Enzyme inhibition/inhibitors irreversible

Another important characteristic of M AOIs is the production of reversible versus irreversible enzyme inhibition. An irreversible inhibitor permanently disables the enzyme. This means that MAO must be resynthesized, in the absence of the drug, before the activity of the enzyme can be reestablished. Resynthesis of the enzyme may take up to 2 weeks. For this reason, an interval of 10-14 days is required after discontinuing irreversible inhibitors and before instituting treatment with other antidepressants or permitting the use of contraindicated drugs or the consumption of contraindicated foods. On the other hand, a reversible inhibitor can move away from the active site of the enzyme, making the enzyme available to metabo-hze other substances. The reversibility and selectivity of the currently available MAOIs are summarized in Table 2-4. 

Enzyme Inhibition. En2yme inhibitors (qv) are reagents that bind to the enzyme and cause a decrease in the reaction rate. Irreversible inhibitors bind to the enzyme by an irreversible reaction, and consequendy cannot dissociate from the enzyme or be removed by dilution or dialysis. Examples of irreversible inhibitors are nerve gases such as diisopropylphosphoduoridate [55-91-4] (DEP). 

Enzyme inhibitors are classified in several ways. The inhibitor may interact either reversibly or irreversibly with the enzyme. Reversible inhibitors interact with the enzyme through noncovalent association/dissociation reactions. In contrast, irreversible inhibitors usually cause stable, covalent alterations in the enzyme. That is, the consequence of irreversible inhibition is a decrease in the concentration of active enzyme. The kinetics observed are consistent with this interpretation, as we shall see later. 

Wortmannin is a fungus-derived inhibitor of PI 3-kinase. The agent binds and inhibits the enzyme covalently and irreversibly. It is very potent and considered to be highly specific (IC5o in most cells in the low nanomolar range). 

CBs, like OPs, act as inhibitors of ChE. They are treated as substrates by the enzyme and carbamylate the serine of the active site (Figure 10.8). Speaking generally, car-bamylated AChE reactivates more rapidly than phosphorylated AChE. After aging has occurred, phosphorylation of the enzyme is effectively irreversible (see Section 10.2.4). Carbamylated AChE reactivates when preparations are diluted with water, a process that is accelerated in the presence of acetylcholine, which competes as a substrate. Thus, the measurement of AChE inhibition is complicated by the fact that reactivation occurs during the course of the assay. Carbamylated AChE is not reactivated by PAM and related compounds that are used as antidotes to OP poisoning (see Box 10.1). 

The acyl-enzyme can eliminate the 4-chlorine atom to generate this reactive intermediate that can then react with a nearby nucleophile such as His57 to give an alkylated acyl-enzyme derivative in which the inhibitor moiety is bound to the enzyme by two covalent bonds (Scheme 11.5). Inhibition is irreversible.59 The mechanism has been confirmed by X-ray structural analysis of protease-isocoumarin complexes. There is a cross-link between the inhibitor and the Serl95 and His57 residues of PPE.60 Human leukocyte elastase is also very efficiently inactivated.61... 

Although substrates may enhance or inhibit their own conversion, as noted in Section 10.4.1, other species may also affect enzyme activity. Inhibitors are compounds that decrease observable enzyme activity, and activators increase activity. The combination of an inhibitor or activator with an enzyme may be irreversible, reversible, or partially... 

Sometimes CYPs can also produce reactive metabolite species that, instead of undergoing the normal detoxification pathway, can act as irreversible CYP inhibitors, thus causing toxicity. Such reactive metabolites that cause CYP inactivation are called MBI and are described in Chapter 9. Mechanism-based enzyme inhibition is associated with irreversible or quasi-irreversible loss of enzyme function, requiring synthesis of new enzymes before activity is restored. The consequences of MBI could be auto-inhibition of the clearance of the inactivator itself or prolonged inhibition of the clearance of other drugs that are cleared by the same isozyme. There may also be serious immunotoxicological consequences if a reactive intermediate is covalently bound to the enzyme. Therefore, screening of new compounds for MBI is now a standard practice within the pharmaceutical industry. 

In addition to the aforementioned allenic steroids, prostaglandins, amino acids and nucleoside analogs, a number of other functionalized allenes have been employed (albeit with limited success) in enzyme inhibition (Scheme 18.56) [154-159]. Thus, the 7-vinylidenecephalosporin 164 and related allenes did not show the expected activity as inhibitors of human leukocyte elastase, but a weak inhibition of porcine pancreas elastase [156], Similarly disappointing were the immunosuppressive activity of the allenic mycophenolic acid derivative 165 [157] and the inhibition of 12-lipoxygenase by the carboxylic acid 166 [158]. In contrast, the carboxyallenyl phosphate 167 turned out to be a potent inhibitor of phosphoenolpyruvate carboxylase and pyruvate kinase [159]. Hydrolysis of this allenic phosphate probably leads to 2-oxobut-3-enoate, which then undergoes an irreversible Michael addition with suitable nucleophilic side chains of the enzyme. 

As a result, the penicillin occupies the active site of the enzyme, and becomes bound via the active-site serine residue. This binding causes irreversible enzyme inhibition, and stops cell-wall biosynthesis. Growing cells are killed due to rupture of the cell membrane and loss of cellular contents. The binding reaction between penicillinbinding proteins and penicillins is chemically analogous to the action of P-lactamases (see Boxes 7.20 and 13.5) however, in the latter case, penicilloic acid is subsequently released from the P-lactamase, and the enzyme can continue to function. Inhibitors of acetylcholinesterase (see Box 7.26) also bind irreversibly to the enzyme through a serine hydroxyl. 

