Human GPR119 Research Articles

YH18421, a novel GPR119 agonist exerts sustained glucose lowering and weight loss in diabetic mouse model.

G-protein-coupled receptor 119 (GPR119) represents a promising target for the treatment of type 2 diabetes as it can increase both GLP-1 secretion from intestinal L cells and glucose-stimulated insulin secretion (GSIS) from pancreatic β cells. Due to this dual mechanism of action, the development of small molecule GPR119 agonists has received much interest for the treatment of type 2 diabetes. Here, we identified a novel small-molecule GPR119 agonist, YH18421 and evaluated its therapeutic potential. YH18421 specifically activated human GPR119 with high potency and potentiated GLP-1 secretion and GSIS in vitro cell based systems. In normal mice, single oral administration of YH18421 improved glucose tolerance. Combined treatment of YH18421 and the DPP-4 inhibitor augmented both plasma active GLP-1 levels and glycemic control. In diet induced obese (DIO) mice model, glucose lowering effect of YH18421 was maintained after 4weeks of repeat dosing and YH18421 acted additively with DPP-IV inhibitor. We also observed that YH18421 inhibited weight gain during 4weeks of administration in DIO mice. These data demonstrate that YH18421 is capable of delivering sustained glucose control and preventing weight gain and combination with the DPP-IV inhibitor maybe an effective strategy for the treatment of type 2 diabetes.

Discovery of Pyrrolidine-Containing GPR40 Agonists: Stereochemistry Effects a Change in Binding Mode

A novel series of pyrrolidine-containing GPR40 agonists is described as a potential treatment for type 2 diabetes. The initial pyrrolidine hit was modified by moving the position of the carboxylic acid, a key pharmacophore for GPR40. Addition of a 4-cis-CF3 to the pyrrolidine improves the human GPR40 binding Ki and agonist efficacy. After further optimization, the discovery of a minor enantiomeric impurity with agonist activity led to the finding that enantiomers (R,R)-68 and (S,S)-68 have differential effects on the radioligand used for the binding assay, with (R,R)-68 potentiating the radioligand and (S,S)-68 displacing the radioligand. Compound (R,R)-68 activates both Gq-coupled intracellular Ca2+ flux and Gs-coupled cAMP accumulation. This signaling bias results in a dual mechanism of action for compound (R,R)-68, demonstrating glucose-dependent insulin and GLP-1 secretion in vitro. In vivo, compound (R,R)-68 significantly lowers plasma glucose levels in mice during an oral glucose challenge, encouraging further development of the series.

Angelica dahurica Extracts Improve Glucose Tolerance through the Activation of GPR119.

G protein-coupled receptor (GPR) 119 is expressed in pancreatic β-cells and intestinal L cells, and is involved in glucose-stimulated insulin secretion and glucagon-like peptide-1 (GLP-1) release, respectively. Therefore, the development of GPR119 agonists is a potential treatment for type 2 diabetes. We screened 1500 natural plant extracts for GPR119 agonistic actions and investigated the most promising extract, that from Angelica dahurica (AD), for hypoglycemic actions in vitro and in vivo. Human GPR119 activation was measured in GeneBLAzer T-Rex GPR119-CRE-bla CHO-K1 cells; intracellular cAMP levels and insulin secretion were measured in INS-1 cells; and GLP-1 release was measured in GLUTag cells. Glucose tolerance tests and serum plasma insulin levels were measured in normal C57BL6 mice and diabetic db/db mice. AD extract-treated cells showed significant increases in GPR119 activation, intracellular cAMP levels, GLP-1 levels and glucose-stimulated insulin secretion as compared with controls. In normal mice, a single treatment with AD extract improved glucose tolerance and increased insulin secretion. Treatment with multiple doses of AD extract or n-hexane fraction improved glucose tolerance in diabetic db/db mice. Imperatorin, phellopterin and isoimperatorin were identified in the active fraction of AD extract. Among these, phellopterin activated GPR119 and increased active GLP-1 and insulin secretion in vitro and enhanced glucose tolerance in normal and db/db mice. We suggest that phellopterin might have a therapeutic potential for the treatment of type 2 diabetes.

Editorial: Obesity and Diabetes: Energy Regulation by Free Fatty Acid Receptors.

