Abstract

The orexins and their receptors are involved in the regulation of arousal and sleep–wake cycle. Clinical investigation with almorexant has indicated that this dual OX antagonist is efficacious in inducing and maintaining sleep. Using site-directed mutagenesis, β₂-adrenergic-based OX₁ and OX₂ modeling, we have determined important molecular determinants of the ligand-binding pocket of OX₁ and OX₂. The conserved residues Asp^45.51, Trp^45.54, Tyr^5.38, Phe^5.42, Tyr^5.47, Tyr^6.48, and His^7.39 were found to be contributing to both orexin-A-binding sites at OX₁ and OX₂. Among these critical residues, five (positions 45.51, 45.54, 5.38, 5.42, and 7.39) were located on the C-terminal strand of the second extracellular loop (ECL2b) and in the top of TM domains at the interface to the main binding crevice, thereby suggesting superficial OX receptor interactions of orexin-A. We found that the mutations W214A^45.54, Y223A^5.38, F227A^5.42, Y317A^6.48, and H350A^7.39 resulted in the complete loss of both [³H]almorexant and [³H]N-ethyl-2-[(6-methoxy-pyridin-3-yl)-(toluene-2-sulfonyl)-amino]-N-pyridin-3-ylmethyl-acetamide (EMPA) binding affinities and also blocked their inhibition of orexin-A-evoked [Ca²⁺]_i response at OX₂. The crucial residues Gln126^3.32, Ala127^3.33, Trp206^45.54, Tyr215^5.38, Phe219^5.42, and His344^7.39 are shared between almorexant and 1-(5-(2-fluoro-phenyl)-2-methyl-thiazol-4-yl)-1-((S)-2-(5-phenyl-(1,3,4)oxadiazol-2-ylmethyl)-pyrrolidin-1-yl)-methanone (SB-674042) binding sites in OX₁. The nonconserved residue at position 3.33 of orexin receptors was identified as occupying a critical position that must be involved in subtype selectivity and also in differentiating two different antagonists for the same receptor. In summary, despite high similarities in the ligand-binding pockets of OX₁ and OX₂ and numerous aromatic/hydrophobic interactions, the local conformation of helix positions 3.32, 3.33, and 3.36 in transmembrane domain 3 and 45.51 in ECL2b provide the structural basis for pharmacologic selectivity between OX₁ and OX₂.