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

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. Importantly, olfaction is a key component of the multimodal approach used by mosquitoes to target and feed on humans. 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. The Orco subunit possesses an allosteric site to which modulators can bind and 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. Here, I characterized Orco antagonists. One compound, OX1w, was shown to inhibit odorant activation of a panel of 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. The addition of the airborne Orco antagonist OX1w to the experimental chamber abolished larval chemotaxis towards ethyl acetate. I then transitioned to a small molecule discovery project. I determined potency of structurally diverse Orco antagonists. Results were used for training machine learning algorithms to rank probable activity of a library of compounds. Testing of a representative subset of predicted active compounds revealed enrichment for Orco antagonists. Two novel Orco antagonists were able to inhibit OR-mediated olfactory behavior in the Drosophila melanogaster larval chemo-attraction assay.</p

Inhibition of insect olfactory behavior by an airborne antagonist of the insect odorant receptor co-receptor subunit.

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. 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. In this study, we 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, we 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 also 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 is the first demonstration that an airborne Orco antagonist can alter olfactory behavior in an insect. These results suggest a new approach to insect control and emphasize the need to develop more potent Orco antagonists

Correction: Inhibition of insect olfactory behavior by an airborne antagonist of the insect odorant receptor co-receptor subunit.

[This corrects the article DOI: 10.1371/journal.pone.0177454.]

Mammalian odorant receptor tuning breadth persists across distinct odorant panels

<div><p>The molecular receptive range (MRR) of a mammalian odorant receptor (OR) is the set of odorant structures that activate the OR, while the distribution of these odorant structures across odor space is the tuning breadth of the OR. Variation in tuning breadth is thought to be an important property of ORs, with the MRRs of these receptors varying from narrowly to broadly tuned. However, defining the tuning breadth of an OR is a technical challenge. For practical reasons, a screening panel that broadly covers odor space must be limited to sparse coverage of the many potential structures in that space. When screened with such a panel, ORs with different odorant specificities, but equal tuning breadths, might appear to have different tuning breadths due to chance. We hypothesized that ORs would maintain their tuning breadths across distinct odorant panels. We constructed a new screening panel that was broadly distributed across an estimated odor space and contained compounds distinct from previous panels. We used this new screening panel to test several murine ORs that were previously characterized as having different tuning breadths. ORs were expressed in <i>Xenopus laevis</i> oocytes and assayed by two-electrode voltage clamp electrophysiology. MOR256-17, an OR previously characterized as broadly tuned, responded to nine novel compounds from our new screening panel that were structurally diverse and broadly dispersed across an estimated odor space. MOR256-22, an OR previously characterized as narrowly tuned, responded to a single novel compound that was structurally similar to a previously known ligand for this receptor. MOR174-9, a well-characterized receptor with a narrowly tuned MRR, did not respond to any novel compounds in our new panel. These results support the idea that variation in tuning breadth among these three ORs is not an artifact of the screening protocol, but is an intrinsic property of the receptors.</p></div

MOR256-17 remains broadly tuned, responding to diverse chemical structures.

<p>(A) Current recordings of oocytes expressing MOR256-17, Gα_olf and CFTR. Odorants were screened in 6 mixtures, with each odorant present at 30 μM. Cyclodecanone (CYD) was screened individually. An application of 2-heptanone (2-HE) is included at the end of each trace for normalization. Representative traces are shown (n = 5–8). (B) Representative current recordings (n = 3–6) of the six odorant mixtures, as well as iodoform (IOD) and cyclodecanone (CYD), applied to sham (water) injected oocytes. Each odorant was present at 30 μM. (C) Representative current recordings of oocytes expressing MOR256-17, Gα_olf and CFTR responding to individual odorants applied at 30 μM. Four of the nine novel odorants we identified as activators of MOR256-17 are shown: methyl hexanoate (MHX), bromobenzene (BRO), amyl methyl sulfide (AMS) and iodoform (IOD). Also shown is the previously identified MOR256-17 ligand, <i>trans</i>-cinnamaldehyde (TCN), that was included in Mixture 4. Several inactive compounds are also shown: isoamyl phenylacetate (IPA), α-hexyl cinnamaldehyde (HC), dimethyl succinate (DMS), isoborneol (IBN), and 1,3,4,6,7,8-hexahydro-4,6,6,7,8,8-hexamethylcyclopenta-[g]-2-benzopyran (HBB). An application of 2-HE is included at the end of each trace for normalization. (D) Responses to nine newly identified odorant ligands were normalized to the response to 2-HE and are presented as mean ± SEM (n = 6–9). In addition to the compounds shown in panel C: 3-octanol (3-OL), toluene (TOL), 2-isobutyl-3-methoxypyrazine (IMP), isophorone (ISO) and piperonal (PIP).</p

MOR174-9 does not respond to the new panel of odorants.

<p>Current recordings of oocytes expressing MOR174-9, Gα_olf and CFTR. Odorants were screened in 6 mixtures, with each odorant present at 30 μM. Cyclodecanone (CYD) and iodoform (IOD) were screened individually. Responses were normalized to the eugenol (EUG) response. Representative traces are shown (n = 4–5).</p

MOR256-22 remains narrowly tuned, responding to structurally similar odorants.

<p>(A) Current recordings of oocytes expressing MOR256-22, Gα_olf and CFTR. Odorants were screened in 6 mixtures, with each odorant present at 30 μM. Cyclodecanone (CYD) and iodoform (IOD) were screened individually. Responses were normalized to the <i>trans</i>-cinnamaldehyde (TCN) response. Representative traces are shown (n = 4–8). (B) Current recording of an oocyte expressing MOR256-22, Gα_olf and CFTR responding to individual odorants. MOR256-22 only responded to the previously identified odorant ligand (<i>trans</i>-cinnamaldehyde) and α,α-dimethylbenzenepropanol (DBP), a structurally similar novel activator. A representative trace is shown (n = 9).</p

Distribution of our new odorant panel in an estimated odor space.

<p>Odor space was estimated using 1595 molecules (blue dots) in a multidimensional space based on 32 physiochemical descriptors and plotted using the first and second principal components. The 60 odorants in our screening panel are indicated with small red circles. One odorant (<i>trans</i>-cinnamaldehyde, green star) was included from our previous panel for reference. (A) The entire odor space is shown, including an extreme outlier (iodoform). The smallest hypersphere (indicated by the large red ellipse) that could encompass all odorants in our screening panel (including iodoform) had a radius of 43.0. (B) A close-up view of the region containing all of the molecules (excluding iodoform) is shown. The smallest hypersphere (indicated by the large red ellipse) that could encompass the odorants in our screening panel (excluding iodoform) had a radius of 15.5.</p