Enzyme inhibitor

An enzyme inhibitor is a molecule that binds to an enzyme and blocks its activity. Enzymes are proteins that speed up chemical reactions necessary for life, in which substrate molecules are converted into products. An enzyme facilitates a specific chemical reaction by binding the substrate to its active site, a specialized area on the enzyme that accelerates the most difficult step of the reaction.

An enzyme inhibitor hinders ("inhibits") this process, either by binding to the enzyme's active site (thus preventing the substrate itself from binding) or by binding to another site on the enzyme such that the enzyme's catalysis of the reaction is blocked. Enzyme inhibitors may bind reversibly or irreversibly. Irreversible inhibitors form a chemical bond with the enzyme such that the enzyme is inhibited until the chemical bond is broken. By contrast, reversible inhibitors bind non-covalently and may spontaneously leave the enzyme, allowing the enzyme to resume its function. Reversible inhibitors produce different types of inhibition depending on whether they bind to the enzyme, the enzyme-substrate complex, or both.

Enzyme inhibitors play an important role in all cells, since they are generally specific to one enzyme and serve to control that enzyme's activity. For example, enzymes in a metabolic pathway may be inhibited by molecules produced later in the pathway, thus curtailing the production of molecules that are no longer needed. This type of negative feedback is an important way to maintain homeostasis in a cell. Enzyme inhibitors also control essential enzymes that, if unchecked, may damage a cell, such as proteases or nucleases. A well-characterised example of this is the ribonuclease inhibitor, which binds to ribonucleases in one of the tightest known protein–protein interactions.[1] Many poisons found in animals are enzyme inhibitors that block the activity of a crucial enzyme in the victim.

Many drug molecules are enzyme inhibitors that inhibit an aberrant human enzyme or an enzyme critical for the survival of a pathogen such as a virus, bacterium or parasite. Examples include methotrexate (used in chemotherapy and in treating rheumatic arthritis) as well as the protease inhibitors used to treat AIDS. Since anti-pathogen inhibitors generally target only one enzyme, such drugs are highly specific and generally produce few side effects in humans, provided that no analogous enzyme is found in humans. (This is often the case, since such pathogens and humans are genetically distant.) Medicinal enzyme inhibitors often have low dissociation constants, meaning that only a minute amount of the inhibitor is required to inhibit the enzyme. A low concentration of the enzyme inhibitor reduces the risk for liver and kidney damage and other adverse drug reactions in humans. Hence the discovery and refinement of enzyme inhibitors is an active area of research in biochemistry and pharmacology.[2]

Reversible inhibitors attach to enzymes with non-covalent interactions such as hydrogen bonds, hydrophobic interactions and ionic bonds. Multiple weak bonds between the inhibitor and the active site combine to produce strong and specific binding. In contrast to irreversible inhibitors, reversible inhibitors generally do not undergo chemical reactions when bound to the enzyme and can be easily removed by dilution or dialysis. A special case are covalent reversible inhibitors that covalently bind to the enzyme, but the chemical reaction that forms the bond is fully reversible.[3]

There are four kinds of reversible enzyme inhibitors. They are classified according to the effect of the inhibitor on the Vmax (maximum reaction rate catalyzed by the enzyme) and Km (the concentration of substrate resulting in half maximal enzyme activity) as the concentration of the enzyme's substrate is varied.[4][5][6]

In competitive inhibition, the substrate and inhibitor cannot bind to the enzyme at the same time,[8]: 134  as shown in the figure on the right. This usually results from the inhibitor having an affinity for the active site of an enzyme where the substrate also binds; the substrate and inhibitor compete for access to the enzyme's active site. This type of inhibition can be overcome by sufficiently high concentrations of substrate (Vmax remains constant), i.e., by out-competing the inhibitor.[8]: 134–135  However, the apparent Km will increase as it takes a higher concentration of the substrate to reach the Km point, or half the Vmax. Competitive inhibitors are often similar in structure to the real substrate (see for example the folic acid methotrexate comparison below).[8]: 134 

In uncompetitive inhibition, the inhibitor binds only to the enzyme-substrate complex.[8]: 139  This type of inhibition causes Vmax to decrease (maximum velocity decreases as a result of removing activated complex) and Km to decrease (due to better binding efficiency as a result of Le Chatelier's principle and the effective elimination of the ES complex thus decreasing the Km which indicates a higher binding affinity).[9]

