Specific Inhibition of Orai1-mediated Calcium Signalling Resolves Inflammation and Clears Bacteria in an Acute Respiratory Distress Syndrome Model.

Rationale: Acute respiratory distress syndrome (ARDS) has an unacceptably high mortality rate (35%) and is without effective therapy. Orai1 is a Ca2+ channel involved in store-operated Ca2+ entry (SOCE), a process that exquisitely regulates inflammation. Orai1 is considered a druggable target, but no Orai1-specific inhibitors exist to date. Objectives: To evaluate whether ELD607, a first-in-class Orai1 antagonist, can treat ARDS caused by bacterial pneumonia in preclinical models. Methods: ELD607 pharmacology was evaluated in HEK293T cells and freshly isolated immune cells from patients with ARDS. A murine acute lung injury model caused by bacterial pneumonia was then used: mice were infected with Pseudomonas aeruginosa, Staphylococcus aureus, methicillin-resistant S. aureus, or multidrug-resistant P. aeruginosa and then treated with ELD607 intranasally. Measurements and Main Results: ELD607 specifically inhibited SOCE in HEK293T cells with a half-maximal inhibitory concentration of 9 nM. ELD607 was stable in ARDS airway secretions and inhibited SOCE in ARDS immune cells. In vivo, inhaled ELD607 significantly reduced neutrophilia and improved survival. Surprisingly, Orai1 inhibition by ELD607 caused a significant reduction in lung bacteria, including methicillin-resistant S. aureus. ELD607 worked as an immunomodulator that reduced cytokine levels, reduced neutrophilia, and promoted macrophage-mediated resolution of inflammation and clearance of bacteria. Indeed, when alveolar macrophages were depleted with inhaled clodronate, ELD607 was no longer able to resolve inflammation or clear bacteria. Conclusions: These data indicate that specific Orai1 inhibition by ELD607 may be a novel approach to reduce multiorgan inflammation and treat antibiotic-resistant bacteria.

Opioid receptor modulation of hedonic taste preference and food intake: a single-dose safety, pharmacokinetic, and pharmacodynamic investigation with GSK1521498, a novel µ-opioid receptor inverse agonist.

Endogenous opioids and µ-opioid receptors have been linked to hedonic and rewarding aspects of palatable food intake. The authors examined the safety, pharmacokinetic, and pharmacodynamic profile of GSK1521498, a µ-opioid receptor inverse agonist that is being investigated primarily for the treatment of overeating behavior in obesity. In healthy participants, GSK1521498 oral solution and capsule formulations were well tolerated up to a dose of 100 mg. After single doses (10-150 mg), the maximum concentration (C(max)) and area under the curve (AUC) in plasma increased in a dose-proportional manner. GSK1521498 selectively reduced sensory hedonic ratings of high-sugar and high-fat dairy products and caloric intake of high-fat/high-sucrose snack foods. These findings provide encouraging data in support of the development of GSK1521498 for the treatment of disorders of maladaptive ingestive behavior or compulsive consumption.

Regulation of ingestive behaviors in the rat by GSK1521498, a novel micro-opioid receptor-selective inverse agonist.

µ-Opioid receptor (MOR) agonism induces palatable food consumption principally through modulation of the rewarding properties of food. N-{[3,5-difluoro-3'-(1H-1,2,4-triazol-3-yl)-4-biphenylyl]methyl}-2,3-dihydro-1H-inden-2-amine (GSK1521498) is a novel opioid receptor inverse agonist that, on the basis of in vitro affinity assays, is greater than 10- or 50-fold selective for human or rat MOR, respectively, compared with κ-opioid receptors (KOR) and δ-opioid receptors (DOR). Likewise, preferential MOR occupancy versus KOR and DOR was observed by autoradiography in brain slices from Long Evans rats dosed orally with the drug. GSK1521498 suppressed nocturnal food consumption of standard or palatable chow in lean and diet-induced obese (DIO) Long Evans rats. Both the dose-response relationship and time course of efficacy in lean rats fed palatable chow correlated with µ receptor occupancy and the plasma concentration profile of the drug. Chronic oral administration of GSK1521498 induced body weight loss in DIO rats, which comprised fat mass reduction. The reduction in body weight was equivalent to the cumulative reduction in food consumption; thus, the effect of GSK1521498 on body weight is related to inhibition of food consumption. GSK1521498 suppressed the preference for sucrose-containing solutions in lean rats. In operant response models also using lean rats, GSK1521498 reduced the reinforcement efficacy of palatable food reward and enhanced satiety. In conclusion, GSK1521498 is a potent, MOR-selective inverse agonist that modulates the hedonic aspects of ingestion and, therefore, could represent a pharmacological treatment for obesity and binge-eating disorders.

