Suicide enzyme-inactivator mechanism-based inhibitor

Mechanism-based inhibitors (also known as suicide inhibitors or as kcat inhibitors) are actually substrates for their target enzymes. A reactive group is only revealed by enzyme action it is therefore not subject to hydrolysis until it has been revealed in the vicinity of the enzyme. The ability of the inhibitor then to inactivate the enzyme will depend upon relative rates of (a) covalent bond formation with the enzyme, (b) diffusion of the reactive entity away from the enzyme, and (c) hydrolysis. 

Mechanism-based inhibitors or suicide substrates seem to be particularly prevalent with CYP3A4. Such compounds are substrates for the enzyme, but metabolism is believed to form products that deactivate the enzyme. Several macrolide antibiotics, generally involving a tertiary amine function, are able to inhibit CYP3A4 in this manner (147,148). Erythromycin is one of the most widely used examples of this type of interaction, although there are other commonly prescribed agents that inactivate CYP3A4 (149-151), and a consideration of this phenomenon partially explains a number of interactions that are not readily explained by the conventional in vitro data (152). 

Fluorouracil is a mechanism-based inhibitor—it inactivates the enzyme by taking part in the normal catalytic mechanism. It is also called a suicide inhibitor because when the enzyme reacts with it, the enzyme commits suicide. The use of 5-fluorouracil illustrates the importance of knowing the mechanism for an enzyme-catalyzed reaction. If you know the mechanism, you may be able to design an inhibitor to turn the reaction off at a certain step. 

Suicide inhibitors, or mechanism-based inhibitors are modified substrates that provide the most specific means to modify an enzyme active site. The inhibitor binds to the enzyme as a substrate and is initially processed by the normal catalytic mechanism. The mechanism of catalysis then generates a chemically reactive intermediate that inactivates the enzyme through covalent modification. The fact that the enzyme participates in its own irreversible inhibition strongly suggests that the covalently modified group on the enzyme is catalytically vital. One example of such an inhibitor is N,N-dimethylpropargylamine. A flavin prosthetic group of monoamine oxidase... 

In this chapter, mechanism-based inhibition is discussed in its broadest sense, where an inhibitor is converted by the enzyme catalytic mechanism to form an enzyme-inhibitor complex. Other terms used in the literature for mechanism-based inhibitors include suicide inhibitors, suicide substrate inhibitors, alternate substrates, substrate inhibitors, and enzyme inactivators, as well as irreversible, catalytic, or cat inhibitors. The terms alternate substrate inhibition and suicide inhibition are used here to describe the two major subclasses of mechanism-based inhibition. 

Suicide substrates and quiescent affinity labels, unlike the other types of inhibitors discussed in this chapter, form covalent bonds with active site nucleophiles and thereby irreversibly inactivate their target enzymes. A suicide substrate,191 also described by Silverman in a comprehensive review1101 as a mechanism-based inactivator, is a molecule that resembles its target enzyme s true substrate but contains a latent (relatively unreactive) electrophile. When the target enzyme attempts to turn over the... 

Mechanistic investigations have shown that these compounds behave as suicide inhibitors (preferably called mechanism-based inactivators) in the sense that they are recognized by /3-lactamases as substrates, but the great stability of the acyl-enzyme intermediate blocks turnover of the enzyme [46] [47]. /3-Lactamase inhibitors can be divided into two classes, class I and class II class-I inhibitors (e.g., clavulanic acid (5.12)), in contrast to those of class II (e.g., olivanic acid (5.15)), have a heteroatom at position 1 that can lead to ring opening at C(5). The mechanistic consequences of this difference in structure are illustrated by the general scheme in Fig. 5.3. 

Aromatase inhibitors may be classified into two types. Type 1 aromatase inhibitors bind to the aromatase enzyme irreversibly, so they are called inactivators. In some cases they are dubbed mechanism-based or suicide inhibitors when they are metabolized by the enzyme into reactive intermediates that bind covalently to the active site. Type 1 aromatase inhibitors are usually steroidal in structure as represented by exemestane (1), formestane (13), and atamestane (14). Formestane (13) was launched by Ciba-Geigy in 1992. As formestane (13) is readily and extensively metabohzed when administered orally, it is used as a depot formulation for injection. 

