor

*m*

*m a K*

*Km*

*Decreases in*

Fig. 13. The equation and the effect of the uncompetitive inhibitor on the double reciprocal

Uncompetitive reversible inhibition is rare, but may occur in multimeric enzymes. Examples of uncompetitive reversible inhibitors include; inhibition of lactate dehydrogenase by oxalate; inhibition of alkaline phosphatase (EC 3.1.3.1) by L-phenylalanine, and, inhibition of the key regulatory heme synthetic enzyme; δ-aminolevulinate synthase and dehydratase and heme synthetase by heavy metal ion, e.g., lead. Heavy metals, e.g., lead, form mercaptides with -SH at the active site of the enzyme (2 R-SH + Pb R-S-Pb-S-R + 2H).

*a'* **[S]**

**1 Vo**

(3)

(4)

*max K V*

*Increases in inhibitor concentration and Decreases in a'/Vmax*

.

, whereas, the line slope stays *<sup>m</sup>*

*Vmax*

*a'*

**1**

*max <sup>o</sup> m V [S] V = K + a'[S]*

*o max max 1 a' <sup>K</sup> <sup>1</sup> =+X V V V [S]*

This gives a number of lines in the Lineweaver-Burk plot that are parallel to the normal line with decreased 1/Vmax and –a'/Km proportional to concentrations of the uncompetitive

and x-intercept is at '

inhibitor. The later is characteristic to uncompetitive inhibition (Figure 13).

type and the inhibitor may be the reaction product or a product analog.

that proportionates with the inhibitor concentration to be:

and in the double-reciprocal equation to be:

*max a' V*

*K-1 Ki* No product <sup>I</sup> **ESI**

plot of the substrate-reaction rate relationship.

while y-intercept is at

*K2* **E + S ES E + P**

*K1*

**4.2 Uncompetitive reversible inhibition** 

Male erectile impotence was a major medical problem. Now a group of chemicals with molecular structural similarity to cGMP is promising that competitively inhibit the cGMPphosphodiesterase-5. They include sildenafil citrate (Viagra; Figure 11), vardenafil (Levitra) and tadalafil (Cialis). The inhibition of this enzyme that has a limited tissue distribution including the penile cavernous tissue spares cGMP. Accumulation of cGMP leads to smooth muscle relaxation (vasodilation) of the intimal cushions of the helicine arteries, resulting in increased inflow of blood and an erection.

Fig. 11. The cGMP substrate and sildenafil a competitive inhibitor of the cGMPphosphodiesterase-5.

Another example of these substrate mimics competitive inhibitors are the peptide-based protease inhibitors, a very successful class of antiretroviral drugs used to treat HIV, e.g., ritonavir that contains three peptide bonds (see Figure 12).

Fig. 12. The peptide-based competitive protease inhibitor ritonavir.

Reversible competitive inhibitors of acetylcholinesterase, such as edrophonium, physostigmine, and neostigmine, are used in the treatment of myasthenia gravis and in anesthesia. The carbamate pesticides are also examples of reversible acetylcholinesterase inhibitors.

Male erectile impotence was a major medical problem. Now a group of chemicals with molecular structural similarity to cGMP is promising that competitively inhibit the cGMPphosphodiesterase-5. They include sildenafil citrate (Viagra; Figure 11), vardenafil (Levitra) and tadalafil (Cialis). The inhibition of this enzyme that has a limited tissue distribution including the penile cavernous tissue spares cGMP. Accumulation of cGMP leads to smooth muscle relaxation (vasodilation) of the intimal cushions of the helicine arteries, resulting in

> **N N**

**Sildenafil cGMP**

Another example of these substrate mimics competitive inhibitors are the peptide-based protease inhibitors, a very successful class of antiretroviral drugs used to treat HIV, e.g.,

Fig. 11. The cGMP substrate and sildenafil a competitive inhibitor of the cGMP-

**HN**

**H2N**

**HN**

**HN**

**O**

**O**

**N**

**S N**

**HN OH**

**O**

Reversible competitive inhibitors of acetylcholinesterase, such as edrophonium, physostigmine, and neostigmine, are used in the treatment of myasthenia gravis and in anesthesia. The

carbamate pesticides are also examples of reversible acetylcholinesterase inhibitors.

**O**

**N S**

Fig. 12. The peptide-based competitive protease inhibitor ritonavir.

**O**

**<sup>N</sup> <sup>N</sup>**

**N**

**O**

**O**

**P O-O**

**OH O H H H H**

increased inflow of blood and an erection.

**N**

phosphodiesterase-5.

**O S O**

**N**

**HN**

**O**

ritonavir that contains three peptide bonds (see Figure 12).

**N**

**O**

#### **4.2 Uncompetitive reversible inhibition**

Uncompetitive inhibitor has no structural similarity to the substrate. It may bind the free enzyme or enzyme substrate complex that exposes the inhibitor binding site (ESI). Its binding, although away from the active site, causes structural distortion of the active and allosteric sites of the complexed enzyme that inactivates the catalysis. This leads to a decrease in both Km and Vmax. Increasing substrate towards a saturating concentration does not reverse this type of inhibition and reversal requires special treatment, e.g., dialysis. This type of inhibition is also encountered in multi-substrate enzymes, where the inhibitor competes with one substrate (S2) to which it has some structural similarity and is uncompetitive for the other (S1). The reaction without the inhibitor would be; **E + S1 ES1 + S2 ES1S2 E + Ps** and with uncompetitive inhibitor becomes; **E + S1 ES1 + I ES1I** (prevents S2 binding)  **no product**. It is a rare type and the inhibitor may be the reaction product or a product analog.

*Kinetically,* uncompetitive inhibition modifies the Michaelis-Menten equation by (a') factor that proportionates with the inhibitor concentration to be:

$$V\_o = \frac{V\_{\max} \text{[S]}}{K\_m + a' \text{[S]}} \tag{3}$$

and in the double-reciprocal equation to be:

$$\frac{1}{V\_o} = \frac{a^\circ}{V\_{\max}} + \frac{K\_m}{V\_{\max}} X \frac{1}{\{S\}} \tag{4}$$

while y-intercept is at *max a' V* and x-intercept is at ' *m a K* , whereas, the line slope stays *<sup>m</sup> max K V* . This gives a number of lines in the Lineweaver-Burk plot that are parallel to the normal line with decreased 1/Vmax and –a'/Km proportional to concentrations of the uncompetitive inhibitor. The later is characteristic to uncompetitive inhibition (Figure 13).

Fig. 13. The equation and the effect of the uncompetitive inhibitor on the double reciprocal plot of the substrate-reaction rate relationship.

Uncompetitive reversible inhibition is rare, but may occur in multimeric enzymes. Examples of uncompetitive reversible inhibitors include; inhibition of lactate dehydrogenase by oxalate; inhibition of alkaline phosphatase (EC 3.1.3.1) by L-phenylalanine, and, inhibition of the key regulatory heme synthetic enzyme; δ-aminolevulinate synthase and dehydratase and heme synthetase by heavy metal ion, e.g., lead. Heavy metals, e.g., lead, form mercaptides with -SH at the active site of the enzyme (2 R-SH + Pb R-S-Pb-S-R + 2H).

Enzyme Inhibition: Mechanisms and Scope 17

*Examples* of noncompetitive inhibitors are mostly poisons because of the crucial role of the targeted enzymes. Cyanide and azide inhibits enzymes with iron or copper as a component of the active site or the prosthetic group, e.g., cytochrome c oxidase (EC 1.9.3.1). They include the inhibition of an enzyme by hydrogen ion at the acidic side and by the hydroxyl ion at the alkaline side of its optimum pH. They also include inhibition of; carbonic anhydrase by acetazolamide; cyclooxygenase by aspirin; and, fructose-1,6 diphosphatase by AMP. Cyanide binds to the Fe3+ in the heme of the cytochrome aa3 component of cytochrome c oxidase and prevents electron transport to O2. Mitochondrial respiration and energy production cease, and cell death rapidly occurs. The central nervous system is the primary target for cyanide toxicity. Acute inhalation of high concentrations of cyanide (e.g., smoke inhalation during a fire and automobile exhaust) provokes a brief central nervous system stimulation rapidly followed by convulsion, coma, and death. Acute exposure to lower amounts can cause lightheadedness, breathlessness, dizziness, numbness, and headaches. Cyanide is present in the air as hydrogen cyanide (HCN), in soil and water as cyanide salts (e.g., NaCN), and in foods as cyanoglycosides. Comparison of the three types of the reversible enzyme inhibitors is

In a special case, the mechanism of *partially competitive inhibition* is similar to that of noncompetitive, except that the EIS complex has catalytic activity, which may be lower or even higher (partially competitive activation) than that of the enzyme-substrate (ES) complex. This inhibition typically displays a lower Vmax, but an unaffected Km value. We compare three main types of inhibitors in terms of reaction properties as shown in Table 1

Competitive inhibitor Uncompetitive inhibitor Mixed

Substrate binding

binding site. Increasing substrate concentration does not reverse the inhibition. The inhibited reaction rate parallel the normal one as reflected on decreased both Vmax and

Km.

Table 1. Comparison of the different types of reversible inhibition is shown in Table with a

exposes the inhibitor binding site away from the catalytic/substrate

(noncompetitive inhibitor)

 The inhibitor binds each of the free enzyme and the substrate-enzyme complex away from the catalytic/substrate binding site. Increasing substrate concentration does not reverse the inhibition. Only Vmax is decreased proportionately to inhibitor concentration, Km is unchanged since increasing substrate concentration is ineffective.

presented in Table 1.

and Figure 15.

 The inhibitor binds the catalytic/substrate binding site. It competes with

substrate for binding. Inhibition is reversible by increasing substrate

> substrate concentration has to be increased as reflected on increased

quick view of mechanism in sketches as below.

concentration. Vmax constant, the

Km.

Oxidizing agents, e.g., ferricyanide also oxidizes -SH into a disulfide linkage (2 R-SH R-S-S-R). Reversion here requires treatment with reducing agents and/or dialysis.

#### **4.3 Mixed (noncompetitive) inhibition**

The mixed type inhibitor does not have structural similarity to the substrate but it binds both of the free enzyme and the enzyme-substrate complex. Thus, its binding manner is not mutually exclusive with the substrate and the presence of a substrate has no influence on the ability of a non-competitive inhibitor to bind an enzyme and *vice versa*. However, its binding - although away from the active site - alters the conformation of the enzyme and reduces its catalytic activity due to changes in the nature of the catalytic groups at the active site. EI and ESI complexes are nonproductive and increasing substrate to a saturating concentration does not reverse the inhibition leading to unaltered Km but reduced Vmax. Reversal of the inhibition requires a special treatment, e.g., dialysis or pH adjustment. Some classifications differentiate between non-competitive inhibition as defined above and *mixed inhibition* in that the EIS-complex has residual enzymatic activity in the mixed inhibition.

*Kinetically,* mixed type inhibition causes changes in the Michaelis-Menten equation so as

$$V\_o = \frac{V\_{\text{max}}\,\text{[S]}}{aK\_m + a'\,\text{[S]}} \tag{5}$$

Mixed type inhibition - as the name imply - has a change in the denominator with Km modified by factor (a) as in competitive inhibition, and [S] modified by factor (a') as in uncompetitive inhibition. In the double reciprocal equation 6,

$$\frac{1}{V\_o} = \frac{a^\circ}{V\_{\max}} + \frac{aK\_m}{V\_{\max}} \ge \frac{1}{\{S\}}\tag{6}$$

A line slope is *<sup>m</sup> max aK V* , and the intercept with y-axis is at *max a' V* and with x-axis is at *m a' aK* . This results in progressive decreases in Vmax and progressive increases in Km proportional to the increase in the mixed inhibitor concentration. The double reciprocal plot shows a number of lines reflecting decreases in Vmax/increases in Km but their intercept is to the left of the yaxis. Mixed type inhibitor would be called non-competitive only if [a = a'], where, it will only lower Vmax without affecting the Km (Figure 14).

Fig. 14. The equation and the effect of the mixed type (noncompetitive) inhibitor on the double reciprocal plot of substrate-reaction rate relationship.

Oxidizing agents, e.g., ferricyanide also oxidizes -SH into a disulfide linkage (2 R-SH R-S-

The mixed type inhibitor does not have structural similarity to the substrate but it binds both of the free enzyme and the enzyme-substrate complex. Thus, its binding manner is not mutually exclusive with the substrate and the presence of a substrate has no influence on the ability of a non-competitive inhibitor to bind an enzyme and *vice versa*. However, its binding - although away from the active site - alters the conformation of the enzyme and reduces its catalytic activity due to changes in the nature of the catalytic groups at the active site. EI and ESI complexes are nonproductive and increasing substrate to a saturating concentration does not reverse the inhibition leading to unaltered Km but reduced Vmax. Reversal of the inhibition requires a special treatment, e.g., dialysis or pH adjustment. Some classifications differentiate between non-competitive inhibition as defined above and *mixed inhibition* in that the EIS-complex has residual enzymatic activity in the mixed inhibition. *Kinetically,* mixed type inhibition causes changes in the Michaelis-Menten equation so as

> *max <sup>o</sup> m V [S] V = aK + a'[S]*

*o max max 1 a' aK <sup>1</sup> =+X V V V [S]*

results in progressive decreases in Vmax and progressive increases in Km proportional to the increase in the mixed inhibitor concentration. The double reciprocal plot shows a number of lines reflecting decreases in Vmax/increases in Km but their intercept is to the left of the yaxis. Mixed type inhibitor would be called non-competitive only if [a = a'], where, it will

, and the intercept with y-axis is at

uncompetitive inhibition. In the double reciprocal equation 6,

only lower Vmax without affecting the Km (Figure 14).

**No product <sup>I</sup>**

double reciprocal plot of substrate-reaction rate relationship.

A line slope is *<sup>m</sup>*

*max aK V*

**E + S ES E + P**

*K2 K1 K-1 Ki*

**ESI <sup>I</sup> EI**

**S**

Mixed type inhibition - as the name imply - has a change in the denominator with Km modified by factor (a) as in competitive inhibition, and [S] modified by factor (a') as in

*m*

*max a' V*

*aKm*

*Increases in*

Fig. 14. The equation and the effect of the mixed type (noncompetitive) inhibitor on the

*a'* **[S]**

**1**

(5)

(6)

*m a' aK*

. This

and with x-axis is at

*Vmax*

*a'*

**Vo** *Increases in inhibitor* 

*concentration and Decreases in a'/Vmax*

**1**

S-R). Reversion here requires treatment with reducing agents and/or dialysis.

**4.3 Mixed (noncompetitive) inhibition** 

*Examples* of noncompetitive inhibitors are mostly poisons because of the crucial role of the targeted enzymes. Cyanide and azide inhibits enzymes with iron or copper as a component of the active site or the prosthetic group, e.g., cytochrome c oxidase (EC 1.9.3.1). They include the inhibition of an enzyme by hydrogen ion at the acidic side and by the hydroxyl ion at the alkaline side of its optimum pH. They also include inhibition of; carbonic anhydrase by acetazolamide; cyclooxygenase by aspirin; and, fructose-1,6 diphosphatase by AMP. Cyanide binds to the Fe3+ in the heme of the cytochrome aa3 component of cytochrome c oxidase and prevents electron transport to O2. Mitochondrial respiration and energy production cease, and cell death rapidly occurs. The central nervous system is the primary target for cyanide toxicity. Acute inhalation of high concentrations of cyanide (e.g., smoke inhalation during a fire and automobile exhaust) provokes a brief central nervous system stimulation rapidly followed by convulsion, coma, and death. Acute exposure to lower amounts can cause lightheadedness, breathlessness, dizziness, numbness, and headaches. Cyanide is present in the air as hydrogen cyanide (HCN), in soil and water as cyanide salts (e.g., NaCN), and in foods as cyanoglycosides. Comparison of the three types of the reversible enzyme inhibitors is presented in Table 1.

In a special case, the mechanism of *partially competitive inhibition* is similar to that of noncompetitive, except that the EIS complex has catalytic activity, which may be lower or even higher (partially competitive activation) than that of the enzyme-substrate (ES) complex. This inhibition typically displays a lower Vmax, but an unaffected Km value. We compare three main types of inhibitors in terms of reaction properties as shown in Table 1 and Figure 15.


Table 1. Comparison of the different types of reversible inhibition is shown in Table with a quick view of mechanism in sketches as below.

Enzyme Inhibition: Mechanisms and Scope 19

An **agonist** or test drug or substrate is similar to natural ligand and binds with receptor to produce a similar biological effect as the natural ligand. Agonist binds at the same binding site in competition with natural ligand to show full or partial response. So, it is called **partial agonist**. If receptor has a basal (or constitutive) activity in the absence of a bound ligand, it is called **inverse agonist**. If either the natural ligand or an agonist binds to the receptor site, the basal activity is increased. If an inverse agonist binds, the activity is decreased. Ro15- 4513 and benzodiazepines (Valium) bind with the GABA receptor. As a result, GABA

cell, inhibiting neuron activation. Ro15-4513 binds to the benzodiazepine site, which leads to the opposite effect of valium, the inhibition of the receptor bound activity - a chloride

Fig. 16. A sketch is shown for membrane receptor binding with ligand (agonist) acting like

A**ntagonist** or test inhibitor can inhibit the effects of the natural ligand (hormone, neurotransmitter), agonist, partial agonist, and inverse agonists. We can think of them as

as enzyme. Reproduced with permission [Jakobowski 2010a].

**4.5 Antagonist** 

into a neural

receptor is "activated" to become a ion channel allowing the inward flow of Cl-

**4.4 Agonist** 

channel as shown in Figure 16.

Fig. 15. Sketch of three different enzyme inhibition by competitive, uncompetitive and noncompetitive types are shown with illustration of enzyme-substrate or inhibitor binding, kinetics and graphs.

In last decade, role of membrane receptors was explored in relation with enzyme inhibition. Membrane receptors or transmembrane proteins bind with natural ligands such as hormones, neurotransmitters in tissue membranes. Receptor-ligand binding modulates the binding of drugs with enzyme. Such ligand binding behavior also influences the analysis of competitive, uncompetitive and noncompetitive inhibition by biological effect of prodrugs on enzymes. It usually involves a shape change in the receptor, a transmembrane protein, which activates intracellular activities. The bound receptor usually does not directly express biological activity, but initiates a cascade of events which leads to expression of intracellular activity. However, occupied receptor actually expresses biological activity itself. For example, the bound receptor can acquire enzymatic activity, or become an active ion channel with similar competitive, noncompetitive behavior. Drugs targeted to membrane receptors can have biological effects similar to the natural ligands, they are called **agonists,** or conversely they may inhibit the biological activity of the receptor, they are called **antagonists** [Jakobowski 2010a]**.**

#### **4.4 Agonist**

18 Enzyme Inhibition and Bioapplications

kinetics and graphs.

**antagonists** [Jakobowski 2010a]**.**

Fig. 15. Sketch of three different enzyme inhibition by competitive, uncompetitive and noncompetitive types are shown with illustration of enzyme-substrate or inhibitor binding,

In last decade, role of membrane receptors was explored in relation with enzyme inhibition. Membrane receptors or transmembrane proteins bind with natural ligands such as hormones, neurotransmitters in tissue membranes. Receptor-ligand binding modulates the binding of drugs with enzyme. Such ligand binding behavior also influences the analysis of competitive, uncompetitive and noncompetitive inhibition by biological effect of prodrugs on enzymes. It usually involves a shape change in the receptor, a transmembrane protein, which activates intracellular activities. The bound receptor usually does not directly express biological activity, but initiates a cascade of events which leads to expression of intracellular activity. However, occupied receptor actually expresses biological activity itself. For example, the bound receptor can acquire enzymatic activity, or become an active ion channel with similar competitive, noncompetitive behavior. Drugs targeted to membrane receptors can have biological effects similar to the natural ligands, they are called **agonists,** or conversely they may inhibit the biological activity of the receptor, they are called An **agonist** or test drug or substrate is similar to natural ligand and binds with receptor to produce a similar biological effect as the natural ligand. Agonist binds at the same binding site in competition with natural ligand to show full or partial response. So, it is called **partial agonist**. If receptor has a basal (or constitutive) activity in the absence of a bound ligand, it is called **inverse agonist**. If either the natural ligand or an agonist binds to the receptor site, the basal activity is increased. If an inverse agonist binds, the activity is decreased. Ro15- 4513 and benzodiazepines (Valium) bind with the GABA receptor. As a result, GABA receptor is "activated" to become a ion channel allowing the inward flow of Cl into a neural cell, inhibiting neuron activation. Ro15-4513 binds to the benzodiazepine site, which leads to the opposite effect of valium, the inhibition of the receptor bound activity - a chloride channel as shown in Figure 16.

Fig. 16. A sketch is shown for membrane receptor binding with ligand (agonist) acting like as enzyme. Reproduced with permission [Jakobowski 2010a].

#### **4.5 Antagonist**

A**ntagonist** or test inhibitor can inhibit the effects of the natural ligand (hormone, neurotransmitter), agonist, partial agonist, and inverse agonists. We can think of them as

Enzyme Inhibition: Mechanisms and Scope 21

Some endothermic or exothermic chemical compounds change the temperature of reaction. Enzyme reaction experiences inhibition at higher or lower than optimal physiological temperature. For example, human body optimal temperature of human body is 37 oC. For most of the enzyme reactions, enzyme activity usually increases at 0 to about 40-50 oC in the absence of catalysts. As a general rule of thumb, reaction velocities double for each increment of 10oC rise. At higher temperatures, the activity decreases dramatically as the

Fig. 18. Figure shows the effect of temperature change on the rate of enzyme reaction. Notice the initial rise of rate of reaction and sudden fall near to optimal temperature 37-42 °C.

Think of all the things that pH changes might affect. Many chemicals such as acids or alkaline chemical compounds if mixed in enzyme reaction medium can change the pH. As a

The easiest assumption is that certain side chains necessary for catalysis must be in the correct protonation state. Thus, some side chain, with an apparent pKa of around 6, must be deprotonated for optimal activity of trypsin which shows an increase in enzyme activity with the increase in range centered at pH 6. Which amino acid side chain would be a likely candidate to participate in enzyme inhibition? It all depends on net charge on active group of each amino acid in the active site chain. The pH of reaction thus depends on net pKa value of amino acids and presence of acid or alkaline nature of substrate effects on enzyme kinetics by formation of EH, ESH as shown in Figure 19. It can be modeled at the chemical and mathematical level to calculate velocity(v), Vm(apparent) and Km(apparent) as shown in Equations 7-9. Different enzymes show different behavior of enzyme catalyzed reactions such

affect E in ways to alter the binding of S to E, which would affect Km

 affect E by globally changing the conformation of the protein affect S by altering the protonation state of the substrate

affect E in ways to alter the actual catalysis of bound S, which would affect kcat

**5. Inhibition by physiological modulators** 

enzyme denatures as shown in Figure 18.

**5.2 Hydrogen ion concentration or pH of reaction** 

result, reaction rate changes. It might

**5.1 Temperature of reaction** 

inhibitors of receptor activity behaving as competitive, noncompetitive and irreversible antagonists as shown in Figure 17. For further details, readers are requested to read advanced text book [Nelson et al. 2008, Dixon and Webb 1979]

Fig. 17. Sketch is shown for membrane receptor binding with ligand (acting as agonist) and antagonist (acting as inhibitor) in competition with agonist to bind with enzyme. Reproduced with permission [Jakobowski 2010a]

#### **5. Inhibition by physiological modulators**

#### **5.1 Temperature of reaction**

20 Enzyme Inhibition and Bioapplications

inhibitors of receptor activity behaving as competitive, noncompetitive and irreversible antagonists as shown in Figure 17. For further details, readers are requested to read

Fig. 17. Sketch is shown for membrane receptor binding with ligand (acting as agonist) and

antagonist (acting as inhibitor) in competition with agonist to bind with enzyme.

Reproduced with permission [Jakobowski 2010a]

advanced text book [Nelson et al. 2008, Dixon and Webb 1979]

Some endothermic or exothermic chemical compounds change the temperature of reaction. Enzyme reaction experiences inhibition at higher or lower than optimal physiological temperature. For example, human body optimal temperature of human body is 37 oC. For most of the enzyme reactions, enzyme activity usually increases at 0 to about 40-50 oC in the absence of catalysts. As a general rule of thumb, reaction velocities double for each increment of 10oC rise. At higher temperatures, the activity decreases dramatically as the enzyme denatures as shown in Figure 18.

Fig. 18. Figure shows the effect of temperature change on the rate of enzyme reaction. Notice the initial rise of rate of reaction and sudden fall near to optimal temperature 37-42 °C.

#### **5.2 Hydrogen ion concentration or pH of reaction**

Think of all the things that pH changes might affect. Many chemicals such as acids or alkaline chemical compounds if mixed in enzyme reaction medium can change the pH. As a result, reaction rate changes. It might


The easiest assumption is that certain side chains necessary for catalysis must be in the correct protonation state. Thus, some side chain, with an apparent pKa of around 6, must be deprotonated for optimal activity of trypsin which shows an increase in enzyme activity with the increase in range centered at pH 6. Which amino acid side chain would be a likely candidate to participate in enzyme inhibition? It all depends on net charge on active group of each amino acid in the active site chain. The pH of reaction thus depends on net pKa value of amino acids and presence of acid or alkaline nature of substrate effects on enzyme kinetics by formation of EH, ESH as shown in Figure 19. It can be modeled at the chemical and mathematical level to calculate velocity(v), Vm(apparent) and Km(apparent) as shown in Equations 7-9. Different enzymes show different behavior of enzyme catalyzed reactions such

Enzyme Inhibition: Mechanisms and Scope 23

Fig. 20. Graphs of different pH effects on enzyme catalyzed reactions as log Vm(app) and Vm/Km(app) are shown on left. Different enzymes such as chymotrypsin, cholinesterase, pepsin and papain are illustrated with different rates of enzyme reaction. Reproduced with

interactions of metal-solvent, oxygen-water molecular bridge, free energy content loss, subunit-subunit biophysical interactions as a result play a significant role in inhibitor-

For more details, readers are requested to read recent reference papers on 3D mechanistic studies on enzymes. Specific example on urease is cited in chapter 11 in this book. Now science is shifting to develop crystallized enzyme molecules, better structural-functional

In following description, factors are discussed on different practical considerations that influence the enzyme reaction rates, enzyme inhibition kinetics, % binding efficiency on enzyme solid support with a glimpse of known theories and concepts on real-time, cheaper,

When actual and practical considerations are analyzed to work in enzyme reactor, the scenario becomes complicated. Several factors such as inhibitor chemical state, substrate structure, enzyme 3D conformation or peptide subunit interactions, physiological reaction

enzyme complex formation and completion of enzyme catalysis.

relationship in enzyme catalysis and immobilized enzyme chips.

economic, user-friendly immobilized enzyme technology.

permission [Jakobowski 2010a]

as chymotrypsin, cholinesterase, papain, and papsin show distinct graphs (see Figure 20). For further details, readers are requested to read text books [Nelson et al. 2008, Berg et al. 2011]

 Vm app S V = Km app +S (7) Vm Vm app = 1+H+/Kes1+Kes2 /H+ (8)

$$\text{Km\,app} = \frac{\text{Km}(1 + \text{H} + /\text{Ke1} + \text{Ke2}/\text{H} +)}{1 + \text{H} + /\text{Ke1} + \text{Ke2}/\text{H} +} \tag{9}$$

Fig. 19. Chemical equations showing the mechanism of pH effects on enzyme catalyzed reactions. Different mathematical equations 7-9 illustrate the modeling pH effects on enzyme catalyzed reactions.

#### **5.2.1 Three dimensional nature of enzyme-inhibitor complex at enzyme active site**

The role of non-covalent interactions such as hydrogen bonding, hydrophobic interaction and orientation of inhibitor and enzyme in an organized fashion was well described in classic paper [Amtul et al., 2002]. 3D nature of enzyme reaction can be understood as following. There are two sites on enzyme molecule: 1. at allosteric site, inhibitor binds with enzyme, and 2. at active site, substrate binds with enzyme. However, substrate and inhibitor interact with each other by non-covalent interactions of their chemical groups. Inhibitors interact at allosteric site and known as 'pharmacohores'. Presently, structure-based design and testing, mechanistic biological approach is a state-of-art to develop new pharmacohores. The non-covalent interactions determine the chemoselectivity of the substrate and enzymes during formation of the ESI complex. In other words, ESI complex provides enzyme as a platform to perform catalysis. 3D geometrical shape and topology of active site match with orientation of chemical groups in substrate molecule that fit together in a 'lock and key' arrangement. Several possibilities happen to make enzyme-inhibitor complexes such as bidentate, tri-, tetra- and polydentate, trigonal, pyramidal, tetrahedral, polyhedral charge transfer complexes due to co-ordinate interactions between metallic co-factor with hydrophilic groups on inhibitor(s). In this process, geometry of amino acid side chains at allosteric site changes due to hydrogen bonding between amino acid residues. Suboptimal

as chymotrypsin, cholinesterase, papain, and papsin show distinct graphs (see Figure 20). For further details, readers are requested to read text books [Nelson et al. 2008, Berg et al. 2011]

Fig. 19. Chemical equations showing the mechanism of pH effects on enzyme catalyzed reactions. Different mathematical equations 7-9 illustrate the modeling pH effects on

**5.2.1 Three dimensional nature of enzyme-inhibitor complex at enzyme active site** 

The role of non-covalent interactions such as hydrogen bonding, hydrophobic interaction and orientation of inhibitor and enzyme in an organized fashion was well described in classic paper [Amtul et al., 2002]. 3D nature of enzyme reaction can be understood as following. There are two sites on enzyme molecule: 1. at allosteric site, inhibitor binds with enzyme, and 2. at active site, substrate binds with enzyme. However, substrate and inhibitor interact with each other by non-covalent interactions of their chemical groups. Inhibitors interact at allosteric site and known as 'pharmacohores'. Presently, structure-based design and testing, mechanistic biological approach is a state-of-art to develop new pharmacohores. The non-covalent interactions determine the chemoselectivity of the substrate and enzymes during formation of the ESI complex. In other words, ESI complex provides enzyme as a platform to perform catalysis. 3D geometrical shape and topology of active site match with orientation of chemical groups in substrate molecule that fit together in a 'lock and key' arrangement. Several possibilities happen to make enzyme-inhibitor complexes such as bidentate, tri-, tetra- and polydentate, trigonal, pyramidal, tetrahedral, polyhedral charge transfer complexes due to co-ordinate interactions between metallic co-factor with hydrophilic groups on inhibitor(s). In this process, geometry of amino acid side chains at allosteric site changes due to hydrogen bonding between amino acid residues. Suboptimal

Km app +S (7)

Vm Vm app = 1+H+/Kes1+Kes2 /H+ (8)

Km(1+H+/Ke1+Ke2/H+) Km app= 1+H+/Kes1+Kes2/H+ (9)

Vm app S V =

enzyme catalyzed reactions.

Fig. 20. Graphs of different pH effects on enzyme catalyzed reactions as log Vm(app) and Vm/Km(app) are shown on left. Different enzymes such as chymotrypsin, cholinesterase, pepsin and papain are illustrated with different rates of enzyme reaction. Reproduced with permission [Jakobowski 2010a]

interactions of metal-solvent, oxygen-water molecular bridge, free energy content loss, subunit-subunit biophysical interactions as a result play a significant role in inhibitorenzyme complex formation and completion of enzyme catalysis.

For more details, readers are requested to read recent reference papers on 3D mechanistic studies on enzymes. Specific example on urease is cited in chapter 11 in this book. Now science is shifting to develop crystallized enzyme molecules, better structural-functional relationship in enzyme catalysis and immobilized enzyme chips.

In following description, factors are discussed on different practical considerations that influence the enzyme reaction rates, enzyme inhibition kinetics, % binding efficiency on enzyme solid support with a glimpse of known theories and concepts on real-time, cheaper, economic, user-friendly immobilized enzyme technology.

When actual and practical considerations are analyzed to work in enzyme reactor, the scenario becomes complicated. Several factors such as inhibitor chemical state, substrate structure, enzyme 3D conformation or peptide subunit interactions, physiological reaction

Enzyme Inhibition: Mechanisms and Scope 25

**Matrix entrapment** is done by mixing enzyme solution with polymer fluid in matrices such as Ca-alginate, agar, polyacrylamide, collagen. **Membrane entrapment** is done by confining enzyme solutions between semi-permeable membrane hollow fibers made of nylon, cellulose, polysulfone, polyacrylate etc. Surface immobilization by **adsorption** is done by attaching enzymes on stationary solids such as alumina, porous glass, cellulose, ionexchange resin, silica, ceramic, clay, starch etc. by physical forces keeping active sites intact. **Covalent bonding** is done by enzyme retention on support surfaces by covalent binding between functional groups such as amino, carboxylic, sulfhydryl, hydroxyl groups on the enzyme and those on the support surface keeping enzyme active site(s) free (see Figure 21)

Fig. 21. Scheme of immobilization of enzyme is shown with chemical groups involved in binding of enzyme on solid surface. Reproduced with permission from reference Lieder et

Diffusional limitations are observed to various degrees in all immobilized enzyme systems. This occurs because substrate must diffuse from the bulk solution up to the surface of the immobilized enzyme prior to reaction. The rate of diffusion relative to enzyme reaction rate determines whether limitations on intrinsic enzyme kinetics is observed or not as shown in Figures 22 [Laider et al.1980]. However, rate of diffusion across and within matrix is

In immobilized enzyme reaction, two major effects due to diffusion and product inhibition are first observed by Lineweaber-Burk plots in classic study [Rees, 1984]. The diffusional effects and product inhibition both influenced the shape of Lineweaver-Burk plot (see Figure 22). In case of substrate inhibition effects binding of more than one substrate molecule(s) lead to inhibition showing same type of curved Lineweaver-Burk plot as those observed for diffusional limitation and product inhibition in immobilized enzymes. Combination of these two effects lead to intermediate behavior, such as normal Michaelis-Menten kinetics as shown

determinant of immobilized enzyme reaction as shown in Figure 22 and 23.

[Laider et al. 1980].

al.1980.

conditions in reactor and enzyme carrier supports also contribute in inhibition kinetics and rates of reaction to form ES,ESI and P. Every year list of new factors grows in new enzyme systems.

Author believes that more and more contributory factors introduced, will influence enzyme reaction rate kinetics and more and more additive kinetic constants are introduced with new variants to define the action of inhibitors on enzyme catalysis.

Other factors to keep in mind for new possibilities are:


For all these factors and details, readers are expected to read advanced text books on enzyme inhibition and enzyme engineering. Readers will experience a wide variation in the scientific analysis of enzyme inhibition data in different enzyme reactors used in different studies. High efficiency with desired results of enzyme inhibitors is the new challenges to optimize reaction, scale-up, and phase out unwanted physiological factors from reaction. In following section, these issues are addressed. Author believes that above mentioned description is just iceberg from a large hidden treasure or unknown factors contributing enzyme inhibition to give desired outcome.

#### **6. Immobilized enzyme systems**

In search of economic, efficient and practical enzyme platforms to test enzyme inhibitors, new user-friendly immobilized enzyme technology is available now. It is based on principle that an enzyme molecule is contained within confined space for the purpose of retaining and re-using enzyme on solid medium in processing system or equipment. There are many advantages of immobilized enzymes and methods of immobilization such as low cost, suitability of reusable model system in membrane-bound enzymes in cell. However, some disadvantages are expansive methods of adsorption or covalent bound or matrix trapping or membrane trapping immobilization methods, low measurement of enzyme activity with mass transfer limitations. For knowledge sake, the entrapment of enzyme molecules on matrix, diffusion phenomenon and kinetics are important to understand. A brief description is given for interested readers on classic concepts and scientific basis of porous or nonporous enzyme supports, theory of enzyme immobilization and efficiency of reaction outcome. For more details of each aspect, readers are requested to read individual research papers.

conditions in reactor and enzyme carrier supports also contribute in inhibition kinetics and rates of reaction to form ES,ESI and P. Every year list of new factors grows in new enzyme

Author believes that more and more contributory factors introduced, will influence enzyme reaction rate kinetics and more and more additive kinetic constants are introduced with new

1. enzyme autoinhibition and enzyme molecular structural-functional factors affecting 3D conformation of active site compatible with active groups of substrate or inhibitor 2. porosity and diffusion across the enzyme support material and availability of exposed

3. real-time recording the instant formation of ESI or ES or EP or EI on solid phase enzyme

5. computer based semi-corrected or averaged calculations of kinetic constants of

6. thermodynamic states of the enzyme reaction in reactor and fluctuating physiological

For all these factors and details, readers are expected to read advanced text books on enzyme inhibition and enzyme engineering. Readers will experience a wide variation in the scientific analysis of enzyme inhibition data in different enzyme reactors used in different studies. High efficiency with desired results of enzyme inhibitors is the new challenges to optimize reaction, scale-up, and phase out unwanted physiological factors from reaction. In following section, these issues are addressed. Author believes that above mentioned description is just iceberg from a large hidden treasure or unknown factors contributing

In search of economic, efficient and practical enzyme platforms to test enzyme inhibitors, new user-friendly immobilized enzyme technology is available now. It is based on principle that an enzyme molecule is contained within confined space for the purpose of retaining and re-using enzyme on solid medium in processing system or equipment. There are many advantages of immobilized enzymes and methods of immobilization such as low cost, suitability of reusable model system in membrane-bound enzymes in cell. However, some disadvantages are expansive methods of adsorption or covalent bound or matrix trapping or membrane trapping immobilization methods, low measurement of enzyme activity with mass transfer limitations. For knowledge sake, the entrapment of enzyme molecules on matrix, diffusion phenomenon and kinetics are important to understand. A brief description is given for interested readers on classic concepts and scientific basis of porous or nonporous enzyme supports, theory of enzyme immobilization and efficiency of reaction outcome. For more details of each aspect, readers are requested to read individual research

4. sustrate-inhibitor interactions, % binding of active site with each additive

and physical states of substrate, inhibitor, enzyme complexes in reactor.

7. synergy of inhibitors, substrate, subunits in enzyme on active site

variants to define the action of inhibitors on enzyme catalysis.

Other factors to keep in mind for new possibilities are:

systems.

active sites to react

support organic chip

inhibition kinetics

enzyme inhibition to give desired outcome.

**6. Immobilized enzyme systems** 

papers.

**Matrix entrapment** is done by mixing enzyme solution with polymer fluid in matrices such as Ca-alginate, agar, polyacrylamide, collagen. **Membrane entrapment** is done by confining enzyme solutions between semi-permeable membrane hollow fibers made of nylon, cellulose, polysulfone, polyacrylate etc. Surface immobilization by **adsorption** is done by attaching enzymes on stationary solids such as alumina, porous glass, cellulose, ionexchange resin, silica, ceramic, clay, starch etc. by physical forces keeping active sites intact. **Covalent bonding** is done by enzyme retention on support surfaces by covalent binding between functional groups such as amino, carboxylic, sulfhydryl, hydroxyl groups on the enzyme and those on the support surface keeping enzyme active site(s) free (see Figure 21) [Laider et al. 1980].

Fig. 21. Scheme of immobilization of enzyme is shown with chemical groups involved in binding of enzyme on solid surface. Reproduced with permission from reference Lieder et al.1980.

Diffusional limitations are observed to various degrees in all immobilized enzyme systems. This occurs because substrate must diffuse from the bulk solution up to the surface of the immobilized enzyme prior to reaction. The rate of diffusion relative to enzyme reaction rate determines whether limitations on intrinsic enzyme kinetics is observed or not as shown in Figures 22 [Laider et al.1980]. However, rate of diffusion across and within matrix is determinant of immobilized enzyme reaction as shown in Figure 22 and 23.

In immobilized enzyme reaction, two major effects due to diffusion and product inhibition are first observed by Lineweaber-Burk plots in classic study [Rees, 1984]. The diffusional effects and product inhibition both influenced the shape of Lineweaver-Burk plot (see Figure 22). In case of substrate inhibition effects binding of more than one substrate molecule(s) lead to inhibition showing same type of curved Lineweaver-Burk plot as those observed for diffusional limitation and product inhibition in immobilized enzymes. Combination of these two effects lead to intermediate behavior, such as normal Michaelis-Menten kinetics as shown

Enzyme Inhibition: Mechanisms and Scope 27

conformational change within the protein structure will initiate enzyme inactivation. As a result, covalent immobilization processes involve an initial freely-reversible stage. Covalent links may form, break and re-form till an unstrained covalently-linked structure is created. However, additional stabilization is derived from maximum enzyme-support compatibility,

least enzyme molecule interactions, least proteolytic and microbiological attacks.

Fig. 24. Effect of one or more inhibitor molecules on enzyme kinetics and their inhibition

Fig. 25. A scheme of immobilized enzyme action is shown on non-porous solid support.

The kinetic constants (e.g. Km, Vmax) of immobilized enzymes may be altered by the process of immobilization due to internal structural changes and restricted access to the active site. Thus, the intrinsic specificity (k./Km) of such enzymes may well be changed relative to the soluble enzyme. An example of trypsin is illustrated in Figure 21, where the freely soluble enzyme hydrolyses fifteen peptide bonds in the protein pepsinogen but the immobilized enzyme hydrolyses only ten. The apparent value of these kinetic parameters, when determined experimentally, may differ from the intrinsic values. This fact may be due to

Notice the dependence of Vm on available immobilized enzyme active sites (EL).

effect dependent on 1/So. Reproduced with permission from Rees et al. 1984.

in Figure 24, 25 by curves [Rees, 1984]. However, immobilized enzyme system also suffers from both diffusion and product inhibition effects. As a consequence, it is important to consider diffusion effects and product inhibition effects while extracting catalytic parameters from kinetic data for immobilized enzyme systems. Use of non-porous support in enzyme immobilization minimizes the diffusion effects to some extent.

Fig. 22. A sketch of porous matrix is shown (on left) and a scheme of substrate mass balance Equation to calculate rate of immobilized enzyme reaction rs is shown (on right)

Fig. 23. A scheme of substrate mass balance is shown to calculate S with boundary conditions.

Enzyme kinetics predicts the efficiency of reaction. Kinetics of immobilized enzymes depends on conformational alterations within the enzyme due to the immobilization procedure, or the presence and nature of the immobilization support. Immobilization can greatly affect the stability of an enzyme such as any strain into the enzyme will inactivate the enzymes under denaturing conditions (e.g. higher temperatures or extremes of pH). An example of unstrained multipoint binding between the enzyme and the support to cause substantial stabilization is illustrated in Figure 20. From mechanistic standpoint, a lesser

in Figure 24, 25 by curves [Rees, 1984]. However, immobilized enzyme system also suffers from both diffusion and product inhibition effects. As a consequence, it is important to consider diffusion effects and product inhibition effects while extracting catalytic parameters from kinetic data for immobilized enzyme systems. Use of non-porous support in enzyme

 Fig. 22. A sketch of porous matrix is shown (on left) and a scheme of substrate mass balance

Equation to calculate rate of immobilized enzyme reaction rs is shown (on right)

Fig. 23. A scheme of substrate mass balance is shown to calculate S with boundary

Enzyme kinetics predicts the efficiency of reaction. Kinetics of immobilized enzymes depends on conformational alterations within the enzyme due to the immobilization procedure, or the presence and nature of the immobilization support. Immobilization can greatly affect the stability of an enzyme such as any strain into the enzyme will inactivate the enzymes under denaturing conditions (e.g. higher temperatures or extremes of pH). An example of unstrained multipoint binding between the enzyme and the support to cause substantial stabilization is illustrated in Figure 20. From mechanistic standpoint, a lesser

conditions.

**ki kp E + S ES EP E + P**

immobilization minimizes the diffusion effects to some extent.

conformational change within the protein structure will initiate enzyme inactivation. As a result, covalent immobilization processes involve an initial freely-reversible stage. Covalent links may form, break and re-form till an unstrained covalently-linked structure is created. However, additional stabilization is derived from maximum enzyme-support compatibility, least enzyme molecule interactions, least proteolytic and microbiological attacks.

Fig. 24. Effect of one or more inhibitor molecules on enzyme kinetics and their inhibition effect dependent on 1/So. Reproduced with permission from Rees et al. 1984.

Fig. 25. A scheme of immobilized enzyme action is shown on non-porous solid support. Notice the dependence of Vm on available immobilized enzyme active sites (EL).

The kinetic constants (e.g. Km, Vmax) of immobilized enzymes may be altered by the process of immobilization due to internal structural changes and restricted access to the active site. Thus, the intrinsic specificity (k./Km) of such enzymes may well be changed relative to the soluble enzyme. An example of trypsin is illustrated in Figure 21, where the freely soluble enzyme hydrolyses fifteen peptide bonds in the protein pepsinogen but the immobilized enzyme hydrolyses only ten. The apparent value of these kinetic parameters, when determined experimentally, may differ from the intrinsic values. This fact may be due to

Enzyme Inhibition: Mechanisms and Scope 29

diffuses through the surrounding layer (external transport) in order to reach the catalytic surface and gets converted to product (P). In order for all immobilized enzyme to be utilized, substrate must diffuse within the pores in the surface of the immobilized enzyme particle (internal transport) [Pryciak 2008]. The degree of stabilization is determined by strength of the gel, and hence the number of non-covalent interactions. As a result, intrinsic parameters of enzyme result with specific apparent parameters dependent on partition and

 The porosity (e) of the particle can be expressed as ratio of the volume of solution contained within the particle to the total volume of the particle. The tortuosity (t) is the average ratio of the path length, via the pores, between any points within the particle to

The tortuosity, which is always greater than or equal to unity, depends on the pore

The concentration of the substrate at the surface of the particles [Sr] depends on radius

geometry. The diagram exaggerates dimensions for the purpose of clarity.

R or internal concentration [Si] at any smaller radius (r) is the lower value.

Fig. 27. Illustration of the use of multipoint interactions for the stabilization of enzymes. (a) **--------** activity of free un-derivatized chymotrypsin. (b) **…..** activity of chymotrypsin derivatized with acryloyl chloride. (c) **-- -- --** activity of acryloyl chymotrypsin copolymerized within a polymethacrylate gel. Up to 12 residues are covalently bound per enzyme molecule. Lower derivatization leads to lower stabilization. (d) **-----** activity of chymotrypsin noncovalently entrapped within a polymethacrylate gel. All reactions were performed at 60°C using low molecular weight artificial substrates. The immobilized chymotrypsin preparations showed stabilization of up to 100,000 fold, most of which is due to their multipoint nature although the consequent prevention of autolytic loss of enzyme activity must be a significant

contributory factor. Reproduced with permission from Martinek et al, 1977a,b.

ecological, environmental, agriculture, health, oceanic, space and earth sciences.

