inhibitor (I) inhibitor (2)

Inhibition of an enzyme on occasions leads to formation of a dead-end complex between the enzyme, co-enzyme and inhibitor rather than straightforward interaction between the inhibitor and the enzyme. 5-Fluorouracil inhibits thymidylate synthetase to form a dead-end complex with the enzyme and coenzyme, tetrahydrofolate, so preventing bacterial growth (Equation [8.7]).

C(i-tactor Z E,Z' Inhibitor'

Inhibitor (Dead end complex)

Ej Et C-D-E (metabolite)

Where product build-up progressively decreases the activity of an enzyme on its substrate, then enhancement of product inhibition (Equation [8.8]) can be achieved by inhibiting an enzyme which disposes of that product. S-Adenosylhomocysteine (SAH), the product of methylating enzymes (e.g. catecholamine methyltransferase, COMT) using S-adenosylmethionine (SAM), and an inhibitor of these enzymes, is removed by the hydrolytic action of its hydrolase (SAH'ase). Inhibitors of SAH'ase should allow a build-up of the product, SAH, leading to a useful clinical effect.

Inhibitors have been used (see Equation [8.9]) as co-drugs to protect an administered drug with a required action from the effects of a metabolizing enzyme. Inhibition of the metabolizing target enzyme permits higher plasma levels of the administered drug to persist, so prolonging its biological half-life and either preserving its effect or resulting in less frequent administration. Clavulanic acid, an inhibitor of certain p-lactamase enzymes produced by bacteria, when administered in conjunction with a p-lactamase-sensitive penicillin preserves the antibacterial action of the penicillin towards the bacteria.

8.1.2 Types of inhibitors

Enzyme inhibiting processes may be divided into two main classes, reversible and irreversible, depending upon the manner in which the inhibitor (or inhibitor residue) is attached to the enzyme.

Reversible inhibition occurs when the inhibitor is bound to the enzyme through a suitable combination of van der Waal's, electrostatic, hydrogen bonding and hydrophobic forces, the extent of the binding being determined by the equilibrium constant, K, for breakdown of the EI or EIS complex for classical inhibitors.

Reversible inhibitors may be competitive, non-competitive or uncompetitive depending upon their point of entry into the enzyme-substrate reaction scheme.

Competitive inhibitors, as their name suggests, compete with the substrate for the active site of the enzyme and by forming an inactive enzyme-inhibitor complex decrease the interaction between the enzyme and the substrate:

inactive enzyme-inhibilor complex b + S k

The rate (u) of the enzyme-catalysed reaction in the presence of a competitive inhibitor is given by;

where Km is the Michaelis constant which is the molar concentration of substrate at u = ±

which 2 . The extent to which the reaction is slowed in the presence of the inhibitor is dependent upon the inhibitor concentration [I], and the dissociation constant, K;, for the enzyme-inhibitor complex. A small value for ^ ^ - JO M) indicates strong binding of the inhibitor to the enzyme. With this type of inhibitor the inhibition may be overcome, for a fixed inhibitor concentration, by increasing the substrate concentration. This fact can be readily established by examination of Equation [8.11].

The value for Ki may be obtained by determining the initial rate of the enzyme-catalysed reaction using a fixed enzyme concentration over a suitable range of substrate concentrations in the presence and absence of a fixed concentration of the inhibitor. Rearrangement of Equation [8.11] gives

A plot of 1/u against 1/[S], known as a Lineweaver-Burk plot for the two series of experiments, gives two regression lines which cut at the same point on the 1/u axis (corresponding to 1/Vmax) but cut the 1/[S] axis at values corresponding to -/Km and -1/Km(1+[I]/Ki) in the absence and presence of the inhibitor, respectively, from which Km and Ki can be calculated.

Very often the inhibitory potency within a series of inhibitors may be expressed as an IC50 value. The IC50 value is the concentration of inhibitor required to halve the enzyme activity and this value should be used with care when comparing inter-laboratory results, since it is dependent on the concentration of substrate used (Equation [8.13]).