Linear furanocoumarins (psoralens) inhibit P450s as mechanism-based inactivators (suicide inhibitors). Thus, species that produce psoralens may have evolved C4H enzymes with enhanced tolerance to these compounds. Recombinant C4H from the psoralen-producing species R. graveolens showed markedly slower inhibition kinetics with psoralens, and possibly biologically significant tolerance, compared to C4H from a species that does not produce the compounds (H. tuberosus) ... 

Irreversible inhibitors combine or destroy a functional group on the enzyme so that it is no longer active. They often act by covalently modifying the enzyme. Thus a new enzyme needs to be synthesized. Examples of irreversible inhibitors include acetylsal-icyclic acid, which irreversibly inhibits cyclooxygenase in prostaglandin synthesis. Organophosphates (e.g., malathion, 8.10) irreversibly inhibit acetylcholinesterase. Suicide inhibitors (mechanism-based inactivators) are a special class of irreversible inhibitors. They are relatively unreactive until they bind to the active site of the enzyme, and then they inactivate the enzyme. 

Because the sulfone looks like the original antibiotic, penicillinase accepts it as a substrate, forming an ester, as it does with penicillin. If the ester were then hydrolyzed, penicillinase would be liberated and, therefore, would be free to react with penicillin. However, the electron-withdrawing sulfone provides an alternative pathway to hydrolysis that forms a stable imine. Because imines are susceptible to nucleophilic attack, an amino group at the active site of penicillinase reacts with the imine, forming a second covalent bond between the enzyme and the inhibitor. The covalently attacked group inactivates penicillinase, thereby wiping out the resistance to penicillin. The sulfone is another example of a mechanism-based suicide inhibitor (Section 25.8). 

The arrow indicates the inhibitor site that is metabolically activated by that P450 isoform resulting in mechanism-based inactivation (MBl) of the enzyme that is either irreversible (suicide, S) or quasi-irreversible (Ql). inhibitor acts competitively by coordinating to the P450 heme-iron and/or ligation to the protein at the active site. 2-PMADA, 2-isopropenyl-2-methyladamantane. 

During the past three decades, besides the rational design of hundreds of molecules that have been synthesized and tested as suicide substrates. It also has come to light that nature Itself has known about this mechanistic mode of enzyme Inhibition and provided us with several extremely potent mechanism-based suicide Inactivators. Below are a few selected examples to demonstrate the mode of action of these Inhibitors. 

The best definition of the third type of irreversible enzyme inhibitor, mechanism-based inactivators, is provided by Dr Richard Silverman, a leading authority on the subject. A mechanism-based inactivator (sometimes, much to my dismay, called a suicide substrate), he writes, is an unreactive compound that has a structural similarity to a substrate or product for an enzyme. Once at the active site of the enzyme, it is converted into a species that generally forms a covalent bond to the enzyme, producing inactivation. Although both quiescent affinity labels and mechanism-based inactivators can be mistaken for... 

Time-dependent inhibition defined mainly by mechanism-based inhibition (MBI), which includes CYP suicide inactivation (irreversible inhibition, the more widely studied process) and metabolite-intermediate (MI) complex formation (quasi-irreversible inhibition), is responsible for most clinically significant DDIs (Silverman, 1995 Waley, 1980 Zhou et al., 2005). Suicide inactivation involves the formation of a reactive intermediate that irreversibly inactivates the CYP in the process of catalytic turnover. Quasi-irreversible inhibition occurs when the CYP produces a metabolite (e.g., nitroso intermediate) with the capacity to bind tightly to the CYP heme. TDI (time-dependent inhibition) can be characterized (1) to be dose dependent, (2) to be preincubation time dependent, (3) to have bioactivation of the inhibitor that is required for inactivation of the target enzyme, (4) to have de novo protein synthesis that is required to recover metabolic capacity, and (5) to have potentially slow onset of the effects but be more profound than reversible inhibition. If present, then TDI is the major component of overall enzyme inhibition and frequently leads to clinically relevant DDIs. Table 4.5 contains a list of inhibitors of TDI observed in vitro and in vivo. 

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