PT - JOURNAL ARTICLE AU - Pari Malherbe AU - Olivier Roche AU - Anne Marcuz AU - Claudia Kratzeisen AU - Joseph G. Wettstein AU - Caterina Bissantz TI - Mapping the binding pocket of dual antagonist almorexant to human orexin 1 and orexin 2 receptors: Comparison with the selective OX<sub>1</sub> (SB-674042) and OX<sub>2</sub> (EMPA) antagonists AID - 10.1124/mol.110.064584 DP - 2010 Apr 19 TA - Molecular Pharmacology PG - mol.110.064584 4099 - http://molpharm.aspetjournals.org/content/early/2010/04/19/mol.110.064584.short 4100 - http://molpharm.aspetjournals.org/content/early/2010/04/19/mol.110.064584.full AB - 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 D45.51, W45.54, Y5.38, F5.42, Y5.47, Y6.48 and H7.39 were found to be contributing to both orexin-A-binding sites at OX1 and OX2. Among these critical residues, five (helix positions 45.51, 45.54, 5.38, 5.42 and 7.39) were located on the 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 W214A45.54, Y223A5.38, F227A5.42, Y317A6.48 and H350A7.39 resulted in the complete loss of both [3H]almorexant and [3H]EMPA binding affinities and also blocked their inhibition of orexin-A-evoked [Ca2+]i response at OX2. The crucial residues Q1263.32, A1273.33, W20645.54, Y2155.38, F2195.42 and H3447.39 are shared between almorexant and SB-674042 binding sites in OX1. The non-conserved 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 TM3 and 45.51 in ECL2b provide the structural basis for pharmacologic selectivity between OX1 and OX2.The American Society for Pharmacology and Experimental Therapeutics