Approaches to design enzyme inhibitors

APPROACHES TO DESIGN ENZYME INHIBITORS : 

APPROACHES TO DESIGN ENZYME INHIBITORS NARESH PANIGRAHI ASST.PROFESSOR GITAM UNIVERSITY

ENZYMES: 

ENZYMES Enzymes are the body's catalysts . Without them, the cell's chemical reactions would be too slow and many would not occur at all. A catalyst is an agent which speeds up a chemical reaction without being changed itself. As an example, let us consider the substrate for lactate dehydrogenase —an enzyme which catalyses the reduction of pyruvic acid to lactic acid

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They increase the rate of a reaction by lowering the activation energy barrier.

How do catalysts lower activation energies?: 

How do catalysts lower activation energies? There are several factors at work. Catalysts provide a reaction surface or environment. Catalysts bring reactants together. Catalysts position reactants correctly so that they easily attain their transition state configurations. Catalysts weaken bonds. Catalysts may participate in the mechanism.

The Mechanism of Enzymatic Action: 

The Mechanism of Enzymatic Action

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The reactant usually binds to the enzyme forming a transition complex that stabilizes the transition state. • The interaction between enzyme and substrate is very specific. Specificity may be for a class of compounds or for a particular compound. Eg. Hexokinase / glucokinase

Enzyme Structure: 

Enzyme Structure All known enzymes are proteins. They are high molecular weight compounds made up principally of chains of amino acids linked together by peptide bonds . Many enzymes require the presence of cofactors Coenzyme= a non-protein organic substance which is loosely attached to the protein part.(NAD + , NADH, FAD) Prosthetic group = an organic substance which is firmly attached to the protein or apoenzyme portion or Metal-ion activators ( K+, Fe++, Fe+++, Cu++, Co++, Zn++, Mn++, Mg++, Ca++ )

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The structure of an ENZYME CONTAINS AN Apoenzyme : Protein part Cofactor : Non-protein component Coenzyme: Organic cofactor Holoenzyme : Apoenzyme plus cofactor

Specificity of Enzymes: 

Specificity of Enzymes One of the properties of enzymes that makes them so important as diagnostic and research tools is the specificity they exhibit relative to the reactions they catalyze. In general, there are four distinct types of specificity: 1. Absolute specificity - the enzyme will catalyze only one reaction. 2 . Group specificity - the enzyme will act only on molecules that have specific functional groups, such as amino, phosphate and methyl groups. 3. Linkage specificity - the enzyme will act on a particular type of chemical bond regardless of the rest of the molecular structure. 4. Stereochemical specificity - the enzyme will act on a particular steric or optical isomer.

Enzyme Classification: 

Enzyme Classification There are 6 main classes based on the type of reaction catalyzed. Oxidoreductase : Oxidation-reduction reactions Transferase : Transfer functional groups Hydrolase : Hydrolysis Lyase : Removal of atoms without hydrolysis Isomerase : Rearrangement of atoms Ligase : Joining of molecules, uses ATP

THE ACTIVE SITE OF AN ENZYME: 

THE ACTIVE SITE OF AN ENZYME ACTIVE SITE: IS a Pocket in the enzyme where substrates bind and catalytic reaction occurs. Active site is a relatively small 3-D region within the enzyme. Substrates bind in active site by weak non-covalent interactions A. hydrogen bonding B. hydrophobic interactions C. ionic interactions

THE ACTIVE SITE OF AN ENZYME: 

THE ACTIVE SITE OF AN ENZYME The amino acids present in the active site play an important role in enzyme function. Amino acids present in the active site can have one of two roles. 1. Binding —the amino acid residue is involved in binding the substrate to the active site. 2. Catalytic —the amino acid is involved in the mechanism of the reaction.