In non-competitive inhibition, the binding of the inhibitor to the enzyme reduces its activity but does not affect the binding of substrate. As a result, the extent of inhibition depends only on the concentration of the inhibitor. Vmax will decrease due to the inability for the reaction to proceed as efficiently, but Km will remain the same as the actual binding of the substrate, by definition, will still function properly.[10]

In mixed inhibition, the inhibitor can bind to the enzyme at the same time as the enzyme's substrate. However, the binding of the inhibitor affects the binding of the substrate, and vice versa.[8]: 136–138  This type of inhibition can be reduced, but not overcome by increasing concentrations of substrate. Although it is possible for mixed-type inhibitors to bind in the active site, this type of inhibition generally results from an allosteric effect where the inhibitor binds to a different site on an enzyme. Inhibitor binding to this allosteric site changes the conformation (i.e., tertiary structure or three-dimensional shape) of the enzyme so that the affinity of the substrate for the active site is reduced.

These types can also be distinguished by the effect of increasing the substrate concentration [S] on the degree of inhibition caused by a given amount of inhibitor. For competitive inhibition the degree of inhibition is reduced by increasing [S], for noncompetitive inhibition the degree of inhibition is unchanged, and for uncompetitive (also called anticompetitive) inhibition the degree of inhibition increases with [S].[11]

Reversible inhibition can be described quantitatively in terms of the inhibitor's binding to the enzyme and to the enzyme-substrate complex, and its effects on the kinetic constants of the enzyme.[12]: 6  In the classic Michaelis-Menten scheme below, an enzyme (E) binds to its substrate (S) to form the enzyme–substrate complex ES. Upon catalysis, this complex breaks down to release product P and free enzyme.[12]: 55  The inhibitor (I) can bind to either E or ES with the dissociation constants Ki or Ki', respectively.[12]: 87 

When an enzyme has multiple substrates, inhibitors can show different types of inhibition depending on which substrate is considered. This results from the active site containing two different binding sites within the active site, one for each substrate. For example, an inhibitor might compete with substrate A for the first binding site, but be a non-competitive inhibitor with respect to substrate B in the second binding site.[14]

Traditionally reversible enzyme inhibitors have been classified as competitive, uncompetitive, or non-competitive, according to their effects on Km and Vmax.[7] These different effects result from the inhibitor binding to the enzyme E, to the enzyme–substrate complex ES, or to both, respectively. The division of these classes arises from a problem in their derivation and results in the need to use two different binding constants for one binding event.[15] The binding of an inhibitor and its effect on the enzymatic activity are two distinctly different things, another problem the traditional equations fail to acknowledge. In noncompetitive inhibition the binding of the inhibitor results in 100% inhibition of the enzyme only, and fails to consider the possibility of anything in between.[15] The common form of the inhibitory term also obscures the relationship between the inhibitor binding to the enzyme and its relationship to any other binding term be it the Michaelis–Menten equation or a dose response curve associated with ligand receptor binding. To demonstrate the relationship the following rearrangement can be made:[16]

This rearrangement demonstrates that similar to the Michaelis–Menten equation, the maximal rate of reaction depends on the proportion of the enzyme population interacting with its substrate.

the effect of the inhibitor is a result of the percent of the enzyme population interacting with inhibitor. The only problem with this equation in its present form is that it assumes absolute inhibition of the enzyme with inhibitor binding, when in fact there can be a wide range of effects anywhere from 100% inhibition of substrate turn over to just >0%. To account for this the equation can be easily modified to allow for different degrees of inhibition by including a delta Vmax term.

This term can then define the residual enzymatic activity present when the inhibitor is interacting with individual enzymes in the population. However the inclusion of this term has the added value of allowing for the possibility of activation if the secondary Vmax term turns out to be higher than the initial term. To account for the possibly of activation as well the notation can then be rewritten replacing the inhibitor "I" with a modifier term (stimulator or inhibitor) denoted here as "X".[16]: eq 13 

While this terminology results in a simplified way of dealing with kinetic effects relating to the maximum velocity of the Michaelis–Menten equation, it highlights potential problems with the term used to describe effects relating to the Km. The Km relating to the affinity of the enzyme for the substrate should in most cases relate to potential changes in the binding site of the enzyme which would directly result from enzyme inhibitor interactions. As such a term similar to the one proposed above to modulate Vmax should be appropriate in most situations:[16]: eq 14 

Lineweaver–Burk diagrams of different types of reversible enzyme inhibitors. The arrow shows the effect of increasing concentrations of inhibitor.

