Suicide enzyme inactivators examples

The biological activity of a compound can often be affected dramatically by the presence of even a single fluorine substituent that is placed in a particular position within the molecule. There are diverse reasons for this, which have been discussed briefly in the preface and introduction of this book. A few illustrative examples of bioactive compounds containing a single fluorine substituent are given in Fig. 3.1. These include what is probably the first example of enhanced bioactivity due to fluorine substitution, that of the corticosteroid 3-1 below wherein Fried discovered, in 1954, that the enhanced acidity of the fluorohydrin enhanced the activity of the compound.1 Also pictured are the antibacterial (3-fluoro amino acid, FA (3-2), which acts as a suicide substrate enzyme inactivator, and the well-known anti-anthrax drug, CIPRO (3-3). 

The catalytic aspect of enzymes has prompted the development of new types of enzyme inactivators, which have been called suicide inhibitors . These inhibitors are substrates which are modified by masking the reactive functions. If they are unmasked by the catalytic action of the target enzyme, reaction of the reactive group with an active site or cofactor is made possible and this leads to the inactivation of the enzyme. Since the chemically reactive group is only liberated at the active site of the target enzyme, reactions with foreign molecules cannot occur. A number of examples of suicide inhibitors (or K -at inhibitors) can be found in a paper by Rando... 

Mechanism-based inactivation of CYP450 (or suicide inhibition) occurs when a non-toxic drug is metabolised by CYP450 to generate a metabolite that can bind irreversibly with the enzyme. The mechanism of inhibition usually involves free-radical alkylation or acylation of the active site and results in destruction of enzyme activity. Examples of drugs that act in this way include the antibiotic chloramphenicol and the anticancer agent cyclophosphamide. 

Certain inferences about the mechanism of inactivation can be made from inactivation kinetics. Stmcture-activity relationships of a series of compounds can lend support to various mechanisms with knowledge of the aetive site of the target enzyme (for examples see Lynas and Walker, 1997). The effect of the inhibitor s chirality can also provide information regarding how the suicide inhibitor is reacting with the enzyme. 

The inactivation kinetics of this type can indeed be treated as a special case of the Michaelis-Menten mechanism in which the turnover of the substrate is too slow compared with the rate (ki) of enzyme inactivation. Penicillins are typical examples of such substrate inhibitors for /3-lactamases. Certain classes of irreversible inhibitors, called suicide inhibitors, are chemically unreactive in the absence of target enzymes. When an enzyme binds the innocuous inhibitor with the same specificity as the substrate, however, the inhibitor is activated into a powerful irreversible inhibitor. 

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. 

True enough, treatment of PAP with FMPP resulted in a time-dependent inactivation of the enzyme. Competitive inhibitors of PAP protected against inactivation. The authors suggest that FMPP represents a useful basic structure which can be incorporated into the design of more specific phosphatase inhibitors for example, the modified tyrosine 77 could be incorporated into a particular peptide to give a suicide substrate that is selective for a protein phosphatase which preferentially hydrolyses that peptide. 

In the preceding section, four diagnostic tests of affinity labeling were listed (inactivation inhibited by substrates, pH dependence of inactivation similar to that of catalysis, labeled inhibitor covalently bound in 1 1 stoichiometry, and saturation kinetics obeyed). The same criteria may be used to diagnose suicide inhibition. In addition, tests must be made to detect any diffusion of the activated intermediate I into solution. For example, the addition of —SH reagents that rapidly react with electrophiles and hence scavenge them should not slow down the rate of reaction. The suicide inhibitor should not, in any case, react with the thiol at an appreciable rate in the absence of enzyme. 

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). 

The compound 5-fluorouridine targets thymidylate synthase. After a nucleoside kinase phosphorylates it, resembles the natural substrate for the enzyme, except that it contains a fluorine where dUMP has a hydrogen. The fluorine isn t removed from the ring by thymidylate synthase, and this causes the ring to remain covalently bound to the enzyme, which means that the enzyme is irreversibly inactivated. The 5-fluorouridine monophosphate is an example of a suicide substrate —a compound whose reaction with an enzyme causes the enzyme to no longer function. 

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). 

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... 

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. 

Halo enol lactones are an example of suicide Inhibitors for serine proteases. These analogues were developed by Katzenellenbogen and coworkers at the University of Illinois (34). On normal catalytic processing by the serine hydroxyl functionality, they give rise to a reactive halo-methyl ketone, which subsequently alkylates a nearby nucleophilic residue on the enzyme (Fig. 5.16). Other suicide Inactivators for the serine proteases have been designed by various researchers (32). 

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