In addition to the assessment of reversible inhibition, the role played by mechanism-based inhibitors (irreversible inhibitors) provides a focus during lead development, as it can result in a more profound and prolonged effect than that suggested by the therapeutic dose or exposure. Mechanism-based inhibition (MBI) occurs as a result of the CYP generating reactive intermediates that bind to the enzyme causing irreversible loss of activity. Oxidative metabolism via that CYP is only restored upon re-synthesis of that enzyme. Three mechanisms have been reported showing how intermediate species act as mechanism-based inhibitors ... 

Reversible inhibition of an enzyme is competitive, uncompetitive, or mixed. Competitive inhibitors compete with substrate by binding reversibly to the active site, but they are not transformed by the enzyme. Uncompetitive inhibitors bind only to the ES complex, at a site distinct from the active site. Mixed inhibitors bind to either E or ES, again at a site distinct from the active site. In irreversible inhibition an inhibitor binds permanently to an active site by forming a covalent bond or a veiy stable noncovalent interaction. 

A closely related E. coli protein is a 79-kDa multifunctional enzyme that catalyzes four different reactions of fatty acid oxidation (Chapter 17). The amino-terminal region contains the enoyl hydratase activity.32 A quite different enzyme catalyzes dehydration of thioesters of (3-hydroxyacids such as 3-hydroxydecanoyl-acyl carrier protein (see Eq. 21-2) to both form and isomerize enoyl-ACP derivatives during synthesis of unsaturated fatty acids by E. coli. Again, a glutamate side chain is the catalytic base but an imidazole group of histidine has also been implicated.33 This enzyme is inhibited irreversibly by the N-acetylcysteamine thioester of 3-decynoic acids (Eq. 13-8). This was one of the first enzyme-activated inhibitors to be studied.34... 

There are two reports that hydroxamic acid inhibition is reversible 59,90) and one that inhibition is irreversible (91). The inhibition appears to be competitive. The rather extensive screening of 36 hydroxamic acids was accomplished with sword bean urease (90), but Proteus urease (92) and jack bean urease (59) also have been found to be inhibited by these specific inhibitors. Using tritium-labeled caprylohydroxamic acid and sword bean urease, Kobashi et al. (94) have shown the formation of an inactive complex containing two moles of inhibitor per mole of enzyme. 

Competitive, noncompetitive, and uncompetitive inhibition are all reversible. If the inhibited enzyme is dialyzed to remove the inhibitor, the enzymatic activity recovers. There are, however, inhibitors that react essentially irreversibly, usually by forming a covalent bond to an amino acid side chain or a bound coenzyme. Examples of such inhibitors are given in table 7.4. Like reversible inhibitors, irreversible inhibitors can change either Umax or Km or both. 

FIGURE 2—47. Some drugs are inhibitors of enzymes. Shown here is an irreversible inhibitor of an enzyme, depicted as binding to the enzyme with chains. The binding is locked so permanently that such irreversible enzyme inhibition is sometimes called the work of a suicide inhibitor, since the enzyme essentially commits suicide by binding to the irreversible inhibitor. Enzyme activity cannot be restored unless another molecule of enzyme is synthesized by the cell s DNA. The enzyme molecule that has bound the irreversible inhibitor is permanently incapable of further enzymatic activity and therefore is essentially dead. ... 

FIGURE 2-51. The consequence of a substrate competing unsuccessfully for reversal of enzyme inhibition is that the substrate is unable to displace the inhibitor. This is depicted as scissors unsuccessfully attempting to cut the chains of the inhibitor. In this case, the inhibition is irreversible. 

Noncompetitive inhibitors interact with enzymes in many different ways. They can bind to the enzymes reversibly or irreversibly at the active site or at some other region. In any case the resultant complex is inactive. The mechanism of noncompetitive inhibition can be expressed as follows ... 

The catalytic rate of an enzyme can be lowered by inhibitor molecules. Many inhibitors exist, including normal body metabolites, foreign drugs and toxins. Enzyme inhibition can be of two main types irreversible or reversible. Reversible inhibition can be subdivided into competitive and noncompetitive. 

Enzyme inhibition Many types of molecule exist which are capable of interfering with the activity of an individual enzyme. Any molecule which acts directly on an enzyme to lower its catalytic rate is called an inhibitor. Some enzyme inhibitors are normal body metabolites that inhibit a particular enzyme as part of the normal metabolic control of a pathway. Other inhibitors may be foreign substances, such as drugs or toxins, where the effect of enzyme inhibition could be either therapeutic or, at the other extreme, lethal. Enzyme inhibition may be of two main types irreversible or reversible, with reversible inhibition itself being subdivided into competitive and noncompetitive inhibition. Reversible inhibition can be overcome by removing the inhibitor from the enzyme, for example by dialysis (see Topic B6), but this is not possible for irreversible inhibition, by definition. 

In unier tu cuklcuuy prevent proteolysis, the inhibitor should fulfill at least two requirements (1) inhibit the target enzyme in an irreversible or pseudo-irreversible manner and (2) suppress the enzyme activity before significant proteolysis has occurred. From a kinetic viewpoint, the classical example of vn irreversible protein inhibitor of NE is ai-PI. Proteolysis can also be prevented by reversible inhibitors if the in vivo inhibitor concentration is much greater than Kj, the equilibrium constant far inhibition (Ujvta >>Kj), resulting in a pseudo-irreversible behavior. 

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