5. Design of inhibitors and activators

Similarly, same kind of analysis can be performed to find out the Vmax and Kb/Vmax by keeping high levels of B and A. The plot of intercept of intercept, i.e. intercept versus 1/[A], will give the values of 1/Vmax and Kb/Vmax. The slope plotted against the 1/[B] was studied for deriving the values of K<sup>a</sup> and Kib by analysing at high levels of B and low concentrations of A (building up the concentration of EB). These secondary plots elaborate various aspects of enzyme kinetics

The data-fitting process can be accomplished by using a software program that provides nonlinear regression-fitting capability. Various programs such as Kaleidagraph are developed wherein the user put the experimental data along with probable fitting function for the prediction of rate constants. Some other software such as dynafit, mathematica, sigmaplot and prism are applied for the estimation of rate expression and effect of inhibition or activator on the enzyme [98]. Alternatively, some of the programs like Enzfitter have a predefined library of equations which can also be used for the prediction kinetics of enzyme. While using the software for data prediction, one should be able to differentiate in errors added due to the lack of fit and pure error as they contribute as a source of error [99]. Although both sources of error normally contribute to the sum of squares of deviations from a model, they can be separated. The inconsistencies between replicate observations are unaffected by the choice of model and thus allow calculations of how much of the total sum of squares is due to the pure

error, and from this one can calculate the contribution of lack of fit [57, 80, 100].

tions that any developed software have done are as follows:

plex.

1. Enzyme to substrate interaction ratio is maintained as 1:1.

inhibitor and enzyme-substrate-inhibitor interaction.

stants of a catalytic reaction.

form enzyme-inhibitor-substrate and/or enzyme-inhibitor complex.

The basic assumption during the development of related software is that the rate of reaction is zero in the absence of substrate. The buffer solution used for the reaction should be selected so that the pKa is not greater than 1 unit of pH that the desired one. Generally, the operational pH should be less than pKa. This will maintain the desired configuration required for the optimal activity of the enzyme. Also, the used buffer system should not react with the enzyme used for the catalysis or the buffer interfering with the analysis method [10, 52, 88]. Primary assump-

2. Inhibitor compete with enzyme active site/or active groups at enzyme allosteric site to

3. The loss of active free energy decides the formation of enzyme-inhibitor-substrate com-

4. Reaction is reversible at every stage of the interaction between enzyme-substrate, enzyme

5. The binding of inhibitor or substrate with enzyme is expressed in terms of kinetic con-

6. The physiological conditions such as pH, temperature, concentration of reactants and

reaction period are kept constant while determining the kinetic constants.

some of which are discussed in the kinetics section.

4.3. Software used in kinetic data analysis

108 Enzyme Inhibitors and Activators

The use of inhibitors usually has two outputs when used against the target enzyme as drug. First, the inhibition of enzyme leads to the accumulation of substrate that it meant to process and second wherein the concentration of metabolite decrease as the enzyme is inactivated by the presence of inhibitors. The inhibition of enzyme results into accumulation of the substrate or the metabolite which then can be detected by the various analytical methods [101]. The reduction of the metabolite due to inhibition of the selective enzyme from the pathway leads to the blocking of further activation sequence and therefore the consequences are resulted due to the same. For example, allopurinol inhibits the action of xanthine oxidase in the treatment of gout disease while inhibition of co-enzymes in the pathway leads to the prevention of undesired cancerous growth. The combination of two inhibitors may work in synergism or may not. It has to be studied thoroughly before using it for the human treatment. Most of the anti-cancerous and antibacterial drugs (e.g. cotrimoxazole) are used in synergism to fight against the targeted disease. Some of the inhibitor (e.g. 5-fluorouracil and doxorubicin) acts on the enzyme-co-enzyme complexes and DNA-enzyme complexes to form dead-end complex instead of binding to the enzyme [85, 102, 103]. Other specific inhibitors can be described in several forms including:


#### 5.1. Rational approach to the design of enzyme inhibitors

#### 5.1.1. General discovery method for lead inhibitor

A lead inhibitor with low potency and selectivity acts as a base scaffold molecule for designing the highly potent and selective inhibitors. The pool of initial molecule (5000–10,000 related compounds) to screen as a lead compound can be decided by two ways. Either by studying the existing drug molecules creating side effects by reacting with proposed enzyme or from the compounds having low potency in pharmacological, anti-bacterial, anti-parasitic, or anti-viral effects in the screening experiments against the proposed target enzyme.

