Structure-activity analysis of aging and reactivation of human butyrylcholinesterase inhibited by analogues of tabun
Résumé
Human butyrylcholinesterase (hBChE) naturally scavenges organophosphorus nerve agents (OPs). This bioscavenger is currently in Clinical Phase I for pretreatment of OP intoxication. Phosphylated cholinesterases (ChEs) can undergo a spontaneous time-dependent process called « aging » during which the conjugate is dealkylated, leading to non-reactivable enzyme. hBChEs inhibited by phosphoramidates like tabun display a peculiar resistance to oximes mediated reactivation. We investigated the basis of oxime resistance of phosphoramidyl-BChE conjugates, by determining the kinetics of inhibition, reactivation (obidoxime, TMB-4, HLö 7, HI-6), aging, and the crystal structures of hBChE inhibited by different N-monoalkyl and N,N-dialkyl tabun analogues. The refined structures of aged hBChE conjugates show that aging proceeds through O-dealkylation of the P(R) enantiomer of N,N-diethyl and N-propyl analogues, with subsequent formation of a salt bridge preventing reactivation, similarly to previous observation made on tabun-ChEs conjugates. Interestingly, the N-methyl analogue projects its amino group towards the choline-binding pocket, so that aging proceeds through deamination. This orientation results from a preference of hBChE's acyl-binding pocket for larger than 2-atoms linear substituents. The correlation between the inhibitory potency and the N-monoalkyl chain length is related to increasingly optimized interactions with the acyl-binding pocket as shown by the X-ray structures. These kinetics and X-ray data lead to a structure-activity relationship that hightlights steric and electronic effects of the amino substituent of phosphoramidate. This study provides structural basis to design new oximes capable of reactivating phosphoramidyl-hBChE conjugates after intoxication, notably when hBChE is used as pretreatment or to design BChE-based catalytic bioscavengers.
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