Mechanism-based enzyme inhibition described

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. 

This article describes various approaches to inhibition of enzyme catalysis. Reversible inhibition includes competitive, uncompetitive, mixed inhibition, noncompetitive inhibition, transition state, and slow tight-binding inhibition. Irreversible inhibition approaches include affinity labeling and mechanism-based enzyme inhibition. The kinetics of the various inhibition approaches are summarized, and examples of each type of Inhibition are presented. 

The starting point for much of the work described in this article is the idea that quinone methides (QMs) are the electrophilic species that are generated from ortho-hydro-xybenzyl halides during the relatively selective modification of tryptophan residues in proteins. Therefore, a series of suicide substrates (a subtype of mechanism-based inhibitors) that produce quinone or quinonimine methides (QIMs) have been designed to inhibit enzymes. The concept of mechanism-based inhibitors was very appealing and has been widely applied. The present review will be focused on the inhibition of mammalian serine proteases and bacterial serine (3-lactamases by suicide inhibitors. These very different classes of enzymes have however an analogous step in their catalytic mechanism, the formation of an acyl-enzyme intermediate. Several studies have examined the possible use of quinone or quinonimine methides as the latent... 

Characterization of a mechanism-based inhibitor may involve the estimation of the constants described in section IV, namely, /qnacI, Kh /ccat, and r. The most common approach has been to incubate inhibitor, enzyme, and cofactors together and to determine the decline in enzyme activity with time (26). In practice, this approach often employs the measurement of residual enzyme activity in a subsequent incubation with a specific substrate under conditions that limit further inactivation and competitive inhibition by the inactivator, usually by an appropriate dilution (10-fold or greater) of the original incubate (5). 

A class of thiazole hydrazide inhibitors of cathepsin K has also been evaluated in references 26 and 27. Similar problems to those described in the preceding paragraph related to enzyme acylation and slow turnover of inhibitor in solution were observed in trying to determine the mechanism of protease inhibition based... 

It should also be noted that the activation of a mechanism-based inhibitor by its target enzyme is, formally, an example of metabolic activation. However, there is a clear distinction between the activation of a mechanism-based inhibitor described above and the metabolic activation of a prodrug. In the latter case, an inactive precursor is metabolized in the body (either chemically or enzymatically) to metabolites that possess the desired activity. For example. Acyclovir (3a) must be metabol-ically converted to the triphosphate (3b) and released into the medium before it will inhibit viral DNA polymerase. Further discussion on prodrugs may be found in volume 2, chapter 14. 

Enzyme inhibition data are often presented as IC50, the concentration of the inhibitor to cause 50 percent inhibition at one chosen substrate concentration Kt, the inhibition constant (dissociation constant from the inhibitor-enzyme complex) determined by enzyme kinetic analysis (e.g., Dixon plot) and /Cin lcl, the time-dependent inhibition constant for mechanism-based inhibitors. IC50 values can be estimated from the study described earlier. A positive inhibition, defined as dose-dependent inhibition, with the inhibited activity lower than 50 percent of that of the negative control, will require further experimentation to define Ki for a better evaluation of in vivo inhibitory potential. Further, a study to determine Klwul may be performed to evaluate if the inhibitor acts via covalent binding to the active site of the enzyme, leading to time-dependent irreversible inhibition. 

Aromatase - Inhibition of aromatase, the cytochrome P-450 enz3rme that oxidizes testosterone to estradiol, offers a means for the control of estrogen-dependent tumors. A number of mechanism-based inhibitors that may act through heme-alkylation have been described for this enzyme. 

As previously described, irreversible enzyme inhibition is defined as time-dependent inactivation of the enzyme, which implies that the enzyme has, in some way or form, been permanently modified, because it can no longer carry out its function. This modification is the result of a covalent bond being formed with the inhibitor and some amino acid residue in the protein. Furthermore, this bond is extremely stable and, for all practical purposes, is not hydrolyzed fo give back the enzyme in its original state or structure. In most examples of irreversible inhibition, a new enzyme must be generated through gene transcription and translation for the enzyme to continue its normal catalytic action. Basically, there are two types of irreversible enzyme inhibitors, the affinity labels or active site-directed irreversible inhibitors and the mechanism-based irreversible enzyme inactivators. 

The initially proposed mechanism based on the n-butylisocyanide inhibited structure has been investigated by two DFT studies. Based on an imposed requirement for Ssumdo C bond formation in the catalytic cycle, results of the first study supported the formation of a highly stable S-C bonded thiocarbonate intermediate in the catalytic cycle. The activation barrier evaluated over two catalytic cycles was computed to be 18.9 kcal mol , which compares with kinetic studies on the enzyme that yield an activation barrier of 11.4 kcal mol at pH 7.2. A subsequent DFT study described a qualitatively similar mechanism with activation barriers calculated to be >33 kcal mol for MeNC and >24 kcal mol for CO as reducing substrates. The lower limits on the activation barriers result from the observation that transition states could not be located for the conversion of... 

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. 

Studies of the mechanism of end-product inhibition have revealed that the configuration and activity of certain enzymes can be modified by combination with substances "effectors ) unrelated to their substrates. The effector binds to a site(s) separate from that involved in substrate-enzyme combination and in so doing alters the configuration of the enzyme, e.g. by altering the degree of aggregation of the sub-units of the enzyme molecule. Such enzymes are described as allosteric enzymes. Aspartic acid can be the precursor of pyrimidine bases and the first reaction involves the allosteric enzyme aspartate carbamyl-transferase. This enzyme is inhibited by one of the end-products, cytidine triphosphate (CTP). Clearly there is consider-... 

Heavy metals stimulate or inhibit a wide variety of enzyme systems (16, 71, 72), sometimes for protracted periods (71, 73). These effects may be so sensitive as to precede overt toxicity as in the case of lead-induced inhibition of 8 ALA dehydrase activity with consequential interference of heme and porphyrin synthesis (15, 16). Urinary excretion of 8 ALA is also a sensitive indicator of lead absorption (74). Another erythrocytic enzyme, glucose-6-phosphatase, when present in abnormally low amounts, may increase susceptibility to lead intoxication (75), and for this reason, screens to detect such affected persons in lead-related injuries have been suggested (76). Biochemical bases for trace element toxicity have been described for the heavy metals (16), selenium (77), fluoride (78), and cobalt (79). Heavy metal metabolic injury, in addition to producing primary toxicity, can adversely alter drug detoxification mechanisms (80, 81), with possible secondary consequences for that portion of the population on medication. 

OPH-based biosensors have been fully discussed in previous reviews [2,165]. AChE-based biosensors are based on the principle that OP pesticides have an inhibitory effect on the activity of AChE that may be permanent or partially reversible. The extent of the inhibition is directly related to the concentration of the pesticide and therefore enzyme activity may be used as a measure of the inhibition [166]. The amperometric measurement of AChE activity can be based on the measurement of any of the following three mechanisms [167] (1) production of hydrogen peroxide from choline, (2) oxygen consumption during the enzyme reaction or (3) production of electroactive compounds directly from the oxidation of acetylthiocholine chloride such as thiocholine. The measurement of hydrogen peroxide and oxygen consumption has been described in more details in other reviews [167]. 

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