A concise synthesis of 1,4-dihydro-[1,4]diazepine-5,7-dione, a novel 7-TM receptor ligand core structure with melanocortin receptor agonist activity.

Finding small non-peptide molecules for G protein-coupled receptors (GPCR) whose endogenous ligands are peptides, is a very important task for medicinal chemists. Over the years, compounds mimicking peptide structures have been discovered, and scaffolds emulating peptide backbones have been designed. In our work on GPCR ligands, including cholecystokinin receptor-1 (CCKR-1) agonists, we have employed benzodiazepines as a core structure. Looking for ways to reduce molecular weight and possibly improve physical properties of GPCR ligands, we embarked on the search for molecules providing similar scaffolds to the benzodiazepine with lower molecular weight. One of our target core structures was 1,4-dihydro-[1,4]diazepine-5,7-dione. There was not, however, a known synthetic route to such molecules. Here we report the discovery of a simple and concise method for synthesis of 2-[6-(1H-indazol-3-ylmethyl)-5,7-dioxo-4-phenyl-4,5,6,7-tetrahydro-[1,4]diazepin-1-yl]-N-isopropyl-N-phenyl-acetamide as an example of a compound containing the tetrahydrodiazepine-5,7-dione core. Compounds from this series were tested in numerous GPCR assays and demonstrated activity at melanocortin 1 and 4 receptors (MC1R and MC4R). Selected compounds from this series were tested in vivo in Peptide YY (PYY)-induced food intake. Compounds dosed by intracerebroventricular and oral routes reduced PYY-induced food intake and this effect was reversed by the cyclic peptide MC4R antagonist SHU9119.

Lysosomes contribute to anomalous pharmacokinetic behavior of melanocortin-4 receptor agonists.

PURPOSE: A series of melanocortin-4 receptor (MC4R) agonists, developed for use as anti-obesity agents, were found to have unusual pharmacokinetic behavior arising from excessive retention in the liver, with nearly undetectable levels in plasma following oral administration in mice. This work investigates the molecular basis of the prolonged liver retention that provided a rational basis for the design of an analog with improved behavior. MATERIALS AND METHODS: The livers of mice were harvested and techniques were utilized to fractionate them into pools differentially enriched in organelles. The distribution of organelles in the fractions was determined using organelle-specific enzymatic assays. Livers from mice dosed with drug were fractionated and comparisons with organelle distributions assisted in determining the subcellular localization of the drug. Further analysis in cell culture systems was used to confirm results from liver fractionation studies and also allowed for more extensive evaluations to examine the mechanism for organelle compartmentalization RESULTS: Fractionation of livers following oral administration of the agonist showed sequestration in lysosomes. Subsequent evaluations in a cell culture system confirmed this finding. Agents used to disrupt acidification of lysosomes led to decreased lysosomal accumulation of the drug, which implicated a pH-partitioning type sequestration mechanism. These findings led to the rational synthesis of an analog of the parent compound with properties that reduced lysosomal sequestration. When this compound was examined in mice, the liver retention was found to be greatly reduced and plasma levels were significantly elevated relative to the parent compound. CONCLUSIONS: Weakly basic drugs with optimal physicochemical properties can be extensively sequestered into lysosomes according to a pH-partitioning type mechanism. When administered orally in animals, this particular sequestration event can manifest itself in long term retention in the liver and negligible levels in blood. This work revealed the mechanism for liver retention and provided a rational platform for the design of a new analog with decreased liver accumulation and better opportunity for pharmacokinetic analysis and therapeutic activity.

Potent, selective, and orally efficacious antagonists of melanin-concentrating hormone receptor 1.