In general, the use of immobilized enzyme can be divided into two major categories of applications: in biosensors and bioreactors. However, list is growing in the other fields of

diffusion as shown in Figure 27.

their absolute distance apart.

changes in the properties of the solution in the immediate vicinity of the immobilized enzyme, or the effects of molecular diffusion within the local environment. The relationship between these intrinsic and apparent parameters is shown below in Figure 26. Typically, nonporous microenvironment consists of the internal solution plus part of the surrounding solution which is influenced by the surface characteristics of the immobilized enzyme. Partitioning of substances occurs between these two environments. Substrate molecule (S)

Fig. 26. A schematic cross-section of an immobilized enzyme particle (a) shows the macroenvironment and microenvironment. Triangular dots represent the enzyme molecules. Courtesy: Pangandai V. Pennirselvam, Ph.D UFRN, Lagoa Nova–Natal/RN Campus Universitário. North East, Brazil.

changes in the properties of the solution in the immediate vicinity of the immobilized enzyme, or the effects of molecular diffusion within the local environment. The relationship between these intrinsic and apparent parameters is shown below in Figure 26. Typically, nonporous microenvironment consists of the internal solution plus part of the surrounding solution which is influenced by the surface characteristics of the immobilized enzyme. Partitioning of substances occurs between these two environments. Substrate molecule (S)

Intrinsic parameters of the soluble enzyme

Intrinsic parameters of the immobilized enzyme

Apparent parameters due to partition and diffusion

Fig. 26. A schematic cross-section of an immobilized enzyme particle (a) shows the macroenvironment and microenvironment. Triangular dots represent the enzyme molecules. Courtesy: Pangandai V. Pennirselvam, Ph.D UFRN, Lagoa Nova–Natal/RN

Campus Universitário. North East, Brazil.

diffuses through the surrounding layer (external transport) in order to reach the catalytic surface and gets converted to product (P). In order for all immobilized enzyme to be utilized, substrate must diffuse within the pores in the surface of the immobilized enzyme particle (internal transport) [Pryciak 2008]. The degree of stabilization is determined by strength of the gel, and hence the number of non-covalent interactions. As a result, intrinsic parameters of enzyme result with specific apparent parameters dependent on partition and diffusion as shown in Figure 27.


Fig. 27. Illustration of the use of multipoint interactions for the stabilization of enzymes. (a) **--------** activity of free un-derivatized chymotrypsin. (b) **…..** activity of chymotrypsin derivatized with acryloyl chloride. (c) **-- -- --** activity of acryloyl chymotrypsin copolymerized within a polymethacrylate gel. Up to 12 residues are covalently bound per enzyme molecule. Lower derivatization leads to lower stabilization. (d) **-----** activity of chymotrypsin noncovalently entrapped within a polymethacrylate gel. All reactions were performed at 60°C using low molecular weight artificial substrates. The immobilized chymotrypsin preparations showed stabilization of up to 100,000 fold, most of which is due to their multipoint nature although the consequent prevention of autolytic loss of enzyme activity must be a significant contributory factor. Reproduced with permission from Martinek et al, 1977a,b.

In general, the use of immobilized enzyme can be divided into two major categories of applications: in biosensors and bioreactors. However, list is growing in the other fields of ecological, environmental, agriculture, health, oceanic, space and earth sciences.

Enzyme Inhibition: Mechanisms and Scope 31

 *Antibodies* against several nonfunctional plasma enzymes have clinical diagnostic importance since they are longer living than the enzyme itself and hence reflect the disease history better. In this respect, autoimmune antibodies are clinically important in diagnosis of autoimmune diseases, e.g., anti-glutamic acid decarboxylase antibodies in

 *Biosensors:* Light inhibits most enzyme activity although some enzymes, e.g., amylase are activated by red or green light and also specific DNA repairing enzymes (e.g., UVspecific endonuclease) are activated by the blue and UV light. Ultraviolet rays and ionizing radiations cause denaturation of most enzymes. Most enzymes contain sulfhydryl (-SH) groups at their active sites which upon oxidation by oxidants and free radicals by oxidants and free radicals inactivate the enzyme. Examples: Effect of

 Other application of membrane bound redox enzymes constitutes them as a scaffolding enzyme arrangement into systems for multi-step catalytic processes. The reconstruction of portions of this redox catalytic machinery, interfaced to an electrical circuit leads to novel biosensing devices or biosensors. An example of nitric oxide synthase enzyme is

 In **EzNET®** water purifying system, nitrate pollution is eliminated. Enzyme is immobilized on "beads" with an electron-carrying dye as shown in Figure 28. Reduction of nitrate to environmentally safe nitrogen gas is driven by a low voltage

Fig. 28. EzNET® system shows immobilized enzyme on "beads" with an electron-carrying dye. In this system, reduction of nitrate generates environmentally safe nitrogen gas driven

by a low voltage direct current. Source: The Nitrate Elimination Co., Inc. 2000.

radiations, light and oxidants on the rate of the enzyme catalyzed reaction.

several side-effects as drugs.

type 1 diabetes mellitus.

cited in this book [Sharma, 2012b].

direct current.

Immunodeficiency Virus (HIV)-induce acquired immunodeficiency syndrome (AIDS). It inhibits the HIV protease that cleaves the large multidomain viral protein into active enzyme subunits. Because these peptide inhibitors may not be specific, they have

#### **7. New developments in art of enzyme inhibition**

Now a day, immobilized enzymes are used in industries and have value as medicinal and industrial enzyme products. Good examples of industrial enzymes are amylase, glucoamylase, trypsin, pepsin, rennet, glucose isomerase, penicillinase, glucose oxidase, lipase, invertase, pectinase, cellulase in medicinal use. With emergence of new inhibitors in the quest of drug discovery, several new inhibition mechanisms are expected in case of new substrate analogues. New substrate–enzyme active site interactions are envisaged due to different binding intricacies. Some examples of emerging concepts are outlined in following description and readers are expected to read advanced literature on these applications.


Now a day, immobilized enzymes are used in industries and have value as medicinal and industrial enzyme products. Good examples of industrial enzymes are amylase, glucoamylase, trypsin, pepsin, rennet, glucose isomerase, penicillinase, glucose oxidase, lipase, invertase, pectinase, cellulase in medicinal use. With emergence of new inhibitors in the quest of drug discovery, several new inhibition mechanisms are expected in case of new substrate analogues. New substrate–enzyme active site interactions are envisaged due to different binding intricacies. Some examples of emerging concepts are outlined in following description and readers are expected to read advanced literature on these applications.

 *Slow-tight inhibition:* Slow-tight inhibition occurs when the initial enzyme-inhibitor complex EI undergoes isomerizing conformational change to a more tightly binding complex. However, the overall inhibition process is reversible. This manifests itself as slowly increasing enzyme inhibition. Under these conditions, traditional Michaelis-Menten kinetics gives a false value of a time-dependent Ki. The true value of Ki can be obtained through more complex analysis of the on (kon) and off (koff) rate constants for

 *Substrate and product inhibition:* Substrate and product inhibition is where either the substrate or product of an enzyme reaction inhibits the enzyme's activity. This inhibition may follow the competitive, uncompetitive or mixed patterns. In substrate inhibition there is a progressive decrease in activity at high substrate concentrations. This may indicate the existence of two substrate-binding sites in the enzyme. At low substrate, the high-affinity site is occupied and normal kinetics is followed. However, at higher concentrations, the second inhibitory site becomes occupied, inhibiting the enzyme. Product inhibition is often a regulatory feature in metabolism and can also be a

form of negative feedback; see allosteric regulation [Pryciak 2008, Bashor 2008]. *Antimetabolites:* They are chemicals that interfere with the normal metabolism of normal biochemical metabolite(s). This in most of case is due to their structural similarity to such physiological substrates and therefore works as competitive enzyme inhibitors. They include antifolates such as methotrexate, hydroxyurea and purine and pyrimidine analogues. They are mainly used as cytotoxic anticancer drugs through inhibiting DNA and RNA synthesis and cell division. An example of nitroimidazole is described in detail on its metabolic effects at cellular level in this book [Sharma 2012a]. *Antienzyme:* Intestinal parasites, e.g., Ascaris, protect themselves from digestion by expressing on their surface substances that are protein in nature which inhibit the action of digestive enzymes, e.g., pepsin and trypsin. The blood plasma and extracellular fluids are containing several types of protease inhibitors particularly important in controlling the blood clot formation and dissolution and matrix and cytokine homeostasis. Most of these inhibitors are peptides and several of them are also isolated from raw egg white, potatoes, tomatoes and Soya bean and other plant sources. Most of the natural peptide protease inhibitors are similar in structure to the amino acid sequence of the peptide substrates of the enzyme. Designed peptide protease inhibitors are important drugs, e.g., captopril that is a metalloprotease angiotensin-converting enzyme peptide inhibitor. Inhibiting this enzyme prevent activation of angiotensin and therefore prevent vasoconstriction to lower blood pressure. Crixivan is an antiretroviral aspartyl protease peptide inhibitor used in the treatment of Human

**7. New developments in art of enzyme inhibition** 

inhibitor association.

Immunodeficiency Virus (HIV)-induce acquired immunodeficiency syndrome (AIDS). It inhibits the HIV protease that cleaves the large multidomain viral protein into active enzyme subunits. Because these peptide inhibitors may not be specific, they have several side-effects as drugs.


Fig. 28. EzNET® system shows immobilized enzyme on "beads" with an electron-carrying dye. In this system, reduction of nitrate generates environmentally safe nitrogen gas driven by a low voltage direct current. Source: The Nitrate Elimination Co., Inc. 2000.

Enzyme Inhibition: Mechanisms and Scope 33

applications in pharmacy industry [Bartolini et al. 2005, 2007].

Source: Kim et al. AIChEngg Annual Meeting 2003, San Francisco, CA.

**9. Limitations and challenges** 

Fig. 30. Inhibition of luciferase activity by increasing the concentration of chloroform.

Recently softwares have popped up to visualize custom visual interface to see curve fits in real-time, graph transforms, equations using kinetic data entry in terms of substrate, inhibitor, activator, velocity, and standard deviation of the velocity. Data tables are directly generated linked to the Fitting Panel of software. The data and results analysis is transferred in userfriendly lay-out, ANOVA window, % inhibition using Monte-Carlos fits, and receptor or ligand binding calculator. For interested readers, VISUALENZYMICS 2010® is available for statistical analysis for enzyme kinetics.[ http://www.softzymics.com/visualenzymics.htm].

Above mentioned description on mechanism and applications shows a clear issue on need of careful analysis for enzyme inhibition factors, presumptions of enzyme reaction, use of new immobilized enzyme support and enzyme recording/monitoring methods. Challenge is that most of times, basic presumptions do not hold true in enzyme reactors and addition of new factors further complicate the calculation of reactor outcome. Most of the times, computer based kinetic calculations average out outcome as less realistic with more chances of variants. Other major challenge is that each time enzyme reactor outcome depends on individual inhibitor and individual enzyme reactor at different times. It is less reproducible

**8. Softwares and computerization in enzyme inhibition kinetics** 

increased data output, reliability and stability to translate into cost reduction for potential

 In biolumescence detection for toxicity of HPV chemicals or drug development, 62 kDa MW oxygenase (yellow green light emitted at 560 nm) enzyme gives 88 photon/cycle light output proportional to [ATP] according to:

#### **Luciferin + luciferase + ATP luciferyl adenylate-luciferase + pyrophosphate**

#### **Luciferyl adenylate-luciferase + O2 Oxyluciferin + luciferase + AMP + light**

Strong inhibition of luciferase by chloroform or HPV chemicals indicates the efficiency of immobilized recombinant luciferase enzyme system as shown in Figure 20. Inhibition by chloroform is much reduced in the mutant Luciferase compared to the wild type Luciferase as shown in Figures 29, 30.

Source: Kim et al. AIChEngg Annual Meeting 2003, San Francisco, CA.

Fig. 29. A sketch of recombinant luciferase is shown illustrating the gene clone.

 In the search for new therapeutics, the high throughput screening (HTS) of ligands for key target proteins, enzymes represent the principal hit identification tool for early drug discovery [Bartolini et al. 2009]. However, output depends on cost-based or amount-based limitation of target availability, need of speed, automation and easy coupling of the enzyme assay with separation systems (affinity chromatography of immobilized proteins) and appropriate detectors. Good example is targeting in drug discovery represented by enzyme inhibition mechanism in monolithic immobilized enzyme reactors (IMERs) to represent different phases of the drug discovery pathway-starting with active compounds (hit) identification, through drug development and lead optimization, early ADMET (absorption, distribution, metabolism, excretion, toxicity) studies and quality control of protein drugs. Some details are described in chapters in this book [Bartolini et al. 2005, 2007]. Interested readers are requested to read advanced text books on these aspects. Different IMER have own requirements for optimal performances to show an

 In biolumescence detection for toxicity of HPV chemicals or drug development, 62 kDa MW oxygenase (yellow green light emitted at 560 nm) enzyme gives 88 photon/cycle

**Luciferin + luciferase + ATP luciferyl adenylate-luciferase + pyrophosphate** 

**Luciferyl adenylate-luciferase + O2 Oxyluciferin + luciferase + AMP + light**  Strong inhibition of luciferase by chloroform or HPV chemicals indicates the efficiency of immobilized recombinant luciferase enzyme system as shown in Figure 20. Inhibition by chloroform is much reduced in the mutant Luciferase compared to the wild type Luciferase

light output proportional to [ATP] according to:

Source: Kim et al. AIChEngg Annual Meeting 2003, San Francisco, CA.

Fig. 29. A sketch of recombinant luciferase is shown illustrating the gene clone.

 In the search for new therapeutics, the high throughput screening (HTS) of ligands for key target proteins, enzymes represent the principal hit identification tool for early drug discovery [Bartolini et al. 2009]. However, output depends on cost-based or amount-based limitation of target availability, need of speed, automation and easy coupling of the enzyme assay with separation systems (affinity chromatography of immobilized proteins) and appropriate detectors. Good example is targeting in drug discovery represented by enzyme inhibition mechanism in monolithic immobilized enzyme reactors (IMERs) to represent different phases of the drug discovery pathway-starting with active compounds (hit) identification, through drug development and lead optimization, early ADMET (absorption, distribution, metabolism, excretion, toxicity) studies and quality control of protein drugs. Some details are described in chapters in this book [Bartolini et al. 2005, 2007]. Interested readers are requested to read advanced text books on these aspects. Different IMER have own requirements for optimal performances to show an

as shown in Figures 29, 30.

increased data output, reliability and stability to translate into cost reduction for potential applications in pharmacy industry [Bartolini et al. 2005, 2007].

Fig. 30. Inhibition of luciferase activity by increasing the concentration of chloroform.

#### **8. Softwares and computerization in enzyme inhibition kinetics**

Recently softwares have popped up to visualize custom visual interface to see curve fits in real-time, graph transforms, equations using kinetic data entry in terms of substrate, inhibitor, activator, velocity, and standard deviation of the velocity. Data tables are directly generated linked to the Fitting Panel of software. The data and results analysis is transferred in userfriendly lay-out, ANOVA window, % inhibition using Monte-Carlos fits, and receptor or ligand binding calculator. For interested readers, VISUALENZYMICS 2010® is available for statistical analysis for enzyme kinetics.[ http://www.softzymics.com/visualenzymics.htm].

#### **9. Limitations and challenges**

Above mentioned description on mechanism and applications shows a clear issue on need of careful analysis for enzyme inhibition factors, presumptions of enzyme reaction, use of new immobilized enzyme support and enzyme recording/monitoring methods. Challenge is that most of times, basic presumptions do not hold true in enzyme reactors and addition of new factors further complicate the calculation of reactor outcome. Most of the times, computer based kinetic calculations average out outcome as less realistic with more chances of variants. Other major challenge is that each time enzyme reactor outcome depends on individual inhibitor and individual enzyme reactor at different times. It is less reproducible

Enzyme Inhibition: Mechanisms and Scope 35

Amtul, Z. Atta, Ur. R., Siddiqui, R.A., Choudhary, M. I. (2002). Chemistry and mechanism of

Bartolini M., Cavrini V., Andrisano V. (2005) *J. Chromatogr A,* Choosing the right

Bartolini M, Greig NH, Yu QS, Andrisano V*.* (2009) Immobilized butyrylcholinesterase in

Bartolini M., Cavrini V., Andrisano V. Characterization of reversible and irreversible

Bartolini M, Andrisano V. (2009) Immobilized enzyme reactors into the drug discovery

Bashor, C.J., Helman,N.C., Yan, S., Lim, W.A. Using Engineered Scaffold Interactions to

Berg, J.M., Tymoczko, J.L., Stryer, L. (2011) Biochemistry ISBN-13: 978-1429231152, Freeman

El-Metwally, T.H., El-Senosi, Y. (2010) Enzyme Inhibition. Medical Enzymology: Simplified

Jakbowski H. (2010a) Personal communication. Online study. Chapter 6- Transport and

http://employees.csbsju.edu/hjakubowski/classes/ch331/transkinetics/olcompli

http://employees.csbsju.edu/hjakubowski/classes/ch331/transkinetics/olinhibiti

Laider, K., Bunting, P. (1980) The kinetics of immonbilized enzyme systems*. Methods* 

Martinek, K., Klibanov, A.M., Goldmacher, V.S. & Berezin, I.V. (1977a) The principles of

Martinek, K., Klibanov, A.M., Goldmacher, V.S., Tchernysheva, A.V., Mozhaev, V.V., Berezin,

Nelson, D.L., Cox, M.M. (2008) Lehninger Principles of Biochemistry. 5th Edition ISBN-13:

polymeric support. *Biochimica et Biophysica Acta* Vol 485, pp 13-28.

enzyme stabilization 1. Increase in thermostability of enzymes covalently bound to a complementary surface of a polymer support in a multipoint fashion. *Biochimica* 

I.V. & Glotov, B.O. (1977b) The principles of enzyme stabilization 2. Increase in the thermostability of enzymes as a result of multipoint noncovalent interaction with a

Kinetics. C. Models of Enzyme Inhibition and D. More complicated Enzymes.

Cleland, W.W.(1979) Substrate inhibition, *Methods Enzymol.* Vol 63, pp 500-513. Dixon,M., Webb,E.C. (1979) Enzymes, 3rd ed., Academic Press, New York.

Approach.Chapter 6, Nova Publishers, NY. pp 57-77.

chromatographic support in making a new acetylcholinesterase microimmobilized

the characterization of new inhibitors that could ease Alzheimer's disease. *J* 

acetylcholinesterase inhibitors by means of an immobilized enzyme reactor. *J.* 

Reshape MAP Kinase Pathway Signaling Dynamics. *Science.*Vol 319 (5869), pp1539-

urease inhibition. *Current Medicinal Chemistry,* Vol 9, pp 1323-1348.

enzyme reactor for drug discovery. Vol 1065, pp 135-144.

*Chromatogr A*. Vol 1216(13), pp 2730-38.

*Chromatogr. A* (2007) Vol 1144, pp 102 –10.

process: The Alzheimer's Disease case. Web Source: http://www.farm.unipi.it/npcf3/pdf/BartoliniManuela.pdf

**13. References** 

1543


WH and Company.

Internet source.

on.html

catedenzyme.html

*Enzymol.* Vol 64, pp 227-248.

*et Biophysica Acta*, Vol 485, pp 1-12.

978071677108, Freeman W.H. and Company.

Pryciak, P. (2008) Customized Signaling Circuits. Science 319, pg 1489.

and unpredictable because of synergy, interplay of known and unknown physical, physiological, biological, molecular factors affecting reaction kinetics.

#### **10. Impact of enzyme inhibition science in business**

The major current and emerging therapeutic markets for enzyme inhibitors used in human therapeutics are very high. New information is available on biochemistry for enzyme inhibitors and classes of enzyme inhibiting products with broad current or potential therapeutic applications in large markets. However, more than 100 enzyme inhibitors are currently marketed and double than those are under development. A better understanding of the emerging enzyme inhibitors on enzyme mechanism is main key. These include selected indications for asthma and chronic obstructive pulmonary disease (COPD), cardiovascular diseases, erectile dysfunction, gastrointestinal disorders, hepatitis B virus infection, hepatitis C virus infection, herpesvirus infections, human immunodeficiency virus (HIV)/acquired immune deficiency syndrome (AIDS) and rheumatoid arthritis and related inflammatory diseases. Key information from the business literature and thorough enzyme inhibition research is the basis of expert opinion on commercial potential and market sizes from enzyme industry professionals. Since initial reports on chemical immobilization of proteins and enzymes first appeared ∼30 years ago, immobilized proteins are now widely used for the processing of products in industries from food business to environmental control. In recent years, use of chemical immobilization was extended to immobilized antibodies or antigens in bioaffinity chromatography. In coming years, it is speculated that immobilization techniques of proteins and enzymes will have greater impact on point-ofcare medical and health business.

#### **11. Conclusion**

Enzyme inhibition is significant biological process to characterize the enzyme reaction, extraction of catalysis parameters in bio-industry and bioengineering. Conceptual models of inhibition define the interactions of substrate-enzyme or inhibitor-enzyme or both substrateenzyme-inhibitor in the moiety of active site. In recent years, application of enzymes and enzyme inhibition science have gone in healthcare, pharmaceutical, bio-industries, environment, and biochemical enzyme chip industries with great impact on healthcare and medical business. Last decade has shown the measurement and accuracy of enzyme detection up to the scale of picometer and enzyme industry is entering in the area of picotechnology. Immobilized enzyme technology has given a new way of economic tools in drug discovery and biosensor industry. Every year new enzyme inhibitors are discovered useful as drugs but success still needs to minimize challenges.

#### **12. Acknowledgements**

Author acknowledges the suggestions of Dr Pagandai V. Pannirselvam, MTech, Ph.D at Centro de Technologia, UFRN, Lagoa Nova–Natal/RN Campus Universitário. North East, Brazil. Author contributed to explain intriguing issues on enzyme inhibition and highlighted the need of better understanding on mechanism of inhibitors before applying them in industries.

#### **13. References**

34 Enzyme Inhibition and Bioapplications

and unpredictable because of synergy, interplay of known and unknown physical,

The major current and emerging therapeutic markets for enzyme inhibitors used in human therapeutics are very high. New information is available on biochemistry for enzyme inhibitors and classes of enzyme inhibiting products with broad current or potential therapeutic applications in large markets. However, more than 100 enzyme inhibitors are currently marketed and double than those are under development. A better understanding of the emerging enzyme inhibitors on enzyme mechanism is main key. These include selected indications for asthma and chronic obstructive pulmonary disease (COPD), cardiovascular diseases, erectile dysfunction, gastrointestinal disorders, hepatitis B virus infection, hepatitis C virus infection, herpesvirus infections, human immunodeficiency virus (HIV)/acquired immune deficiency syndrome (AIDS) and rheumatoid arthritis and related inflammatory diseases. Key information from the business literature and thorough enzyme inhibition research is the basis of expert opinion on commercial potential and market sizes from enzyme industry professionals. Since initial reports on chemical immobilization of proteins and enzymes first appeared ∼30 years ago, immobilized proteins are now widely used for the processing of products in industries from food business to environmental control. In recent years, use of chemical immobilization was extended to immobilized antibodies or antigens in bioaffinity chromatography. In coming years, it is speculated that immobilization techniques of proteins and enzymes will have greater impact on point-of-

Enzyme inhibition is significant biological process to characterize the enzyme reaction, extraction of catalysis parameters in bio-industry and bioengineering. Conceptual models of inhibition define the interactions of substrate-enzyme or inhibitor-enzyme or both substrateenzyme-inhibitor in the moiety of active site. In recent years, application of enzymes and enzyme inhibition science have gone in healthcare, pharmaceutical, bio-industries, environment, and biochemical enzyme chip industries with great impact on healthcare and medical business. Last decade has shown the measurement and accuracy of enzyme detection up to the scale of picometer and enzyme industry is entering in the area of picotechnology. Immobilized enzyme technology has given a new way of economic tools in drug discovery and biosensor industry. Every year new enzyme inhibitors are discovered

Author acknowledges the suggestions of Dr Pagandai V. Pannirselvam, MTech, Ph.D at Centro de Technologia, UFRN, Lagoa Nova–Natal/RN Campus Universitário. North East, Brazil. Author contributed to explain intriguing issues on enzyme inhibition and highlighted the need of better understanding on mechanism of inhibitors before applying

physiological, biological, molecular factors affecting reaction kinetics.

**10. Impact of enzyme inhibition science in business** 

useful as drugs but success still needs to minimize challenges.

care medical and health business.

**12. Acknowledgements** 

them in industries.

**11. Conclusion** 


http://www.farm.unipi.it/npcf3/pdf/BartoliniManuela.pdf


 http://employees.csbsju.edu/hjakubowski/classes/ch331/transkinetics/olcompli catedenzyme.html


 http://employees.csbsju.edu/hjakubowski/classes/ch331/transkinetics/olinhibiti on.html


**Section 2** 

**Applications of Enzyme Inhibition** 


## **Section 2**

**Applications of Enzyme Inhibition** 

36 Enzyme Inhibition and Bioapplications

Rees, D.C. (1984) A general solution for the steady state kinetics of immobilized enzyme

Sami, A.J., Shakoor, A.R.. (2011) Cellulase activity inhibition and growth retardation of

Sharma,R. (1990) The effect of nitroimidazoles on isolated liver cell metabolism during

Sharma, R. (2012a) Mechanisms of Hepatocellular Dysfunction and Regeneration: Enzyme

Sharma, R. (2012b) Inhibition of Nitric Oxide Synthase Gene Expression: *In Vivo* Imaging

associated bacterial strains of Aulacophora foviecollis by two glycosylated flavonoids isolated from Mangifera indica leaves. *Journal of Medicinal Plants* 

development of amoebic liver abscess. Dissertation submitted to Indian institute of

Inhibition by Nitroimidazole and Human Liver Regeneration. In: *Enzyme Inhibition: Concepts and Bioapplications*. Chapter 7, InTech Web Publishers, Croatia. ISBN 979-

Approaches of Nitric Oxide with Multimodal Imaging. In: *Enzyme Inhibition: Concepts and Bioapplications*. Chapter 8, InTech Web Publishers, Croatia. ISBN 979-

systems. *Bulletin of Mathematical Biology*, Vol 46, 2,pp 229-234.

*Research* (2011) Vol. 5(2), pp. 184-190.

Technology, Delhi and CCS University.

953-307-301-8.

953-307-301-8.

**2** 

*Jamaica* 

**Cytochrome P450 Enzyme** 

Simone Badal, Mario Shields and Rupika Delgoda

Cytochrome P450 (CYP) is a heme containing enzyme superfamily that catalyzes the oxidative biotransformation of lipophilic substrates to hydrophilic metabolites facilitating their removal from cells. The CYPs were first recognized by Martin Klingenberg (Klingenberg, 1958) while studying the spectrophotometric properties of pigments in a microsomal fraction prepared from rat livers. When a diluted microsomal preparation was reduced by sodium dithionite and exposed to carbon monoxide gas, a unique spectral absorbance band with a maximum at 450nm appeared. The ferric ion in the resting heme, binds easily with CO following reduction, and the complex's maximal absorbance band,

CYPs are mostly located in the endoplasmic reticulum, and to some extent in mitochondrial fractions of hepatic and extra-hepatic tissues. Even though these enzymes are ubiquitous in the body (Table 1), of the 18 families in mammals identified, 11 are expressed in a typical human liver (CYP1A2, CYP2A6, CYP2B6, CYP2C8/9/18/19, CYP2D6, CYP2E1, and CYP3A4/5). In addition, five of these enzymes (CYPs 1A2, 2C9, 2C19, 2D6 and 3A4) expressed at high levels in the liver demonstrate a broad substrate selectivity which

The metabolism of a drug can be altered by another drug or foreign chemical and such interactions can often be clinically significant. As a result, the FDA (Food and Drug Administration) and other regulatory agencies such as the Department of Health and Human Services (DHHS), Centers for Disease Control and Prevention (CDS) and Hazard Analysis Critical Control Point (HACCP) among others expect information on the relationship between each new drug to CYP enzymes (substrate, inhibitor and or inducer) making these enzymes vital in the process of drug discovery. One of the major concerns is avoiding drug interactions, an issue whose importance increases with the aging of population (Guengerich, 2003) along with the increase in the practice of

unique amongst hemeproteins, serves as the signature of CYP enzymes.

accounts for about 95% of drug metabolism (Nelson, 2009; Treasure, 2000).

**1. Introduction** 

polypharmacy.

**1.1 Cytochrome P450** 

**Inhibitors from Nature** 

*University of the West Indies/ Natural Products Institute* 

## **Cytochrome P450 Enzyme Inhibitors from Nature**

Simone Badal, Mario Shields and Rupika Delgoda *University of the West Indies/ Natural Products Institute Jamaica* 

#### **1. Introduction**

#### **1.1 Cytochrome P450**

Cytochrome P450 (CYP) is a heme containing enzyme superfamily that catalyzes the oxidative biotransformation of lipophilic substrates to hydrophilic metabolites facilitating their removal from cells. The CYPs were first recognized by Martin Klingenberg (Klingenberg, 1958) while studying the spectrophotometric properties of pigments in a microsomal fraction prepared from rat livers. When a diluted microsomal preparation was reduced by sodium dithionite and exposed to carbon monoxide gas, a unique spectral absorbance band with a maximum at 450nm appeared. The ferric ion in the resting heme, binds easily with CO following reduction, and the complex's maximal absorbance band, unique amongst hemeproteins, serves as the signature of CYP enzymes.

CYPs are mostly located in the endoplasmic reticulum, and to some extent in mitochondrial fractions of hepatic and extra-hepatic tissues. Even though these enzymes are ubiquitous in the body (Table 1), of the 18 families in mammals identified, 11 are expressed in a typical human liver (CYP1A2, CYP2A6, CYP2B6, CYP2C8/9/18/19, CYP2D6, CYP2E1, and CYP3A4/5). In addition, five of these enzymes (CYPs 1A2, 2C9, 2C19, 2D6 and 3A4) expressed at high levels in the liver demonstrate a broad substrate selectivity which accounts for about 95% of drug metabolism (Nelson, 2009; Treasure, 2000).

The metabolism of a drug can be altered by another drug or foreign chemical and such interactions can often be clinically significant. As a result, the FDA (Food and Drug Administration) and other regulatory agencies such as the Department of Health and Human Services (DHHS), Centers for Disease Control and Prevention (CDS) and Hazard Analysis Critical Control Point (HACCP) among others expect information on the relationship between each new drug to CYP enzymes (substrate, inhibitor and or inducer) making these enzymes vital in the process of drug discovery. One of the major concerns is avoiding drug interactions, an issue whose importance increases with the aging of population (Guengerich, 2003) along with the increase in the practice of polypharmacy.

Cytochrome P450 Enzyme Inhibitors from Nature 41

competitive, noncompetitive, or uncompetitive inhibition of drug metabolizing enzymes or

Several epidemiological surveys including ones conducted by our laboratory (Delgoda *et al*., 2004; Delgoda *et al*., 2010; Picking *et al*., 2011) have indicated high usage of herbal medicines along with prescription medicines with low physician awareness. With over 80% of the prescription medicine users also seeking some form of herbal remedy in Jamaica, the chances of drug interactions rises and this prompted investigations into likely pharamacokinetic, metabolism based interactions between the two types of medicines.

The CYP enzymes, responsible for the metabolism of over 90% of drugs in the market is unsurprisingly associated with numerous metabolism related drug interactions (Guengerich, 1997), including those of drugs and herbs (Ioannides, 2002; Delgoda and Westlake, 2004). The inhibition of CYP3A4 by fucocoumarins found in grapefruit juice leading to clinically observable toxicities with drugs and the induction of the same CYP3A4 enzyme by ingredients found in St. John's wort leading to subtherapeutic interferences with cycloporin provide suitable examples for the involvement of CYP enzymes in drug herb interactions. While clinical studies provide the ultimate proof for relevant drug interactions, *in-vitro* laboratory evaluations with CYP enzymes, has provided a convenient, economical and useful starting point for screening those herbs that may ultimately cause clinically observable drug interactions. Human liver microsomes, heterologously expressed enzymes and hepatocytes although with limitations, have provided convenient means for such initial

In this chapter, we describe for the first time, the initial inhibitory impact of four commonly consumed infusions on six major CYP enzymes. Our findings support that the teas are moderate to weak CYP inhibitors and so we postulate that they would unlikely result in

Approximately five decades of systematic drug discovery and development have established a reliable collection of chemotherapeutic agents (Yarbro, 1992; Chabner, 1991). These chemotherapeutic agents have assisted with numerous successes in the treatment and

Chemoprevention is the ability of compounds to protect healthy tissues via the prevention, inhibition or reversal of caricnogenesis. The inhibition of CYP1 enzymes is one such route among others that include the induction of cell cycle arrest, the induction of phase II enzymes and the inhibition of inflammatory. The CYP1 family has been linked with the activation of pro-carcinogens which is facilitated by the regulation of the aryl hydrocarbon receptor. As such research has shown that inhibiting CYP1 enzymes plays a key role in

Among the polycyclic hydrocarbons that are activated into reactive metabolites by CYPs 1A1 and 1B1 is benzo-a-pyrene [BaP]. Metabolites from BaP include phenols, polyphenols, quinines, epoxides and dihydrodiols. Among these dihydrodiols; (-)-benzo[a]pyrene-trans-7,8-dihydrodiol (BPD) and (+)-anti-benzo[a]pyrene-trans-7,8-dihydrodiol-9,10-epoxide (anti-

protecting healthy cells from the harmful effects of activated carcinogens.

**1.3.2 CYP inhibition and its relation to chemoprevention** 

management of human cancers (Chabner *et al*., 1991).

enzyme induction by the phytopharmaceutical (Delgoda and Westlake, 2004).

assessements.

drug interactions.


Table 1. Human cytochrome P450 genes expressed in different parts of the respiratory and gastrointestinal tracts (adopted from Ding and Kaminsky, 2003).

#### **1.2 Classification of CYP enzymes**

All eukaryotic CYPs except fungal CYP55s are membrane bound; 18 mammalian CYP enzyme structures are known and 15 of these are of human origin; [1A2, 2A6, 2A13, 2B4 rabbit, 2B6, 2C5 rabbit, 2C8, 2C9, 2D6, 2E1, 2R1, 3A4, 7A1, 8A1, 19A1, 24A1 rat, 46A1, 51A1, (Nelson and Nebert, 2011)]. CYPs sharing >40% sequence identity are categorised within the same family while those with >55% sequence identity are placed within the same subfamily. The CYP superfamily members are named according to a nomenclature system that was established in the mid-1980s (Nebert *et al*., 1987), however, the last comprehensive revision was published in 1996 (Nelson *et al*., 1996).

CYP2 is the largest CYP450 family in mammals with 13 subfamilies and 16 genes in humans. CYPs2C8, 2C9, 2C18 and 2C19 jointly metabolise more than 50 drugs whilst CYP2D6 metabolises more than 70 drugs (Meyer and Zanger, 1997). CYP3A is the most abundantly expressed CYP450 gene in the human liver and gastrointestinal tract (Nelson, 1999) and is known to metabolise more than 120 commonly prescribed pharmaceutical agents.

CYPs1Al and 1B1 are predominately expressed in extra-hepatic tissues (Guengerich and Shimada, 1991; Shimada *et al*., 1992) while CYP1A2 is expressed primarily in the liver. As a result, constitutive levels of CYP1A2 are much greater than those of CYPs1A1 and 1B1 (Shimada *et al*., 1992; Shimada *et al*., 1994b) whose levels are usually induced by PAHs. All 3 members of the CYP1 family are upregulated by halogenated and polycyclic aromatic hydrocarbons such as those found in cigarette smoke and charred food.

#### **1.3 Importance of CYP enzyme inhibition**

#### **1.3.1 Involvement in drug interactions**

The metabolism of a drug can be altered by another drug or foreign chemical and such interactions can often be clinically significant. The observed induction and inhibition of CYP enzymes by various traditional remedies have led to the general acceptance that natural therapies can have adverse effects. This is contrary to the popular beliefs in countries where there is an active practice of ethnomedicine. Drug-herb interactions may involve

Lung 1A1, 1A2, 1B1, 2A6, 2A13, 2B6, 2C8, 2D6, 2E1, 2F1, 2J2, 2S1,

Table 1. Human cytochrome P450 genes expressed in different parts of the respiratory and

All eukaryotic CYPs except fungal CYP55s are membrane bound; 18 mammalian CYP enzyme structures are known and 15 of these are of human origin; [1A2, 2A6, 2A13, 2B4 rabbit, 2B6, 2C5 rabbit, 2C8, 2C9, 2D6, 2E1, 2R1, 3A4, 7A1, 8A1, 19A1, 24A1 rat, 46A1, 51A1, (Nelson and Nebert, 2011)]. CYPs sharing >40% sequence identity are categorised within the same family while those with >55% sequence identity are placed within the same subfamily. The CYP superfamily members are named according to a nomenclature system that was established in the mid-1980s (Nebert *et al*., 1987), however, the last comprehensive revision

CYP2 is the largest CYP450 family in mammals with 13 subfamilies and 16 genes in humans. CYPs2C8, 2C9, 2C18 and 2C19 jointly metabolise more than 50 drugs whilst CYP2D6 metabolises more than 70 drugs (Meyer and Zanger, 1997). CYP3A is the most abundantly expressed CYP450 gene in the human liver and gastrointestinal tract (Nelson, 1999) and is

CYPs1Al and 1B1 are predominately expressed in extra-hepatic tissues (Guengerich and Shimada, 1991; Shimada *et al*., 1992) while CYP1A2 is expressed primarily in the liver. As a result, constitutive levels of CYP1A2 are much greater than those of CYPs1A1 and 1B1 (Shimada *et al*., 1992; Shimada *et al*., 1994b) whose levels are usually induced by PAHs. All 3 members of the CYP1 family are upregulated by halogenated and polycyclic aromatic

The metabolism of a drug can be altered by another drug or foreign chemical and such interactions can often be clinically significant. The observed induction and inhibition of CYP enzymes by various traditional remedies have led to the general acceptance that natural therapies can have adverse effects. This is contrary to the popular beliefs in countries where there is an active practice of ethnomedicine. Drug-herb interactions may involve

known to metabolise more than 120 commonly prescribed pharmaceutical agents.

hydrocarbons such as those found in cigarette smoke and charred food.

**Organ CYPs detected** 

**1.2 Classification of CYP enzymes** 

was published in 1996 (Nelson *et al*., 1996).

**1.3 Importance of CYP enzyme inhibition 1.3.1 Involvement in drug interactions** 

Trachea 2A6, 2A13, 2B6, 2S1

Nasal mucosa 2A6, 2A13, 2B6, 2C, 2J2, 3A

Oesophagus 1A1, 1A2, 2A, 2E1, 2J2, 3A5 Stomach 1A1, 1A2, 2C, 2J2, 2S1, 3A4

Colon 1A1, 1A2, 1B1, 2J2, 3A4, 3A5

gastrointestinal tracts (adopted from Ding and Kaminsky, 2003).

3A4, 3A5, 4B1

Small Intestine 1A1, 1B1, 2C9, 2C19, 2D6, 2E1,2J2, 2S1, 3A4, 3A5

competitive, noncompetitive, or uncompetitive inhibition of drug metabolizing enzymes or enzyme induction by the phytopharmaceutical (Delgoda and Westlake, 2004).

Several epidemiological surveys including ones conducted by our laboratory (Delgoda *et al*., 2004; Delgoda *et al*., 2010; Picking *et al*., 2011) have indicated high usage of herbal medicines along with prescription medicines with low physician awareness. With over 80% of the prescription medicine users also seeking some form of herbal remedy in Jamaica, the chances of drug interactions rises and this prompted investigations into likely pharamacokinetic, metabolism based interactions between the two types of medicines.

The CYP enzymes, responsible for the metabolism of over 90% of drugs in the market is unsurprisingly associated with numerous metabolism related drug interactions (Guengerich, 1997), including those of drugs and herbs (Ioannides, 2002; Delgoda and Westlake, 2004). The inhibition of CYP3A4 by fucocoumarins found in grapefruit juice leading to clinically observable toxicities with drugs and the induction of the same CYP3A4 enzyme by ingredients found in St. John's wort leading to subtherapeutic interferences with cycloporin provide suitable examples for the involvement of CYP enzymes in drug herb interactions. While clinical studies provide the ultimate proof for relevant drug interactions, *in-vitro* laboratory evaluations with CYP enzymes, has provided a convenient, economical and useful starting point for screening those herbs that may ultimately cause clinically observable drug interactions. Human liver microsomes, heterologously expressed enzymes and hepatocytes although with limitations, have provided convenient means for such initial assessements.

In this chapter, we describe for the first time, the initial inhibitory impact of four commonly consumed infusions on six major CYP enzymes. Our findings support that the teas are moderate to weak CYP inhibitors and so we postulate that they would unlikely result in drug interactions.

#### **1.3.2 CYP inhibition and its relation to chemoprevention**

Approximately five decades of systematic drug discovery and development have established a reliable collection of chemotherapeutic agents (Yarbro, 1992; Chabner, 1991). These chemotherapeutic agents have assisted with numerous successes in the treatment and management of human cancers (Chabner *et al*., 1991).

Chemoprevention is the ability of compounds to protect healthy tissues via the prevention, inhibition or reversal of caricnogenesis. The inhibition of CYP1 enzymes is one such route among others that include the induction of cell cycle arrest, the induction of phase II enzymes and the inhibition of inflammatory. The CYP1 family has been linked with the activation of pro-carcinogens which is facilitated by the regulation of the aryl hydrocarbon receptor. As such research has shown that inhibiting CYP1 enzymes plays a key role in protecting healthy cells from the harmful effects of activated carcinogens.

Among the polycyclic hydrocarbons that are activated into reactive metabolites by CYPs 1A1 and 1B1 is benzo-a-pyrene [BaP]. Metabolites from BaP include phenols, polyphenols, quinines, epoxides and dihydrodiols. Among these dihydrodiols; (-)-benzo[a]pyrene-trans-7,8-dihydrodiol (BPD) and (+)-anti-benzo[a]pyrene-trans-7,8-dihydrodiol-9,10-epoxide (anti-

Cytochrome P450 Enzyme Inhibitors from Nature 43

to be an agonist of the aryl hydrocarbon receptor and consequently was responsible for an increased level of CYP1A1 expression, however this effect was counteracted by its ability to inhibit the enzyme directly and so is deemed an effective chemo-preventive agent (Ciolino and Yeh, 1999). Resveratrol was also found to exhibit chemo-preventive properties via the inhibition of CYP1A1 expression *in vivo* by preventing the binding of the AhR to promoter sequences that regulate the CYP1A1 transcription and also by the direct potent inhibition of

Bioactivity of isolates from the Jamaica plants, *Amyris plumieri*, *Peperomia amplexicaulis*, *Spathelia sorbifolia* and *Picrasma excelsa* are reported in this chapter. *Amyris plumieri* is found in the Caribbean, Central America and Venezuela and plants of this genus have been used in folk medicine against skin irritation while isolates have been found to exhibit anticancer and antimycobacterial properties (Fuente *et al*., 1991, Hartwell, 1968). Even though both *Peperomia amplexicaulis* and *Spathelia sorbifolia* are not commonly consumed in Jamaica, isolates from these plants have been shown to exhibit antiprotozoal, chemopreventive and anti-cancer activity (Mota *et al*., 2009; Cassady *et al*., 1990) and previously examined for CYP inhibitions (Badal *et al*., 2011; Shields *et al*., 2009) and overviewed in this chapter. Infusions of the plant *Picrasma excelsa*, known as Jamaican bitterwood tea, are commonly consumed to lower blood sugar levels in diabetics who are already on prescription medicines. All other plants investigated in this chapter; *Rhytidophyllum tomentosa*, *Psidium guajava, Symphytium officinale, Momordica charantia* are frequently consumed in the form of teas or the fruits of the appropriate plants. We therefore investigated the inhibition properties of these teas against a panel of CYP450 enzymes in order to assess the potential for drug interactions with co-

All CYP substrates and metabolites were purchased from Gentest Corporation (Woburn,

*Escherichia coli* membranes expressing human CYP2D6, CYP3A4, CYP1A1, CYP1A2 and each containing P450 reductase, were a gift from Dr. Mark Paine and Prof. Roland Wolfe (University of Dundee, UK). CYP2C19 expressed in baculovirus-insect cells (supersomes)

The selection of the plants for screening and method of preparation were based on the survey conducted by Delgoda *et al* (Delgoda *et al*., 2010). The teas were prepared by infusing 100ml of boiling deionized water per 1g of dried, finely ground material (leaf, bark or wood chips), for 10 minutes. The resulting liquor was suctioned filtered through type 1 Watman

MA, U.S.A.). All other chemicals were purchased from Sigma-Aldrich (MO, U.S.A.).

were purchased from Gentest Corporation, Woburn, MA

**2.3 Preparation of infusions from medicinal plants** 

CYPs1A1 and 1B1 (Ciolino *et al.,* 1998; Chen *et al.,* 2004).

medicated pharmaceuticals.

**2. Materials and methods** 

**2.1 Chemicals** 

**2.2 CYP microsomes** 

**1.4 CYP inhibition and its relation to chemoprevention** 

BPDE) are carcinogenic, however the latter is the ultimate carcinogen as it has been shown to bind DNA predominantly at the N2-position of guanine to produce primarily N2-guanine lesions, benzo-a-pyrene 7,8-diol-9,10-epoxide-N2-deoxyguanosine (BPDE-N2-dG) adduct (Osborn *et al*., 1976). It is proposed that BPDE-N2-dG is linked to the high frequency of p53 G→T transversions observed in lung cancer of smokers (Hainaut and Pfeifer, 2001; Pfeifer *et al*., 2002). Further mutations in the p53 gene have also been found and these include transversions, G→A and G→C (Shukla *et al.,* 1997; Schiltz *et al*., 1999). Similar to the role of CYP1A1 in the activation of BaP is that of the aromatic amines; amino-3-methylimidazo[4,5 *f*]quinoline (IQ), 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and 2-amino-3,8 dimethylimidazo-[4,5-flquinoxaline (MeIQx). CYP1A2 plays an important role in the Noxidation of these aromatic amines which have been linked to colon and urothelium cancers (Landi *et al.,* 1999), thus highlighting the role of CYP1 enzymes in carcinogenic activation and thus their potential as preventative targets. Fig.1 is a schematic representation of the process of carcinogenesis at the cellular level.

Fig. 1. A schematic representation of carcinogenesis via the activation of CYP1 enzymes. Upon the activation of the pro-carcinogens by the CYP1 enzymes, they have the ability to bind to DNA, which can lead to mutations and then the formation of cancer cells.

