Lorcaserin, a novel selective human 5-hydroxytryptamine2C agonist: in vitro and in vivo pharmacological characterization.

5-Hydroxytryptamine (5-HT)(2C) receptor agonists hold promise for the treatment of obesity. In this study, we describe the in vitro and in vivo characteristics of lorcaserin [(1R)-8-chloro-2,3,4,5-tetrahydro-1-methyl-1H-3 benzazepine], a selective, high affinity 5-HT(2C) full agonist. Lorcaserin bound to human and rat 5-HT(2C) receptors with high affinity (K(i) = 15 +/- 1 nM, 29 +/- 7 nM, respectively), and it was a full agonist for the human 5-HT(2C) receptor in a functional inositol phosphate accumulation assay, with 18- and 104-fold selectivity over 5-HT(2A) and 5-HT(2B) receptors, respectively. Lorcaserin was also highly selective for human 5-HT(2C) over other human 5-HT receptors (5-HT(1A), 5-HT(3), 5-HT(4C), 5-HT5(5A), 5-HT(6), and 5-HT(7)), in addition to a panel of 67 other G protein-coupled receptors and ion channels. Lorcaserin did not compete for binding of ligands to serotonin, dopamine, and norepinephrine transporters, and it did not alter their function in vitro. Behavioral observations indicated that unlike the 5-HT(2A) agonist (+/-)-1-(2,5-dimethoxy-4-phenyl)-2-aminopropane, lorcaserin did not induce behavioral changes indicative of functional 5-HT(2A) agonist activity. Acutely, lorcaserin reduced food intake in rats, an effect that was reversed by pretreatment with the 5-HT(2C)-selective antagonist 6-chloro-5-methyl-1-[6-(2-methylpyridin-3-yloxy)pyridin-3-yl-carbamoyl]indoline (SB242,084) but not the 5-HT(2A) antagonist (R)-(+)-alpha-(2,3-dimethoxyphenyl)-1-[2-(4-fluorophenylethyl)]-4-piperidine-methanol (MDL 100,907), demonstrating mediation by the 5-HT(2C) receptor. Chronic daily treatment with lorcaserin to rats maintained on a high fat diet produced dose-dependent reductions in food intake and body weight gain that were maintained during the 4-week study. Upon discontinuation, body weight returned to control levels. These data demonstrate lorcaserin to be a potent, selective, and efficacious agonist of the 5-HT(2C) receptor, with potential for the treatment of obesity.

Neurotransmission- and cellular stress-related gene expression associated with prepulse inhibition in mice.

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.

Ligand specific up-regulation of a Renilla reniformis luciferase-tagged, structurally unstable muscarinic M3 chimeric G protein-coupled receptor.

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.

The G-protein-coupled receptor GCR1 regulates DNA synthesis through activation of phosphatidylinositol-specific phospholipase C.

Different lines of evidence suggest that specific events during the cell cycle may be mediated by a heterotrimeric G-protein activated by a cognate G-protein coupled receptor. However, coupling between the only known Galpha-subunit of the heterotrimeric G-protein (GPA1) and the only putative G-protein coupled receptor (GCR1) of plants has never been shown. Using a variety of approaches, we show here that GCR1-enhanced thymidine incorporation into DNA depends on an increase in phosphatidylinositol-specific phospholipase C activity and an elevation of inositol 1,4,5-trisphosphate levels in the cells. Tobacco (Nicotiana tabacum) cells that overexpress either Arabidopsis GCR1 or GPA1 display this phenomenon. We suggest on the basis of these results that GCR1-controlled events during the cell cycle involve phosphatidylinositol-specific phospholipase C as an effector of GCR1 and inositol 1,4,5-trisphosphate as a second messenger, and that GCR1 and GPA1 are both involved in this particular signaling pathway.

Global analysis of G-protein-coupled receptor signaling in human tissues.

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.

The use of constitutively active GPCRs in drug discovery and functional genomics.

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.

Generation and analysis of constitutively active and physically destabilized mutants of the human beta(1)-adrenoceptor.

Constitutive activity of wild-type and mutant forms of human beta(1)- and beta(2)-adrenoceptors was measured by guanosine 5'-O-(3-[(35)S]thio)triphosphate ([(35)S]GTPgammaS) binding assays using fusion proteins between these receptors and G(s)alpha. Constitutive activity of the beta(1)-adrenoceptor is enhanced by mutation of Leu(322). The ability of ligands to suppress receptor instability and produce up-regulation is often associated with constitutively active mutants. Leu(322)Lysbeta(1)-adrenoceptor, but not wild type, was up-regulated by exposure to the beta(1)-adrenoceptor selective blocker betaxolol. More extensive sequence alterations of the beta(1)-adrenoceptor were generated to mimic the initially described constitutively active mutant (CAM) of the beta(2)-adrenoceptor that is up-regulated strongly by betaxolol. Substitution of amino acids 316 to 324 of the beta(1)-adrenoceptor with the equivalent alpha(1b)-adrenoceptor sequence did not result in up-regulation by betaxolol. However, these forms of both beta(1)- and beta(2)-adrenoceptors displayed substantial and equivalent constitutive activity. The addition of the Leu(322)Lys mutation into the alpha(1b)-adrenoceptor substituted beta(1)-adrenoceptor to produce the CAMKbeta(1)-adrenoceptor allowed substantially greater levels of up-regulation by betaxolol without enhancement of constitutive [(35)S]GTPgammaS binding. Arg(156)Alabeta(1)-adrenoceptor was up-regulated strongly by betaxolol but displayed lower constitutive activity than did other mutants. Binding of [(35)S]GTPgammaS binding to all the fusion proteins was increased substantially by isoprenaline. Despite the ability of betaxolol to cause up-regulation of many mutants, only for the CAMbeta(2)-adrenoceptor-G(s)alpha and CAMKbeta(1)-adrenoceptor-G(s)alpha fusion proteins was the basal binding of [(35)S]GTPgammaS decreased by betaxolol. Clear resolution between receptor constitutive activity and ligand suppression of receptor instability can be obtained for mutant beta-adrenoceptors, and potential inverse agonists do not function equally at phenotypically apparently equivalent CAM receptors.

GCR1, the putative Arabidopsis G protein-coupled receptor gene is cell cycle-regulated, and its overexpression abolishes seed dormancy and shortens time to flowering.

Although signaling through heterotrimeric G proteins has been extensively studied in eukaryotes, there is little information about this important signaling pathway in plants. We observed that expression of GCR1, the gene encoding the only known (but still putative) G protein-coupled receptor of Arabidopsis thaliana, is modulated during the cell cycle and during plant development. Overexpression of GCR1 in tobacco (Nicotiana tabacum) BY-2 cells caused an increase in thymidine incorporation and in the mitotic index of aphidicolin synchronized cells. Overexpression of GCR1 in Arabidopsis caused two remarkable phenotypes: seed dormancy was abolished and time to flowering was reduced. Molecular markers of these two developmental processes (phosphatase PP2A and MYB65 in germination; LFY during flowering) were up-regulated in GCR1 overexpressors. These data are consistent with the hypothesis that GCR1 may be a regulator of the cell cycle and that this regulation underlies the developmental changes observed in the GCR1 transformants.

Constitutively activated G protein-coupled receptors: a novel approach to CNS drug discovery.

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.