**2.1 Enzyme inhibitors**

Enzyme inhibitors are compounds that interact with enzymes (either temporarily or permanently) in some way and minimize the rate of an enzyme-catalyzed

**Figure 2.** *Therapeutic inhibitors.*

reaction or stop enzymes from working in a normal manner [13]. In therapeutic, enzyme inhibitors bind to enzymes and lower their activity [14] and achieve a therapeutic benefit. Some molecules are used as drugs today because of their ability to cause correction of metabolic imbalance, the correction which is due to the effectiveness of the molecules in causing blockage of enzyme activity. Therefore, the search and discovery of molecules with inhibitory enzyme ability is an active area of research in biochemistry and pharmacology [14]. It is noteworthy to state that not all molecules that bind to enzymes are inhibitors; some could be enzyme activators; in this case, the molecules bind to enzymes and elevate their enzymatic activity [15], which can also cause therapeutic benefit. The binding of inhibitors to enzymes can either be reversible or irreversible.

A molecule is a reversible inhibitor if it binds non-covalently to the enzyme's active site to produce an inhibition. The binding could be direct with the enzyme, the enzyme-substrate complex, or both [15].

A reversible inhibitor is described as one that, once removed from the enzyme, the enzyme returns to its normal function pre-inhibition. It exerts no permanent effects on the enzyme and does not change the shape of the active site of the enzyme [16]. There are different types of reversible inhibition. They include competitive, non-competitive and uncompetitive types, although a mixed type sometimes arises [15].

The underlying principle of competitive inhibition is that, at a single active or binding site of a drug-metabolizing enzyme, there is the mutually exclusive binding of either the substrate or the inhibitor [17]. Competitive enzyme inhibitors possess a comparable shape to that of the substrate molecule. These two drugs compete for binding to a single active site of an enzyme. Substrates are compounds or molecules upon which enzymes act. The interaction of a substrate and an enzyme occurs at the active site of the enzyme or in a binding site that can, in turn, alter the active site. This brings about competition for binding/active sites between a substrate and an inhibitor.

The second type of reversible inhibition, non-competitive reversible inhibition, utilizes inhibitors that do not have similarity with the substrate and so do not bind to the active site but rather to a separate site on the enzyme. The outcome of an interaction of a non-competitive inhibitor with an enzyme appreciably differs from an interaction with a competitive inhibitor due to the non-existence of antagonism. In the case of an antagonistic inhibition, the inhibitory effect could be minimized and subsequently overcome with escalating concentrations of substrate. With non-competitive inhibition, growing the quantity of substrate does not affect the percentage of an enzyme that is active. Indeed, in non-competitive inhibition, the

#### *Therapeutic Inhibitors: Natural Product Options through Computer-Aided Drug Design DOI: http://dx.doi.org/10.5772/intechopen.104412*

percentage of enzyme inhibited remains the same through all ranges of a substrate. The implication of this is that non-competitive inhibition will efficiently diminish the concentration of enzyme by equal, fixed concentration in a typical experiment at every substrate concentration used [18].

The third type of reversible inhibition, uncompetitive reversible inhibition, utilizes inhibitors that bind to the already formed enzyme-substrate complex and not to the free enzyme. In this type of reversible inhibition, the interaction of the substrate with an enzyme could trigger a conformational modification that leads to the revelation of an inhibitor binding site on the enzyme, or the inhibitor could bind and interact directly to the enzyme-bound substrate. The underlining outcome in this type of reversible inhibition is that it does not compete with the substrate for the same active site in either case and so the increasing concentration of substrate cannot overcome the effect of the inhibitor [19].

As opposed to reversible inhibition, there is irreversible inhibition. In irreversible inhibition, the inhibitor no longer separates from the enzyme after binding and interaction and the enzyme reaction is reduced. The reduction rate is dependent on the enzyme and inhibitor concentrations only and independent of the concentration of the substrate. This implies one inhibitor molecule can ideally minimize to zero the activity of one enzyme molecule [20]. Irreversible inhibition could be of two forms. The first occurs when an inhibitor is strongly bound and complex with an enzyme and fail to dissociate under physiological conditions from the enzyme. There are two types of irreversible inhibitors. The first type is so strongly complexed to the enzyme that it fails to dissociate from the enzyme under physiological conditions but can be dissociated through the method of dialysis or by chromatographic techniques [20]. The second type of irreversible inhibition is one in which the inhibitor forms a covalent bond with the enzyme; in a situation whereby the formation of the covalent bond terminates the conversion of substrate to product, then the enzyme has been irreversibly terminated. The irreversible inhibitors that function through the formation of covalent bonds are of two main types. The first type involves the reaction of an inhibitor with an essential functional group by a bimolecular process on the enzyme [21]. The biomolecular process is a reaction that involves the combination of two molecular entities. In the second type of irreversible inhibition that occurs through the formation of a covalent bond, the inhibitor which bears a leaving group forms a reversible complex with an enzyme. As this occurs, the presence of a nucleophilic group on the enzyme of the leaving group, juxtaposed within the reversible enzyme-inhibitor complex formed on the enzyme of the leaving group, could lead to a rapid neighboring group reaction within the complex in which a covalent bond is formed. Such formation of a covalent bond can be highly specific since properly positioned neighboring groups can react more rapidly than the identical bimolecular reaction [21]. A leaving group is an atom or group of atoms that dissociates from the rest of the molecule, taking with it the electron pair, which was previously the bond between the leaving group and the rest of the molecule.