One of the first reported chemoprotectants was disulfiram (Stoner *et al.,* 1997) which inhibited the action of dimethylhydrazine via the inhibition of CYP1 enzymes. Other chemopreventive agents are discussed by Chang and others (Chang *et al*., 2002) who report that Ginseng decreases the incidence of 7,12 dimethyldenz(a)anthracence (DMBA)-initiated tumorigenesis in mice via the inhibition of CYPs1A1, 1A2 and 1B1. Also, the flavanoid, galangin was found

BPDE) are carcinogenic, however the latter is the ultimate carcinogen as it has been shown to bind DNA predominantly at the N2-position of guanine to produce primarily N2-guanine lesions, benzo-a-pyrene 7,8-diol-9,10-epoxide-N2-deoxyguanosine (BPDE-N2-dG) adduct (Osborn *et al*., 1976). It is proposed that BPDE-N2-dG is linked to the high frequency of p53 G→T transversions observed in lung cancer of smokers (Hainaut and Pfeifer, 2001; Pfeifer *et al*., 2002). Further mutations in the p53 gene have also been found and these include transversions, G→A and G→C (Shukla *et al.,* 1997; Schiltz *et al*., 1999). Similar to the role of CYP1A1 in the activation of BaP is that of the aromatic amines; amino-3-methylimidazo[4,5 *f*]quinoline (IQ), 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and 2-amino-3,8 dimethylimidazo-[4,5-flquinoxaline (MeIQx). CYP1A2 plays an important role in the Noxidation of these aromatic amines which have been linked to colon and urothelium cancers (Landi *et al.,* 1999), thus highlighting the role of CYP1 enzymes in carcinogenic activation and thus their potential as preventative targets. Fig.1 is a schematic representation of the

Fig. 1. A schematic representation of carcinogenesis via the activation of CYP1 enzymes. Upon the activation of the pro-carcinogens by the CYP1 enzymes, they have the ability to

One of the first reported chemoprotectants was disulfiram (Stoner *et al.,* 1997) which inhibited the action of dimethylhydrazine via the inhibition of CYP1 enzymes. Other chemopreventive agents are discussed by Chang and others (Chang *et al*., 2002) who report that Ginseng decreases the incidence of 7,12 dimethyldenz(a)anthracence (DMBA)-initiated tumorigenesis in mice via the inhibition of CYPs1A1, 1A2 and 1B1. Also, the flavanoid, galangin was found

bind to DNA, which can lead to mutations and then the formation of cancer cells.

process of carcinogenesis at the cellular level.

to be an agonist of the aryl hydrocarbon receptor and consequently was responsible for an increased level of CYP1A1 expression, however this effect was counteracted by its ability to inhibit the enzyme directly and so is deemed an effective chemo-preventive agent (Ciolino and Yeh, 1999). Resveratrol was also found to exhibit chemo-preventive properties via the inhibition of CYP1A1 expression *in vivo* by preventing the binding of the AhR to promoter sequences that regulate the CYP1A1 transcription and also by the direct potent inhibition of CYPs1A1 and 1B1 (Ciolino *et al.,* 1998; Chen *et al.,* 2004).

#### **1.4 CYP inhibition and its relation to chemoprevention**

Bioactivity of isolates from the Jamaica plants, *Amyris plumieri*, *Peperomia amplexicaulis*, *Spathelia sorbifolia* and *Picrasma excelsa* are reported in this chapter. *Amyris plumieri* is found in the Caribbean, Central America and Venezuela and plants of this genus have been used in folk medicine against skin irritation while isolates have been found to exhibit anticancer and antimycobacterial properties (Fuente *et al*., 1991, Hartwell, 1968). Even though both *Peperomia amplexicaulis* and *Spathelia sorbifolia* are not commonly consumed in Jamaica, isolates from these plants have been shown to exhibit antiprotozoal, chemopreventive and anti-cancer activity (Mota *et al*., 2009; Cassady *et al*., 1990) and previously examined for CYP inhibitions (Badal *et al*., 2011; Shields *et al*., 2009) and overviewed in this chapter. Infusions of the plant *Picrasma excelsa*, known as Jamaican bitterwood tea, are commonly consumed to lower blood sugar levels in diabetics who are already on prescription medicines. All other plants investigated in this chapter; *Rhytidophyllum tomentosa*, *Psidium guajava, Symphytium officinale, Momordica charantia* are frequently consumed in the form of teas or the fruits of the appropriate plants. We therefore investigated the inhibition properties of these teas against a panel of CYP450 enzymes in order to assess the potential for drug interactions with comedicated pharmaceuticals.

#### **2. Materials and methods**

#### **2.1 Chemicals**

All CYP substrates and metabolites were purchased from Gentest Corporation (Woburn, MA, U.S.A.). All other chemicals were purchased from Sigma-Aldrich (MO, U.S.A.).

#### **2.2 CYP microsomes**

*Escherichia coli* membranes expressing human CYP2D6, CYP3A4, CYP1A1, CYP1A2 and each containing P450 reductase, were a gift from Dr. Mark Paine and Prof. Roland Wolfe (University of Dundee, UK). CYP2C19 expressed in baculovirus-insect cells (supersomes) were purchased from Gentest Corporation, Woburn, MA

#### **2.3 Preparation of infusions from medicinal plants**

The selection of the plants for screening and method of preparation were based on the survey conducted by Delgoda *et al* (Delgoda *et al*., 2010). The teas were prepared by infusing 100ml of boiling deionized water per 1g of dried, finely ground material (leaf, bark or wood chips), for 10 minutes. The resulting liquor was suctioned filtered through type 1 Watman

Cytochrome P450 Enzyme Inhibitors from Nature 45

*Spathelia sorbifolia* and chroman 6 isolated from *Peperomia amplexicaulis*. Structures for these can be seen in Figs. 2.1, 2.2 and 2.3 and in addition obtained IC50s can be seen in Table 2. Both CA1 and quassin exhibited the most potency against CYP1A1. Both Anhydrosorbifolin and chroman 6 and CAs, 1, 2 and 3moderately (IC50 between 1 and 10μM) inhibited the

activities of CYP1 family.

tyramine

Fig. 2.1. Chromene amides

H3CO

Fig. 2.2. Quassinoids

HO

O

chromene ring

R = CH3

NH2

H N O R

R = CH2CH2CH3 n-butanamide

R = CH2CH(CH3 )2 3-methylbutanamide

R = benzamide

O H

H

O O

H

acetamide

R = CH(CH3)2 2-methylpropanamide or isobutanamide

R = CH=C(CH3 )2 3-methyl-2-butenamide or -dimethylacrylamide

H3CO

Quassin Neoquassin

acyl residue

CA1

CA3

CA4

CA6

H

H

OH H

O O

O O

H

CA2

CA5

filter paper. A portion of the filtrate was then centrifuged at 13000 × g for 5 minutes to remove suspended solids.

#### **2.4 Separation of active ingredients from medicinal plants**

Infusions were freeze dried and re-dissolved in water just prior to use, unless otherwise stated. 25µl infusions were loaded onto a microsorb C18 column (ID 4.6mm, 25cm, 5m) and separated using the appropriate solvent systems using Varian Prostar HPLC system (Varian Inc. USA).

#### **2.5 CYP inhibition assays**

Routinely, appropriate volumes of potassium phosphate buffer (KPB), test inhibitor, CYP, and the substrates were added to a NADPH regenerating mixture and made up to 400 µL, and monitored fluorometrically on a continuous basis for 10mins as described elsewhere (Shields, 2009), using CYP450 substrates,3-[2-(N,N-Diethyl-N-methylamino)ethyl]-7 methoxy-4methylcoumarin (AMMC), 7-Benzyloxy-4-trifluoromethylcoumarin (BFC), as substrates for CYP3A4 and CYP2D6 respectively and 7-ethoxy-3-cyanocoumarin (CEC) as substrate for CYPs 1A1, 1A2, 2C19 and 2C9. In other instances (as specified in each case), a 96-well plate assay was employed as detailed in (Badal *et al*., 2011). Fluoroscence was monitored using a Varian Cary Eclipse Fluorescence spectrophotometer.

Positive control experiments were conducted with varying concentrations of furafaylline (≥98%) (0.5-10µM), quinidine (≥90%, 1-50nM) and ketoconazole (≥98%) (2-100nM) with CYP1A2, CYP2D6 and CYP3A4 respectively.

#### **2.6 Data analysis**

IC50 and Ki values were determined by fitting the data in Sigma Plot (version 10.0) and enzyme kinetics module, using non linear regression analysis. The data listed represent the average values from three different determinations.

#### **3. Results**

#### **3.1 Optimising experimental conditions**

To verify the accuracy of experimental techniques employed to detect CYP inhibition, assays with known inhibitors were carried out with furafylline (against CYP1A2), ketoconazole (against CYP1A1, CYP1B1 and CYP3A4), (−)-N-3-benzyl-phenobarbital (NBPB, against CYP2C19) and quinidine (against CYP2D6) and the obtained IC50 values (0.8±0.2, 0.04±0.01, 6.3±1.7, 0.06±0.01, 0.3±191 0.01, and 0.03±0.01μM respectively) compared well with published values (0.99, b10, b10, 0.06, 0.25 and 0.01μM respectively; Shields, 2009; Badal *et al*., 2011; Powrie, 2010; Stresser *et al.,* 2004; Cali, 2003 and McLaughlin *et al*., 2008).

#### **3.2 Natural products as CYP inhibitors**

Several classes of natural products were examined in our laboratory for their inhibitory properties towards CYP450 enzymes. Chromene amides (CAs) isolated from *Amyris plumieri*, quassinoids isolated from *Picrasma excelsa*, anhydrosorbifolin isolated from

filter paper. A portion of the filtrate was then centrifuged at 13000 × g for 5 minutes to

Infusions were freeze dried and re-dissolved in water just prior to use, unless otherwise stated. 25µl infusions were loaded onto a microsorb C18 column (ID 4.6mm, 25cm, 5m) and separated using the appropriate solvent systems using Varian Prostar HPLC system (Varian Inc. USA).

Routinely, appropriate volumes of potassium phosphate buffer (KPB), test inhibitor, CYP, and the substrates were added to a NADPH regenerating mixture and made up to 400 µL, and monitored fluorometrically on a continuous basis for 10mins as described elsewhere (Shields, 2009), using CYP450 substrates,3-[2-(N,N-Diethyl-N-methylamino)ethyl]-7 methoxy-4methylcoumarin (AMMC), 7-Benzyloxy-4-trifluoromethylcoumarin (BFC), as substrates for CYP3A4 and CYP2D6 respectively and 7-ethoxy-3-cyanocoumarin (CEC) as substrate for CYPs 1A1, 1A2, 2C19 and 2C9. In other instances (as specified in each case), a 96-well plate assay was employed as detailed in (Badal *et al*., 2011). Fluoroscence was

Positive control experiments were conducted with varying concentrations of furafaylline (≥98%) (0.5-10µM), quinidine (≥90%, 1-50nM) and ketoconazole (≥98%) (2-100nM) with

IC50 and Ki values were determined by fitting the data in Sigma Plot (version 10.0) and enzyme kinetics module, using non linear regression analysis. The data listed represent the

To verify the accuracy of experimental techniques employed to detect CYP inhibition, assays with known inhibitors were carried out with furafylline (against CYP1A2), ketoconazole (against CYP1A1, CYP1B1 and CYP3A4), (−)-N-3-benzyl-phenobarbital (NBPB, against CYP2C19) and quinidine (against CYP2D6) and the obtained IC50 values (0.8±0.2, 0.04±0.01, 6.3±1.7, 0.06±0.01, 0.3±191 0.01, and 0.03±0.01μM respectively) compared well with published values (0.99, b10, b10, 0.06, 0.25 and 0.01μM respectively; Shields, 2009; Badal *et* 

Several classes of natural products were examined in our laboratory for their inhibitory properties towards CYP450 enzymes. Chromene amides (CAs) isolated from *Amyris plumieri*, quassinoids isolated from *Picrasma excelsa*, anhydrosorbifolin isolated from

*al*., 2011; Powrie, 2010; Stresser *et al.,* 2004; Cali, 2003 and McLaughlin *et al*., 2008).

**2.4 Separation of active ingredients from medicinal plants** 

monitored using a Varian Cary Eclipse Fluorescence spectrophotometer.

CYP1A2, CYP2D6 and CYP3A4 respectively.

**3.1 Optimising experimental conditions** 

**3.2 Natural products as CYP inhibitors** 

average values from three different determinations.

remove suspended solids.

**2.5 CYP inhibition assays** 

**2.6 Data analysis** 

**3. Results** 

*Spathelia sorbifolia* and chroman 6 isolated from *Peperomia amplexicaulis*. Structures for these can be seen in Figs. 2.1, 2.2 and 2.3 and in addition obtained IC50s can be seen in Table 2. Both CA1 and quassin exhibited the most potency against CYP1A1. Both Anhydrosorbifolin and chroman 6 and CAs, 1, 2 and 3moderately (IC50 between 1 and 10μM) inhibited the activities of CYP1 family.

Fig. 2.1. Chromene amides

Fig. 2.2. Quassinoids

Cytochrome P450 Enzyme Inhibitors from Nature 47

% in h ib itio n o f CYP a c tiv ity

A B

% in h ib itio n o f C Y P a ctivity

C D

Due to the potency displayed against the activities of CYP450 enzymes, *Psidium guajava* was selected for further characterization. Preliminary separation of the freeze-dried extract of *Psidium guajava* by reverse phase HPLC (see Fig.4) revealed several resolved peaks and LC-MS analysis at the same time and the results are summarized in Table 3. Two peaks were identified as quercetin and hyperin whose structures are shown in Fig.5 and these displayed

0

20

40

60

80

100

120

0

2 0

4 0

6 0

8 0

100

120

[te a ] ( g /m L ) 1 1 0 100

[tea] (g/mL)

10 100 1000

CYP3A4 CYP2D6 CYP1A2 CYP2C19 CYP1A1

> CYP1A1 CYP1A2 CYP2D6 CYP2C19

CYP3A4 CYP2D6 CYP1A2 CYP2C19 CYP1A1

[te a ] ( g /m L )

[tea] (g/mL)

**3.4 Identification of active ingredients** 

10 100 1000

Fig. 3. Inhibition of CYP activity by medicinal plant infusions.

CYP1A1 CYP3A4 CYP2C19

1 1 0 100

% in h ib itio n o f CYP a c tiv ity

0

%

0

20

40

60

80

100

120

inhibition of C

 Y P activity

2 0

4 0

6 0

8 0

100

120

#### Fig. 2.3. Others


Table 2. Summary of IC50 and Ki values (µM) obtained from the interaction of isomers of chromene amides, quassinoids along with anhydrosorbifolin and chroman 6 using heterologously expressed CYP microsomes. ND: Not determined due to intrinsic fluorescence and quenching/enhancement of the metabolite nd: not done

#### **3.3 Herbal infusions with CYP inhibitors**

Hot water infusions of five popular herbs; *Rhytidophyllum tomentosa*, *Psidium guajava, Symphytium officinale, Momordica charantia* and *Picrasma excelsa* were characterized for impact as shown in Fig.3 and calculated IC50 values on the activities of CYP enzymes are shown in Table 3.

O

2.22 ± 0.69

359.88 ± 144.55

11.70 ± 5.40

84.40 ± 3.5

1.14±0.4 8

15.48±0. 45

122.93±5 .95

7.63±1.2 6

OH O

O

1A1 1A2 1B1 2C9 2C19 2D6 3A4

15.36 ± 0.42 nd 0.77 ±

37.04 ± 1.51 nd 1.09 ±

179.30 ± 20.5 nd 2.43 ±

18.14 ± 1.02 nd 2.55 ±

4.9 1.9 1.4 nd nd nd nd

Chroman 6 2.1 5.8 5.6 nd nd nd nd

Hot water infusions of five popular herbs; *Rhytidophyllum tomentosa*, *Psidium guajava, Symphytium officinale, Momordica charantia* and *Picrasma excelsa* were characterized for impact as shown in Fig.3 and calculated IC50 values on the activities of CYP enzymes are shown in

Table 2. Summary of IC50 and Ki values (µM) obtained from the interaction of isomers of chromene amides, quassinoids along with anhydrosorbifolin and chroman 6 using heterologously expressed CYP microsomes. ND: Not determined due to intrinsic fluorescence and quenching/enhancement of the metabolite nd: not done

Anhydrosorbifolin

0.39

0.52

0.28

1.85

57.6 ND 92.5 262.5 217.8 47.0

85.3 ND 80.6 113.4 184.1 24.5

O

5-Hydroxy-2,7-dimethyl-8-(3-methylbut-2-enyl)-2-(4-methyl-penta-1,3 dienyl)-chroman-6-carboxylic acid

Compounds CYP isoforms

32.80 ± 4.45

6.25 ± 1.85

189.84 ± 7.60

18.59 ± 0.67

HO

Fig. 2.3. Others

O OH

CA1 1.31 ± 0.42

CA2 1.63 ± 0.53

CA3 2.43 ± 0.62

CA4 14.39 ±

Quassin 9.2

Neoquassin 11.9

Anhydrosorbifolin

Table 3.

Ki =0.37

Ki=2.40

Ki=1.39

7.40

Ki=10.8

Ki=11.3

**3.3 Herbal infusions with CYP inhibitors** 

Fig. 3. Inhibition of CYP activity by medicinal plant infusions.

#### **3.4 Identification of active ingredients**

Due to the potency displayed against the activities of CYP450 enzymes, *Psidium guajava* was selected for further characterization. Preliminary separation of the freeze-dried extract of *Psidium guajava* by reverse phase HPLC (see Fig.4) revealed several resolved peaks and LC-MS analysis at the same time and the results are summarized in Table 3. Two peaks were identified as quercetin and hyperin whose structures are shown in Fig.5 and these displayed

Cytochrome P450 Enzyme Inhibitors from Nature 49

HO

Quercetin Hyperin

O

OH O

O

OH

O

HO OH

OH

OH

OH

OH

O

O

OH

HO

OH

Fig. 5. Structures of quercetin and hyperin (quercetin-3-D-galactoside).

Fig. 6. HPLC profile of *Psidium guajava* (adapted from Shields *et al*., 2009).

OH

50% inhibition against the activity of CYP2D6 enzymes as shown in Fig.6. Previously modelled active site of CYP1A1 with bound quassin is displayed in Fig.7 where key residues in the enzyme are identified; Asp313, Thr11, Ser124, Phe123, Ile386 and Leu496 in the interaction between quassin and neoquassin.

Inhibition of CYP activity by *Rhytidophyllum tomentosa infusion* (A); *Psidium guajava* (B); *Momordica charantia* (C); *and Symphytium officinale* (D). Different volumes of reconstituted freeze-dried infusion were added to the incubation mixture, along with the CYP isoform, substrate, and 6GPDH, and monitored fluorometrically over time, as described in Materials and Methods. Control enzyme activity (mean ± SEM) for CYP3A4, CYP1A1, CYP2D6, CYP1A2, CYP2C19, and CYP2C9 was 0.147 ± 0.037, 0.907 ± 0.095, 0.005 ± 0.000, 1.45 ± 0.04, 0.054 ± 0.016, and 0.057 ± 0.004μM/min/pmol of CYP, respectively. Curves for CYP2D6 and CYP1A2 in for *Memordica charantia* (C) and for CYP3A4 in by *Symphytium officinale* (D) were not included because their IC50 values exceeded 200g/mL.


\*Values for *Picrasma excelsa* were obtained from Shields *et al*, 2008.

Table 3. Summary of IC50 values obtained for the extracts from Fig. 2

Fig. 4. HPLC profile of *Psidium guajava* extract

50% inhibition against the activity of CYP2D6 enzymes as shown in Fig.6. Previously modelled active site of CYP1A1 with bound quassin is displayed in Fig.7 where key residues in the enzyme are identified; Asp313, Thr11, Ser124, Phe123, Ile386 and Leu496 in the

Inhibition of CYP activity by *Rhytidophyllum tomentosa infusion* (A); *Psidium guajava* (B); *Momordica charantia* (C); *and Symphytium officinale* (D). Different volumes of reconstituted freeze-dried infusion were added to the incubation mixture, along with the CYP isoform, substrate, and 6GPDH, and monitored fluorometrically over time, as described in Materials and Methods. Control enzyme activity (mean ± SEM) for CYP3A4, CYP1A1, CYP2D6, CYP1A2, CYP2C19, and CYP2C9 was 0.147 ± 0.037, 0.907 ± 0.095, 0.005 ± 0.000, 1.45 ± 0.04, 0.054 ± 0.016, and 0.057 ± 0.004μM/min/pmol of CYP, respectively. Curves for CYP2D6 and CYP1A2 in for *Memordica charantia* (C) and for CYP3A4 in by *Symphytium officinale* (D) were

> *Momordica charantia*

8 10 12 14 16 18 20 22 m

*Symphytium officinale*

*Picrasma excelsa\** 

interaction between quassin and neoquassin.

*Rhytidophyllum tomentosa*

Norm.

0

Fig. 4. HPLC profile of *Psidium guajava* extract

10

20

30

40

50

60

not included because their IC50 values exceeded 200g/mL.

Isoform IC50 (g/mL)

\*Values for *Picrasma excelsa* were obtained from Shields *et al*, 2008.

10.068

10.525 10.795

11.727

12.271

13.817

15.139

17.093

18.341

19.397

17.472

15.924

Table 3. Summary of IC50 values obtained for the extracts from Fig. 2

*Psidium guajava*

CYP1A1 10.2 4.9 137.1 24.0 15.0 CYP1A2 28.3 24.0 >200.0 40.3 19.1 CYP2D6 158.0 26.3 >200.0 127.9 >200 CYP2C19 93.8 23.3 91.0 172.7 199.9 CYP3A4 178.1 48.7 82.3 >200.0 122.8

Fig. 5. Structures of quercetin and hyperin (quercetin-3-D-galactoside).

Fig. 6. HPLC profile of *Psidium guajava* (adapted from Shields *et al*., 2009).

Cytochrome P450 Enzyme Inhibitors from Nature 51

from *Peperomia amplexicaulis.* We also report for the first time bioactive screening of CYP enzymes in the presence of five aqueous infusions of popularly used herbs; *Rhytidophyllum tomentosa*, *Psidium guajava, Symphytium officinale, Momordica charantia* and *Picrasma excelsa.*

Potent and selective inhibition of the CYP1 enzymes were found amongst the investigated natural compounds in particular chromene amides and quassinoids. CA1 displayed potent inhibition against the activity of CYP1A1, with a Ki of 0.37μM and an IC50 value of 1.31μM while quassin inhibited this enzyme with an IC50 value of 9.2μM and Ki of 10.8μM with selectivity extended throughout all CYP enzymes investigated except CYP2C19 for CA1. The degree of potency and selectivity with which both compounds inhibited this enzyme warrants further research as possible chemoprotectants. Previously known and studied natural compounds deemed to possess chemoprotective properties due to their ability to inhibit CYP1A1 include; quercetin (IC50=1.36μM, Leung *et al*., 2007), curcumin (IC50=20μM), demethoxycurcumin (IC50=21μM), ε-viniferin (IC50=1μM), resveratrol (IC50=30μM), and sanguinarine (*K*i =2μM). Both test compounds compare well with these known

Both CYPs 1A1 and 1A2 share approximately 70% similarity in amino acid and the specificity with which inhibition targeted towards CYP1A1 activity is noticeable in the compounds CA1, CA3 and quassin. As such we unlocked the interaction between quassin and CYP1A1 in previous publication (Shields *et al*., 2009). One of the first active site models for CYP1A1 was demonstrated with quasin and important residues were highlighted; Asp313, Thr11, Ser124, Phe123, Ile386 and Leu496 as shown in Fig.7 as being critical for

CYP1B1 has been drawing keen interest for novel and anticancer therapeutics. Findings of the over-expression of CYP1B1 in many tumour tissues compared with normal surrounding cells, have led to the search for pro-drugs reliant on CYP1B1 metabolism for the conversion into cytotoxic therapeutics. Although the role of such over- expression is yet to be fully understood, it has been linked with drug resistance and in the promotion of cell survival (Martinez *et al*., 2008). The modification in the expression levels of CYP1B1 has been shown to modulate tumour progression (Castro *et al*., 2008) and thus specific inhibitors are expected to be of therapeutic/preventive benefit. Although the potency and the specificity of the chromene amides examined this chapter against CYP1B1 is not particularly high, structure-activity relations may guide towards chromene amides with putative improvement. Anhydrosorbifolin and chroman 6 displayed the most potency against this

Little impact towards the CYP1 family was observed in the presence of CA2 which could be due to the isopropyl group on this chromene amide, compared with CA1. Even though the Ki against CYP1A1 was increased (to 2.63μM), the IC50 value remained more or less the same as CA1 (1.63μM), displayed moderate to low potency against CYPs 1A2 and 1B1. The structural change made to CA2 was more significant in binding CYP2D6 as the inhibition dropped over a 100 times (IC50=360μM for CA2 vs 2μM for CA1). Hence, CA2 displayed characteristics of a useful molecular probe where all significant drug metabolizing enzymes can be inhibited except for the activity of CYP2D6. Chain elongation and the loss of branching in the n-propyl end unit to form CA3, have a

chemoprotectants and thus warrant further research.

enzyme deeming further investigations worthwhile.

binding quassinoids.

Fig. 7. Interaction of quassinoids with CYP1A1

#### **4. Discussion**

Cytochrome P450 enzymes have been of particular interest in the field of drug discovery for numerous reasons including the involvement of these enzymes in the metabolism of over 95% of the drugs on the market and the potential of drug-drug interaction through metabolism. CYP1 family which is under the regulation of the aryl hydrocarbon receptor have been extensively researched and implicated in drug resistance as well as carcinogenesis. CYP1B1 in particular, found in elevated levels in cancer tissues such as those in colon is thought to provide a novel pathway for drug discovery and optimisation for cancer treatment. Inhibitors of the activities of CYP1 enzymes are now accepted as potential chemoprotectants by preventing the activation of polycyclic aromatic hydrocarbons such as benzo-a-pyrene. Catalysed by CYP1A1 and CYP1B1, metabolites of this pro-carcinogen, (+)-anti-benzo[a]pyrene-trans-7,8-dihydrodiol-9,10-epoxide (anti-BPDE) and that 3-hydroxybenzo [a]pyrene (3HBaP) have been shown to bind to DNA predominantly at the N2-position of guanine to produce N2-guanine lesions, benzo-apyrene 7,8-diol-9,10-epoxide-N2-deoxyguanosine (BPDE-N2-dG) adduct (King *et al*., 1976). Thus inhibitors of CYP1 enzymes hold the potential to prevent the formation of such damaging precursors that initiate malignant cancers of the breast, colon, lung and urothelium among others.

In this chapter we highlight the potential of a few natural products abundant in the Caribbean: chromene amides isolated from *Amyris plumieri*, quassinoids isolated from *Picrasma excelsa*, anhydrosorbifolin isolated from *Spathelia sorbifolia* and chroman 6 isolated

Cytochrome P450 enzymes have been of particular interest in the field of drug discovery for numerous reasons including the involvement of these enzymes in the metabolism of over 95% of the drugs on the market and the potential of drug-drug interaction through metabolism. CYP1 family which is under the regulation of the aryl hydrocarbon receptor have been extensively researched and implicated in drug resistance as well as carcinogenesis. CYP1B1 in particular, found in elevated levels in cancer tissues such as those in colon is thought to provide a novel pathway for drug discovery and optimisation for cancer treatment. Inhibitors of the activities of CYP1 enzymes are now accepted as potential chemoprotectants by preventing the activation of polycyclic aromatic hydrocarbons such as benzo-a-pyrene. Catalysed by CYP1A1 and CYP1B1, metabolites of this pro-carcinogen, (+)-anti-benzo[a]pyrene-trans-7,8-dihydrodiol-9,10-epoxide (anti-BPDE) and that 3-hydroxybenzo [a]pyrene (3HBaP) have been shown to bind to DNA predominantly at the N2-position of guanine to produce N2-guanine lesions, benzo-apyrene 7,8-diol-9,10-epoxide-N2-deoxyguanosine (BPDE-N2-dG) adduct (King *et al*., 1976). Thus inhibitors of CYP1 enzymes hold the potential to prevent the formation of such damaging precursors that initiate malignant cancers of the breast, colon, lung and

In this chapter we highlight the potential of a few natural products abundant in the Caribbean: chromene amides isolated from *Amyris plumieri*, quassinoids isolated from *Picrasma excelsa*, anhydrosorbifolin isolated from *Spathelia sorbifolia* and chroman 6 isolated

Fig. 7. Interaction of quassinoids with CYP1A1

**4. Discussion** 

urothelium among others.

from *Peperomia amplexicaulis.* We also report for the first time bioactive screening of CYP enzymes in the presence of five aqueous infusions of popularly used herbs; *Rhytidophyllum tomentosa*, *Psidium guajava, Symphytium officinale, Momordica charantia* and *Picrasma excelsa.*

Potent and selective inhibition of the CYP1 enzymes were found amongst the investigated natural compounds in particular chromene amides and quassinoids. CA1 displayed potent inhibition against the activity of CYP1A1, with a Ki of 0.37μM and an IC50 value of 1.31μM while quassin inhibited this enzyme with an IC50 value of 9.2μM and Ki of 10.8μM with selectivity extended throughout all CYP enzymes investigated except CYP2C19 for CA1. The degree of potency and selectivity with which both compounds inhibited this enzyme warrants further research as possible chemoprotectants. Previously known and studied natural compounds deemed to possess chemoprotective properties due to their ability to inhibit CYP1A1 include; quercetin (IC50=1.36μM, Leung *et al*., 2007), curcumin (IC50=20μM), demethoxycurcumin (IC50=21μM), ε-viniferin (IC50=1μM), resveratrol (IC50=30μM), and sanguinarine (*K*i =2μM). Both test compounds compare well with these known chemoprotectants and thus warrant further research.

Both CYPs 1A1 and 1A2 share approximately 70% similarity in amino acid and the specificity with which inhibition targeted towards CYP1A1 activity is noticeable in the compounds CA1, CA3 and quassin. As such we unlocked the interaction between quassin and CYP1A1 in previous publication (Shields *et al*., 2009). One of the first active site models for CYP1A1 was demonstrated with quasin and important residues were highlighted; Asp313, Thr11, Ser124, Phe123, Ile386 and Leu496 as shown in Fig.7 as being critical for binding quassinoids.

CYP1B1 has been drawing keen interest for novel and anticancer therapeutics. Findings of the over-expression of CYP1B1 in many tumour tissues compared with normal surrounding cells, have led to the search for pro-drugs reliant on CYP1B1 metabolism for the conversion into cytotoxic therapeutics. Although the role of such over- expression is yet to be fully understood, it has been linked with drug resistance and in the promotion of cell survival (Martinez *et al*., 2008). The modification in the expression levels of CYP1B1 has been shown to modulate tumour progression (Castro *et al*., 2008) and thus specific inhibitors are expected to be of therapeutic/preventive benefit. Although the potency and the specificity of the chromene amides examined this chapter against CYP1B1 is not particularly high, structure-activity relations may guide towards chromene amides with putative improvement. Anhydrosorbifolin and chroman 6 displayed the most potency against this enzyme deeming further investigations worthwhile.

Little impact towards the CYP1 family was observed in the presence of CA2 which could be due to the isopropyl group on this chromene amide, compared with CA1. Even though the Ki against CYP1A1 was increased (to 2.63μM), the IC50 value remained more or less the same as CA1 (1.63μM), displayed moderate to low potency against CYPs 1A2 and 1B1. The structural change made to CA2 was more significant in binding CYP2D6 as the inhibition dropped over a 100 times (IC50=360μM for CA2 vs 2μM for CA1). Hence, CA2 displayed characteristics of a useful molecular probe where all significant drug metabolizing enzymes can be inhibited except for the activity of CYP2D6. Chain elongation and the loss of branching in the n-propyl end unit to form CA3, have a

Cytochrome P450 Enzyme Inhibitors from Nature 53

CYP1B1. *In vitro* and *in silico* models as demonstrated for the first time in this chapter are useful tools in the process of drug development to approximate the risk of drug interactions and in the process of target improvement of key enzymes in chemoprevention. In particular, for herbal remedies they confer useful models for evaluating the risks of adverse effects arising from interactions with co-administered prescription medicines, for which drug-interaction information is not mandated by the regulatory agencies. Once the initial risk is estimated, clinical drug-interaction studies can be launched, thus providing a cost-effective sieving process prior to embarking on

We are grateful to the International Foundation for Science (IFS), Sweden, the University of the West Indies post graduate fund, the Forestry Conservation fund and the Luther Speare Scholarship for financial support. We are also grateful to Professor Helen Jacobs for

Badal, S.; Williams, S. G. Huang, G. Francis, S. Vedantam, P. Dunbar, O. Jacobs, H. Tzeng, J.

Cassady, J.M.; Baird, W.M. Chang, C.J. (1990). Natural Products as a Source of Potential

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Chabner, B.; (1991). Anti-cancer drugs. Principles and Practice, 4th Edition. Philadelphia,

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Chang, S.; Johnston Jr, P. Frokjaer-Jensen, C. Lockery, S. and Hobert, O. (2004). Hobert,

Chen, Z.H.; Hurh, Y.J. Na, H.K. Kim, J.H., Chun, Y.J. Kim, D.H. Kang, K.S. Cho, M.H. Surh,

Ciolino, H.; Daschner, P. and Yeh, G. (1998). Resveratrol inhibits transcription of CYP1A1 in

CYP1A2, and CYP1B1. *Drug Metab. Dispo,s* Vol.30 pp. 378-384.

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in the nematode. *Nature,* Vol.430 pp.785-789.

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Cancer Chemotherapeutic and Chemopreventive Agents. *Journal of natural products*.

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rigorous and expensive investigations.

provision of select natural products.

pp. 230-236.

Vol.53 pp 23-41.

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pp. 5707-5712.

Lippincott pp. 325-417

**6. Acknowledgments** 

**7. References** 

dramatic impact on the affinity to CYP1A2 and CYP1B1. The inhibition potency dropped 6 folds against CYP1A2 (from 32.8μM for CA1 to 189.8μM for CA3) and 10 folds against CYP1B1 (from15.4μM to 179.3μM). Thus, CA3 appears to show increased selectivity in its inhibition against CYP1A1. Exploring other side groups as well as shifting the position of the existing side groups can be explored in hopes of increasing the potency of CAs towards the activity of CYP1B1

There is a large consumption of natural medicines alone and concurrently with prescription medicines in the Caribbean, as in many parts of the world. This is confirmed in a recent pilot study done in which 80% of prescription medicine consumers also take natural remedies (Delgoda *et al*., 2004; Picking *et al*., 2011). Also, adverse drug reactions (ADRs) accumulates to over 2 million per year in the United States alone (Gurwitz *et al*., 2000), therefore, the ability to predict drug interactions involving the CYP enzymes has become a key component of the drug discovery process (Forti and Wahlstrom, 2008). Providing the FDA with the metabolic profile of a new drug entity with CYP enzymes is the first step towards avoiding adverse reactions (Delgoda and Westlake, 2004). Known drug –herb interactions with clinical impact include grapefruit juice with felopidium, tricyclic anti depressants which is medicated through CYP3A4 inhibition.

We report for the first time hot water infusions of *Rhytidophyllum tomentosa*, *Psidium guajava, Symphytium officinale, Momordica charantia* in comparison with reported *Picrasma excelsa* being tested against CYP enzymes activity. The greatest potency was observed in the presence of *Psidium guajava* that inhibited CYP1A1 with an IC50 of 4.9µg/mL. All of the infusions are commonly consumed, be it, the fruit or as teas. With the high levels of polypharmacy practise that exist among Caribbean people and the world, having a metabolic profile on teas or commonly consumed plants become of grave importance. Results displayed in Table 3 point to is minimal risk through CYP mediated drug interactions as the teas weakly inhibited the main drug metabolising enzymes. Because, the activity of *Psidium guajava* towards CYP1A1 was the most potent we further evaluated the identification of the active ingredients that could be responsible for the observed bioactivity towards the CYP1A1 activity. The active ingredients were found to be quercetin and hyperin (see Figs. 4 & 5), compounds known as inhibitors of CYP enzymes, where quercetin isolated from St. John's Wort has been previoulsy shown to inhibit activities of CYPs 1A2, 2C19 and CYP2D6 with IC50s of 3.87 µM, 6.23 µM and 20.99 µM respectively; while hyperforin isolated from *Ginko biloba* shown to inhibit the activity of CYP3A4 with an IC50 of 4.30µM (Moltke *et al*., 2004; Zou *et al*., 2002). Inhibitions against the CYP enzymes appear to be between moderate and weak which confirms data in our lab, thus evoking moderate concern for potential interactions with co-medicated pharmaceuticals through CYP mediated metabolism.

#### **5. Conclusion**

Review of compounds that have potent and selective inhibitory properties against the activities of CYP1 family in particular CYPs 1A1 and 1B1 aid in identification of useful chemoprotectors. CA1 and quassin warrant further research because they were both potent against the activity of CYP1A1 while anhydrosorbifolin and chroma 6 targeted CYP1B1. *In vitro* and *in silico* models as demonstrated for the first time in this chapter are useful tools in the process of drug development to approximate the risk of drug interactions and in the process of target improvement of key enzymes in chemoprevention. In particular, for herbal remedies they confer useful models for evaluating the risks of adverse effects arising from interactions with co-administered prescription medicines, for which drug-interaction information is not mandated by the regulatory agencies. Once the initial risk is estimated, clinical drug-interaction studies can be launched, thus providing a cost-effective sieving process prior to embarking on rigorous and expensive investigations.

#### **6. Acknowledgments**

We are grateful to the International Foundation for Science (IFS), Sweden, the University of the West Indies post graduate fund, the Forestry Conservation fund and the Luther Speare Scholarship for financial support. We are also grateful to Professor Helen Jacobs for provision of select natural products.

#### **7. References**

52 Enzyme Inhibition and Bioapplications

dramatic impact on the affinity to CYP1A2 and CYP1B1. The inhibition potency dropped 6 folds against CYP1A2 (from 32.8μM for CA1 to 189.8μM for CA3) and 10 folds against CYP1B1 (from15.4μM to 179.3μM). Thus, CA3 appears to show increased selectivity in its inhibition against CYP1A1. Exploring other side groups as well as shifting the position of the existing side groups can be explored in hopes of increasing the potency of CAs

There is a large consumption of natural medicines alone and concurrently with prescription medicines in the Caribbean, as in many parts of the world. This is confirmed in a recent pilot study done in which 80% of prescription medicine consumers also take natural remedies (Delgoda *et al*., 2004; Picking *et al*., 2011). Also, adverse drug reactions (ADRs) accumulates to over 2 million per year in the United States alone (Gurwitz *et al*., 2000), therefore, the ability to predict drug interactions involving the CYP enzymes has become a key component of the drug discovery process (Forti and Wahlstrom, 2008). Providing the FDA with the metabolic profile of a new drug entity with CYP enzymes is the first step towards avoiding adverse reactions (Delgoda and Westlake, 2004). Known drug –herb interactions with clinical impact include grapefruit juice with felopidium, tricyclic anti depressants which is

We report for the first time hot water infusions of *Rhytidophyllum tomentosa*, *Psidium guajava, Symphytium officinale, Momordica charantia* in comparison with reported *Picrasma excelsa* being tested against CYP enzymes activity. The greatest potency was observed in the presence of *Psidium guajava* that inhibited CYP1A1 with an IC50 of 4.9µg/mL. All of the infusions are commonly consumed, be it, the fruit or as teas. With the high levels of polypharmacy practise that exist among Caribbean people and the world, having a metabolic profile on teas or commonly consumed plants become of grave importance. Results displayed in Table 3 point to is minimal risk through CYP mediated drug interactions as the teas weakly inhibited the main drug metabolising enzymes. Because, the activity of *Psidium guajava* towards CYP1A1 was the most potent we further evaluated the identification of the active ingredients that could be responsible for the observed bioactivity towards the CYP1A1 activity. The active ingredients were found to be quercetin and hyperin (see Figs. 4 & 5), compounds known as inhibitors of CYP enzymes, where quercetin isolated from St. John's Wort has been previoulsy shown to inhibit activities of CYPs 1A2, 2C19 and CYP2D6 with IC50s of 3.87 µM, 6.23 µM and 20.99 µM respectively; while hyperforin isolated from *Ginko biloba* shown to inhibit the activity of CYP3A4 with an IC50 of 4.30µM (Moltke *et al*., 2004; Zou *et al*., 2002). Inhibitions against the CYP enzymes appear to be between moderate and weak which confirms data in our lab, thus evoking moderate concern for potential interactions with co-medicated pharmaceuticals through CYP

Review of compounds that have potent and selective inhibitory properties against the activities of CYP1 family in particular CYPs 1A1 and 1B1 aid in identification of useful chemoprotectors. CA1 and quassin warrant further research because they were both potent against the activity of CYP1A1 while anhydrosorbifolin and chroma 6 targeted

towards the activity of CYP1B1

medicated through CYP3A4 inhibition.

mediated metabolism.

**5. Conclusion** 


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523-528.

194-204.


**3** 

*France* 

**Pharmacomodulation of Broad Spectrum** 

Janos Sapi1, Alain Jean-Paul Alix3 and Gautier Moroy4

*de Pharmacie, Université de Reims-Champagne-Ardenne* 

*UFR de Médecine, Université de Reims-Champagne-Ardenne* 

**Regulation of Gelatinases** 

Erika Bourguet1, William Hornebeck2,

**Matrix Metalloproteinase Inhibitors Towards** 

*1CNRS UMR 6229, Institut de Chimie Moléculaire de Reims, IFR 53 Biomolécules, UFR* 

*4INSERM UMR 973, Molécules thérapeutiques in silico (MTi), Université Paris Diderot* 

Matrix metalloproteinases (MMP) constitute a family of 23 zinc- and calcium-dependent endopeptidases that play pivotal functions in several physiological processes such as embryogenesis, wound healing, vasculogenesis or stem cell mobilization (Nagase et al., 2006). These enzymes were originally defined as matrix-degrading proteases, but a myriad of other substrates have been discovered including cytokines, chemokines, growth factors and their receptors, cell adhesion molecules and angiogenic factors. MMP were first described to exert their degradative function extracellularly against matrix macromolecules or at the pericellular microenvironment. Recently, MMP proved to cleave intracellular substrates belonging to any subcellular compartments (Cauwe & Opdenakker, 2010). Among them were notably apoptotic regulators, signal transducers, molecular chaperones or transcriptional and translational regulators. Therefore, MMP can be considered as proteases mainly controlling signaling events through processing cytokines, chemokines and degrading matrix, liberating matrikines in the extracellular space, or in turn cleaving enzymes involved in signal transduction inside the cells. MMP are regulated at distinct levels including gene expression, compartmentalization, proenzyme activation, enzyme inhibition, endocytosis, and finally substrate availability and affinity. MMP up-regulation participates in tumor progression and metastasis, inflammatory disorders, cardiovascular and autoimmune diseases (Hu et al., 2007; López-Otín & Matrisian, 2007; Mandal et al.,

All MMP are produced as proenzymes *i.e.* zymogen; enzyme latency is due to the formation of a coordinated bond between the zinc atom in the active site and an amino

**1. Introduction** 

2003; Murphy & Nagase, 2008).

*2CNRS UMR 6237, Laboratoire de Biochimie Médicale, MéDyc, IFR 53 Biomolécules,* 

*3Laboratoire de Spectroscopies et Structures Biomoléculaires (EA4303), IFR 53* 

*Biomolécules, UFR Sciences, Université de Reims-Champagne-Ardenne* 


## **Pharmacomodulation of Broad Spectrum Matrix Metalloproteinase Inhibitors Towards Regulation of Gelatinases**

Erika Bourguet1, William Hornebeck2, Janos Sapi1, Alain Jean-Paul Alix3 and Gautier Moroy4 *1CNRS UMR 6229, Institut de Chimie Moléculaire de Reims, IFR 53 Biomolécules, UFR de Pharmacie, Université de Reims-Champagne-Ardenne 2CNRS UMR 6237, Laboratoire de Biochimie Médicale, MéDyc, IFR 53 Biomolécules, UFR de Médecine, Université de Reims-Champagne-Ardenne 3Laboratoire de Spectroscopies et Structures Biomoléculaires (EA4303), IFR 53 Biomolécules, UFR Sciences, Université de Reims-Champagne-Ardenne 4INSERM UMR 973, Molécules thérapeutiques in silico (MTi), Université Paris Diderot France* 

#### **1. Introduction**

56 Enzyme Inhibition and Bioapplications

Zou, L.; Hrakey, M.R. Henderson, G.L. (2002). Effects of herbal components on cDNA-

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1579-1589.

expressed cytochrome P450 enzyme catalytic activity. Life Sciences, Vol.71 pp.

Matrix metalloproteinases (MMP) constitute a family of 23 zinc- and calcium-dependent endopeptidases that play pivotal functions in several physiological processes such as embryogenesis, wound healing, vasculogenesis or stem cell mobilization (Nagase et al., 2006). These enzymes were originally defined as matrix-degrading proteases, but a myriad of other substrates have been discovered including cytokines, chemokines, growth factors and their receptors, cell adhesion molecules and angiogenic factors. MMP were first described to exert their degradative function extracellularly against matrix macromolecules or at the pericellular microenvironment. Recently, MMP proved to cleave intracellular substrates belonging to any subcellular compartments (Cauwe & Opdenakker, 2010). Among them were notably apoptotic regulators, signal transducers, molecular chaperones or transcriptional and translational regulators. Therefore, MMP can be considered as proteases mainly controlling signaling events through processing cytokines, chemokines and degrading matrix, liberating matrikines in the extracellular space, or in turn cleaving enzymes involved in signal transduction inside the cells. MMP are regulated at distinct levels including gene expression, compartmentalization, proenzyme activation, enzyme inhibition, endocytosis, and finally substrate availability and affinity. MMP up-regulation participates in tumor progression and metastasis, inflammatory disorders, cardiovascular and autoimmune diseases (Hu et al., 2007; López-Otín & Matrisian, 2007; Mandal et al., 2003; Murphy & Nagase, 2008).

All MMP are produced as proenzymes *i.e.* zymogen; enzyme latency is due to the formation of a coordinated bond between the zinc atom in the active site and an amino

Pharmacomodulation of Broad Spectrum Matrix

S H

Pre Pro

Pre Pro

Pre Pro

Pre Pro S H

S H

S H

S H

S H

F u

F u

V n Fu

S H

c ataly tic Z n

c ataly tic Z n

catalytic Zn

F n F n F n

c atalyt ic Zn

ca taly tic Z n

catalytic Z n

MMP-23 Pre Pro F u cata ly tic C A Ig-lik e Type II transm em brane Femalysin

Table 1. MMP family. Pre: signal peptide, Pro: propeptide, Fn: fibronectin type II domain, Fu: furin recognition site, Vn: vitronectin-like domain, TM: transmembrane domain, Cy: cytoplasmic domain, GPI: glycosylphosphatidylinositol, CA: cysteine array, Ig-like:

F u Tm

MMP-7

MMP-1 MMP-3 MMP-8 MMP-10 MMP-12 MMP-13 MMP-18 MMP-19 MMP-20

MMP-2 MMP-9

MMP-11 MMP-28

MMP-14 MMP-15 MMP-16 MMP-24

MMP-17

MMP-25 P re Pro

MMP-21 Pre Pro

immunoglobulin-like domain.

