Anenzyme inhibitor is amolecule that binds to anenzyme and blocks itsactivity. Enzymes areproteins that speed upchemical reactions necessary forlife, in whichsubstrate molecules are converted intoproducts.[1] An enzymefacilitates a specific chemical reaction by binding the substrate to itsactive site, a specialized area on the enzyme that accelerates themost difficult step of the reaction.
An enzyme inhibitor stops ("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'scatalysis of the reaction is blocked. Enzyme inhibitors may bindreversibly or irreversibly. Irreversible inhibitors form achemical bond with the enzyme such that the enzyme is inhibited until the chemical bond is broken. By contrast, reversible inhibitors bindnon-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 each and serve to control that enzyme's activity. For example, enzymes in ametabolic pathway may be inhibited by molecules produced later in the pathway, thus curtailing the production of molecules that are no longer needed. This type ofnegative feedback is an important way to maintainbalance in acell.[2] Enzyme inhibitors also control essential enzymes such asproteases ornucleases that, if left unchecked, may damage a cell. Manypoisons produced by animals or plants are enzyme inhibitors that block the activity of crucial enzymes in prey orpredators.
Manydrug molecules are enzyme inhibitors that inhibit an aberrant human enzyme or an enzyme critical for the survival of apathogen such as avirus,bacterium orparasite. Examples includemethotrexate (used inchemotherapy and in treatingrheumatic arthritis) and theprotease inhibitors used to treatHIV/AIDS. Since anti-pathogen inhibitors generally target only one enzyme, such drugs are highlyspecific and generally produce few side effects in humans, provided that noanalogous enzyme is found in humans. (This is often the case, since suchpathogens andhumans aregenetically distant.) Medicinal enzyme inhibitors often have lowdissociation 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 forliver andkidney damage and otheradverse drug reactions in humans. Hence the discovery and refinement of enzyme inhibitors is an active area of research inbiochemistry andpharmacology.
Enzyme inhibitors are a chemically diverse set of substances that range in size from organicsmall molecules to macromolecularproteins.
Small molecule inhibitors include essentialprimary metabolites that inhibit upstream enzymes that produce those metabolites. This provides a negative feedback loop that prevents over production of metabolites and thus maintains cellularhomeostasis (steady internal conditions).[3][2] Small molecule enzyme inhibitors also includesecondary metabolites, which are not essential to the organism that produces them, but provide the organism with an evolutionary advantage, in that they can be used to repel predators or competing organisms or immobilize prey.[4] In addition, many drugs are small molecule enzyme inhibitors that target either disease-modifying enzymes in the patient[1]: 5 or enzymes in pathogens which are required for the growth and reproduction of the pathogen.[5]
In addition to small molecules, some proteins act as enzyme inhibitors. The most prominent example areserpins (serineproteaseinhibitors) which are produced by animals to protect against inappropriate enzyme activation and by plants to prevent predation.[6] Another class of inhibitor proteins is theribonuclease inhibitors, which bind toribonucleases in one of the tightest knownprotein–protein interactions.[7] A special case of protein enzyme inhibitors arezymogens that contain an autoinhibitoryN-terminal peptide that binds to the active site of enzyme thatintramolecularly blocks its activity as a protective mechanism against uncontrolled catalysis. The N‑terminal peptide is cleaved (split) from the zymogen enzyme precursor by another enzyme to release an active enzyme.[8]
Thebinding site of inhibitors on enzymes is most commonly the same site that binds thesubstrate of the enzyme. Theseactive site inhibitors are known asorthosteric ("regular" orientation) inhibitors.[9] The mechanism of orthosteric inhibition is simply to prevent substrate binding to the enzyme through direct competition which in turn prevents the enzyme from catalysing the conversion of substrates into products. Alternatively, the inhibitor can bind to a site remote from the enzyme active site. These are known asallosteric ("alternative" orientation) inhibitors.[9] The mechanisms of allosteric inhibition are varied and include changing theconformation (shape) of the enzyme such that it can no longer bind substrate (kinetically indistinguishable from competitive orthosteric inhibition)[10] or alternatively stabilise binding of substrate to the enzyme but lock the enzyme in a conformation which is no longer catalytically active.[11]