Non-competitive inhibitors combine with the enzyme-substrate complex and prevent the breakdown of the complex to products (Equation [8.14]). These

inhibitors do not compete with the substrate for the active site and only change the Vmax parameter for the reaction. The kinetics for this type of inhibitor are given by

The extent of the inhibition, by a fixed concentration of inhibitor, cannot be reversed by increasing the substrate concentration (contrast competitive inhibition) since substrate and inhibitor bind at different sites. A Lineweaver-Burk plot of 1/u against 1/[S] gives a straight line which cuts the 1/[S] axis at -/Km and the 1/u axis at (1+[I]/Ki)/Vmax. Other classes of reversible inhibitors are the uncompetitive and mixed inhibitors where Km and Vmax are both altered.

Nearly all reversible inhibitors which have been designed as potential drugs, as well as drugs in current use, are competitive inhibitors, one notable exception being the cardiac glycosides, which are non-competitive inhibitors of Na+, K+, -ATPase. One reason for this is that competitive inhibitors of the enzyme bear some resemblance to the substrate, since they bind at the same site, and this knowledge has provided a starting point in design, whereas the other types bind elsewhere on the enzyme and need not resemble the substrate, so removing an obvious design aspect.

A special type of competitive inhibitor is a transition state analogue. This is a stable compound which resembles in structure the substrate portion of the enzymic transition state for chemical change. An organic reaction between two types of molecules is considered to proceed through a high energy activated complex known as the transition state which is formed by collision of molecules with greater kinetic energy than the majority present in the reaction. The energy required for formation of the transition state is the activation energy for the reaction and is the barrier to the reaction occurring spontaneously. The transition state may break down to give either the components from which it was formed or the products of the reaction. The transition state for the reaction between hydroxyl ion and methyl iodide is shown in Equation [8.16]. The transition state shown depicts both commencement of formation of a C-OH bond and the breaking of the C-I bond. Enzymes catalyse organic reactions by lowering the activation energy for the reaction and one view is that they accomplish this by straining or distorting the bound substrate towards the transition state.

Equation [8.17] shows a single substrate-enzymatic reaction and the corresponding non-enzymatic reaction where ES* and S*' represent the transition states for the enzymatic and non-enzymatic reaction, respectively, and KN* and KE* are equilibrium constants, respectively, for their formation. KS is the association constant for formation of ES from

Table 8.1 Some reversible inhibitors used clinically (after Sandler and Smith,

Drug_Enzyme inhibited Clinical use_


Acetazolamide, methazolamide, dichlorphenamide, ethoxzolamide

Trimethoprim, methotrexate, pyrimethamine

Cardiac glycosides

6-Mercaptopurine, azathioprine Captopril, enalapril, cilazapril


Sodium valproate


Xanthine oxidase Gout Carbonic anhydrase Glaucoma, anti-II convulsants

Cytosine arabinoside (Ara-C), 5-fluoro-2',5'-anhydro-cytosine arabinoside

N-(Phosphonoacetyl)-L-aspartate (PALA) Indomethacin, ibuprofen, naproxen

Miconazole, clotrimazole, Ketoconazole, ticonazole Benzserazide

Dihydrofolate reductase Na+, K+,-ATP'ase Riboxyl amidotransferase Angiotensin-converting enzyme Carbonic anhydrase Succinic semialdehyde dehydrogenase Thymidine kinase and thymidylate kinase DNA, RNA polymerases

Anti-bacterial, anticancer, antiprotozoal agents Cardiac disorders Anti-cancer therapy Anti-hypertensive agent




Anti-viral agent

Anti-viral and anticancer agent

Aspartate transcarbamylase Prostaglandin synthetase cyclooxygenase I and II Sterol 14a-demethylase of fungi

AADC (peripheral)

Anti-cancer agent Anti-inflammatory


Aminoglutethimide, fadrozole, vorozole, letrozole Saquinavir Zidovudine, ddI, zalcitabine,


HIV protease HIV reverse transcriptase

Co-drug with L-dopa in

Parkinson's disease Oestrogen-mediated breast cancer

HIV infections HIV infections

TIBO derivatives Acyclovir, vidarabine, ganciclovir Naftifine terbinafine Finasteride

Mevinolin, pravastatin, synvinolim

Adriamycin, etoposide viral DNA polymerase fungal squalene epoxidase 5a-reductase HMG-CoA reductase Topoisomerase II