THEORIES OF ENZYME ACTION: 

THEORIES OF ENZYME ACTION

INDUCED FIT MODEL: 

INDUCED FIT MODEL

Allosteric Enzymes : 

Allosteric Enzymes • Allosteric enzymes have one or more allosteric sites • Allosteric sites are binding sites distinct from an enzyme’s active site or substrate-binding site • Molecules that bind to allosteric sites are called effectors or modulators

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Binding to allosteric sites alters the activity of the enzyme . This is called cooperative binding . Effectors may be positive or negative Effectors may be homotropic or heterotropic Regulatory enzymes of metabolic pathways are allosteric enzymes ( eg : feedback inhibition)

Enzyme Inhibitors: Competitive Inhibition: 

Enzyme Inhibitors: Competitive Inhibition

Example: 

Example

Enzyme Inhibitors: Noncompetitive Inhibition: 

Enzyme Inhibitors: Noncompetitive Inhibition

Enzyme inhibitors in Medicine: 

Enzyme inhibitors in Medicine The effectiveness of an enzyme inhibitor as a therapeutic agent will depend on The potency of the inhibitor, Its specificity toward its target enzyme, The choice of metabolic pathway targeted for disruption, and The inhibitor or a derivative possessing appropriate pharmacokinetic characteristics.

Enzyme inhibitors in Basic Research: 

Enzyme inhibitors in Basic Research Classification of Enzyme Inhibitors

RATIONAL DESIGN OF NONCOVALENTLY BINDING ENZYME INHIBITORS: 

RATIONAL DESIGN OF NONCOVALENTLY BINDING ENZYME INHIBITORS Rapid, Reversible lnhibitors :- This class of inhibitors acts by binding to the target enzyme's active site in a rapid, reversible, and non-covalent fashion. The net result is that the active site is blocked and the substrate is prevented from binding .

Types of Rapid, Reversible Inhibitors.: 

Types of Rapid, Reversible Inhibitors. Binding of these inhibitors follows simple Michaelis-Menten kinetics and, depending on their preference of binding to the free enzyme and/or the enzyme-substrate complex, again they are divided in to 3 types Competitive, Uncompetitive, and Noncompetitive inhibition

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Uncompetitive inhibition is rarely observed in single-substrate reactions but is frequently observed in multisubstrate reactions. An uncompetitive inhibitor can provide information about the order of binding of the different substrates. Km appears lower But.. Vmax and kcat are equally lower Therefore… Specificity constant ( kcat /Km is unchanged)

Noncompetitive Inhibition: 

Noncompetitive Inhibition Classi cal noncompetitive inhibitors have no effect on substrate binding and vice versa, given that they bind randomly and reversibly to different sites on the enzyme.

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They also bind with the same affinity to the free enzyme and to the enzymesubstrate complex. Both the enzyme- inhibitor complex E . I and the enzyme-substrate-inhibitor complex E . S .I are catalytically inactive. Simple Michaelis-Menten kinetics of noncompetitive inhibitors are described in following Equation

Examples of Rapid Reversible Inhibitors: 

Examples of Rapid Reversible Inhibitors Competitive inhibitors are often structurally similar to one of the substrates of the reaction.

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This reaction is competitively inhibited by malonate (-00C--CH 2 --C00-) that has, like succinate , two carboxylate groups. It is therefore able to bind to the enzyme's active site but, with only one carbon atom between the carboxylates , further reaction is impossible. Chemotherapy : reduction of dihydrofolate to tetrahydrofolate by dihydrofolate reductase (DHFR) (e.g. methotrexate ) ii) Treatment of gout : xanthine to uric acid by xanthine oxidase

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Phenyl-ethanolamine N- methyltransferase (PNMT) catalyzes the terminal step in epinephrine (adrenaline) biosynthesis, Inhibitors of phenylethanolamine N- methyltransferase are

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Kinetic analyses showed that SAH was a competitive inhibitor of SAM and a noncompetitive inhibitor of norepinephrine , whereas (2) was a competitive inhibitor of norepinephrine and an uncompetitive inhibitor of SAM.

