Chen S and Luetje CW (2012), Identification of new agonists and antagonists ...

XB-ART-45298

PLoS One 2012 Jan 01;75:e36784. doi: 10.1371/journal.pone.0036784.

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Identification of new agonists and antagonists of the insect odorant receptor co-receptor subunit.

Chen S , Luetje CW .

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Insects detect attractive and aversive chemicals using several families of chemosensory receptors, including the OR family of olfactory receptors, making these receptors appealing targets for the control of insects. Insect ORs are odorant-gated ion channels, comprised of at least one common subunit (the odorant receptor co-receptor subunit, Orco) and at least one variable odorant specificity subunit. Each of the many ORs of an insect species is activated or inhibited by an unique set of odorants that interact with the variable odorant specificity subunits, making the development of OR directed insect control agents complex and laborious. However, several N-,2-substituted triazolothioacetamide compounds (VUAA1, VU0450667 and VU0183254) were recently shown to act directly on the highly conserved Orco subunit, suggesting that broadly active compounds can be developed. We have explored the chemical space around the VUAA1 structure in order to identify new Orco ligands. We screened ORs from several insect species, using heterologous expression in Xenopus oocytes and an electrophysiological assay, with a panel of 22 compounds structurally related to VUAA1. By varying the nitrogen position in the pyridine ring and altering the moieties decorating the phenyl ring, we identified two new agonists and a series of competitive antagonists. Screening smaller compounds, similar to portions of the VUAA1 structure, also yielded competitive antagonists. Importantly, we show that Orco antagonists inhibit odorant activation of ORs from several insect species. Detailed examination of one antagonist demonstrated inhibition to be through a non-competitive mechanism. A similar pattern of agonist and antagonist sensitivity displayed by Orco subunits from different species suggests a highly conserved binding site structure. The susceptibility to inhibition of odorant activation by Orco antagonism is conserved across disparate insect species, suggesing the ligand binding site on Orco as a promising target for the development of novel, broadly active insect repellants.

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Species referenced: Xenopus laevis
Genes referenced: hhex tcf3

