sb674042 and almorexant

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, beta(2)-adrenergic-based OX(1) and OX(2) modeling, we have determined important molecular determinants of the ligand-binding pocket of OX(1) and OX(2). 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 OX(1) and OX(2). 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 [(3)H]almorexant and [(3)H]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 [Ca(2+)](i) response at OX(2). 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 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).

Topics: Acetamides; Amino Acid Sequence; Aminopyridines; Binding Sites; Cell Line; Humans; Isoquinolines; Molecular Sequence Data; Mutagenesis, Site-Directed; Orexin Receptors; Pyrrolidines; Radioligand Assay; Receptors, G-Protein-Coupled; Receptors, Neuropeptide; Sequence Homology, Amino Acid; Sulfonamides; Thiazoles

Recent preclinical and clinical research has shown that almorexant promotes sleep in animals and humans without disrupting the sleep architecture. Here, the pharmacology and kinetics of [(3)H]almorexant binding to human orexin 1 receptor (OX(1))- and human orexin 2 receptor (OX(2))-human embryonic kidney 293 membranes were characterized and compared with those of selective OX(1) and OX(2) antagonists, including 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), 1-(6,8-difluoro-2-methyl-quinolin-4-yl)-3-(4-dimethylamino-phenyl)-urea (SB-408124), and N-ethyl-2-[(6-methoxy-pyridin-3-yl)-(toluene-2-sulfonyl)-amino]-N-pyridin-3-ylmethyl-acetamide (EMPA). The effect of these antagonists was also examined in vitro on the spontaneous activity of rat ventral tegmental area (VTA) dopaminergic neurons. [(3)H]Almorexant bound to a single saturable site on hOX(1) and hOX(2) with high affinity (K(d) of 1.3 and 0.17 nM, respectively). In Schild analyses using the [(3)H]inositol phosphates assay, almorexant acted as a competitive antagonist at hOX(1) and as a noncompetitive-like antagonist at hOX(2). In binding kinetic analyses, [(3)H]almorexant had fast association and dissociation rates at hOX(1), whereas it had a fast association rate and a remarkably slow dissociation rate at hOX(2). In the VTA, orexin-A potentiated the basal firing frequency to 175 +/- 17% of control in approximately half of the neurons tested. In the presence of 1 microM SB-674042 or SB-408124, the effect of orexin-A was only partially antagonized. However, in the presence of 1 microM EMPA or 1 microM almorexant, the effect of orexin-A was completely antagonized. In conclusion, almorexant exhibited a noncompetitive and long-lasting pseudo-irreversible mode of antagonism as a result of its very slow rate of dissociation from OX(2). The electrophysiology data suggest that OX(2) might be more important than OX(1) in mediating the effect of orexin-A on slow-firing of VTA dopaminergic neurons.

Topics: Acetamides; Animals; Evoked Potentials; Humans; Isoquinolines; Kinetics; Molecular Structure; Neurons; Orexin Receptors; Phenylurea Compounds; Pyrrolidines; Rats; Receptors, G-Protein-Coupled; Receptors, Neuropeptide; Sleep; Thiazoles