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Reversible inhibitors attach to enzymes withnon-covalent interactions such ashydrogen bonds,hydrophobic interactions andionic bonds.[12] Multiple weak bonds between the inhibitor and the enzymeactive 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 ordialysis. A special case iscovalent reversible inhibitors that form a chemical bond with the enzyme, but the bond can be cleaved so the inhibition is fully reversible.[13]
Reversible inhibitors are generally categorized into four types, as introduced byCleland in 1963.[14] They are classified according to the effect of the inhibitor on theVmax (maximum reaction rate catalysed by the enzyme) andKm (the concentration of substrate resulting in half maximal enzyme activity) as the concentration of the enzyme's substrate is varied.[15][16]
Incompetitive inhibition the substrate and inhibitor cannot bind to the enzyme at the same time.[17]: 134 This usually results from the inhibitor having an affinity for the active site of an enzyme where the substrate also binds; the substrate and inhibitorcompete 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.[17]: 134–135 However, the apparentKm will increase as it takes a higher concentration of the substrate to reach theKm point, or half theVmax. Competitive inhibitors are often similar in structure to the real substrate (see for example the "methotrexate versus folate" figure in the"Drugs" section).[17]: 134
Inuncompetitive inhibition the inhibitor binds only to the enzyme-substrate complex.[17]: 139 This type of inhibition causesVmax to decrease (maximum velocity decreases as a result of removing activated complex) andKm to decrease (due to better binding efficiency as a result ofLe Chatelier's principle and the effective elimination of the ES complex thus decreasing theKm which indicates a higher binding affinity).[18] Uncompetitive inhibition is rare.[17]: 139 [19]
Innon-competitive inhibition the binding of the inhibitor to the enzyme reduces itsactivity but does not affect the binding of substrate.[16] This type of inhibitor binds with equal affinity to the free enzyme as to the enzyme-substrate complex. It can be thought of as having the ability of competitive and uncompetitive inhibitors, but with no preference to either type. 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, butKm will remain the same as the actual binding of the substrate, by definition, will still function properly.[20]
Inmixed inhibition the inhibitor may bind to the enzyme whether or not the substrate has already bound. Hence mixed inhibition is a combination of competitive and noncompetitive inhibition.[16] Furthermore, the affinity of the inhibitor for the free enzyme and the enzyme-substrate complex may differ.[17]: 136–139 By increasing concentrations of substrate [S], this type of inhibition can be reduced (due to the competitive contribution), but not entirely overcome (due to the noncompetitive component).[21]: 381–382 Although it is possible for mixed-type inhibitors to bind in the active site, this type of inhibition generally results from anallosteric effect where the inhibitor binds to a different site on an enzyme. Inhibitor binding to thisallosteric site changes theconformation (that is, thetertiary structure or three-dimensional shape) of the enzyme so that the affinity of the substrate for the active site is reduced.[22]
These four types of inhibition 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].[23]
Reversible inhibition can be described quantitatively in terms of the inhibitor'sbinding to the enzyme and to the enzyme-substrate complex, and its effects on thekinetic constants of the enzyme.[24]: 6 In the classicMichaelis-Menten scheme (shown in the "inhibition mechanism schematic" diagram), 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.[24]: 55 The inhibitor (I) can bind to either E or ES with thedissociation constantsKi orKi', respectively.[24]: 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.[26]
Traditionally reversible enzyme inhibitors have been classified as competitive, uncompetitive, or non-competitive, according to their effects onKm andVmax.[14] These three types of inhibition result respectively from the inhibitor binding only to the enzyme E in the absence of substrate S, to the enzyme–substrate complex ES, or to both. 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.[27] It is further assumed that binding of the inhibitor to the enzyme results in 100% inhibition and fails to consider the possibility of partial inhibition.[27] 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:[28]
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.
