Functional and genetic validation for indazapyroxamet as an antagonist of insect TRPV channels

Transient receptor potential vanilloid (TRPV) channels formed by Nanchung (Nan) and Inactive (Iav) subunits are essential for mechanosensation and coordinated locomotion in insects and represent validated targets of several commercial insecticides that act as channel agonists. Indazapyroxamet is a recently developed insecticide with high efficacy against piercing-sucking pests; however, its molecular target and mode of action remain unclear. Here, we investigated the mode of action of indazapyroxamet using molecular pharmacology, Drosophila genetics and structural modeling. Calcium imaging in Drosophila S2 cells expressing the brown planthopper (Nilaparvata lugens) TRPV channel revealed that while indazapyroxamet exhibits low-efficacy partial agonism, it potently antagonizes nicotinamide-induced calcium influx with an IC₅₀ of 8.1 nM. Consistently, dietary exposure to indazapyroxamet caused dose-dependent impairment of negative geotaxis in wild-type Drosophila melanogaster, whereas this behavioral effect was completely abolished in nan and iav loss-of-function mutants, demonstrating that its in vivo effects require functional TRPV channels. Molecular docking analysis showed that indazapyroxamet occupies a binding pocket overlapping that of known TRPV agonists and forms aromatic interactions with a conserved residue W322 in the Iav subunit, but lacks interactions with key residue Y178 in Nan subunit required for stabilizing the open-channel conformation. Together, these results identify indazapyroxamet as a TRPV antagonist and reveal a previously unrecognized mode of action for TRPV-targeting insecticides, with important implications for insecticide development and resistance management.