Structural Modifications Redirect Phenylpyrazole Ectoparasiticides from the Ion Channel Pore to an Allosteric Site on GABA Receptors

Phenylpyrazole ectoparasiticides, exemplified by fipronil, act as noncompetitive antagonists of arthropod GABA receptors by blocking the ion channel pore. Here, we investigated the molecular mechanism of the newer phenylpyrazole nicofluprole using Drosophila melanogaster. CRISPR/Cas9-mediated mutagenesis and pharmacological assays showed that A301 substitutions in the pore-forming TM2 helix conferred strong resistance to fipronil but had little effect on nicofluprole sensitivity. In contrast, G335 mutations in TM3 markedly reduced nicofluprole activity. Molecular docking supported these findings, indicating that nicofluprole binds at the transmembrane subunit interface rather than within the central pore, consistent with allosteric modulation. These results demonstrate that structural modification of the phenylpyrazole scaffold can redirect target-site engagement from the pore to an allosteric site, explaining the lack of cross-resistance with traditional pore blockers and expanding strategies for GABA receptor-targeted insecticide design.