fraction of the enzyme population bound by substrate
fraction of the enzyme population bound by inhibitor
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 no inhibition. To account for this the equation can be easily modified to allow for different degrees of inhibition by including a deltaVmax term.[29]: 361
or
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 secondaryVmax 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".[28]: 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 theKm. TheKm 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 deltaVmax term proposed above to modulateVmax should be appropriate in most situations:[28]: eq 14
![2D plots of 1/[S] concentration (x-axis) and 1/V (y-axis) demonstrating that as inhibitor concentration is changed, competitive inhibitor lines intersect at a single point on the y-axis, non-competitive inhibitors intersect at the x-axis, and mixed inhibitors intersect a point that is on neither axis](/mt/?noimg=&dark=on&url=http%3a%2f%2fen.wikipedia.org%2fwiki%2fFile%3aInhibition_diagrams-1-.png)
An enzyme inhibitor is characterised by itsdissociation constantKi, the concentration at which the inhibitor half occupies the enzyme. In non-competitive inhibition the inhibitor can also bind to the enzyme-substrate complex, and the presence of bound substrate can change the affinity of the inhibitor for the enzyme, resulting in a second dissociation constantKi'. HenceKi andKi' are the dissociation constants of the inhibitor for the enzyme and to the enzyme-substrate complex, respectively.[30]: Glossary The enzyme-inhibitor constantKi can be measured directly by various methods; one especially accurate method isisothermal titration calorimetry, in which the inhibitor is titrated into a solution of enzyme and the heat released or absorbed is measured.[31] However, the other dissociation constantKi' 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 theenzyme activity under various substrate and inhibitor concentrations, and fitting the data vianonlinear regression[32] to a modifiedMichaelis–Menten equation.[21]
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 effectiveKm andVmax 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 the inhibition becomes effectively irreversible, hence it is more practical to treat such tight-binding inhibitors as irreversible (seebelow).
The effects of different types of reversible enzyme inhibitors on enzymatic activity can be visualised using graphical representations of the Michaelis–Menten equation, such asLineweaver–Burk,Eadie-Hofstee orHanes-Woolf plots.[17]: 140–144 An illustration is provided by the three Lineweaver–Burk plots depicted in theLineweaver–Burk diagrams figure. In the top diagram the competitive inhibition lines intersect on they-axis, illustrating that such inhibitors do not affectVmax. In the bottom diagram the non-competitive inhibition lines intersect on thex-axis, showing these inhibitors do not affectKm. However, since it can be difficult to estimateKi andKi' accurately from such plots,[33] it is advisable to estimate these constants using more reliable nonlinear regression methods.[33]
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 lowerVmax, but an unaffectedKm value.[18]
Substrate or product inhibition is where either an enzymes substrate or product also act as an inhibitor. This inhibition may follow the competitive, uncompetitive or mixed patterns. In substrate inhibition there is a progressive decrease in activity at high substrate concentrations, potentially from an enzyme having two competing substrate-binding sites. At low substrate, the high-affinity site is occupied and normalkinetics are followed. However, at higher concentrations, the second inhibitory site becomes occupied, inhibiting the enzyme.[34] Product inhibition (either the enzyme's own product, or a product to an enzyme downstream in its metabolic pathway) is often aregulatory feature in metabolism and can be a form ofnegative feedback.[2]
Slow-tight inhibition occurs when the initial enzyme–inhibitor complex EI undergoesconformational isomerism (a change in shape) 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 forKi, which is time–dependent. The true value ofKi can be obtained through more complex analysis of the on (kon) and off (koff) rate constants for inhibitor association with kinetics similar toirreversible inhibition.[17]: 168