An enzyme inhibitor is characterised by its two dissociation constants, Ki and Ki', to the enzyme and to the enzyme-substrate complex, respectively.[17]: Glossary  The enzyme-inhibitor constant Ki can be measured directly by various methods; one especially accurate method is isothermal titration calorimetry, in which the inhibitor is titrated into a solution of enzyme and the heat released or absorbed is measured.[18] However, the other dissociation constant Ki' is difficult to measure directly, since the enzyme-substrate complex is short-lived and undergoing a chemical reaction to form the product. Hence, Ki' is usually measured indirectly, by observing the enzyme activity under various substrate and inhibitor concentrations, and fitting the data[19] to a modified Michaelis–Menten equation[20]

where the modifying factors α and α' are defined by the inhibitor concentration and its two dissociation constants

Thus, in the presence of the inhibitor, the enzyme's effective Km and Vmax become (α/α')Km and (1/α')Vmax, respectively. However, the modified Michaelis-Menten equation assumes that binding of the inhibitor to the enzyme has reached equilibrium, which may be a very slow process for inhibitors with sub-nanomolar dissociation constants. In these cases, it is usually more practical to treat the tight-binding inhibitor as an irreversible inhibitor (see below); however, it can still be possible to estimate Ki' kinetically if Ki is measured independently.

The effects of different types of reversible enzyme inhibitors on enzymatic activity can be visualised using graphical representations of the Michaelis–Menten equation, such as Lineweaver–Burk plots, Eadie-Hofstee plots or Hanes-Woolf plots. An illustration is provided by the three Lineweaver–Burk plots depicted in the adjacent figure on the right. In the top diagram, the competitive inhibition lines intersect on the y-axis, illustrating that such inhibitors do not affect Vmax. In the bottom diagram, the non-competitive inhibition lines intersect on the x-axis, showing these inhibitors do not affect Km. However, since it can be difficult to estimate Ki and Ki' accurately from such plots,[21] it is advisable to estimate these constants using more reliable nonlinear regression methods.

The mechanism of partially competitive inhibition is similar to that of non-competitive, 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.[9]

Substrate and product inhibition is where either the substrate or product of an enzyme reaction inhibit 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.[2] At low substrate, the high-affinity site is occupied and normal kinetics are followed. However, at higher concentrations, the second inhibitory site becomes occupied, inhibiting the enzyme.[22] Product inhibition is often a regulatory feature in metabolism and can be a form of negative feedback.

Peptide-based HIV-1 protease inhibitor ritonavir with substrate binding sites located in enzyme labeled as S2, S1, S1', and S2'

Slow-tight inhibition occurs when the initial enzyme–inhibitor complex EI undergoes isomerisation to a second more tightly held complex, EI*, but the overall inhibition process is reversible. This manifests itself as slowly increasing enzyme inhibition. Under these conditions, traditional Michaelis–Menten kinetics give a false value for Ki, which is time–dependent.[2] The true value of Ki can be obtained through more complex analysis of the on (kon) and off (koff) rate constants for inhibitor association. See irreversible inhibition below for more information.

Bi-substrate analogue inhibitors are high affinity and selectivity inhibitors that can be prepared for enzymes that catalyze bi-molecular reactions by capturing the binding energy of each substrate into one molecule.[23][24] For example, in the formyl transfer reactions of purine biosynthesis, a potent multi-substrate adduct inhibitor (MAI) to GAR TFase was prepared synthetically by linking analogues of the glycinamide ribonucleotide (GAR) substrate and the N-10-formyl tetrahydrofolate cofactor together to produce thioglycinamide ribonucleotide dideazafolate (TGDDF),[25] or enzymatically from the natural GAR substrate to yield GDDF.[26] Here the subnanomolar dissociation constant (KD) of TGDDF was greater than predicted presumably due to entropic advantages gained and/or positive interactions acquired through the atoms linking the components. MAIs have also been observed to be produced in cells by reactions of pro-drugs such as isoniazid[27] or enzyme inhibitor ligands (e.g., PTC124) [28] with cellular cofactors such as NADH and ATP respectively.

