Suicide inhibition

To identify a compound as a suicide inhibitor, the inhibition must be established as time dependent, irreversible, active-site directed, requiring catalytic conversion of inhibitor, and have 1 1 stoichiometry for E and X in the EyX complex. To assess the potency and efficacy of a suicide inhibitor, the kinetics of the inactivation and the partition ratio should be determined. Identification of both X and the amino acid/cofactor labeled in the EyX complex is useful in establishing the actual mechanism of inactivation. 

As with alternate substrate inhibitors, a progress curve or continuous enzyme assay is the most useful to begin to characterize the kinetics of inhibition. There can be immediate, or diffusion-limited inhibition of the 

Because continuous assays monitor only free enzyme, they do not distinguish between E-I, E-I, or the E()X complex. Therefore, obs represents the apparent inactivation rate, a combination of inhibition and inactivation. As with alternate substrate inhibition, the second-order apparent inactivation rate can be determined from one of the following equations, depending on whether or not saturation kinetics are observed and the concentration of substrate  

Incubation/dilution assays or rescue assays can help distinguish between the reversible and irreversible steps in the inactivation. In incubation/dilution assays, enzyme and inhibitor are incubated in the absence of substrate under assay conditions. At various time points, At, an aliquot of this incubation is diluted into an assay mixture containing substrate, and the activity monitored. A rescue assay is a standard progress assay in which the inhibitor is removed in situ, at various time points. At, by the addition of a chemical nucleophile, which consumes free inhibitor (Fig. 13.2). In both cases, either by dilution or by chemical modification, the free inhibitor is effectively removed from the reaction. Any time-dependent recovery of activity should represent ks, as shown in Fig. 13.2 (although in the rescue assay, the rate of disappearance of the inhibitor will also effect enzyme recovery). Any decrease in the final steady-state rate of activity as compared to the initial enzyme activity is due to inactivated enzyme, E X. 

By varying At for each inhibitor concentration, obs for each assay can be determined as the negative slope of n vt/vo) versus At. Repeating this for a series of [I] and using Eq. (13.2), (13.3), or (13.4), depending on whether or not the system is saturating in inhibitor or substrate, the actual 

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SERINE PROTEASES MINIMAL SCHEMES CATALYTIC MECHANISMS SUICIDE INHIBITION... 

Irreversible CYP inhibition can arise from different chemical mechanisms. However, a common initial step is the metabolic activation of a substrate into a reactive metabolite that is trapped within the active site of the CYP to form a tightly bound complex causing a long-lasting inactivation of enzyme activity. Enzymatic activity can be restored only through the new synthesis of the enzyme. For this reason, irreversible CYP inhibition is often referred to as mechanism-based inhibition , metabolite-based inhibition or suicide inhibition . 

When reactive metabolites are formed by metabolic activation, some of them can escape from the active site and bind to external protein residues or be trapped by reduced glutathione (GSH) or other nucleophiles. The remaining molecules that are not released from the active site will cause the suicide inhibition [7]. The ratio of the number of reactive molecules remaining in the active site and those escaping is a measure of the reactivity of the intermediates formed. The addition of scavengers or GSH to the incubation mixture does not affect and cannot prevent the CYP mechanism-based inhibition. However, GSH can reduce the extent of the nonspecific covalent binding to proteins by those reactive molecules that escape from the active site. In contrast, addition of substrates or inhibitors that compete for the same catalytic center usually results in reduction of the extent of inhibition. 

Scheme 3.6 Piperonyl butoxide (PBO), a pesticide synergist, and PBO suicide inhibition of CYP. Modified from [49].

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. 

Enzymes containing pyridoxal phosphate are prime targets for suicide inhibition because the chemistry is so naturally suitable. As discussed in Chapter 2, section C2, the pyridoxal ring acts as an electron sink that facilitates the formation of carbanions and also forms part of an extended system of conjugated double bonds. For example, vinyl glycine, CH2=CHCH(NH3+)C02, condenses with the pyridoxal phosphate of aspartate aminotransferase to form a Schiff base, as described in Chapter 2, equation 2.42.19 The a proton may be abstracted (as in equation 2.43) so that the isomerization shown in equation 9.13 readily occurs. 