Multi-substrate analogue inhibitors are high affinity selective inhibitors that can be prepared for enzymes that catalyse reactions with more than one substrate by capturing the binding energy of each of those substrate into one molecule.[35][36] For example, in theformyl transfer reactions ofpurine biosynthesis, a potent Multi-substrate Adduct Inhibitor (MAI) to glycinamide ribonucleotide (GAR)TFase was prepared synthetically by linking analogues of the GAR substrate and theN-10-formyl tetrahydrofolate cofactor together to produce thioglycinamide ribonucleotide dideazafolate (TGDDF),[37] or enzymatically from the natural GAR substrate to yield GDDF.[38] Here the subnanomolar dissociation constant (KD) of TGDDF was greater than predicted presumably due toentropic 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 asisoniazid[39] or enzyme inhibitor ligands (for example,PTC124)[40] with cellular cofactors such asnicotinamide adenine dinucleotide (NADH) andadenosine triphosphate (ATP) respectively.[41]
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 ofdihydrofolate reductase (DHFR) are prominent examples.[42] Other examples of these substrate mimics are theprotease inhibitors, a therapeutically effective class ofantiretroviral drugs used to treatHIV/AIDS.[43][44] The structure ofritonavir, apeptidomimetic (peptide mimic) protease inhibitor containing threepeptide bonds, as shown in the"competitive inhibition" figure above. As this drug resembles the peptide that is the substrate of the HIV protease, it competes with the substrate in the enzyme's active site.[45]
Enzyme inhibitors are often designed to mimic thetransition state or intermediate of an enzyme-catalysed reaction.[46] This ensures that the inhibitor exploits the transition state stabilising effect of the enzyme, resulting in a better binding affinity (lowerKi) than substrate-based designs. An example of such a transition state inhibitor is the antiviral drugoseltamivir; this drug mimics the planar nature of the ringoxonium ion in the reaction of the viral enzymeneuraminidase.[47]
However, not all inhibitors are based on the structures of substrates. For example, the structure of another HIV protease inhibitortipranavir 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 forpeptidases and are less likely to be degraded.[48]
In drug design it is important to consider the concentrations of substrates to which the target enzymes are exposed. For example, someprotein kinase inhibitors have chemical structures that are similar to ATP, one of the substrates of these enzymes.[49] 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.[50]
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Irreversible inhibitorscovalently bind to an enzyme, and this type of inhibition can therefore not be readily reversed.[51] Irreversible inhibitors often contain reactive functional groups such asnitrogen mustards,aldehydes,haloalkanes,alkenes,Michael acceptors,phenyl sulfonates, orfluorophosphonates.[52] Theseelectrophilic groups react with amino acid side chains to form covalentadducts.[51] The residues modified are those with side chains containingnucleophiles such ashydroxyl orsulfhydryl groups; these include the amino acidsserine (that reacts withDFP, see the "DFP reaction" diagram), and alsocysteine,threonine, ortyrosine.[53]
Irreversible inhibition is different from irreversible enzyme inactivation.[54] Irreversible inhibitors are generally specific for one class of enzyme and do not inactivate all proteins; they do not function by destroyingprotein structure but by specifically altering the active site of their target. For example, extremes of pH or temperature usually causedenaturation of all protein structure, but this is a non-specific effect. Similarly, some non-specific chemical treatments destroy protein structure: for example, heating in concentratedhydrochloric acid will hydrolyse thepeptide bonds holding proteins together, releasing free amino acids.[55]
Irreversible inhibitors display time-dependent inhibition and their potency therefore cannot be characterised by an IC50 value. 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,[56] wherekobs is the observed pseudo-first order rate of inactivation (obtained by plotting the log of % activity versus time) and [I] is the concentration of inhibitor. Thekobs/[I] parameter is valid as long as the inhibitor does not saturate binding with the enzyme (in which casekobs =kinact) wherekinact is the rate of inactivation.
