Potent inhibition and limited reactivation of human cholinesterases by cholesterol- and quinoline-based oximes.

Current oxime antidotes exhibit poor blood-brain barrier (BBB) penetration due to their permanent positive charge, limiting the reactivation of organophosphorus (OP)-inhibited cholinesterases in the central nervous system. To address this limitation, the present study investigates nine novel cholesterol- and quinoline-based oximes as potential reactivators of human acetylcholinesterase (hAChE) and butyrylcholinesterase (hBChE) inhibited by OP compounds. Several oximes exhibited strong reversible inhibition with pronounced potency toward hBChE (Ki as low as 4.6nM). However, despite the favourable binding affinities, reactivation assays using sarin- and cyclosarin-inhibited enzymes revealed negligible recovery of enzymatic activity compared with the standard antidote pralidoxime (2-PAM). Molecular modelling of near-attack conformations showed that the oxime group of the most potent inhibitor adopted an unproductive orientation relative to the catalytic serine. These results highlighted that binding affinity alone is insufficient for effective reactivation; precise positioning of the oxime moiety to enable nucleophilic access to the phosphorus centre is critical. Therefore, a strategic framework for designing next-generation oxime reactivators based on such structures is needed to improve functional efficacy. On the other hand, cholesterol- and quinoline-based oximes with the previously described neuroprotective properties may provide a foundation for further investigation of these compounds as valuable adjuvants in OP therapy. Moreover, the observed cytotoxicity in hepatocarcinoma (HepG2) and neuroblastoma (SH-SY5Y) cell lines occurred in the 50-100µM range, indicating moderate in vitro cytotoxicity. However, further studies are needed to determine their significance for the compounds' therapeutic potential and safety profile.