A classic example of suicide inhibition is that of /3-hydroxy l-decanoyl-dehydrase by 3 -decenoy 1-N-acetylcy steamine.12 The enzyme catalyzes the reaction... 

The intermediate 9.14 is probably generated during the suicide inhibition of /3-aspartate decarboxylase,21 aspartate aminotransferase,22 and alanine racemase23,24 by /3-chloroalanine. These enzymes are inactivated by the intermediate, since they have not evolved to cope with it during the normal course of reaction. 

As illustrated in Scheme 6.1, once the covalent intermediate is formed, the complex can either follow a normal catalytic cycle or go through a suicide event leading to the irreversible labeling that is necessary for selection. The suicide inhibition efficiency depends on the ratio k /k. This ratio depends on the nature of the suicide substrate and of the enzyme. Therefore, a large excess of suicide substrate as compared to the displayed enzyme is recommended for selection experiments. 

Inhibition can be reversible when it simply complexes at the active site preventing further catalysis. The active enzyme under these conditions can be recovered by dialysis. Another form of inhibition is the irreversible type where the active enzyme cannot be recovered by dialysis. A variant of this type of inhibition is suicide inhibition a substrate of the enzyme reacts at the active site to yield an irreversible inhibitor which then reacts directly with groups at the active site [18]. A technique, in situ click chemistry , is related to that of suicide inhibition and involves click chemistry components which complex at the active site of an enzyme and combine to form femtomolar inhibitors. The technique can be used to synthesise inhibitors or by selection from a library of click chemistry components to search structure space of the inhibitor for the drug target [ 19]. 

L Hazen, LA Zupan, RH Weiss, DP Getman, RW Gross. Suicide inhibition of canine myocardial cytosolic calcium-independent phospholipase A2. Mechanism-based discrimination between calcium-dependent and independent phospholipases A2. J Biol... 

Keys DA, Schultz IR, Mahle DA, Fisher JW. 2004. A quantitative description of suicide inhibition of dichloroacetic acid in rats and mice. Toxicol Sci 82 381-393. 

Scheme 5.2. Kinetic scheme for a suicide inhibition arising from a covalent intermediate (Y).

Schallreuter, K.U., Gibbons, N.C.J., Elwary, S.M., Parkin, S.M., Wood, J.M. (2007). Calcium-activated butyrylcholinesterase in human skin protects acetylcholinesterase against suicide inhibition by neurotoxic organophosphates. Biochem. Biophys. Res. Commun. 355 1069-74. 

Irreversible or quasi-irreversible metabolic inhibition occurs when either the parent compound or a metabolic intermediate binds to the reduced ferrous heme portion of the P450 enzyme/ thereby inactivating it (51). In irreversible inhibition, or "suicide inhibition/" the intermediate forms a covalent bond with the CYP protein or its heme component/ causing permanent... 

Danse tte PM, Delaforge M, Sartori E, Beaune P, Jaouen M, Mansuy D. Drug interactions with macrolide antibiotics Specificity of pseudo-suicide inhibition and induction of cytochrome P-450. Adv Exp Med Biol 1986 197 155-62. 

Figure 8.23. Mechanism-Based (Suicide) Inhibition. Monoamine oxidase, an enzyme important for neurotransmitter synthesis, requires the cofactor FAD (flavin adenine dinucleotide). AA -Dimethylpropargylamine inhibits monoamine oxidase by covalently modifying the flavin prosthetic group only after the inhibitor is first oxidized. The N-5 flavin adduct is stabilized by the addition of a proton.

Figure 25.15. Suicide Inhibition. Fluorodeoxyuridylate (generated from fluorouracil) traps thymidylate synthase in a form that cannot proceed down the reaction pathway.

MPO from H. niger was isolated and its molecular weight determined by gel filtration (135 kDa) and the SDS-PAGE analysis showed that the enzyme is a dimmer [140]. MPO has also been obtained from other plant speeies such as D. stramonium [141] and N. tabacum [142], Inhibition studies of Hyoscyamus MPO demonstrated the quinoprotein characteristic of this enzyme [140], a feature previously reported for N. tabacum MPO, after conducting a suicidal inhibition of the enzyme with phenyUiydrazine [143]. 

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