Food intake regulates energy balance, and its dysregulation leads to metabolic disorders, such as obesity and type 2 diabetes (T2D). During feeding, free fatty acids (FFAs) are not only essential nutrients but also act as signaling molecules in various cellular processes. Recently, several G protein-coupled receptors (GPCRs) that act as FFA receptors (FFARs) have been identified; GPR41/FFAR3 and GPR43/FFAR2 are activated by short-chain FFAs. GPR40/FFAR1, GPR119, and GPR120/FFAR4 are activated by medium- and long-chain FFAs. FFARs are widely expressed and contribute to many important physiological functions in order to maintain energy homeostasis. Hence, these FFARs have come to be regarded as new drug targets for metabolic disorder such as obesity and T2D. All articles in this topic highlight the interconnection between FFARs and the regulation of energy homeostasis. They also focused on essential role of FFARs in the pathogenesis of metabolic syndromes, such as obesity, insulin resistance, and T2D and discussed the potential of FFARs as drug target. These articles give valuable insight into unanswered questions in relation to this topic. First, recent studies demonstrate that short-chain free fatty acids (SCFAs) produced by microbiota fermentation act as signaling molecules through SCFAs receptors (SCFARs), such as GPR41 and GPR43 and influence the host’s metabolism (1–3). Hence, the gut microbiota can influence and play important roles in host physiology and pathology via these receptors. GPR41, which is expressed in adipose tissue, gut, and the peripheral nervous system, contributes SCFAs-dependent systemic energy regulation (1). In particular, GPR41 regulates host energy balance by modulating sympathetic activity and intestinal gluconeogenesis. GPR43, which is expressed in the adipose tissue, intestines, and immune tissues, also contributes the regulation of energy homeostasis depends on SCFAs produced by gut microbiota (2). GPR43 deficiency induced obesity in mice, while mice that overexpress GPR43 only in adipose tissue were lean under normal conditions; both of these strains did not exhibit either phenotype under germ-free conditions or after antibiotic treatment. Furthermore, SCFA-mediated GPR43 activation suppressed adipose insulin signaling, leading to inhibition of fat accumulation in the adipose tissues, while unincorporated lipids and glucose were primarily utilized in muscles. The GPR43-insulin pathway has a key role in adipose tissue acting as an important physiological mechanism through which metabolic fuels regulate body energy balance (2, 3). These studies clearly showed the importance of SCFAs produced by microbiota and their receptors (1–3). Based on the importance and dynamic roles of microbiota in host physiology, Pluznick pointed out a complex interplay between the genetics of the microbiota and that of the host organism (4). Researchers should consider the contribution of these microorganisms and their metabolites because there are many examples of phenotypes that were not easily to replicated by other groups may be due to the influence of variations of gut microbiota (4). Second, medium-chain fatty acids (MCFAs) and long-chain fatty acids (LCFAs) are not only essential nutrient, but also act as ligands of GPR40/FFAR1 and GPR120/FFAR4 and regulate systemic energy homeostasis (5–8). GPR40 is highly expressed in pancreatic β cells and intestine. GPR40 augment glucose-stimulated insulin secretion after acute exposure to LCFAs by stimulation of not only insulin secretion from pancreatic β cells directly, but also incretin hormones, such as glucagon like peptide-1 (GLP-1), gastric inhibitory polypeptide (GIP) and cholecystokinin (CCK) from intestine (5, 8). The activation of GPR120 by ω-3 FFAs mediated anti-inflammatory effect of ω-3 FFAs as described in the articles by Oh et al. (7). This effect is associated with the suppression of Toll-like receptor via β-arrestin2 signaling pathway and transforming growth factor-β activated kinase 1 (TAK1) involved in TNF-α inflammation signaling pathway. Furthermore, both a gene deficiency in mice and non-synonymous functional-loss mutation of human GPR120 are associated with obesity, which was accompanied with decreased differentiation and lipogenesis (6). Hence, selective synthetic ligands for FFARs have consequently been developed as potential treatments for metabolic syndrome (9). Particularly, clinical studies show that TAK875/Fasiglifam, an agonist of GPR40 improved glucose metabolism with a reduced risk of hypoglycemia, although this ligand was dropped from clinical trials due to potential liver toxicity. Activation of each of GPR41, 43, and 120 has also been suggested to have potential benefits for metabolic function (9). Overall, all the review articles provided a comprehensive overview of the energy regulation by FFARs and a new prospect for treatment of metabolic disorder such as obesity and type 2 diabetes.

ChemInform Abstract: Rhodium/Chiral Diene Complexes in the Catalytic Asymmetric Arylation of β‐Pyrazol‐1‐yl Acrylates.

AbstractThe reaction proceeds with high regio‐ and enantioselectivity and can be applied in the formal synthesis of the bioactive compound (XIII), an agonist toward the human GPR40 G‐protein‐coupled receptor.

Rhodium/chiral diene complexes in the catalytic asymmetric arylation of β-pyrazol-1-yl acrylates.