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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 orkinact.[13] 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 followingexponential decay. Fitting these data to arate 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 givekinact andKi.[57]
Another method that is widely used in these analyses ismass 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.[58] This is usually done using aMALDI-TOF mass spectrometer.[59] In a complementary technique,peptide mass fingerprinting involves digestion of the native and modified protein with aprotease such astrypsin. This will produce a set ofpeptides 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.[60]
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 the "irreversible inhibition mechanism" diagram). This kinetic behaviour is called slow-binding.[62] This slow rearrangement after binding often involves aconformational change as the enzyme "clamps down" around the inhibitor molecule. Examples of slow-binding inhibitors include some important drugs, suchmethotrexate,[63]allopurinol,[64] and the activated form ofacyclovir.[65]
Diisopropylfluorophosphate (DFP) is an example of an irreversibleprotease inhibitor (see the "DFP reaction" diagram). The enzyme hydrolyses the phosphorus–fluorine bond, but the phosphate residue remains bound to the serine in theactive site, deactivating it.[67] Similarly, DFP also reacts with the active site ofacetylcholine esterase in thesynapses of neurons, and consequently is a potent neurotoxin, with a lethal dose of less than 100 mg.[68]
Suicide inhibition is an unusual type of irreversible inhibition where the enzyme converts the inhibitor into a reactive form in its active site.[69] An example is the inhibitor ofpolyamine biosynthesis,α-difluoromethylornithine (DFMO), which is an analogue of the amino acidornithine, and is used to treatAfrican trypanosomiasis (sleeping sickness).Ornithine decarboxylase can catalyse the decarboxylation of DFMO instead of ornithine (see the "DFMO inhibitor mechanism" diagram). However, this decarboxylation reaction is followed by the elimination of a fluorine atom, which converts this catalytic intermediate into a conjugatedimine, 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.[61]
Since irreversible inhibition often involves the initial formation of a non-covalent enzyme inhibitor (EI) complex,[13] it is sometimes possible for an inhibitor to bind to an enzyme in more than one way. For example, in the figure showingtrypanothione reductase from the human protozoan parasiteTrypanosoma cruzi, two molecules of an inhibitor calledquinacrine 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 itsnitrogen mustard group.[70]
Enzyme inhibitors are found in nature[71] and also produced artificially in the laboratory.[72] Naturally occurring enzyme inhibitors regulate manymetabolic processes and are essential for life.[3][1] In addition, naturally producedpoisons are often enzyme inhibitors that have evolved for use as toxic agents against predators, prey, and competing organisms.[4] These natural toxins include some of the most poisonous substances known.[73] Artificial inhibitors are often used as drugs, but can also beinsecticides such asmalathion,herbicides such asglyphosate,[74] ordisinfectants such astriclosan. Other artificial enzyme inhibitors blockacetylcholinesterase, an enzyme which breaks downacetylcholine, and are used asnerve agents inchemical warfare.[75]
Enzyme inhibition is a common feature ofmetabolic pathway control in cells.[3]Metabolic flux through a pathway is often regulated by a pathway'smetabolites acting as inhibitors and enhancers for the enzymes in that same pathway. Theglycolytic pathway is a classic example.[76] Thiscatabolic pathway consumesglucose and producesATP,NADH andpyruvate. A key step for the regulation of glycolysis is an early reaction in the pathway catalysed byphosphofructokinase‑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. Thisnegative 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 andADP are examples of metabolites that are allosteric activators.[77]
Physiological enzyme inhibition can also be produced by specific protein inhibitors. This mechanism occurs in thepancreas, which synthesises many digestive precursor enzymes known aszymogens. Many of these are activated by thetrypsin 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 potenttrypsin inhibitor protein in the pancreas. This inhibitor binds tightly to trypsin, preventing the trypsin activity that would otherwise be detrimental to the organ.[78] 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.[79] Other examples of physiological enzyme inhibitor proteins include thebarstar inhibitor of the bacterial ribonucleasebarnase.[80]