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	Figure 1. Identification of OLC3 and OLC12 as Orco agonists. A) VUAA1 activates both heteromeric (top traces) and homomeric (bottom traces) insect ORs expressed in Xenopus oocytes and assayed by two electrode voltage clamp electrophysiology. Compounds were applied for 60 sec with a 9 min wash between applications. VUAA1 was applied at 100 ÂµM. Oocytes expressing Cqui\Orco + Cqui\Or10 or Cqui\Orco were also exposed to 30 nM 3-methylindole (3-MI). Oocytes expressing Dmel\Orco + Dmel\Or35a or Dmel\Orco were also exposed to 3 ÂµM Hexanol (HEX). Oocytes expressing Onub\Orco + Onub\Or1 or Onub\Orco were also exposed to 1 ÂµM E12â14:OAc (E12). B) OLC3 activates both heteromeric and homomeric insect ORs. Oocytes expressing Dmel\Orco + Dmel\Or35a (top trace) or Dmel\Orco (bottom trace) were challenged with 60 sec applications of 100 ÂµM OLC2, OLC3, OLC4, OLC5 and VUAA1, with 9 min washes between applications. C) Results from a screen of 22 compounds (each applied at 100 ÂµM) for Orco agonist activity. Responses are normalized to the response of the same oocyte to 100 ÂµM OLC3 and presented as the mean of 3â8 oocytes (SEM values may be found in Table S1). D) Structures of VUAA1, OLC3 and OLC12.
	Figure 2. OLC12 is a more potent Orco agonist than VUAA1 or OLC3.Concentration-response analysis for VUAA1, OLC3 and OLC12 activation of Dmel\Orco + Dmel\Or35a (A) and Dmel\Orco (B). Each response was normalized to the response of the same oocyte to 30 ÂµM OLC3. EC50 values can be found in Supplementary Table 2.
	Figure 3. Identification of new Orco antagonists. A) Co-application of 100 ÂµM OLC2 inhibits activation of Dmel\Orco by 30 ÂµM OLC12. B) Co-application of 100 ÂµM OLC8 fails to block activation of Dmel\Orco by 30 ÂµM OLC12. CâD) Results of a screen of 20 compounds for Orco antagonism. Responses of Dmel\Orco + Dmel\Or35a to 10 ÂµM OLC12 (EC25) (C) or of Dmel\Orco to 30 ÂµM OLC12 (EC39) (D) in the presence of 100 ÂµM of each candidate antagonist are presented as a percentage of the average of the two preceding responses to OLC12 alone (meanÂ±SEM, nâ=â3â6). Sh, sham. E) Structures of Orco antagonists.
	Figure 4. Competitive antagonism of Orco activation. A-B) Concentration-inhibition analysis for OLC2, OLC9, OLC14, OLC15, OLC20 and OLC22 inhibition of Dmel\Orco + Dmel\Or35a activated by 10 ÂµM OLC12 (EC25) (A) and Dmel\Orco activated by 30 ÂµM OLC12 (EC39) (B). IC50 values may be found in Table 1. C) Increasing the concentration of agonist (OLC12) decreases the effectiveness of Orco antagonists. 100 ÂµM OLC2, OLC9, OLC14, OLC15, OLC20, or OLC22 was co-applied with 10 ÂµM OLC12 or 100 ÂµM OLC12 to oocytes expressing Dmel\Orco + Dmel\Or35a. Responses in the presence antagonist are presented as a percentage of the average of the two preceding responses to OLC12 alone (meanÂ±SEM, nâ=â3â5). Statistical significance (t-test): p<0.05; p<0.01; **p<0.001. D, Altering agonist (OLC12) concentration, shifts the OLC15 inhibition curve. The IC50 values for OLC15 inhibition of 10 ÂµM OLC12 (15Â±1 ÂµM) and 100 ÂµM OLC12 (67Â±7 ÂµM) are significantly different (p<0.0001, F test).
	Figure 5. Non-competitive inhibition of odorant activation of insect ORs by an Orco antagonist.A) Oocytes expressing Dmel\Orco + Dmel\Or35a were exposed to 60 sec applications of 3 ÂµM hexanol (HEX), with 20 min washes between applications. OLC2, OLC15 or OLC20 (100 ÂµM) were applied for 90 sec preceding the second application of HEX and then co-applied during the HEX application. Top trace, OLC2 fails to inhibit HEX activation of Dmel\Orco + Dmel\Or35a. Middle trace, OLC15 inhibits HEX activation of Dmel\Orco + Dmel\Or35a. Bottom trace, OLC20 inhibits HEX activation of Dmel\Orco + Dmel\Or35a. B) OLC compounds inhibit odorant activation of ORs from a variety of insect species. Oocytes expressing Cqui\Orco+Cqui\Or10 were activated by 100 nM 3-methylindole (3-MI), oocytes expressing Dmel\Orco + Dmel\Or35a were activated by 3 ÂµM HEX, oocytes expressing Agam\Orco + Agam\Or65 were activated by 100 nM eugenol (Eug) and oocytes expressing Onub\Orco + Onub\Or1 were activated E12â14:OAc (E12). Current responses in the presence of OLC compounds (100 ÂµM) were compared to the preceding response to odorant alone and are presented as meanÂ±SEM (nâ=â3â14). C) Altering odorant concentration fails to alter the inhibition curve for OLC15 antagonism of Cqui/Orco + Cqui\Or10 activation by 3-MI. The IC50 values for OLC15 inhibition of responses to 10 nM 3-MI (1.5Â±0.2 ÂµM) and 100 nM 3-MI (2.0Â±0.3 ÂµM) do not differ (pâ=â0.13, F test). D) Co-application of 3 ÂµM OLC15 significantly reduces the maximal response to 3-MI, but does not alter the EC50 for 3-MI activation. The maximal response to 3-MI in the presence of 3 ÂµM OLC15 was significantly lower (62Â±2%) than the maximal response in the absence of OLC15 (p<0.05, F test). The EC50 for 3-MI activation of Cqui\Orco + Cqui\Or10 was 313Â±103 nM, while the EC50 in presence of 3 ÂµM OLC15 was 275Â±41 nM (pâ=â0.81, F test).

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