GABA Rdl receptors as targets for novel negative allosteric modulators with pore-blocking potential

GABA type A receptors (GABAARs) are ligand-gated pentameric ion channels found throughout vertebrate and invertebrate nervous systems. Although their structure and function are highly conserved, they can be selectively modulated. Among insect GABA receptors, the Rdl variant has received significant attention, and recent studies have shown its ability to form functional channels with LCCH3 subunits. Mutations like A302S on GABA Rdl of Drosophila confer resistance to various insecticides. Thus, the search for novel compounds able to avoid this resistance mechanism becomes urgent. Due to a lack of structural information on the Rdl/LCCH3 receptor, we built an RdlA302S/LCCH3 model using the human GABAAR beta3 structure as a template. We performed molecular dynamics to evaluate conformational changes and stability and validated the model by docking protein-ligand simulations with known insecticides to assess the ability to mimic the development of resistance. Next, we carried out a virtual screening using the Molport database (5,000,000 molecules), positioning the interaction grid inside the non-competitive antagonist IA binding site. Complexes were ranked based on docking scores (DS) and deltaGbind, yielding a set with 3-fold higher predicted interaction capabilities. To experimentally validate, GABA Rdl receptors were expressed in HEK293 cells. GABA dose-response curves showed EC50 values of 53.7 μM for the wild type and 34.4 μM for the A302S mutant, which displayed reduced sensitivity to insecticides, serving as a suitable test model. These results highlight the value of our models in identifying more selective compounds with negative allosteric modulation.Supported by FONDECYT 11221211 (CFB), 1211082 (GEY), 1211095 (GM-C)