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

Christopher J. Langmead, Jeffrey C. Jerman, Stephen J. Brough, Claire Scott, Rod A. Porter, Hugh J. Herdon

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1. 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-((5)-2-(5-phenyl-(l,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 [ 3H]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 ∼ 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 [ 3H]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.

Original languageEnglish
Pages (from-to)340-346
Number of pages7
JournalBritish Journal of Pharmacology
Issue number2
Publication statusPublished - 1 Jan 2004


  • [ H]SB-674042
  • Hypocretin
  • Orexin
  • Radioligand binding
  • SB-334867
  • SB-408124
  • SB-410220

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