Animals and plants have evolved to synthesise a vast array of poisonous products includingsecondary metabolites,[81] peptides and proteins[82] that can act as inhibitors. Natural toxins are usuallysmall organic molecules and are so diverse that there are probably natural inhibitors for most metabolic processes.[83] 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 thePacific yew tree, binds tightly totubulindimers and inhibits their assembly intomicrotubules in thecytoskeleton.[84]
Many natural poisons act asneurotoxins that can causeparalysis leading to death and function for defence against predators or in hunting and capturing prey. Some of these natural inhibitors,[85] despite their toxic attributes, are valuable for therapeutic uses at lower doses.[86] An example of a neurotoxin are theglycoalkaloids, from the plant species in the familySolanaceae (includespotato,tomato andeggplant), that areacetylcholinesterase 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 acompetitive antagonist of themuscarinic acetylcholine receptors.[87]
Although many natural toxins are secondary metabolites, these poisons also include peptides and proteins. An example of a toxic peptide isalpha-amanitin, which is found in relatives of thedeath cap mushroom. This is a potent enzyme inhibitor, in this case preventing theRNA polymerase II enzyme from transcribing DNA.[88] The algal toxinmicrocystin is also a peptide and is an inhibitor ofprotein phosphatases.[89] This toxin can contaminate water supplies afteralgal blooms and is a known carcinogen that can also cause acute liver haemorrhage and death at higher doses.[90]
Proteins can also be natural poisons orantinutrients, such as thetrypsin inhibitors (discussed in the "metabolic regulation" section above) that are found in somelegumes.[91] 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 isricin, an extremely potent protein toxin found incastor oil beans.[92] This enzyme is aglycosidase that inactivates ribosomes.[93] Since ricin is a catalytic irreversible inhibitor, this allows just a single molecule of ricin to kill a cell.[94]
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 asuicide inhibitor of thecyclooxygenase enzyme.[95] This inhibition in turn suppresses the production of proinflammatoryprostaglandins and thus aspirin may be used to reduce pain, fever, and inflammation.[95]
As of 2017,[update] an estimated 29% of approved drugs are enzyme inhibitors[96] of which approximately one-fifth arekinase inhibitors.[96] A notable class of kinase drug targets is thereceptor tyrosine kinases which are essential enzymes that regulatecell growth; their over-activation may result in cancer. Hencekinase inhibitors such asimatinib are frequently used to treat malignancies.[97]Janus kinases are another notable example of drug enzyme targets.Inhibitors of Janus kinases block the production ofinflammatory cytokines and hence these inhibitors are used to treat a variety ofinflammatory diseases in includingarthritis,asthma, andCrohn's disease.[98]
An example of the structural similarity of some inhibitors to the substrates of the enzymes they target is seen in the figure comparing the drugmethotrexate tofolic acid. Folic acid is the oxidised form of the substrate ofdihydrofolate reductase, an enzyme that is potently inhibited by methotrexate. Methotrexate blocks the action of dihydrofolate reductase and thereby haltsthymidine biosynthesis.[42] This block ofnucleotide biosynthesis is selectively toxic to rapidly growing cells, therefore methotrexate is often used in cancer chemotherapy.[99]
A common treatment forerectile dysfunction issildenafil (Viagra).[100] This compound is a potent inhibitor ofcGMP specific phosphodiesterase type 5, the enzyme that degrades the signalling moleculecyclic guanosine monophosphate.[101] This signalling molecule triggers smooth muscle relaxation and allows blood flow into thecorpus 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.
Drugs are also used to inhibit enzymes needed for the survival of pathogens. For example, bacteria are surrounded by a thickcell wall made of a net-like polymer calledpeptidoglycan. Manyantibiotics such aspenicillin andvancomycin inhibit the enzymes that produce and then cross-link the strands of this polymer together.[102][103] 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, thetranspeptidase from the bacteriaStreptomyces R61 (the protein is shown as aribbon diagram).
