Discovery of 6,7-dihydro-5H-pyrrolo[2,3-a]pyrimidines as orally available G protein-coupled receptor 119 agonists.

GPR119 is a 7-transmembrane receptor that is expressed in the enteroendocrine cells in the intestine and in the islets of Langerhans in the pancreas. Indolines and 6,7-dihydro-5H-pyrrolo[2,3-a]pyrimidines were discovered as G protein-coupled receptor 119 (GPR119) agonists, and lead optimization efforts led to the identification of 1-methylethyl 4-({7-[2-fluoro-4-(methylsulfonyl)phenyl]-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-4-yl}oxy)-1-piperidinecarboxylate (GSK1104252A) (3), a potent and selective GPR119 agonist. Compound 3 showed excellent pharmacokinetic properties and sufficient selectivity with in vivo studies supporting a role for GPR119 in glucose homeostasis in the rodent. Thus, 3 appeared to modulate the enteroinsular axis, improve glycemic control, and strengthen previous suggestions that GPR119 agonists may have utility in the treatment of type 2 diabetes.

Ligand modulation of the Epstein-Barr virus-induced seven-transmembrane receptor EBI2: identification of a potent and efficacious inverse agonist.

The Epstein-Barr virus-induced receptor 2 (EBI2) is a constitutively active seven-transmembrane receptor, which was recently shown to orchestrate the positioning of B cells in the follicle. To date, no ligands, endogenously or synthetic, have been identified that modulate EBI2 activity. Here we describe an inverse agonist, GSK682753A, which selectively inhibited the constitutive activity of EBI2 with high potency and efficacy. In cAMP-response element-binding protein-based reporter and guanosine 5'-3-O-(thio)triphosphate (GTPγS) binding assays, the potency of this compound was 2.6-53.6 nm, and its inhibitory efficacy was 75%. In addition, we show that EBI2 constitutively activated extracellular signal-regulated kinase (ERK) in a pertussis toxin-insensitive manner. Intriguingly, GSK682753A inhibited ERK phosphorylation, GTPγS binding, and cAMP-response element-binding protein activation with similar potency. Overexpression of EBI2 profoundly potentiated antibody-stimulated ex vivo proliferation of murine B cells compared with WT cells, whereas this was equivalently reduced for EBI2-deficient B cells. Inhibition of EBI2 constitutive activity suppressed the proliferation in all cases. Importantly, the suppression was of much higher potency (32-fold) in WT or EBI2-overexpressing B cells compared with EBI2-deficient counterparts. Finally, we screened GSK682753A against an EBI2 mutant library to determine putative molecular binding determinants in EBI2. We identified Phe(111) at position III:08/3.32 as being crucial for GSK682753A inverse agonism because Ala substitution resulted in a >500-fold decrease in IC(50). In conclusion, we present the first ligand targeting EBI2. In turn, this molecule provides a useful tool for further characterization of EBI2 as well as serving as a potent lead compound.

2,5-Disubstituted pyridines as potent GPR119 agonists.

A series of 2-piperazinyl-5-alkoxypyridines were synthesized and screened against human GPR119 receptor. Through SAR analysis, compounds containing 2-alkylsulfonylpiperazinyl-5-alkoxypyridines were discovered and found to be potent agonists of the human GPR119 receptor.

Functional screening in the melanophore bioassay.

The melanophore bioassay is a robust, sensitive, and versatile procedure for screening G protein-coupled receptors in a variety of formats. Because melanophores contain a wide variety of G proteins, they can be employed as a sensitive, real-time response system for studying transfected receptors and for defining equilibria for drug effects. This assay can be run in 96-well microtiter plates or in open-lawn 1536 format, and can yield conventional agonist-antagonist as well as constitutive assays.

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.