Slow-, Tight-, and Slow-Tight-Binding Inhibitors: 

Slow-, Tight-, and Slow-Tight-Binding Inhibitors Not all reversible inhibitors have an instantaneous effect on the rate of an enzymatic reaction. Some inhibitors, known as slow-binding enzyme inhibitors, can take a considerable time to establish the equilibrium between the free enzyme and inhibitor, and the enzymeinhibitor complex. This time period may be on the scale of seconds, minutes, or even longer.

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Slow binding does not necessarily indicate a slow binding of inhibitor to enzyme but rather the fact that reaching equilibrium is a slow process . Tight-binding inhibitors , bind their target enzyme with such high affinity that the population of free inhibitor molecules is significantly depleted when the enzyme-inhibitor complex is formed. Often, tight-binding inhibitors also have a slow onset of action, and are termed slow-tight-binding inhibitors. These three types of inhibitors have in common is that, generally, the major assumptions of Michaelis-Menten kinetics do not hold true.

Example of slow-binding inhibition : 

Example of slow-binding inhibition Slow binding inhibitors of arginase

Multi-Substrate Analogs: 

Multi-Substrate Analogs A large number of enzymatic reactions involve the simultaneous binding of two or more substrates at the active site. The bound substrates must be in close proximity to each other and positioned in such a way as to facilitate covalent bond formation or the transfer of a functional group from one substrate to another. Thus Multisubstrate analog inhibitors mimic the simultaneous binding of two or more substrates at the active site of the enzyme.

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There are two ways the two substrates, A and B, may bind to the enzyme to form an E - A B complex . First, and most likely, they bind individually (in either a random or an ordered fashion) with dissociation constants of K A and K B . Second, the substrates may come together, positioned in such a way as to facilitate their subsequent reaction with a dissociation constant of K Bi . This reactive complex A .B then binds to the enzyme with a dissociation constant of K Ms

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A multisubstrate analog inhibitor will bind more tightly than substrate analog inhibitor. Inhibitors that combine two substrates are termed bisubstrate analogs , whereas those combining three substrates are termed trisubstrate analogs and so on. The design of a bisubstrate analog inhibitor ordinarily requires the development of two single-substrate analog inhibitors of reasonable affinity. The two single-substrate inhibitors are then connected by an appropriate linker , and the optimal length of the linker is determined experimentally.

Multi-Substrate Analog inhibitors : 

Multi-Substrate Analog inhibitors

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Glycinamide ribonucleotide transformylase (GAR- TFase ) catalyzes the transfer of a formyl group from N 1O -formyltetrahydrofolate to glycinamide ribonucleotide . This is a crucial step in de novo purine biosynthesis, which is essential for cell division, and GAR TFase has become a target enzyme for the deveIopment of antineopIastic agents.

Bisubstrate analog inhibitors of GAR-TFase.: 

Bisubstrate analog inhibitors of GAR- TFase . BW1476U89 β - thioGARdideazafolate ( β -TGDDF)

Inhibitors of AspartateTranscarbamoylase (ATCase): 

Inhibitors of AspartateTranscarbamoylase (ATCase) The condensation of carbamyl phosphate and L- aspartate , catalyzed by aspartate transcarbamoylase (ATCase), produces N- carbamyl - L- aspartate . This is one of the early steps in de novo pyrimidine biosynthesis, also a requirement for cell division, making ATCase also a target for potential anticancer agents.

Inhibitors of AspartateTranscarbamoylase (ATCase): 

Inhibitors of AspartateTranscarbamoylase (ATCase) N- Phosphonoacetyl -L- aspartate (PALA) Carbamyl phosphate and Succinate

HMG-CoA reductase inhibitors: 

HMG- CoA reductase inhibitors The statins are a group of cholesterol-lowering agents that have become some of the largest selling drugs in the world. They lower serum cholesterol levels by competitively inhibiting 3-hydroxy-3-methylglutaryl-coenzyme A (HMG- CoA ) reductase , a key enzyme in cholesterol biosynthesis.

