Reactivation screening of A-234-inhibited human recombinant acetylcholinesterase in vitro

The nerve agent A-234, classified among the so-called A-series agents or "Novichoks", represents a significant challenge in the treatment of nerve agent poisoning due to its potent and irreversible inhibition of human recombinant acetylcholinesterase (HssAChE). In this study, we screened 22 structurally diverse oxime reactivators for their efficacy against A-234-inhibited HssAChE in vitro after a 10-min incubation. The initial screening was extended to GB- and VX-inhibited enzymes under the same conditions for comparison. Then, the 22 oximes were tested against A-234-inhibited HssAChE for up to 24 h. Finally, we evaluated the reactivation kinetics of the two most effective oximes over a 240-min period. While most reactivators demonstrated considerable reactivation against GB- and VX-inhibited enzymes within 10 min, none showed efficacy against A-234 in this time frame. Extended incubation over 24 h revealed that only HLö-7, MMB-4, HI-6, K027, K868, TMB-4, GM415, and LüH-6 achieved efficient reactivation (set at a 10 % threshold for a 24-h interval), with HLö-7 and methoxime (MMB-4) being the most effective. Kinetic analysis indicated that HLö-7 exhibited a superior second-order reactivation rate constant compared to MMB-4, highlighting differences in binding affinity and catalytic efficacy. In the case of A-234 poisonings, our findings indicate the most effective oxime antidotes and emphasize the need for prolonged therapy to improve clinical outcomes.