The high expression of MCH in the hypothalamus with the lean hypophagic phenotype coupled with increased resting metabolic rate and resistance to high fat diet-induced obesity of MCH KO mice has spurred considerable efforts to develop small molecule MCHR1 antagonists. Starting from a lead thienopyrimidinone series, structure-activity studies at the 3- and 6-positions of the thienopyrimidinone core afforded potent and selective MCHR1 antagonists with representative examples having suitable pharmacokinetic properties. Based on structure-activity relationships, a structural model for MCHR1 was constructed to explain the binding mode of these antagonists. In general, a good correlation was observed between pKas and activity in the right-hand side of the template, with Asp123 playing an important role in the enhancement of binding affinity. A representative example when evaluated chronically in diet-induced obese mice resulted in good weight loss effects. These antagonists provide a viable lead series in the discovery of new therapies for the treatment of obesity.

6-(4-chlorophenyl)-3-substituted-thieno[3,2-d]pyrimidin-4(3H)-one-based melanin-concentrating hormone receptor 1 antagonist.

Genetic manipulation studies in mice at both the MCH receptor 1 (MCHR1) as well as the MCH peptide levels have implicated MCHR1 as a key player in energy homeostasis. The phenotype exhibited by these studies, that is, increased metabolic rate, resistance to high fat diet, and subsequent weight loss, has spurred considerable efforts to develop antagonists of MCHR1. In continuation of efforts directed toward this goal, the present work capitalizes on the putative binding mode of an MCH antagonist, resulting in the identification of several novel chemotypes that are potent and selective MCHR1 antagonists. In addition, the favorable pharmacokinetics of representative examples has allowed for the evaluation of an MCHR1 antagonist in a high fat diet-induced obese rodent model of obesity. The tolerability of the right-hand side of the template for diverse chemotypes accompanied by favorable effects on weight loss enhances the attractiveness of this template in the pursuit toward development of effective anti-obesity agents.

Novel benzimidazole-based MCH R1 antagonists.

The identification of an MCH R1 antagonist screening hit led to the optimization of a class of benzimidazole-based MCH R1 antagonists. Structure-activity relationships and efforts to optimize pharmacokinetic properties are detailed along with the demonstration of the effectiveness of an MCH R1 antagonist in an animal model of obesity.

The discovery and optimization of pyrimidinone-containing MCH R1 antagonists.

Optimization of a series of constrained melanin-concentrating hormone receptor 1 (MCH R1) antagonists has provided compounds with potent and selective MCH R1 activity. Details of the optimization process are provided and the use of one of the compounds in an animal model of diet-induced obesity is presented.

Pharmacological regulation of insulin secretion in MIN6 cells through the fatty acid receptor GPR40: identification of agonist and antagonist small molecules.

1. Long chain fatty acids have recently been identified as agonists for the G protein-coupled receptors GPR40 and GPR120. Here, we present the first description of GW9508, a small-molecule agonist of the fatty acid receptors GPR40 and GPR120. In addition, we also describe the pharmacology of GW1100, a selective GPR40 antagonist. These molecules were used to further investigate the role of GPR40 in glucose-stimulated insulin secretion in the MIN6 mouse pancreatic beta-cell line. 2. GW9508 and linoleic acid both stimulated intracellular Ca2+ mobilization in human embryonic kidney (HEK)293 cells expressing GPR40 (pEC50 values of 7.32+/-0.03 and 5.65+/-0.06, respectively) or GPR120 (pEC50 values of 5.46+/-0.09 and 5.89+/-0.04, respectively), but not in the parent HEK-293 cell line. 3. GW1100 dose dependently inhibited GPR40-mediated Ca2+ elevations stimulated by GW9508 and linoleic acid (pIC50 values of 5.99+/-0.03 and 5.99+/-0.06, respectively). GW1100 had no effect on the GPR120-mediated stimulation of intracellular Ca2+ release produced by either GW9508 or linoleic acid. 4. GW9508 dose dependently potentiated glucose-stimulated insulin secretion in MIN6 cells, but not in primary rat or mouse islets. Furthermore, GW9508 was able to potentiate the KCl-mediated increase in insulin secretion in MIN6 cells. The effects of GW9508 on insulin secretion were reversed by GW1100, while linoleic acid-stimulated insulin secretion was partially attenuated by GW1100. 5. These results add further evidence to a link between GPR40 and the ability of fatty acids to acutely potentiate insulin secretion and demonstrate that small-molecule GPR40 agonists are glucose-sensitive insulin secretagogues.

