Suicide inhibition mechanism

Figure 3. Proposed Suicide Inhibition Mechanism for the p-Chloromethylphenyl Carbonates.

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. 

Irreversible inhibitors combine or destroy a functional group on the enzyme so that it is no longer active.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. 

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... 

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.

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. 

R. A. Engh, R. Huber, W. Bode, and A. J. Schulze Divining the serpin inhibition mechanism, a suicide substrate spring Trends in Biotechnology 13,503(1995). 

Administration of allopurinol, an analog of hypoxanthine, is one treatment for gout. The mechanism of action of allopurinol is interesting it acts first as a substrate and then as an inhibitor of xanthine oxidase. The oxidase hydroxylates allopurinol to alloxanthine (oxipurinol), which then remains tightly bound to the active site. The binding of alloxanthine keeps the molybdenum atom of xanthine oxidase in the + 4 oxidation state instead of it returning to the + 6 oxidation state as in a normal catalytic cycle. We see here another example of suicide inhibition. 

A unique mechanism-based mode of action ( suicide inhibition ) for some fluoropharmaceuticals involves direct chemical reaction of a fluorinated substructure with the target protein. 

Scheme 4.33 Mechanism of the suicide inhibition of tymidylate synthase by 5-fluorouracil. The reaction pathway with the natural substrate (dUMP) is depicted on the left, the analogous sequence with 5-fluoro-dUMP on the right. The key to the irreversible blocking of the enzyme reaction site is the inability of fluorine to functionally replace hydrogen in proton-transfer reactions, for example the -elimination liberating the enzyme thiolate group [10], In addition, the transient positive charge on the methylene group during hydride transfer is destabilized by the jff-fluorine.

Nagahisa, A., W.H. Orme-Johnson, and S.R. Wilson (1984). Silicon mediated suicide inhibition An efficient mechanism-based inhibitor of cytochrome P-450j,j, oxidation of cholesterol. J. Am. Chem. Soc. 106, 1166-1167. 

As already mentioned, 2-ethynylnaphthalene was a weak inhibitor of cytochrome P450 1A but exhibited potent inhibitory activity of cytochrome P450 2B1. It was also shown to be a potent inhibitor of 2-naphthylamine N-oxidation in vitro, a reaction mediated by cytochrome P450 1A2. Of the two isomers, 2-ethynylnaphthalene was more potent than 1-ethynylnaphthalene. The mechanism of inhibition of cytochrome P450 1A2 proved to be suicide inhibition ". ... 

The acetylenic analog of Mead Acid, 5,8,11-eicosatriynoic acid, was reported to be a selective inhibitor of platelet 12-lipoxygenase. Acetylenic fatty acids also inhibited 15-lipoxygenases of plant (soybean) as well as of animal (rabbit reticulocyte) origin. The nature of the acetylenic compound significantly affected its activity on the soybean enzyme, but not the rabbit enzyme. For the former, 7,10,13-eicosatriynoic acid was the most powerful inactivator. Addition of a fourth triple bond at position 4 or 5 strongly reduced the rate of inactivation. On the other hand, the rabbit reticulocyte enzyme was inactivated almost equally well by the various acetylenic fatty acids that were tried. The mechanism of inactivation of lipoxygenases was also suicide inhibition . ... 

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