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Brett Lauring,
Andrew K P Taggart,
James R Tata,
Richard Dunbar,
Luzelena Caro,
Kang Cheng,
Jayne Chin,
Steven L Colletti,
Josee Cote,
Sauzanne Khalilieh, [......],
Daniel T Connolly,
Eseng Lai,
John A Wagner,
Samuel D Wright,
Cynthia Cuffie,
Yale B Mitchel,
Daniel J Rader,
John F Paolini,
M Gerard Waters,
Andrew Plump
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ABSTRACT: Nicotinic acid (niacin) induces beneficial changes in serum lipoproteins and has been associated with beneficial cardiovascular effects. Niacin reduces low-density lipoprotein, increases high-density lipoprotein, and decreases triglycerides. It is well established that activation of the seven-transmembrane G(i)-coupled receptor GPR109A on Langerhans cells results in release of prostaglandin D₂, which mediates the well-known flushing side effect of niacin. Niacin activation of GPR109A on adipocytes also mediates the transient reduction of plasma free fatty acid (FFA) levels characteristic of niacin, which has been long hypothesized to be the mechanism underlying the changes in the serum lipid profile. We tested this "FFA hypothesis" and the hypothesis that niacin lipid efficacy is mediated via GPR109A by dosing mice lacking GPR109A with niacin and testing two novel, full GPR109A agonists, MK-1903 and SCH900271, in three human clinical trials. In mice, the absence of GPR109A had no effect on niacin's lipid efficacy despite complete abrogation of the anti-lipolytic effect. Both MK-1903 and SCH900271 lowered FFAs acutely in humans; however, neither had the expected effects on serum lipids. Chronic FFA suppression was not sustainable via GPR109A agonism with niacin, MK-1903, or SCH900271. We conclude that the GPR109A receptor does not mediate niacin's lipid efficacy, challenging the long-standing FFA hypothesis.
Science translational medicine 08/2012; 4(148):148ra115. · 7.80 Impact Factor
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Zhi-Liang Chu,
Chris Carroll,
Ruoping Chen,
Jean Alfonso,
Veronica Gutierrez,
Hongmei He,
Annette Lucman,
Charles Xing,
Kristen Sebring,
Jinyao Zhou,
Brandee Wagner,
David Unett,
Robert M Jones, Dominic P Behan,
James Leonard
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ABSTRACT: G protein-coupled receptor 119 (GPR119) is largely restricted to pancreatic insulin-producing beta-cells and intestinal glucagon-like peptide-1-producing L-cells. Synthetic agonists of this receptor elicit glucose-dependent release of these endocrine factors, thereby enhancing glycemic control. Oleoylethanolamide also activates GPR119, but it remains unclear whether endogenous production of this lipid modulates GPR119 activity under normal or dysglycemic conditions. We show here that a relatively diverse set of lipid amides activate GPR119. Among these, the endovallinoid N-oleoyldopamine (OLDA) stimulated cAMP accumulation in GPR119-transfected cells as effectively as oleoylethanolamide and the previously described synthetic agonist AR231453. None of these lipid amides increased cAMP in control-transfected cells or in cells transfected with a number of other G protein-coupled receptors. OLDA stimulated both cAMP accumulation and insulin release in HIT-T15 cells, which express GPR119 endogenously, and in GPR119-transfected RIN-5F cells. Oral administration of OLDA to C57bl/6 mice elicited significant improvement in glucose tolerance, whereas GPR119-deficient mice were essentially unresponsive. OLDA also acutely elevated plasma gastric inhibitory peptide levels, a known hallmark of GPR119 activation. OLDA represents a possible paracrine modulator of GPR119 in pancreatic islets, where markers of dopamine synthesis correlated well with GPR119 expression. However, no such correlation was seen in the colon. Collectively, these studies indicate that multiple, distinct classes of lipid amides, acting via GPR119, may be important modulators of glucose homeostasis.