Antibioticdrug design is facilitated when an enzyme that is essential to the pathogen's survival is absent or very different in humans.[104] Humans do not make peptidoglycan, therefore antibiotics that inhibit this process are selectively toxic to bacteria.[105] Selective toxicity is also produced in antibiotics by exploiting differences in the structure of theribosomes in bacteria,[106] or how they makefatty acids.[107]
Drugs that inhibit enzymes needed for thereplication of viruses are effective in treating viral infections.[108]Antiviral drugs includeprotease inhibitors used to treatHIV/AIDS[109] andHepatitis C,[110]reverse-transcriptase inhibitors targeting HIV/AIDS,[111]neuraminidase inhibitors targetinginfluenza,[112] and terminase inhibitors targetinghuman cytomegalovirus.[113]
Manypesticides are enzyme inhibitors.[114]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 neurotransmitteracetylcholine into its constituents,acetate andcholine.[115] This is somewhat unusual among neurotransmitters as most, includingserotonin,dopamine, andnorepinephrine, are absorbed from thesynaptic cleft rather than cleaved. A large number of AChE inhibitors are used in both medicine and agriculture.[116] Reversible competitive inhibitors, such asedrophonium,physostigmine, andneostigmine, are used in the treatment ofmyasthenia gravis[117] and in anaesthesia to reverse muscle blockade.[118] Thecarbamate pesticides are also examples of reversible AChE inhibitors. Theorganophosphate pesticides such asmalathion,parathion, andchlorpyrifos irreversibly inhibit acetylcholinesterase.[119]
The herbicideglyphosate is an inhibitor of3-phosphoshikimate 1-carboxyvinyltransferase,[120] other herbicides, such as thesulfonylureas inhibit the enzymeacetolactate synthase.[121] Both enzymes are needed for plants to make branched-chainamino acids. Many other enzymes are inhibited by herbicides, including enzymes needed for the biosynthesis oflipids andcarotenoids and the processes ofphotosynthesis andoxidative phosphorylation.[122]
New drugs are the products of a longdrug development process, the first step of which is often the discovery of a new enzyme inhibitor.[123] There are two principle approaches of discovering these inhibitors.[124]
The first general method isrational drug design based on mimicking thetransition state of the chemical reaction catalysed by the enzyme.[125] The designed inhibitor often closely resembles the substrate, except that the portion of the substrate that undergoes chemical reaction is replaced by a chemically stablefunctional group that resembles the transition state. Since the enzyme has evolved to stabilise the transition state,transition state analogues generally possess higher affinity for the enzyme compared to the substrate, and therefore are effective inhibitors.[46]
The second way of discovering new enzyme inhibitors ishigh-throughput screening of large libraries of structurally diverse compounds to identify hit molecules that bind to the enzyme. This method has been extended to includevirtual screening of databases of diverse molecules using computers,[126][127] which are then followed by experimental confirmation of binding of the virtual screening hits.[128] Complementary approaches that can provide new starting points for inhibitors includefragment-based lead discovery[129] andDNA Encoded Chemical Libraries (DEL).[130]
Hits from any of the above approaches can beoptimised to high affinity binders that efficiently inhibit the enzyme.[131]Computer-based methods for predicting the binding orientation and affinity of an inhibitor for an enzyme such asmolecular docking[132] andmolecular mechanics can be used to assist in the optimisation process.[133] New inhibitors are used to obtaincrystallographic structures 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 optimise binding in a process known asstructure-based drug design.[1]: 66 This test and improve cycle is repeated until a sufficiently potent inhibitor is produced.
In some cases, the inhibitor may bind to a distinct site on the enzyme that is in allosteric communication with the substrate binding pocket. In many cases, allosteric, substrate competitive compounds result in conformational changes to the enzyme that change the ability of the enzyme to bind substrate.
Enzyme inactivation is generally explained as a chemical process involving several phenomena like aggregation, dissociation into subunits, or denaturation (conformational changes), which occur simultaneously during the inactivation of a specific enzyme.
Figure 1C: Clinical success of privileged protein family classes (% approved drugs targeting each target class): Reductase 7.62, Kinase 5.94, Protease 3.35, Hydrolase 2.76, NPTase 2.09, Transferase 1.92, Lyase 1.59, Isomerase 1.51, Phosphodiesterase 1.50, Cytochrome p450 0.84, Epigenetic eraser 0.33, Total enzyme targets of approved drugs = 29.45%
Chapter 10.2.1: Sulfonylurea acetolactate synthase inhibitors
