Discovery process and pharmacological characterization of a novel dual orexin 1 and orexin 2 receptor antagonist useful for treatment of sleep disorders.

The hypothalamic peptides orexin-A and orexin-B are potent agonists of two G-protein coupled receptors, namely the OX(1) and the OX(2) receptor. These receptors are widely distributed, though differentially, in the rat brain. In particular, the OX(1) receptor is highly expressed throughout the hypothalamus, whilst the OX(2) receptor is mainly located in the ventral posterior nucleus. A large body of compelling evidence, both pre-clinical and clinical, suggests that the orexin system is profoundly implicated in sleep disorders. In particular, modulation of the orexin receptors activation by appropriate antagonists was proven to be an efficacious strategy for the treatment of insomnia in man. A novel, drug-like bis-amido piperidine derivative was identified as potent dual OX(1) and OX(2) receptor antagonists, highly effective in a pre-clinical model of sleep.

Synthesis and pharmacological characterization of 5-phenyl-2-[2-(1-piperidinylcarbonyl)phenyl]-2,3-dihydro-1H-pyrrolo[1,2-c]imidazol-1-ones: a new class of Neuropeptide S antagonists.

A new class of selective NPS antagonist was developed starting from a commercially available product identified by screening activities. Experimental NMR observations and computational experiments allowed the discovery of a new class of derivatives. 5-Phenyl-2-[2-(1-piperidinylcarbonyl)phenyl]-2,3-dihydro-1H-pyrrolo[1,2-c]imidazol-1-one represents a new lead compound in the NPS antagonist field.

The identification of novel orally active mGluR5 antagonist GSK2210875.

The identification of novel orally active mGluR5 antagonist GSK2210875 is described.

The identification of structurally novel, selective, orally bioavailable positive modulators of mGluR2.

The optimisation of an HTS hit series (1) leading to the identification of structurally novel, selective, orally bioavailable mGluR2 positive modulators GSK1331258 and GSK1331268 is described. Structure-activity relationships, attenuation of dopaminergic activity, and potentiation of mGluR2 responses in rat hippocampal MPP-DG synapses are also reported.

Altered dimerization of metabotropic glutamate receptor 3 in schizophrenia.

BACKGROUND: Metabotropic glutamate receptors (mGlus) may be involved in the pathophysiology of schizophrenia. Group II mGlus (mGlu2 and mGlu3) have attracted considerable interest since the development of potent specific agonists that exhibit atypical antipsychotic-like activity and reports of a genetic association between the mGlu3 gene and schizophrenia. METHODS: In this postmortem study, mGlu3 protein levels in Brodmann area 10 of prefrontal cortex from schizophrenic (n = 20) and control (n = 35) subjects were analyzed by western immunoblotting using a novel specific mGlu3 antibody and an antibody for the vesicular glutamate transporter 1 (VGluT1). RESULTS: We report a significant decrease in the dimeric/oligomeric forms of mGlu3 in schizophrenic patients compared with control subjects, whereas total mGlu3 and VGluT1 levels were not altered significantly. CONCLUSIONS: This is the first experimental evidence that mGlu3 receptor levels are altered in schizophrenia and supports the hypothesis that neurotransmission involving this particular excitatory amino acid receptor is impaired in schizophrenia.

In vitro and in vivo pharmacological characterization of the novel UT receptor ligand [Pen5,DTrp7,Dab8]urotensin II(4-11) (UFP-803).

The novel urotensin-II (U-II) receptor (UT) ligand, [Pen(5),DTrp(7),Dab(8)]U-II(4-11) (UFP-803), was pharmacologically evaluated and compared with urantide in in vitro and in vivo assays. In the rat isolated aorta, UFP-803 was inactive alone but, concentration dependently, displaced the contractile response to U-II to the right, revealing a competitive type of antagonism and a pA(2) value of 7.46. In the FLIPR [Ca(2+)](i) assay, performed at room temperature in HEK293(hUT) and HEK293(rUT) cells, U-II increased [Ca(2+)](i) with pEC(50) values of 8.11 and 8.48. Urantide and UFP-803 were inactive as agonists, but antagonized the actions of U-II by reducing, in a concentration-dependent manner, the agonist maximal effects with apparent pK(B) values in the range of 8.45-9.05. In a separate series of experiments performed at 37 degrees C using a cuvette-based [Ca(2+)](i) assay and CHO(hUT) cells, urantide mimicked the [Ca(2+)](i) stimulatory effect of U-II with an intrinsic activity (alpha) of 0.80, while UFP-803 displayed a small (alpha=0.21) but consistent residual agonist activity. When the same experiments were repeated at 22 degrees C (a temperature similar to that in FLIPR experiments), urantide displayed a very small intrinsic activity (alpha=0.11) and UFP-803 was completely inactive as an agonist. In vivo in mice, UFP-803 (10 nmol kg(-1)) antagonized U-II (1 nmol kg(-1))-induced increase in plasma extravasation in various vascular beds, while being inactive alone. In conclusion, UFP-803 is a potent UT receptor ligand which displays competitive/noncompetitive antagonist behavior depending on the assay. While UFP-803 is less potent than urantide, it displayed reduced residual agonist activity and as such may be a useful pharmacological tool.

The opioid receptor pharmacology of GSK1521498 compared to other ligands with differential effects on compulsive reward-related behaviours.