Herpes infections

Anti-fungals Benign prostatic hyperplasia Hyperlipidaemia

Anti-cancer agents

E and S, and KT is the association constant for the hypothetical reaction involving the binding of S*' to E. Analysis of the relationships between these equilibrium constants shows the KTKN*=KSKE*. Since the equilibrium constant for a reaction is equal to the rate constant multiplied by h/kT, where h is Planck's constant and k is Boltzmann's constant, then KT=KS(kE/kN), where kE and kN are the first-order rate constants for breakdown of the ES complex and the non-enzymatic reaction, respectively. Since the ratio kE/KN is usually of the order 1010 or greater, it follows that KT KS. This means that the transition state S*' tightly than the substrate.

is considered to bind to the enzyme at least 1010 times more

A transition state analogue is a stable compound that structurally resembles the substrate portion of the unstable transition state of an enzymic reaction. Since the bond-breaking and bond-making mechanism of the enzyme-catalysed and non-enzymatic reaction are similar, then the analogue will resemble S*' and have an enormous affinity for the enzyme compared to the substrate and consequently will be bound more tightly. It would not be possible to design a stable compound which mimics the transition state closely, since the transition state itself is unstable by possessing partially broken and/or made covalent bonds. Even crude transition state analogues of substrate reactions would be expected to be sufficiently tightly bound to the enzyme to be excellent reversible inhibitors. This expectation has been borne out in practice.

Design of a transition state analogue for a specific enzyme requires a knowledge of the mechanism of the enzymatic reaction. Fortunately, the main structural features of the transition states for the majority of enzymatic reactions are either known or can be predicted with some confidence.

Another class of competitive inhibitor which binds tightly to the enzyme is the slow, tight-binding inhibitor. These may be bound either noncovalently or covalently and are released very slowly from the enzyme because of the tight interaction. The slow binding is a time-dependent process and is believed to be due either to an enforced conformational change in the enzyme structure or reversible, covalent bond formation. Coformycin, methotrexate and allopurinol belong to this class and are useful drugs. Tight binding, where the dissociation from the complex takes days, is not distinguishable in effect from covalent bonding and this type of inhibitor may be classed as an irreversible inhibitor.

Compounds producing irreversible enzyme inhibition fall into two groups; active site-directed (affinity labelling) inhibitors and mechanism-based inactivators (¿cat inhibitors, suicide substrates).

Active site-directed irreversible inhibitors resemble the substrate sufficiently to form a reversible enzyme-inhibitor complex, analogous to the enzyme-substrate complex, within which reaction occurs between functional groups on the inhibitor and enzyme. A stable covalent bond is formed with irreversible inhibition of the enzyme. Active site-directed irreversible inhibitors are designed to exhibit specificity towards their target enzymes, since they are structurally modelled on the specific substrate of the enzyme concerned.

In the previous discussion on reversible inhibitors, the potency of an inhibitor was shown to be reflected in the Ki value, which is characteristic of the inhibitor and independent of inhibitor concentration. However, the actual level of inhibition achieved in an enzyme system involves the use of equations into which inhibitor and substrate concentrations, as well as the Km value for the substrate, need to be incorporated. Similarly, the potency of an irreversible inhibitor is given by binding and rate constants which are both independent of inhibitor concentration. This allows a precise comparison of the relative potency of inhibitors, which is necessary in the design and development of more effective inhibitors of an enzyme.

Irreversible inhibition of an enzyme by an active site-directed inhibitor can be represented by

provided that complex formation between the inhibitor and the enzyme is ignored here for the present time. The reaction is bimolecular, but, since the inhibitor is usually present in large excess of the enzyme concentration, the kinetics for inactivation of the enzyme follow a pseudo first-order reaction.

In the general case of a bimolecular reaction between two compounds A and B, the rate of reaction is given by

where k2 is the second-order rate constant, a and b are the initial concentrations of A and B, respectively, and the concentration of product is x at time t. Integration and rearrangement of Equation [8.19] gives

In the situation where a b, this simplifies to

Since k2a=k1 where k1 is the pseudo first-order reaction rate constant, then k^^llcg. "

A plot of log (b-x) versus t for the reaction as it proceeds, using a known concentration of the inhibitor, gives a regression line with slope=-k1/2.303, from which k1 and k2 may be obtained.

