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

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. 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 Bfive-fold higher affinity than orexin-B (K i values of 3187158 and 15167597 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 99718, 5778.3 and 1974.5 nM, respectively) and membrane (K i values 3873.6, 2774.1 and 4.570.2 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.

The Terminal (Catalytic) Adenosine of the HIV LTR Controls the Kinetics of Binding and Dissociation of HIV Integrase Strand Transfer Inhibitors

“…For example, the binding and dissociation rate constants (43) for [ 3 H]-nitroarginine binding to the N-terminal heme-binding domain of rat neuronal nitric oxide synthase were the same as those determined by inhibition of catalysis (44) or by a precipitation method (45). Also, the binding kinetics of a nonpeptide antagonist, [ 3 H]-SB-674042, to the human or- exin-1 receptor immobilized on SPA beads gave comparable rate constants to those obtained by measuring binding kinetics to the receptor on whole cells (46).…”

Neurofizjologiczne uwarunkowania procesów snu, czuwania, świadomości i przytomności. Część 2

“…During REM sleep, vlPAG/LPT nuclei can be inhibited by GABA and glycinoergic neurons, which are activated by the SLD nucleus. Similarly, inhibition of SLD nuclei could occur during NREM through increased melatonin concentration and the production of transmitters (mainly dopamine) by vlPAG/LPT nuclei [23]. This hypothesis is currently the topic of vigorous discussion and investigation.…”

“…These peptides were discovered in 1998 by two independent scientific groups [23,24]. The first group is called the peptides hypocretins because they were detected in the hypothalamus and had a chemical structure similar to secretin.…”

“…Two peptides, called hypocretin/orexin A and B, were identified. These have, respectively, 33 and 28 amino acids, and show different levels of biological activity; orexin A is almost 100 times more powerful than orexin B [23].…”

Acute Aerobic Exercise and Plasma Levels of Orexin A, Insulin, Glucose, and Insulin Resistance in Males With Type 2 Diabetes

“…Orexin is found in two types: orexin A (OXA) and orexin B (OXB), which are 33 and 28 amino acid residues long, respectively, and are made by the activity of the prepro-orexin (PPO) genes. Researches showed that OXA may be of greater biological importance than OXB (7). OXA has an inevitable role in glucose metabolism, so that decrease in OXA leads to dysfunction, glucose tolerance and insulin resistance in non-obese male mice and almost obese female mice.…”