Target identification and mechanism of action of the neonicotinoid Cycloxaprid

Neonicotinoid insecticides targeting nicotinic acetylcholine receptors (nAChRs) are critical for agricultural pest control but face challenges from rapid resistance and environmental toxicity. Cycloxaprid, a cis-nitro-configured neonicotinoid analog, shows unique pharmacological properties that may circumvent cross-resistance. Here, we elucidated its mode of action in Drosophila melanogaster through integrated genetic, electrophysiological, and computational approaches. Cycloxaprid induced excitatory neurotoxicity, including hyperactivity, uncoordinated locomotion, and tremors, consistent with nAChR overactivation. Bioassays with Drosophila nAChR subunit mutants demonstrated that the α1, α2, β1, and β2 subunits are essential for insecticidal activity. Heterologous expression of the α1α2β1β2 nAChR in Xenopus oocytes revealed cycloxaprid acts as a partial agonist (EC₅₀ = 34.7 nM; 37.7 % maximal acetylcholine response) and enhances acetylcholine-evoked currents at sub-saturating doses. AI-driven structural modeling (Chai-1) localized cycloxaprid within the canonical agonist-binding site at the α1-β1 interface, forming key electrostatic interactions with residues like Arg81 in the β1 subunit. Collectively, these results elucidate the molecular basis of cycloxaprid's insecticidal activity and underscore its potential as a valuable tool for resistance management.