Molecular Endocrinology 11/2009; 24(1):161-70. · 4.54 Impact Factor
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John W Adams,
Juan Ramirez,
Yunqing Shi,
William Thomsen,
John Frazer,
Michael Morgan,
Jeffrey E Edwards,
Weichao Chen,
Bradley R Teegarden,
Yifeng Xiong,
Hussien Al-Shamma, Dominic P Behan,
Daniel T Connolly
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ABSTRACT: We have evaluated the receptor pharmacology, antiplatelet activity, and vascular pharmacology of APD791 [3-methoxy-N-(3-(1-methyl-1H-pyrazol-5-yl)-4-(2-morpholinoethoxy)phenyl)benzamide] a novel 5-hydroxytryptamine 2A (5-HT(2A)) receptor antagonist. APD791 displayed high-affinity binding to membranes (K(i) = 4.9 nM) and functional inverse agonism of inositol phosphate accumulation (IC(50) = 5.2 nM) in human embryonic kidney cells stably expressing the human 5-HT(2A) receptor. In competition binding assays, APD791 was greater than 2000-fold selective for the 5-HT(2A) receptor versus 5-HT(2C) and 5-HT(2B) receptors, and was inactive when tested against a wide panel of other G-protein-coupled receptors. APD791 inhibited 5-HT-mediated amplification of ADP-stimulated human and dog platelet aggregation (IC(50) = 8.7 and 23.1 nM, respectively). Similar potency was observed for inhibition of 5-HT-stimulated DNA synthesis in rabbit aortic smooth muscle cells (IC(50) = 13 nM) and 5-HT-mediated vasoconstriction in rabbit aortic rings. Oral administration of APD791 to dogs resulted in acute (1-h) and subchronic (10-day) inhibition of 5-HT-mediated amplification of collagen-stimulated platelet aggregation in whole blood. Two active metabolites, APD791-M1 and APD791-M2, were generated upon incubation of APD791 with human liver microsomes and were also indentified in dogs after oral administration of APD791. The affinity and selectivity profiles of both metabolites were similar to APD791. These results demonstrate that APD791 is an orally available, high-affinity 5-HT(2A) receptor antagonist with potent activity on platelets and vascular smooth muscle.
Journal of Pharmacology and Experimental Therapeutics 08/2009; 331(1):96-103. · 3.83 Impact Factor
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ABSTRACT: Regulation of cellular metabolism by the citric acid cycle occurs in the mitochondria. However, the citric acid cycle intermediate succinate was shown recently to be a ligand for the G-protein-coupled receptor GPR91. Here, we describe a role for succinate and its receptor in the stimulation of hematopoietic progenitor cell (HPC) growth. GPR91 mRNA and protein expression were detected in human bone marrow CD34+ progenitor cells, as well as in erythroid and megakaryocyte cultures and the erythroleukemic cell line TF-1. Treatment of these cell cultures with succinate resulted in increased proliferation rates. The proliferation response of TF-1 cells was pertussis toxin (PTX)-sensitive, suggesting a role for Gi signaling. Proliferation was also blocked when TF-1 cells were transfected with small interfering RNA specific for GPR91. Succinate stimulated activation of the Erk MAPK pathway and inositol phosphate accumulation in a PTX-sensitive manner. Pretreatment of TF-1 cells with the Erk1/2 kinase (MEK) inhibitor PD98059 blocked the proliferation response. Succinate treatment additionally protected TF-1 cells from cell death induced by serum deprivation. Finally, in vivo administration of succinate was found to elevate the levels of hemoglobin, platelets, and neutrophils in a mouse model of chemotherapy-induced myelosuppression. These results suggest that succinate-GPR91 signaling is capable of promoting HPC development.
Journal of leukocyte biology 03/2009; 85(5):837-43. · 4.99 Impact Factor
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Graeme Semple,
Philip J Skinner,
Tawfik Gharbaoui,
Young-Jun Shin,
Jae-Kyu Jung,
Martin C Cherrier,
Peter J Webb,
Susan Y Tamura,
P Douglas Boatman,
Carleton R Sage, [......],
Steven L Colletti,
James R Tata,
M Gerard Waters,
Kang Cheng,
Andrew K Taggart,
Tian-Quan Cai,
Ester Carballo-Jane, Dominic P Behan,
Daniel T Connolly,
Jeremy G Richman
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ABSTRACT: The discovery and profiling of 3-(1H-tetrazol-5-yl)-1,4,5,6-tetrahydro-cyclopentapyrazole (5a, MK-0354), a partial agonist of GPR109a, is described. Compound 5a retained the plasma free fatty acid lowering effects in mice associated with GPR109a agonism, but did not induce vasodilation at the maximum feasible dose. Moreover, preadministration of 5a blocked the flushing effect induced by nicotinic acid but not that induced by PGD2. This profile made 5a a suitable candidate for further study for the treatment of dyslipidemia.