#### **2.2 Kinase inhibitors**

Kinase is a type of enzyme that acts to add phosphates to other molecules, such as sugars or proteins. The addition of phosphate may cause other molecules in a cell or system to become either active, overactive, or inactive. Kinases facilitate the transmission of a phosphate moiety from a high-energy molecule to its substrate molecule. Kinases are widely utilized to convey signs and control multifaceted procedures in cells. Phosphorylation of compounds can boost or impede their effectiveness and regulate their capability to interrelate with other compounds.

The presence and absence of phosphoryl groups offer the cell a means of control because various kinases can react to diverse situations or signals. There are 518 kinases encoded in the human genome are 518 kinases. These kinases are known to phosphorylate about one-third of the proteome [22, 23]. Nearly all signal transduction route occurs through a phosphotransfer process. This indicates that kinases offer several nodes for therapeutic mediation in numerous abnormally controlled biological processes [24]. Kinase function deregulation has been shown to perform an essential role in cancer immunological, inflammatory, degenerative, metabolic, cardiovascular and infectious diseases [25, 26].

Kinases are of three main categories depending on the substrate type of kinase: protein kinase, lipid kinase, carbohydrate kinase. Protein and lipid kinases represent one of the most important target classes for treating human disorders after G-protein-coupled receptors (GPCRs) and proteases. As a matter of fact, one-third of the protein targets currently undergoing investigation by pharmaceutical companies consist of protein or lipid kinases [27].

Kinase inhibitors are molecules with the ability to alter the activities of kinases. The recognized druggability and the therapeutic safety profile of standard kinase inhibitors make kinases attractive targets for drug development. Nevertheless, there are many kinases yet to be studied effectively; this shows that the discovery of kinase inhibitors is still the majority of kinases that have been historically understudied, indicating that the field of kinase inhibitor discovery is still not fully harnessed [28–30]. There are some significant challenges in drug discovery as regard kinase inhibitors. These challenges are obstacles to the full potential of kinases as drug targets. The challenges include validating novel kinase targets, utilization of kinase inhibitors in non-oncology therapeutic areas, overcoming drug resistance, obtaining target selectivity to minimize off-target-mediated toxicity and to develop effective compound screening and profiling technologies [31]. Nevertheless, some progress has been made in towards overcoming these challenges, and also research in the field of kinase inhibitors have Over the course of the past 5 years, immense progress has been made towards these goals, and also studies the field of kinase inhibitor discovery is expanding rapidly in oncology and into different disease areas, including autoimmune and inflammatory disease as well as degenerative disorders.

The estimated current spending in research and development by pharmaceutical companies towards the development of new kinase inhibitors is about 30%. In all these, one of the most important classes of drugs targeted by pharmaceutical industrial researchers is protein kinases. To date, 89 drugs targeting protein kinases have clinically received approval. It is estimated that the current global market for kinase therapies is about US\$20 billion per annum, projection to rise distinctly. Over 100 active small-molecule kinase inhibitors are currently in an advanced stage of clinical development, and many more are expected to be approved in the years ahead [32].

#### **2.3 Protease inhibitors**

Proteases, which are also known as proteinases or proteolytic enzymes, are a large class of enzymes that catalyzes the hydrolysis of peptide bonds in proteins and polypeptides. Proteases control the fortune, localization, and numerous protein actions. Proteases are important aspects in the well-being and viability of cells, participating in several procedures, such as replication, transcription, cell multiplication, differentiation, extracellular matrix remodeling, and processing of hormones and biologically active peptides. Proteases are greatly controlled (e.g. transcriptionally, post-translationally, stimulated, inhibited, and classified) [33]. Protease action has been found to play a role in the pathogenesis of vascular diseases, including

#### *Therapeutic Inhibitors: Natural Product Options through Computer-Aided Drug Design DOI: http://dx.doi.org/10.5772/intechopen.104412*

atherosclerosis, thrombosis, and aneurysm. Broad diversity of proteases representing various proteolytic groups and their corresponding inhibitors are involved. These proteases play a role(s) vascular ailment through a sequence of overlapping pathways that upset the overall inflammatory status and structural integrity of the vessel wall. By triggering PARs (protease-activated receptors), these enzymes cause inflammatory signaling, cytokine production, and inflammatory cell recruitment. Furthermore, proteases can destroy components of the extracellular matrix (ECM), elastic lamina, and fibrous cap in the atheroma. The fundamental paradigm is that excessive proteolytic action is an important contributor to the start and progression of vascular disease. Recent approaches to the treatment of vascular pathologies have attempted to modulate protease activity in an effort to reduce inflammation and preserve the structural integrity of the vessel wall [34]. Proteases can be divided into six broad classes based on proteolytic mechanism: serine proteases, threonine proteases, cysteine proteases, aspartic proteases, metalloproteases, and glutamic acid proteases.