In practice in enzyme inhibition reactions it is sometimes found that k1 is not directly proportional to a so that the value of k2 is not constant with a change in the concentration of the inhibitor a. This is due to initial binding of the inhibitor to the active site of the enzyme before the irreversible inhibition reaction occurs.

The rate of the inactivation reaction is given by

where x represents the concentration of the inhibited enzyme (EI), K; is the dissociation constant for the enzyme-inhibitor complex and k+2 is the first-order rate constant for the breakdown of the complex into products. Integration of Equation [8.24] gives k,t = !t) E - In (E - 5t)

where ki is the observed first-order rate constant and

When Equation [8.26] is written in the reciprocal form (8.27)

A plot of 1/k1 against 1/[I] gives a regression line from which k+2 and Ki may be evaluated, since the intercepts on the 1/k1 and 1/[I] axes give the values for 1/k+2 and -1/Ki, respectively.

Many irreversible inhibitors of certain enzymes have previously been recognized in which the range of electrophilic centres normally associated with active site-directed irreversible inhibitors, e.g. -COCH2Cl, -COCHN2, -OCONHR, -SO2F, are absent so that the means by which they inhibited the enzyme was not understood. The action of these inhibitors has now become understandable since they have been characterized as mechanism-based enzyme inactivators. Mechanism-based enzyme inactivators bind to the enzyme through the Ks parameter and are modified by the enzyme in such a way as to generate a reactive group which irreversibly inhibits the enzyme by forming a covalent bond with a functional group present at the active site. On occasion, catalysis leads not to a reactive species but an enzyme-intermediate complex which is partitioned away from the catalytic pathway to a more stable complex by bond rearrangement (e.g P-lactamase inhibitors).

These inhibitors are substrates of the enzyme, as suggested by their alternative name, kcat inhibitors, where kcat is the overall rate constant for the decomposition of the enzyme-substrate complex in an enzyme-catalysed reaction. Mechanism-based inactivators do not generate a reactive electrophilic centre until acted upon by the target enzyme. Reaction may then occur with a nucleophile on the enzyme surface, or alternatively the species may be released and either react with external nucleophiles or decompose (Equation [8.28]).

The ratio of the rate constants i.e. k+4/k+3 gives the partition ratio (r) for the reaction and where this approaches zero the mechanism-based inactivation will proceed with little turnover of the inhibitor and release of the active species as shown in Equation [8.29] where the non-covalent enzyme-inhibitor complex (EI) is transformed into an activated species (EI*) which then irreversibly inhibits the enzyme.

Consequently, the reactive electrophilic species, by not being free to react with other molecules in the biological media, has a high degree of specificity for its target enzyme and exhibits low toxicity.

The inactivation rate constant for a mechanism-based enzyme inactivation is termed kinact and is a complex mixture of the rate constants k2, k3 and k4 (Equation [8.28]). However the kinetic form of Equation [8.28] and that for active-site directed inhibition are identical so that Equation [8.27] becomes,

which, since becomes,


A plot of t1/2 versus the reciprical of the inhibitor concentration for the inactivation process using various concentration of the inactivator gives a regression line which cuts the y axis at 0.693/kinact and the x axis at -1/Ki. The meaning of Ki described here and Ki the dissociation for the enzyme—reversible inhibitor complex may not be the same under certain conditions e.g. when k3 becomes rate deterining. Certain criteria need to be fulfilled before an irreversible inhibitor can be classified as a 'mechanism' based enzyme inactivator (see Silverman).


8.2.1 Target enzyme and inhibitor selection

Occasionally, drugs in current use for one therapeutic purpose have exhibited side-effects indicative of potential usefulness for another, subsequent work establishing that the newly-discovered drug effect is due to inhibition of a particular enzyme. Although the drug may possess minimal therapeutic usefulness in its newly found role, it does constitute an important 'lead' compound for the development of analogues with improved clinical characteristics.

