Novel Anosmia-Inducing Compounds for Environmentally Friendly Mosquito Vector Control: Structural Determinants of ORco Ligands Antagonizing Odorant Receptor Function.

Insect repellents are important means of personal protection against bites from mosquitoes carrying various pathogens. Odorant-dependent behaviours are triggered by odor ligands bound to odorant receptors. Insect odorant receptors are heterotetrameric ligand-gated cation channels composed of an obligatory subunit, ORco, and one of many variable subunits, ORx, to which odorant ligands bind. Upon expression ex vivo, ORco forms homomeric channels gated by specific ligands acting as channel agonists. In past studies, we identified ligands of natural origin that bind to the African malaria mosquito vector Anopheles gambiae ORco (AgamORco). Some of these ligands function as specific ORco channel antagonists, orthosteric or allosteric relative to a predicted ORco agonist binding site, and cause severe inhibition of the olfactory function in a variety of mosquito species. Given that ORco is evolutionarily conserved, we hypothesized that it should be feasible to develop an AgamORco-based screening platform allowing fast identification of new ORco ligands acting as mosquito odorant receptor antagonists causing anosmia-like deficits to mosquitoes. We are now reporting on the compilation of common structural features of previously identified orthosteric AgamORco antagonists and generation of a ligand-based pharmacophore. In silico screening of a collection of volatile compounds of natural origin resulted in identification of several AgamORco ligand hits. Parallel cell-based screening of the same compounds against AgamORco was also employed in order to identify ligands acting as AgamORco antagonists. The screening resulted in identification of several ligands inhibiting AgamORco channel function ex vivo by at least 40% and inducing anosmic behaviors to Aedes albopictus mosquitoes in vivo. To test the pharmacophore model for binding site specificity, binding competition assays were undertaken, which distinguished the identified antagonists into orthosteric and allosteric ones. Direct comparisons of the pharmacophore predictions with the results of the cell-based screen revealed that the pharmacophore predicted correctly 100% of the orthosteric antagonists and none of the allosteric ones. These results confirm the pharmacophore's value for in silico prediction of AgamORco orthosteric ligands. Upon combination with the AgamORco-specific cell-based screening platform, the pharmacophore provides a valuable tool for fast identification of multiple, ecologically friendly vector control agents. Finally, to confirm the predicted AgamORco agonist binding site and determine the binding orientation of orthosteric antagonists in it, we are assessing the interactions of these ligands with specific amino acids in the AgamORco binding pocket by site-specific mutagenesis.