ABSTRACT: 1. This study characterises the binding of a novel nonpeptide antagonist radioligand, [(3)H]SB-674042 (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), to the human orexin-1 (OX(1)) receptor stably expressed in Chinese hamster ovary (CHO) cells in both a whole cell assay and in a cell membrane-based scintillation proximity assay (SPA) format. 2. Specific binding of [(3)H]SB-674042 was saturable in both whole cell and membrane formats. Analyses suggested a single high-affinity site, with K(d) values of 3.76+/-0.45 and 5.03+/-0.31 nm, and corresponding B(max) values of 30.8+/-1.8 and 34.4+/-2.0 pmol mg protein(-1), in whole cell and membrane formats, respectively. Kinetic studies yielded similar K(d) values. 3. Competition studies in whole cells revealed that the native orexin peptides display a low affinity for the OX(1) receptor, with orexin-A displaying a approximately five-fold higher affinity than orexin-B (K(i) values of 318+/-158 and 1516+/-597 nm, respectively). 4. SB-334867, SB-408124 (1-(6,8-difluoro-2-methyl-quinolin-4-yl)-3-(4-dimethylamino-phenyl)-urea) and SB-410220 (1-(5,8-difluoro-quinolin-4-yl)-3-(4-dimethylamino-phenyl)-urea) all displayed high affinity for the OX(1) receptor in both whole cell (K(i) values 99+/-18, 57+/-8.3 and 19+/-4.5 nm, respectively) and membrane (K(i) values 38+/-3.6, 27+/-4.1 and 4.5+/-0.2 nm, respectively) formats. 5. Calcium mobilisation studies showed that SB-334867, SB-408124 and SB-410220 are all functional antagonists of the OX(1) receptor, with potencies in line with their affinities, as measured in the radioligand binding assays, and with approximately 50-fold selectivity over the orexin-2 receptor. 6. These studies indicate that [(3)H]SB-674042 is a specific, high-affinity radioligand for the OX(1) receptor. The availability of this radioligand will be a valuable tool with which to investigate the physiological functions of OX(1) receptors.