The use of sulphanilamide as an antibacterial was associated with acidosis in the body due to its inhibition of renal carbonic anhydrase. This observation led to the development of the currently little used acetazolamide and subsequently the important chlorthiazide group of diuretics although these have a different mode of action.

The anticonvulsant aminoglutethide was withdrawn from the market due to inhibition of steroidogenesis and an insufficiency of 110-hydroxy steroids. Aminoglutethimide, in conjunction with supplementary hydrocortisone, is now in clinical use for the treatment of oestrogen-dependent breast cancer in postmenopausal women due to its ability to inhibit aromatase, which is responsible for the production of oestrogens from androstenedione. Other more potent aromatase inhibitors have subsequently been developed (see Section 8.6.3).

Iproniazid, initially used as a drug in the treatment of tuberculosis, was observed to be a central nervous stimulant due to a mild inhibitory effect on MAO. This observation eventually led to the discovery of more potent inhibitors of MAO, such as phenelzine, tranylcypromine, selegiline ((-)-deprenyl) and chlorgyline.

Many drugs introduced into therapy following detection of biological activity by pharmacological or microbiological screening experiments have subsequently been shown to exert their action by inhibition of a specific enzyme in the animal or parasite. This knowledge has helped in the development of clinically more useful drugs by limiting screening tests to involve only the isolated pure or partially purified target enzyme concerned and so introducing a more rapid screening protocol. However translation of in vitro potency to the in vivo situation and finally the clinic is thwart with difficulties as will be seen later (also see Chapter 6).

The rational design of an enzyme inhibitor for a particular disease or condition in the absence of a lead compound presents a challenging task to the drug designer, since selection of a suitable target enzyme is a necessary first step in the process of drug design. A priori examination of the biochemical or physiological processes responsible for a disease or condition, where these are known or can be guessed at, may point to a suitable target enzyme in its biochemical environment, the inhibition of which would rationally be expected to lead to alleviation or removal of the disease or condition.

In a chain of reactions with closely packed enzymes in a steady state (see Equation [8.32]), where the initial substrate A does not undergo a change in concentration as a consequence of changes effected elsewhere in the chain, then any type of reversible inhibitor which inhibits the first step of the chain will effectively block that sequence of reactions.

It is a general misconception that the overall rate in a linear chain can be depressed only by inhibiting the rate limiting reaction, i.e. the one with lowest velocity at saturation with its substrate. Since individual enzymes will not be saturated with their substrates, the overall rate is determined largely by the concentration of the initial substrate, so that the first enzyme will often be rate limiting, irrespective of its potential rate due to a low concentration of its substrate. Inhibitors acting at later points in the chain of closely bound enzymes may not block the metabolic pathway. If E;

the reaction 0-C (Equation [8.32]) is considered, competitive inhibition of E2

will initially decrease the rate of formation of C but eventually the original velocity (u2) of the step will be attained as the concentration of B rises due to the difference between its rates of formation and consumption. However, selection of a target enzyme within a metabolic chain which does not inhibit the first step may lead successfully to translation of in vitro results, with the isolated target enzyme, to the in vivo situation due to additional changes. These changes relate to an increase in concentration of B which may have secondary effects on the chain due to product inhibition (B on Ei) or product reversal ^ B); either of these effects can slow u1, so leading to a slowing of the overall pathway.

This view is well illustrated by studies on inhibitors of the noradrenaline biosynthetic pathway. These were intended to decrease production of noradrenaline at the nerve-capillary junction in hypertensive patients, with an associated reduction in blood pressure. The selected target enzyme aromatic amino acid decarboxylase (AADC) catalyses the conversion of dopa to dopamine in the second step of the biosynthesis of noradrenaline from tyrosine. Many reversible inhibitors, although active in vitro against this enzyme, fail to lower noradrenaline production in vivo although they may slow decarboxylation of dopa in peripheral tissues. Irreversible inhibitors of AADC successfully lower noradrenaline levels (see later).

However, competitive inhibitors have proved useful clinical agents, as examination of Table 8.1 illustrates, especially where the target enzyme has a degradative role on a substrate and is not part of the metabolic pathway in which the substrate is produced.

Examples here are the anticholinesterases (Equation [8.3]) and AADC inhibitors as L—dopa protecting agents in the treatment of Parkinson's disease.