RATIONALE: The novel opioid receptor antagonist, GSK1421498, has been shown to attenuate reward-driven compulsive behaviours, such as stimulant drug seeking or binge eating, in animals and humans. Here, we report new data on the receptor pharmacology of GSK121498, in comparison to naltrexone, naloxone, 6-ß-naltrexol and nalmefene. OBJECTIVES: To determine whether the novel opioid antagonist, GSK1521498, is an orthosteric or allosteric antagonist at the µ opioid receptor (MOPr) and whether it has neutral antagonist or inverse agonist properties. METHODS: A combination of radioligand binding assays and [(35)S]GTPγS binding assays was employed. RESULTS: GSK1521498 completely displaced [(3)H]naloxone binding to MOPr and did not alter the rate of [(3)H]naloxone dissociation from MOPr observations compatible with it binding to the orthosteric site on MOPr. GSK1521498 exhibited inverse agonism when MOPr was overexpressed but not when the level of MOPr expression was low. In parallel studies under conditions of high receptor expression density, naloxone, naltrexone, 6-ß-naltrexol and nalmefene exhibited partial agonism, not inverse agonism as has been reported previously for naloxone and naltrexone. In brain tissue from mice receiving a prolonged morphine pre-treatment, GSK1521498 exhibited slight inverse agonism. CONCLUSIONS: Differences between GSK1521498 and naltrexone in their effects on compulsive reward seeking are arguably linked to the more selective and complete MOPr antagonism of GSK1521498 versus the partial MOPr agonism of naltrexone. GSK1521498 is also pharmacologically differentiated by its inverse agonist efficacy at high levels of MOPr expression, but this may be less likely to contribute to behavioural differentiation at patho-physiological levels of expression.

Large clusters of alpha7-containing nicotinic acetylcholine receptors on chick spinal cord neurons.

Nicotinic acetylcholine receptors containing the alpha7 gene product are widely expressed in the nervous system and have high calcium permeabilities that allow them to influence numerous calcium-dependent processes. Though often found at presynaptic locations, where they enhance transmitter release, the receptors can also occupy postsynaptic sites. Highest levels have been reported for chick ciliary ganglion neurons, where the postsynaptic receptors are concentrated on somatic spines arranged in clumps and appear as large receptor clusters. We show here that subpopulations of chick spinal cord neurons also express high levels of alpha7-containing receptors and arrange them in large clusters. The populations include peripheral motoneurons, presumptive preganglionic neurons, neurons adjacent to the lateral motor column, and possible interneurons in the ventral horn. In many cases, the receptor clusters codistribute with filamentous actin, as do clusters on ciliary ganglion neurons, where the actin represents a somatic spine constituent. In other respects, the spinal cord clusters differ. Those on motoneurons codistribute with the actin-associated component drebrin, as do the clusters on ciliary ganglion neurons, but the clusters on preganglionic neurons do not. Preganglionic neurons do, however, stain for lipid raft components as found for ciliary ganglion neurons, where the rafts embed the receptor-enriched spines. The results demonstrate that CNS neurons can configure alpha7-containing nicotinic receptors into large clusters but also suggest that the clusters are not likely to reflect a common molecular substructure on all neurons.

Paradoxical response to the sedative effects of diazepam and alcohol in C57BL/6J mice lacking the neuropeptide S receptor.

The neuropeptide S (NPS) system is characterized by a unique pharmacology because it has anxiolytic-like effects and promotes arousal and wakefulness. To shed light on this peptidergic system, we tested the sedative effect of the central depressants diazepam and ethanol on the loss of righting reflex in mice lacking the neuropeptide S receptor (NPSR), NPSR(-/-). Furthermore, we tested the effect of the intracerebroventricular (ICV) administration of NPS on the sedative effect of diazepam and ethanol in NPSR(-/-) and their wild type counterpart NPSR(+/+). Finally, we evaluated the effect of the pro-arousal neuropeptides CRF and Hcrt-1/Ox-A in NPSR-deficient mice. Contrary to our expectations, the results showed that the NPSR(-/-) were less sensitive to the hypnotic effects of both diazepam and ethanol compared with their wild type littermates. ICV NPS was able to attenuate the sedative effect of both alcohol and diazepam in wild type mice, but not in the NPSR(-/-) line. The administration of CRF and Hcrt-1/Ox-A, two classic pro-arousal peptides, elicited the same effects in both NPSR(-/-) and wild type mice, ruling out the possibility that adaptive mechanisms occurring at the level of these two systems could have occurred during NPSR(-/-) development to compensate for the lack of NPSR receptors. Our findings demonstrated that the deletion of NPSR leads to minor changes in the arousal behavior of mice. Moreover, we demonstrated that the deletion of NPSR did not lead to compensatory changes in the vigilance-promoting effects of the CRF and Hcrt-1/Ox-A systems.

Structure-activity studies on neuropeptide S: identification of the amino acid residues crucial for receptor activation.

Neuropeptide S (NPS) has been recently recognized as the endogenous ligand for the previous orphan G-protein-coupled receptor GPR154, now referred to as the NPS receptor (NPSR). The NPS-NPSR receptor system regulates important biological functions such as sleeping/wakening, locomotion, anxiety, and food intake. To collect information on the mechanisms of interaction between NPS and its receptor, a classical structure-activity relationship study was performed. Human (h) NPS derivatives obtained by Ala and d-scan and N- and C-terminal truncation were assessed for their ability to stimulate calcium release in HEK293 cells expressing the human recombinant NPSR. The results of this study indicate that (i) the effect of hNPS is mimicked by the fragment hNPS-(1-10); (ii) Phe(2), Arg(3), and Asn(4) are crucial for biological activity; (iii) the sequence Thr(8)-Gly(9)-Met(10) is important for receptor activation, although with non-stringent chemical requirements; and (iv) the sequence Val(6)-Gly(7) acts as a hinge region between the two above-mentioned domains. However, the stimulatory effect of hNPS given intracerebroventricularly on mouse locomotor activity was not fully mimicked by hNPS-(1-10), suggesting that the C-terminal region of the peptide maintains importance for in vivo activity. In conclusion, this study identified the amino acid residues of this peptide most important for receptor activation.