MMP-26 Pre Pro

Metalloproteinase Inhibitors Towards Regulation of Gelatinases 59

c ataly tic Z n M inim al-d om ain

S S

S S

S S

S S

he mop e xin

he mo pe xin

GPI

S S

hemopexin

h em o pe xin

he mo pe xin

S im ple h em op exin-dom a in

Matrilysin Matrilysin-2

Collagenase-1 Stromelysin-1 Collagenase-2 Stromelysin-2 Metalloelastase Collagenase-3 Collagenase-4

Enamelysin

Gelatinase B

Epilysin

MT1-MMP MT2-MMP MT3-MMP MT5-MMP

MT4-MMP MT6-MMP

(*Xenopus*)

G elatin bin ding Gelatinase A

F urin ac tiv ated Stromelysin-3

C y T rans m em b ra ne

GP I-a nch ored

Vitronec tin -lik e XMMP

**Designation Main structure Name** 

S S

h emo pe xin

acid residue cysteine present in a consensus PRCGXPD sequence in MMP prodomain. Proteolysis of the prodomain, action of reactive oxygen species (O2 , NO) on the amino acid residue cysteine and allosteric perturbation (Sela-Passwell et al., 2010) of the prodomain can disrupt this Cys-Zn bond, a process named "cysteine switch" (Van Wart & Birkedal-Hansen, 1990). In the active enzyme, the zinc atom is linked to three histidine residues and a water molecule. A conserved glutamic acid residue (Glu) in the catalytic domain HEBXHXBGBXHS polarizes the water molecule (Gomis-Rüth, 2009; Lovejoy et al., 1994). This ligated water molecule attacks the carbonyl carbon of the scissile bond and transfers a proton to Glu and then to the scissile nitrogen atom. Then Glu releases the second proton from the water molecule to the scissile nitrogen atom and the peptide bond is cleaved (Figure 1).

Fig. 1. Mechanism of action of MMP (adapted from Lovejoy et al., 1994).

Historically, MMP were named according to their preferential action on matrix components: collagenases (MMP-1, MMP-8, MMP-13), gelatinases (MMP-2, MMP-9), proteoglycanases or stromelysins (MMP-3, MMP-10), macrophage elastase (MMP-12).

To date, a classification based on their domain organization is favoured: five of them are secreted and the others are transmembrane proteins (MT-MMP) based on their structure similarities (Table 1) (Egeblad & Werb, 2002).

MMP family is constituted by: a pre-domain involved in enzyme secretion, a pro-domain including a cysteine residue interacting with the zinc atom in the catalytic domain that maintains the inactive enzyme form. The catalytic domain is responsible of the MMP activity. All MMP, except MMP-7, MMP-26 (28 kDa) and MMP-23 (56 kDa) possess a hemopexin-like domain involved in the substrate interactions. The gelatinases (MMP-2 (72 kDa) and MMP-9 (92 kDa)) contain a gelatin-binding type II domain with three fibronectin (Fn(II))-like repeats. MMP-11 (51 kDa) and MMP-28 (59 kDa) contain a furin motif for recognition by furin-like serine proteinases. This motif is also present in MMP containing a vitronectin-like domain (MMP-21 (70 kDa)) and membrane-type MMP (MT-MMP). In addition, MT-MMP have a transmembrane domain and a short cytoplasmic domain or a glycosylphosphatidylinositol anchored (MMP-17 (57 kDa) and MMP-25 (63 kDa)). Finally, MMP-23 is a type II transmembrane MMP with a cysteine array and immunoglobulin-like domain.

acid residue cysteine present in a consensus PRCGXPD sequence in MMP prodomain.

acid residue cysteine and allosteric perturbation (Sela-Passwell et al., 2010) of the prodomain can disrupt this Cys-Zn bond, a process named "cysteine switch" (Van Wart & Birkedal-Hansen, 1990). In the active enzyme, the zinc atom is linked to three histidine residues and a water molecule. A conserved glutamic acid residue (Glu) in the catalytic domain HEBXHXBGBXHS polarizes the water molecule (Gomis-Rüth, 2009; Lovejoy et al., 1994). This ligated water molecule attacks the carbonyl carbon of the scissile bond and transfers a proton to Glu and then to the scissile nitrogen atom. Then Glu releases the second proton from the water molecule to the scissile nitrogen atom and the peptide bond

, NO) on the amino

N

H H

H

O O

Glu

O

O

**Zn2+** His His His

Proteolysis of the prodomain, action of reactive oxygen species (O2

N O

**Zn2+** His His His

stromelysins (MMP-3, MMP-10), macrophage elastase (MMP-12).

similarities (Table 1) (Egeblad & Werb, 2002).

Fig. 1. Mechanism of action of MMP (adapted from Lovejoy et al., 1994).

O H H

O O

Glu

H

N O

**Zn2+** His His His

O O

Historically, MMP were named according to their preferential action on matrix components: collagenases (MMP-1, MMP-8, MMP-13), gelatinases (MMP-2, MMP-9), proteoglycanases or

To date, a classification based on their domain organization is favoured: five of them are secreted and the others are transmembrane proteins (MT-MMP) based on their structure

MMP family is constituted by: a pre-domain involved in enzyme secretion, a pro-domain including a cysteine residue interacting with the zinc atom in the catalytic domain that maintains the inactive enzyme form. The catalytic domain is responsible of the MMP activity. All MMP, except MMP-7, MMP-26 (28 kDa) and MMP-23 (56 kDa) possess a hemopexin-like domain involved in the substrate interactions. The gelatinases (MMP-2 (72 kDa) and MMP-9 (92 kDa)) contain a gelatin-binding type II domain with three fibronectin (Fn(II))-like repeats. MMP-11 (51 kDa) and MMP-28 (59 kDa) contain a furin motif for recognition by furin-like serine proteinases. This motif is also present in MMP containing a vitronectin-like domain (MMP-21 (70 kDa)) and membrane-type MMP (MT-MMP). In addition, MT-MMP have a transmembrane domain and a short cytoplasmic domain or a glycosylphosphatidylinositol anchored (MMP-17 (57 kDa) and MMP-25 (63 kDa)). Finally, MMP-23 is a type II transmembrane MMP with a cysteine array and immunoglobulin-like

Glu

O <sup>H</sup> <sup>H</sup>

H

is cleaved (Figure 1).

**Zn2+** His His His

O O

Glu

<sup>O</sup> <sup>H</sup> <sup>H</sup>

domain.

N O

H


Table 1. MMP family. Pre: signal peptide, Pro: propeptide, Fn: fibronectin type II domain, Fu: furin recognition site, Vn: vitronectin-like domain, TM: transmembrane domain, Cy: cytoplasmic domain, GPI: glycosylphosphatidylinositol, CA: cysteine array, Ig-like: immunoglobulin-like domain.

Pharmacomodulation of Broad Spectrum Matrix

Metalloproteinase Inhibitors Towards Regulation of Gelatinases 61

Table 2. Overview of favorable ligand properties and conserved domains of gelatinases

(adapted from Cuniasse et al., 2005; Nicolotti et al., 2007; Terp et al., 2002).

### **2. Structures and properties of gelatinases**

#### **2.1 Structure of gelatinases active sites**

Gelatinases A (MMP-2) and B (MMP-9), as classified as both collagenases and elastases, are involved to a great extent in pathologies affecting major elastic tissues (lung, arteries). Among the MMP family members, gelatinases subclan, MMP-2 and MMP-9, do exhibit several originalities that could be taken into account for the design of inhibitors.

MMP family proved to have a great homology of sequence and the zinc-containing catalytic site is surrounded by subsite pockets named S1, S2, S3 for non-primed and S'1, S'2, S'3 for the primed side (Terp et al., 2002).

The conserved amino acid residues in gelatinases active-site region (Cuniasse et al., 2005; Kontogiorgis et al., 2005; Nicolotti et al., 2007; Rao, 2005) are given in Table 2.

The structural amino acid sequence of MMP is mainly similar except for the loop region (S'1 pocket), which displays different length and is composed of distinct amino acid composition. The similarities are ordered as S'1 > S2 > S'3 > S1, S3 > S'2.

Selective and/or combined occupancy of these pockets were believed to direct selectivity of inhibitor. More generally, it has been determined that such subsites display distinct potency in driving selectivity in order S'1 > S2, S'3, S3 > S1 > S'2.

S'1 pocket located immediately to "the right" of the catalytic site differs notably in size and shape among MMP and has been named specific pocket.

The S'1 pocket is deep, presenting an elongated and hydrophobic shape with an amino acid residue Leu at position 197 for all MMP except MMP-1 and MMP-7. The variation of amino acid residues among MMP, within this pocket, might be important. It adopts an extended shape in both gelatinases, but S'1 pocket in MMP-2 forms a large channel nearly bottomless, while it is slightly flexible in MMP-9 presenting a real pocket-like subsite.

The S'2 pocket is shallow, partly solvent-exposed and delimited on the top face by the amino acid residue 158 and on the bottom face by the amino acid residue 218. Its size is affected by the amino acid residues 162 (Asn), which is a Leu for both gelatinases, 163 (Val) which is an Ala for both gelatinases and 164 (Leu).

The S'3 pocket is neutral and partly solvent-exposed and delimited by the amino acid residues 222 (Leu) and 223 (Tyr). The size of this pocket is dependent on the amino acid residue 193, which is a Tyr for both gelatinases (Table 2).

As a rule, the substrates bind weakly with the unprimed subsites; however, some differences could be assigned between gelatinases.

The S1 pocket is shallow and hydrophobic. The same triad is pinpointed for MMP-2 and MMP-9 (His166-Phe168-Tyr155 and His183-Phe185-Tyr172, respectively). The amino acid residue 163 and to a lower extent the amino acid residue 155 influence the S1 subsite interactions with an inhibitor. The amino acid residue 163 is a Leu for both gelatinases.

The S2 pocket is solvent-exposed and the amino acid residues 86, 169 and 210 are poorly conserved in MMP family and affect the shape and the properties of this pocket. Its shape is


Gelatinases A (MMP-2) and B (MMP-9), as classified as both collagenases and elastases, are involved to a great extent in pathologies affecting major elastic tissues (lung, arteries). Among the MMP family members, gelatinases subclan, MMP-2 and MMP-9, do exhibit

MMP family proved to have a great homology of sequence and the zinc-containing catalytic site is surrounded by subsite pockets named S1, S2, S3 for non-primed and S'1, S'2, S'3 for

The conserved amino acid residues in gelatinases active-site region (Cuniasse et al., 2005;

The structural amino acid sequence of MMP is mainly similar except for the loop region (S'1 pocket), which displays different length and is composed of distinct amino acid

Selective and/or combined occupancy of these pockets were believed to direct selectivity of inhibitor. More generally, it has been determined that such subsites display distinct potency

S'1 pocket located immediately to "the right" of the catalytic site differs notably in size and

The S'1 pocket is deep, presenting an elongated and hydrophobic shape with an amino acid residue Leu at position 197 for all MMP except MMP-1 and MMP-7. The variation of amino acid residues among MMP, within this pocket, might be important. It adopts an extended shape in both gelatinases, but S'1 pocket in MMP-2 forms a large channel nearly bottomless,

The S'2 pocket is shallow, partly solvent-exposed and delimited on the top face by the amino acid residue 158 and on the bottom face by the amino acid residue 218. Its size is affected by the amino acid residues 162 (Asn), which is a Leu for both gelatinases, 163 (Val)

The S'3 pocket is neutral and partly solvent-exposed and delimited by the amino acid residues 222 (Leu) and 223 (Tyr). The size of this pocket is dependent on the amino acid

As a rule, the substrates bind weakly with the unprimed subsites; however, some

The S1 pocket is shallow and hydrophobic. The same triad is pinpointed for MMP-2 and MMP-9 (His166-Phe168-Tyr155 and His183-Phe185-Tyr172, respectively). The amino acid residue 163 and to a lower extent the amino acid residue 155 influence the S1 subsite interactions with an inhibitor. The amino acid residue 163 is a Leu for both gelatinases.

The S2 pocket is solvent-exposed and the amino acid residues 86, 169 and 210 are poorly conserved in MMP family and affect the shape and the properties of this pocket. Its shape is

several originalities that could be taken into account for the design of inhibitors.

Kontogiorgis et al., 2005; Nicolotti et al., 2007; Rao, 2005) are given in Table 2.

composition. The similarities are ordered as S'1 > S2 > S'3 > S1, S3 > S'2.

while it is slightly flexible in MMP-9 presenting a real pocket-like subsite.

in driving selectivity in order S'1 > S2, S'3, S3 > S1 > S'2.

shape among MMP and has been named specific pocket.

which is an Ala for both gelatinases and 164 (Leu).

differences could be assigned between gelatinases.

residue 193, which is a Tyr for both gelatinases (Table 2).

**2. Structures and properties of gelatinases** 

**2.1 Structure of gelatinases active sites** 

the primed side (Terp et al., 2002).

Table 2. Overview of favorable ligand properties and conserved domains of gelatinases (adapted from Cuniasse et al., 2005; Nicolotti et al., 2007; Terp et al., 2002).

Pharmacomodulation of Broad Spectrum Matrix

Metalloproteinase Inhibitors Towards Regulation of Gelatinases 63

It needs to be emphasized that interaction of PEX domain in MMP-2 with MT3-MMP also leads to enzyme activation that can be enhanced by the prior binding of pro-MMP-2 to chondroitin-4 sulfate chains-containing proteoglycan. PEX domain of MMP-2 was also reported to bind to v3, CC chemokine as monocyte chemoattractant protein-3 (MCP-3),

Such PEX domain is also important in driving the pro-MMP-9 activation; in human neutrophils formation of pro-MMP-9-lipocalin complex favours enzyme activation by kallikrein. It is also important for localizing the enzyme at the cell periphery through interaction with low density lipoprotein-receptor related protein (LRP), CD-91 or different isomer forms of CD-44, protein Ku, and is involved in the formation of covalent complexes

An important property of this carboxy terminal domain, in association with an unstructured, hydrophilic and flexible long-O-glycosylated domain (OG) in pro-MMP-9 which contains 11 repeats of the sequence T/SXXP (Figure 2), relies on its ability to catalyze the intracellular formation of enzyme dimers (Van den Steen et al., 2001). Importantly, the

Gelatinases also appear unique in MMP family in exhibiting fibronectin type II domains which are also designated as collagen binding domains (CBD) (Shipley et al., 1996). Indeed, deletion of these domains in both enzymes led to protease devoided of collagen(s) gelatin(s)- or elastin-degrading capacity (Allan et al., 1995). Recent data also indicated that OG could also mediate MMP-9 gelatin interaction by allowing the independent movement

The most important studies focus on the combinations of diverse structural modifications; three classes of compounds have been developed: combined inhibitors, right hand side and left hand side inhibitors based on the scissile bond in the catalytic site (Skiles et al., 2004; Whittaker et al., 1999). Some of them have been used as potential therapeutic agents to limit tumor progression. Instead of using MMP inhibitors as therapeutic treatment, they might be also useful as preventive drugs or as biomarkers in early stage of cancer. Up to now, most of the clinical trials in cancer were rather disappointing (Abbenante & Fairlie, 2005; Dormán et

The first generation of MMPI was based on peptidomimetic skeleton containing a succinic acid motif and a hydroxamic acid as zinc binding group (ZBG) (batimastat®, marimastat®, solimastat®, galardin®, trocade®). Hydroxamic acid is a bidentate chelator, but it has also a good binding affinity to other ions (Cu2+, Fe2+ and Ni2+). They are broad spectrum inhibitors and led to musculoskeletal syndrome side effect mainly due to the presence of hydroxamic

Prinomastat® contains a ring embedded sulfo-succinic acid motif, which increases oral availability and water solubility. Nevertheless, clinical trials have been discontinued after

CXC chemokine as stromal cell derived factor-1 (SDF-1) and fibrinogen.

with proteoglycans (Malla et al., 2008; Monferran et al., 2004).

dimer form of pro-MMP-9 is more resistant to MMP-3 activation.

Up to now, a myriad of MMPI has already been synthesized (Table 3).

of enzyme terminal domain (Vandooren et al., 2011).

**3. Design of MMP inhibitors (MMPI)** 

al., 2010; Fingleton, 2007; Gialeli et al. 2011).

phase III for musculoskeletal toxicity and poor survival rate.

acid.

dependent on the amino acid residue Pro at the position 87, then the MMP-2 has its Phe87 leading to a small and hydrophobic pocket and MMP-2 interacts with positive charge probes. When the Pro87 is lacking, another conformation was observed. The amino acid residue at the position 169 is a Pro for MMP-9 and defines a large hydrophobic pocket.

Finally, the amino acid residue 210, Asn in MMP-9 and Glu in MMP-2, leads to a less exposed pocket and notably plays a crucial role in enzyme selectivity. The S2 pocket is important and differentiates both gelatinases.

The S3 pocket is composed of a hydrophobic cleft delimited by the amino acid residues 155 and 168. The pocket shape and size are influenced by the amino acid residue 155 which is a Tyr and 168 which is a Phe for both gelatinases.

Although these subsites might direct enzyme specificities, interaction of gelatinases with macromolecular substrates also relies on the presence of remote binding sites named exosites that also notably act in driving enzyme action (Figure 2).

Fig. 2. Functions of gelatinases domains.

#### **2.2 Biological properties of gelatinases**

Gelatinases are most often associated to the cell plasma membrane of normal or transformed cells, thus targeting the proteolytic activity of invasive cells. Enzyme tethering to cell periphery requires the carboxy terminal hemopexin-like domain, designated as PEX of 200 amino acid residues on average forming a four bladed -propeller structure.

In pro-MMP-2, the PEX domain interacts with the C-terminal domain of TIMP-2 (Tissue Inhibitors of Matrix MetalloProteinase-2 (Brew & Nagase, 2010)) which allowed the complex to tether to plasma membrane through interaction of the N-terminal part of the inhibitor to a MT1-MMP homodimer: one molecule of MT1-MMP acting as a docking molecule, the other catalyzing pro-MMP-2 activation (Itoh et al., 2006, 2011; Sato & Takino, 2010).

Of note, this PEX domain also reacts with TIMP-3 and TIMP-4 but no MMP-2 activation was noted in such case.

dependent on the amino acid residue Pro at the position 87, then the MMP-2 has its Phe87 leading to a small and hydrophobic pocket and MMP-2 interacts with positive charge probes. When the Pro87 is lacking, another conformation was observed. The amino acid residue at the position 169 is a Pro for MMP-9 and defines a large hydrophobic pocket.

Finally, the amino acid residue 210, Asn in MMP-9 and Glu in MMP-2, leads to a less exposed pocket and notably plays a crucial role in enzyme selectivity. The S2 pocket is

The S3 pocket is composed of a hydrophobic cleft delimited by the amino acid residues 155 and 168. The pocket shape and size are influenced by the amino acid residue 155 which is a

Although these subsites might direct enzyme specificities, interaction of gelatinases with macromolecular substrates also relies on the presence of remote binding sites named

Pre

Gelatinases are most often associated to the cell plasma membrane of normal or transformed cells, thus targeting the proteolytic activity of invasive cells. Enzyme tethering to cell periphery requires the carboxy terminal hemopexin-like domain, designated as PEX of 200

In pro-MMP-2, the PEX domain interacts with the C-terminal domain of TIMP-2 (Tissue Inhibitors of Matrix MetalloProteinase-2 (Brew & Nagase, 2010)) which allowed the complex to tether to plasma membrane through interaction of the N-terminal part of the inhibitor to a MT1-MMP homodimer: one molecule of MT1-MMP acting as a docking molecule, the other

Of note, this PEX domain also reacts with TIMP-3 and TIMP-4 but no MMP-2 activation was

Proteolytic activation by MMP-3

Pro

Zn2+ catalytic

Degradation of substrates

**MMP-9**

His

Fn Fn Fn CBD

> Collagen Elastin Gelatin Fibrillin Fibrinogen Aggrecan Vitronectin Laminine Entactin...

S

PEX-like

Homodimerization TIMP-1 Ku CD44 Proteoglycan...

OGOG

His <sup>S</sup> His

OG

Hinge domain (444-521)

OG

S

important and differentiates both gelatinases.

Tyr and 168 which is a Phe for both gelatinases.

**MMP-2**

Collagen Elastin Gelatin Fibrillin Fibrinogen Aggrecan Vitronectin Fibronectin Laminine Tenascin-C...

His

Fn FnFn

(CBD)

Zn2+ catalytic

Fig. 2. Functions of gelatinases domains.

**2.2 Biological properties of gelatinases** 

Proteolytic activation by MT1MMP

Pre

Pro

noted in such case.

Degradation of substrates

exosites that also notably act in driving enzyme action (Figure 2).

Hinge domain (465-475)

His <sup>S</sup> His

S

Gelatin binding domain Collagen binding domain

> PEX-like S

Dimerization TIMP-2 TIMP-3 TIMP-4 v3 CC (MCP-3) CXC (SDF-1) Proteoglycan Fibrinogen...

amino acid residues on average forming a four bladed -propeller structure.

catalyzing pro-MMP-2 activation (Itoh et al., 2006, 2011; Sato & Takino, 2010).

It needs to be emphasized that interaction of PEX domain in MMP-2 with MT3-MMP also leads to enzyme activation that can be enhanced by the prior binding of pro-MMP-2 to chondroitin-4 sulfate chains-containing proteoglycan. PEX domain of MMP-2 was also reported to bind to v3, CC chemokine as monocyte chemoattractant protein-3 (MCP-3), CXC chemokine as stromal cell derived factor-1 (SDF-1) and fibrinogen.

Such PEX domain is also important in driving the pro-MMP-9 activation; in human neutrophils formation of pro-MMP-9-lipocalin complex favours enzyme activation by kallikrein. It is also important for localizing the enzyme at the cell periphery through interaction with low density lipoprotein-receptor related protein (LRP), CD-91 or different isomer forms of CD-44, protein Ku, and is involved in the formation of covalent complexes with proteoglycans (Malla et al., 2008; Monferran et al., 2004).

An important property of this carboxy terminal domain, in association with an unstructured, hydrophilic and flexible long-O-glycosylated domain (OG) in pro-MMP-9 which contains 11 repeats of the sequence T/SXXP (Figure 2), relies on its ability to catalyze the intracellular formation of enzyme dimers (Van den Steen et al., 2001). Importantly, the dimer form of pro-MMP-9 is more resistant to MMP-3 activation.

Gelatinases also appear unique in MMP family in exhibiting fibronectin type II domains which are also designated as collagen binding domains (CBD) (Shipley et al., 1996). Indeed, deletion of these domains in both enzymes led to protease devoided of collagen(s) gelatin(s)- or elastin-degrading capacity (Allan et al., 1995). Recent data also indicated that OG could also mediate MMP-9 gelatin interaction by allowing the independent movement of enzyme terminal domain (Vandooren et al., 2011).

#### **3. Design of MMP inhibitors (MMPI)**

Up to now, a myriad of MMPI has already been synthesized (Table 3).

The most important studies focus on the combinations of diverse structural modifications; three classes of compounds have been developed: combined inhibitors, right hand side and left hand side inhibitors based on the scissile bond in the catalytic site (Skiles et al., 2004; Whittaker et al., 1999). Some of them have been used as potential therapeutic agents to limit tumor progression. Instead of using MMP inhibitors as therapeutic treatment, they might be also useful as preventive drugs or as biomarkers in early stage of cancer. Up to now, most of the clinical trials in cancer were rather disappointing (Abbenante & Fairlie, 2005; Dormán et al., 2010; Fingleton, 2007; Gialeli et al. 2011).

The first generation of MMPI was based on peptidomimetic skeleton containing a succinic acid motif and a hydroxamic acid as zinc binding group (ZBG) (batimastat®, marimastat®, solimastat®, galardin®, trocade®). Hydroxamic acid is a bidentate chelator, but it has also a good binding affinity to other ions (Cu2+, Fe2+ and Ni2+). They are broad spectrum inhibitors and led to musculoskeletal syndrome side effect mainly due to the presence of hydroxamic acid.

Prinomastat® contains a ring embedded sulfo-succinic acid motif, which increases oral availability and water solubility. Nevertheless, clinical trials have been discontinued after phase III for musculoskeletal toxicity and poor survival rate.

Pharmacomodulation of Broad Spectrum Matrix

limited oral bioavailability.

**gelatinases inhibitors** 

H O <sup>N</sup> H

Z n 2 +

broad spectrum MMPI (Figure 3).

O

Fig. 3. Galardin® and analogues.

H <sup>N</sup> <sup>N</sup> H M e

S '1

N H

**4.1 Influence of the S'1 subsite: Modulation of galardin®**

S '2

The modifications have been focused on the P'1, P'2, P'3 groups and ZBG.

O

O

G a lard in ®

disease.

brain barrier. Actually, it is the most promising MMPI.

Metalloproteinase Inhibitors Towards Regulation of Gelatinases 65

The thiol zinc binding group of rebimastat® is a weak monodentate ligand and the

Finally, metastat® is a second generation of tetracyclines still in phase II clinical trials (Acne, AIDS-related Kaposi's sarcoma and mainly used in cancer). The only detected side effect is its photosensibility. This inhibitor is selective of MMP-2 and MMP-9 and crosses the blood-

These failures are mainly due to their broad spectrum MMP inhibitory activity, the similarity of their active sites with those of other metalloproteinases (ADAM, ADAMT…), the poor selectivity of the chelating group, the administration of MMPI in late disease stage, their poor pharmacokinetics, unavoidable side effects (musculoskeletal pain), toxicity and

However, these efforts led to pinpoint the importance of MMPI selectivity and allowed the identification of MMP as target and anti-target in various diseases progression (Overall & Kleifeld, 2006). Certain MMP are both targets and anti-targets depending on the stage of the

For a decade, we have been involved in the pharmacomodulation of galardin®, a powerful

ZBG

P2

S electivity

By the beginning of 1994, galardin® was in phase I clinical trials for age related macular degeneration (ARMD) and as chronic obstructive pulmonary disease (COPD) by Glycomed. In order to increase selectivity, the synthesis of analogues of galardin®, has been achieved.

Our first experiments started with the insertion of one unsaturation in P'1 position to increase the hydrophobicity of the new compounds and to study the effect of substitution on S'1 pocket specificity (Figure 4). For this purpose, replacement of isobutyl group by an isobutylidene group of *E* geometry enhanced by 100-fold MMP-2 selectivity *versus* MMP-3

H <sup>N</sup> <sup>N</sup> <sup>H</sup> <sup>O</sup>

P '2

Analogues

P '1 O

N H P '3

A nchor to other target

 **in gelatinases inhibition** 

**4. Galardin® pharmacomodulation as a tool for designing specific** 

S '3

musculoskeletal toxicity and its poor response led clinicians to stop treatment.

Tanomastat® has a thioether function increasing the oral activity and selectivity for MMP-2, MMP-3 and MMP-9. Unfortunately, clinical trials are discontinued for haematological toxicity and poor survival rate.


Table 3. Main MMPI in clinical development.

Tanomastat® has a thioether function increasing the oral activity and selectivity for MMP-2, MMP-3 and MMP-9. Unfortunately, clinical trials are discontinued for haematological

> Phase I (Discontinued)

> > Phase III

Phase I

Phase I (Discontinued)

Phase III

Phase III

Phase II Phase III (Discontinued)

Phase III

(Discontinued) <sup>N</sup>

(Discontinued) <sup>N</sup>

C l

(Discontinued) <sup>N</sup>

O

N N

O

(Discontinued) <sup>N</sup>

(Discontinued) <sup>N</sup>

**development Structure** 

N H H O

H H O

H H O O

N H H O O

H N O HO

O

S S

<sup>O</sup> <sup>H</sup> N O

<sup>O</sup> <sup>H</sup> N O

> H N O

> > H N O

S

S O O

H H O O

O

N N H

O

O

H

O H

O O H O

H H

O

S H

O H

N H O

> N H

> > N

O

N H O

> N H O

> > N H

O

N O

> S O O H

> > N H

O

N H <sup>2</sup> O

O H

H N O

**Inhibitors Indication Clinical trials**

Cancer Broad spectrum (MMP-1, -2, -3, -7, -9)

Cancer Broad spectrum (MMP-1, -2, -7, -9)

Cancer Broad spectrum (MMP-1, -7)

Eye disease, COPD Broad spectrum (MMP-1, -2, -9, -12)

Cancer Macular degeneration Broad spectrum (MMP-2, -3, -7, -9, -13)

Rheumatoid arthritis (MMP-1, -8, -13)

Cancer Arthritis (MMP-2, -3, -8, -9)

Cancer Broad spectrum (MMP-1, -2, -7, -9, -14)

Cancer

Table 3. Main MMPI in clinical development.

(MMP-2, -9) Phase II

toxicity and poor survival rate.

**MMP** 

Batimastat® (BB-94) British Biotech

Marimastat® (BB-2516) British Biotech

Solimastat® (BB-3644) British Biotech

> Galardin® (GM-6001) Glycomed

Prinomastat® (AG-3340) Agouron

Trocade® (Ro32-3555) Roche

Tanomastat® (BAY 12-9566) Bayer

Rebimastat® (BMS-275291) Bristol-Myers Squibb

Metastat® (CMT-3) Collagenex The thiol zinc binding group of rebimastat® is a weak monodentate ligand and the musculoskeletal toxicity and its poor response led clinicians to stop treatment.

Finally, metastat® is a second generation of tetracyclines still in phase II clinical trials (Acne, AIDS-related Kaposi's sarcoma and mainly used in cancer). The only detected side effect is its photosensibility. This inhibitor is selective of MMP-2 and MMP-9 and crosses the bloodbrain barrier. Actually, it is the most promising MMPI.

These failures are mainly due to their broad spectrum MMP inhibitory activity, the similarity of their active sites with those of other metalloproteinases (ADAM, ADAMT…), the poor selectivity of the chelating group, the administration of MMPI in late disease stage, their poor pharmacokinetics, unavoidable side effects (musculoskeletal pain), toxicity and limited oral bioavailability.

However, these efforts led to pinpoint the importance of MMPI selectivity and allowed the identification of MMP as target and anti-target in various diseases progression (Overall & Kleifeld, 2006). Certain MMP are both targets and anti-targets depending on the stage of the disease.

#### **4. Galardin® pharmacomodulation as a tool for designing specific gelatinases inhibitors**

For a decade, we have been involved in the pharmacomodulation of galardin®, a powerful broad spectrum MMPI (Figure 3).

Fig. 3. Galardin® and analogues.

By the beginning of 1994, galardin® was in phase I clinical trials for age related macular degeneration (ARMD) and as chronic obstructive pulmonary disease (COPD) by Glycomed.

In order to increase selectivity, the synthesis of analogues of galardin®, has been achieved. The modifications have been focused on the P'1, P'2, P'3 groups and ZBG.

#### **4.1 Influence of the S'1 subsite: Modulation of galardin® in gelatinases inhibition**

Our first experiments started with the insertion of one unsaturation in P'1 position to increase the hydrophobicity of the new compounds and to study the effect of substitution on S'1 pocket specificity (Figure 4). For this purpose, replacement of isobutyl group by an isobutylidene group of *E* geometry enhanced by 100-fold MMP-2 selectivity *versus* MMP-3

Pharmacomodulation of Broad Spectrum Matrix

Graphics System, Palo Alto, CA. http://www.pymol.org).

Metalloproteinase Inhibitors Towards Regulation of Gelatinases 67

input files necessary to the docking procedures and to analyze the docking results. The figures have been done using Pymol program (DeLano, W. L. 2002. PyMol Molecular

Molecular modelling experiments confirmed the importance of the insertion of the alkyl

Fig. 5. Complex between MMP-2 and analogue **2a**. The MMP-2 secondary structure was represented in cartoon. The analogue **2a** is shown with sticks in which the C atoms are

The S'1 pocket of MMP-2 is sufficiently deep to accommodate the long alkyl chain. On the contrary, the S'1 pocket of MMP-9 at the end of the tunnel is restrained, like a funnel by the amino acid residues Glu233, Arg241, Thr246 and Pro247. The S'1 pocket of MMP-9 is large enough to accept a phenyl group at the entrance of the pocket such as compounds **2b** and

In order to increase hydrophobicity, aiming at a better MMP-2 selectivity, the alkyl chain was elongated with n = 8 to 20 carbon atoms as described in the literature (Levy et al., 1998; Miller et al., 1997; Whittaker et al., 1999). Using galardin® as template, the activity and selectivity were not really increased with the length of the linear chain. Batimastat® inhibitors increase activity for MMP-2 with C8 long alkyl chain (IC50 = 0.6 nM) and a C12 analogue displays a MMP-2 selectivity comparing to MMP-1 (IC50 = 1 and 50000 nM, respectively). Finally, a good activity for MMP-2 is obtained for C9 long chain marimastat® analogues (IC50 < 0.15 nM) and a maximal selectivity occurs with a C16 for MMP-2 *versus*

colored in magenta. Zn atom is displayed as a grey sphere.

MMP-1 (IC50 = 0.6 and 5000 nM, respectively).

**2d**.

chains in the S'1 pocket, supporting the observed biological data for **2a** (Figure 5).

(IC50 = 1.3 and 179 nM, respectively) (Marcq et al., 2003). The double bond geometry was found important for potency and selectivity as shown with the equimolar *E/Z* mixture which displayed lower activity.

Pursuing these pharmacomodulations aiming at better MMP-2 selectivities, we planned to increase hydrophobicity and rigidity with the dehydro and didehydro analogues which were synthesized (analogue **2a-d** and **3a-h**).

Fig. 4. Pharmacomodulation of the P'1 group.

Introduction of either one or two unsaturations decreased their potent MMP inhibitory activity as compared to parent molecule over all MMP (Moroy et al., 2007). However, the presence of a phenyl group at the end of alkyl chain (**2b** and **2d**) led to inhibitors with a good activity and selectivity for MMP-9 (IC50 = 38 and 45 nM, respectively).

In parallel, C7 long alkyl chain containing galardin*®* **2a** displayed a MMP-2 selectivity comparing to MMP-9 (IC50 = 123 nM) (Table 4).


Table 4. Influence of the P'1 chain length on the selectivity and potency of MMP inhibitors. IC50 values are expressed in nM.

AutoDock 4.0 program (Huey et al., 2007; Morris et al., 1998) was used to perform the computational molecular docking. AutoDockTools package was employed to prepare the

(IC50 = 1.3 and 179 nM, respectively) (Marcq et al., 2003). The double bond geometry was found important for potency and selectivity as shown with the equimolar *E/Z* mixture

Pursuing these pharmacomodulations aiming at better MMP-2 selectivities, we planned to increase hydrophobicity and rigidity with the dehydro and didehydro analogues which

> H <sup>N</sup> <sup>N</sup> H Me

> > N H

Introduction of either one or two unsaturations decreased their potent MMP inhibitory activity as compared to parent molecule over all MMP (Moroy et al., 2007). However, the presence of a phenyl group at the end of alkyl chain (**2b** and **2d**) led to inhibitors with a

In parallel, C7 long alkyl chain containing galardin*®* **2a** displayed a MMP-2 selectivity

Compounds ZBG P'1 MMP-1 MMP-2 MMP-9 MMP-14 **Galardin®** -NH-OH 1.5 1.1 0.5 13.4 **1** -NH-OH *-i*Pr 18 9.2 17 10 **2a** -NH-OH -(CH2)5-Me 32000 123 > 104 2660 **2b** -NH-OH -(CH2)2-Ph 9130 280 45 53100 **2c** -NH-OH -CH=CH-Me 984 78500 974 913 **2d** -NH-OH -CH=CH-Ph 1240 120 38 2490 **3a** -OH -(CH2)6-Me > 105 7570 838 > 105 **3b** -OH -(CH2)7-Me > 105 458 241 > 105 **3c** -OH -(CH2)8-Me > 105 247 173 > 105 **3d** -OH -(CH2)9-Me > 105 249 450 > 105 **3e** -OH -(CH2)10-Me > 105 351 211 > 105 **3f** -OH -(CH2)11-Me > 105 655 673 > 105 **3g** -OH -(CH2)14-Me > 105 762 582 > 105 **3h** -OH -(CH2)18-Me > 105 > 105 > 105 > 105

Table 4. Influence of the P'1 chain length on the selectivity and potency of MMP inhibitors.

AutoDock 4.0 program (Huey et al., 2007; Morris et al., 1998) was used to perform the computational molecular docking. AutoDockTools package was employed to prepare the

O

R = -NH-OH

P'1 = -(CH2)n-CH3

**2a-d**

**3a-h** R = -OH

P'1 = -(CH2)5-CH3, -(CH2)2-Ph, -CH=CH-Me, -CH=CH-Ph

O

which displayed lower activity.

H <sup>N</sup> <sup>N</sup> H Me

**1**

IC50 values are expressed in nM.

O

HO <sup>N</sup> H O

were synthesized (analogue **2a-d** and **3a-h**).

R

O

P'1

good activity and selectivity for MMP-9 (IC50 = 38 and 45 nM, respectively).

N H

Fig. 4. Pharmacomodulation of the P'1 group.

comparing to MMP-9 (IC50 = 123 nM) (Table 4).

O

input files necessary to the docking procedures and to analyze the docking results. The figures have been done using Pymol program (DeLano, W. L. 2002. PyMol Molecular Graphics System, Palo Alto, CA. http://www.pymol.org).

Molecular modelling experiments confirmed the importance of the insertion of the alkyl chains in the S'1 pocket, supporting the observed biological data for **2a** (Figure 5).

Fig. 5. Complex between MMP-2 and analogue **2a**. The MMP-2 secondary structure was represented in cartoon. The analogue **2a** is shown with sticks in which the C atoms are colored in magenta. Zn atom is displayed as a grey sphere.

The S'1 pocket of MMP-2 is sufficiently deep to accommodate the long alkyl chain. On the contrary, the S'1 pocket of MMP-9 at the end of the tunnel is restrained, like a funnel by the amino acid residues Glu233, Arg241, Thr246 and Pro247. The S'1 pocket of MMP-9 is large enough to accept a phenyl group at the entrance of the pocket such as compounds **2b** and **2d**.

In order to increase hydrophobicity, aiming at a better MMP-2 selectivity, the alkyl chain was elongated with n = 8 to 20 carbon atoms as described in the literature (Levy et al., 1998; Miller et al., 1997; Whittaker et al., 1999). Using galardin® as template, the activity and selectivity were not really increased with the length of the linear chain. Batimastat® inhibitors increase activity for MMP-2 with C8 long alkyl chain (IC50 = 0.6 nM) and a C12 analogue displays a MMP-2 selectivity comparing to MMP-1 (IC50 = 1 and 50000 nM, respectively). Finally, a good activity for MMP-2 is obtained for C9 long chain marimastat® analogues (IC50 < 0.15 nM) and a maximal selectivity occurs with a C16 for MMP-2 *versus* MMP-1 (IC50 = 0.6 and 5000 nM, respectively).

Pharmacomodulation of Broad Spectrum Matrix

group (Rouffet et al., 2009).

In all cases, interaction between

properties and also biodisponibility and stability.

Br

the S'2 solvent-exposed pocket.

inhibitors. IC50 values are expressed in nM.

or tri-dentate mode.

(Figure 6).

Metalloproteinase Inhibitors Towards Regulation of Gelatinases 69

The hydroxamate acts as a bidentate ligand with the zinc ion to form the distorded trigonalbipyramidal coordination geometry. With respect to the design of new ZBG, a DFT (Density Functional Theory) study revealed different modes of chelation of the sulfonylhydrazide

The zinc ion was found to be ligated to three 4-Me-imidazoles used as mimetics of histidine imidazole moieties located in the MMP catalytic site in physiological conditions. The sulfonylhydrazide group could chelate the zinc ion in two different manners either in a bi-

In the case of the tridentate mode, the third interaction involves one of the sulfonyl oxygen atom and the Zn2+ ion. Consequently, the bidentate conformation was more favourable (4 to 5 kcal/mol) and the sulfonylhydrazide function seemed to possess ideal zinc binding

Following these investigations, the sulfonylhydrazide group was incorporated into the galardin*®* backbone as zinc binding group. Among the synthesized subtituents, the *p*bromobiphenyl group displayed a good potency for MMP-2 (LeDour et al., 2008). Then, based on our preliminary results further modifications of the P'1 (long alkyl chain) and the P'2 (phenyl group) substituents were introduced to increase the selectivity for MMP-2

> <sup>N</sup> <sup>N</sup> H

Zn2+

R1 = H, Ph **5**

Finally, a high potency for MMP-9 was obtained with the compound **5a** (or **5b**) with a small group such as an isobutyl (Table 5). Introduction of a phenyl group at the position 2 of the indole ring did not modify the activity. This could easily be explained taking into account

Compounds R1 MMP-1 MMP-2 MMP-3 MMP-9 MMP-14 **5a** H 30 98 5800 3 20000 **5b** Ph 350 247 53 18 1237

Table 5. Influence of the P'2 group and ZBG on the selectivity and potency of MMP

S O

Fig. 6. Modifications of the P'2 group and sulfonylhydrazide function as ZBG.

O

O

H <sup>N</sup> <sup>N</sup> H Me

R1

N H

O

O

i. the Zn2+ ion and the sulfonamide nitrogen was observed as well as, ii. the Zn2+ ion with the oxygen atom of the sulfonylhydrazide carbonyl.

Nevertheless, in our case no better IC50 were found for MMP-2 with increasing chain length when an unsaturation was incorporated. However, a good selectivity for MMP-2 *versus* MMP-1 and MMP-14 is observed. Also, carboxylic derivatives **3c** and **3e** displayed a good selectivity for MMP-9 *versus* MMP-1 and MMP-14.

As stated previously, S'1 pocket is generally quite large in all MMP. However, the amino acid residue Arg214 redefines the bottom of the pocket in MMP-1 leading to a small and restricted S'1 pocket. The amino acid residue Arg214 can be flexible but in most cases, a large P'1 group inhibitor is expected to bind weakly to MMP-1. Thus, it is not surprising, to find that most of the MMP-1 inhibitors have relatively small P'1 groups.

MMP-14 appears as one key-proteolytic enzyme to promote cancer invasion and metastasis (Hernandez-Barrantes et al., 2002). Up to now, only one pentacyclic sterol sulphate MMP-14 inhibitor was described to display MMP-14 selectivity while it exhibits only low potency (Fujita et al., 2001).

S'1 pocket of MMP-14 was found to be two amino acid residues longer than those of gelatinases. The amino acid residue Met237 allows favourable interactions with the hydrophobic substituents at the bottom of the pocket, but unfavourable interaction with the positively charged substituents.

Therefore, lack of inhibition of this enzyme by long alkyl chain (Table 4) is rather surprising. The unsaturation might disturb the entrance of the S'1 pocket, but that is purely speculative. Finally, it seems to be more difficult to fit MMP-14 pockets, perhaps in keeping with its transmembrane localization and domain structure.

#### **4.2 Influence of the S'2 subsite on gelatinases inhibition**

Introduction of an alkyl chain in the P'2 position of the indole ring leads to lower gelatinase inhibitory activity. Nevertheless, the selectivity for MMP-2 was pinpointed (Marcq et al., 2003).

On the contrary, the introduction of a phenyl group at the P'2 position (analogue **4**) enhanced the selectivity towards MMP-2 maintaining a high potency (IC50 = 0.092 nM). To the best of our knowledge, the large and solvent-exposed S'2 pocket could accommodate large and hydrophobic groups (LeDour et al., 2008). A good activity was found for MMP-1 and MMP-14 (IC50 = 0.244 and 0.601 nM, respectively).

#### **4.3 Influence of the unprimed subsites on gelatinases inhibition**

It is well documented that the hydroxamic acid is one of the most powerful ZBG, but its toxicity and low bioavailability triggered tremendous efforts to design other ZBG (Jacobsen et al., 2007, 2010). Of note, the zing binding group affinity is as follows: hydroxamate > retrohydroxamate > sulfhydryl > phosphinate > carboxylate > heterocyclic core. Nevertheless, a zinc binding group with lower affinity may be advantageous.

In this line, we have proposed various hydrazide and sulfonylhydrazide-type functions as potential ZBG. The sulfonylhydrazide derivative is responsible for the increased acidity of the NH close to SO2 function allowing the H-bond to be formed with the catalytic glutamate residue (Augé et al., 2003, 2004).

The hydroxamate acts as a bidentate ligand with the zinc ion to form the distorded trigonalbipyramidal coordination geometry. With respect to the design of new ZBG, a DFT (Density Functional Theory) study revealed different modes of chelation of the sulfonylhydrazide group (Rouffet et al., 2009).

The zinc ion was found to be ligated to three 4-Me-imidazoles used as mimetics of histidine imidazole moieties located in the MMP catalytic site in physiological conditions. The sulfonylhydrazide group could chelate the zinc ion in two different manners either in a bior tri-dentate mode.

In all cases, interaction between

68 Enzyme Inhibition and Bioapplications

Nevertheless, in our case no better IC50 were found for MMP-2 with increasing chain length when an unsaturation was incorporated. However, a good selectivity for MMP-2 *versus* MMP-1 and MMP-14 is observed. Also, carboxylic derivatives **3c** and **3e** displayed a good

As stated previously, S'1 pocket is generally quite large in all MMP. However, the amino acid residue Arg214 redefines the bottom of the pocket in MMP-1 leading to a small and restricted S'1 pocket. The amino acid residue Arg214 can be flexible but in most cases, a large P'1 group inhibitor is expected to bind weakly to MMP-1. Thus, it is not surprising, to

MMP-14 appears as one key-proteolytic enzyme to promote cancer invasion and metastasis (Hernandez-Barrantes et al., 2002). Up to now, only one pentacyclic sterol sulphate MMP-14 inhibitor was described to display MMP-14 selectivity while it exhibits only low potency

S'1 pocket of MMP-14 was found to be two amino acid residues longer than those of gelatinases. The amino acid residue Met237 allows favourable interactions with the hydrophobic substituents at the bottom of the pocket, but unfavourable interaction with the

Therefore, lack of inhibition of this enzyme by long alkyl chain (Table 4) is rather surprising. The unsaturation might disturb the entrance of the S'1 pocket, but that is purely speculative. Finally, it seems to be more difficult to fit MMP-14 pockets, perhaps in keeping with its

Introduction of an alkyl chain in the P'2 position of the indole ring leads to lower gelatinase inhibitory activity. Nevertheless, the selectivity for MMP-2 was pinpointed (Marcq et al.,

On the contrary, the introduction of a phenyl group at the P'2 position (analogue **4**) enhanced the selectivity towards MMP-2 maintaining a high potency (IC50 = 0.092 nM). To the best of our knowledge, the large and solvent-exposed S'2 pocket could accommodate large and hydrophobic groups (LeDour et al., 2008). A good activity was found for MMP-1

It is well documented that the hydroxamic acid is one of the most powerful ZBG, but its toxicity and low bioavailability triggered tremendous efforts to design other ZBG (Jacobsen et al., 2007, 2010). Of note, the zing binding group affinity is as follows: hydroxamate > retrohydroxamate > sulfhydryl > phosphinate > carboxylate > heterocyclic core.

In this line, we have proposed various hydrazide and sulfonylhydrazide-type functions as potential ZBG. The sulfonylhydrazide derivative is responsible for the increased acidity of the NH close to SO2 function allowing the H-bond to be formed with the catalytic glutamate

find that most of the MMP-1 inhibitors have relatively small P'1 groups.

selectivity for MMP-9 *versus* MMP-1 and MMP-14.

transmembrane localization and domain structure.

**4.2 Influence of the S'2 subsite on gelatinases inhibition** 

and MMP-14 (IC50 = 0.244 and 0.601 nM, respectively).

**4.3 Influence of the unprimed subsites on gelatinases inhibition** 

Nevertheless, a zinc binding group with lower affinity may be advantageous.

(Fujita et al., 2001).

2003).

positively charged substituents.

residue (Augé et al., 2003, 2004).


