Structure-Activity Approach in the Reactivation of Tabun-Phosphorylated Human Acetylcholinesterase with Bispyridinium para-Aldoximes

Abstract

We investigated interactions of bispyridinium para-aldoximes N,N'-(propano)bis(4-hydroxyiminomethyl)pyridinium bromide N,bis(4-hydroxyiminomethyl)pyridinium methanosulphonate (DMB-4), and N,N'-(methano)bis(4-hydroxyiminomethyl)pyridinium chloride (MMB-4) with human erythrocyte acetylcholinesterase phosphorylated by tabun. We analysed aldoxime conformations to determine the flexibility of aldoxime as an important feature for binding to the acetylcholinesterase active site. Tabun-inhibited human erythrocyte acetylcholinesterase was completely reactivated only by the most flexible bispyridinium aldoxime -TMB-4 with a propylene chain between two rings. Shorter linkers than propylene (methylene or ethylene) as in MMB-4 and DMB-4 did not allow appropriate orientation in the active site, and MMB-4 and DMB-4 were not efficient reactivators of tabun-phosphorylated acetylcholinesterase. Since aldoximes are also reversible inhibitors of native acetylcholinesterase, we determined dissociation constants and their protective index against acetylcholinesterase inactivation by tabun.

Evaluation of Oxime K203 as Antidote in Tabun Poisoning

“…AChE and BChE reversible inhibition, protection against tabun progressive inhibition, and reactivation of tabun-inhibited cholinesterases by K203 were measured spectrophotometrically as described earlier (13,14). Kinetic parameters were calculated from experimental data obtained in at least three experiments using known equations (13)(14)(15)20). We evaluated the following kinetic parameters: secondorder rate constant of the non-enzymatic reaction (k NE ), enzyme-oxime dissociation constant (K i ), enzyme-substrate dissociation constant corresponding to Michaelis constant (K m ), protection by oxime against phosphorylation in terms of calculated and experimentally obtained protective index (PI calc , PI exp ), maximum fi rst-order rate constant of reactivation (k max ), phosphorylated enzyme-oxime dissociation constant (K OX ), and overall second-order rate constant of reactivation (k r ).…”

Changes of rat plasma total low molecular weight antioxidant level after tabun exposure and consequent treatment by acetylcholinesterase reactivators

“…Their potency to reactivate AChE was evaluated by in vitro studies and seemed to be sufficient [16]. On the other hand, their efficacy is limited by the safe dosage and this constrains their reactivation efficacy [17]. Hence, the measured AChE activity might appear higher than the real AChE activity due to oximolysis [18].…”

Differential binding of bispyridinium oxime drugs with acetylcholinesterase

“…Currently, strategies for the development of effective oximes can be broadly classified into two categories: the kinetic approach [37,39,[41][42][43] and a structural approach [35,36,38,40] . Kinetics studies on oxime function have revealed that reactivation proceeds via a twostep reaction that consists of the formation of a fully reversible Michaelis-type enzyme-inhibitor-oxime complex followed by the splitting of the complex into a phosphorylated oxime and reactivated enzyme.…”

“…Intentional (in a suicidal attempt [28] ) or unintentional (through trace dietary contaminants [29] or through chemical warfare [30] ) OP intoxication is a major threat to human kind. While intoxication by OPs accounts for up to 80% of pesticide-related hospitalization [31] , the possibilities of terrorist attacks (like the Iraqi weaponized sarin attack against the Kurdish [32] or the Tokyo subway sarin attack [33] ) with nerve agents looms large, and thus the discovery of effective OP antidotes is necessary and urgent [34][35][36][37][38][39][40][41][42][43][44][45][46] . Thus, fundamental studies [16,17] elucidating the passage of molecules through the AChE gorge into the active triad of the enzyme is of paramount importance for the discovery of new drugs.…”

“…However, prior to aging, the inhibited enzyme can be reactivated by strong nucleophiles, such as oximes. The medical management of an OP victim includes drugs such as atropine (a muscarinic receptor antagonist that artificially maintains the respiration capacity), diazepam (or similar anticonvulsant drug, commonly administered to treat seizure) and oxime reactivators [34][35][36][37][38][39][40][41][42][43][44][45][46] (which liberate the inhibited enzyme). Since aging of an inhibited enzyme occurs within minutes to hours and results in the permanent disability of the enzymatic activity of AChE, the victim should be administered these drugs as soon as possible [49] .…”