Chen S and Luetje CW (2013), Phenylthiophenecarboxamide antagonists of the o...

XB-ART-48415

PLoS One 2013 Jan 01;812:e84575. doi: 10.1371/journal.pone.0084575.

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Phenylthiophenecarboxamide antagonists of the olfactory receptor co-receptor subunit from a mosquito.

Chen S , Luetje CW .

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Insects detect environmental chemicals using chemosensory receptors, such as the ORs, a family of odorant-gated ion channels. Insect ORs are multimeric complexes of unknown stoichiometry, formed by a common subunit (the odorant receptor co-receptor subunit, Orco) and one of many variable subunits that confer odorant specificity. The recent discovery of Orco directed ligands, including both agonists and antagonists, suggests Orco as a promising target for chemical control of insects. In addition to competitively inhibiting OR activation by Orco agonists, several Orco antagonists have been shown to act through a non-competitive mechanism to inhibit OR activation by odorants. We previously identified a series of Orco antagonists, including N-(4-ethylphenyl)-2-thiophenecarboxamide (OX1a, previously referred to as OLC20). Here, we explore the chemical space around the OX1a structure to identify more potent Orco antagonists. Cqui\Orco+Cqui\Or21, an OR from Culex quinquefasciatus (the Southern House Mosquito) that responds to 3-methylindole (skatole) and is thought to mediate oviposition behavior, was expressed in Xenopus oocytes and receptor function assayed by two-electrode voltage clamp electrophysiology. 22 structural analogs of OX1a were screened for antagonism of OR activation by an Orco agonist. By varying the moieties decorating the phenyl and thiophene rings, and altering the distance between the rings, we were able to identify antagonists with improved potency. Detailed examination of three of these compounds (N-mesityl-2-thiophenecarboxamide, N-(4-methylbenzyl)-2-thiophenecarboxamide and N-(2-ethylphenyl)-3-(2-thienyl)-2-propenamide) demonstrated competitive inhibition of receptor activation by an Orco agonist and non-competitive inhibition of receptor activation by an odorant. The ability to inhibit OR activation by odorants may be a general property of this class of Orco antagonist, suggesting that odorant mediated behaviors can be manipulated through Orco antagonism. The high conservation of Orco across insect species and previous demonstrations that various Orco ligands are active at ORs derived from several different insect orders suggests that Orco antagonists may have broad applicability.