Journal of Medicinal Chemistry 09/2008; 51(16):5101-8. · 4.80 Impact Factor
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John W Adams,
Juan Ramirez,
Danny Ortuno,
Yunqing Shi,
William Thomsen,
Jeremy G Richman,
Michael Morgan,
Peter Dosa,
Bradley R Teegarden,
Hussien Al-Shamma, Dominic P Behan,
Daniel T Connolly
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ABSTRACT: We have evaluated the anti-platelet and vascular pharmacology of AR246686, a novel 5-hydroxytryptamine2A (5-HT2A) receptor antagonist. AR246686 displayed high affinity binding to membranes of HEK cells stably expressing recombinant human and rat 5-HT2A receptors (Ki=0.2 nM and 0.4 nM, respectively). Functional antagonism (IC50=1.9 nM) with AR246686 was determined by inhibition of ligand-independent inositol phosphate accumulation in the 5-HT2A stable cell line. We observed 8.7-fold and 1360-fold higher affinity of AR246686 for the 5-HT2A receptor vs. 5-HT2C and 5-HT2B receptors, respectively. AR246686 inhibited 5-HT-induced amplification of ADP-stimulated human platelet aggregation (IC50=21 nM). Similar potency was observed for inhibition of 5-HT stimulated DNA synthesis in rat aortic smooth muscle cells (IC(50)=10 nM) and 5-HT-mediated contraction in rat aortic rings. Effects of AR246686 on arterial thrombosis and bleeding time were studied in a rat model of femoral artery occlusion. Oral dosing of AR246686 to rats resulted in prolongation of time to occlusion at 1 mg/kg, whereas increased bleeding time was observed at a dose of 20 mg/kg. In contrast, both bleeding time and time to occlusion were increased at the same dose (10 mg/kg) of clopidogrel. These results demonstrate that AR246686 is a high affinity 5-HT2A receptor antagonist with potent activity on platelets and vascular smooth muscle. Further, oral administration results in anti-thrombotic effects at doses that are free of significant effects on traumatic bleeding time.
European Journal of Pharmacology 06/2008; 586(1-3):234-43. · 2.52 Impact Factor
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Zhi-Liang Chu,
Chris Carroll,
Jean Alfonso,
Veronica Gutierrez,
Hongmei He,
Annette Lucman,
Melinda Pedraza,
Helen Mondala,
Hui Gao,
Didier Bagnol,
Ruoping Chen,
Robert M Jones, Dominic P Behan,
James Leonard
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ABSTRACT: We recently showed that activation of G protein-coupled receptor 119 (GPR119) (also termed glucose dependent insulinotropic receptor) improves glucose homeostasis via direct cAMP-mediated enhancement of glucose-dependent insulin release in pancreatic beta-cells. Here we show that GPR119 also stimulates incretin hormone release and thus may regulate glucose homeostasis by this additional mechanism. GPR119 mRNA was found to be expressed at significant levels in intestinal subregions that produce glucose-dependent insulinotropic peptide and glucagon-like peptide (GLP)-1. Furthermore, in situ hybridization studies indicated that most GLP-1-producing cells coexpress GPR119 mRNA. In GLUTag cells, a well-established model of intestinal L-cell function, the potent GPR119 agonist AR231453 stimulated cAMP accumulation and GLP-1 release. When administered in mice, AR231453 increased active GLP-1 levels within 2 min after oral glucose delivery and substantially enhanced total glucose-dependent insulinotropic peptide levels. Blockade of GLP-1 receptor signaling with exendin(9-39) reduced the ability of AR231453 to improve glucose tolerance in mice. Conversely, combined administration of AR231453 and the DPP-4 inhibitor sitagliptin to wild-type mice significantly amplified both plasma GLP-1 levels and oral glucose tolerance, relative to either agent alone. In mice lacking GPR119, no such enhancement was seen. Thus, GPR119 regulates glucose tolerance by acting on intestinal endocrine cells as well as pancreatic beta-cells. These data also suggest that combined stimulation of incretin hormone release and protection against incretin hormone degradation may be an effective antidiabetic strategy.
