Mechanistic characterization of tigolaner reveals allosteric inhibition of insect GABA receptors

Tigolaner is a recently developed long-acting ectoparasiticide with potent activity against fleas and ticks, but its molecular mode of action remains poorly defined. In this study, we investigated how tigolaner interacts with insect γ-aminobutyric acid (GABA)-gated chloride channels by combining receptor pharmacology, insecticide bioassays using Drosophila point mutants, and molecular docking. The pore-associated A301S substitution markedly reduced the inhibitory activity of the classical non-competitive antagonist α-endosulfan but caused only an approximately two-fold decrease in tigolaner potency in membrane-potential assays. Consistently, Drosophila strains carrying A301S or A301N substitutions exhibited little or only modest resistance to tigolaner, indicating that tigolaner does not primarily act through the canonical central ion-pore site. In contrast, the G335M substitution at the transmembrane subunit interface substantially reduced tigolaner potency in vitro and conferred a 12.5-fold increase in resistance in Drosophila mutants. Similar resistance profiles were observed for the established transmembrane allosteric modulators broflanilide and isocycloseram. Molecular docking further predicted that tigolaner occupies the transmembrane subunit-interface pocket rather than the ion pore, where multiple hydrophobic residues may contribute to ligand recognition. Collectively, our results provide pharmacological and in vivo genetic evidence that tigolaner inhibits insect GABA receptors primarily through a transmembrane subunit-interface-associated allosteric mechanism. These findings provide a mechanistic foundation for understanding tigolaner action and may facilitate the development and resistance management of next-generation ectoparasiticides.