In the case of the tridentate mode, the third interaction involves one of the sulfonyl oxygen atom and the Zn2+ ion. Consequently, the bidentate conformation was more favourable (4 to 5 kcal/mol) and the sulfonylhydrazide function seemed to possess ideal zinc binding properties and also biodisponibility and stability.

Following these investigations, the sulfonylhydrazide group was incorporated into the galardin*®* backbone as zinc binding group. Among the synthesized subtituents, the *p*bromobiphenyl group displayed a good potency for MMP-2 (LeDour et al., 2008). Then, based on our preliminary results further modifications of the P'1 (long alkyl chain) and the P'2 (phenyl group) substituents were introduced to increase the selectivity for MMP-2 (Figure 6).

Fig. 6. Modifications of the P'2 group and sulfonylhydrazide function as ZBG.

Finally, a high potency for MMP-9 was obtained with the compound **5a** (or **5b**) with a small group such as an isobutyl (Table 5). Introduction of a phenyl group at the position 2 of the indole ring did not modify the activity. This could easily be explained taking into account the S'2 solvent-exposed pocket.


Table 5. Influence of the P'2 group and ZBG on the selectivity and potency of MMP inhibitors. IC50 values are expressed in nM.

Pharmacomodulation of Broad Spectrum Matrix

through proteolytic cascades.

al., 2011).

d-hPI

d-hPI

Metalloproteinase Inhibitors Towards Regulation of Gelatinases 71

Elastolysis requires the participation of serine- and metallo-elastases (Figure 8) which act

M M P-3

Fig. 8. Control of the serine (HLE)- and metallo-elastases (MMP-2, MMP-9) crosstalk in

Besides, a serine elastase as human leucocyte elastase (HLE) can degrade TIMP, and reversely MMP can hydrolyse serine protease inhibitor as 1 proteinase inhibitor (Nunes et

To control elastolysis, we thus attempted to design substances that could interfere with all actors of the depicted cascade. For that purpose, long chain-unsaturated fatty acids, as oleic acid, have been described to inhibit HLE (Hornebeck et al., 1985; Shock et al., 1990; Tyagi & Simon, 1990) and to impede plasmin-mediated prostromelysin-1 activation (Huet et al., 2004) as well as gelatinases activities (Berton et al., 2001). To that respect, we envisaged the synthesis of a double-headed protease-MMP inhibitor (d-hPI) able to block elastase and MMP activities. To that end, an oleoyl group was incorporated to galardin® at the P'3

pro-M M P-3 pro-M M P-2

d-hPI

d-hPI

d-hPI

plasmin

M M P-2

d-hPI

d-hPI

d-hPI

d-hPI

HLE 1P i

TIM P-1 :pro-M M P-9 pro-M M P-9 M M P-9

elastolysis by double-headed protease-MMP inhibitor (d-hPI).

position (Figure 9) (Moroy et al., 2011).

R = OH, **6** R = NH-OH, **7**

H <sup>N</sup> <sup>N</sup> H

Fig. 9. Double-headed protease-MMP inhibitor (d-hPI).

O

O

N H

O NH

MMP HLE, plasmin

R

O

Docking studies of **5a** confirmed the occupancy of the MMP-9 S'1 pocket by the isobutyl group, the S2 subsite by the *p*-bromobiphenyl group and the chelation of the sulfonylhydrazide to the catalytic site (Figure 7). It is known that the amino acid residues Glu412 and Asp410, located in the S2 subsite control the selectivity of MMP-2 and MMP-9, respectively.

In MMP-2, the amino acid residue Glu412 is able to form an H-bond with the substrate which could not be formed in MMP-9 presenting the amino acid residue Asp410 (Chen et al., 2003).

In our case, no H-bond can be formed with the hydrophobic *p*-bromobiphenyl group and the compound **5a** displayed selectivity for MMP-9.

Fig. 7. Complex between MMP-9 and analogue **5a.** The MMP-9 secondary structure was represented in cartoon. The analogue **5a** is shown with sticks in which the C atoms are colored in magenta. Zn atom is displayed as a grey sphere.

Unfortunately, none of the P'1 (with an unsaturation and long alkyl chain or bulky substituent) and P'2 (with a phenyl group) modified galardin*®* derivatives exhibited increased inhibitory capacity and selectivity. Our docking data indicated that these compounds adopted a conformation in which sp2-hybridized carbon atom of the alkylidene side-chain led to steric hindrance impeding the entrance in the S'1 subsite. Consequently, when the S2 pocket is occupied, the primed subsites could not tolerate any large substituent and no synergistic effect could be obtained.

#### **5. Control of elastolytic cascade by oleoyl-galardin®**

Elastin degradation is at the genesis of cardiovascular disease as athero-arteriosclerosis and aneurysm formation, and pulmonary diseases as chronic obstructive pulmonary disease or lung cancer (Moroy et al., 2012; Muroski et al., 2008; Thompson & Parks, 1996).

Docking studies of **5a** confirmed the occupancy of the MMP-9 S'1 pocket by the isobutyl group, the S2 subsite by the *p*-bromobiphenyl group and the chelation of the sulfonylhydrazide to the catalytic site (Figure 7). It is known that the amino acid residues Glu412 and Asp410, located in the S2 subsite control the selectivity of MMP-2 and MMP-9,

In MMP-2, the amino acid residue Glu412 is able to form an H-bond with the substrate which could not be formed in MMP-9 presenting the amino acid residue Asp410 (Chen et al., 2003). In our case, no H-bond can be formed with the hydrophobic *p*-bromobiphenyl group and

Fig. 7. Complex between MMP-9 and analogue **5a.** The MMP-9 secondary structure was represented in cartoon. The analogue **5a** is shown with sticks in which the C atoms are

Unfortunately, none of the P'1 (with an unsaturation and long alkyl chain or bulky substituent) and P'2 (with a phenyl group) modified galardin*®* derivatives exhibited increased inhibitory capacity and selectivity. Our docking data indicated that these compounds adopted a conformation in which sp2-hybridized carbon atom of the alkylidene side-chain led to steric hindrance impeding the entrance in the S'1 subsite. Consequently, when the S2 pocket is occupied, the primed subsites could not tolerate any large substituent

Elastin degradation is at the genesis of cardiovascular disease as athero-arteriosclerosis and aneurysm formation, and pulmonary diseases as chronic obstructive pulmonary disease or

lung cancer (Moroy et al., 2012; Muroski et al., 2008; Thompson & Parks, 1996).

colored in magenta. Zn atom is displayed as a grey sphere.

**5. Control of elastolytic cascade by oleoyl-galardin®**

and no synergistic effect could be obtained.

respectively.

the compound **5a** displayed selectivity for MMP-9.

Elastolysis requires the participation of serine- and metallo-elastases (Figure 8) which act through proteolytic cascades.

Fig. 8. Control of the serine (HLE)- and metallo-elastases (MMP-2, MMP-9) crosstalk in elastolysis by double-headed protease-MMP inhibitor (d-hPI).

Besides, a serine elastase as human leucocyte elastase (HLE) can degrade TIMP, and reversely MMP can hydrolyse serine protease inhibitor as 1 proteinase inhibitor (Nunes et al., 2011).

To control elastolysis, we thus attempted to design substances that could interfere with all actors of the depicted cascade. For that purpose, long chain-unsaturated fatty acids, as oleic acid, have been described to inhibit HLE (Hornebeck et al., 1985; Shock et al., 1990; Tyagi & Simon, 1990) and to impede plasmin-mediated prostromelysin-1 activation (Huet et al., 2004) as well as gelatinases activities (Berton et al., 2001). To that respect, we envisaged the synthesis of a double-headed protease-MMP inhibitor (d-hPI) able to block elastase and MMP activities. To that end, an oleoyl group was incorporated to galardin® at the P'3 position (Figure 9) (Moroy et al., 2011).

Fig. 9. Double-headed protease-MMP inhibitor (d-hPI).

Pharmacomodulation of Broad Spectrum Matrix

not compound **7** (not shown).

Arg512 of plasmin (not shown).

**6.1 Fn(II) domains** 

domain.

Metalloproteinase Inhibitors Towards Regulation of Gelatinases 73

was already demonstrated. The heterocycle is inserted into the S1 subsite where it interacts *via* an H-bond with the carbonyl group of the amino acid residue Ala194 peptide bond.

We analyzed the inhibitory capacity of oleic acid, analogues **6** and **7** towards HLE and plasmin activities. The compound **6** displayed high potency (IC50 = 0.6 M) against HLE, but lower inhibition was observed with oleic acid and the analogue **7** (IC50 = 3.0 and 8.7 M, respectively). Molecular docking computations indicated that the carboxylic function of compound **6** and oleic acid can form a salt bridge with the amino acid residue Arg217, but

Almost the same values are found in the same order for the plasmin–mediated pro-MMP-3 activation. The lowest energy model of oleic acid with the kringle 5 domain is characterized by the presence of a salt bridge between the carboxylic function and the amino acid residue

**6. Control of gelatinases through impeding enzyme-substrate interaction** 

intact the potential of gelatinases to cleave proteoglycans or several growth factors.

90% gelatinolysis catalyzed by MMP-2 (Xu et al., 2009).

Phe355, Trp374, Tyr381 and Trp387 (Figure 11).

Another approach to control the activity of those enzymes consists in the regulation of exosite protein-ligand interaction. To that respect both Fn(II) [or CBD] and PEX domains are involved and, in keeping with data presented on Figure 2, blocking either Fn(II) or PEX function will prevent the catalytic function of gelatinases on protein substrates selectively. For instance, the proteolysis of collagen and elastin might be inhibited while maintaining

Using recombinant Fn(II) domain as bait, a one bead one-peptide combinatorial peptide library was screened (Xu et al., 2007). A peptide displaying high sequence identity with the segment 715-721 in human 1(I) collagen chain was identified and proved to inhibit by >

The unsaturated fatty acid such as oleic acid inhibited MMP-2 with Ki = 4.3 M. Molecular modelling studies focus on the interactions localized at two sites on MMP-2: the fatty chain filled the S'1 pocket while the carboxylic acid group was exposed to the solvent. This result is in agreement with our previous works showing that the S'1 pocket could accommodate long alkyl side chains (LeDour et al., 2008; Moroy et al., 2007). The molecular docking computations identified the second site of the oleic acid interactions as the 3rd Fn(II) domain. The carboxylic acid function interacts *via* an H-bond with the phenolic group from the amino acid residue Tyr381, *via* a salt bridge with the guanidinium group of the amino acid residue Arg385 and *via* van der Waals interactions with the amino acid residue Leu356 while the unsaturated bond forms van der Waals interaction with the amino acid residues

Another approach could be the use of a more specific inhibitor directed against the 3rd Fn(II)

For this latter, an inhibitor with two carboxyl groups at each end of the alkyl chain should be efficient for targeting respectively the amino acid residue Arg385, as it was observed for

Oleoyl analogues (carboxylic **6** or hydroxamic **7** acids) are more potent than oleic acid to inhibit MMP (Table 6). The hydroxamic acid **7** was found to improve the inhibitory capacity toward MMP-2 comparing to oleic acid.


Table 6. Inhibition of MMP and serine elastases by oleic acid and oleoyl-galardin® derivatives. MMP (*Kis* values are expressed in M), HLE and plasmin (IC50 values are expressed in M).

The molecular docking computations indicated that compound **7** is able to bind MMP-2 active site and unable to chelate Zn2+ ion in the active site (Figure 10).

Fig. 10. Complex between MMP-2 and analogue **7**. The MMP-2 secondary structure was represented in cartoon. The analogue **7** is shown with sticks in which the C atoms are colored in magenta. Zn atom is displayed as a grey sphere.

Instead, the hydroxamic acid function forms a salt bridge with the N-terminal end of the amino acid residue Tyr110, while the long alkyl chain was inserted into the S'1 pocket as was already demonstrated. The heterocycle is inserted into the S1 subsite where it interacts *via* an H-bond with the carbonyl group of the amino acid residue Ala194 peptide bond.

We analyzed the inhibitory capacity of oleic acid, analogues **6** and **7** towards HLE and plasmin activities. The compound **6** displayed high potency (IC50 = 0.6 M) against HLE, but lower inhibition was observed with oleic acid and the analogue **7** (IC50 = 3.0 and 8.7 M, respectively). Molecular docking computations indicated that the carboxylic function of compound **6** and oleic acid can form a salt bridge with the amino acid residue Arg217, but not compound **7** (not shown).

Almost the same values are found in the same order for the plasmin–mediated pro-MMP-3 activation. The lowest energy model of oleic acid with the kringle 5 domain is characterized by the presence of a salt bridge between the carboxylic function and the amino acid residue Arg512 of plasmin (not shown).

### **6. Control of gelatinases through impeding enzyme-substrate interaction**

Another approach to control the activity of those enzymes consists in the regulation of exosite protein-ligand interaction. To that respect both Fn(II) [or CBD] and PEX domains are involved and, in keeping with data presented on Figure 2, blocking either Fn(II) or PEX function will prevent the catalytic function of gelatinases on protein substrates selectively. For instance, the proteolysis of collagen and elastin might be inhibited while maintaining intact the potential of gelatinases to cleave proteoglycans or several growth factors.

#### **6.1 Fn(II) domains**

72 Enzyme Inhibition and Bioapplications

Oleoyl analogues (carboxylic **6** or hydroxamic **7** acids) are more potent than oleic acid to inhibit MMP (Table 6). The hydroxamic acid **7** was found to improve the inhibitory capacity

Compounds MMP-2 MMP-3 MMP-7 MMP-9 HLE Plasmin **Oleic acid** 4.3 - 6.5 6.4 3.0 3.5

**6** 0.5 0.5 0.07 0.7 0.6 0.7 **7** 0.1 0.05 0.6 0.04 8.7 8.3

The molecular docking computations indicated that compound **7** is able to bind MMP-2

Fig. 10. Complex between MMP-2 and analogue **7**. The MMP-2 secondary structure was represented in cartoon. The analogue **7** is shown with sticks in which the C atoms are

Instead, the hydroxamic acid function forms a salt bridge with the N-terminal end of the amino acid residue Tyr110, while the long alkyl chain was inserted into the S'1 pocket as

colored in magenta. Zn atom is displayed as a grey sphere.

Table 6. Inhibition of MMP and serine elastases by oleic acid and oleoyl-galardin® derivatives. MMP (*Kis* values are expressed in M), HLE and plasmin (IC50 values are

active site and unable to chelate Zn2+ ion in the active site (Figure 10).

toward MMP-2 comparing to oleic acid.

expressed in M).

Using recombinant Fn(II) domain as bait, a one bead one-peptide combinatorial peptide library was screened (Xu et al., 2007). A peptide displaying high sequence identity with the segment 715-721 in human 1(I) collagen chain was identified and proved to inhibit by > 90% gelatinolysis catalyzed by MMP-2 (Xu et al., 2009).

The unsaturated fatty acid such as oleic acid inhibited MMP-2 with Ki = 4.3 M. Molecular modelling studies focus on the interactions localized at two sites on MMP-2: the fatty chain filled the S'1 pocket while the carboxylic acid group was exposed to the solvent. This result is in agreement with our previous works showing that the S'1 pocket could accommodate long alkyl side chains (LeDour et al., 2008; Moroy et al., 2007). The molecular docking computations identified the second site of the oleic acid interactions as the 3rd Fn(II) domain. The carboxylic acid function interacts *via* an H-bond with the phenolic group from the amino acid residue Tyr381, *via* a salt bridge with the guanidinium group of the amino acid residue Arg385 and *via* van der Waals interactions with the amino acid residue Leu356 while the unsaturated bond forms van der Waals interaction with the amino acid residues Phe355, Trp374, Tyr381 and Trp387 (Figure 11).

Another approach could be the use of a more specific inhibitor directed against the 3rd Fn(II) domain.

For this latter, an inhibitor with two carboxyl groups at each end of the alkyl chain should be efficient for targeting respectively the amino acid residue Arg385, as it was observed for

Pharmacomodulation of Broad Spectrum Matrix

cyan, respectively.

**6.3 PEX domains** 

**7. Conclusion** 

design of substances able to regulate MMP activity.

Metalloproteinase Inhibitors Towards Regulation of Gelatinases 75

Fig. 12. Hypothetical model of an inhibitor able to bind both the catalytic site and the Fn(II) domain of the human MMP-2. The MMP-2 is displayed with its accessible surface area. The active site and the binding pocket on the 3rd Fn(II) domain are coloured in orange and in

Since gelatinases are critically involved in directing cellular invasion, interfering with PEXintegrins (receptors) interaction might be a nice alternative. As an example, the use of phage display identified a peptide that inhibits the association of MMP-9 PEX domain with the v5 integrin, preventing proenzyme activation and cell migration (Björklund et al., 2004). More recently, a 20 mers peptide encompassing the PEX-binding tail region of C-TIMP-2 was found to inhibit the membrane-mediated activation in HT-1080 cells (Xu et al., 2011).

PEX domains play a crucial function in the non proteolytic function of gelatinases. As example, the PEX domain of MMP-9 is directly involved in the modulation of epithelial cell migration in a transwell chamber assay (Dufour et al., 2008). This domain also promotes B cell survival by interacting with 41 and CD-44 receptors (Redondo-Muñoz et al., 2010).

General considerations need to be pinpointed at aims to give a novel expansion to the

First, MMP as gelatinases are produced by nearly all cell types, but their cellular source may intervene in their function and activity. Proteolytic activity liberated by activated neutrophils is one pivotal element in the genesis and progression of aneurysms or chronic

obstructive pulmonary diseases (Muroski et al., 2008; Thompson & Parks, 1996).

oleic acid, and the amino acid residue Arg368 that is also present on the rim of the hydrophobic pocket.

Interestingly, in the full MMP-2, the amino acid residue Arg368 forms a salt bridge with the amino acid residue Asp40 belonging to the propeptide domain. According to the binding mode of oleic acid, the size of the alkyl chain should be composed by 15 or 16 atoms of carbon such as (7*Z*)-hexadec-7-enedioic acid and (6*Z*)-pentadec-6-enedioic acid.

Fig. 11. Complex between MMP-2 and oleic acid. Oleic acid is anchored in the binding pocket at the surface of the 3rd Fn(II) domain.

#### **6.2 Dual occupancy of the enzyme active site and Fn(II) domains**

Another more complex strategy relies on the design of a dual occupancy of the enzyme active site and the exosite as Fn(II). That has been originally attempted with a coupled hydroxamate-based inhibitor to gelatin-like structures (Jani et al., 2005). No increase in selectivity or potency of those compounds towards gelatinases could be attained; it was attributed to the possibility that the Fn(II) and catalytic domains of enzyme are tumbling independently.

Thus, we have built a hypothetical inhibitor from the inhibitors previously studied. We have added alkyl groups until the hydrophobic pocket of the 3rd Fn(II) was reached: 19 are needed to interact with its rims, *i.e*. the amino acid residues Arg368 and Trp387. If we want to reproduce the binding mode of the oleic acid, 32 alkyl groups should be added (Figure 12). However, the size and the high flexibility of this kind of inhibitor could be problematic.

Fig. 12. Hypothetical model of an inhibitor able to bind both the catalytic site and the Fn(II) domain of the human MMP-2. The MMP-2 is displayed with its accessible surface area. The active site and the binding pocket on the 3rd Fn(II) domain are coloured in orange and in cyan, respectively.

#### **6.3 PEX domains**

74 Enzyme Inhibition and Bioapplications

oleic acid, and the amino acid residue Arg368 that is also present on the rim of the

Interestingly, in the full MMP-2, the amino acid residue Arg368 forms a salt bridge with the amino acid residue Asp40 belonging to the propeptide domain. According to the binding mode of oleic acid, the size of the alkyl chain should be composed by 15 or 16 atoms of

carbon such as (7*Z*)-hexadec-7-enedioic acid and (6*Z*)-pentadec-6-enedioic acid.

Fig. 11. Complex between MMP-2 and oleic acid. Oleic acid is anchored in the binding

Another more complex strategy relies on the design of a dual occupancy of the enzyme active site and the exosite as Fn(II). That has been originally attempted with a coupled hydroxamate-based inhibitor to gelatin-like structures (Jani et al., 2005). No increase in selectivity or potency of those compounds towards gelatinases could be attained; it was attributed to the possibility that the Fn(II) and catalytic domains of enzyme are tumbling

Thus, we have built a hypothetical inhibitor from the inhibitors previously studied. We have added alkyl groups until the hydrophobic pocket of the 3rd Fn(II) was reached: 19 are needed to interact with its rims, *i.e*. the amino acid residues Arg368 and Trp387. If we want to reproduce the binding mode of the oleic acid, 32 alkyl groups should be added (Figure 12). However, the size and the high flexibility of this kind of inhibitor could be problematic.

**6.2 Dual occupancy of the enzyme active site and Fn(II) domains** 

pocket at the surface of the 3rd Fn(II) domain.

independently.

hydrophobic pocket.

Since gelatinases are critically involved in directing cellular invasion, interfering with PEXintegrins (receptors) interaction might be a nice alternative. As an example, the use of phage display identified a peptide that inhibits the association of MMP-9 PEX domain with the v5 integrin, preventing proenzyme activation and cell migration (Björklund et al., 2004). More recently, a 20 mers peptide encompassing the PEX-binding tail region of C-TIMP-2 was found to inhibit the membrane-mediated activation in HT-1080 cells (Xu et al., 2011).

PEX domains play a crucial function in the non proteolytic function of gelatinases. As example, the PEX domain of MMP-9 is directly involved in the modulation of epithelial cell migration in a transwell chamber assay (Dufour et al., 2008). This domain also promotes B cell survival by interacting with 41 and CD-44 receptors (Redondo-Muñoz et al., 2010).

#### **7. Conclusion**

General considerations need to be pinpointed at aims to give a novel expansion to the design of substances able to regulate MMP activity.

First, MMP as gelatinases are produced by nearly all cell types, but their cellular source may intervene in their function and activity. Proteolytic activity liberated by activated neutrophils is one pivotal element in the genesis and progression of aneurysms or chronic obstructive pulmonary diseases (Muroski et al., 2008; Thompson & Parks, 1996).

Pharmacomodulation of Broad Spectrum Matrix

inhibitory activity (Bourguet et al., 2009).

tumor do we need to incorporate MMPI in cancer treatment?

and near infrared FRET pairs (Scherer et al., 2008).

of Mrs. M. Decarme is greatfully acknowledged.

**8. Acknowledgment** 

**9. References** 

MMP-9(PEX)-CD-44 interactions.

Metalloproteinase Inhibitors Towards Regulation of Gelatinases 77

Peptide or chemical libraries can be developed at aims to impede MMP-2(PEX)-v3 or

As one example, a bivalent derivatized dilysine tetraamide was isolated which proved to interfere with MMP-2/v3 interaction and inhibit angiogenesis (Silletti et al., 2001). Possibly, this compound could be chemically modified to confer it additionally MMP-2

One main problem related with the control of MMP in general and gelatinases in particular relies on the kinetics of production of those enzymes during the cancer course from initiation to metastasis formation. In other words, at what stage for one particular type of

The development of imaging MMP activity using derivatized selective inhibitor will probably answer to this question. Several techniques have been already developed using Positron Emission Tomography (PET) with 18F-labelled MMP-2 inhibitor (Furumoto et al., 2003), Single Photon Emission Computed Tomography (SPECT) with a 123I gelatinases inhibitor (Schaffers et al., 2004), or the use of fluorogenic substrates bearing self quenched

In our Federal Research Institute (IFR), we aim to develop hybrid nanoprobes build from MMPI and fluorescent nanocrystal quantum dots (QDs). Design and chemical synthesis of derivatives of galardin®, selective inhibitors of MMP-2, will be followed by their tagging with QDs. Photo- and chemical stability of QDs will enable long-term spatiotemporal tracking of the process of inhibition of MMP-2 with developed nanoprobe thus permitting

Authors thank *Université de Reims Champagne-Ardenne, IFR53 "Biomolécules", Région Champagne-Ardenne*, *EU* (Fonds Feder) and *CNRS* for financial support. Technical assistance

Abbenante, G. & Fairlie, D.P. (2005). Protease inhibitors in the clinic. *Medicinal Chemistry*,

Allan, J.A.; Docherty, A.J.; Barker, P.J.; Huskisson, N.S.; Reynolds, J.J. & Murphy, G. (1995).

*Biochemical Journal*, Vol.309, Pt.1, (July 1995), pp. 299-306, ISSN 0264-6021 Ardi, V.C.; Kupriyanova, T.A.; Deryugina, E.I. & Quigley, J.P. (2007). Human neutrophils

*America*, Vol.104, No.51, (December 2007), pp. 20262-20267, ISSN 0027-8424 Ardi, V.C.; Van den Steen, P.E.; Opdenakker, G.; Schweighofer, B.; Deryugina, E.I. &

Binding of gelatinases A and B to type-I collagen and other matrix components. *The* 

uniquely release TIMP-free MMP-9 to provide a potent catalytic stimulator of angiogenesis. *Proceedings of the National Academy of Sciences of the United States of* 

Quigley, J.P. (2009). Neutrophil MMP-9 proenzyme, unencumbered by TIMP-1, undergoes efficient activation in vivo and catalytically induces angiogenesis via a basic fibroblast growth factor (FGF-2)/FGFR-2 pathway. *The Journal of Biological Chemistry*, Vol.284, No.38, (September 2009), pp. 25854-25866, ISSN 0021-9258

understanding of physiological process of invasion of melanoma for example.

Vol.1, No.1, (January 2005), pp. 71-104, ISSN 1573-4064

It has been demonstrated that pro-MMP-9 is produced by neutrophils as a free form *i.e*. not associated with TIMP-1 molecule, more readily activatable by enzyme as stromelysin-1 (Ardi et al., 2007). In addition, following activation, those cells release extracellular traps *i.e*. neutrophil extracellular traps (NET) formed by the association of chromatin and granule proteins; NET are enriched in neutral endopeptidases as neutrophil elastase and MMP-9 (Brinkmann et al., 2004). To that respect, the use of double-headed (HLE-MMP-9) inhibitors as oleoyl-galardin® might be of therapeutic value. Advantageously, as we recently documented, oleoyl moiety might be replaced by -lactam (Moroy et al., 2012), a more potent and selective HLE inhibitor.

Although inflammation can orchestrate cancer (Kessenbrock et al., 2010), MMP-2 and MMP-9 intervene in several other stages of cancer progression. Both enzymes have been involved in promoting cell growth; MMP-2 is more linked to cancer cell invasiveness while MMP-9 may contribute to cell survival.

Up to now, one selective MMP-9 inhibitor is a monoclonal antibody binding to N-terminal part of catalytic domain (Martens et al., 2007). Thus, intuitively, in keeping with those distinct functions, the concept of selective inhibitor among gelatinases is emerging.

Their role in establishing a "metastatic niche" has also been delineated and their contribution in angiogenesis has been widely underlined.

However, paradoxically, MMP-9 can generate either pro- or anti-angiogenic signals (Figure 13).

On one side, it can


**PRO-ANGIOGENIC SIGNALS ANTI-ANGIOGENIC SIGNALS**

Fig. 13. The paradoxical function of MMP-9 in angiogenesis.

At the opposite, proteolysis of plasminogen and 3 chain of collagen IV leads to the formation of angiostatin and tumstatin (Cornelius et al., 1998; Kessenbrock et al., 2010). Importantly, mice deficient in MMP-9 evidenced an increased-tumor growth which was attributed to lack of tumstatin formation (Hamano et al., 2003).

As mentioned, both gelatinases exerted their action at the pericellular environment, following binding of their PEX domain to receptors as v3 for MMP-2 or CD-44 for MMP-9. Peptide or chemical libraries can be developed at aims to impede MMP-2(PEX)-v3 or MMP-9(PEX)-CD-44 interactions.

As one example, a bivalent derivatized dilysine tetraamide was isolated which proved to interfere with MMP-2/v3 interaction and inhibit angiogenesis (Silletti et al., 2001). Possibly, this compound could be chemically modified to confer it additionally MMP-2 inhibitory activity (Bourguet et al., 2009).

One main problem related with the control of MMP in general and gelatinases in particular relies on the kinetics of production of those enzymes during the cancer course from initiation to metastasis formation. In other words, at what stage for one particular type of tumor do we need to incorporate MMPI in cancer treatment?

The development of imaging MMP activity using derivatized selective inhibitor will probably answer to this question. Several techniques have been already developed using Positron Emission Tomography (PET) with 18F-labelled MMP-2 inhibitor (Furumoto et al., 2003), Single Photon Emission Computed Tomography (SPECT) with a 123I gelatinases inhibitor (Schaffers et al., 2004), or the use of fluorogenic substrates bearing self quenched and near infrared FRET pairs (Scherer et al., 2008).

In our Federal Research Institute (IFR), we aim to develop hybrid nanoprobes build from MMPI and fluorescent nanocrystal quantum dots (QDs). Design and chemical synthesis of derivatives of galardin®, selective inhibitors of MMP-2, will be followed by their tagging with QDs. Photo- and chemical stability of QDs will enable long-term spatiotemporal tracking of the process of inhibition of MMP-2 with developed nanoprobe thus permitting understanding of physiological process of invasion of melanoma for example.

#### **8. Acknowledgment**

Authors thank *Université de Reims Champagne-Ardenne, IFR53 "Biomolécules", Région Champagne-Ardenne*, *EU* (Fonds Feder) and *CNRS* for financial support. Technical assistance of Mrs. M. Decarme is greatfully acknowledged.

#### **9. References**

76 Enzyme Inhibition and Bioapplications

It has been demonstrated that pro-MMP-9 is produced by neutrophils as a free form *i.e*. not associated with TIMP-1 molecule, more readily activatable by enzyme as stromelysin-1 (Ardi et al., 2007). In addition, following activation, those cells release extracellular traps *i.e*. neutrophil extracellular traps (NET) formed by the association of chromatin and granule proteins; NET are enriched in neutral endopeptidases as neutrophil elastase and MMP-9 (Brinkmann et al., 2004). To that respect, the use of double-headed (HLE-MMP-9) inhibitors as oleoyl-galardin® might be of therapeutic value. Advantageously, as we recently documented, oleoyl moiety might be replaced by -lactam (Moroy et al., 2012), a more

Although inflammation can orchestrate cancer (Kessenbrock et al., 2010), MMP-2 and MMP-9 intervene in several other stages of cancer progression. Both enzymes have been involved in promoting cell growth; MMP-2 is more linked to cancer cell invasiveness while MMP-9

Up to now, one selective MMP-9 inhibitor is a monoclonal antibody binding to N-terminal part of catalytic domain (Martens et al., 2007). Thus, intuitively, in keeping with those

Their role in establishing a "metastatic niche" has also been delineated and their

However, paradoxically, MMP-9 can generate either pro- or anti-angiogenic signals

iii. generate elastin fragments *i.e*. elastokines with potent angiogenic activity (Robinet et al.,

**PRO-ANGIOGENIC SIGNALS ANTI-ANGIOGENIC SIGNALS**

PEX-like

His S His

OG

At the opposite, proteolysis of plasminogen and 3 chain of collagen IV leads to the formation of angiostatin and tumstatin (Cornelius et al., 1998; Kessenbrock et al., 2010). Importantly, mice deficient in MMP-9 evidenced an increased-tumor growth which was

As mentioned, both gelatinases exerted their action at the pericellular environment, following binding of their PEX domain to receptors as v3 for MMP-2 or CD-44 for MMP-9.

OGOG

Hinge

OG

S

Plasminogen

ANGIOSTATIN

TUMSTATIN

Collagen IV3

distinct functions, the concept of selective inhibitor among gelatinases is emerging.

i. regulate VEGF bioavailability for VEGF-R2 receptor (Bergers et al., 2000), ii. activate the basic fibroblast growth factor-2 (FGF-2) pathway (Ardi et al., 2009),

Zn2+ catalytic

**MMP-9**

Fig. 13. The paradoxical function of MMP-9 in angiogenesis.

attributed to lack of tumstatin formation (Hamano et al., 2003).

His

FnFnFn CBD

contribution in angiogenesis has been widely underlined.

potent and selective HLE inhibitor.

may contribute to cell survival.

(Figure 13).

On one side, it can

2005).

VEGF pathway activation

> FGF-2 pathway activation

Elastokines Elastin


Pharmacomodulation of Broad Spectrum Matrix

ISSN 0012-6667

0021-9258

No.5, p 1011)

Metalloproteinase Inhibitors Towards Regulation of Gelatinases 79

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Dufour, A.; Sampson, N.S.; Zucker, S. & Cao, J. (2008). Role of the hemopexin domain of

Egeblad, M. & Werb, Z. (2002). New functions for the matrix metalloproteinases in cancer

Fingleton, B. (2007). Matrix Metalloproteinases as Valid Clinical Targets. *Current* 

Fujita, M.; Nakao, Y.; Matsunaga, S.; Seiki, M.; Itoh, Y.; van Soest, R.W.M.; Heubes, M.;

Furumoto, S.; Takashima, K.; Kubota, K.; Ido, T.; Iwata, R. & Fukuda, H. (2003). Tumor

Gomis-Rüth, F.X. (2009). Catalytic domain architecture of metzincin metalloproteases. *The* 

Hamano, Y.; Zeisberg, M.; Sugimoto, H.; Lively, J.C.; Maeshima, Y.; Yang, C.; Hynes, R.O.;

Hernandez-Barrantes, S.; Bernardo, M.; Toth, M. & Fridman, R. (2002). Regulation of

Hornebeck, W.; Moczar, E.; Szecsi, J. & Robert, L. (1985). Fatty acid peptide derivatives as

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**4** 

**Non-Enzymatic Glycation** 

*Faculty of Pharmacy in Hradec Králové* 

*Czech Republic* 

**of Aminotransferases and the Possibilities of Its Modulation** 

Iva Boušová, Lenka Srbová and Jaroslav Dršata

*Department of Biochemical Sciences, Charles University in Prague,* 

Enzymes are catalytic protein molecules performing specific functions in their native form. Native structure is one of basic conditions for normal function of proteins including enzymes. Catalytic activity of enzyme may be decreased by both non-covalent modulation by true inhibitors and covalent modifications by metabolites in the body or natural products. One example of such modulation is non-enzymatic glycation of proteins by

Enzymes are very good models for studies of protein interactions with other molecules, including sugars (e.g., Arai et al., 1987; Dolhofer & Wieland, 1978; Okada et al., 1994; Okada et al., 1997; Sakurai et al., 1987). Advantage of these studies is the fact that enzyme interactions may be investigated not only by common methods of studies of protein properties like changes in spectral characteristics, molecular weight, charge, solubility etc. but also by measurement of their catalytic activity as the most characteristic property of enzyme molecules. For such studies, it is important to use the enzyme with known structure and reaction mechanism, available in sufficient purity, and measurable by simple assay method. Aminotransferases belong to such enzymes. That is the reason why research activities of our laboratory devote to inhibition of aminotransferases by low-molecular compounds for many years (e.g. Dršata & Veselá, 1984; Netopilová et al., 1991; Netopilová et al., 2001). Our studies of aminotransferase glycation belong to the efforts made also by several other research groups (see e.g. Okada & Ayabe, 1997; Fitzgerald et al., 2000; Seidler

Aspartate aminotransferase (AST, EC 2.6.1.1) and alanine aminotransferase (ALT, EC 2.6.1.2), enzymes frequently assessed in clinical laboratories, catalyse reversible transfer of α-amino group from amino acids aspartate and alanine to the acceptor 2-oxoglutarate, respectively. The resulting products are oxaloacetate, pyruvate and glutamate. In metabolism, AST activity mediates the connection between the metabolism of amino acids

**1. Introduction** 

& Kowalewski 2003; Hobart et al., 2004).

**1.1 Aminotransferases** 

sugars.


## **Non-Enzymatic Glycation of Aminotransferases and the Possibilities of Its Modulation**

Iva Boušová, Lenka Srbová and Jaroslav Dršata *Department of Biochemical Sciences, Charles University in Prague, Faculty of Pharmacy in Hradec Králové Czech Republic* 

#### **1. Introduction**

84 Enzyme Inhibition and Bioapplications

Xu, X.; Mikhailova, M.; Ilangovan, U.; Chen, Z.; Yu, A.; Pal, S.; Hinck, A.P. & Steffensen, B.

Xu, X.; Mikhailova, M.; Chen, Z.; Pal, S.; Robichaud, T.K.; Lafer, E.M.; Baber, S. & Steffensen,

(June 2009), pp. 5822-5831, ISSN 0006-2960

053X

(2009). Nuclear magnetic resonance mapping and functional confirmation of the collagen binding sites of matrix metalloproteinase-2. *Biochemistry*, Vol.48, No.25,

B. (2011). Peptide from the C-terminal domain of tissue inhibitor of matrix metalloproteinases-2 (TIMP-2) inhibits membrane activation of matrix metalloproteinase-2 (MMP-2). *Matrix biology: journal of the International Society for Matrix Biology*, *Biology*, Vol.30, No.7-8, (September 2011), pp. 404-412, ISSN 0945-

> Enzymes are catalytic protein molecules performing specific functions in their native form. Native structure is one of basic conditions for normal function of proteins including enzymes. Catalytic activity of enzyme may be decreased by both non-covalent modulation by true inhibitors and covalent modifications by metabolites in the body or natural products. One example of such modulation is non-enzymatic glycation of proteins by sugars.

> Enzymes are very good models for studies of protein interactions with other molecules, including sugars (e.g., Arai et al., 1987; Dolhofer & Wieland, 1978; Okada et al., 1994; Okada et al., 1997; Sakurai et al., 1987). Advantage of these studies is the fact that enzyme interactions may be investigated not only by common methods of studies of protein properties like changes in spectral characteristics, molecular weight, charge, solubility etc. but also by measurement of their catalytic activity as the most characteristic property of enzyme molecules. For such studies, it is important to use the enzyme with known structure and reaction mechanism, available in sufficient purity, and measurable by simple assay method. Aminotransferases belong to such enzymes. That is the reason why research activities of our laboratory devote to inhibition of aminotransferases by low-molecular compounds for many years (e.g. Dršata & Veselá, 1984; Netopilová et al., 1991; Netopilová et al., 2001). Our studies of aminotransferase glycation belong to the efforts made also by several other research groups (see e.g. Okada & Ayabe, 1997; Fitzgerald et al., 2000; Seidler & Kowalewski 2003; Hobart et al., 2004).

#### **1.1 Aminotransferases**

Aspartate aminotransferase (AST, EC 2.6.1.1) and alanine aminotransferase (ALT, EC 2.6.1.2), enzymes frequently assessed in clinical laboratories, catalyse reversible transfer of α-amino group from amino acids aspartate and alanine to the acceptor 2-oxoglutarate, respectively. The resulting products are oxaloacetate, pyruvate and glutamate. In metabolism, AST activity mediates the connection between the metabolism of amino acids

Non-Enzymatic Glycation of Aminotransferases and the Possibilities of Its Modulation 87

Fig. 1. Characteristic UV-VIS absorption and CD spectra of AST (panel A and C) and of the free coenzyme (panel B). (A) UV-VIS spectra of AST (1 mg/mL; 0.1 M sodium phosphate buffer, pH 7.4) alone or in the presence of L-Asp (1 mM), (B) UV-VIS spectra of free PLP (0.1 mM) and PMP (0.1 mM) in sodium phosphate buffer (0.1 M; pH 7.4) were recorded using a diode array spectrophotometer HP 8453 (Hewlett Packard, USA) in a 0.5 cm quartz cuvette against sodium phosphate buffer (0.1 M; pH 7.4). (C) CD spectra of AST (1 mg/mL; 0.1 M sodium phosphate buffer, pH 7.4) alone or in the presence of L-Asp (1 mM) were recorded using a dichrograph CD6 (Jobin Yvon, France) in a 0.5 cm quartz cuvette against sodium

extracellular concentration of glucose including a hallmark of the disease - hyperglycemia. There is increasing evidence that Maillard reaction plays an important role in the onset and progression of some other diseases, such as atherosclerosis and Alzheimer's disease (Nursten, 2005; Yegin et al., 1995). Plenty of studies have been devoted to investigation of protein structural and functional changes caused by glycation process (e.g. Jabeen & Saleemuddin, 2006; Seidler & Seibel, 2000; Yan & Harding 1997, 2006; Zeng et al., 2006; Zhao

The early stage of the Maillard reaction is initiated by non-enzymatic condensation of a reducing sugar or a certain related compound (e.g. ascorbate) with a free amino group of a

phosphate buffer (0.1 M; pH 7.4). For further details on experimental work see

**1.2.1 Formation of advanced glycation end-products** 

(Dršata et al., 2005)

et al., 2000).

and saccharides and participates in the transportation of reduced equivalents across the membrane of mitochondria as a part of the malate–aspartate shuttle. The catalytic activities of AST in cells are implemented by two isoenzymes—the cytosolic and the mitochondrial one, which differ in primary structure and in some properties (Yagi et al., 1985; Metzler et al., 1979).

Porcine heart cytosolic AST, which was used in further described experiments of our research group, is a homodimer with molecular mass of about 93,150 Da. Both subunits are composed of 412 amino acid residues. This dimer contains 38 lysine and 52 arginine residues with six Lys–Arg and four Arg–Lys sequence pairs (Seidler & Kowalewski, 2003). Presence of these amino acid residues makes glycation of aminotransferases *in vitro* as well as *in vivo* possible. The Lys258 binding coenzyme PLP could be one of the possible targets of glycating agents as can be seen from the loss of enzymatic activity due to the effect of methylglyoxal as well as other glycating agents (e.g. Boušová et al., 2009; Seidler & Kowalewski, 2003).

Aminotransferases are characterised by the presence of coenzyme pyridoxal-5'-phosphate (PLP) and its direct participation in catalysis. The protein part of aminotransferase molecules consists of two identical, non-covalently bound subunits, which are composed of one small and one large domain. PLP coenzyme forms a Schiff base with ε-amino group of lysine residue (Lys313 in ALT and Lys258 in AST) located in an active site of the larger domain of each subunit (Kirsch et al., 1984). The PLP form of AST shows, depending on pH, a major absorption peak at 360 nm (an active, unprotonized form of the coenzyme, prevailing at lower pH values), and/or a peak at 430 nm (an inactive, protonized form, increasing at lower pH values) (Kirsch et al., 1984). After a reaction with L-aspartate during the first part of a ping-pong transaminating reaction, the pyridoxamine-5'-phosphate (PMP) form of the coenzyme appears and the original absorption maximum shifts to 325–330 nm (Fig. 1A). While free PLP or PMP are not optically active substances, the coenzyme bound in the active site of AST shows circular dichroism (CD) spectra in the range of 300–500 nm, which are similar to absorption spectra (Fig. 1C). The CD effect is caused by the change in the electronic configuration of the molecule (Kelly & Price, 2000). Circular dichroism clears away absorption characteristics of optically inactive components, which facilitates identification of the specific coenzyme signal and its changes (Dršata et al., 2005). This method is also a powerful tool for characterizing secondary (190-240 nm, chromophore is peptide bond) and tertiary (250-320 nm, chromophores are aromatic amino acids and disulfide bonds) structures of studied proteins as well as determining whether the protein is folded (Kelly & Price, 2000).

#### **1.2 Non-enzymatic glycation of proteins**

Non-enzymatic glycation, also called Maillard reaction, was first described in 1912 by Louis Camille Maillard (Monnier, 1989). This non-enzymatic browning process had been first extensively studied by food chemists and later has become the center of attention of geological or agricultural sciences. Much later it was recognized that the process is important for medical science (Monnier, 1989; Singh et al., 2001). A very common but serious human disease is diabetes mellitus, which in its untreated or unsuccessfully treated form is accompanied with development of chronic complications due to increased intra- and

and saccharides and participates in the transportation of reduced equivalents across the membrane of mitochondria as a part of the malate–aspartate shuttle. The catalytic activities of AST in cells are implemented by two isoenzymes—the cytosolic and the mitochondrial one, which differ in primary structure and in some properties (Yagi et al., 1985; Metzler et

Porcine heart cytosolic AST, which was used in further described experiments of our research group, is a homodimer with molecular mass of about 93,150 Da. Both subunits are composed of 412 amino acid residues. This dimer contains 38 lysine and 52 arginine residues with six Lys–Arg and four Arg–Lys sequence pairs (Seidler & Kowalewski, 2003). Presence of these amino acid residues makes glycation of aminotransferases *in vitro* as well as *in vivo* possible. The Lys258 binding coenzyme PLP could be one of the possible targets of glycating agents as can be seen from the loss of enzymatic activity due to the effect of methylglyoxal as well as other glycating agents (e.g. Boušová et al., 2009; Seidler &

Aminotransferases are characterised by the presence of coenzyme pyridoxal-5'-phosphate (PLP) and its direct participation in catalysis. The protein part of aminotransferase molecules consists of two identical, non-covalently bound subunits, which are composed of one small and one large domain. PLP coenzyme forms a Schiff base with ε-amino group of lysine residue (Lys313 in ALT and Lys258 in AST) located in an active site of the larger domain of each subunit (Kirsch et al., 1984). The PLP form of AST shows, depending on pH, a major absorption peak at 360 nm (an active, unprotonized form of the coenzyme, prevailing at lower pH values), and/or a peak at 430 nm (an inactive, protonized form, increasing at lower pH values) (Kirsch et al., 1984). After a reaction with L-aspartate during the first part of a ping-pong transaminating reaction, the pyridoxamine-5'-phosphate (PMP) form of the coenzyme appears and the original absorption maximum shifts to 325–330 nm (Fig. 1A). While free PLP or PMP are not optically active substances, the coenzyme bound in the active site of AST shows circular dichroism (CD) spectra in the range of 300–500 nm, which are similar to absorption spectra (Fig. 1C). The CD effect is caused by the change in the electronic configuration of the molecule (Kelly & Price, 2000). Circular dichroism clears away absorption characteristics of optically inactive components, which facilitates identification of the specific coenzyme signal and its changes (Dršata et al., 2005). This method is also a powerful tool for characterizing secondary (190-240 nm, chromophore is peptide bond) and tertiary (250-320 nm, chromophores are aromatic amino acids and disulfide bonds) structures of studied proteins as well as determining whether the protein is

Non-enzymatic glycation, also called Maillard reaction, was first described in 1912 by Louis Camille Maillard (Monnier, 1989). This non-enzymatic browning process had been first extensively studied by food chemists and later has become the center of attention of geological or agricultural sciences. Much later it was recognized that the process is important for medical science (Monnier, 1989; Singh et al., 2001). A very common but serious human disease is diabetes mellitus, which in its untreated or unsuccessfully treated form is accompanied with development of chronic complications due to increased intra- and

al., 1979).

Kowalewski, 2003).

folded (Kelly & Price, 2000).

