Mapping the Binding Pocket of Dual Antagonist Almorexant to Human Orexin 1 and Orexin 2 Receptors: Comparison with the Selective OX₁ Antagonist SB-674042 and the Selective OX₂ Antagonist N-Ethyl-2-[(6-methoxy-pyridin-3-yl)-(toluene-2-sulfonyl)-amino]-N-pyridin-3-ylmethyl-acetamide (EMPA)

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, ␤ 2 -adrenergic-based OX 1 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 OX 1 . 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 OX 1 and OX 2 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 OX 1 and OX 2 .

International Union of Basic and Clinical Pharmacology. LXXXVI. Orexin Receptor Function, Nomenclature and Pharmacology

“…Transmembrane domain 3 seems to be the most critical, where exchange of OX 2 receptor sequence with that from OX 1 receptor switches the ligand binding properties of a chimeric receptor (Putula et al, 2011). The extracellular portion of transmembrane domain 3 also contains Gln134 and Thr135, residues essential for peptide ligand and small molecule interactions, respectively (Malherbe et al, 2010;Tran et al, 2011). It is noteworthy that substitution of Thr135 with an alanine in the OX 2 receptor, which matches the Ala135 naturally found in the OX 1 receptor, substantially attenuates small-molecule binding but induces a small increase in activity toward OX-B (Tran et al, 2011).…”

“…Homology models were created using MOE software (Chemical Computing Group, Montreal, QC, Canada) based on the crystal structure of carazolol binding to the ␤ 2 -adrenergic receptor (Protein Date Bank code 2rh1) Rosenbaum et al, 2007) used as the template. Sequence alignment of the transmembrane helices and the extracellular loop 2 (ECL2) of OX 1 and OX 2 receptors with the 2rh1 structure was performed according to Malherbe et al (2010) and the best model selected from 10 intermediates. Backbone coordinates remained identical to the crystal structure such that no minimization was performed (structural data comparison and figure generated by M. Katharine Holloway, Ph.D., Chemical Modeling and Informatics, Merck Research Laboratories).…”

Orexin/hypocretin receptor chimaeras reveal structural features important for orexin peptide distinction

“…For further analysis of receptor determinants, modeling of the receptor structure would probably be necessary. However, this is not a trivial task, despite development of the algorithms and the four already available crystal structures, as also illustrated by the results of a recent study on orexin receptors [16]. Receptor mutagenesis is also not a straight-forward approach, as the correct folding may be compromised in the mutant receptors.…”

“…A number of single nucleotide polymorphisms of orexin receptors are known [6], but have not been consequently investigated. No mutagenesis studies, except for a recent point mutation study [16], have been performed. In this study, several amino acids predicted to contribute to antagonist binding were mutated to alanine.…”

“…In this study, several amino acids predicted to contribute to antagonist binding were mutated to alanine. Many of the mutations caused dramatic reduction in the antagonist binding [16].…”