Irreversible inhibition progressively decreases the titre of the target enzyme to a low level and the biochemical environment of the enzyme is unimportant. For example a-monofluoromethyldopa is a mechanism-based inactivator of AADC and produces a metabolite which irreversibly inhibits and decreases the level of the enzyme by >99% (see Section This leads to a near complete depletion of catecholamine levels in brain, heart and kidney despite the occurrence of the enzyme in the second step of the noradrenaline biosynthetic pathway as discussed earlier.

The production of inhibited enzyme must be faster than the generation of new enzyme by resynthesis to maintain the target enzyme titre at a low level so that dosing is infrequent. For mechanism based inactivators, not only is the turnover rate of the enzyme important because of enzyme resynthesis, and this rate may be 103-105 slower than for natural substrates, but the partition ratio for the reaction should ideally be close to zero when every turnover should result in inhibition. A list of drugs which act by irreversible inhibition of the enzyme is given in Table 8.2.

8.2.2 Specificity and toxicity

Inhibitors used in therapy must show specificity towards the target enzyme. Inhibition of closely related enzymes with different biological roles (e.g. trypsin-like enzymes such as thrombin, plasmin and kallikrein), or reaction with constituents essential for the well-being of the body (e.g. DNA, glutathione, liver P-450 metabolizing enzymes) could lead to serious side-effects.

Table 8.2 Some irreversible inhibitors used clinically (after Sandler and Smith 1989).


Enzyme inhibited Clinical use

Omeprazole Sulphonamides

H+, K+-ATPase Dihydropteroate synthetase Iproniazid, phenelzine, MAO

isocarboxazid, tranylcypromine Neostigmine, eserine, dyflos, Acetylcholinesterase benzpyrinium, ecothiopate, tacrine

Penicillins, cephalosporins, cephamycins, carbapenems, monobactams Organic-arsenicals



Pyruvate dehydrogenase Alanine racemase

Anti-ulcer agent Anti-bacterial


Glaucoma, myasthenia gravis Alzheimers disease Antibiotics

Anti-protozoal agents


D-Cycloserine Azaserine

Y-Vinyl GABA (Vigabatrin) Clavulanic acid, sulbactam a-Difluoromethylornithine,


Alanine racemase

Formylglycinamide ribonucleotide aminotransferase

GABA transaminase


Antibiotic Anti-cancer

L-Ornithine decarboxylase

Selegiline ((-)-deprenyl) MAO-B

4-Hydroxyandrostendione Aromatase

Thymidilate synthetase

Epilepsy Adjuvant to penicillin antibiotic Trypanosomal and other parasitic diseases

Co-drug with L-dopa in Parkinson's disease Oestrogen-mediated breast cancer Anti-cancer

Active site-directed irreversible inhibitors are alkylating or acylating agents and would be expected to react with a range of tissue constituents containing amino or thiol groups besides the target enzyme, with potentially serious side-effects. They are mainly used in in vitro studies for labelling of amino acid residues at the active site.

Mechanism-based inactivators do not possess a biologically reactive functional group until after they have been modified by the target enzyme and, consequently, would be expected to demonstrate high specificity of action and low incidence of adverse reactions. It is these features which have encouraged their active application in inhibitor design studies.

In the situation where the target enzyme is common to the host's normal cells as well as to cancerous or parasitic cells, chemotherapy can be successful when host and parasitic cells contain different isoenzymes, e.g. DHFR, with that of the parasite being more susceptible to carefully designed inhibitors. Alternatively, the target enzyme may be absent from the host cell. Sulphonamides are toxic to bacterial cells by inhibiting dihydropteroate synthetase, an enzyme on the biosynthetic pathway to folic acid. The host cell is unaffected, since it utilizes preformed folic acid whilst the susceptible bacterial cannot. Sulphonamides (8.1) are toxic to bacterial cells by inhibiting the utilization of p-aminobenzoate (8.2) by dihydropteroated synthetase, an enzyme in the biosynthetic pathway to dihydrofolic acid.

Normal and cancerous cells contain the same form of the target enzyme, DHFR, but the faster rate of growth of the tumour cells makes them more susceptible to the effects of h£N



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