Enzyme inhibitor

Enzyme inhibitors are a chemically diverse set of substances that range in size from organic small molecules to macromolecular proteins.

Small molecule inhibitors include essential primary metabolites that inhibit upstream enzymes that produce those metabolites. This provides a negative feedback loop that prevents over production of metabolites and thus maintains cellular homeostasis (steady internal conditions).^[3][2] Small molecule enzyme inhibitors also include secondary 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 are

Inhibition mechanism schematic

Kinetic mechanisms for reversible inhibition. Substrate (S) binding to enzyme (E) in blue, catalysis releasing product (P) in red, inhibitor (I) binding to enzyme in green.

Schematics for reversible inhibition. Binding site in blue, substrate in black, inhibitor in green, and allosteric site in light green.

Competitive inhibitors usually bind to the active site. Non-competitive bind to a remote (allosteric) site. Uncompetitive inhibitors only bind once the substrate is bound, fully disrupting catalysis, and mixed inhibition is similar but with only partial disruption of catalysis.

Reversible inhibitors attach to enzymes with

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 is covalent 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 by Cleland in 1963.^[14] They are classified according to the effect of the inhibitor on the V_max (maximum reaction rate catalysed by the enzyme) and K_m (the concentration of substrate resulting in half maximal enzyme activity) as the concentration of the enzyme's substrate is varied.^[15][16]

Competitive

In competitive 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 inhibitor compete for access to the enzyme's active site. This type of inhibition can be overcome by sufficiently high concentrations of substrate (V_max remains constant), i.e., by out-competing the inhibitor.^{[17]: 134–135} However, the apparent K_m will increase as it takes a higher concentration of the substrate to reach the K_m point, or half the V_max. 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

Uncompetitive

In uncompetitive inhibition the inhibitor binds only to the enzyme-substrate complex.^[17]: 139 This type of inhibition causes V_max to decrease (maximum velocity decreases as a result of removing activated complex) and K_m 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 K_m which indicates a higher binding affinity).^[18] Uncompetitive inhibition is rare.^{[17]: 139 [19]}

Non-competitive

In

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]

Quantitative description

Reversible inhibition can be described quantitatively in terms of the inhibitor's

• Competitive inhibitors can bind to E, but not to ES. Competitive inhibition increases K_m (i.e., the inhibitor interferes with substrate binding), but does not affect V_max (the inhibitor does not hamper catalysis in ES because it cannot bind to ES).[24]^: 102
• Uncompetitive inhibitors bind to ES. Uncompetitive inhibition decreases both K_m and V_max. The inhibitor affects substrate binding by increasing the enzyme's affinity for the substrate (decreasing K_m) as well as hampering catalysis (decreases V_max).^[24]: 106
• Non-competitive inhibitors have identical affinities for E and ES (K_i = K_i'). Non-competitive inhibition does not change K_m (i.e., it does not affect substrate binding) but decreases V_max (i.e., inhibitor binding hampers catalysis).^[24]: 97
• Mixed-type inhibitors bind to both E and ES, but their affinities for these two forms of the enzyme are different (K_i ≠ K_i'). Thus, mixed-type inhibitors affect substrate binding (increase or decrease K_m) and hamper catalysis in the ES complex (decrease V_max).^{[25]: 63–64}

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 on K_m and V_max.^[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]

${\displaystyle {\begin{aligned}{\cfrac {V_{\max }}{1+{\cfrac {\ce {[I]}}{K_{i}}}}}&={V_{\max }}\left({\cfrac {K_{i}}{K_{i}+[{\ce {I}}]}}\right)&&{\text{multiply by }}{\cfrac {K_{i}}{K_{i}}}=1\\&={V_{\max }}\left({\cfrac {K_{i}+[{\ce {I}}]-[{\ce {I}}]}{K_{i}+[{\ce {I}}]}}\right)&&{\text{add }}[{\ce {I}}]-[{\ce {I}}]=0{\text{ to numerator}}\\&={V_{\max }}\left(1-{\cfrac {[{\ce {I}}]}{K_{i}+[{\ce {I}}]}}\right)&&{\text{simplify }}{\cfrac {K_{i}+[{\ce {I}}]}{K_{i}+[{\ce {I}}]}}=1\\&=V_{\max }-V_{\max }{\cfrac {\ce {[I]}}{K_{i}+[{\ce {I}}]}}&&{\text{multiply out by }}V_{\max }\end{aligned}}}$

