Bavan S et al. (2014), Discovery of novel ligands for mouse olfactory ...

XB-ART-48704

PLoS One 2014 Jan 01;93:e92064. doi: 10.1371/journal.pone.0092064.

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Discovery of novel ligands for mouse olfactory receptor MOR42-3 using an in silico screening approach and in vitro validation.

Bavan S , Sherman B , Luetje CW , Abaffy T .

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The ligands for many olfactory receptors remain largely unknown despite successful heterologous expression of these receptors. Understanding the molecular receptive range of olfactory receptors and deciphering the olfactory recognition code are hampered by the huge number of odorants and large number of olfactory receptors, as well as the complexity of their combinatorial coding. Here, we present an in silico screening approach to find additional ligands for a mouse olfactory receptor that allows improved definition of its molecular receptive range. A virtual library of 574 odorants was screened against a mouse olfactory receptor MOR42-3. We selected the top 20 candidate ligands using two different scoring functions. These 40 odorant candidate ligands were then tested in vitro using the Xenopus oocyte heterologous expression system and two-electrode voltage clamp electrophysiology. We experimentally confirmed 22 of these ligands. The candidate ligands were screened for both agonist and antagonist activity. In summary, we validated 19 agonists and 3 antagonists. Two of the newly identified antagonists were of low potency. Several previously known ligands (mono- and dicarboxylic acids) are also confirmed in this study. However, some of the newly identified ligands were structurally dissimilar compounds with various functional groups belonging to aldehydes, phenyls, alkenes, esters and ethers. The high positive predictive value of our in silico approach is promising. We believe that this approach can be used for initial deorphanization of olfactory receptors as well as for future comprehensive studies of molecular receptive range of olfactory receptors.

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Species referenced: Xenopus laevis
Genes referenced: cftr hhex

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	Figure 2. In vitro validation of in silico results.A. Representative traces of agonists, their structures and corresponding screen numbers tested at 100 μM for 15sec. Responses to 100 μM nononedioic acid (NDA) before and after application of each odorant was used for normalization. B. Histogram showing relative activity of the top 20 candidate ligands listed according to their score, tested at 100 μM and normalized to nonanedioic acid (mean±SEM, n = 5–10). Compound 18 full name is * 3-(4-(2-carboxyethyl)-phenyl)-propanoic acid. C. Histogram showing responses of ligands tested as antagonists at 1mM concentration and in the presence of 10 μM NDA. Responses are expressed as % of the responses to 100 μM nonanedioic acid, mean±SEM, n = 5–10. Compound 13 (dodecanoic acid) is an antagonist, *p<0.05. This compound (underlined) was previously identified as an antagonist for MOR42-3 [18].
	Figure 3. In vitro validation of in silico results.A. Histogram showing relative activity of the top 20 candidate ligands listed according to their mfscore, tested at 100 μM and normalized to the nonanedioic acid, mean±SEM, n = 5–10. Full name of the compounds: Compound 1 = 3,7,11,15-tetramethyl-hexadec-1-en-3-ol (1); Compound 2 = 1-methyl-ethyl-2-phenylethanoate (2); Compound 6 = ethyl-3-methyl-3-phenyl-oxirane-2-carboylate (6); compound 7 = 5-methyl-2-phenyl-hex-2-enal (7) and compound 13 = 4-(4-hydroxy-4-methylpentyl)-cyclohexene-1-carbaldehyde (13). B. Histogram showing response of ligands tested as antagonists at 1mM concentration, in the presence of 10 μM NDA. Compounds 3 and 6 were identified as low potency antagonists, *p<0.0025. Previously identified ligands for MOR42-3 confirmed in our in silico screen are underlined.
	Figure 4. Clustering of the ligands based on their molecular properties.A. VLS dot plot of score values vs molecular volume. Compounds acting as ligands from the Table 1 are labeled. Color scheme represents scoring as indicated. B. VLS dot plot of score values vs log P for compounds from Table 1 are labeled. C. VLS dot plot of mfscore values vs molecular volume. Compounds acting as ligands from the Table 2 are labeled. D. VLS dot plot of score values vs log P for compounds from Table 2 are labeled.
	Figure 5. Ligands for MOR42-3 identified by VLS screening and their structural similarity to nonanedioic acid expressed as a fingerprint distance from nonanedioic acid.(Antagonists are labeled in grey).
	Figure 6. Agonist binding pocket and additional ligands for MOR42-3.A. α-hexyl cinnamaldehyde docked in MOR42-3. Ligand binding pocket is colored by binding property with green representing hydrophobic areas, red hydrogen bond acceptors and blue hydrogen bond donors. Hydrogen bond between carbonyl oxygen from α-hexyl cinnamaldehyde (ranked 17, Table 1) and guanidinuim group of arginine 179 (R179) is presented with an interatomic distance of 1.57Å. B. Residues within 5Å distance from the best docking conformation of α-hexyl cinnamaldehyde. C. Structural relatives of α-hexyl cinnamaldehyde and their responses. Oocytes expressing MOR42-3, Gαolf and CFTR were screened with 15 sec application of 100 μM indicated odorants. Responses were normalized to the average 100 μM NDA-evoked responses and results are presented as a mean±SEM, n = 5–10.
	Figure 1. The experimental study design.List of procedures used in this study for in silico and in vitro approach of deorphanizing olfactory receptors.

References [+] :

Abaffy, Functional analysis of a mammalian odorant receptor subfamily. 2006, Pubmed, Xenbase