HMG-CoA reductase inhibitors: 

HMG- CoA reductase inhibitors Mevastatin Simvastatin fluvastatin

Transition-State Analogs: 

Transition-State Analogs As a chemical reaction proceeds from substrates to products, it will pass through one or more transition states. The energy barrier imposed by the highest energy transition state controls the overall rate of the reaction. Enzymes bring about rate enhancements of 10 10 -10 15 by lowering this energy barrier. They do this by having a greater affinity to the structure of the transition state than to the structures of either substrates or products.

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Compounds that can take advantage of this enhanced binding to the transition state can prove to be potent and selective enzyme inhibitors .

Design of transition-state inhibitors : 

Design of transition-state inhibitors The design of a good transition-state inhibitors requires the knowledge of the mechanism of the target enzyme to predict transition-state structure(s). This is why transition-state analogs are sometimes referred to as mechanism-based inhibitors.

Transition state analog inhibitors: 

Transition state analog inhibitors Phosphonic acid peptides were transition-state analog inhibitors of pepsin. Adenosine deaminase (ADA), which catalyzes the conversion of adenosine to inosine is an extremely proficient enzyme, and the enzyme-catalyzed reaction is thought to pass through an unstable hydrated intermediate.

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The structures of several inhibitors of ADA are synthesized Of these, the antibiotics coformycin and (R)- deoxycoformycin ( pentostatin ) were found to be potent ADA inhibitors, Coformycin (R)- deoxycoformycin

RATIONAL DESIGN OF COVALENTLY BINDING ENZYME INHIBITORS: 

RATIONAL DESIGN OF COVALENTLY BINDING ENZYME INHIBITORS

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The targets for COVALENTLY BINDING ENZYME INHIBITORS are the chemically reactive groups found within the enzyme's active site. These groups, in the majority of cases, are nucleophiles such as the –OH groups of serine, threonine , and tyrosine. The --SH group of cysteine , and The –COOH groups of aspartic and glutamic acid residues. Other nucleophilic groups include the amino – NH 2 group of lysine and The imidazole ring of histidine .

CHEMICAL MODIFIERS: 

CHEMICAL MODIFIERS The chemical modifiers, are small organic molecules, generally electrophiles , that are used to modify the enzyme's side chains in such a way as to produce a stable covalent bond. These are often used to study enzyme inactivation and to identify residues potentially involved in binding and catalysis.

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They first bind to the enzyme's active site in a noncovalent fashion , like rapid reversible inhibitors. However, upon formation of the enzyme-inhibitor complex (E . I), they react by various mechanisms with one or more amino acid residues in close proximity in the enzyme's active site. This results in covalent bond formation between the enzyme and the inhibitor (E-I) Usually the inhibitor contains an electrophilic moiety that labels amino acids containing nucleophilic groups.

Affinity labels: 

Affinity labels Affinity labels do not require activation by catalysis at the enzyme's active site. The covalent bond formation occurs by an SN2 alkylation-type mechanism, Schiff base formation, or acylation . Affinity labels are potentially good drugs. The presence of a reactive functional group can make them somewhat nonselective and prone toward reaction with other proteins and metabolites . If the affinity label is highly selective toward its target enzyme and has a great affinity for the enzyme's active site, this drawback can be overcome kinetically.

Example Of An Affinity Label Inhibitor Inhibition of chymotrypsin by TPCK: 

Example Of An Affinity Label Inhibitor Inhibition of chymotrypsin by TPCK TPCK N- tosyl -L- phenylalanylchloromethyl ketone ( TPCK ),

INHIBITION OF MANDELATE RACEMASE: 

INHIBITION OF MANDELATE RACEMASE ( R,S)- α - phenylglycidate MANDELATE RACEMASE is reversibly inhibited by the substrate analog atrolactate and irreversibly inhibited by (R,S)- α - phenylglycidate

