Rational design to enhance the catalytic activity of acetylcholinesterase and mitigate trichlorfon toxicity in vitro.

Trichlorfon (TCF) is a widely used organophosphate pesticide whose inhibition of acetylcholinesterase (AChE) results in neurotoxicity and significant biosafety risks. Addressing these concerns requires effective strategies to mitigate TCF-induced toxicity and safeguard exposed organisms. In this study, we explored the potential of a catalytic activity enhanced Culex pipiens AChE mutant to mitigate TCF-induced cytotoxicity through rational design. A double-point mutant, M5 (I198M/Y249F), was developed by combining molecular dynamics (MD) simulations with structural feature analysis to reshape the active pocket, which demonstrated enhanced catalytic efficiency and maintained thermostability. Its functional activity and improved catalytic performance were further confirmed by activity staining on non-denaturing gels. The analysis of the catalytic mechanism and the reduction in Molecular Mechanics-Generalized Born Surface Area (MM/GBSA) free energy revealed an increase in substrate affinity for M5. Additionally, the application of exogenous M5 not only restored endogenous AChE activity in NIH/3T3 cells exposed to TCF but also reduced reactive oxygen species (ROS) accumulation and apoptosis, thereby improving cell viability. In silico studies indicate that the stable interaction between M5 and TCF promotes the targeted depletion of TCF, effectively neutralizing its toxic effects. These findings indicate that M5 has potential as an enzyme-based antidote for organophosphate pesticide, offering a novel strategy for protecting non-target species from pesticide-induced damage.