The selection of molecules to study for primary screening of lead compound can be decided on the basis of prior knowledge of (1) modification of the structural scaffold of a lead inhibitor, (2) information of substrate enzyme and inhibitor interactions and their catalytic mechanism and (3) use of molecular modelling software. Among this pool, the compounds are then studied for their inhibitory action. The molecules showing dissociation constant less than 1 μM against the desired enzyme are designated as lead compound. These molecules are then studied with various permutation and combination of approaches to enhance their activity against the selective target. Some of the approaches that can be used for the redesigning of the molecules are replacing existing functional group, enhancing the hydrophobic characters, removal of steric hindrances as well as stereogenic centres. While applying the approaches, it needs to understand the solubility of the drug molecule and its adsorption within the body. After modifications, if the selected molecules are designed with the intent of pharmaceutical applications, then they tested and studied on the animal model. After clearing the animal studies, it the molecules fail in the clinical trials, it requires further structural and manipulation of to improve in vivo profiling [55].

#### 5.1.1.1. Process of development of lead inhibitor

The complementary structure of the molecule to the active site of the enzyme forms noncovalent bonds such as hydrogen bonds, ionic bonds and hydrophobic interaction to bind with the enzyme. The structure specific requirements of the molecules are maintained by replacing the isosteric replacement of groups. Isosteric replacement is done by replacing the atoms or the group with similar electronic and steric configurations which can be the elements within the same vertical group of the periodic table. The replacement of groups is done on the basis of the outer electronic configuration as well as by the ring equivalents without disturbing the spatial arrangement of the lead molecule. In the process of improving potency of the lead molecule, the atoms or the groups can be replaced by the non-specific substitute which may increase the hydrophobicity or hydrophilicity of the molecule to enhance the membrane penetration or the solubility. It is necessary to understand the dissociation state of the functional group at the physiological pH during the process of modification of the lead compound [68, 102].

The strategy of replacement leads compound group with the analogous group (]F, ]OH, ] NH2 and ]CH3) and is successfully applied to design the hypoglycemic agents, inhibitory compounds such as aminopterin, 6-mercaptopurine, 6-thioguanine, etc. The cis- and transconfigurational similarities can also serve as the way to design better lead compound. For example, replacement of ester functionality of the anaesthetics prolongs their action in body which led to the group of drugs such as anticholinergic spasmolytics and anti-depressant drugs. The pyridyl containing compound has more hydrophilicity as compared to the benzene because of the presence of p-electron deficiency which improve the reactivity of most of the drugs. This concept has already applied to the development of tricyclic, anti-histaminic and neuroleptic (major tranquilizing) drugs as well as in sulfide-containing groups [69, 104].

Ring replacement of ]N< by ]HC< and its subsequent modification to >Cd have resulted in a variety of useful drugs. This is seen in the development of psychotherapeutics (chlorprothixene), the anti-inflammatory drug (sulindac). Several other modifications such as reversal of group, ring opening and closure and groups with similar polar effect can lead to the development of more potent lead compound [97].

#### 5.2. Design from a knowledge of the catalytic mechanism

3. Prosthetic group inhibitors: e.g. cyanide that inhibits the heme-prosthetic group of cyto-

4. Apoenzyme inhibitors that the molecules which attack the apoenzyme component of the

5. Physiological modulators of reaction such as pH and temperature denature the enzyme

A lead inhibitor with low potency and selectivity acts as a base scaffold molecule for designing the highly potent and selective inhibitors. The pool of initial molecule (5000–10,000 related compounds) to screen as a lead compound can be decided by two ways. Either by studying the existing drug molecules creating side effects by reacting with proposed enzyme or from the compounds having low potency in pharmacological, anti-bacterial, anti-parasitic, or anti-viral