As enzymes have evolved to bind their substrates tightly, and most reversible inhibitors bind in the active site of enzymes, it is unsurprising that some of these inhibitors are strikingly similar in structure to the substrates of their targets. Inhibitors of DHFR are prominent examples. Other example of these substrate mimics are the protease inhibitors, a very successful class of antiretroviral drugs used to treat HIV.[29] The structure of ritonavir, a protease inhibitor based on a peptide and containing three peptide bonds, is shown on the right. As this drug resembles the protein that is the substrate of the HIV protease, it competes with this substrate in the enzyme's active site.

Enzyme inhibitors are often designed to mimic the transition state or intermediate of an enzyme-catalyzed reaction. This ensures that the inhibitor exploits the transition state stabilising 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 the antiviral drug oseltamivir; this drug mimics the planar nature of the ring oxonium ion in the reaction of the viral enzyme neuraminidase.[30]

However, not all inhibitors are based on the structures of substrates. For example, the structure of another HIV protease inhibitor tipranavir is shown on the left. This molecule is not based on a peptide and has no obvious structural similarity to a protein substrate. These non-peptide inhibitors can be more stable than inhibitors containing peptide bonds, because they will not be substrates for peptidases and are less likely to be degraded.[31]

In drug design it is important to consider the concentrations of substrates to which the target enzymes are exposed. For example, some protein kinase inhibitors have chemical structures that are similar to adenosine triphosphate, one of the substrates of these enzymes. However, drugs that are simple competitive inhibitors will have to compete with the high concentrations of ATP in the cell. Protein kinases can also be inhibited by competition at the binding sites where the kinases interact with their substrate proteins, and most proteins are present inside cells at concentrations much lower than the concentration of ATP. As a consequence, if two protein kinase inhibitors both bind in the active site with similar affinity, but only one has to compete with ATP, then the competitive inhibitor at the protein-binding site will inhibit the enzyme more effectively.[32]

Reaction of the irreversible inhibitor diisopropylfluorophosphate (DFP) with a serine protease

Irreversible inhibitors covalently bind to an enzyme, and this type of inhibition can therefore not be readily reversed.[33] Irreversible inhibitors often contain reactive functional groups such as nitrogen mustards, aldehydes, haloalkanes, alkenes, Michael acceptors, phenyl sulfonates, or fluorophosphonates. These electrophilic groups react with amino acid side chains to form covalent adducts.[33] The residues modified are those with side chains containing nucleophiles such as hydroxyl or sulfhydryl groups; these include the amino acids serine (as in DFP, right), cysteine, threonine, or tyrosine.[34]

Irreversible inhibition is different from irreversible enzyme inactivation. Irreversible inhibitors are generally specific for one class of enzyme and do not inactivate all proteins; they do not function by destroying protein structure but by specifically altering the active site of their target. For example, extremes of pH or temperature usually cause denaturation of all protein structure, but this is a non-specific effect. Similarly, some non-specific chemical treatments destroy protein structure: for example, heating in concentrated hydrochloric acid will hydrolyse the peptide bonds holding proteins together, releasing free amino acids.[35]

Irreversible inhibitors display time-dependent inhibition and their potency therefore cannot be characterised by an IC50 value.[2][36] This is because the amount of active enzyme at a given concentration of irreversible inhibitor will be different depending on how long the inhibitor is pre-incubated with the enzyme. Instead, kobs/[I] values are used,[37] where kobs is the observed pseudo-first order rate of inactivation (obtained by plotting the log of % activity vs. time) and [I] is the concentration of inhibitor. The kobs/[I] parameter is valid as long as the inhibitor does not saturate binding with the enzyme (in which case kobs = kinact).

Irreversible inhibitors first form a reversible non-covalent complex with the enzyme (EI or ESI). Subsequently a chemical reaction occurs between the enzyme and inhibitor to produce the covalently modified "dead-end complex" EI* (an irreversible covalent complex). The rate at which EI* is formed is called the inactivation rate or kinact.[3] Since formation of EI may compete with ES, binding of irreversible inhibitors can be prevented by competition either with substrate or with a second, reversible inhibitor. This protection effect is good evidence of a specific reaction of the irreversible inhibitor with the active site.

