engleski

Acetylcholinesterase interactions with highly toxic organophosphorus compounds

In the cholinergic system, acetylcholinesterase (AChE) is the primary target of organophosphorus compound nerve agent intoxication. Nerve agents inhibit AChE activity resulting in accumulation of excess acetylcholine at vital cholinergic sites, thereby causing toxic manifestation. Reaction of organophosphates with AChE is characterized by the formation of the active site serine-conjugated phosphates, which react very slowly with water. Strong nucleophiles, such as oximes, are particularly effective in reactivating AChE regenerating the active enzyme. The effectiveness of oxime reactivators is primarily attributed to the enchanced rate of nucleophilic displacement of conjugated organophosphates, but efficiency varies with the structure of the bound organophosphate, the source of enzyme, and the oxime. The active site gorge of AChE is lined largely by aromatic side chains contributing to a well-defined acyl pocket and choline binding site at the base of the gorge. Therefore, the orientation of the associated and conjugated ligands within narrow confines of the gorge and the rates of nucleophilic attack by oxime at the conjugated phosphorus atom become important determinants of the reactivation mechanism. Phosphorylation and oxime reactivation measurements were conducted to investigate structural and kinetic bases for these reactions using a combined structure-activity approach where inhibitor, enzyme and oxime were modified systematically. AChE was modified within the active site domains in various mutation permutations. By using a congeneric series of SP and RP enantiomeric pairs of alkyl methylphosphonates, nerve agents analogs, and two related oximes of different dimensions, HI-6 and 2-PAM, subtle differences in reactivation capability were analyzed with the objective of enhancing reactivation rates. Obtained phosphorylation and reactivation rates reach values sufficient for consideration of mixtures of a mutant enzyme and an oxime as a scavenging strategy in prophylaxis and treatment of organophosphate exposure. The phosphoramide class of nerve agents (e.g. tabun) is known to be quite resistant to oxime-mediated reactivation showing limited reactivity and slow rates of regeneration of active enzyme. Therefore, a series of bispirydinium oximes were tested as reactivators of tabun-phosphylated AChE and as protectors of AChE against phosphorylation by tabun. Oximes with oxime group in para position efficiently reactivated tabun-inhibited AChE by micromolar concentrations. Beside acting as reactivators, all oximes protected the AChE catalytic site against phosphorylation due to a direct competition between the oxime and tabun by at least factor 2. Supported by the grant under the Croatian-Slovenian Bilateral programme on Corporation in Science and Technology

acetylcholinesterase; antidotes; inhibition; nerve agents; oxime; reactivation

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