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	Figure 2. Compounds tested in this study.
	Figure 3. A screen of OX1a analogs for Orco antagonist activity.A. An example of the antagonist screening protocol. An oocyte expressing Cqui\Orco+Cqui\Or21 was exposed to 60 sec applications of 3 µM OLC12 with 4 min washed between applications. 100 µM OX1l was applied for 90 sec preceding the third application of OLC12 and then co-applied during the OLC12 application. B. Responses of Cqui\Orco+Cqui\Or21 to 3 µM OLC12 (~EC25) in the presence of 100 µM of each analog are presented as a percentage of the average of the two preceding responses to OLC12 alone (mean ± SEM, n = 3-7). Inhibition by OX1d and OX2 did not differ from inhibition by OX1a. Inhibition by all other compounds was significantly different from inhibition by OX1a: OX1e (p<0.05); OX1b-c,f-n, OX3a (p<0.001). C. A screen of additional OX1a analogs for Orco antagonist activity. Responses of Cqui\Orco+Cqui\Or21 to 3 µM OLC12 in the presence of 30 µM of each candidate antagonist are presented as a percentage of the average of two preceding responses to OLC12 alone (mean ± SEM, n=3-5). Inhibition by all compounds was significantly different from inhibition by OX1a (p<0.001). Inhibition by OX1i was significantly different (p<0.001) from values for OX1r and OX1s. Inhibition by OX1l was significantly different from values for OX1o (p<0.001) and OX1p (p<0.05). Inhibition by OX1m was significantly different (p<0.001) from values for OX1o and OX1p. Inhibition values that did not differ are: OX1g and OX1q, OX1j and OX4, OX1m and OX1q.
	Figure 4. Concentration-inhibition curves for OX1a, OX1g, OX1i, OX1j, OX1k, OX1l, OX1o, OX1p, OX1q, OX3a, OX3b and OX4 inhibition of Cqui\Orco+Cqui\Or21 activated by 3 µM OLC12.IC50 and nH values may be found in Table 1.
	Figure 5. The potent Orco antagonists OX1l, OX3a and OX4 non-competitively inhibit odorant activation of a mosquito OR.A. Altering the concentration of Orco agonist (OLC12) shifts the OX1l inhibition curve. The IC50 for OX1l inhibition of Cqui\Orco+Cqui\Or21 activation by 10 µM OLC12 (8.3 ± 0.5 µM, n = 4) is significantly different from the IC50 for OX1l inhibition of Cqui\Orco+Cqui\Or21 activation by 3 µM OLC12 (3.8 ± 0.3 µM, n = 9) (p<0.0001, F-test). The IC50 for OX1l inhibition of Cqui\Orco+Cqui\Or21 activation by 30 µM OLC12 (24 ± 3 µM, n = 6) is significantly different from the IC50 for OX1l inhibition of Cqui\Orco+Cqui\Or21 activation by 3 µM OLC12 (p<0.0001, F-test) and from the IC50 for OX1l inhibition of Cqui\Orco+Cqui\Or21 activation by 10 µM OLC12 (p<0.0001, F-test). B. Altering odorant (3MI) concentration fails to alter the inhibition curve for OX1l antagonism of Cqui\Orco+Cqui\Or21 activated by 3MI. The IC50 values for OX1l inhibition of responses to 10 nM 3MI (17 ± 1 µM, n = 3), 100 nM 3MI (16 ± 1 µM, n = 3), and 1µM 3MI (19 ± 2 µM, n = 3) did not differ (p=0.3605, F-test). C. Altering the concentration of Orco agonist (OLC12) shifts the OX3a inhibition curve. The IC50 for OX3a inhibition of Cqui\Orco+Cqui\Or21 activation by 10 µM OLC12 (12 ± 1 µM, n = 3) is significantly different from the IC50 for OX3a inhibition of Cqui\Orco+Cqui\Or21 activation by 3 µM OLC12 (1.7 ± 0.2 µM, n = 4) (p<0.0001, F-test). The IC50 for OX3a inhibition of Cqui\Orco+Cqui\Or21 activation by 30 µM OLC12 (28 ± 2 µM, n = 3) is significantly different from the IC50 for OX3a inhibition of Cqui\Orco+Cqui\Or21 activation by 3 µM OLC12 (p<0.0001, F-test) and from the IC50 for OX3a inhibition of Cqui\Orco+Cqui\Or21 activation by 10 µM OLC12 (p<0.0001, F-test). D. Altering odorant (3MI) concentration fails to alter the inhibition curve for OX3a antagonism of Cqui\Orco+Cqui\Or21 activated by 3MI. The IC50 values for OX3a inhibition of responses to 10 nM 3MI (2.6 ± 0.3 µM, n = 3), 100 nM 3MI (2.3 ± 0.2 µM, n = 4), and 1µM 3MI (1.9 ± 0.1 µM, n = 3) did not differ (p=0.07, F-test). E. Altering the concentration of Orco agonist (OLC12) shifts the OX4 inhibition curve. The IC50 for OX4 inhibition of Cqui\Orco+Cqui\Or21 activation by 10 µM OLC12 (8.8 ± 1.2 µM, n = 3) is significantly different from the IC50 for OX4 inhibition of Cqui\Orco+Cqui\Or21 activation by 3 µM OLC12 (2.1 ± 0.3 µM, n = 5) (p<0.0001, F-test). The IC50 for OX4 inhibition of Cqui\Orco+Cqui\Or21 activation by 30 µM OLC12 (73 ± 5 µM, n = 3) is significantly different from the IC50 for OX4 inhibition of Cqui\Orco+Cqui\Or21 activation by 3 µM OLC12 (p<0.0001, F-test) and from the IC50 for OX4 inhibition of Cqui\Orco+Cqui\Or21 activation by 10 µM OLC12 (p<0.0001, F-test). F. Altering odorant (3MI) concentration fails to alter the inhibition curve for OX4 antagonism of Cqui\Orco+Cqui\Or21 activated by 3MI. The IC50 values for OX4 inhibition of responses to 10 nM 3MI (3.4 ± 0.6 µM, n = 3), 100 nM 3MI (2.7 ± 0.4 µM, n = 3), and 1µM 3MI (2.5 ± 0.3 µM, n = 3) did not differ (p=0.42, F-test).
	Figure 1. Non-competitive inhibition of odorant activation of an insect OR by an Orco antagonist. A. Structure of N-(4-ethylphenyl)-2-thiophenecarboxamide (OX1a). B. Increasing the concentration of Orco agonist (OLC12) decreases the effectiveness of OX1a. Oocytes expressing Cqui\Orco+Cqui\Or21 were activated with 3 µM or 30 µM OLC12, in the absence or presence of 30 µM or 100 µM OX1a. Responses in the presence of OX1a are presented as a percentage of the average of the two preceding responses to OLC12 alone (mean ± SEM, n=3). Statistical significance: p<0.01, *p<0.001 (two-tailed, unpaired t-test). C. OX1a inhibition of Cqui\Orco+Cqui\Or21 activation by 3-methylindole (3MI) is not altered by changes in odorant (3MI) concentration. IC50 values for OX1a inhibition of responses to 10nM 3MI (47 ± 5 µM), 100nM 3MI (42 ± 7 µM) and 1uM 3MI (50 ± 4 µM) did not differ (p=0.2970, F-test). D. Co-application of 50 µM OX1a significantly reduces the maximal response to 3MI, as compared to the response to 3MI in the absence of OX1a (p<0.0001, F-test). The EC50 for 3MI activation of Cqui\Orco+Cqui\Or21 in presence of 50 µM OX1a (37 ± 7 nM) did not differ (p=0.57, F-test) from the EC50 for activation of Cqui\Orco+Cqui\Or21 by 3MI alone (32 ± 7 nM).

References [+] :

Benton, Atypical membrane topology and heteromeric function of Drosophila odorant receptors in vivo. 2006, Pubmed