Publication Date

2014-08-26

Availability

Embargoed

Embargo Period

2016-08-25

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PHD)

Department

Molecular and Cellular Pharmacology (Medicine)

Date of Defense

2014-07-28

First Committee Member

Charles W. Luetje

Second Committee Member

Stephan C. Schürer

Third Committee Member

Michael S. Kapiloff

Fourth Committee Member

H. Peter Larsson

Fifth Committee Member

Alexandra C. C. Wilson

Abstract

Human health is threatened by insect borne diseases, such as dengue fever, malaria, yellow fever and epidemic typhus. Insects can also cause significant destruction of crops and food stores. Insects depend on olfaction to perceive their surroundings and guide activities; including feeding, mating and oviposition. Understanding the molecular structure and biological activities of insect odorant receptors (ORs) is paramount to developing novel and highly effective insect repellants. Each insect olfactory receptor is composed of two functionally essential parts: a highly conserved co-receptor subunit (Orco) and one of a large number of variable odorant-binding (or “tuning”) subunits. These subunits associate in an unknown stoichiometry to form an odorant-gated ion channel. While the odorant-binding subunit is responsible for interacting with odorants, both the odorant-binding subunits and Orco are involved in forming the ion channel pore. Recently, the identification of VUAA1, a novel OR agonist that acts directly on Orco, suggested the possible functional involvement of Orco in OR ligand binding, and that manipulation of insect behavior might be achieved by targeting Orco. However, little was known about Orco pharmacological properties and its functional interactions with odorant-binding subunits. In this project I identified two new Orco agonists and a large series of Orco antagonists. I showed that a similar pattern of agonist and antagonist sensitivity is displayed by Orco subunits from different species, suggesting a highly conserved binding site structure on Orco. This is consistent with the high level of protein sequence identity among Orco subunits of different species. I explored an expanded panel of structures for Orco antagonism and revealed a relatively broad Orco antagonist chemical space, compared to the less tolerant structure for Orco agonists. I examined in detail five Orco antagonists over two insect species (Drosophila and Culex), and demonstrated competitive inhibition of receptor activation by an Orco agonist and non-competitive inhibition of receptor activation by an odorant, suggesting a potential general antagonist action of this class of Orco antagonist on OR activation. I screened biogenic and trace amines for Orco ligand activity, and identified tryptamine, a plant-produced amine, as a potent antagonist of Orco. Tryptamine was able to competitively block Orco activation by an Orco agonist and to allosterically inhibit activation of ORs by odorants. I also identified a less potent Orco antagonist tyramine, an amine endogenous to the insect nervous system. Both trypatmine and tyramine displayed broad activity, inhibiting odorant activation of ORs from species of three different insect orders (Diptera, Lepidoptera and Coleoptera), as well as odorant activation of six diverse ORs from a single species (the human malaria vector mosquito, Anopheles gambiae). In all, my efforts in this project achieved comprehensive information of insect Orco agonist and antagonist pharmacological properties, providing useful compounds for future insect OR studies, as well as basic knowledge of Orco ligand structural features that will be useful for developing Orco-oriented insect repellent.

Keywords

insect olfactory receptor

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