Endocrinology 06/2008; 149(5):2038-47. · 4.46 Impact Factor
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Zhi-Liang Chu,
Robert M Jones,
Hongmei He,
Chris Carroll,
Veronica Gutierrez,
Annette Lucman,
Molly Moloney,
Hui Gao,
Helen Mondala,
Didier Bagnol,
David Unett,
Yin Liang,
Keith Demarest,
Graeme Semple, Dominic P Behan,
James Leonard
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ABSTRACT: Pancreatic beta-cell dysfunction is a hallmark event in the pathogenesis of type 2 diabetes. Injectable peptide agonists of the glucagon-like peptide 1 (GLP-1) receptor have shown significant promise as antidiabetic agents by virtue of their ability to amplify glucose-dependent insulin release and preserve pancreatic beta-cell mass. These effects are mediated via stimulation of cAMP through beta-cell GLP-1 receptors. We report that the Galpha(s)-coupled receptor GPR119 is largely restricted to insulin-producing beta-cells of pancreatic islets. Additionally, we show here that GPR119 functions as a glucose-dependent insulinotropic receptor. Unlike receptors for GLP-1 and other peptides that mediate enhanced glucose-dependent insulin release, GPR119 was suitable for the development of potent, orally active, small-molecule agonists. The GPR119-specific agonist AR231453 significantly increased cAMP accumulation and insulin release in both HIT-T15 cells and rodent islets. In both cases, loss of GPR119 rendered AR231453 inactive. AR231453 also enhanced glucose-dependent insulin release in vivo and improved oral glucose tolerance in wild-type mice but not in GPR119-deficient mice. Diabetic KK/A(y) mice were also highly responsive to AR231453. Orally active GPR119 agonists may offer significant promise as novel antihyperglycemic agents acting in a glucose-dependent fashion.
Endocrinology 07/2007; 148(6):2601-9. · 4.46 Impact Factor
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Jeremy G Richman,
Martha Kanemitsu-Parks,
Ibragim Gaidarov,
Jill S Cameron,
Peter Griffin,
Hong Zheng,
Nuvia C Guerra,
Linda Cham,
Dominique Maciejewski-Lenoir, Dominic P Behan,
Doug Boatman,
Ruoping Chen,
Philip Skinner,
Pricilla Ornelas,
M Gerard Waters,
Samuel D Wright,
Graeme Semple,
Daniel T Connolly
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ABSTRACT: Nicotinic acid remains the most effective therapeutic agent for the treatment and prevention of atherosclerosis resulting from low high density lipoprotein cholesterol. The therapeutic actions of nicotinic acid are mediated by GPR109A, a Gi protein-coupled receptor, expressed primarily on adipocytes, Langerhans cells, and macrophage. Unfortunately, a severe, cutaneous flushing side effect limits its use and patient compliance. The mechanism of high density lipoprotein elevation is not clearly established but assumed to be influenced by an inhibition of lipolysis in the adipose. The flushing side effect appears to be mediated by the release of prostaglandin D2 from Langerhans cells in the skin. We hypothesized that the signal transduction pathways mediating the anti-lipolytic and prostaglandin D2/flushing pathways are distinct and that agonists may be identified that are capable of selectively eliciting the therapeutic, anti-lipolytic pathway while avoiding the activation of the parallel flush-inducing pathway. We have identified a number of GPR109A pyrazole agonists that are capable of fully inhibiting lipolysis in vitro and in vivo and not only fail to elicit a flushing response but can antagonize the ability of nicotinic acid to elicit a flush response in vivo. In contrast to flushing agonists, exposure of cells expressing GPR109A to the non-flushing agonists fails to induce internalization of the receptor or to activate ERK 1/2 mitogen-activated protein kinase phosphorylation.