The asymmetric conjugate addition of arylboronic acids to substituted and unsubstituted β-pyrazol-1-yl (E)-tert-butyl acrylates 4 catalyzed by 5 mol % of the Rh(I)/diene 2a catalyst provided the corresponding addition products in 44-98% yield and 91->99.5% ee. The method was applied to the formal synthesis of (3S)-3-aryl-3-(pyrazol-1-yl)propanoic acid 1b with agonistic activity toward the human GPR40 G-protein coupled receptor.

Cloning and characterization of spliced variants of the porcine G protein coupled receptor 120.

The polyunsaturated fatty acids (PUFAs) receptor GPR120 exerts a significant impact on systemic nutrient homeostasis in human and rodents. However, the porcine GPR120 (pGPR120) has not been well characterized. In the current study, we found that pGPR120 had 3 spliced variants. Transcript 1 encoded 362-amino acids (aa) wild type pGPR120-WT, which shared 88% homology with human short form GPR120. Transcript 1 was the mainly expressed transcript of pGPR120. It was expressed predominantly in ileum, jejunum, duodenum, spleen, and adipose. Transcript 3 (coding 320-aa isoform) was detected in spleen, while the transcript 2 (coding 310-aa isoform) was only slightly expressed in spleen. A selective agonist for human GPR120 (TUG-891) and PUFAs activated SRE-luc and NFAT-luc reporter in HEK293T cells transfected with construct for pGPR120-WT but not pGPR120-V2. However, 320-aa isoform was not a dominant negative isoform. The extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation levels in cells transfected with construct for pGPR120-WT were well activated by PUFAs, especially n-3 PUFA. These results showed that although pGPR120 had 3 transcripts, transcript 1 which encoded pGPR120-WT was the mainly expressed transcript. TUG-891 and PUFAs, especially n-3 PUFA, well activated pGPR120-WT. The current study contributed to dissecting the molecular regulation mechanisms of n-3 PUFA in pigs.

A Gut Microbial Metabolite of Linoleic Acid, 10-Hydroxy-cis-12-octadecenoic Acid, Ameliorates Intestinal Epithelial Barrier Impairment Partially via GPR40-MEK-ERK Pathway

Gut microbial metabolites of polyunsaturated fatty acids have attracted much attention because of their various physiological properties. Dysfunction of tight junction (TJ) in the intestine contributes to the pathogenesis of many disorders such as inflammatory bowel disease. We evaluated the effects of five novel gut microbial metabolites on tumor necrosis factor (TNF)-α-induced barrier impairment in Caco-2 cells and dextran sulfate sodium-induced colitis in mice. 10-Hydroxy-cis-12-octadecenoic acid (HYA), a gut microbial metabolite of linoleic acid, suppressed TNF-α and dextran sulfate sodium-induced changes in the expression of TJ-related molecules, occludin, zonula occludens-1, and myosin light chain kinase. HYA also suppressed the expression of TNF receptor 2 (TNFR2) mRNA and protein expression in Caco-2 cells and colonic tissue. In addition, HYA suppressed the protein expression of TNFR2 in murine intestinal epithelial cells. Furthermore, HYA significantly up-regulated G protein-coupled receptor (GPR) 40 expression in Caco-2 cells. It also induced [Ca(2+)]i responses in HEK293 cells expressing human GPR40 with higher sensitivity than linoleic acid, its metabolic precursor. The barrier-recovering effects of HYA were abrogated by a GPR40 antagonist and MEK inhibitor in Caco-2 cells. Conversely, 10-hydroxyoctadacanoic acid, which is a gut microbial metabolite of oleic acid and lacks a carbon-carbon double bond at Δ12 position, did not show these TJ-restoring activities and down-regulated GPR40 expression. Therefore, HYA modulates TNFR2 expression, at least partially, via the GPR40-MEK-ERK pathway and may be useful in the treatment of TJ-related disorders such as inflammatory bowel disease.

3D-QSAR studies of 8-substituted chromen-4-one-2-carboxylic acid derivatives as potent agonists for the orphan G protein-coupled receptor 35

