Characterisation of the binding of [3H]‐SB‐674042, a novel nonpeptide antagonist, to the human orexin‐1 receptor

This study characterises the binding of a novel nonpeptide antagonist radioligand, [3H]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 (OX1) 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. Specific binding of [3H]SB‐674042 was saturable in both whole cell and membrane formats. Analyses suggested a single high‐affinity site, with Kd values of 3.76±0.45 and 5.03±0.31 nM, and corresponding Bmax 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 Kd values. Competition studies in whole cells revealed that the native orexin peptides display a low affinity for the OX1 receptor, with orexin‐A displaying a ∼five‐fold higher affinity than orexin‐B (Ki values of 318±158 and 1516±597 nM, respectively). 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 OX1 receptor in both whole cell (Ki values 99±18, 57±8.3 and 19±4.5 nM, respectively) and membrane (Ki values 38±3.6, 27±4.1 and 4.5±0.2 nM, respectively) formats. Calcium mobilisation studies showed that SB‐334867, SB‐408124 and SB‐410220 are all functional antagonists of the OX1 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. These studies indicate that [3H]SB‐674042 is a specific, high‐affinity radioligand for the OX1 receptor. The availability of this radioligand will be a valuable tool with which to investigate the physiological functions of OX1 receptors. British Journal of Pharmacology (2004) 141, 340–346. doi:10.1038/sj.bjp.0705610