The binding and inactivation steps of this reaction are investigated by incubating the enzyme with inhibitor and assaying the amount of activity remaining over time. The activity will be decreased in a time-dependent manner, usually following exponential decay. Fitting these data to a rate equation gives the rate of inactivation at this concentration of inhibitor. This is done at several different concentrations of inhibitor. If a reversible EI complex is involved the inactivation rate will be saturable and fitting this curve will give kinact and Ki.[38]

Another method that is widely used in these analyses is mass spectrometry. Here, accurate measurement of the mass of the unmodified native enzyme and the inactivated enzyme gives the increase in mass caused by reaction with the inhibitor and shows the stoichiometry of the reaction.[39] This is usually done using a MALDI-TOF mass spectrometer. In a complementary technique, peptide mass fingerprinting involves digestion of the native and modified protein with a protease such as trypsin. This will produce a set of peptides that can be analysed using a mass spectrometer. The peptide that changes in mass after reaction with the inhibitor will be the one that contains the site of modification.

Not all irreversible inhibitors form covalent adducts with their enzyme targets. Some reversible inhibitors bind so tightly to their target enzyme that they are essentially irreversible. These tight-binding inhibitors may show kinetics similar to covalent irreversible inhibitors. In these cases, some of these inhibitors rapidly bind to the enzyme in a low-affinity EI complex and this then undergoes a slower rearrangement to a very tightly bound EI* complex (see figure above). This kinetic behaviour is called slow-binding.[41] This slow rearrangement after binding often involves a conformational change as the enzyme "clamps down" around the inhibitor molecule. Examples of slow-binding inhibitors include some important drugs, such methotrexate,[42] allopurinol,[43] and the activated form of acyclovir.[44]

Trypanothione reductase with the lower molecule of an inhibitor bound irreversibly and the upper one reversibly. Created from .

Diisopropylfluorophosphate (DFP) is shown as an example of an irreversible protease inhibitor in the figure above right. The enzyme hydrolyses the phosphorus–fluorine bond, but the phosphate residue remains bound to the serine in the active site, deactivating it.[45] Similarly, DFP also reacts with the active site of acetylcholine esterase in the synapses of neurons, and consequently is a potent neurotoxin, with a lethal dose of less than 100 mg.[46]

Suicide inhibition is an unusual type of irreversible inhibition where the enzyme converts the inhibitor into a reactive form in its active site.[47] An example is the inhibitor of polyamine biosynthesis, α-difluoromethylornithine or DFMO, which is an analogue of the amino acid ornithine, and is used to treat African trypanosomiasis (sleeping sickness). Ornithine decarboxylase can catalyse the decarboxylation of DFMO instead of ornithine, as shown above. However, this decarboxylation reaction is followed by the elimination of a fluorine atom, which converts this catalytic intermediate into a conjugated imine, a highly electrophilic species. This reactive form of DFMO then reacts with either a cysteine or lysine residue in the active site to irreversibly inactivate the enzyme.[40]

Since irreversible inhibition often involves the initial formation of a non-covalent EI complex,[3] it is sometimes possible for an inhibitor to bind to an enzyme in more than one way. For example, in the figure showing trypanothione reductase from the human protozoan parasite Trypanosoma cruzi, two molecules of an inhibitor called quinacrine mustard are bound in its active site. The top molecule is bound reversibly, but the lower one is bound covalently as it has reacted with an amino acid residue through its nitrogen mustard group.[48]

Enzyme inhibitors are found in nature[49] and also produced artificially in the laboratory.[50] Naturally occurring enzyme inhibitors regulate many metabolic processes and are essential for life.[51] In addition, naturally produced poisons are often enzyme inhibitors that have evolved for use as toxic agents against predators, prey, and competing organisms.[52] These natural toxins include some of the most poisonous substances known. Artificial inhibitors are often used as drugs, but can also be insecticides such as malathion, herbicides such as glyphosate,[53] or disinfectants such as triclosan. Other artificial enzyme inhibitors block acetylcholinesterase, an enzyme which breaks down acetylcholine, and are used as nerve agents in chemical warfare.[54]

Enzyme inhibitors are also important in metabolic control.[51] Many metabolic pathways in the cell are inhibited by metabolites that control enzyme activity through allosteric regulation or substrate inhibition. A good example is the allosteric regulation of the glycolytic pathway. This catabolic pathway consumes glucose and produces ATP, NADH and pyruvate. A key step for the regulation of glycolysis is an early reaction in the pathway catalysed by phosphofructokinase-1 (PFK1). When ATP levels rise, ATP binds an allosteric site in PFK1 to decrease the rate of the enzyme reaction; glycolysis is inhibited and ATP production falls. This negative feedback control helps maintain a steady concentration of ATP in the cell. However, metabolic pathways are not just regulated through inhibition since enzyme activation is equally important. With respect to PFK1, fructose 2,6-bisphosphate and ADP are examples of metabolites that are allosteric activators.[55]

