High-potency olfactory receptor agonists discovered by virtual high-throughput screening: molecular probes for receptor structure and olfactory function

Abstract

The detection of diverse chemical structures by the vertebrate olfactory system is accomplished by the recognition of odorous ligands by their cognate receptors. In the present study, we used computational screening to discover novel high-affinity agonists of an olfactory G protein-coupled receptor that recognizes amino acid ligands. Functional testing of the top candidates validated several agonists with potencies higher than any of the receptor's known natural ligands. Computational modeling revealed molecular interactions involved in ligand binding and further highlighted interactions that have been conserved in evolutionarily divergent amino acid receptors. Significantly, the top compounds display robust activities as odorants in vivo and include a natural product that may be used to signal the presence of bacteria in the environment. Our virtual screening approach should be applicable to the identification of new bioactive molecules for probing the structure of chemosensory receptors and the function of chemosensory systems in vivo.

Figure 1

Molecular models of selected vHTS hits docked in the receptor 5.24 binding pocket. Lysine and arginine analogues: (A) L-oxalysine (hit # 244) and (B) L-canavanine (hit # 677); di-amino acids: (C) L-cystathionine (hit # 380) and (D) LL-α,ε-diaminopimelic acid (hit # 299); glutamine and glutamine analogues: (E) L-glutamine and (F) L-glutamic acid-γ-p-nitroanilide (hit # 338). Compounds were computationally docked in the receptor 5.24 binding pocket. For clarity, only distal binding pocket residues are shown (thin lines); in panels E and F, far distal lobe 1 residues are displayed in blue, amide binding residues are shown in purple, and residues highlighted in yellow comprise a hydrophobic ring proposed previously (Luu et al., 2004). Carbon atoms are depicted in grey, oxygens in red, nitrogens in blue, sulphurs in yellow, and hydrogens in white (hydrogens shown only on ligands). Predicted hydrogen bonds are displayed as dashed green lines. Novel receptor-ligand interactions with the oxygen heteroatoms of oxalysine and canavanine as compared to their natural amino acid counterparts (lysine and arginine, respectively) are noted with green arrowheads (panels A and B). Note the different binding of each di-amino acid’s distal carboxylate to distal pocket residues (far right of panels C and D). Comparison of the dockings in panels E and F reveals the participation of L-glutamic acid-γ-p-nitroanilide’s nitro substituent in a distal hydrogen bond network, which may explain this ligand’s enhanced potency relative to glutamine.

Figure 2

Conservation of agonist–helix interactions in the LIVBP-like family of amino acid binding proteins. (A) Sequence alignment of helices αI and αIX (red) of the LIVBP, mGlu1 and receptor 5.24 binding pockets. Residues participating in ligand-helix bridging interactions are highlighted in grey. (B) Crystal structure of L-glutamate bound to mGlu1 (PDB code 1ewk) reveals coordination of the ligand’s terminal carboxylate by R78 (helix αI) and K409 (helix αIX). (C) Crystal structure of L-leucine bound to LIVBP (PDB code 1z16) similarly shows hydrophobic interactions between the bound ligand’s side chain and Y18 (helix αI) and F276 (helix αIX). (D) Molecular modeling of L-arginine docked in a three-dimensional model of the receptor 5.24 binding pocket predicts interactions of the ligand’s side chain with M389 and D388 in helix αIX, but not with residues in helix αI. (E) By contrast, the terminal nitro substituent of L-glutamic acid-γ-p-nitroanilide (hit 338) is predicted to interact via hydrogen bonding with Q78 (helix αI) and D388 (helix αIX). A hydrophobic interaction is also predicted between the ligand’s phenyl ring and M389 (helix αIX). In addition, an orthogonal interaction (Paulini et al., 2005) may occur between the distal nitro group and D388. Polar interactions are depicted by dashed green lines (small red sphere in panel B represents a bridging water molecule); hydrophobic contacts are shown as transparent grey spheres.

Figure 3

Compounds identified by vHTS function as odorants in vivo. Electro-olfactogram (EOG) recordings were performed on goldfish olfactory epithelium to measure olfactory responses to L-arginine and selected top vHTS hits. Representative traces are shown in which each compound was applied at 10⁻⁵ M concentration. Each compound elicited a negative potential characteristic of an excitatory olfactory neuronal response. H₂0, water control; L-Arg, L-arginine; 338, L-glutamic acid-γ-p-nitroanilide; 244, L-oxalysine; 299, diaminopimelic acid; 677, L-canavanine.