Ligand-Specific Remodeling of the Anopheles gambiae RDL GABA Receptor by the A296S Mutation Differentiates Recognition of Fipronil and Fluralaner

The γ-aminobutyric acid (GABA)-gated chloride channel Resistance to Dieldrin (RDL) receptor is a major molecular target of insecticides, yet the structural basis by which resistance-associated mutations differentially influence chemically distinct ligands remains incompletely understood. Here, we investigated the effects of the classical A296S substitution on the interactions of the Anopheles gambiae RDL receptor with the phenylpyrazole fipronil and the isoxazoline fluralaner using homology modeling, molecular docking, 200 ns molecular dynamics simulations (apo receptors and three independent replicas of each ligand-bound complex), and MM/PBSA free-energy calculations that include an interaction-entropy (IE) term. Apo-state molecular dynamics simulations showed that A296S preserved the overall structural stability of the receptor while locally modifying the environment of the pore-lining M2 transmembrane helix. Although docking scored fluralaner more favorably than fipronil in both receptor variants, the replicated molecular dynamics simulations revealed ligand- and genotype-dependent behavior. In the wild-type receptor, fluralaner remained stably bound and had a favorable MM/PBSA free-energy estimate (−18.3 ± 4.2 kcal/mol), whereas the ligand RMSD of fipronil increased progressively and its estimate was close to zero (+1.2 ± 9.5 kcal/mol). In the A296S model, the fipronil estimate was similar (+1.6 ± 4.3 kcal/mol), but the fluralaner estimate became unfavorable (+10.0 ± 1.8 kcal/mol), and Ser296 itself contributed slightly unfavorably to both ligands. Together, these findings indicate that A296S does not act as a universal determinant of ligand recognition but is associated with ligand-specific changes in the modeled interaction landscape, with the larger predicted effect on fluralaner. These predictions derive from a homology model, were not tested by electrophysiology or binding assays, and do not measure functional resistance. They may nonetheless help inform the interpretation of cross-resistance patterns and structure-guided efforts targeting GABA receptor-based insecticides.