The selection of molecules to study for primary screening of lead compound can be decided on the basis of prior knowledge of (1) modification of the structural scaffold of a lead inhibitor, (2) information of substrate enzyme and inhibitor interactions and their catalytic mechanism and (3) use of molecular modelling software. Among this pool, the compounds are then studied for their inhibitory action. The molecules showing dissociation constant less than 1 μM against the desired enzyme are designated as lead compound. These molecules are then studied with various permutation and combination of approaches to enhance their activity against the selective target. Some of the approaches that can be used for the redesigning of the molecules are replacing existing functional group, enhancing the hydrophobic characters, removal of steric hindrances as well as stereogenic centres. While applying the approaches, it needs to understand the solubility of the drug molecule and its adsorption within the body. After modifications, if the selected molecules are designed with the intent of pharmaceutical applications, then they tested and studied on the animal model. After clearing the animal studies, it the molecules fail in the clinical trials, it requires further structural and manipulation of to

The complementary structure of the molecule to the active site of the enzyme forms noncovalent bonds such as hydrogen bonds, ionic bonds and hydrophobic interaction to bind with the enzyme. The structure specific requirements of the molecules are maintained by replacing the isosteric replacement of groups. Isosteric replacement is done by replacing the atoms or the group with similar electronic and steric configurations which can be the elements within the same vertical group of the periodic table. The replacement of groups is done on the basis of the outer electronic configuration as well as by the ring equivalents without disturbing the spatial arrangement of the lead molecule. In the process of improving potency of the lead

chrome oxidase.

110 Enzyme Inhibitors and Activators

holoenzyme and

improve in vivo profiling [55].

5.1.1.1. Process of development of lead inhibitor

and disturb the catalytic activity drastically.

5.1.1. General discovery method for lead inhibitor

5.1. Rational approach to the design of enzyme inhibitors

effects in the screening experiments against the proposed target enzyme.

In most of the examples, the prior data on the enzyme active site are not available. The new lead compounds are designed based on the knowledge of substrate and catalytic mechanism followed by the reaction. The design of the molecules and their interaction with predicted structure of enzymes can also serve as better option to design the lead compound wherein molecular modelling plays an essential role [105–107]. The enzymes about which no data are available, structure of substance acts as a guiding line. For example, before discovery of the structural data of the angiotensin-converting enzyme (ACE), the inhibitors are decided based on the knowledge of substrate and poison (snake venom) and its similarity with the carboxypeptidase A enzyme. The discovery that D-benzylsuccinic acid was a potent inhibitor of carboxypeptidase A was challenged to the molecule similar to snake venom with the attachment of terminal. Ala-Pro sequence leads to the development of inhibitor methyl glutamyl proline. For the further improvements to enhance similarity between the substrates, increasing the solubility of the compound and reduction of side effect caused the drug molecule, it undergoes a series of changes to develop highly potent and efficient drug [78, 94, 108, 109].

Nowadays, application of computer-based molecular modelling techniques has gained the relative importance over the conventional techniques. The highly resolved crystal structure of the enzyme and enzyme-inhibitor complex reveals the change structural configurations, the spatial arrangement of the inhibitor within the active site of the enzyme along with the binding site of the enzyme and inhibitors [110–112]. On the basis of analysis of these data, the molecular modelling software showed the best match for the given enzyme from the large pool for chemical molecules in its database. These might be the completely new structures which need to explore for their catalytic activity. It can also suggest the best modification that can serve for the developing potent inhibitor from the existing drugs. By using data from various sources such as structure of inhibitors from the various crystallographic databases and crystal structures of enzymes, enzyme-substrate and enzyme-inhibitor complex from the various PBD databases can be imported and analysed for the prediction of inhibition capacities of the various molecules [113]. Various ab initio and drawing softwares are used for the prediction of the structure of the inhibitors which are not present in the database. There spatial arrangement and group and chain positions are studied and model is minimized to confirm with lowest energy status. This is then docked inside the active site of the enzyme and positioned to have a maximum favourable interaction and tried for the energy minimization of the system. Thus, the finalized inhibitor then modified by various methods discussed earlier (Section 7.1) and analysed again in the software [77, 84, 114]. The use of high-resolution NMR studies or by homology modelling (minimum 30%) can give the relative desired protein structure. This can be used for the further docking experiments against the library of the inhibitors using computerized modelling software. The prediction of the active site on the surface of enzyme (pharmacophore) is done by docking the multiple inhibitor molecules. Because of the basic assumption of the rigid structure of the protein, the predicted inhibitor may differ in activity with the dynamic enzymes molecule [115, 116]. Thus, the developed lead compounds then can be applied in pharmaceutical, agrichemical industries and in the study of metabolic flux, genetic engineering and molecular biology for the betterment of life.