**1.2 Non-enzymatic glycation of proteins** 

Fig. 1. Characteristic UV-VIS absorption and CD spectra of AST (panel A and C) and of the free coenzyme (panel B). (A) UV-VIS spectra of AST (1 mg/mL; 0.1 M sodium phosphate buffer, pH 7.4) alone or in the presence of L-Asp (1 mM), (B) UV-VIS spectra of free PLP (0.1 mM) and PMP (0.1 mM) in sodium phosphate buffer (0.1 M; pH 7.4) were recorded using a diode array spectrophotometer HP 8453 (Hewlett Packard, USA) in a 0.5 cm quartz cuvette against sodium phosphate buffer (0.1 M; pH 7.4). (C) CD spectra of AST (1 mg/mL; 0.1 M sodium phosphate buffer, pH 7.4) alone or in the presence of L-Asp (1 mM) were recorded using a dichrograph CD6 (Jobin Yvon, France) in a 0.5 cm quartz cuvette against sodium phosphate buffer (0.1 M; pH 7.4). For further details on experimental work see (Dršata et al., 2005)

extracellular concentration of glucose including a hallmark of the disease - hyperglycemia. There is increasing evidence that Maillard reaction plays an important role in the onset and progression of some other diseases, such as atherosclerosis and Alzheimer's disease (Nursten, 2005; Yegin et al., 1995). Plenty of studies have been devoted to investigation of protein structural and functional changes caused by glycation process (e.g. Jabeen & Saleemuddin, 2006; Seidler & Seibel, 2000; Yan & Harding 1997, 2006; Zeng et al., 2006; Zhao et al., 2000).

#### **1.2.1 Formation of advanced glycation end-products**

The early stage of the Maillard reaction is initiated by non-enzymatic condensation of a reducing sugar or a certain related compound (e.g. ascorbate) with a free amino group of a

Non-Enzymatic Glycation of Aminotransferases and the Possibilities of Its Modulation 89

Fig. 2. Three stages of non-enzymatic glycation reaction. CML, *N*-*ε*-(carboxymethyl)lysine; GOLD, glyoxal-lysine dimer; CEL, *N*-*ε*-(carboxyethyl)lysine; MOLD, methylglyoxal-lysine

The advanced glycation end-products found under physiological conditions can be classified according to their fluorescent properties and their ability to form cross-links. The first group is represented by fluorescent AGE cross-links, which are thought to be responsible for a major share of the deleterious effects of AGEs in diabetes and aging. Fluorescence is a good qualitative indicator used to estimate AGEs formation. Pentosidine, crossline, and various vesperlysines are members of this group. However, also nonfluorescent AGE cross-links are found *in vivo*. Their isolation and identification is more complicated than in the case of fluorescent AGE cross-links. It is thought that they account just for 1% of all cross-links rising under physiological conditions. Various imidazolium dilysine cross-links (GOLD, MOLD), arginine-lysine cross-links, and glucosepan belong to this group. Last but not least, a group of non-cross-linking protein bound AGE structures have been identified *in vivo*. These structures may exert deleterious effects as precursors of cross-links or as biological receptor ligands inducing a variety of adverse cellular and tissue changes. The well-known members of this group are pyrraline, carboxyalkyllysines (CML, CEL), imidazolones, and argpyrimidine (Ulrich & Cerami, 2001). Classification and

dimer; 3-DG, 3-deoxyglucosone; DOLD, deoxyglucosone-lysine dimer

examples of each above mentioned group are shown in Fig. 3.

**1.2.2 Structure of AGEs** 

protein, a lipid or a nucleic acid. In the case of glucose, the reaction first leads to the formation of acid-labile Schiff base, which undergoes a rearrangement to a relatively stable Amadori product, e.g. fructosamine. Only a small portion of these Amadori-adducts experiences further rearrangements leading to an irreversible formation of advanced glycation end-products (AGEs) (Monnier, 1989). The reaction with fructose proceeds in a similar way, but it is called Heyns rearrangement and two separate Heyns products are generated (Suarez et al., 1989). The formation of Schiff base proceeds in the range of hours and it is fully reversible, while Amadori rearrangement takes days and is reversible only to a certain extent.

In the intermediate stage, Amadori product subsequently degrades and various reactive intermediates are formed. These products are known as α-dicarbonyls or α-oxoaldehydes and are represented by products like methylglyoxal (MGO), 3-deoxyglucosone (3-DG), and glyoxal (GO). Also a Schiff base is a potential source of reactive α-dicarbonyls, because it can be fragmented to MGO and GO. These dicarbonyls possess higher reactivity towards proteins than the parent monosaccharide. They are capable of forming various cross-links as well as chromo/fluorophoric adducts called AGEs, upon reaction with proteins (Schalkwijk et al., 2004; Wolff et al., 1991). Both MGO and 3-DG form adducts with proteins and nucleic acids up to 10,000 times more readily than glucose (Beisswenger et al., 2003). The accumulation of α-dicarbonyl compounds is termed carbonyl stress (Miyata et al., 1999). The other process proceeding during the intermediate stage of glycation is metal catalyzed autoxidation of glucose, in which the carbonyl compounds (arabinose and glyoxal), H2O2 and free radicals are formed (Hunt et al., 1988; Wolff & Dean, 1987). The generated free radicals initiate further oxidative steps. The glycation process accompanied by oxidation steps is called glycoxidation (Baynes, 1991). Various pathways incorporated in the formation of AGEs are shown in Fig. 2.

The advanced glycation end-products are formed during the late stage of glycation over a period of weeks, thereby affecting predominantly long-lived proteins, such as collagen and lens crystallins. They represent a heterogeneous group of compounds rising from different precursors. The chemical structures of AGEs have not been fully described yet. These compounds are formed either by oxidative pathway (pentosidine and CML) or by nonoxidative pathway (pyrraline, DOLD, GOLD, MOLD, and CEL) as can be seen in Fig. 2. Proteins modified by advanced glycation are characterized by a much higher molecular weight than the original protein, a yellow-brown pigmentation, a typical fluorescent spectra (λex/λem: 370/440 nm), an ability to form various cross-links, and by their biological half-life, which is comparable to the half-lives of parent proteins (Lapolla et al., 2005; Singh et al., 2001).

Glucose is the least reactive of the common sugars and that is probably the reason for its evolutionary selection as the principal sugar *in vivo* (Bunn et al., 1978). Because of its low reactivity towards proteins, AGEs have been thought to form only at long-lived extracellular proteins, such as collagen, crystallines, and myelin. Recently also rapid intracellular AGE formation by various intracellular sugars (e.g. fructose, ribose, glyceraldehyde, dihydroxyacetone phosphate, glyceraldehyde-3-phosphate, glyoxal, methylglyoxal, and 3 deoxyglucosone) *in vivo* has been described. The rate of glycation is directly proportional to the percentage of sugar in the open-chain form and the rate for fructose (0.7% open-chain) is 7.5-fold faster than that of glucose (0.002% open-chain). More strikingly, the glycolytic intermediate glyceraldehyde-3-phosphate (100% open-chain) forms 200-fold more glycated proteins than do equimolar amounts of glucose (Schalkwijk et al., 2004).

Fig. 2. Three stages of non-enzymatic glycation reaction. CML, *N*-*ε*-(carboxymethyl)lysine; GOLD, glyoxal-lysine dimer; CEL, *N*-*ε*-(carboxyethyl)lysine; MOLD, methylglyoxal-lysine dimer; 3-DG, 3-deoxyglucosone; DOLD, deoxyglucosone-lysine dimer

#### **1.2.2 Structure of AGEs**

88 Enzyme Inhibition and Bioapplications

protein, a lipid or a nucleic acid. In the case of glucose, the reaction first leads to the formation of acid-labile Schiff base, which undergoes a rearrangement to a relatively stable Amadori product, e.g. fructosamine. Only a small portion of these Amadori-adducts experiences further rearrangements leading to an irreversible formation of advanced glycation end-products (AGEs) (Monnier, 1989). The reaction with fructose proceeds in a similar way, but it is called Heyns rearrangement and two separate Heyns products are generated (Suarez et al., 1989). The formation of Schiff base proceeds in the range of hours and it is fully reversible, while

In the intermediate stage, Amadori product subsequently degrades and various reactive intermediates are formed. These products are known as α-dicarbonyls or α-oxoaldehydes and are represented by products like methylglyoxal (MGO), 3-deoxyglucosone (3-DG), and glyoxal (GO). Also a Schiff base is a potential source of reactive α-dicarbonyls, because it can be fragmented to MGO and GO. These dicarbonyls possess higher reactivity towards proteins than the parent monosaccharide. They are capable of forming various cross-links as well as chromo/fluorophoric adducts called AGEs, upon reaction with proteins (Schalkwijk et al., 2004; Wolff et al., 1991). Both MGO and 3-DG form adducts with proteins and nucleic acids up to 10,000 times more readily than glucose (Beisswenger et al., 2003). The accumulation of α-dicarbonyl compounds is termed carbonyl stress (Miyata et al., 1999). The other process proceeding during the intermediate stage of glycation is metal catalyzed autoxidation of glucose, in which the carbonyl compounds (arabinose and glyoxal), H2O2 and free radicals are formed (Hunt et al., 1988; Wolff & Dean, 1987). The generated free radicals initiate further oxidative steps. The glycation process accompanied by oxidation steps is called glycoxidation (Baynes, 1991). Various pathways incorporated in the formation

The advanced glycation end-products are formed during the late stage of glycation over a period of weeks, thereby affecting predominantly long-lived proteins, such as collagen and lens crystallins. They represent a heterogeneous group of compounds rising from different precursors. The chemical structures of AGEs have not been fully described yet. These compounds are formed either by oxidative pathway (pentosidine and CML) or by nonoxidative pathway (pyrraline, DOLD, GOLD, MOLD, and CEL) as can be seen in Fig. 2. Proteins modified by advanced glycation are characterized by a much higher molecular weight than the original protein, a yellow-brown pigmentation, a typical fluorescent spectra (λex/λem: 370/440 nm), an ability to form various cross-links, and by their biological half-life, which is comparable to the half-lives of parent proteins (Lapolla et al., 2005; Singh et al., 2001). Glucose is the least reactive of the common sugars and that is probably the reason for its evolutionary selection as the principal sugar *in vivo* (Bunn et al., 1978). Because of its low reactivity towards proteins, AGEs have been thought to form only at long-lived extracellular proteins, such as collagen, crystallines, and myelin. Recently also rapid intracellular AGE formation by various intracellular sugars (e.g. fructose, ribose, glyceraldehyde, dihydroxyacetone phosphate, glyceraldehyde-3-phosphate, glyoxal, methylglyoxal, and 3 deoxyglucosone) *in vivo* has been described. The rate of glycation is directly proportional to the percentage of sugar in the open-chain form and the rate for fructose (0.7% open-chain) is 7.5-fold faster than that of glucose (0.002% open-chain). More strikingly, the glycolytic intermediate glyceraldehyde-3-phosphate (100% open-chain) forms 200-fold more glycated

proteins than do equimolar amounts of glucose (Schalkwijk et al., 2004).

Amadori rearrangement takes days and is reversible only to a certain extent.

of AGEs are shown in Fig. 2.

The advanced glycation end-products found under physiological conditions can be classified according to their fluorescent properties and their ability to form cross-links. The first group is represented by fluorescent AGE cross-links, which are thought to be responsible for a major share of the deleterious effects of AGEs in diabetes and aging. Fluorescence is a good qualitative indicator used to estimate AGEs formation. Pentosidine, crossline, and various vesperlysines are members of this group. However, also nonfluorescent AGE cross-links are found *in vivo*. Their isolation and identification is more complicated than in the case of fluorescent AGE cross-links. It is thought that they account just for 1% of all cross-links rising under physiological conditions. Various imidazolium dilysine cross-links (GOLD, MOLD), arginine-lysine cross-links, and glucosepan belong to this group. Last but not least, a group of non-cross-linking protein bound AGE structures have been identified *in vivo*. These structures may exert deleterious effects as precursors of cross-links or as biological receptor ligands inducing a variety of adverse cellular and tissue changes. The well-known members of this group are pyrraline, carboxyalkyllysines (CML, CEL), imidazolones, and argpyrimidine (Ulrich & Cerami, 2001). Classification and examples of each above mentioned group are shown in Fig. 3.

Non-Enzymatic Glycation of Aminotransferases and the Possibilities of Its Modulation 91

RAGE or by suppression of post-receptor signaling using antioxidants (Hudson et al., 2003; Stuchbury & Münch, 2005). The secreted RAGE form, named soluble RAGE (sRAGE), acts as a decoy to trap ligands and prevent interaction with cell surface receptors (Bucciarelli et al., 2002). Soluble RAGE was shown to have important inhibitory effects in several cell culture and transgenic mouse models, in which it prevented or reversed full-length RAGE signaling. The administration of sRAGE has been shown to suppress accelerated diabetic

Aminoguanidine, also known by its trade name Pimagedine (Alteon Inc.), is a prototype therapeutic agent for prevention of the AGEs formation. It is a low-molecular, highly nucleophilic hydrazine compound that rapidly reacts with α-dicarbonyl compounds such as MGO, GO, and 3-DG to prevent formation of AGE cross-links. The products of the scavenging reaction are substituted 3-amino-1,2,4-triazines. Aminoguanidine does not affect the formation of the Schiff base and Amadori products (Thornalley et al., 2000). Clinical trials of aminoguanidine in overt diabetic nephropathy (ACTION) were performed, but they were early terminated due to safety concerns. Reported side effects of aminoguanidine in clinical therapy were gastrointestinal disturbance, abnormalities in liver function tests, flulike symptoms, and a rare vasculitis (Bolton et al., 2004; Thornalley, 2003). Other nucleophilic compounds, which are designed to trap reactive carbonyl intermediates in AGE formation, are for example OPB-9195, diaminophenazine, tenilsetam, and pyridoxamine (Baynes & Thorpe, 2000; De La Cruz et al., 2004). With regard to the presence of free radicals and oxidative steps in the course of glycoxidation, compounds with antioxidant effect such as α-lipoic acid, α-tocopherol, ascorbic acid, and ß-carotene were tested. Dipeptide carnosine, pyridoindole derivative stobadine, hypolipidemic drug probucol, and mucolytic remedy *N*-acetylcysteine are just a few more examples of the compounds with described antioxidant properties, which were tested in order to estimate their potential protective effect in the process of glycation. Also some antioxidant enzymes such as superoxide dismutase, catalase, and selenium-dependent glutathione peroxidase may protect proteins against impairment caused by non-enzymatic glycation (De La Cruz et

Aminoguanidine and other compounds mentioned before can inhibit the formation of new AGE cross-links, but they are not able to cleave those already formed. Vasan et al. (1996) reported the first of cross-link breakers, phenyl thiazolium bromide (PTB). This anti-AGE agent chemically breaks α-dicarbonyl compounds by cleaving the carbon-carbon bond between the carbonyls. Under physiological conditions, PTB is not stable and therefore its analogs were tested and alagebrium chloride (ALT-711), a highly potent cross-link breaker with higher stability, has been discovered. This compound successfully completed preclinical studies and Phase II clinical study on healthy volunteers (Yamagishi et al., 2008). Unfortunately, the specific types of AGEs affected by alagebrium are more important in rats than humans; hence the promising results in animals were never repeated in human studies. Randomized Phase II clinical trial (BENEFICIAL) in patients with chronic heart failure

The objective of current study was to evaluate potential antiglycation activity of two mitochondrial antioxidants, α-phenyl *N*-*tert*-butyl nitrone (PBN) and *N*-*tert*-butyl hydroxylamine (NtBHA). PBN is a nitrone that traps free radicals and protects against

(Willemsen et al., 2010) has been terminated early due to financial constraints.

atherosclerosis (Park et al., 1998).

al., 2004; Kyselova et al., 2004).

Fig. 3. Classification of AGEs formed under physiological conditions including several examples to each group. [Lys] represents a desamino-lysine residue; [Arg] stands for a desguanidino-arginine residue; R represents either hydrogen atom (GOLD), methyl group (MOLD), 1,2,3-trihydroxypropyl (DOLD) or 2,3,4-trihydroxybutyl group (imidazolone A and B).

#### **1.2.3 Therapeutic strategies targeting the AGEs**

The therapeutic intervention to the glycation process has followed three main approaches. A first approach follows inhibition of RAGE by neutralizing antibodies or suppression of postreceptor signaling using antioxidants. A second one is inhibition of AGE formation process by carbonyl-blocking agents (aminoguanidine) or by antioxidants. The last approach is reducing AGE deposition by using cross-link breakers or by enhancing cellular uptake and degradation.

Interactions of AGEs with the receptor for AGEs (RAGE) have been implicated in the development of diabetic vascular complications, which cause various disabilities and shortened life expectancy, and reduced quality of life in patients with diabetes. These undesirable effects can be suppressed by the use of specific antibodies to RAGE, soluble

Fig. 3. Classification of AGEs formed under physiological conditions including several examples to each group. [Lys] represents a desamino-lysine residue; [Arg] stands for a desguanidino-arginine residue; R represents either hydrogen atom (GOLD), methyl group (MOLD), 1,2,3-trihydroxypropyl (DOLD) or 2,3,4-trihydroxybutyl group (imidazolone A and B).

The therapeutic intervention to the glycation process has followed three main approaches. A first approach follows inhibition of RAGE by neutralizing antibodies or suppression of postreceptor signaling using antioxidants. A second one is inhibition of AGE formation process by carbonyl-blocking agents (aminoguanidine) or by antioxidants. The last approach is reducing AGE deposition by using cross-link breakers or by enhancing cellular uptake and

Interactions of AGEs with the receptor for AGEs (RAGE) have been implicated in the development of diabetic vascular complications, which cause various disabilities and shortened life expectancy, and reduced quality of life in patients with diabetes. These undesirable effects can be suppressed by the use of specific antibodies to RAGE, soluble

**1.2.3 Therapeutic strategies targeting the AGEs** 

degradation.

RAGE or by suppression of post-receptor signaling using antioxidants (Hudson et al., 2003; Stuchbury & Münch, 2005). The secreted RAGE form, named soluble RAGE (sRAGE), acts as a decoy to trap ligands and prevent interaction with cell surface receptors (Bucciarelli et al., 2002). Soluble RAGE was shown to have important inhibitory effects in several cell culture and transgenic mouse models, in which it prevented or reversed full-length RAGE signaling. The administration of sRAGE has been shown to suppress accelerated diabetic atherosclerosis (Park et al., 1998).

Aminoguanidine, also known by its trade name Pimagedine (Alteon Inc.), is a prototype therapeutic agent for prevention of the AGEs formation. It is a low-molecular, highly nucleophilic hydrazine compound that rapidly reacts with α-dicarbonyl compounds such as MGO, GO, and 3-DG to prevent formation of AGE cross-links. The products of the scavenging reaction are substituted 3-amino-1,2,4-triazines. Aminoguanidine does not affect the formation of the Schiff base and Amadori products (Thornalley et al., 2000). Clinical trials of aminoguanidine in overt diabetic nephropathy (ACTION) were performed, but they were early terminated due to safety concerns. Reported side effects of aminoguanidine in clinical therapy were gastrointestinal disturbance, abnormalities in liver function tests, flulike symptoms, and a rare vasculitis (Bolton et al., 2004; Thornalley, 2003). Other nucleophilic compounds, which are designed to trap reactive carbonyl intermediates in AGE formation, are for example OPB-9195, diaminophenazine, tenilsetam, and pyridoxamine (Baynes & Thorpe, 2000; De La Cruz et al., 2004). With regard to the presence of free radicals and oxidative steps in the course of glycoxidation, compounds with antioxidant effect such as α-lipoic acid, α-tocopherol, ascorbic acid, and ß-carotene were tested. Dipeptide carnosine, pyridoindole derivative stobadine, hypolipidemic drug probucol, and mucolytic remedy *N*-acetylcysteine are just a few more examples of the compounds with described antioxidant properties, which were tested in order to estimate their potential protective effect in the process of glycation. Also some antioxidant enzymes such as superoxide dismutase, catalase, and selenium-dependent glutathione peroxidase may protect proteins against impairment caused by non-enzymatic glycation (De La Cruz et al., 2004; Kyselova et al., 2004).

Aminoguanidine and other compounds mentioned before can inhibit the formation of new AGE cross-links, but they are not able to cleave those already formed. Vasan et al. (1996) reported the first of cross-link breakers, phenyl thiazolium bromide (PTB). This anti-AGE agent chemically breaks α-dicarbonyl compounds by cleaving the carbon-carbon bond between the carbonyls. Under physiological conditions, PTB is not stable and therefore its analogs were tested and alagebrium chloride (ALT-711), a highly potent cross-link breaker with higher stability, has been discovered. This compound successfully completed preclinical studies and Phase II clinical study on healthy volunteers (Yamagishi et al., 2008). Unfortunately, the specific types of AGEs affected by alagebrium are more important in rats than humans; hence the promising results in animals were never repeated in human studies. Randomized Phase II clinical trial (BENEFICIAL) in patients with chronic heart failure (Willemsen et al., 2010) has been terminated early due to financial constraints.

The objective of current study was to evaluate potential antiglycation activity of two mitochondrial antioxidants, α-phenyl *N*-*tert*-butyl nitrone (PBN) and *N*-*tert*-butyl hydroxylamine (NtBHA). PBN is a nitrone that traps free radicals and protects against

Non-Enzymatic Glycation of Aminotransferases and the Possibilities of Its Modulation 93

Fructose, ribose and glyceraldehyde have been found more potent glycating agents than glucose. The strongest glycation effect was exerted by D,L-glyceraldehyde. Complete enzyme inhibition was reached after 6 days but most enzymatic activity (about 75%) was reduced in the course of the first 2 days of incubation. Moreover, a decrease in ALT activity to 88% of the relevant control was apparent at the zero-time determination of the enzymatic activity in the glyceraldehyde sample. Similar results were obtained with AST (Beránek et

Fig. 5. Inhibitory effect of glycation on the catalytic activity of aminotransferases incubated in sodium phosphate buffer (50 mM, pH 7.4) at 4 °C (―――), 25 °C (––––) and 37 °C (–·–·–) for up to 56 days. The values show residual catalytic activities of the enzymes related to the appropriate control. (A) ALT incubated with 50 mM D-fructose; (B) AST with 50 mM D-

In our further experiments, aspartate aminotransferase (AST, EC 2.6.1.1) has been chosen as a model protein due to its relative stability during *in vitro* incubation and availability of the enzyme preparations of high purity (for further experimental details see Dršata et al., 2005). The purity of the enzyme preparation was crucial for experiments with prolonged incubation. While AST activity of the rat liver 20 000 x g supernatant was very unstable *in vitro* and declined rapidly independently of presence or absence of sugar during incubation even at 25 °C, the purified preparation enabled experiments using incubation for many days

fructose; (C) ALT with 500 mM D-fructose; (D) AST with 500 mM D-fructose.

(Tupcová, 1996). Data are presented in Fig. 6.

al., 2002). These data are presented in Fig. 5.

damage in different models such as inflammation, ischemia reperfusion, and aging. Its decomposition product NtBHA mimics PBN and is much more effective in delaying senescence of human lung fibroblasts IMR90 (Atamna et al., 2000). NtBHA appears to act on mitochondria to delay alterations in function (Atamna et al., 2001). Supplementation with NtBHA improved the respiratory control ratio of mitochondria from liver of old rats (Atamna et al., 2001).

#### **1.3 Summary of existing results**

Our laboratory deals with research of the protein glycation *in vitro* for many years. Aminotransferases were chosen as suitable model proteins, because they are commercially available in a highly purified and stable form, their structures and mechanism of catalysis are well known, and at least two simple methods for their enzyme activity assessment are in use. This subchapter summarizes results, which have been obtained by our research group up to now.

In our laboratory, we found decrease in alanine aminotransferase (ALT, EC 2.6.1.2) activities in the presence of several reducing monosaccharides *in vitro*. The decrease in the catalytic activity of ALT from porcine heart after 20 days of incubation with D-glucose, D-fructose, Dribose or D,L-glyceraldehyde varied and was dependent on the nature of glycating agent (the percentage of sugar present in open-chain form). The dependence of enzyme inactivation on the presence of these sugars and time of incubation is presented in Fig. 4 (Beránek et al., 2001). As was described earlier, the rate of glycation is directly proportional to the percentage of sugar in the open chain form and the rate for fructose (0.7% open-chain) is 7.5-fold faster than that of glucose (0.002% open-chain). More strikingly, the glycolytic intermediate glyceraldehyde 3-phosphate (100% open-chain) forms 200-fold more glycated proteins than do equimolar amounts of glucose (Schalkwijk et al., 2004).

Fig. 4. An effect of glycation on ALT activity. ALT was incubated with 50 mM sugars in sodium phosphate buffer (0.1 M, pH 7.4) at 25 °C. Aliquots of samples were taken at days 0, 2, 6, 9, 13, 20 and remaining enzyme activities in samples were determined. Activity was expressed as a percentage of the activity of the control sample (without sugars). ● ALT with D-glucose; ALT with D-ribose; ALT with D-fructose; ■ ALT with D,L-glyceraldehyde

damage in different models such as inflammation, ischemia reperfusion, and aging. Its decomposition product NtBHA mimics PBN and is much more effective in delaying senescence of human lung fibroblasts IMR90 (Atamna et al., 2000). NtBHA appears to act on mitochondria to delay alterations in function (Atamna et al., 2001). Supplementation with NtBHA improved the respiratory control ratio of mitochondria from liver of old rats

Our laboratory deals with research of the protein glycation *in vitro* for many years. Aminotransferases were chosen as suitable model proteins, because they are commercially available in a highly purified and stable form, their structures and mechanism of catalysis are well known, and at least two simple methods for their enzyme activity assessment are in use. This subchapter summarizes results, which have been obtained by our research group

In our laboratory, we found decrease in alanine aminotransferase (ALT, EC 2.6.1.2) activities in the presence of several reducing monosaccharides *in vitro*. The decrease in the catalytic activity of ALT from porcine heart after 20 days of incubation with D-glucose, D-fructose, Dribose or D,L-glyceraldehyde varied and was dependent on the nature of glycating agent (the percentage of sugar present in open-chain form). The dependence of enzyme inactivation on the presence of these sugars and time of incubation is presented in Fig. 4 (Beránek et al., 2001). As was described earlier, the rate of glycation is directly proportional to the percentage of sugar in the open chain form and the rate for fructose (0.7% open-chain) is 7.5-fold faster than that of glucose (0.002% open-chain). More strikingly, the glycolytic intermediate glyceraldehyde 3-phosphate (100% open-chain) forms 200-fold more glycated

proteins than do equimolar amounts of glucose (Schalkwijk et al., 2004).

Fig. 4. An effect of glycation on ALT activity. ALT was incubated with 50 mM sugars in sodium phosphate buffer (0.1 M, pH 7.4) at 25 °C. Aliquots of samples were taken at days 0, 2, 6, 9, 13, 20 and remaining enzyme activities in samples were determined. Activity was expressed as a percentage of the activity of the control sample (without sugars). ● ALT with D-glucose; ALT with D-ribose; ALT with D-fructose; ■ ALT with D,L-glyceraldehyde

(Atamna et al., 2001).

up to now.

**1.3 Summary of existing results** 

Fructose, ribose and glyceraldehyde have been found more potent glycating agents than glucose. The strongest glycation effect was exerted by D,L-glyceraldehyde. Complete enzyme inhibition was reached after 6 days but most enzymatic activity (about 75%) was reduced in the course of the first 2 days of incubation. Moreover, a decrease in ALT activity to 88% of the relevant control was apparent at the zero-time determination of the enzymatic activity in the glyceraldehyde sample. Similar results were obtained with AST (Beránek et al., 2002). These data are presented in Fig. 5.

Fig. 5. Inhibitory effect of glycation on the catalytic activity of aminotransferases incubated in sodium phosphate buffer (50 mM, pH 7.4) at 4 °C (―――), 25 °C (––––) and 37 °C (–·–·–) for up to 56 days. The values show residual catalytic activities of the enzymes related to the appropriate control. (A) ALT incubated with 50 mM D-fructose; (B) AST with 50 mM Dfructose; (C) ALT with 500 mM D-fructose; (D) AST with 500 mM D-fructose.

In our further experiments, aspartate aminotransferase (AST, EC 2.6.1.1) has been chosen as a model protein due to its relative stability during *in vitro* incubation and availability of the enzyme preparations of high purity (for further experimental details see Dršata et al., 2005).

The purity of the enzyme preparation was crucial for experiments with prolonged incubation. While AST activity of the rat liver 20 000 x g supernatant was very unstable *in vitro* and declined rapidly independently of presence or absence of sugar during incubation even at 25 °C, the purified preparation enabled experiments using incubation for many days (Tupcová, 1996). Data are presented in Fig. 6.

Non-Enzymatic Glycation of Aminotransferases and the Possibilities of Its Modulation 95

Fig. 7. Influence of substrates on AST activity during glycation by D-ribose *in vitro*. Concentration of the enzyme (purified Serva preparation) in the incubation mixture: 0.413 mg/ml, final catalytic concentration in the mixture 7.83 µkat/mg. Values are mean ± S.D. of 6 (control) or 3 (with D-ribose) independent samples. Each sample was measured three-

Fig. 8. Effect of glycation on AST activity and intervention of uric acid (UA) in the process. AST (1.33 mg/ml) was incubated with and without fructose (Frc) in 0.1 mM phosphate buffer, pH 7.4 at 37 °C in the presence or absence of 1.2 mM uric acid. AST activity was expressed as percentage of that of control sample (without sugar), which was considered as 100% ± S.D. (%) at every interval. Each point represents an average of three experiments in interval 0-12 days, and an average of two experiments on the 15th and 21st day. Each

experiment was performed in triplicate (\*data with *P*<0.05, Student's *t*-test).

times and the mean was used to calculate the value presented.

L-Asp = L-aspartate; 2-OG = 2-oxoglutarate

Fig. 6. Comparison of stability of AST in rat liver supernatant and in purified preparation from pig heart during *in vitro* incubation at 25 °C. The incubation mixture was diluted before the assay in order to obtain activities within the analytical range of the method. Values are mean ± S.D. of three independent samples. Each sample was measured three-times and the mean was used to calculate the value presented.

Moreover, glycation of AST was accompanied by a decrease in its catalytic activity in dependence on the concentration and activity of the glycating agent, while the concentration effect was not clearly demonstrated in case of ALT (Dršata et al., 2002). The effect of substrates (2-oxoglutarate and L-aspartate) on AST stability and on glycation process was assessed as well. There was no effect of 2-oxoglutarate on the control AST activity throughout the experiment, which demonstrated a high stability of the pyridoxal form both in the presence and absence of this substrate. On the other hand, the presence of 25 mM L-aspartate (inducing the pyridoxamine form of the enzyme) caused a rapid decrease in AST activity even in the control reaction (Dršata et al., 2002). The results of AST incubation with D-ribose and Dfructose in presence of 0.5 mM 2-oxoglutarate suggested that AST glycation could be partly prevented by this substrate. This finding supports the idea that Lys258 in the active center of AST is involved in glycation of the free enzyme (Fig. 7, taken from Dršata et al., 2002).

The *in vitro* model of protein glycation (AST) by D-fructose has been then established and experimentally used. (Boušová et al., 2005a). As mentioned above, attempts have been made by researchers to investigate various chemical compounds as potential antiglycating agents. With this model, influence of potential antiglycating compounds with antioxidant activities was investigated. In an attempt to reduce glycoxidation process and formation of AGEs, influence of endogenous antioxidant uric acid (0.2-1.2 mM) on glycoxidation process of AST by 50 mM and 500 mM D-fructose *in vitro* was studied. Uric acid at 1.2 mM concentration reduced AST activity decrease caused by incubation of the enzyme with 50 mM sugar up to 25 days at 37 °C (Fig. 8), as well as formation of total fluorescent AGE products (Fig. 9). The results obtained supported the hypothesis that uric acid has beneficial effects in controlling protein glycoxidation (Fig. 8 and 9).

Fig. 6. Comparison of stability of AST in rat liver supernatant and in purified preparation from pig heart during *in vitro* incubation at 25 °C. The incubation mixture was diluted before the assay in order to obtain activities within the analytical range of the method. Values are mean ± S.D. of three independent samples. Each sample was measured

Moreover, glycation of AST was accompanied by a decrease in its catalytic activity in dependence on the concentration and activity of the glycating agent, while the concentration effect was not clearly demonstrated in case of ALT (Dršata et al., 2002). The effect of substrates (2-oxoglutarate and L-aspartate) on AST stability and on glycation process was assessed as well. There was no effect of 2-oxoglutarate on the control AST activity throughout the experiment, which demonstrated a high stability of the pyridoxal form both in the presence and absence of this substrate. On the other hand, the presence of 25 mM L-aspartate (inducing the pyridoxamine form of the enzyme) caused a rapid decrease in AST activity even in the control reaction (Dršata et al., 2002). The results of AST incubation with D-ribose and Dfructose in presence of 0.5 mM 2-oxoglutarate suggested that AST glycation could be partly prevented by this substrate. This finding supports the idea that Lys258 in the active center of

AST is involved in glycation of the free enzyme (Fig. 7, taken from Dršata et al., 2002).

The *in vitro* model of protein glycation (AST) by D-fructose has been then established and experimentally used. (Boušová et al., 2005a). As mentioned above, attempts have been made by researchers to investigate various chemical compounds as potential antiglycating agents. With this model, influence of potential antiglycating compounds with antioxidant activities was investigated. In an attempt to reduce glycoxidation process and formation of AGEs, influence of endogenous antioxidant uric acid (0.2-1.2 mM) on glycoxidation process of AST by 50 mM and 500 mM D-fructose *in vitro* was studied. Uric acid at 1.2 mM concentration reduced AST activity decrease caused by incubation of the enzyme with 50 mM sugar up to 25 days at 37 °C (Fig. 8), as well as formation of total fluorescent AGE products (Fig. 9). The results obtained supported the hypothesis that uric acid has beneficial

three-times and the mean was used to calculate the value presented.

effects in controlling protein glycoxidation (Fig. 8 and 9).

Fig. 7. Influence of substrates on AST activity during glycation by D-ribose *in vitro*. Concentration of the enzyme (purified Serva preparation) in the incubation mixture: 0.413 mg/ml, final catalytic concentration in the mixture 7.83 µkat/mg. Values are mean ± S.D. of 6 (control) or 3 (with D-ribose) independent samples. Each sample was measured threetimes and the mean was used to calculate the value presented. L-Asp = L-aspartate; 2-OG = 2-oxoglutarate

Fig. 8. Effect of glycation on AST activity and intervention of uric acid (UA) in the process. AST (1.33 mg/ml) was incubated with and without fructose (Frc) in 0.1 mM phosphate buffer, pH 7.4 at 37 °C in the presence or absence of 1.2 mM uric acid. AST activity was expressed as percentage of that of control sample (without sugar), which was considered as 100% ± S.D. (%) at every interval. Each point represents an average of three experiments in interval 0-12 days, and an average of two experiments on the 15th and 21st day. Each experiment was performed in triplicate (\*data with *P*<0.05, Student's *t*-test).

Non-Enzymatic Glycation of Aminotransferases and the Possibilities of Its Modulation 97

Fig. 10. The effect of hydroxycitric acid (HCA) on the glycation of AST by fructose *in vitro*. Concentration of AST in incubation mixtures was 1.33 mg/ml. AST was incubated with or without D-fructose 50 mM in sodium phosphate buffer (0.1 M, pH 7.4, 0.05% sodium azide) at 37 °C in the presence or absence of HCA up to 21 days. The samples were diluted before the assay to fit to the analytical range of the method. Values are expressed as a percentage of the activity of respective control (AST) ± S.D. of six independent samples (\*data with *P*<0.05, Student's *t*-test). AST; AST + Frc 50 mM; ▲ AST + Frc + HCA 2.5 mM; ∆ AST + Frc +

HCA 5.0 mM; AST + Frc + HCA 7.5 mM; o AST + Frc + HCA 10.0 mM.

Fig. 11. Direct effect of baicalin on the AST activity *in vitro*. Concentration of AST in incubation mixtures was 1.33 mg/ml in 0.1M sodium phosphate buffer (pH 7.4; 0.05% sodium azide). Incubation at 37 °C. The samples were diluted before the assay to fit to the analytical range of the method. Values are expressed as mean ± S.D. of six (control and AST + Frc 50 mM) or three (with baicalin) independent samples (\*data with *P*<0.05, Student's *t*test). AST; AST + Frc 50 mM; ▲ AST + baicalin 0.5 mM; ∆ AST + baicalin 1.0 mM; AST

+ baicalin 1.5 mM; o AST + baicalin 3.0 mM.

Fig. 9. Formation of fluorescent products of glycation under the conditions of long lasting incubation. AST (1.33 mg/ml) was incubated with or without D-fructose in sodium phosphate buffer (0.1 M, pH 7.4) at 37 °C in the presence or absence of 1.2 mM uric acid up to 25 days. Aliquots of samples were taken on days 0, 1, 3, 5, 25, and arising fluorescent AGE products in samples were determined at specific wavelengths of excitation and emission (λex/λem) corresponding to total AGEs (370/440 nm). Data of relative fluorescence were expressed in arbitrary units per mg of protein ± S.D., with 1 AU corresponding to the fluorescence of BSA 1.0 mg/ml. Every point in days 0 and 5 represents an average of four experiments (10 samples), in days 1 and 3 an average of three experiments for mixtures with 50 mM fructose (7 samples) and of two experiments for mixtures with 500 mM fructose (4 samples), and in day 25 an average of three experiments (7 samples), (\*data with *P*<0.05, Student's *t*-test).

Also further studies of our research group have been pointed at possible protective activity of selected natural compounds (e.g. hydroxycinnamic acids, flavonoids, arbutin, hydroxycitric acid) on AST glycation by fructose. The results have shown that these compounds can exert also negative effects on enzyme activity but some of them have been able to slow down the course of AST modification by glycating agent. The compound with overall positive activity has been hydroxycitric acid (Fig. 10), which is major active component in the fruit rinds of certain species of the plant *Garcinia*. The effect of this compound was concentration-depended and its positive activity was most pronounced at 2.5 mM concentration. On the other hand, flavonoid baicalin (Fig. 11) and its aglycone baicalein rapidly decreased the *in vitro* activity of the enzyme in all concentrations used (0.5– 3 mM), and no beneficial effects of these compounds on glycation of the enzyme by fructose were found (Boušová et al., 2005b).

Fig. 9. Formation of fluorescent products of glycation under the conditions of long lasting incubation. AST (1.33 mg/ml) was incubated with or without D-fructose in sodium phosphate buffer (0.1 M, pH 7.4) at 37 °C in the presence or absence of 1.2 mM uric acid

up to 25 days. Aliquots of samples were taken on days 0, 1, 3, 5, 25, and arising fluorescent AGE products in samples were determined at specific wavelengths of excitation and emission (λex/λem) corresponding to total AGEs (370/440 nm). Data of relative fluorescence were expressed in arbitrary units per mg of protein ± S.D., with 1 AU

corresponding to the fluorescence of BSA 1.0 mg/ml. Every point in days 0 and 5 represents an average of four experiments (10 samples), in days 1 and 3 an average of three experiments for mixtures with 50 mM fructose (7 samples) and of two experiments

for mixtures with 500 mM fructose (4 samples), and in day 25 an average of three

Also further studies of our research group have been pointed at possible protective activity of selected natural compounds (e.g. hydroxycinnamic acids, flavonoids, arbutin, hydroxycitric acid) on AST glycation by fructose. The results have shown that these compounds can exert also negative effects on enzyme activity but some of them have been able to slow down the course of AST modification by glycating agent. The compound with overall positive activity has been hydroxycitric acid (Fig. 10), which is major active component in the fruit rinds of certain species of the plant *Garcinia*. The effect of this compound was concentration-depended and its positive activity was most pronounced at 2.5 mM concentration. On the other hand, flavonoid baicalin (Fig. 11) and its aglycone baicalein rapidly decreased the *in vitro* activity of the enzyme in all concentrations used (0.5– 3 mM), and no beneficial effects of these compounds on glycation of the enzyme by fructose

experiments (7 samples), (\*data with *P*<0.05, Student's *t*-test).

were found (Boušová et al., 2005b).

Fig. 10. The effect of hydroxycitric acid (HCA) on the glycation of AST by fructose *in vitro*. Concentration of AST in incubation mixtures was 1.33 mg/ml. AST was incubated with or without D-fructose 50 mM in sodium phosphate buffer (0.1 M, pH 7.4, 0.05% sodium azide) at 37 °C in the presence or absence of HCA up to 21 days. The samples were diluted before the assay to fit to the analytical range of the method. Values are expressed as a percentage of the activity of respective control (AST) ± S.D. of six independent samples (\*data with *P*<0.05, Student's *t*-test). AST; AST + Frc 50 mM; ▲ AST + Frc + HCA 2.5 mM; ∆ AST + Frc + HCA 5.0 mM; AST + Frc + HCA 7.5 mM; o AST + Frc + HCA 10.0 mM.

Fig. 11. Direct effect of baicalin on the AST activity *in vitro*. Concentration of AST in incubation mixtures was 1.33 mg/ml in 0.1M sodium phosphate buffer (pH 7.4; 0.05% sodium azide). Incubation at 37 °C. The samples were diluted before the assay to fit to the analytical range of the method. Values are expressed as mean ± S.D. of six (control and AST + Frc 50 mM) or three (with baicalin) independent samples (\*data with *P*<0.05, Student's *t*test). AST; AST + Frc 50 mM; ▲ AST + baicalin 0.5 mM; ∆ AST + baicalin 1.0 mM; AST + baicalin 1.5 mM; o AST + baicalin 3.0 mM.

Non-Enzymatic Glycation of Aminotransferases and the Possibilities of Its Modulation 99

The enzyme suspension was centrifuged at 5000 rpm at 4 °C for 20 minutes, the supernatant was removed, and protein pellet was reconstituted in 0.1 M sodium phosphate buffer (pH 7.4, 0.05% sodium azide) and the stock solution of 1.0 mg/ml was prepared. This stock solution was used for the preparation of four different types of incubation mixtures: (a) control samples (with buffer only), (b) methylglyoxal-modified samples (with MGO in a final concentration of 0.5 mM), (c) direct protein-antioxidant interaction samples (with individual antioxidants in a final concentration 0.5-10 mM), (d) antiglycation samples (with individual antioxidants in a final concentration of 0.5-10 mM and MGO in a final concentration 0.5 mM). The inhibitory effect of α-phenyl *N*-*tert*-butyl nitrone and *N*-*tert*butyl hydroxylamine on protein glycation was compared to the effect of aminoguanidine (AG) in a concentration of 1.0 mM and Trolox in a concentration of 2.5 mM. The final concentrations of the enzyme were 5 µg/ml for catalytic activity assessment and 0.5 mg/ml for electrophoresis, amine content and fluorescence measurements. All incubation mixtures were incubated in the dark at 37 °C for up to 14 days. The low-molecular compounds were removed using Amicon centrifugal filtration device with 0.1 M sodium phosphate buffer (pH 7.4), protein content was measured using Bradford assay, and adjusted to the concentration 0.5 mg/ml. Aliquots were stored frozen at -20 °C until analysis. All samples were assessed in triplicates and experiments were repeated twice if not stated otherwise.

Catalytic activity of AST was assessed spectrophotometrically using kinetic UV method with addition of PLP (Bergmeyer et al., 1986). Sample aliquots were diluted by 0.1 M sodium phosphate buffer (pH 7.4) to obtain enzyme activities within the analytical range of the method used. Sampling and measuring was carried out at 37 °C in the intervals 0, 120, and 240 minutes using Helios ß spectrophotometer. Absorbance changes at 340 nm were

Fig. 13. Structures of tested compounds

**2.1 Sample preparation and incubation** 

**2.2 Enzyme assay** 

Following experiments were conducted using methylglyoxal (MGO) as a glycating agent, because this compound has higher glycating potential than reducing monosaccharides and the incubation period has been shortened to one week only. Changes in the catalytic activity of AST caused by MGO were observable even after 120 min of incubation at 37 °C. Antiglycating activity of hydroxycitric (Fig. 12) and uric acid has been studied in this modified model (Boušová et al., 2009).

Fig. 12. Effect of glycation on AST activity and its intervention by hydroxycitric acid. AST (5 µg/ml) was incubated with or without methylglyoxal (0.5 mM) in 0.1 M sodium phosphate buffer (pH 7.4) at 37 °C in the presence or absence of hydroxycitric acid (1.0 and 2.5 mM). Catalytic activity of AST was expressed as percentage of each sample activity at the time 0, which was 100% ± S.D. (%). Every point represents an average of two independent experiments, in which assays were performed in triplicates († data with *P*<0.05 and \*data with *P*<0.01, Student's *t*-test).

#### **2. Methods**

Following parameters have been assessed: enzyme activity, fluorescence (total AGEs and argpyrimidine), amount of primary amino groups, molecular charge of AST (using native polyacrylamide gel electrophoresis), and protein cross-linking and aggregation (using polyacrylamide gel electrophoresis under reducing conditions with subsequent western blotting). Structures of all tested compounds are shown in Fig. 13.

Fig. 13. Structures of tested compounds

Following experiments were conducted using methylglyoxal (MGO) as a glycating agent, because this compound has higher glycating potential than reducing monosaccharides and the incubation period has been shortened to one week only. Changes in the catalytic activity of AST caused by MGO were observable even after 120 min of incubation at 37 °C. Antiglycating activity of hydroxycitric (Fig. 12) and uric acid has been studied in this

Fig. 12. Effect of glycation on AST activity and its intervention by hydroxycitric acid. AST (5 µg/ml) was incubated with or without methylglyoxal (0.5 mM) in 0.1 M sodium phosphate buffer (pH 7.4) at 37 °C in the presence or absence of hydroxycitric acid (1.0 and 2.5 mM). Catalytic activity of AST was expressed as percentage of each sample activity at the time 0,

Following parameters have been assessed: enzyme activity, fluorescence (total AGEs and argpyrimidine), amount of primary amino groups, molecular charge of AST (using native polyacrylamide gel electrophoresis), and protein cross-linking and aggregation (using polyacrylamide gel electrophoresis under reducing conditions with subsequent western

which was 100% ± S.D. (%). Every point represents an average of two independent experiments, in which assays were performed in triplicates († data with *P*<0.05 and \*data

blotting). Structures of all tested compounds are shown in Fig. 13.

modified model (Boušová et al., 2009).

with *P*<0.01, Student's *t*-test).

**2. Methods** 

#### **2.1 Sample preparation and incubation**

The enzyme suspension was centrifuged at 5000 rpm at 4 °C for 20 minutes, the supernatant was removed, and protein pellet was reconstituted in 0.1 M sodium phosphate buffer (pH 7.4, 0.05% sodium azide) and the stock solution of 1.0 mg/ml was prepared. This stock solution was used for the preparation of four different types of incubation mixtures: (a) control samples (with buffer only), (b) methylglyoxal-modified samples (with MGO in a final concentration of 0.5 mM), (c) direct protein-antioxidant interaction samples (with individual antioxidants in a final concentration 0.5-10 mM), (d) antiglycation samples (with individual antioxidants in a final concentration of 0.5-10 mM and MGO in a final concentration 0.5 mM). The inhibitory effect of α-phenyl *N*-*tert*-butyl nitrone and *N*-*tert*butyl hydroxylamine on protein glycation was compared to the effect of aminoguanidine (AG) in a concentration of 1.0 mM and Trolox in a concentration of 2.5 mM. The final concentrations of the enzyme were 5 µg/ml for catalytic activity assessment and 0.5 mg/ml for electrophoresis, amine content and fluorescence measurements. All incubation mixtures were incubated in the dark at 37 °C for up to 14 days. The low-molecular compounds were removed using Amicon centrifugal filtration device with 0.1 M sodium phosphate buffer (pH 7.4), protein content was measured using Bradford assay, and adjusted to the concentration 0.5 mg/ml. Aliquots were stored frozen at -20 °C until analysis. All samples were assessed in triplicates and experiments were repeated twice if not stated otherwise.