Characterisation of olanzapine-induced weight gain and effect of aripiprazole vs olanzapine on body weight and prolactin secretion in female rats.

RATIONALE: Atypical antipsychotic drug (APD)-induced weight gain causes non-compliance, increasing the risk of relapse and medical complications. OBJECTIVES: In an animal model, we assessed body weights, food intake, body fat/lean body mass contents and blood serum levels of glucose and lipids in female rats treated with olanzapine (Experiment 1). Also, we investigated the effect of aripiprazole vs olanzapine treatment on weight gain (WG) and plasma prolactin secretion in two strains (Wistar and Sprague-Dawley) and in two housing conditions (singly and group housed; Experiment 2). METHODS: In Experiment 1, Wistar females received either vehicle or olanzapine (5.0 mg kg(-1), p.o.) twice daily for 14 days. In Experiment 2, female rats (Wistar or Sprague-Dawley), housed singly or in groups, received either vehicle, aripiprazole (2.0-8.0 mg kg(-1), p.o.), or olanzapine (1.0-10 mg kg(-1), p.o.) twice daily for 7 days. Body weights and food intake were assessed daily. Body composition and blood assays were analyzed at the end of the treatment. RESULTS: WG induced by chronic olanzapine treatment was characterised by hyperphagia, increased body fat, and serum free fatty acid content and reduced lean tissue and serum glucose content. Subchronic aripiprazole treatment resulted in rapid and robust WG similar to those observed with olanzapine. In spite of similar effects on body weight, aripiprazole and olanzapine stimulated markedly different patterns of prolactin secretion. Body weight changes and prolactin secretion induced by these APDs were significantly modulated by housing and by strain. CONCLUSION: Assessment of body weight in the present model may not have predictive validity, and other measures may be needed to differentiate between WG-inducing and weight-neutral drugs.

A coding polymorphism in the CYSLT2 receptor with reduced affinity to LTD4 is associated with asthma.

BACKGROUND: Cysteinyl leukotrienes (CYSLTR) are potent biological mediators in the pathophysiology of asthma for which two receptors have been characterized, CYSLTR1 and CYSLTR2. The leukotriene modifying agents currently used to control bronchoconstriction and inflammation in asthmatic patients are CYSLTR1-specific leukotriene receptor antagonists. In this report, we investigated a possible role for therapeutic modulation of CYSLTR2 in asthma by investigating genetic association with asthma and further characterization of the pharmacology of a coding polymorphism. METHODS: The association of CYSLTR2 polymorphisms with asthma was assessed by transmission disequilibrium test in two family-based collections (359 families from Denmark and Minnesota, USA and 384 families from the Genetics of Asthma International Network). RESULTS: A significant association of the coding polymorphism, 601A>G, with asthma was observed (P = 0.003). We replicated these findings in a collection of 384 families from the Genetics of Asthma International Network (P = 0.04). The G allele is significantly under-transmitted to asthmatics, indicating a possible role for this receptor in resistance to asthma. The potency of cysteinyl leukotrienes at the wild-type CYSLTR2 and the coding polymorphism 601A>G were assessed using a calcium mobilization assay. The potency of LTC4 and LTE4 was similar for both forms of the receptor and LTB4 was inactive, however, LTD4 was approximately five-fold less potent on 601A>G compared to wild-type CYSLTR2. CONCLUSIONS: Since 601A>G alters the potency of LTD4 and this variant allele may be associated with resistance to asthma, it is possible that modulation of the CYSLTR2 may be useful in asthma pharmacotherapy.

Regulation of TNF-alpha secretion by a specific melanocortin-1 receptor peptide agonist.

The lack of specific pharmacological tools has impeded the evaluation of the role of each melanocortin receptor (MCR) subtype in the myriad physiological effects of melanocortins. 154N-5 is an octapeptide (MFRdWFKPV-NH(2)) that was first identified as an MC1R antagonist in Xenopus melanophores [J. Biol. Chem. 269 (1994) 29846]. In this manuscript, we show that 154N-5 is a specific agonist for human and murine MC1R. The peptide has negligible activity at MC3R and MC4R and is 25-fold less potent and a weak agonist at MC5R. 154N-5 was tested in both a cellular and an animal model of tumor necrosis factor-alpha (TNF-alpha) secretion. The inhibitory efficacy of 154N-5 on TNF-alpha secretion in both models was similar to the nonselective agonist NDP-alpha-melanocyte stimulating hormone (NDP-alphaMSH), thus, we conclude that inhibition of TNF-alpha secretion by melanocortin peptides is mediated by MC1R. 154N-5 is a valuable new tool for the evaluation of specific contribution of MC1R agonism to physiological and pathological processes.

