Structural Optimization and Behavioral Analysis of Antagonists of the Insect Odorant Receptor Co-receptor Subunit

of a dissertation at the University of Miami. Dissertation supervised by Professor Charles W. Luetje. No. of pages in text. (97) Response to volatile environmental chemosensory cues is essential for insect survival. The odorant receptor (OR) family is an important class of receptors that detects volatile molecules; guiding insects towards food, mates, and oviposition sites. Importantly, olfaction is a key component of the multimodal approach used by mosquitoes to target and feed on humans, a process by which various diseases are spread. Current repellents have drawbacks, necessitating development of more effective agents. Insect ORs are odorant-gated ion channels, consisting of a variable odorant specificity subunit and a conserved odorant receptor co-receptor (Orco) subunit, in an unknown stoichiometry. The Orco subunit possesses an allosteric site to which modulators can bind and noncompetitively inhibit odorant activation of ORs. All insect ORs contain Orco, and Orco is highly conserved across diverse insect species, making this subunit an attractive target for repellent development. In this project, I characterized several halogen-substituted versions of a phenylthiophenecarboxamide Orco antagonist structure. Orco antagonist activity was assessed on ORs from Drosophila melanogaster flies and Culex quinquefasciatus mosquitoes, expressed in Xenopus laevis oocytes and assayed by two-electrode voltage clamp electrophysiology. One compound, OX1w, was also shown to inhibit odorant activation of a panel of Anopheles gambiae mosquito ORs activated by diverse odorants. Next, I asked whether Orco antagonist OX1w could affect insect olfactory behavior. A Drosophila melanogaster larval chemotaxis assay was utilized to address this question. Larvae were robustly attracted to highly diluted ethyl acetate in a closed experimental chamber. Attraction to ethyl acetate was Orco dependent and required the odorant specificity subunit Or42b. The addition of the airborne Orco antagonist OX1w to the experimental chamber abolished larval chemotaxis towards ethyl acetate. The Orco antagonist was not a general inhibitor of sensory behavior, as behavioral repulsion from a light source was unaffected. This was the first demonstration that an airborne Orco antagonist can alter olfactory behavior in an insect. I then transitioned to a detailed small molecule discovery project. I determined potency of 58 structurally diverse compounds against Orco from Anopheles gambiae, and additionally compiled a series of 25 diverse inactive compounds. Results were used for training machine learning algorithms to rank probable activity of a library of 1280 odorant molecules. Functional testing of a representative subset of predicted active compounds revealed enrichment for Orco antagonists, many structurally distinct from previously known Orco antagonists. Two novel Orco antagonists, 2-tert-butyl-6-methylphenol and linalyl formate, were able to inhibit OR-mediated olfactory behavior in the Drosophila melanogaster larval chemo-attraction assay. These results provide a new approach to the discovery of behaviorally active Orco antagonists for the eventual use as insect repellents/deterrents.