Protease inhibitors are synthetic drugs that prevent the activity of HIV-1 protease, an enzyme that cleaves two precursor proteins into smaller fragments. These fragments are essential for viral growth, infectivity, and replication. It is important to mention that proteases are not limited to HIV. Protease inhibitors interact with protease at the active site, thereby thwarting the growth and development of the freshly formed virions; this makes them stay non-infectious. Protease inhibitors are utilized in taking care of individuals with human immunodeficiency virus (HIV infection) and acquired immune deficiency syndrome (AIDS) [35]. Also, protease inhibitors are useful medically as angiotensin-converting enzyme inhibitors for blood pressure, proteasome inhibitors for myeloma, dipeptidyl peptidase IV inhibitors for type II diabetes [33]. Currently, there are many studies in progress targeting SAR-COV-2 main protease (Mpro) [36–41]. Mpro, also termed 3CL protease, is a 33.8 kDa cysteine protease that mediates the maturation of functional polypeptides involved in the assembly of replication-transcription machinery [42]. Due to the significant role of this main protease, it is considered a promising drug target, as it is dissimilar to human proteases.

### **2.4 Protein synthesis inhibitors**

The process of making a protein molecule using DNA, RNA, and various enzymes by cells is termed protein synthesis. In biological systems, it takes place inside the cell and involves amino acid synthesis, transcription, translation, and post-translational events. It takes place in the cytoplasm of prokaryotes, while in eukaryotes, it takes place usually in the nucleus and aids the generation of a transcript (mRNA) of the coding region of the DNA. The transcript departs the nucleus and gets to the ribosomes, where translation into a protein molecule takes place with a specific sequence of amino acids [43].

A protein synthesis inhibitor is a molecule with the ability to terminate or reduce the growth rate of cells by interrupting the progressions that directly leads to the production of new proteins [44]. Even though a wide description of this definition could be utilized in closely describing any compound depending on the amount present, in reality, it classically denotes compounds that exert their molecular effect level on translational machinery. Protein synthesis inhibitors are another major group of therapeutically useful antibacterials, such as erythromycin, tetracycline, chloramphenicol, and aminoglycosides. They specifically interact with the 70S bacterial ribosome and spare the 80S eukaryotic ribosome particle. Macrolide, lincosamide, and streptogramins (MLS) antibiotics represent three classes of structurally diverse protein biosynthesis inhibitors used clinically [45]. Generally, protein

synthesis inhibitors work at different stages of bacterial mRNA translation into proteins, like initiation, elongation (including aminoacyl tRNA entry, proofreading, peptidyl transfer, and bacterial translocation) and termination.

## **2.5 Protein-protein interactions inhibitors**

The protein-protein interaction (PPI) can be described as a substantial network linking a protein and its partner(s) [46–48]. These networks may exhibit a variety of heterogeneities and complexities in large molecular structures, leading to the formation of protein dimers, multi-constituent complexes, or lengthy chains [49]. The contact between subunits of protein can be transitory or constant, similar or dissimilar, and precise or imprecise [48, 50, 51]. There are closely 650,000 protein-protein interactions in humans, and this figure keeps on increasing as additional interaction networks are being discovered [48, 52]. Protein-protein interactions (PPIs) play pivotal roles in biological processes [53]. Mutations or compromised regulation of PPIs affect cellular networks and have a role to play in the development of diseases. The discovery and development of new PPI inhibitors with the intention to control abnormal pathways have therefore aroused substantial interest from the pharmaceutical industry [54]. Almost half of the dry mass of a cell is composed of proteins, and disorder in PPIs often causes diseases, including cancer [55, 56]. Hence, research and studies on PPI play a vital role in advancing our understanding of molecular biology and human diseases, as well as for developing new therapeutic agents in drug discovery [51, 57, 58].

Generally, protein-protein interactions were used to being seen as a nondruggable target. This standing is likely due to the lack of or limited knowledge on high-throughput assessment assays, as well as the consideration that most protein-protein interactions are held to by big, chemically noncomplex surfaces with a deficiency of easily druggable pockets [59]. While such tough protein-protein interaction targets indisputably exist, it is now understood that many protein-protein interactions use minimal interfaces for their interaction, regularly consisting of an unstructured peptide bound to a distinct groove [54]. Additionally, mutagenesis analyses of numerous PPIs have shown that surfaces causing the affinity of a given PPI are not steadily spread across the whole interface. Rather, there tends to be a "hot spot" or a small number of important residues that anchor two proteins together [60]. This implies that a putative inhibitor would not need to dislodge the entirety of a given PPI but rather only occupy the hot spot, a more tractable problem.

Currently, researches in the area of SARS-COV-2 also include inhibition of Spike-ACE2 interaction, which is a protein-protein interaction [61, 62].