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

${\displaystyle {\cfrac {\ce {[S]}}{[{\ce {S}}]+K_{m}}}}$

fraction of the enzyme population bound by inhibitor

${\displaystyle {\cfrac {\ce {[I]}}{[{\ce {I}}]+K_{i}}}}$

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 delta V_max term.^[29]: 361

${\displaystyle V_{\max }-\Delta V_{\max }{\cfrac {\ce {[I]}}{[{\ce {I}}]+K_{i}}}}$

or

${\displaystyle V_{\max 1}-(V_{\max 1}-V_{\max 2}){\cfrac {\ce {[I]}}{[{\ce {I}}]+K_{i}}}}$

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 V_max 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}

${\displaystyle V_{\max 1}-(V_{\max 1}-V_{\max 2}){\cfrac {\ce {[X]}}{[{\ce {X}}]+K_{x}}}}$

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 K_m. The K_m 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 delta V_max term proposed above to modulate V_max should be appropriate in most situations:^{[28]: eq 14}

${\displaystyle K_{m1}-(K_{m1}-K_{m2}){\cfrac {\ce {[X]}}{[{\ce {X}}]+K_{x}}}}$

Dissociation constants

An enzyme inhibitor is characterised by its

Thus, in the presence of the inhibitor, the enzyme's effective K_m and V_max become (α/α')K_m and (1/α')V_max, 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 (see below).

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

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 V_max, but an unaffected K_m value.[18]

Substrate or product

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 normal kinetics 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 a regulatory feature in metabolism and can be a form of negative feedback.^[2]

Slow-tight

Slow-tight inhibition occurs when the initial enzyme–inhibitor complex EI undergoes

TGDDF/GDDF multi-substrate adduct inhibitor. Substrate analogue in black, cofactor analogue in blue, non-cleavable linker in red.

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

Nonpeptidic HIV-1 protease inhibitor tipranavir

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.

Enzyme inhibitors are often designed to mimic the transition 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 (lower K_i) 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.^[47]

However, not all inhibitors are based on the structures of substrates. For example, the structure of another HIV protease inhibitor

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 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]

Irreversible inhibitors

Types

DFP reaction

diisopropylfluorophosphate (DFP) with a serine protease

Irreversible inhibitors bind to the enzyme's binding site then undergo a chemical reaction to form a covalent enzyme-inhibitor complex (EI*). Binding site in blue, inhibitor in green.

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 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.^[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, k_obs/[I] values are used,^[56] where k_obs 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. The k_obs/[I] parameter is valid as long as the inhibitor does not saturate binding with the enzyme (in which case k_obs = k_inact) where k_inact is the rate of inactivation.

Measuring

Irreversible inhibition mechanism

Kinetic mechanism for irreversible inhibition. Substrate binding in blue, catalysis in red, inhibitor binding in green, inactivation reaction in dark green.

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 k_inact.^[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 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 k_inact and K_i.^[57]

Another method that is widely used in these analyses is

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.

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 of polyamine biosynthesis, α-difluoromethylornithine (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 (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 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.^[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 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.^[70]

Applications

Enzyme inhibitors are found in nature

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.^[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 the barstar inhibitor of the bacterial ribonuclease barnase.^[80]

Natural poisons

Animals and plants have evolved to synthesise a vast array of poisonous products including secondary metabolites,^[81] peptides and proteins^[82] 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.^[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 the Pacific yew tree, binds tightly to tubulin dimers and inhibits their assembly into microtubules in the cytoskeleton.^[84]

Many natural poisons act as

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,

As of 2017,[update] an estimated 29% of approved drugs are enzyme inhibitors

Drugs are also 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.^[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, 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.^[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 the ribosomes in bacteria,^[106] or how they make fatty acids.^[107]

Antivirals

Drugs that inhibit enzymes needed for the

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.^[123] There are two principle approaches of discovering these inhibitors.^[124]

The first general method is

• Activity-based proteomics – a branch of proteomics that uses covalent enzyme inhibitors as reporters to monitor enzyme activity.
• Antimetabolite – an enzyme inhibitor that is used to interfere with cell growth and division
• Transition state analogue
  – a type of enzyme inhibitor that mimics the transition state of the chemical reaction catalysed by the enzyme

References