Molecular identification of high and low affinity receptors for nicotinic acid.

Nicotinic acid has been used clinically for over 40 years in the treatment of dyslipidemia producing a desirable normalization of a range of cardiovascular risk factors, including a marked elevation of high density lipoprotein and a reduction in mortality. The precise mechanism of action of nicotinic acid is unknown, although it is believed that activation of a G(i)-G protein-coupled receptor may contribute. Utilizing available information on the tissue distribution of nicotinic acid receptors, we identified candidate orphan receptors. The selected orphan receptors were screened for responses to nicotinic acid, in an assay for activation of G(i)-G proteins. Here we describe the identification of the G protein-coupled receptor HM74 as a low affinity receptor for nicotinic acid. We then describe the subsequent identification of HM74A in follow-up bioinformatics searches and demonstrate that it acts as a high affinity receptor for nicotinic acid and other compounds with related pharmacology. The discovery of HM74A as a molecular target for nicotinic acid may facilitate the discovery of superior drug molecules to treat dyslipidemia.

The Orphan G protein-coupled receptors GPR41 and GPR43 are activated by propionate and other short chain carboxylic acids.

GPR41 and GPR43 are related members of a homologous family of orphan G protein-coupled receptors that are tandemly encoded at a single chromosomal locus in both humans and mice. We identified the acetate anion as an agonist of human GPR43 during routine ligand bank screening in yeast. This activity was confirmed after transient transfection of GPR43 into mammalian cells using Ca(2+) mobilization and [(35)S]guanosine 5'-O-(3-thiotriphosphate) binding assays and by coexpression with GIRK G protein-regulated potassium channels in Xenopus laevis oocytes. Other short chain carboxylic acid anions such as formate, propionate, butyrate, and pentanoate also had agonist activity. GPR41 is related to GPR43 (52% similarity; 43% identity) and was activated by similar ligands but with differing specificity for carbon chain length, with pentanoate being the most potent agonist. A third family member, GPR42, is most likely a recent gene duplication of GPR41 and may be a pseudogene. GPR41 was expressed primarily in adipose tissue, whereas the highest levels of GPR43 were found in immune cells. The identity of the cognate physiological ligands for these receptors is not clear, although propionate is known to occur in vivo at high concentrations under certain pathophysiological conditions.

The orphan G protein-coupled receptor GPR40 is activated by medium and long chain fatty acids.

GPR40 is a member of a subfamily of homologous G protein-coupled receptors that include GPR41 and GPR43 and that have no current function or ligand ascribed. Ligand fishing experiments in HEK293 cells expressing human GPR40 revealed that a range of saturated and unsaturated carboxylic acids with carbon chain lengths greater than six were able to induce an elevation of [Ca(2+)](i), measured using a fluorometric imaging plate reader. 5,8,11-Eicosatriynoic acid was the most potent fatty acid tested, with a pEC(50) of 5.7. G protein coupling of GPR40 was examined in Chinese hamster ovary cells expressing the G alpha(q/i)-responsive Gal4-Elk1 reporter system. Expression of human GPR40 led to a constitutive induction of luciferase activity, which was further increased by exposure of the cells to eicosatriynoic acid. Neither the constitutive nor ligand-mediated luciferase induction was inhibited by pertussis toxin treatment, suggesting that GPR40 was coupled to G alpha(q/11.) Expression analysis by quantitative reverse transcription-PCR showed that GPR40 was specifically expressed in brain and pancreas, with expression in rodent pancreas being localized to insulin-producing beta-cells. These data suggest that some of the physiological effects of fatty acids in pancreatic islets and brain may be mediated through a cell-surface receptor.

Characterization of melanocortin receptors.

This unit describes a Scintillation Proximity Assay (SPA) for the measurement of ligand binding to melanocortin receptors (MCRs) using membranes prepared from cell lines stably expressing recombinant MCRs. It provides a facile method for determining the affinity of compounds at MC1R, MC3R, MC4R, or MC5R.