Physiological enzyme inhibition can also be produced by specific protein inhibitors. This mechanism occurs in the pancreas, which synthesises many digestive precursor enzymes known as zymogens. Many of these are activated by the trypsin protease, so it is important to inhibit the activity of trypsin in the pancreas to prevent the organ from digesting itself. One way in which the activity of trypsin is controlled is the production of a specific and potent trypsin inhibitor protein in the pancreas. This inhibitor binds tightly to trypsin, preventing the trypsin activity that would otherwise be detrimental to the organ.[56] Although the trypsin inhibitor is a protein, it avoids being hydrolysed as a substrate by the protease by excluding water from trypsin's active site and destabilising the transition state.[57] Other examples of physiological enzyme inhibitor proteins include the barstar inhibitor of the bacterial ribonuclease barnase.[58]

Animals and plants have evolved to synthesise a vast array of poisonous products including secondary metabolites, peptides and proteins that can act as inhibitors. Natural toxins are usually small organic molecules and are so diverse that there are probably natural inhibitors for most metabolic processes.[59] The metabolic processes targeted by natural poisons encompass more than enzymes in metabolic pathways and can also include the inhibition of receptor, channel and structural protein functions in a cell. For example, paclitaxel (taxol), an organic molecule found in the Pacific yew tree, binds tightly to tubulin dimers and inhibits their assembly into microtubules in the cytoskeleton.[60]

Many natural poisons act as neurotoxins that can cause paralysis leading to death and have functions for defence against predators or in hunting and capturing prey. Some of these natural inhibitors, despite their toxic attributes, are valuable for therapeutic uses at lower doses.[61] An example of a neurotoxin are the glycoalkaloids, from the plant species in the family Solanaceae (includes potato, tomato and eggplant), that are acetylcholinesterase inhibitors. Inhibition of this enzyme causes an uncontrolled increase in the acetylcholine neurotransmitter, muscular paralysis and then death. Neurotoxicity can also result from the inhibition of receptors; for example, atropine from deadly nightshade (Atropa belladonna) that functions as a competitive antagonist of the muscarinic acetylcholine receptors.[62]

Although many natural toxins are secondary metabolites, these poisons also include peptides and proteins. An example of a toxic peptide is alpha-amanitin, which is found in relatives of the death cap mushroom. This is a potent enzyme inhibitor, in this case preventing the RNA polymerase II enzyme from transcribing DNA.[63] The algal toxin microcystin is also a peptide and is an inhibitor of protein phosphatases.[64] This toxin can contaminate water supplies after algal blooms and is a known carcinogen that can also cause acute liver haemorrhage and death at higher doses.[65]

Proteins can also be natural poisons or antinutrients, such as the trypsin inhibitors (discussed above) that are found in some legumes, as shown in the figure above. A less common class of toxins are toxic enzymes: these act as irreversible inhibitors of their target enzymes and work by chemically modifying their substrate enzymes. An example is ricin, an extremely potent protein toxin found in castor oil beans. This enzyme is a glycosidase that inactivates ribosomes. Since ricin is a catalytic irreversible inhibitor, this allows just a single molecule of ricin to kill a cell.[66]

The coenzyme folic acid (top) compared to the anti-cancer drug methotrexate (bottom)

The most common uses for enzyme inhibitors are as drugs to treat disease. Many of these inhibitors target a human enzyme and aim to correct a pathological condition. For instance, aspirin is a widely used drug that acts as a suicide inhibitor of the cyclooxygenase enzyme.[67] This inhibition in turn suppresses the production of proinflammatory prostaglandins and thus apirin may be used to reduce pain, fever, and inflammation.[67]

An estimated 29% of currently approved drugs are enzyme inhibitors[68] of which approximately 1/3 are kinase inhibitors.[69] A notable class of kinase drug targets are the receptor tyrosine kinases which are essential enzymes that regulate cell growth and their over-activation may result in cancer. Hence kinase inhibitors such as imatinib are frequently used to treat malignancies.[70] Janus kinases are another notable example of drug enzyme targets. Inhibitors of Janus kinases block the production of inflammatory cytokines and hence these inhbitors are used to treat a variety of inflammatory diseases in including arthritis, asthma, and Crohn's disease.[71]