Journal of Biological Chemistry 07/2007; 282(25):18028-36. · 4.77 Impact Factor
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ABSTRACT: Nicotinic acid, used for atherosclerosis treatment, has an adverse effect of skin flushing. The flushing mechanism, thought to be caused by the release of prostaglandin D(2) (PGD(2)), is not well understood. We aimed to identify which cells mediate the flushing effect. Nicotinic acid receptor (GPR109A) gene expression was assessed in various tissues and cell lines. Cells expressing GPR109A mRNA were further assayed for PGD(2) release in response to nicotinic acid. Of all samples, only skin was able to release PGD(2) upon stimulation with nicotinic acid. The responsive cells were localized to the epidermis, and immunocytochemical studies revealed the presence of GPR109A on epidermal Langerhans cells. CD34+ cells isolated from human blood and differentiated into Langerhans cells (hLC-L) also showed GPR109A expression. IFNgamma treatment increased both mRNA and plasma membrane expression of GPR109A. IFNgamma-stimulated hLC-Ls released PGD(2) in response to nicotinic acid in a dose-dependant manner (effector concentration for half-maximum response=1.2 mM+/-0.7). Acifran, a structurally distinct GPR109A ligand, also increased PGD(2) release, whereas isonicotinic acid, a nicotinic acid analog with low affinity for GPR109A, had no effect. These results suggest that nicotinic acid mediates its flushing side effect by interacting with GPR109A on skin Langerhans cells, resulting in release of PGD(2).
Journal of Investigative Dermatology 01/2007; 126(12):2637-46. · 6.31 Impact Factor
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ABSTRACT: Prepulse inhibition (PPI) is a cross-species measure of sensorimotor gating. PPI deficits have been associated with a number of neuropsychiatric disorders, including schizophrenia. Differential PPI has been demonstrated also across various inbred mouse strains; however, the molecular mechanisms underlying these differences in sensorimotor gating remain unclear. Here, we sought to identify gene expression in the medial prefrontal cortex (mPFC) of mice associated with PPI using a laser microdissection and microarray analysis-based approach. C57BL/6 mouse substrains were used for the study as they have dramatically different PPI. Transcriptional analysis of closely related substrains was predicted to reduce the detection of genetic variation incidental to the phenotype. Microarray analysis comparing the mPFC of C57BL/6J to C57BL/6NHsd mice revealed neurotransmission- and cellular stress-related transcriptional responses associated with lower PPI. Down-regulation of metabotropic glutamate receptor 5, phospholipase C, and inositol monophosphatase 1 gene expression suggest altered phosphoinositide signaling, while decreased expression of a gamma-amino-butyric acid (GABA)A receptor subunit implies changes in GABAergic signaling. Genes involved in neuronal excitation and protection were also differentially expressed, including up-regulation of five immediate early genes and anti-apoptotic/survival factors as Bcl2-associated athanogene 3 and brain-derived neurotrophic factor. These data support previous findings of genetic influences on PPI, and provide novel insights into the molecular mechanisms regulating sensorimotor gating.
Molecular Brain Research 10/2005; 139(1):153-62. · 2.00 Impact Factor
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ABSTRACT: Nearly half of the identified non-olfactory G protein-coupled receptors (GPCRs) have no identified cognate ligand. These 'orphan' receptors are likely to have important physiological roles that could potentially be exploited therapeutically. However, by definition, such receptors are not immediately open for pharmacological investigation of their function. Here we summarize several strategic approaches to facilitate the discovery of orphan GPCR biology.