#### **2.2 Enzyme assay**

Catalytic activity of AST was assessed spectrophotometrically using kinetic UV method with addition of PLP (Bergmeyer et al., 1986). Sample aliquots were diluted by 0.1 M sodium phosphate buffer (pH 7.4) to obtain enzyme activities within the analytical range of the method used. Sampling and measuring was carried out at 37 °C in the intervals 0, 120, and 240 minutes using Helios ß spectrophotometer. Absorbance changes at 340 nm were

Non-Enzymatic Glycation of Aminotransferases and the Possibilities of Its Modulation 101

membranes were washed six times with TBST and incubated with secondary antibody for 45 minutes (dilution 1:1000). The blots were extensively washed in 0.1 M TRIS buffer containing 5 mM MgCl2.6H2O (pH 9.5), covered with chemiluminescent substrate DuoLux (Vector Laboratories) and incubated for 5 minutes. The membranes were then exposed to Xray film (CL-XPosure film, Thermo Fisher Scientific), developed by standard developing process, and images were recorded with a GelDoc XR system. The blots were

Values of catalytic activity are given as means ± S.D. and mostly expressed in % of the time 0 of individual samples ± relative S.D. Values of fluorescence (AU) are given as means ± S.D. Statistical significance was determined using Student's *t*-test and differences were regarded

The activity of tested compounds was compared to the effect of known carbonyl-blocking agent aminoguanidine and to the effect of Trolox (water-soluble derivative of vitamin E), which is often used in various methods for assessing antioxidant/antiradical properties of

Activity of control sample (AST alone) was stable throughout the experiment. Some of tested compounds had a more or less pronounced negative direct effect on enzyme activity, which was probably due to a direct interaction of their molecules with the molecule of the enzyme. PBN itself had no harmful influence on stability and catalytic activity of AST in the concentrations used (Fig. 14B), while NtBHA caused concentration-dependent decrease in AST catalytic activity, which was statistically significant at concentrations of NtBHA 1 mM and higher (Fig. 14A). Aminoguanidine 1.0 mM caused significant decrease of enzyme activity by 21.7% after 240 min of incubation. This inhibitory effect of AG may be explained by its binding to PLP coenzyme forming a Schiff base, which disturbs tissue distribution of PLP *in vivo* and decreases its concentration in liver (Okada & Ayabe, 1995; Taguchi et al.,

Following incubation of enzyme with MGO 0.5 mM, a rapid decline of AST activity was observed. The enzymatic activity decreased to 53.1 and 30.1% of control sample after 120 and 240 min, respectively. In addition, positive antiglycation effects were observed with some compounds. NtBHA exerted antiglycation influence only at 5 mM concentration after 120 and 240 min and at 1 mM concentration after 240 min of incubation (Fig. 15A). Negative direct effect of this compound observed at 10 mM concentration probably outweighed its positive antiglycation activity. The catalytic activity of AST was by 33.1% and 16.5% higher in the presence of PBN 10 mM and 1 mM after 120 min of incubation with MGO compared to the activity of sample containing AST + MGO only (Fig. 15B), respectively. PBN 1-10 mM protected AST against MGO-induced glycation also after 240 min of incubation, when all three concentrations increased activity of AST by 20%. In comparison, aminoguanidine 1.0

densitometrically quantified using Quantity One software.

as significant when *P*<0.05 and *P*<0.01, respectively.

potential antioxidants as reference compound.

1998). Trolox 2.5 mM did not influence AST activity.

**2.7 Statistical analysis** 

**3. Results and discussion** 

**3.1 Enzyme assay** 

used to calculate enzyme activities. All results of enzyme assays were expressed in µkat/l and usually recalculated as activities relative to those of the value of individual sample at time 0.

#### **2.3 Fluorescence measurements**

Formation of fluorescent AGEs and argpyrimidine were measured using the method of Wu & Yen (2005) with some modifications. Briefly, samples were incubated for 7 days at 37 °C. The aliquots were taken away at time 0, 3 and 7 days and stored frozen at -20 °C. Aliquots of time 0 were used as unincubated blanks. Fluorescence of samples was measured at excitation and emission wavelengths of 330 nm/410 nm (fluorescent AGEs) and 320 nm/380 nm (argpyrimidine) against corresponding blanks in 96-well-plate by microplate reader (Tecan Infinity M200) using 0.1 mg of protein per well. The percentage inhibition of AGEs and argpyrimidine formation were calculated according to following formula: % inhibition = [1 - (fluorescence of test group/fluorescence of glycated control)] x 100%.

#### **2.4 Determination of primary amino groups**

Amine content, which is a measure of protein glycation, was estimated spectrophotometrically with trinitrobenzenesulfonic acid (Steinbrecher, 1987) using ß-alanine as the standard. Sample containing 50 µg of AST was incubated with 0.1% trinitrobenzenesulfonic acid in alkaline conditions for 2 h at 37 °C. The reaction was stopped by acidification (1 M HCl) and addition of 10% sodium dodecyl sulfate. The absorbance of trinitrophenyl-amino acid complex was measured at 340 nm. The standard curve was linear in the range 5–100 nmol of NH2.

#### **2.5 Effect of glycation on molecular charge of AST**

Native polyacrylamide gel electrophoresis (PAGE) was used to investigate the changes in the molecular charge of AST due to glycation. Electrophoresis was performed in discontinuous system with 4% stacking gel and 7.5% separating non-denaturating gel (Ornstein, 1964; Davies, 1964). All lanes were loaded with 9 µg of protein. Electrophoresis was performed at 30 mA for 2 hours using Mini ProteanIII apparatus. The gel was then stained by colloidal Coomassie Blue G250, scanned, and relative migration distances were calculated from Rf using Quantity One software. Electrophoretic mobilities were expressed as a rise in percentage mobility compared to the native enzyme (control).

#### **2.6 SDS-PAGE and western blotting**

Protein cross-linking and aggregation were assessed using a sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE) on Mini ProteanIII apparatus (BioRad). SDS-PAGE was performed using discontinuous system with 4% stacking gel and 10% separating gel (Laemmli, 1970). Lanes were loaded with 4 µg of protein. Proteins after electrophoretic separation were transferred to PVDF membrane (0.2 µm, Bio-Rad) at a constant voltage 100 V for 90 minutes (Mini Trans-Blot Electrophoretic Transfer Cell, Bio-Rad). After blotting, membranes were blocked with 8% non-fat dry milk in Tris buffered saline-Tween-20 buffer (TBST) overnight at 4°C, then washed in TBST and reacted with primary antibody (dilution 1:1000) for 45 minutes at room temperature. Subsequently, membranes were washed six times with TBST and incubated with secondary antibody for 45 minutes (dilution 1:1000). The blots were extensively washed in 0.1 M TRIS buffer containing 5 mM MgCl2.6H2O (pH 9.5), covered with chemiluminescent substrate DuoLux (Vector Laboratories) and incubated for 5 minutes. The membranes were then exposed to Xray film (CL-XPosure film, Thermo Fisher Scientific), developed by standard developing process, and images were recorded with a GelDoc XR system. The blots were densitometrically quantified using Quantity One software.

#### **2.7 Statistical analysis**

100 Enzyme Inhibition and Bioapplications

used to calculate enzyme activities. All results of enzyme assays were expressed in µkat/l and usually recalculated as activities relative to those of the value of individual sample at

Formation of fluorescent AGEs and argpyrimidine were measured using the method of Wu & Yen (2005) with some modifications. Briefly, samples were incubated for 7 days at 37 °C. The aliquots were taken away at time 0, 3 and 7 days and stored frozen at -20 °C. Aliquots of time 0 were used as unincubated blanks. Fluorescence of samples was measured at excitation and emission wavelengths of 330 nm/410 nm (fluorescent AGEs) and 320 nm/380 nm (argpyrimidine) against corresponding blanks in 96-well-plate by microplate reader (Tecan Infinity M200) using 0.1 mg of protein per well. The percentage inhibition of AGEs and argpyrimidine formation were calculated according to following formula: % inhibition

Amine content, which is a measure of protein glycation, was estimated spectrophotometrically with trinitrobenzenesulfonic acid (Steinbrecher, 1987) using ß-alanine as the standard. Sample containing 50 µg of AST was incubated with 0.1% trinitrobenzenesulfonic acid in alkaline conditions for 2 h at 37 °C. The reaction was stopped by acidification (1 M HCl) and addition of 10% sodium dodecyl sulfate. The absorbance of trinitrophenyl-amino acid complex was

Native polyacrylamide gel electrophoresis (PAGE) was used to investigate the changes in the molecular charge of AST due to glycation. Electrophoresis was performed in discontinuous system with 4% stacking gel and 7.5% separating non-denaturating gel (Ornstein, 1964; Davies, 1964). All lanes were loaded with 9 µg of protein. Electrophoresis was performed at 30 mA for 2 hours using Mini ProteanIII apparatus. The gel was then stained by colloidal Coomassie Blue G250, scanned, and relative migration distances were calculated from Rf using Quantity One software. Electrophoretic mobilities were expressed

Protein cross-linking and aggregation were assessed using a sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE) on Mini ProteanIII apparatus (BioRad). SDS-PAGE was performed using discontinuous system with 4% stacking gel and 10% separating gel (Laemmli, 1970). Lanes were loaded with 4 µg of protein. Proteins after electrophoretic separation were transferred to PVDF membrane (0.2 µm, Bio-Rad) at a constant voltage 100 V for 90 minutes (Mini Trans-Blot Electrophoretic Transfer Cell, Bio-Rad). After blotting, membranes were blocked with 8% non-fat dry milk in Tris buffered saline-Tween-20 buffer (TBST) overnight at 4°C, then washed in TBST and reacted with primary antibody (dilution 1:1000) for 45 minutes at room temperature. Subsequently,

measured at 340 nm. The standard curve was linear in the range 5–100 nmol of NH2.

as a rise in percentage mobility compared to the native enzyme (control).

= [1 - (fluorescence of test group/fluorescence of glycated control)] x 100%.

time 0.

**2.3 Fluorescence measurements** 

**2.4 Determination of primary amino groups** 

**2.5 Effect of glycation on molecular charge of AST** 

**2.6 SDS-PAGE and western blotting** 

Values of catalytic activity are given as means ± S.D. and mostly expressed in % of the time 0 of individual samples ± relative S.D. Values of fluorescence (AU) are given as means ± S.D. Statistical significance was determined using Student's *t*-test and differences were regarded as significant when *P*<0.05 and *P*<0.01, respectively.

### **3. Results and discussion**

The activity of tested compounds was compared to the effect of known carbonyl-blocking agent aminoguanidine and to the effect of Trolox (water-soluble derivative of vitamin E), which is often used in various methods for assessing antioxidant/antiradical properties of potential antioxidants as reference compound.

#### **3.1 Enzyme assay**

Activity of control sample (AST alone) was stable throughout the experiment. Some of tested compounds had a more or less pronounced negative direct effect on enzyme activity, which was probably due to a direct interaction of their molecules with the molecule of the enzyme. PBN itself had no harmful influence on stability and catalytic activity of AST in the concentrations used (Fig. 14B), while NtBHA caused concentration-dependent decrease in AST catalytic activity, which was statistically significant at concentrations of NtBHA 1 mM and higher (Fig. 14A). Aminoguanidine 1.0 mM caused significant decrease of enzyme activity by 21.7% after 240 min of incubation. This inhibitory effect of AG may be explained by its binding to PLP coenzyme forming a Schiff base, which disturbs tissue distribution of PLP *in vivo* and decreases its concentration in liver (Okada & Ayabe, 1995; Taguchi et al., 1998). Trolox 2.5 mM did not influence AST activity.

Following incubation of enzyme with MGO 0.5 mM, a rapid decline of AST activity was observed. The enzymatic activity decreased to 53.1 and 30.1% of control sample after 120 and 240 min, respectively. In addition, positive antiglycation effects were observed with some compounds. NtBHA exerted antiglycation influence only at 5 mM concentration after 120 and 240 min and at 1 mM concentration after 240 min of incubation (Fig. 15A). Negative direct effect of this compound observed at 10 mM concentration probably outweighed its positive antiglycation activity. The catalytic activity of AST was by 33.1% and 16.5% higher in the presence of PBN 10 mM and 1 mM after 120 min of incubation with MGO compared to the activity of sample containing AST + MGO only (Fig. 15B), respectively. PBN 1-10 mM protected AST against MGO-induced glycation also after 240 min of incubation, when all three concentrations increased activity of AST by 20%. In comparison, aminoguanidine 1.0

Non-Enzymatic Glycation of Aminotransferases and the Possibilities of Its Modulation 103

argpyrimidine and the effect of NtBHA on this process. Sample containing AST + MGO exerted 12.5 times higher fluorescence intensity than the control sample without MGO after 7 days of incubation. NtBHA caused statistically significant decrease in the formation of argpyrimidine during incubation (inhibition by 42.3–83.1%). The most remarkable decline in argpyrimidine formation was observed at 10 mM concentration of NtBHA, which exhibited inhibition by 83.1%. Effect of PBN on the argpyrimidine formation was less remarkable (inhibition by 35.2–55.8%) but still highly significant (Fig. 16A). The influence of AG 1.0 mM was well-pronounced (93.2%), while the effect of Trolox 2.5 mM was much weaker (53.2%)

The effect of NtBHA on the formation of ''non-specific'' AGE products is presented in Fig. 16D. Methylglyoxal caused almost 13 fold increase in concentration of AGEs with fluorescent properties compared to the control sample (AST alone) after 7 days of incubation. Positive effect of NtBHA reached almost the same extent as in the case of argpyrimidine formation with statistically significant inhibition of glycation (34.3–76.6%). Little bit lower rate of inhibition (8.9–48.2%) was obtained also with PBN (Fig. 16C). Aminoguanidine and Trolox

As for fluorescence measurement, control sample showed stable but not negligible fluorescence, since the start of the experiment. Most of this fluorescence is probably constituted by general fluorescence properties of this protein. Presence of pyridoxal-5' phosphate coenzyme in the molecule of AST also contributes to basal fluorescence of the enzyme. Results of fluorescence measurements clearly show an inhibiting effect of NtBHA and PBN on the formation of AGE products. NtBHA was also quite effective in the inhibition of argpyrimidine generation. Apart from these findings, the use of fluorescence method for evaluation of protein glycation is limited by its imprecision. The measurement of some well-identified AGEs (e.g., pentosidine and carboxymethyllysine) by techniques as HPLC or ELISA could give more precise information on this matter (Boušová et al., 2009).

Following incubation of AST with methylglyoxal, there was a decrease in amine content compared to control (Table 1). Unmodified AST exhibited 25.2 ± 0.4 nmol NH2/mol AST versus 13.9 ± 1.1 nmol NH2/mol AST for sample containing AST + MGO (*P*<0.001, using Student's *t*-test). This difference represents a 45% decrease in amine content due to chemical modification of the primary amines (α-amino group of *N*-terminal amino acids and ε-amino group of Lys residues). Native AST in the dimer form contains 40 primary amines (38 Lys residues and 2 *N*-terminal amino acids), suggesting that approximately 18 amines were modified by methylglyoxal. Modification of primary amino group of Lys258 in AST molecule by MGO may be also responsible for the loss of its catalytic activity, because this Lys residue binds coenzyme PLP in the active centre of AST and thus directly participates in

PBN as well as NtBHA significantly protected AST against the loss of primary amino groups induced by MGO. Their effect was concentration-dependent and more pronounced in the case of NtBHA. Sample containing AST + MGO + NtBHA 10 mM exhibited 21.8 ± 0.7 nmol NH2/mol AST (*P*<0.001, using Student's *t*-test) suggesting that about 5 amines were

and comparable to the activity of NtBHA 1 mM and PBN 1-10 mM.

showed 88.7 and 44.4% suppressing effect on AGEs generation, respectively.

**3.3 Determination of primary amino groups** 

the enzymatic catalysis.

mM almost completely reversed negative effect of MGO and the AST activity of the sample containing AST + MGO + AG was 86.2 and 76.6% of control sample activity after 120 and 240 min, respectively. The effect of Trolox 2.5 mM was slightly higher than that of NtBHA 5 mM. The AST activity was by 15.1 and 13.7% higher in the presence of Trolox after 120 and 240 min, respectively.

Fig. 14. Direct effect of NtBHA (panel A) and PBN (panel B) on AST catalytic activity. AST (0.5 µg/ml) was incubated with or without methylglyoxal (0.5 mM) in 0.1 M sodium phosphate buffer (pH 7.4) at 37 °C in the presence or absence of NtBHA (1-10 mM) or PBN (1-10 mM). Catalytic activity of AST was expressed as percentage of each sample activity at the time 0, which was 100% ± S.D. (%). Every point represents an average of eighteen (AST and AST + MGO) or six independent samples (\*data with *P*<0.01, Student's *t*-test)

Fig. 15. Antiglycating activity of NtBHA (panel A) and PBN (panel B) towards methylglyoxal-induced deactivation of AST. AST (0.5 µg/ml) was incubated with or without methylglyoxal (0.5 mM) in 0.1 M sodium phosphate buffer (pH 7.4) at 37 °C in the presence or absence of NtBHA (1-10 mM) or PBN (1-10 mM). Catalytic activity of AST was expressed as percentage of each sample activity at the time 0, which was 100% ± S.D. (%). Every point represents an average of eighteen (AST and AST + MGO) or six independent samples (\*data with *P*<0.01, Student's *t*-test)

#### **3.2 Fluorescence measurements**

The inhibition of MGO-mediated protein glycation by several antioxidants was determined by measuring of AGEs with fluorescent properties. Figure 16B shows formation of

mM almost completely reversed negative effect of MGO and the AST activity of the sample containing AST + MGO + AG was 86.2 and 76.6% of control sample activity after 120 and 240 min, respectively. The effect of Trolox 2.5 mM was slightly higher than that of NtBHA 5 mM. The AST activity was by 15.1 and 13.7% higher in the presence of Trolox after 120 and

Fig. 14. Direct effect of NtBHA (panel A) and PBN (panel B) on AST catalytic activity. AST (0.5 µg/ml) was incubated with or without methylglyoxal (0.5 mM) in 0.1 M sodium phosphate buffer (pH 7.4) at 37 °C in the presence or absence of NtBHA (1-10 mM) or PBN (1-10 mM). Catalytic activity of AST was expressed as percentage of each sample activity at the time 0, which was 100% ± S.D. (%). Every point represents an average of eighteen (AST

and AST + MGO) or six independent samples (\*data with *P*<0.01, Student's *t*-test)

Fig. 15. Antiglycating activity of NtBHA (panel A) and PBN (panel B) towards methylglyoxal-induced deactivation of AST. AST (0.5 µg/ml) was incubated with or without methylglyoxal (0.5 mM) in 0.1 M sodium phosphate buffer (pH 7.4) at 37 °C in the presence or absence of NtBHA (1-10 mM) or PBN (1-10 mM). Catalytic activity of AST was expressed as percentage of each sample activity at the time 0, which was 100% ± S.D. (%). Every point represents an average of eighteen (AST and AST + MGO) or six independent

The inhibition of MGO-mediated protein glycation by several antioxidants was determined by measuring of AGEs with fluorescent properties. Figure 16B shows formation of

samples (\*data with *P*<0.01, Student's *t*-test)

**3.2 Fluorescence measurements** 

240 min, respectively.

argpyrimidine and the effect of NtBHA on this process. Sample containing AST + MGO exerted 12.5 times higher fluorescence intensity than the control sample without MGO after 7 days of incubation. NtBHA caused statistically significant decrease in the formation of argpyrimidine during incubation (inhibition by 42.3–83.1%). The most remarkable decline in argpyrimidine formation was observed at 10 mM concentration of NtBHA, which exhibited inhibition by 83.1%. Effect of PBN on the argpyrimidine formation was less remarkable (inhibition by 35.2–55.8%) but still highly significant (Fig. 16A). The influence of AG 1.0 mM was well-pronounced (93.2%), while the effect of Trolox 2.5 mM was much weaker (53.2%) and comparable to the activity of NtBHA 1 mM and PBN 1-10 mM.

The effect of NtBHA on the formation of ''non-specific'' AGE products is presented in Fig. 16D. Methylglyoxal caused almost 13 fold increase in concentration of AGEs with fluorescent properties compared to the control sample (AST alone) after 7 days of incubation. Positive effect of NtBHA reached almost the same extent as in the case of argpyrimidine formation with statistically significant inhibition of glycation (34.3–76.6%). Little bit lower rate of inhibition (8.9–48.2%) was obtained also with PBN (Fig. 16C). Aminoguanidine and Trolox showed 88.7 and 44.4% suppressing effect on AGEs generation, respectively.

As for fluorescence measurement, control sample showed stable but not negligible fluorescence, since the start of the experiment. Most of this fluorescence is probably constituted by general fluorescence properties of this protein. Presence of pyridoxal-5' phosphate coenzyme in the molecule of AST also contributes to basal fluorescence of the enzyme. Results of fluorescence measurements clearly show an inhibiting effect of NtBHA and PBN on the formation of AGE products. NtBHA was also quite effective in the inhibition of argpyrimidine generation. Apart from these findings, the use of fluorescence method for evaluation of protein glycation is limited by its imprecision. The measurement of some well-identified AGEs (e.g., pentosidine and carboxymethyllysine) by techniques as HPLC or ELISA could give more precise information on this matter (Boušová et al., 2009).

#### **3.3 Determination of primary amino groups**

Following incubation of AST with methylglyoxal, there was a decrease in amine content compared to control (Table 1). Unmodified AST exhibited 25.2 ± 0.4 nmol NH2/mol AST versus 13.9 ± 1.1 nmol NH2/mol AST for sample containing AST + MGO (*P*<0.001, using Student's *t*-test). This difference represents a 45% decrease in amine content due to chemical modification of the primary amines (α-amino group of *N*-terminal amino acids and ε-amino group of Lys residues). Native AST in the dimer form contains 40 primary amines (38 Lys residues and 2 *N*-terminal amino acids), suggesting that approximately 18 amines were modified by methylglyoxal. Modification of primary amino group of Lys258 in AST molecule by MGO may be also responsible for the loss of its catalytic activity, because this Lys residue binds coenzyme PLP in the active centre of AST and thus directly participates in the enzymatic catalysis.

PBN as well as NtBHA significantly protected AST against the loss of primary amino groups induced by MGO. Their effect was concentration-dependent and more pronounced in the case of NtBHA. Sample containing AST + MGO + NtBHA 10 mM exhibited 21.8 ± 0.7 nmol NH2/mol AST (*P*<0.001, using Student's *t*-test) suggesting that about 5 amines were

Non-Enzymatic Glycation of Aminotransferases and the Possibilities of Its Modulation 105

with the formation of fluorescent AGEs (r = 0.991, *P*<0.005). In addition, the amine content measured in samples containing AST + MGO + NtBHA (0.5-10 mM) was plotted as a function of AGEs fluorescence, indicating that MGO-induced formation of AGEs was directly proportional to an irreversible loss of primary amino groups in AST molecule (r = 0.946, *P*<0.027). Similar results were obtained also in samples containing AST + MGO + PBN

Sample Amine content Number of primary NH2

AST 25.2 ± 0.4a 40 0 AST + MGO 0.5 mM 13.9 ± 1.1b 22 18 AST + MGO + PBN 0.5 mM 17.9 ± 0.9c 28 12 AST + MGO + PBN 1 mM 18.2 ± 1.1cd 29 11 AST + MGO + PBN 5 mM 18.6 ± 1.7cd 30 10 AST + MGO + PBN 10 mM 19.8 ± 1.2d 31 9 AST + MGO + NtBHA 0.5 mM 17.3 ± 0.4c 27 13 AST + MGO + NtBHA 1 mM 19.5 ± 0.4d 31 9 AST + MGO + NtBHA 5 mM 20.8 ± 0.8de 32 8 AST + MGO + NtBHA 10 mM 21.8 ± 0.7ef 35 5 AST + MGO + AG 1 mM 22.0 ± 0.4f 35 5

Table 1. Effect of PBN, NtBHA and AG on the changes in AST primary amine content

NtBHA had significant inhibitory effect on the middle stage of glycation process.

Native PAGE was run several times, and the representative native PAGE gel is presented in Fig. 17. Mobility of MGO-modified protein to the positive pole significantly increased (by 41%) after 7 days of incubation compared to the mobility of control sample (AST alone). This result indicates the progressive loss of the positive charge in the MGO-modified AST during the glycation reaction. NtBHA showed concentration-dependent protective effect against changes in AST molecular change induced by MGO, when the relative mobility of sample containing AST + MGO + NtBHA 10 mM was increased only by 9.2% compared to the mobility of control. The enzyme incubated in the presence of both MGO and PBN showed a smaller rise in mobility, up to 21.6% in the case of PBN 10 mM. The effect of this compound on the protein electrophoretic mobility ranged from 21.6 to 26.3%. Aminoguanidine 1 mM completely reversed effect of MGO and the relative mobility of sample containing AST + MGO + AG was only slightly increased (by 0.35%) against the mobility of control, whereas Trolox 2.5 mM showed similar effect on molecular charge of AST as PBN, i.e., the mobility was increased by 27% compared to control sample (data not shown). These data indicated that the molecule of enzyme became more anionic due to glycation and that PBN as well as

a,b,c,d,e,f Groups with different letters vary significantly (*P*<0.05, Student's *t*-test)

**3.4 Effect of glycation on molecular charge of AST** 

(nmol NH2/mol AST) remaining modified

(0.5-10 mM).

induced by MGO 0.5 mM

modified by MGO. In the sample containing AST + MGO + PBN 10 mM was found 19.8 ± 1.2 nmol NH2/mol AST (*P*<0.001, using Student's *t*-test), which means that approximately 9 amines were lost. This result is comparable to the effect of NtBHA at 1 mM concentration. Moreover, AG 1 mM exerted the same effect as NtBHA 10 mM. In the sample containing AST + MGO + AG was detected 22.0 ± 0.4 nmol NH2/mol AST suggesting that 5 amines was lost in its presence.

Fig. 16. Formation of argpyrimidine (panel A and B) and fluorescent AGEs (panel C and D). AST (0.5 mg/ml) was incubated with or without methylglyoxal (0.5 mM) in 0.1 M sodium phosphate buffer (pH 7.4) at 37 °C in the presence or absence of PBN (0.5-10 mM), NtBHA (0.5–10 mM), aminoguanidine (1 mM) or Trolox (2.5 mM) for 7 days. Aliquots of samples were taken on days 0 and 7 and stored frozen at -20 °C. Fluorescence of samples was measured at specific excitation and emission wavelengths (λex/λem) corresponding to argpyrimidine (335/385 nm) and AGEs (330/410 nm) versus the unincubated blanks. Data of fluorescence were expressed in relative fluorescence units ± S.D. Every point represents an average of two independent experiments (6 samples). Groups with different letters are significantly different (*P*<0.01, Student's *t*-test).

To determine whether the changes in fluorescence of argpyrimidine observed in the samples containing various concentrations of NtBHA (0.5–10 mM) and MGO 0.5 mM correlated with the loss of primary amino groups in these samples, the fluorescence intensity was plotted as a function of amine content (data not shown). The decrease in fluorescence emission (λex/λem = 320/380 nm) varied directly with the loss of primary amino groups (*r* = 0.963, *P*<0.019). Changes in fluorescence of argpyrimidine in these samples also correlated well

modified by MGO. In the sample containing AST + MGO + PBN 10 mM was found 19.8 ± 1.2 nmol NH2/mol AST (*P*<0.001, using Student's *t*-test), which means that approximately 9 amines were lost. This result is comparable to the effect of NtBHA at 1 mM concentration. Moreover, AG 1 mM exerted the same effect as NtBHA 10 mM. In the sample containing AST + MGO + AG was detected 22.0 ± 0.4 nmol NH2/mol AST suggesting that 5 amines

Fig. 16. Formation of argpyrimidine (panel A and B) and fluorescent AGEs (panel C and D). AST (0.5 mg/ml) was incubated with or without methylglyoxal (0.5 mM) in 0.1 M sodium phosphate buffer (pH 7.4) at 37 °C in the presence or absence of PBN (0.5-10 mM), NtBHA (0.5–10 mM), aminoguanidine (1 mM) or Trolox (2.5 mM) for 7 days. Aliquots of samples were taken on days 0 and 7 and stored frozen at -20 °C. Fluorescence of samples was measured at specific excitation and emission wavelengths (λex/λem) corresponding to argpyrimidine (335/385 nm) and AGEs (330/410 nm) versus the unincubated blanks. Data of fluorescence were expressed in relative fluorescence units ± S.D. Every point represents an average of two independent experiments (6 samples). Groups with different letters are

To determine whether the changes in fluorescence of argpyrimidine observed in the samples containing various concentrations of NtBHA (0.5–10 mM) and MGO 0.5 mM correlated with the loss of primary amino groups in these samples, the fluorescence intensity was plotted as a function of amine content (data not shown). The decrease in fluorescence emission (λex/λem = 320/380 nm) varied directly with the loss of primary amino groups (*r* = 0.963, *P*<0.019). Changes in fluorescence of argpyrimidine in these samples also correlated well

significantly different (*P*<0.01, Student's *t*-test).

was lost in its presence.

with the formation of fluorescent AGEs (r = 0.991, *P*<0.005). In addition, the amine content measured in samples containing AST + MGO + NtBHA (0.5-10 mM) was plotted as a function of AGEs fluorescence, indicating that MGO-induced formation of AGEs was directly proportional to an irreversible loss of primary amino groups in AST molecule (r = 0.946, *P*<0.027). Similar results were obtained also in samples containing AST + MGO + PBN (0.5-10 mM).


a,b,c,d,e,f Groups with different letters vary significantly (*P*<0.05, Student's *t*-test)

Table 1. Effect of PBN, NtBHA and AG on the changes in AST primary amine content induced by MGO 0.5 mM

#### **3.4 Effect of glycation on molecular charge of AST**

Native PAGE was run several times, and the representative native PAGE gel is presented in Fig. 17. Mobility of MGO-modified protein to the positive pole significantly increased (by 41%) after 7 days of incubation compared to the mobility of control sample (AST alone). This result indicates the progressive loss of the positive charge in the MGO-modified AST during the glycation reaction. NtBHA showed concentration-dependent protective effect against changes in AST molecular change induced by MGO, when the relative mobility of sample containing AST + MGO + NtBHA 10 mM was increased only by 9.2% compared to the mobility of control. The enzyme incubated in the presence of both MGO and PBN showed a smaller rise in mobility, up to 21.6% in the case of PBN 10 mM. The effect of this compound on the protein electrophoretic mobility ranged from 21.6 to 26.3%. Aminoguanidine 1 mM completely reversed effect of MGO and the relative mobility of sample containing AST + MGO + AG was only slightly increased (by 0.35%) against the mobility of control, whereas Trolox 2.5 mM showed similar effect on molecular charge of AST as PBN, i.e., the mobility was increased by 27% compared to control sample (data not shown). These data indicated that the molecule of enzyme became more anionic due to glycation and that PBN as well as NtBHA had significant inhibitory effect on the middle stage of glycation process.

Non-Enzymatic Glycation of Aminotransferases and the Possibilities of Its Modulation 107

Western blotting with specific antibody against advanced glycation end products derived from MGO (anti-MGO [3C]) was used to confirm formation of protein aggregates as a result of MGO activity. The presence of high molecular weight protein cross-links in samples containing AST + MGO, AST + MGO + NtBHA, and AST + MGO + PBN was observed (data not shown). These protein aggregates had molecular weight about 85, 107, and 145 kDa corresponding to AST dimer, trimer, and tetramer, respectively. Quantitative differences between bands of samples with and without PBN or Trolox were not observed. These compounds are not able to prevent formation of protein cross-links. On the other hand, some reduction in the amount of AST tetramer was observed in samples containing NtBHA. These results suggest that NtBHA possesses, at least in part, antiglycation properties. Nevertheless, aminoguanidine 1 mM completely inhibited formation of protein aggregates,

The electrophoretic techniques confirmed the results obtained by other methods; i.e., changes in protein molecule caused by the presence of methylglyoxal and positive antiglycating effect of NtBHA. Methylglyoxal-induced chemical modifications led to a change in molecular charge of AST, which became more anionic as revealed by native PAGE. The SDS-PAGE and subsequent western blotting clearly showed formation of protein cross-links with higher molecular weight than native enzyme. NtBHA partially protected native AST from glycation

Fig. 18. Formation of protein cross-links on reaction of AST with methylglyoxal. AST (0.5 mg/ml) was incubated with or without methylglyoxal (0.5 mM) in sodium phosphate buffer (0.1 M, pH 7.4) at 37 °C in the presence or absence of *N*-*tert*-butyl hydroxylamine (0.5–10 mM) for 7 days and then subjected to SDS-PAGE. Electrophoretic separation was performed on 4% stacking and 10% resolving polyacrylamide gels under reducing conditions. Bands were visualized with silver staining. Each lane was loaded with 4 µg of protein. MM = Mw marker; AST = aspartate aminotransferase; MGO = methylglyoxal;

since no bands of AST dimer, trimer or tetramer were present.

by MGO and also exhibited mild anti-cross-linking activity.

NtBHA = *N*-*tert*-butyl hydroxylamine

Fig. 17. Protective effect of NtBHA and PBN on changes in molecular change of AST caused by MGO-induced glycation. AST (0.5 mg/ml) was incubated with or without MGO (0.5 mM) and NtBHA (0.5-10 mM) or PBN (0.5-10 mM) in sodium phosphate buffer (0.1 M, pH 7.4) at 37 °C for 7 days and then subjected to native PAGE. Proteins were visualized by Coomassie Blue G250. Gels were scanned and Rf was obtained using Quantity One software

Methylglyoxal-induced chemical modifications led to a change in molecular charge of AST, which became more anionic as revealed by native PAGE. These results indicate the progressive loss of the positive charge in the glycation-modified AST molecule, which is caused by the irreversible modification of Arg and Lys residues (Kang, 2006; Nagai et al., 2000) as was confirmed by determination of amine content. Both PBN and NtBHA partially protected native AST against glycation by MGO. The antiglycation activity was more pronounced in the case of NtBHA mainly at higher concentrations tested. The antiglycation activity of NtBHA 10 mM was a little bit lower than that of AG 1 mM.

#### **3.5 SDS-PAGE and western blotting**

The ability of aggregation and cross-link formation of tested antioxidants was determined by SDS-PAGE under denaturing conditions (Fig. 18). MGO readily reacts with lysine and arginine residues to produce high molecular weight protein products. Incubation of AST with MGO 0.5 mM at 37 °C for 7 days resulted in the formation of protein aggregates with molecular weight about 85, 107, and 145 kDa corresponding to protein dimer, trimer, and tetramer, respectively. No presence of protein dimer and tetramer, and lower concentration of protein trimer were observed in samples containing AST alone (lane 2), AST + NtBHA 10 mM (lane 7), and AST + AG 1 mM (data not shown). Also lower concentrations of NtBHA were able to partially protect formation of protein tetramer, although they had no effect on formation of protein dimer and trimer. On the other hand, PBN as well as Trolox were not able to prevent formation of protein cross-links and high molecular weight aggregates. Additional bands with molecular weight 20–35, 57 and 63 kDa were constituted of several contaminating proteins present in commercial preparation (Fig. 18).

Fig. 17. Protective effect of NtBHA and PBN on changes in molecular change of AST caused by MGO-induced glycation. AST (0.5 mg/ml) was incubated with or without MGO (0.5 mM) and NtBHA (0.5-10 mM) or PBN (0.5-10 mM) in sodium phosphate buffer (0.1 M, pH 7.4) at 37 °C for 7 days and then subjected to native PAGE. Proteins were visualized by Coomassie Blue G250. Gels were scanned and Rf was obtained using Quantity One software

Methylglyoxal-induced chemical modifications led to a change in molecular charge of AST, which became more anionic as revealed by native PAGE. These results indicate the progressive loss of the positive charge in the glycation-modified AST molecule, which is caused by the irreversible modification of Arg and Lys residues (Kang, 2006; Nagai et al., 2000) as was confirmed by determination of amine content. Both PBN and NtBHA partially protected native AST against glycation by MGO. The antiglycation activity was more pronounced in the case of NtBHA mainly at higher concentrations tested. The antiglycation

The ability of aggregation and cross-link formation of tested antioxidants was determined by SDS-PAGE under denaturing conditions (Fig. 18). MGO readily reacts with lysine and arginine residues to produce high molecular weight protein products. Incubation of AST with MGO 0.5 mM at 37 °C for 7 days resulted in the formation of protein aggregates with molecular weight about 85, 107, and 145 kDa corresponding to protein dimer, trimer, and tetramer, respectively. No presence of protein dimer and tetramer, and lower concentration of protein trimer were observed in samples containing AST alone (lane 2), AST + NtBHA 10 mM (lane 7), and AST + AG 1 mM (data not shown). Also lower concentrations of NtBHA were able to partially protect formation of protein tetramer, although they had no effect on formation of protein dimer and trimer. On the other hand, PBN as well as Trolox were not able to prevent formation of protein cross-links and high molecular weight aggregates. Additional bands with molecular weight 20–35, 57 and 63 kDa were constituted of several

activity of NtBHA 10 mM was a little bit lower than that of AG 1 mM.

contaminating proteins present in commercial preparation (Fig. 18).

**3.5 SDS-PAGE and western blotting** 

Western blotting with specific antibody against advanced glycation end products derived from MGO (anti-MGO [3C]) was used to confirm formation of protein aggregates as a result of MGO activity. The presence of high molecular weight protein cross-links in samples containing AST + MGO, AST + MGO + NtBHA, and AST + MGO + PBN was observed (data not shown). These protein aggregates had molecular weight about 85, 107, and 145 kDa corresponding to AST dimer, trimer, and tetramer, respectively. Quantitative differences between bands of samples with and without PBN or Trolox were not observed. These compounds are not able to prevent formation of protein cross-links. On the other hand, some reduction in the amount of AST tetramer was observed in samples containing NtBHA. These results suggest that NtBHA possesses, at least in part, antiglycation properties. Nevertheless, aminoguanidine 1 mM completely inhibited formation of protein aggregates, since no bands of AST dimer, trimer or tetramer were present.

The electrophoretic techniques confirmed the results obtained by other methods; i.e., changes in protein molecule caused by the presence of methylglyoxal and positive antiglycating effect of NtBHA. Methylglyoxal-induced chemical modifications led to a change in molecular charge of AST, which became more anionic as revealed by native PAGE. The SDS-PAGE and subsequent western blotting clearly showed formation of protein cross-links with higher molecular weight than native enzyme. NtBHA partially protected native AST from glycation by MGO and also exhibited mild anti-cross-linking activity.

Fig. 18. Formation of protein cross-links on reaction of AST with methylglyoxal. AST (0.5 mg/ml) was incubated with or without methylglyoxal (0.5 mM) in sodium phosphate buffer (0.1 M, pH 7.4) at 37 °C in the presence or absence of *N*-*tert*-butyl hydroxylamine (0.5–10 mM) for 7 days and then subjected to SDS-PAGE. Electrophoretic separation was performed on 4% stacking and 10% resolving polyacrylamide gels under reducing conditions. Bands were visualized with silver staining. Each lane was loaded with 4 µg of protein. MM = Mw marker; AST = aspartate aminotransferase; MGO = methylglyoxal; NtBHA = *N*-*tert*-butyl hydroxylamine

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**6. References** 

6638

Modification of proteins caused by methylglyoxal can be accompanied by formation of free radicals. Lee et al. (1998) identified three types of free radical species in samples containing methylglyoxal and bovine serum albumin by electron spin resonance spectroscopy. These radicals (methylglyoxal dialkylimine radical cation, methylglyoxal radical anion, and superoxide anion radical) were formed by direct 1-electron transfer process. Scavenging ability of NtBHA and PBN were already described (Lee et al., 2004; Atamna et al., 2001). It can be assumed that the positive antiglycation activity of these compounds may be at least partly attributed to their scavenging ability.

#### **4. Conclusion**



#### **5. Acknowledgment**

This study was supported by the Charles University in Prague (Project SVV 263 004).

#### **6. References**

108 Enzyme Inhibition and Bioapplications

Modification of proteins caused by methylglyoxal can be accompanied by formation of free radicals. Lee et al. (1998) identified three types of free radical species in samples containing methylglyoxal and bovine serum albumin by electron spin resonance spectroscopy. These radicals (methylglyoxal dialkylimine radical cation, methylglyoxal radical anion, and superoxide anion radical) were formed by direct 1-electron transfer process. Scavenging ability of NtBHA and PBN were already described (Lee et al., 2004; Atamna et al., 2001). It can be assumed that the positive antiglycation activity of these compounds may be at least








partly attributed to their scavenging ability.

compounds (e.g., MGO).

and aggregation.

antiglycation activity.

protein, which possesses catalytic properties.

duration of action (see Fig. 4, Fig. 5 and Fig. 8).

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**5** 

*India* 

Rakesh Sharma1,2

**Inhibition of Nitric Oxide Synthase Gene** 

**Nitric Oxide with Multimodal Imaging** 

*2Amity Institute of Nanotechnology, Amity University, NOIDA, U.P.* 

**Expression:** *In vivo* **Imaging Approaches of** 

*1Center of Nanomagnetics and Biotechnology, Florida State University, Tallahassee, FL* 

Nitric oxide is an uncharged free radical abundant in nanomolar quantities and detected by measuring nitric oxide synthase (NOS). Nitric oxide is a gas highly reactive, short lived free radical generated enzymatically by NOS involved in diverse physiological (neurotransmission, immune system) and pathophysiological (tumor progression) mechanisms. Nitric oxide is a biological mediator for its role as EDRF (endothelial-derived relaxing factor) responsible for the regulation of blood vessel relaxation and blood pressure maintenance [Labet et al. 2009]. In recent years, inhibition of NOS gene expression has become a scientific interest to measure NO in tissues and synthetic NOS/NO inhibitors have revolutionized molecular imaging of tissues. Present chapter presents a journey from major mechanistic concepts to the detection of NO,

 Several genes involve in NOS enzyme for its synthase activity (NOS1, NOS2A, NOS3); oxidoreductase activity (NOS1, NOS2A, NOS3, NQO1); as positive regulators(HSP90AB1 or HSPCB, INS); as negative regulators (DNCL1, GLA, IL10);other AKT1, ARG2, DDAH2,

 NO diffuses freely across cell membranes. It is short lived but combines with metallic aromatic 'spin traps' to make stable compounds. NO acts in a paracrine or autocrine

NO is synthesized within cells by a flavocytochrome enzyme NO synthase (NOS). The

 nNOS (NOS-1) is found in neurons (hence the "n"); iNOS (inducible NOS-2), triggered by inflammatory cytokines found in macrophages; and eNOS (NOS-3): constitutively distributed in the vascular endothelium lining the lumen of blood vessels, lung, and platelets (called constitutive NOS or *c*NOS) [Perrier, et al. 2009]. All types of NOS produce NO from arginine with the aid of molecular oxygen and NADPH as shown in following redox reaction and Figure 1. Inhibition of NOS gene expression in cells may

 NO is generated in vascular endothelium cells (NO plays role in the regulation of vascular tone), peripheral and central neurons (NO acts as synaptic neuronal

NOS and their multimodal bioimaging applications with limitations.

manner influencing only cells near its point of synthesis.

DNCL1, EGFR, GCH1, GCHFR genes

human contains 3 different NO synthases:

detect NO [Nie et al.2008; Terashima et al. 2010].

NADH-diphorase stain the NOS expression to detect NO in tissues

**1. Introduction** 


## **Inhibition of Nitric Oxide Synthase Gene Expression:** *In vivo* **Imaging Approaches of Nitric Oxide with Multimodal Imaging**

Rakesh Sharma1,2

*1Center of Nanomagnetics and Biotechnology, Florida State University, Tallahassee, FL 2Amity Institute of Nanotechnology, Amity University, NOIDA, U.P. India* 

#### **1. Introduction**

114 Enzyme Inhibition and Bioapplications

Yan, H. & Harding, J.J. (2006). Carnosine inhibits modifications and decreased molecular

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Nitric oxide is an uncharged free radical abundant in nanomolar quantities and detected by measuring nitric oxide synthase (NOS). Nitric oxide is a gas highly reactive, short lived free radical generated enzymatically by NOS involved in diverse physiological (neurotransmission, immune system) and pathophysiological (tumor progression) mechanisms. Nitric oxide is a biological mediator for its role as EDRF (endothelial-derived relaxing factor) responsible for the regulation of blood vessel relaxation and blood pressure maintenance [Labet et al. 2009]. In recent years, inhibition of NOS gene expression has become a scientific interest to measure NO in tissues and synthetic NOS/NO inhibitors have revolutionized molecular imaging of tissues. Present chapter presents a journey from major mechanistic concepts to the detection of NO, NOS and their multimodal bioimaging applications with limitations.


Inhibition of Nitric Oxide Synthase Gene Expression:

map cellular events.

**1.1 Nitric oxide as a second messenger in cellular signaling** 

3. Colocalization of target proteins with a source of NO;

4. Reversibility of protein S-nitrosylation;

reductase (Janssen-Heininger et al., 2008).

*In vivo* Imaging Approaches of Nitric Oxide with Multimodal Imaging 117

Nitric oxide signal transduction through induced-nitric-oxide modifications relies on the system of Cys-based posttranslational modifications. Accordingly, S-nitrosylation of proteins plays an essential role in downstream cascades (Do et al., 1996). Nitric oxide exerts an ubiquitous influence on cellular signaling in large part by means of Snitrosylation/denitrosylation of protein cysteine residues. S-NO undergo a regulated posttranslational protein modification specific to NO-derived effects. These NO-dependent modifications influence protein activity, protein-protein interactions, and protein location. Snitrosylation thus serves as the prototypical redox-based signal (Janssen-Heininger et al., 2008). S-Nitrosylation has been implicated in transmitting signals downstream of all classes of receptors, including G-protein-coupled receptor (GPCR), receptor tyrosine kinase, tumor necrosis factor, Toll-receptors, and glutaminergic receptors, acting locally within subcellular signaling domains as well conveying signals from the cell surface to intracellular compartments, including the mitochondria and the nucleus (Janssen-Heininger et al., 2008).

