Pseudo-irreversible inhibitors

Rivastigmine is a pseudo-irreversible inhibitor of both acetyl and butyryl cholinesterases. Thus although the drug initially blocks the enzymes, it is metabolized by them thereby giving the drug a relatively short half-life. The top dose is often necessary to achieve therapeutic efficacy, at which dose the central and peripheral cholinergic side effects become apparent. 

As may be expected, criteria for the study of pseudo irreversible inhibitors are very similar to those for both affinity labels and mechanism-based inhibitors. However, because of the inherent reversibility of pseudoirrevers-ible inhibitors, it may be more difficult to obtain structural evidence for the covalent enzyme inhibitor adduct. Further, determination of the rate of reactivation and characterization of the products of the recovery process will also be of major importance in designating an inhibitor as pseudoirreversible. 

Acetylcholinesterase (AChE) inhibitors are successfully used as drugs for the treatment of Alzheimer s disease. Physostigmine, also named eserine, has been isolated from the Calabar bean Physostigmina Venenosum). It is classified as a pseudo-irreversible inhibitor because it reacts with acetyl- as well as butyryl-cholinesterase (BChE) to form a carbamylated serine which is hydrolyzed again with a fi/2 of 40 min [73]. Physostigmine inhibits AChE with an IC50 of 27.9 nM and BChE with an IC50 of 16 nM. Its enantiomer is 350 times less active on AChE and 150 times less active on BchE [74]. 

Other unsaturated substrate analogs that have been tried as enzyme inhibitors include allyl amine and allyl alcohol. Allylamine is a pseudo-irreversible inhibitor of flavin-linked monoamine oxidase i.e., in the presence of allylamine, the enzyme shows a time-dependent inactivation that cannot be reversed by dialysis. When radiolabeled allylamine is used, radioactivity is incorporated at the same rate as the enzyme is inhibited. However, inhibition is relieved and radioactivity is removed from the enzyme upon incubation with the substrate, benzylamine. 

In unier tu cuklcuuy prevent proteolysis, the inhibitor should fulfill at least two requirements (1) inhibit the target enzyme in an irreversible or pseudo-irreversible manner and (2) suppress the enzyme activity before significant proteolysis has occurred. From a kinetic viewpoint, the classical example of vn irreversible protein inhibitor of NE is ai-PI. Proteolysis can also be prevented by reversible inhibitors if the in vivo inhibitor concentration is much greater than Kj, the equilibrium constant far inhibition (Ujvta >>Kj), resulting in a pseudo-irreversible behavior. 

Based on this premise, y-acetylenic GABA (IV) waB synthesized (11) and found to be an irreversible inhibitor of GABA-T, in vitro and in vivo (12). Thus, when GABA-T, partially purified from pig brain, is incubated for varying time periods with y-acetylenic GABA, a time-dependent inactivation process is observed which follows pseudo first-order kinetics. Enzyme half lives range from 28 minutes to 9 minutes with concentrations of inhibitor between 0.029 mM and 0.29 mM. Time dependent inactivation is... 

Figure 17.26. Pseudo first-order inactivation kinetics of an active-site directed irreversible inhibitor.

One of the most important mechanistic characteristics of COX-2 inhibition exhibited by many COX-2-selective inhibitors is the observed time-dependent inhibition of COX-2 but not of COX-1. A slow, noncovalent, pseudo-irreversable conformational change in the protein occurs after binding of the inhibitor, but this is only observed in binding with the COX-2 enzyme (353,398,399). As a result, a much lower concentration of inhibitor is required for effective inactivation of the COX-2 enzyme after a longer incubation, even for a... 

The active-site-directed inhibitor tosylphenylalanine chloromethyl ketone that specifically and irreversibly inhibits chymotrypsin. This chloroketone inhibitor relies on its toluene sulfonyl (or tosyl) group for binding into the aromatic binding pocket of chymotrypsin s active site. Inactivation occurs by alkylation of histidine-57 (pseudo-first order rate constant 0.2 min ). See Chymo-trypsin... 

In vitro studies with human term placental microsomes showed that PED displayed the typical properties of a mechanism-based inhibitor of cytochrome P450, namely (a) NADPH-dependent inhibition (b) time-dependent loss of enzymatic activity, followed by pseudo-first-order kinetics and (c) natural substrates delayed the irreversible inhibition. The irreversibility of inhibition was further substantiated by dialysis experiments ... 

Fia. 1. Irreversible inactivation of A -3-ketosteroid isomerase by 5,10-secoestr-6-yne-3,10,17-trione (O) or 5,10-seco-19-norpregn-5-yne-3,10,20-trione ( ). Double reciprocal plots are shown of the apparent pseudo-first-order rate constant of inactivation (fcapp) with respect to inhibitor concentration. Values of Ki and k, were determined from slopes and intercepts. [F. H. Batzold and C. H. Robinson, J. Am. Chem. Soc. 97, 2576 (1975)]. 

Park C, Koh JS, Son YC, Choi H, Lee CS, Choy N, Moon KY, Jung WH, Kim SC, Yoon H (1995) Rational design of irreversible, pseudo-C2-symmetric hiv-1 protease inhibitors. Bioorg Med Chem Lett 5(16) 1843-1848. doi 10.1016/0960-894X(95)00306-E... 

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