Acetylcholinesterase (AChE) stands
as a primary target of commercial
insecticides, notably organophosphates and carbamates. Despite their
widespread use in agricultural and indoor pest control, concerns over
their high toxicity and the emergence of resistance have restricted
their efficacy. In this study, we conducted high-throughput virtual
screening against both wild-type (WT) and resistant Cimex lectularius AChE utilizing a library encompassing
1 270 000 compounds. From this screening, we identified
100 candidate compounds and subsequently assessed their inhibitory
effects on purified AChE enzymes. Among these candidates, AE027 emerged
as a potent inhibitor against both WT and resistant AChE, exhibiting
IC50 values of 10 and 43 μM, respectively. Moreover,
the binding of AE027 significantly stabilized AChE, elevating its
melting temperature by approximately 7 °C. Through molecular
docking and molecular dynamics simulation, we delineated the binding
mode of AE027, revealing its interaction with a site adjacent to the
catalytic center, which is distinct from known inhibitors, with differing
poses observed between WT and resistant AChE. Notably, the resistance
mutation F348Y, positioned at a site directly interfacing with AE027,
impedes ligand binding through steric hindrance. Furthermore, we evaluated
the toxicity and pharmacokinetic properties of AE027 utilizing bioinformatics
tools. These findings lay a crucial foundation for the development
of a novel generation of insecticides that can combat both WT and
resistant pest populations effectively and safely.