BEST-KNOWN AFFINITY LABELING REAGENT IS ASPIRIN: 

BEST-KNOWN AFFINITY LABELING REAGENT IS ASPIRIN

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Prostaglandin synthase , which catalyzes the first step in the arachidonic acid cascade, is a heme protein and possesses two activities. 1) cyclooxygenase activity is used in the conversion of arachidonic acid to the bicyclic endoperoxide PGG, 2) whereas a Peroxidase activity catalyzes the subsequent reduction of PGG, to prostaglandin H 2 . Aspirin (acetylsalicylic acid) was ultimately confirmed as an inhibitor of prostaglandin synthetase . Enzymatic digest of the labeled enzyme provided evidence for the mechanism of asprin that binds to a serine residue, later identified as Ser530 , was acetylated.

Mechanism of asprin binding: 

Mechanism of asprin binding

Mechanism-Based lnhibitors: 

Mechanism-Based lnhibitors Mechanism-based inactivators have great potential as drugs because they are designed to be specific toward their target enzyme . These compounds are unreactive until activated within their target enzyme , they are expected to show little or no cellular toxicity . The design of mechanism based inhibitors requires an understanding of the binding specificity requirements for the ligand -recognition site of the enzyme.

GABA Transaminase inhibitors: 

GABA Transaminase inhibitors Vigabatrin GABA Transaminase inhibitors

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gama -Aminobutyric acid (GABA) is one of the major inhibitory neurotransmitters in the mammalian central nervous system. A decrease in the concentration of GABA had been shown to lead to convulsions. Therefore it was suggested that inhibitors of GABA transaminase , may act as antiepileptic agents, by providing an increase in the concentration of GABA in the brain. Vigabatrin , currently used as an antiepileptic drug, provides an excellent example of this approach.

Ornithine decarboxylase (ODC) inhibitors : 

Ornithine decarboxylase (ODC) inhibitors Ornithine decarboxylase (ODC), another PLP-dependent enzyme, catalyzes the ratelimiting step in the biosynthesis of polymines , i.e., the conversion of ornithine to putrescine . The enzyme is a target for drugs against African sleeping sickness caused by Typanosoma brucei . One of the currently used drugs, eflomithine (a- difluoromethylornithine , DFMO) is a mechanism-based inhibitor of ODC.

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Pyridoxal phosphate (PLP)-dependent enzymes difluoromethylornithine

Pseudoirreversible inhibitors: 

Pseudoirreversible inhibitors Pseudoirreversible inhibitors are the least common of the covalently binding enzyme inhibitors. They have some features in common with both affinity labels and mechanism-based inhibitors but they have one distinguishing feature; that is, the covalent bond formed between the enzyme and the inhibitor is reversible. Pseudoirreversible inhibitors can be broken into two classes, depending on how the active enzyme is regenerated

Class-1 Pseudoirreversible inhibitors: 

Mechanism Class-1 Pseudoirreversible inhibitors In the first class , exemplified by inhibitors of acetylcholinesterase , the enzyme is regenerated as the covalent E-I' bond is hydrolyzed.

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parathion serin

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Acetylcholine is a neurotransmitter that relays nerve impulses across the neuromuscular junction. Acetylcholinesterase ( AcChE ) rapidly breaks down acetylcholine, thereby lowering its concentration in the synaptic cleft. Agents such as parathion and sarin have found utility as insecticides and nerve gases , respectively, because they react with the enzyme to form the active-site serine-phosphate esters, and These esters are hydrolyzed extremely slowly by water , making the inhibition effectively irreversible although the inhibition can be overcome with high concentrations of strong nucleophiles such as hydroxylamine.

Class-2 Pseudoirreversible inhibitors: 

Class-2 Pseudoirreversible inhibitors The enzyme is regenerated by the inhibitor simply dissociating from the enzyme ; that is, the binding is covalent but reversible . For example, the trifluoromethyl ketone binds to AcChE as a slow-binding inhibitor.

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