Current opinion in molecular therapeutics 01/2005; 6(6):640-56. · 3.68 Impact Factor
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ABSTRACT: The rat muscarinic acetylcholine receptor subtype 3 was modified by swapping the third intracellular loop with the corresponding region of a constitutively active mutant human beta2-adrenergic receptor and attaching Renilla reniformis luciferase to its C terminus. The chimeric fusion receptor displayed constitutive Gq- and Gs-coupled activity as demonstrated in nuclear factor of activated T cell and cAMP response element reporter gene assays. The chimeric receptor displayed a pharmacological binding profile comparable with that of the wild-type receptor for agonists, antagonists, and inverse agonists but showed a large decrease in expression in both human embryonic kidney 293 and COS-7 cells. Long-term treatment of cells expressing the chimeric receptor with agonists, antagonists, and inverse agonists resulted in a concentration-dependent up-regulation in the steady-state levels that was not observed for the wild-type receptor. The EC50 of neutral antagonists and inverse agonists was significantly correlated to their binding affinities at the wild-type receptor, whereas agonists demonstrated greater EC50 values for the chimeric receptor. To validate the approach as a means of discovering novel receptor modulators, a cell-based, high-throughput screening assay was developed and used to screen a small molecule compound collection against the chimeric fusion receptor. Several novel hits were identified and confirmed by ligand binding assay and functional assays using the wild-type rat muscarinic acetylcholine receptor subtype 3.
Molecular Pharmacology 01/2004; 64(6):1474-84. · 4.88 Impact Factor
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ABSTRACT: The G-protein-coupled receptor (GPCR) family mediates a host of cell-cell communications upon activation by diverse ligands. Numerous GPCRs have been shown to display anatomically selective patterns of gene expression, however, our understanding of the complexity of GPCR signaling within human tissues remains unclear. In an effort to characterize global patterns of GPCR signaling in the human body, microarray analysis was performed on a large panel of tissues to monitor the gene expression levels of the receptors as well as related signaling and regulatory molecules. Analysis of the data revealed complex signaling networks in many tissue types, with tissue-specific patterns of gene expression observed for the majority of the receptors and a number of components and regulators of GPCR signaling.
FEBS Letters 09/2003; 550(1-3):11-7. · 3.54 Impact Factor
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ABSTRACT: The complete sequencing of the human genome has afforded researchers the opportunity to identify novel G-protein-coupled receptors (GPCRs) that are expressed in human tissues. The successful identification of hundreds of GPCRs represents the single greatest opportunity for novel drug development today. However, the lack of identified ligands for these GPCRs has limited their utility for traditional drug discovery approaches that focus on ligand-based assay methods to discover and pharmacologically characterize drug candidates. Here, we review the use of constitutively activated GPCRs in the discovery pathway, both as a means to overcome the limitations of traditional drug discovery at novel GPCRs and as a tool to investigate the functionality of these receptors.
dressNature Reviews Drug Discovery 09/2002; 1(8):599-608. · 29.01 Impact Factor
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ABSTRACT: G protein-coupled receptors (GPCRs) represent a major class of signal transduction proteins that modulate various biological functions. GPCRs are one of the most common targets for drug development-currently, 39 of the top 100 marketed drugs in use act directly or indirectly through activation or blockade of GPCR-mediated receptors. Nearly 160 GPCRs have been identified based on their gene sequence and their ability to interact with known endogenous ligands. However, an estimated 500-800 additional GPCRs have been classified as "orphan" receptors (oGPCRs) because their endogenous ligands have not yet been identified. Given that known GPCRs have proven to be such clinically useful drug targets, these oGPCRs represent a rich group of receptor targets for the development of novel and improved medicines. To develop ligands for these potential drug targets requires the ability to identify groups or pools of GPCRs that are likely to be involved in a specific disease process (obesity, schizophrenia, depression, etc.) and to dissect out the pharmacological and signal transduction differences between these GPCR subtypes. It also requires the development of assays to detect ligands of GPCRs even when the endogenous ligands are unidentified. This paper will review novel strategies to identify clinically interesting oGPCRs and to screen for small molecules that act as ligands without prior knowledge of endogenous ligands. This involves the use of constitutively activated GPCRs, a technology that provides a unique opportunity to identify several classes of pharmacological agents, including agonists, inverse agonists and allosteric modulators.
Current Drug Targets - CNS & Neurological Disorders 03/2002; 1(1):105-21.
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Zhi-Liang Chu,
Chris Carroll,
Jean Alfonso,
Veronica Gutierrez,
Hongmei He,
Annette Lucman,
Melinda Pedraza,
Helen Mondala,
Hui Gao,
Didier Bagnol,
Ruoping Chen,
Robert M Jones, Dominic P Behan,
James Leonard
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