Cell signaling through S-nitrosylation is useful tool in signaling transduction for: 1. Temporal regulation of response through a rapid and controlled stimulation; 2. The existence of motifs within proteins that provides S-nitrosylation specificity;

**1.2 Why inhibition of NOS expression as bioimaging of NO technique** 

5. Enzymatic control of S-nitrosylation through the action of S-nitrosoglutathione

Mapping the distribution of NO generation at different locations in different tissues and organs by *in vivo* perfusion reveals the physiological function of NO (gas or ionic form) in different tissue physiological conditions [Thatte, et al.2009]. Major nitric oxide imaging techniques utilize mapping NO in tissue using NO specific imaging contrast agents sensitive to fluorescence, magnetic resonance and electron spin resonance. Imaging *in vivo* physical properties of tissue cells such as cell calcium signaling and NO-biomarker is new way by proton magnetic resonance techniques to achieve nanomolar range of nitric oxide mapping

**structural-functional or conformational dynamics as switch OFF and ON**) may allow us to understand the real physiological function of NO gas or ionic form under different tissue physiological conditions [Thatte et al. 2009]. Using *in vivo* nitric oxide direct detection by stabilizing NO with suitable spin-trapping reagents is a challenge to estimate the *in vivo* NO concentration by MRI techniques [Hong et al. 2009; Sari-Sarraf, et al. 2009; Liu, et al. 2009; Ny et al. 2008; Fujii, et al, 2007; Vandsburger, et al.2007; Samouilov et al. 2007; Flögel, et al. 2007; Bobko, et al. 2005; Day, et al. 2005; Waller, et al. 2005; Sirmatel, et al. 2007; Itoh, et al. 2004; Berliner, et al. 2004; Haga,et al. 2003; Li, et al. 2003; Hsiao, et al. 2008; Kuppusamy, et al. 2001; Fichtlscherer, et al. 2000; Fujii,et al.1999; Fujii, et al. 2002]. Other nitric oxide bioimaging technique is flourimetry using fluorescent biomarkers in EPR spectroscopy [Kojima, et al.2001; Fuji, et al.1997; Yoshimura, et al. 1996]. Our immediate focus is to highlight the existing multimodal mechanisms of NO sensitive MRI/EPR signal generation using NOS enzyme expression inhibitors and biosensors to

messenger) by three isoforms of NO synthase, endothelial, neuronal, and inducible form [Perrier, et al. 2009, Claudette, et al. 2005]. Detection and in vivo monitoring of NO is very difficult. Spin traps, fluorescent dyes, chemi/bioluminescent sensors detect and image NO by ESR,NMR,PET,US,and optical methods.

Fig. 1. Redox potentials and direction of electron flow in nNOS enzyme action are shown. The electron flow in the NOS dimer goes via NADPHFADFMN in the reductase of one monomer to the haem iron in the oxygenase domain of a separate monomer. The redox potentials are poised thermodynamically to make this occur. The potentials for the twoelectron oxidation of NADPH and the one-electron oxidations of FADH2, FMNH2 and ferric haem are illustrated at the bottom of the Scheme, with the red arrows indicating the direction of electron flow. Note that given the variety of redox couples and the closeness of the FADH2 and FMNH2 potentials the detailed picture is more complex than that illustrated. For example the FMNH+/FMN couple has a redox potential of -49 mV and is likely to donate electrons to the high-potential ferric superoxide species to form the ferryl intermediate. Reduction of the FMN back to FMNH+ would then require an electron from FMNH+. Adapted from Alderton, et al. 2001.

 Mapping the distribution of NO generation or formation of stable spin-trapping species at different locations in tissues or organs by *in vivo* perfusion (**3D visualization of NO** 

Fig. 1. Redox potentials and direction of electron flow in nNOS enzyme action are shown. The electron flow in the NOS dimer goes via NADPHFADFMN in the reductase of one monomer to the haem iron in the oxygenase domain of a separate monomer. The redox potentials are poised thermodynamically to make this occur. The potentials for the twoelectron oxidation of NADPH and the one-electron oxidations of FADH2, FMNH2 and ferric

intermediate. Reduction of the FMN back to FMNH+ would then require an electron from

 Mapping the distribution of NO generation or formation of stable spin-trapping species at different locations in tissues or organs by *in vivo* perfusion (**3D visualization of NO** 

haem are illustrated at the bottom of the Scheme, with the red arrows indicating the direction of electron flow. Note that given the variety of redox couples and the closeness of the FADH2 and FMNH2 potentials the detailed picture is more complex than that illustrated. For example the FMNH+/FMN couple has a redox potential of -49 mV and is likely to donate electrons to the high-potential ferric superoxide species to form the ferryl

FMNH+. Adapted from Alderton, et al. 2001.

image NO by ESR,NMR,PET,US,and optical methods.

messenger) by three isoforms of NO synthase, endothelial, neuronal, and inducible form [Perrier, et al. 2009, Claudette, et al. 2005]. Detection and in vivo monitoring of NO is very difficult. Spin traps, fluorescent dyes, chemi/bioluminescent sensors detect and **structural-functional or conformational dynamics as switch OFF and ON**) may allow us to understand the real physiological function of NO gas or ionic form under different tissue physiological conditions [Thatte et al. 2009]. Using *in vivo* nitric oxide direct detection by stabilizing NO with suitable spin-trapping reagents is a challenge to estimate the *in vivo* NO concentration by MRI techniques [Hong et al. 2009; Sari-Sarraf, et al. 2009; Liu, et al. 2009; Ny et al. 2008; Fujii, et al, 2007; Vandsburger, et al.2007; Samouilov et al. 2007; Flögel, et al. 2007; Bobko, et al. 2005; Day, et al. 2005; Waller, et al. 2005; Sirmatel, et al. 2007; Itoh, et al. 2004; Berliner, et al. 2004; Haga,et al. 2003; Li, et al. 2003; Hsiao, et al. 2008; Kuppusamy, et al. 2001; Fichtlscherer, et al. 2000; Fujii,et al.1999; Fujii, et al. 2002].

 Other nitric oxide bioimaging technique is flourimetry using fluorescent biomarkers in EPR spectroscopy [Kojima, et al.2001; Fuji, et al.1997; Yoshimura, et al. 1996]. Our immediate focus is to highlight the existing multimodal mechanisms of NO sensitive MRI/EPR signal generation using NOS enzyme expression inhibitors and biosensors to map cellular events.

#### **1.1 Nitric oxide as a second messenger in cellular signaling**

Nitric oxide signal transduction through induced-nitric-oxide modifications relies on the system of Cys-based posttranslational modifications. Accordingly, S-nitrosylation of proteins plays an essential role in downstream cascades (Do et al., 1996). Nitric oxide exerts an ubiquitous influence on cellular signaling in large part by means of Snitrosylation/denitrosylation of protein cysteine residues. S-NO undergo a regulated posttranslational protein modification specific to NO-derived effects. These NO-dependent modifications influence protein activity, protein-protein interactions, and protein location. Snitrosylation thus serves as the prototypical redox-based signal (Janssen-Heininger et al., 2008). S-Nitrosylation has been implicated in transmitting signals downstream of all classes of receptors, including G-protein-coupled receptor (GPCR), receptor tyrosine kinase, tumor necrosis factor, Toll-receptors, and glutaminergic receptors, acting locally within subcellular signaling domains as well conveying signals from the cell surface to intracellular compartments, including the mitochondria and the nucleus (Janssen-Heininger et al., 2008). Cell signaling through S-nitrosylation is useful tool in signaling transduction for:


#### **1.2 Why inhibition of NOS expression as bioimaging of NO technique**

Mapping the distribution of NO generation at different locations in different tissues and organs by *in vivo* perfusion reveals the physiological function of NO (gas or ionic form) in different tissue physiological conditions [Thatte, et al.2009]. Major nitric oxide imaging techniques utilize mapping NO in tissue using NO specific imaging contrast agents sensitive to fluorescence, magnetic resonance and electron spin resonance. Imaging *in vivo* physical properties of tissue cells such as cell calcium signaling and NO-biomarker is new way by proton magnetic resonance techniques to achieve nanomolar range of nitric oxide mapping

Inhibition of Nitric Oxide Synthase Gene Expression:

Some examples of NOS ihibitors are given below.

50-fold selectivity are useful as `partially selective inhibitors.

tissues.

inhibitor

**1.2.2 NOS Inhibitors** 

*In vivo* Imaging Approaches of Nitric Oxide with Multimodal Imaging 119

myristoylation, palmitoylation, caveolin, at different domain located in NOS or spilce variants: nNOSβ and nNOSγ, nNOSµ, nNOS-2, iNOS/eNOS splice variants. However, cross-activities of all three isoforms of NOS and their dependence on calcium, common locations have put challenge to specificity and gave way to a new way of specific NO/NOS inhibitor compounds. In recent years, NOS gene expression and its regulation have got attention for two reasons:

NOS inhibition occurs at arginine site, tetrahydrobiopterin site, pteridine site, and heme domain by partially selective or highly selective inhibitors [Alderton, et al. 2001, Matter et a. 2004]. Excellent information of binding site interactions of NOS with relationships at different sites is available [Alderton, et al. 2001, Matter et al. 2004]. To understand better the NOS inhibitor structural-activity relationship, X-Ray analysis, 3D QUSAR, comparative molecular field analysis (CoMFA) analysis, GRID/PCA interpretations have enhanced the scope of NOS in pharmacology as shown in Figures 3,4,5 [Matter et al.2004]. There are array of NOS inhibitors described in the literature as drug testing tools. Table 1 shows efficacy of some of these in inhibiting the three human NOS isoforms.. Of these the most widely used have been l-NMMA, l-NNA and its methyl ester prodrug (NG-nitro-l-arginine methyl ester, `l-NAME' and aminoguanidine. However, inhibitors show pitfalls on selectivity to NOS, types of interactions with iNOS and nNOS isoforms. **Selective** NOS inhibitors may be selective in the physiological range (l-arginine concentration etc). Inhibitor agents with 10-

iNOS nNOS eNOS iNOS vs

L-NNA\* 3.1 0.29 0.35 0.09 0.11 1.2 L-NMMA 6.6 4.9 3.5 0.7 0.5 0.7 7-NI\* 9.7 8.3 11.8 0.9 1.2 1.4 ARL17477\* 0.33 0.07 1.6 0.2 5 23. Aminoguanidine\* 31 170 330 5.5 11 1.9 L-NIL 1.6 37 49 23. 49. 1.3 1400W 0.23. 7.3 1000 32. 4000\*. 130\* GW273629 8.0. 630 1000 78. 125\*. 1.6\* GW274150 1.4. 145 466 104. 333 3.2 The data shown are for inhibition of the human NOS isoforms in the presence of 30 µM L-arginine at 37 °C over 15 min after a 15 min pre-incubation with inhibitor under turnover. The data serves as progressive inhibitory mechanisms for the NOS assay. Data are from Young et al. 2000. Table 1. Selectivity of inhibitors of NOSs is compared for different inhibitors

IC50 (µM) Selectivity (fold)

nNOS

iNOS vs eNOS

nNOS vs eNOS

 NOS gene expression can be stained, imaged for multimodal molecular imaging The NOS inhibitors inhibit NOS as potent anti-inflammatory agents in recent years. Visualization of NOS inhibition is believed to serve as in vivo or in vitro biomarkers of the proinflammatory status of NOS inhibitors or in vivo proinflammation bioimaging of

without any toxic effects[Thatte, et al.2009, Hong et al.2009]. Fluorescent nitric oxide cheletropic traps are currently available choices in nitric oxide imaging but all of these have pitfalls of causing neurotoxicity [Reif et al.2009]. In this chapter, we display evidence of the NO sensitive fluorescent probes as cell calcium signaling indicator and possibility of NO specific perfusion MRI tool to visualize physiological nanomolar dynamics of NO in living cells and tissues up to the detection limit of 0.1 nM. The cell signaling indicators such as intracellular calcium revealed that ~1 nM of NO was enough to detect apoptosis events such as caspase 3 activation [Li et al.2009].

Fig. 2. Domain structure of human nNOS, eNOS and iNOS(on left), Overall reaction catalysed and cofactors of NOS(on right). Adapted from Alderton, et al. 2001.

#### **1.2.1 Feasibility of NOS expression inhibitors as imaging Contrast Agents**

NO is a gaseous and highly active neuronal messenger, short lived free radical generated enzymatically by NOS in the brain[ Perrier et al.2009]. NOS is a flavocytochrome that is constitutively distributed in the vascular endothelium, brain, lung, and platelets (called constitutive NOS or *c*NOS), but is also found as an inducible form (called inducible NOS or *i*NOS) in many cells and organs, triggered by several factors such as inflammatory cytokines [Claudette et al.2009]. However, NOS is abundant in three isoforms cNOS, iNOS, eNOS as shown in Figure 2. NOS undergo dimerization in presence of BH4 or heme or arginine binding sites. Different roles of flabin, heme, pterin cofactors in NOS are described in detail [Alderton, et al. 2001, Matter et a. 2004]. Authors reviewed NOS activity regulation by calmodulin, phosphorylation, protein inhibitors of NOS(PIN), heat shock protein 90(Hsp 90), myristoylation, palmitoylation, caveolin, at different domain located in NOS or spilce variants: nNOSβ and nNOSγ, nNOSµ, nNOS-2, iNOS/eNOS splice variants. However, cross-activities of all three isoforms of NOS and their dependence on calcium, common locations have put challenge to specificity and gave way to a new way of specific NO/NOS inhibitor compounds. In recent years, NOS gene expression and its regulation have got attention for two reasons:


Some examples of NOS ihibitors are given below.

#### **1.2.2 NOS Inhibitors**

118 Enzyme Inhibition and Bioapplications

without any toxic effects[Thatte, et al.2009, Hong et al.2009]. Fluorescent nitric oxide cheletropic traps are currently available choices in nitric oxide imaging but all of these have pitfalls of causing neurotoxicity [Reif et al.2009]. In this chapter, we display evidence of the NO sensitive fluorescent probes as cell calcium signaling indicator and possibility of NO specific perfusion MRI tool to visualize physiological nanomolar dynamics of NO in living cells and tissues up to the detection limit of 0.1 nM. The cell signaling indicators such as intracellular calcium revealed that ~1 nM of NO was enough to detect apoptosis events such

Fig. 2. Domain structure of human nNOS, eNOS and iNOS(on left), Overall reaction catalysed and cofactors of NOS(on right). Adapted from Alderton, et al. 2001.

NO is a gaseous and highly active neuronal messenger, short lived free radical generated enzymatically by NOS in the brain[ Perrier et al.2009]. NOS is a flavocytochrome that is constitutively distributed in the vascular endothelium, brain, lung, and platelets (called constitutive NOS or *c*NOS), but is also found as an inducible form (called inducible NOS or *i*NOS) in many cells and organs, triggered by several factors such as inflammatory cytokines [Claudette et al.2009]. However, NOS is abundant in three isoforms cNOS, iNOS, eNOS as shown in Figure 2. NOS undergo dimerization in presence of BH4 or heme or arginine binding sites. Different roles of flabin, heme, pterin cofactors in NOS are described in detail [Alderton, et al. 2001, Matter et a. 2004]. Authors reviewed NOS activity regulation by calmodulin, phosphorylation, protein inhibitors of NOS(PIN), heat shock protein 90(Hsp 90),

**1.2.1 Feasibility of NOS expression inhibitors as imaging Contrast Agents** 

as caspase 3 activation [Li et al.2009].

NOS inhibition occurs at arginine site, tetrahydrobiopterin site, pteridine site, and heme domain by partially selective or highly selective inhibitors [Alderton, et al. 2001, Matter et a. 2004]. Excellent information of binding site interactions of NOS with relationships at different sites is available [Alderton, et al. 2001, Matter et al. 2004]. To understand better the NOS inhibitor structural-activity relationship, X-Ray analysis, 3D QUSAR, comparative molecular field analysis (CoMFA) analysis, GRID/PCA interpretations have enhanced the scope of NOS in pharmacology as shown in Figures 3,4,5 [Matter et al.2004]. There are array of NOS inhibitors described in the literature as drug testing tools. Table 1 shows efficacy of some of these in inhibiting the three human NOS isoforms.. Of these the most widely used have been l-NMMA, l-NNA and its methyl ester prodrug (NG-nitro-l-arginine methyl ester, `l-NAME' and aminoguanidine. However, inhibitors show pitfalls on selectivity to NOS, types of interactions with iNOS and nNOS isoforms. **Selective** NOS inhibitors may be selective in the physiological range (l-arginine concentration etc). Inhibitor agents with 10- 50-fold selectivity are useful as `partially selective inhibitors.


The data shown are for inhibition of the human NOS isoforms in the presence of 30 µM L-arginine at 37 °C over 15 min after a 15 min pre-incubation with inhibitor under turnover. The data serves as progressive inhibitory mechanisms for the NOS assay. Data are from Young et al. 2000.

Table 1. Selectivity of inhibitors of NOSs is compared for different inhibitors

Inhibition of Nitric Oxide Synthase Gene Expression:

Bryk and Wolff, 1999.

thiocitrulline) [Raman, et al.1998].

inhibition.

**1.2.3 NOS inhibitor interactions with the NOS enzymes** 

*In vivo* Imaging Approaches of Nitric Oxide with Multimodal Imaging 121

The mechanistic nature of three human NOS isoforms was expressed in the baculovirus expression system, and cell lysates as the enzyme source. iNOS potency and selectivity underestimates progressive inhibition of iNOS but not eNOS or nNOS; e.g. for 1400W the steady-state values of iNOS IC50 and selectivity have been estimated to be 0.1 µM, 250 fold (versus nNOS) and 5000-fold (versus eNOS) as reported by Young, et al. 2000. Highly selective compounds of over 50- or 100-fold selectivity, inhibit the NOS activity of a single isoform without affecting others. They have potential as selective therapeutic agents without side effects. Recently, 7-nitroindazole (7-NI) was reported as non selective nNOS inhibitor, of isolated NOS enzyme (Table 1). 7-NI showed inhibition of nNOS independent of increases in blood pressure but showed eNOS-dependent celltype specificity (neuronal verus endothelial), intracellular BH4 concentration, or depending on specific cellular transport or metabolism [Handy, et al. 1998]. All three NOS isoforms can be expressed in neurons and both eNOS and iNOS in endothelial cells. Cell-type specificity is clearly a very distinct phenomenon from isoform selectivity. Other pitfall is particular dose. For example, in humans, the non-selective NOS inhibitor l-NMMA causes a five-fold increase in vascular resistance with only a 10% change in blood pressure, because of reflex decreases in cardiac output 185±187 [Ross et al. 1998] Suppression of inhibitor-induced plasma nitrate (mediated predominantly by iNOS) and no effects on blood pressure has led to inhibitors as non selective, e.g. *S*-ethylisothiourea [Raman, et al. 1998]. Selectivity for iNOS versus eNOS distinct enzyme targets is another pitfall. Time-dependent NOS inhibition in assessing efficacy and selectivity of NOS inhibitors were first reviewed by

Inhibitors of NOS have been described which interact with the NOS enzymes in a variety of ways: different sites, differing time- and substrate-dependence, and mechanism of

**L-Arginine site inhibitors** identified so far as competitive with the substrate L-arginine binding at the arginine-binding site inhibitors (aminoguanidine, *S*-ethylisothiourea,

Mechanism-based inhibitors of iNOS require active enzyme and NADPH substrate to permit inhibition to proceed through multiple enzyme covalent modification by a complex formation pathway: EI-EI\* complex where E is iNOS, I is the inhibitor, EI is the initial non-covalent complex and EI\* is a modified complex, either with a conformational change to tight binding or with covalent changes to the enzyme, inhibitor or both. The interactions of some of these pterin-site inhibitors with NOS reveal unexpected complexity. An example of a mechanism based iNOS inhibition was cited by aminoguanidine [197±199] and by the acetamidine inhibitors N-α-iminoethyl-l-ornithine (l-NIO) and *N*'-iminoethyl-lysine (l-NIL) [200±203], GW273629 (*S*-[2-[(1-iminoethyl) amino]ethyl]-4,4-dioxo-l-cysteine) and GW274150 (*S*-[2-[(1-iminoethyl)amino]ethyl]-lhomocysteine) (see Table 1). **Heme-binding inhibitors** have been shown to bind with each NOS monomer, one to the haem iron and one to the arginine-binding region (Glu ) in competition with CaM. These compounds affect the assembly of iNOS monomers into active dimer inhibiting the dimerization [Sennequier, et al. 1999]. A class of substituted

Fig. 3. Contour maps for NOS-I comparative molecular field analysis (CoMFA) analysis with a 4-amino-pteridine inhibitor.A: Steric contour map, green contours indicate sterically favored regions, yellow contours indicate unfavored areas. B: Samethan, A: with NOS-I binding site. C: Electrostatic contour map, blue contours refer to regions, where negatively charged substituents are unfavorable, red contours indicate regions, where negatively charged substituents are favorable. D: Same as C with NOS-I binding site. Reproduced with permission from Matter et al. 2004.

Fig. 4. A: Score plot from GRID/principal componentanalysis (PCA) based on13 conformations and 3NOS isoforms. B: SuperpositionofNOS-III (1nse, blue) toNOS-IX-ray (magenta) andhumanbrainNOS-I (homology, purple).C:Scoreplot fromGRID/CPCA for hydrophobic interactions (GRID dry probe). D:GRID/CPCA differential plots highlighting differences between NOS-I/NOS-II and NOS-I/NOS-III.Favorable interactions toachieveNOS-I isoformselectivities are shownincyancontours, unfavorable interactions are displayed in yellow with respect to H4Bipfromthe NOS-III1nse X-ray structure. Reproduced with permission from reference [Matter et al. 2004]

Fig. 3. Contour maps for NOS-I comparative molecular field analysis (CoMFA) analysis with a 4-amino-pteridine inhibitor.A: Steric contour map, green contours indicate sterically favored regions, yellow contours indicate unfavored areas. B: Samethan, A: with NOS-I binding site. C: Electrostatic contour map, blue contours refer to regions, where negatively charged substituents are unfavorable, red contours indicate regions, where negatively charged substituents are favorable. D: Same as C with NOS-I binding site. Reproduced with

Fig. 4. A: Score plot from GRID/principal componentanalysis (PCA) based on13

differences between NOS-I/NOS-II and NOS-I/NOS-III.Favorable interactions

with permission from reference [Matter et al. 2004]

conformations and 3NOS isoforms. B: SuperpositionofNOS-III (1nse, blue) toNOS-IX-ray (magenta) andhumanbrainNOS-I (homology, purple).C:Scoreplot fromGRID/CPCA for hydrophobic interactions (GRID dry probe). D:GRID/CPCA differential plots highlighting

toachieveNOS-I isoformselectivities are shownincyancontours, unfavorable interactions are displayed in yellow with respect to H4Bipfromthe NOS-III1nse X-ray structure. Reproduced

permission from Matter et al. 2004.

The mechanistic nature of three human NOS isoforms was expressed in the baculovirus expression system, and cell lysates as the enzyme source. iNOS potency and selectivity underestimates progressive inhibition of iNOS but not eNOS or nNOS; e.g. for 1400W the steady-state values of iNOS IC50 and selectivity have been estimated to be 0.1 µM, 250 fold (versus nNOS) and 5000-fold (versus eNOS) as reported by Young, et al. 2000. Highly selective compounds of over 50- or 100-fold selectivity, inhibit the NOS activity of a single isoform without affecting others. They have potential as selective therapeutic agents without side effects. Recently, 7-nitroindazole (7-NI) was reported as non selective nNOS inhibitor, of isolated NOS enzyme (Table 1). 7-NI showed inhibition of nNOS independent of increases in blood pressure but showed eNOS-dependent celltype specificity (neuronal verus endothelial), intracellular BH4 concentration, or depending on specific cellular transport or metabolism [Handy, et al. 1998]. All three NOS isoforms can be expressed in neurons and both eNOS and iNOS in endothelial cells. Cell-type specificity is clearly a very distinct phenomenon from isoform selectivity. Other pitfall is particular dose. For example, in humans, the non-selective NOS inhibitor l-NMMA causes a five-fold increase in vascular resistance with only a 10% change in blood pressure, because of reflex decreases in cardiac output 185±187 [Ross et al. 1998] Suppression of inhibitor-induced plasma nitrate (mediated predominantly by iNOS) and no effects on blood pressure has led to inhibitors as non selective, e.g. *S*-ethylisothiourea [Raman, et al. 1998]. Selectivity for iNOS versus eNOS distinct enzyme targets is another pitfall. Time-dependent NOS inhibition in assessing efficacy and selectivity of NOS inhibitors were first reviewed by Bryk and Wolff, 1999.

#### **1.2.3 NOS inhibitor interactions with the NOS enzymes**

Inhibitors of NOS have been described which interact with the NOS enzymes in a variety of ways: different sites, differing time- and substrate-dependence, and mechanism of inhibition.

**L-Arginine site inhibitors** identified so far as competitive with the substrate L-arginine binding at the arginine-binding site inhibitors (aminoguanidine, *S*-ethylisothiourea, thiocitrulline) [Raman, et al.1998].

Mechanism-based inhibitors of iNOS require active enzyme and NADPH substrate to permit inhibition to proceed through multiple enzyme covalent modification by a complex formation pathway: EI-EI\* complex where E is iNOS, I is the inhibitor, EI is the initial non-covalent complex and EI\* is a modified complex, either with a conformational change to tight binding or with covalent changes to the enzyme, inhibitor or both. The interactions of some of these pterin-site inhibitors with NOS reveal unexpected complexity. An example of a mechanism based iNOS inhibition was cited by aminoguanidine [197±199] and by the acetamidine inhibitors N-α-iminoethyl-l-ornithine (l-NIO) and *N*'-iminoethyl-lysine (l-NIL) [200±203], GW273629 (*S*-[2-[(1-iminoethyl) amino]ethyl]-4,4-dioxo-l-cysteine) and GW274150 (*S*-[2-[(1-iminoethyl)amino]ethyl]-lhomocysteine) (see Table 1). **Heme-binding inhibitors** have been shown to bind with each NOS monomer, one to the haem iron and one to the arginine-binding region (Glu ) in competition with CaM. These compounds affect the assembly of iNOS monomers into active dimer inhibiting the dimerization [Sennequier, et al. 1999]. A class of substituted

Inhibition of Nitric Oxide Synthase Gene Expression:

pathway [Kolodziejski et al.2002].

of isoform-selective inhibitors. [Bretscher, et al. 2003]

and competitive with L-arginine[Narayanan, et al. 1995].

[Ratovitski, et al. 1999].

*In vivo* Imaging Approaches of Nitric Oxide with Multimodal Imaging 123

1. AMP Kinase protein kinase induced inhibition of inducible nitric-oxide synthase (iNOS) Inducible NOS inhibition in endotoxic shock in chronic inflammatory states was observed in several cell types (myocytes, adipocytes, macrophages) and primarily resulted from post-transcriptional regulation of the iNOS protein. Best example is inhibition of inducible nitric oxide synthase by activators of AMP activated protein kinase to explain a new mechanism of insulin sensitizing drug action [Pilon, et al.2004].. Inflammatory cytokines and LPS trigger the iNOS transcription through a complex network of intracellular pathways including NF-κB, Janus kinase/signal transducers and activators of transcription, and mitogen-activated AMPK protein kinase by reducing the transcription of iNOS and mRNA expression [Blanchette et al. 2003]. AMPK switches off protein synthesis either through suppression of the mTOR-p70S6 kinase pathway or by direct activation of eukaryotic elongation factor 2 kinase, resulting in the phosphorylation and inactivation of eukaryotic elongation factor 2 [Horman et al. 2002]. AMPK reduces iNOS protein content by promoting its ubiquitination, required for targeting iNOS through the proteasome proteolysis

2. Expression of exogenous Kalirin in pituitary cells dramatically reduces iNOS inhibition of ACTH secretion. Kalirin inhibits iNOS activity by affecting iNOS homodimerization, which is required for iNOS activity. Thus Kalirin may play a neuroprotective role during inflammation of the central nervous system by inhibiting iNOS activity

3. *N*5-(Iminoalkyl)- and *N*5-(Iminoalkenyl)-ornithines (VNIO) and several L-VNIO analogs showed minor structural changes to produce inhibitors either iNOS-selective or nonselective [Bretscher et al. 2003]. Furthermore, derivatives having a methyl group added to the butenyl moiety of L-VNIO and L-VNIO derivatives display slow-on, slowoff kinetics rather than irreversible inactivation. These results elucidate isoformselective inhibition by L-VNIO and may provide information useful in rational design

4. Constitutive and inducible isoforms of NOS are inhibited by S-alkyl-L-thiocitrullines with n-alkyl groups of any one carbon. The NOS inhibition is reversible, stereoselective,

5. Autoinhibition of endothelial NOS was reported by presence of an electron transfer control element in the NOS. [Nishida, et al. 1999]. Investigators examined the role of the insert in its native protein context by deleting the insert from both wild-type eNOS and from chimeras obtained by swapping the reductase domains of the three NOS isoforms. The Ca2+ concentrations required to activate the enzymes decrease significantly when the insert is deleted, consistent with suppression of autoinhibition. Furthermore, removal of the insert greatly enhances the maximal activity of wild-type eNOS, the least active of the three isoforms. Despite the correlation between reductase and overall enzymatic activity for the wild-type and chimeric NOS proteins, the loop-free eNOS still requires CaM to synthesize zNO. However, the reductive activity of the CaM-free, loop-deleted eNOS is enhanced significantly over that of CaM-free wild-type eNOS and approaches the same level as that of CaMbound wild-type eNOS. Thus, the inhibitory effect of the loop on both the eNOS reductase and zNO-synthesizing activities may have an origin distinct from the loop's

pyrimidine inidazoles have been identified which inhibit dimerization of iNOS during its synthesis and assembly.

#### **1.2.4 Flavoprotein and CaM inhibitors**

A range of flavoproteins (e.g. diphenylene iodonium) or CaM (e.g. trifluoperazine) has been shown to inhibit NOS. NOS inhibitors display selectivity of their isoforms as partially selective, highly selective.

#### **Partially and highly isoform-selective NOS inhibitors**

Identification of selective inhibitors of iNOS and nNOS "100-fold selectivity for iNOS versus eNOS are reported [Anon, 1999a, 1999b]. Partially-selective nNOS inhibitor amino acids for nNOS versus eNOS and iNOS were reported. For example, *S*-ethyl- and *S*-methyl-1 thiocitrulline, vinyl-l-NIO showed timedependent inhibition of nNOS with significant selectivities versus isolated eNOS and iNOS enzymes [Babu and Griffith, 1998]. Other partially-selective iNOS inhibitors such as acetamidine-containing analogues of arginine, l-NIO and l-NIL have been widely used to probe the effects of iNOS inhibition. For example, some 2-iminohomopiperidines and 2-iminopyrrolidines with high (100±900-fold) selectivity for iNOS versus eNOS, but similar potency was observed on iNOS and nNOS (1±13-fold selectivity), with dual action iNOS-nNOS inhibitors.

#### **Highly-selective iNOS inhibitors**

The `highly selective ' iNOS inhibitors versus eNOS are mostly bis-isothioureas. Of these, *S*,*S*- [1,3-phenylene bis-(1,2-ethanediyl)bis-thiourea (`PBITU') is an l-arginine-competitive, rapidly reversible inhibitor of human iNOS with a *K*i of 47 nM, and a selectivity (in *K*i terms) of 190 fold versus eNOS showing substrate-binding sites of full-length human iNOS and eNOS in solution or in cells and tissues. Inhibition of human iNOS by 1400W was competitive with larginine, NADPH-dependent either an irreversible, or reversible. Mechanism-based inhibitor action was reported as *K*d value %7 nM and steady-state selectivity against eNOS and nNOS of 5000 and 250-fold respective effects on vascular leakage [Lazlo, et al.1997]. GW273629 and GW274150 are two novel NOS inhibitors for iNOS versus both eNOS and nNOS. Both are sulphur-substituted acetamidine amino acids acting in competition with l-arginine as NADPH-dependent, whereas the inhibition of human eNOS and nNOS is rapidly reversible. The heme-binding substituted pyrimidine imidazoles inhibit assembly of active dimeric iNOS during its synthesis. It will be interesting to see what the pharmacology and utility of such compounds will be, and whether other compound series are discovered with this NOS inhibition mechanism of action.

#### **1.3 The pharmacological inhibition of inducible nitric-oxide synthase (iNOS) gene expression**

Presently, inhibition of NOS gene expression approach is used in testing inhibitors of pharmacological value. In past, inhibition of NOS was ideal assay to measure less stable NO in tissues but less specificity of NOS was discouragement and spin trap agents have emerged as a new approach of detection and measurement of NO in both in vivo and in vitro assays and bioimaging. Some examples of NOS inhibitors are cited in following section.

pyrimidine inidazoles have been identified which inhibit dimerization of iNOS during its

A range of flavoproteins (e.g. diphenylene iodonium) or CaM (e.g. trifluoperazine) has been shown to inhibit NOS. NOS inhibitors display selectivity of their isoforms as partially

Identification of selective inhibitors of iNOS and nNOS "100-fold selectivity for iNOS versus eNOS are reported [Anon, 1999a, 1999b]. Partially-selective nNOS inhibitor amino acids for nNOS versus eNOS and iNOS were reported. For example, *S*-ethyl- and *S*-methyl-1 thiocitrulline, vinyl-l-NIO showed timedependent inhibition of nNOS with significant selectivities versus isolated eNOS and iNOS enzymes [Babu and Griffith, 1998]. Other partially-selective iNOS inhibitors such as acetamidine-containing analogues of arginine, l-NIO and l-NIL have been widely used to probe the effects of iNOS inhibition. For example, some 2-iminohomopiperidines and 2-iminopyrrolidines with high (100±900-fold) selectivity for iNOS versus eNOS, but similar potency was observed on iNOS and nNOS (1±13-fold

The `highly selective ' iNOS inhibitors versus eNOS are mostly bis-isothioureas. Of these, *S*,*S*- [1,3-phenylene bis-(1,2-ethanediyl)bis-thiourea (`PBITU') is an l-arginine-competitive, rapidly reversible inhibitor of human iNOS with a *K*i of 47 nM, and a selectivity (in *K*i terms) of 190 fold versus eNOS showing substrate-binding sites of full-length human iNOS and eNOS in solution or in cells and tissues. Inhibition of human iNOS by 1400W was competitive with larginine, NADPH-dependent either an irreversible, or reversible. Mechanism-based inhibitor action was reported as *K*d value %7 nM and steady-state selectivity against eNOS and nNOS of 5000 and 250-fold respective effects on vascular leakage [Lazlo, et al.1997]. GW273629 and GW274150 are two novel NOS inhibitors for iNOS versus both eNOS and nNOS. Both are sulphur-substituted acetamidine amino acids acting in competition with l-arginine as NADPH-dependent, whereas the inhibition of human eNOS and nNOS is rapidly reversible. The heme-binding substituted pyrimidine imidazoles inhibit assembly of active dimeric iNOS during its synthesis. It will be interesting to see what the pharmacology and utility of such compounds will be, and whether other compound series are discovered with this NOS

**1.3 The pharmacological inhibition of inducible nitric-oxide synthase (iNOS) gene** 

Presently, inhibition of NOS gene expression approach is used in testing inhibitors of pharmacological value. In past, inhibition of NOS was ideal assay to measure less stable NO in tissues but less specificity of NOS was discouragement and spin trap agents have emerged as a new approach of detection and measurement of NO in both in vivo and in vitro assays and bioimaging. Some examples of NOS inhibitors are cited in following

synthesis and assembly.

selective, highly selective.

**1.2.4 Flavoprotein and CaM inhibitors** 

**Partially and highly isoform-selective NOS inhibitors** 

selectivity), with dual action iNOS-nNOS inhibitors.

**Highly-selective iNOS inhibitors** 

inhibition mechanism of action.

**expression** 

section.


Inhibition of Nitric Oxide Synthase Gene Expression:

Use of cytochrome proteins sensitive to EPR effect;

imaging contrast agents in MRI to image nitric oxide in tissues.

*In vivo* Imaging Approaches of Nitric Oxide with Multimodal Imaging 125

MRI and fluorescence arising out from paramagnetic metals, dithiacarbamates or

 Use of paramagnetic metals (SPIO) in MRI and dithiacarbamates (DTC) or lipopolysaccharides (LPS) complexes sensitive to MRI and fluorescence effects;

Use of NO synthase inhibitors to measure the reduced NO concentrations for

The following sections are focused on dithiacarbamates in fluorometry and less known

The first evidence of dithiacarbamates (DTC) reported them as electron Fe(II)-chelate spin trap agents. Examples are N-methyl-D-glucamine dithiocarbamate (MGD), (MGD)2-Fe(II)- NO and NO-Fe-DTC metal complexes as multimodal imaging agents. These were initially verified for EPR with possibility of visualizing the radical distribution by MR images [Kubrina, et al.1992]. The (MGD)2-Fe(II)-NO complex enhanced the contrast in the vascular structures such as hepatic vein and inferior vena cava. The paramagnetic NO-Fe-DTC metal complex is also a potential MRI signal enhancer and acts as contrast agent. These contrast agents showed the magnetic relaxation changes of neighboring protons to visualize the NO generated in living animal tumors [Jordan et al. 2000]. Other contrast enhancement effect showed an impact of short NO exposure to hemoglobin during MRI signal recording as source of *in vitro* MRI and *in vivo* functional MRI (fMRI) [Di Salle, et al. 1997]. fMRI signal intensity of venous blood in T1-, T2-, and T2\*-weighted images proportionately changed with NO real-time generation in brain. Later, different approaches of blood hemoglobin and NO interaction were attempted to monitor fMRI signal sensitive to NO: mainly metHb and NO-Hb enhanced the MRI signal intensity. These observations suggested a blood flowindependent effect and less utility [Di Salle, et al. 1997]. Still it is hope that NO sensitive fMRI techniques can detect slow epithelial intracellular processes such as metabolic integrity, vascular tonicity, stress, shear and inflammatory effects at early stages of the disease processs, allowing precise monitoring of onset in intact biological systems at cellular level. Other approaches are also emerging to use NO biosensors for multimodal imaging. Currently, use of fMRI as a non-invasive NO sensitive technique has emerged as potential and remarkable tool to detect apoptosis *in vivo*. The NO sensitive fMRI techniques can detect slow epithelial intracellular processes such as metabolic integrity, vascular tonicity, stress, shear and inflammatory effects at early stages of the process, allowing the onset in intact biological systems, providing a useful tool for monitoring at cellular level.

lipopolysaccharides complexes. The mechanisms depend on three approaches:

fluorometry and blood oxygen sensitivity to reduced NO concentrations.

**2.2 The source of intracellular NO and metabolic integrity- feasibility of MRI** 

The nitric oxide is released from the L-arginine in tissue along with molecular oxygen in the oxidative L-arginine degradation reaction of L-arginine pathway catalyzed by either of any three different NO synthase (NOS) isoenzymes. NO controls the intracellular redox state in tissue and protects the metabolic integrity in two ways [Kuppusamy, et al. 1994]. First, anions and cations in intracellular space or cellular redox state prevent apoptosis for example, NO in hepatocytes, neurons, glial cells and fibroblasts controls the release of mitochondrial apoptogenic factors and induces apoptosis by activation of caspases [Hortelano, et al.2005 ]. Second, peroxynitrites accumulate as product of nitric oxide and

inhibitory effects on the binding of CaM and the concomitant activation of the reductase and zNO-synthesizing activities.


#### **2. NO inhibitors in imaging**

Major nitric oxide imaging techniques utilize mapping NO in tissue using NO specific imaging contrast agents sensitive to fluorescence, magnetic resonance and electron spin resonance. Recently, focus is diverted towards imaging *in vivo* physical properties of tissue cells such as cell calcium signaling and NO-biomarker based proton magnetic resonance techniques to achieve nanomolar range of nitric oxide mapping without any toxic effect. Fluorescent nitric oxide cheletropic traps are currently available choices in nitric oxide imaging but all of these have pitfalls of causing neurotoxicity [Reif, et al.2009]. In following sections, we put evidence of the NO sensitive fluorescent probes as cell calcium signaling indicator in tissues and possibility of NO specific perfusion MRI tool to visualize physiological nanomolar dynamics of NO in living cells up to the detection limit of 0.1 nM. The cell signaling indicators such as intracellular calcium revealed that ~1 nM of NO was enough to detect apoptosis events such as caspase-3 activation [Green, 1998, Nagata, 1997, Kwon et al.2009]. Furthermore, the possibility of superparamagnetic iron oxide nanoparticle bound complexes serve as MRI imaging contrast agents based on dephasing contrast. The iron oxide chelates are still in active evaluation phase to test their toxicity. The nanomolar range of basal endothelial NO appears to be fundamental to vascular homeostasis, hypoxia, apoptosis and inflammation.


#### **2.1 Dithiacarbamates, LPS and MGD complexes for bioimaging of NO**

In following sections we highlight the existing mechanisms and description to NO sensitive signal generation. Existing mechanisms of NO sensitive signal generation are explored by

6. A mechanism was reported as Ca2+ triggers cross-talk signal transduction between CaM kinase and NO and CaM-K IIa phosphorylating nNOS on Ser847, which in turn decreases the gaseous second messenger NO in neuronal cells by Calcium/Calmodulin-dependent Protein Kinase IIa in NG108-15 neuronal Cells.

7. Nitric oxide (NO) is moderately produced under control of iNOS gene expression. In recent years, scientists solved the problem of visualizing very unstable NO by mapping iNOS inhibition using gene expression array, *in vivo* nitric oxide direct detection (by stabilizing NO with suitable spin-trapping reagents), bioluminesence and MRI techniques to estimate *in vivo* NO concentration [Hong et al. 2009]. New approaches are in the direction of multimodal bioimaging iNOS gene expression and NO bioimaging as biomarkers. Success depends on NOS gene sensitive MR relaxation signal in cells and

Major nitric oxide imaging techniques utilize mapping NO in tissue using NO specific imaging contrast agents sensitive to fluorescence, magnetic resonance and electron spin resonance. Recently, focus is diverted towards imaging *in vivo* physical properties of tissue cells such as cell calcium signaling and NO-biomarker based proton magnetic resonance techniques to achieve nanomolar range of nitric oxide mapping without any toxic effect. Fluorescent nitric oxide cheletropic traps are currently available choices in nitric oxide imaging but all of these have pitfalls of causing neurotoxicity [Reif, et al.2009]. In following sections, we put evidence of the NO sensitive fluorescent probes as cell calcium signaling indicator in tissues and possibility of NO specific perfusion MRI tool to visualize physiological nanomolar dynamics of NO in living cells up to the detection limit of 0.1 nM. The cell signaling indicators such as intracellular calcium revealed that ~1 nM of NO was enough to detect apoptosis events such as caspase-3 activation [Green, 1998, Nagata, 1997, Kwon et al.2009]. Furthermore, the possibility of superparamagnetic iron oxide nanoparticle bound complexes serve as MRI imaging contrast agents based on dephasing contrast. The iron oxide chelates are still in active evaluation phase to test their toxicity. The nanomolar range of basal endothelial NO appears to be fundamental to vascular homeostasis, hypoxia,

 different contrast mechanisms and contrast characteristics of known nitrosyl-iron complexes display possibility of potential multimodal MRI and EPR probes specific to NO with examples of dithiacarbamates and Fe(MGD)2 complexs in different

fMRI detects the interaction of paramagnetic species with NO in blood but possibility is

In following sections we highlight the existing mechanisms and description to NO sensitive signal generation. Existing mechanisms of NO sensitive signal generation are explored by

still controversial. We review different applications of NO bioimaging.

**2.1 Dithiacarbamates, LPS and MGD complexes for bioimaging of NO** 

specificity to inflammation [Nie et al.2008; Terashima et al. 2010].

reductase and zNO-synthesizing activities.

[Komeima, et al. 2000].

**2. NO inhibitors in imaging** 

apoptosis and inflammation.

applications.

inhibitory effects on the binding of CaM and the concomitant activation of the

MRI and fluorescence arising out from paramagnetic metals, dithiacarbamates or lipopolysaccharides complexes. The mechanisms depend on three approaches:


The following sections are focused on dithiacarbamates in fluorometry and less known imaging contrast agents in MRI to image nitric oxide in tissues.

The first evidence of dithiacarbamates (DTC) reported them as electron Fe(II)-chelate spin trap agents. Examples are N-methyl-D-glucamine dithiocarbamate (MGD), (MGD)2-Fe(II)- NO and NO-Fe-DTC metal complexes as multimodal imaging agents. These were initially verified for EPR with possibility of visualizing the radical distribution by MR images [Kubrina, et al.1992]. The (MGD)2-Fe(II)-NO complex enhanced the contrast in the vascular structures such as hepatic vein and inferior vena cava. The paramagnetic NO-Fe-DTC metal complex is also a potential MRI signal enhancer and acts as contrast agent. These contrast agents showed the magnetic relaxation changes of neighboring protons to visualize the NO generated in living animal tumors [Jordan et al. 2000]. Other contrast enhancement effect showed an impact of short NO exposure to hemoglobin during MRI signal recording as source of *in vitro* MRI and *in vivo* functional MRI (fMRI) [Di Salle, et al. 1997]. fMRI signal intensity of venous blood in T1-, T2-, and T2\*-weighted images proportionately changed with NO real-time generation in brain. Later, different approaches of blood hemoglobin and NO interaction were attempted to monitor fMRI signal sensitive to NO: mainly metHb and NO-Hb enhanced the MRI signal intensity. These observations suggested a blood flowindependent effect and less utility [Di Salle, et al. 1997]. Still it is hope that NO sensitive fMRI techniques can detect slow epithelial intracellular processes such as metabolic integrity, vascular tonicity, stress, shear and inflammatory effects at early stages of the disease processs, allowing precise monitoring of onset in intact biological systems at cellular level. Other approaches are also emerging to use NO biosensors for multimodal imaging. Currently, use of fMRI as a non-invasive NO sensitive technique has emerged as potential and remarkable tool to detect apoptosis *in vivo*. The NO sensitive fMRI techniques can detect slow epithelial intracellular processes such as metabolic integrity, vascular tonicity, stress, shear and inflammatory effects at early stages of the process, allowing the onset in intact biological systems, providing a useful tool for monitoring at cellular level.

#### **2.2 The source of intracellular NO and metabolic integrity- feasibility of MRI**

The nitric oxide is released from the L-arginine in tissue along with molecular oxygen in the oxidative L-arginine degradation reaction of L-arginine pathway catalyzed by either of any three different NO synthase (NOS) isoenzymes. NO controls the intracellular redox state in tissue and protects the metabolic integrity in two ways [Kuppusamy, et al. 1994]. First, anions and cations in intracellular space or cellular redox state prevent apoptosis for example, NO in hepatocytes, neurons, glial cells and fibroblasts controls the release of mitochondrial apoptogenic factors and induces apoptosis by activation of caspases [Hortelano, et al.2005 ]. Second, peroxynitrites accumulate as product of nitric oxide and

Inhibition of Nitric Oxide Synthase Gene Expression:

Biosensor# T1 (600/15)

DNIC (240 µmol/L) 931 ±3 7

MGD 55.5 52.4 (MGD)2-Fe(II)-NO T1 13.3\* 13.9\*\* (100 nmol/g) T2 8.3\* 8.7\*\* (MGD)2-Fe(II) 24.9 25

aqueous solutions.

msec

DNIC-cysteine 0.23 ± 0.06 0.33 ± 0.03 DNIC-GSH(230 µM) 850 ± 24 0.11 ± 0.02 0.19 ± 0.03 DNIC-BSA(160 µM) 247 ± 13 0.71 ± 0.09 1.37 ± 0.30 MNIC-MGD 0.97 ± 0.09 1.37 ± 0.03
