Mechanistic insights into the enantioselective activity of Fluxametamide in Tetranychus cinnabarinus.

BACKGROUND: Fluxametamide is a chiral isoxazoline insecticide widely used for pest control. However, the enantioselective biological activity and underlying mechanisms of its enantiomers remain poorly understood. This study aimed to systematically evaluate the enantioselective acaricidal activity of fluxametamide against Tetranychus cinnabarinus. RESULTS: Bioassays revealed pronounced enantioselectivity, with S-(+)-fluxametamide exhibiting significantly higher acaricidal activity than the racemate. Detoxification enzyme assays indicated significant induction of cytochrome P450 activity following sublethal exposure. Transcriptomic and RT-qPCR analyses further identified multiple P450 genes responsive to fluxametamide, among which CYP392E7 and CYP392A16 were strongly up-regulated. Molecular docking and molecular dynamics simulations demonstrated that R-(-)-fluxametamide exhibited stronger and more stable binding to these P450 enzymes than the S-(+) enantiomer. Functional assays using recombinant proteins confirmed that CYP392E7 preferentially metabolized R-(-)-fluxametamide, whereas S-(+)-fluxametamide showed lower metabolic susceptibility. Electrophysiological analyses using two-electrode voltage clamp recordings revealed that fluxametamide acts as a noncompetitive antagonist of RDL2-mediated GABA currents without direct activation, with S-(+)-fluxametamide producing the strongest inhibitory effect. CONCLUSION: The enantioselective acaricidal activity of fluxametamide is governed by a dual mechanism involving preferential metabolic clearance of the R-(-)-enantiomer and stronger target-site inhibition by the S-(+) enantiomer. These findings provide mechanistic insights into the stereoselective action of fluxametamide and highlight the importance of considering enantioselectivity in the development and risk assessment of chiral acaricides. © 2026 Society of Chemical Industry.