An example of the structural similarity of some inhibitors to the substrates of the enzymes they target is seen in the figure comparing the drug methotrexate to folic acid. Folic acid is the oxidised form of the substrate of dihydrofolate reductase, an enzyme that is potently inhibited by methotrexate. Methotrexate blocks the action of dihydrofolate reductase and thereby halts thymidine biosynthesis. This block of nucleotide biosynthesis is selectively toxic to rapidly growing cells, therefore methotrexate is often used in cancer chemotherapy.[72]

A common treatment for male erectile dysfunction is sildenafil (Viagra), (figure, right). This compound is a potent inhibitor of cGMP specific phosphodiesterase type 5, the enzyme that degrades the signalling molecule cyclic guanosine monophosphate.[73] This signalling molecule triggers smooth muscle relaxation and allows blood flow into the corpus cavernosum, which causes an erection. Since the drug decreases the activity of the enzyme that halts the signal, it makes this signal last for a longer period of time.

The structure of a complex between penicillin G and the Streptomyces transpeptidase. Generated from .

Drugs also are used to inhibit enzymes needed for the survival of pathogens. For example, bacteria are surrounded by a thick cell wall made of a net-like polymer called peptidoglycan. Many antibiotics such as penicillin and vancomycin inhibit the enzymes that produce and then cross-link the strands of this polymer together.[74][75] This causes the cell wall to lose strength and the bacteria to burst. In the figure, a molecule of penicillin (shown in a ball-and-stick form) is shown bound to its target, the transpeptidase from the bacteria Streptomyces R61 (the protein is shown as a ribbon diagram).

Antibiotic drug design is facilitated when an enzyme that is essential to the pathogen's survival is absent or very different in humans.[76] In the example above, humans do not make peptidoglycan, therefore inhibitors of this process are selectively toxic to bacteria.[77] Selective toxicity is also produced in antibiotics by exploiting differences in the structure of the ribosomes in bacteria,[78] or how they make fatty acids.[79]

Many pesticides are enzyme inhibitors.[80] Acetylcholinesterase (AChE) is an enzyme found in animals, from insects to humans. It is essential to nerve cell function through its mechanism of breaking down the neurotransmitter acetylcholine into its constituents, acetate and choline.[81] This is somewhat unusual among neurotransmitters as most, including serotonin, dopamine, and norepinephrine, are absorbed from the synaptic cleft rather than cleaved. A large number of AChE inhibitors are used in both medicine and agriculture. Reversible competitive inhibitors, such as edrophonium, physostigmine, and neostigmine, are used in the treatment of myasthenia gravis[82] and in anaesthesia to reverse muscle blockade.[83] The carbamate pesticides are also examples of reversible AChE inhibitors. The organophosphate pesticides such as malathion, parathion, and chlorpyrifos irreversibly inhibit acetylcholinesterase.[84]

The herbicide glyphosate is an inhibitor of 3-phosphoshikimate 1-carboxyvinyltransferase,[85] other herbicides, such as the sulfonylureas inhibit the enzyme acetolactate synthase. Both these enzymes are needed for plants to make branched-chain amino acids. Many other enzymes are inhibited by herbicides, including enzymes needed for the biosynthesis of lipids and carotenoids and the processes of photosynthesis and oxidative phosphorylation.[86]

Robots used for the high-throughput screening of chemical libraries to discover new enzyme inhibitors

New drugs are the products of a long drug development process, the first step of which is often the discovery of a new enzyme inhibitor. In the past the only way to discover these new inhibitors was by trial and error: screening huge libraries of compounds against a target enzyme and hoping that some useful leads would emerge. This brute force approach is still successful and has even been extended by combinatorial chemistry approaches that quickly produce large numbers of novel compounds and high-throughput screening technology to rapidly screen these huge chemical libraries for useful inhibitors.[87]

More recently, an alternative approach has been applied: rational drug design uses the three-dimensional structure of an enzyme's active site to predict which molecules might be inhibitors.[88] These predictions are then tested and one of these tested compounds may be a novel inhibitor. This new inhibitor is then used to try to obtain a structure of the enzyme in an inhibitor/enzyme complex to show how the molecule is binding to the active site, allowing changes to be made to the inhibitor to try to optimise binding. This test and improve cycle is then repeated until a sufficiently potent inhibitor is produced.[89] Computer-based methods of predicting the affinity of an inhibitor for an enzyme are also being developed, such as molecular docking[90] and molecular mechanics.