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 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.
Footnotes
- Received March 9, 2010.
- Revision received April 16, 2010.
- Accepted April 16, 2010.
- The American Society for Pharmacology and Experimental Therapeutics