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

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 ∼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

Characterization of the binding of [(125)I]-human prolactin releasing peptide (PrRP) to GPR10, a novel G protein coupled receptor

1. GPR10 is a novel G-protein coupled receptor that is the human orthologue of rat Unknown Hypothalamic Receptor-1 (UHR-1). Human prolactin-releasing peptide (PrRP) has been identified as an endogenous ligand for GPR10, and occurs as 31 and 20 amino acid forms. The present study characterizes the binding of [(125)I]-PrRP-20 to HEK293 cells stably expressing GPR10 receptors. 2. Specific binding of [(125)I]-PrRP-20 was saturable, and analysis suggested evidence of both high and low affinity sites, with K(D) values of 0.026±0.006 and 0.57±0.14 nM respectively, and B(max) values of 3010±400 and 8570±2240 fmol mg protein(−1) respectively. Kinetic studies were unable to distinguish two sites, but single site analysis of association and dissociation data produced a K(D) of 0.012 nM. 3. Competition studies revealed that human and rat PrRP-20 and PrRP-31 all display high affinity for GPR10. A range of other drugs which are known ligands at receptors which share limited homology with GPR10 were also tested. None of the drugs tested, including the RF-amide neuropeptide FF, demonstrated any affinity for GPR10. 4. Human PrRP-20 failed to alter basal or forskolin-stimulated levels of intracellular cyclic AMP in HEK293-GPR10 cells, suggesting that GPR10 does not couple via either G(s) or G(i). 5. Functional studies using measurements of intracellular calcium confirmed that human and rat PrRP-20 and PrRP-31 are all potent, full agonists at the GPR10 receptor. The response was blocked both by thapsigargin, indicating mobilization of intracellular Ca(2+) stores. 6. These studies indicate that [(125)I]-PrRP-20 is a specific, high affinity radioligand for GPR10. The availability of this radioligand binding assay will be a valuable tool for the investigation of the key features involved in PrRP binding and studies on the localization and function of GPR10

Profile of SB-204269, a mechanistically novel anticonvulsant drug, in rat models of focal and generalized epileptic seizures

1. Earlier optimization of structure-activity relationships in a novel series of 4-(benzoylamino)-benzopyrans, led to the discovery of SB-204269 (trans-(+)-6-acetyl-4S-(4-fluorobenzoylamino)-3,4-dihydro-2,2-dimethyl-2H-benzo[b]pyran-3R-ol, hemihydrate), a potent orally-active anticonvulsant in the mouse maximal electroshock seizure threshold (MEST) test. 2. Studies have now been undertaken to determine the effects of SB-204269 in a range of seizure models and tests of neurological deficits in rats. In addition, the compound has been evaluated in a series of in vitro mechanistic assays. 3. SB-204269 proved to be an orally-effective anticonvulsant agent, at doses (0.1–30 mg kg(−1)) devoid of overt behavioural depressant properties, in models of both electrically (MEST and maximal electroshock (MES)) and chemically (i.v. pentylenetetrazol (PTZ) infusion)-evoked tonic extension seizures. However, the compound did not inhibit PTZ-induced myoclonic seizures at doses up to 30 mg kg(−1), p.o. 4. SB-204269 also selectively reduced focal electrographic seizure activity in an in vitro elevated K(+) rat hippocampal slice model at concentrations (0.1–10 μM) that had no effect on normal synaptic activity and neuronal excitability. 5. In all of these seizure models, SB-204269 was equivalent or better than the clinically established antiepileptic drugs carbamazepine and lamotrigine, in terms of anticonvulsant potency and efficacy. 6. Unlike SB-204269, the corresponding trans 3S,4R enantiomer, SB-204268, did not produce marked anticonvulsant effects, an observation in accord with previous findings for other related pairs of trans enantiomers in the benzopyran series. 7. In the rat accelerating rotarod test, a sensitive paradigm for the detection of neurological deficits such as sedation and motor incoordination, SB-204269 was inactive even at doses as high as 200 mg kg(−1), p.o. This was reflected in the excellent therapeutic index (minimum significantly effective dose in the rotarod test/ED(50) in the MES test) for SB-204269 of >31, as compared to equivalent values of only 7 and 13 for carbamazepine and lamotrigine, respectively. 8. At concentrations (⩾10 μM) well above those required to produce anticonvulsant activity in vivo (i.e. 0.1 μM in brain), SB-204269 did not interact with many of the well known mechanistic targets for established antiepileptic drugs (e.g. Na(+) channels or GABAergic neurotransmission). Subsequent studies have shown that the anticonvulsant properties of SB-204269 are likely to be mediated by a novel stereospecific binding site present in the CNS. 9. The overall efficacy profile in rodent seizure models, together with a minimal liability for inducing neurological impairment and an apparently unique mechanism of action, highlight the therapeutic potential of SB-204269 for the treatment of refractory partial and generalized tonic-clonic seizures