GPR35, an orphan G protein-coupled receptor, has attracted much attention as a novel therapeutic target for the treatment of diabetes, hypertension, etc. Recently, 8-substituted chromen-4-one-2-carboxylic acid derivatives were identified as potent and selective agonists for human GPR35. In the present study, the three-dimensional quantitative structure–activity relationship (3D-QSAR) models were developed for a series of 8-substituted chromen-4-one-2-carboxylic acid derivatives using comparative molecular field analysis (CoMFA), comparative molecular similarity indices analysis (CoMSIA), and Topomer CoMFA techniques implemented in the SYBYL software packages. The statistically significant models were obtained with 30 compounds in training set by ligand-based atom-by-atom matching alignment, which were further validated by a test set of eight compounds. The CoMFA model resulted in cross-validated coefficient (q 2) value of 0.610 using 4 components, non-cross-validated coefficient (r 2) value of 0.918 with estimated F value of 69.917, and standard error of estimate (SEE) of 0.352. While the CoMSIA model combined with steric, electrostatic and hydrophobic fields were finally selected (q 2 = 0.646, r 2 = 0.800, F = 53.852, SEE = 0.489, N = 2). For the Topomer CoMFA model, the better statistics were obtained based on fragment units (q 2 = 0.746, r 2 = 0.979, F = 146.294, SEE = 0.175, N = 7). Furthermore, the contour maps obtained from 3D-QSAR studies were appraised for activity trends for the compounds analyzed. The results indicate that steric, electrostatic, and hydrophobic substituents play a significant role in the agonist activity. The data generated from the present study will further help design novel, potent, and selective agonists for GPR35.

HD047703, a New Promising Anti-Diabetic Drug Candidate: In Vivo Preclinical Studies.

G-protein coupled receptor 119 (GPR119) has emerged as a novel target for the treatment of type 2 diabetes mellitus. GPR119 is involved in glucose-stimulated insulin secretion (GSIS) from the pancreatic β-cells and intestinal cells. In this study, we identified a novel small-molecule GPR119 agonist, HD047703, which raises intracellular cAMP concentrations in pancreatic β-cells and can be expected to potentiate glucose-stimulated insulin secretion from human GPR119 receptor stably expressing cells (CHO cells). We evaluated the acute efficacy of HD047703 by the oral glucose tolerance test (OGTT) in normal C57BL/6J mice. Then, chronic administrations of HD047703 were performed to determine its efficacy in various diabetic rodent models. Single administration of HD047703 caused improved glycemic control during OGTT in a dose-dependent manner in normal mice, and the plasma GLP-1 level was also increased. With respect to chronic efficacy, we observed a decline in blood glucose levels in db/db, ob/ob and DIO mice. These results suggest that HD047703 may be a potentially promising anti-diabetic agent.

High-resolution structure of the human GPR40 receptor bound to allosteric agonist TAK-875.

Human GPR40 receptor (hGPR40), also known as free fatty-acid receptor 1 (FFAR1), is a G-protein-coupled receptor that binds long-chain free fatty acids to enhance glucose-dependent insulin secretion. Novel treatments for type-2 diabetes mellitus are therefore possible by targeting hGPR40 with partial or full agonists. TAK-875, or fasiglifam, is an orally available, potent and selective partial agonist of hGPR40 receptor, which reached phase III clinical trials for the potential treatment of type-2 diabetes mellitus. Data from clinical studies indicate that TAK-875, which is an ago-allosteric modulator of hGPR40 (ref. 3), demonstrates improved glycaemic control and low hypoglycaemic risk in diabetic patients. Here we report the crystal structure of hGPR40 receptor bound to TAK-875 at 2.3Å resolution. The co-complex structure reveals a unique binding mode of TAK-875 and suggests that entry to the non-canonical binding pocket most probably occurs via the lipid bilayer. The atomic details of the extensive charge network in the ligand binding pocket reveal additional interactions not identified in previous studies and contribute to a clear understanding of TAK-875 binding to the receptor. The hGPR40-TAK-875 structure also provides insights into the plausible binding of multiple ligands to the receptor, which has been observed in radioligand binding and Ca(2+) influx assay studies. Comparison of the transmembrane helix architecture with other G-protein-coupled receptors suggests that the crystallized TAK-875-bound hGPR40 complex is in an inactive-like state.

Synthesis and structure–activity relationship of dihydrobenzofuran derivatives as novel human GPR119 agonists

Through appropriate medicinal chemistry design tactics and computer-assisted conformational modeling, the initial lead A was evolved into a series of dihydrobenzofuran derivatives 3 as potent GPR119 agonists. This Letter describes the optimization of general structure 3, including the substituent(s) on dihydrobenzofuran, the R1 attachment on right-hand piperidine nitrogen, and the left-hand piperidine/piperazine and its attachment R2. The efforts led to the identification of compounds 13c and 24 as potent human GPR119 modulators with favorable metabolic stability, ion channel activity, and PXR profiles.

Molecular Evolution of Kiss2 Genes and Peptides in Vertebrates

The kiss1 peptide (kisspeptin), a product of the kiss1 gene, is one of the key neuropeptides regulating vertebrate reproduction. In 2009, we identified a paralogous gene of kiss1 in the brain of amphibians and named it kiss2. Currently, the presence of the kiss2 gene and the kiss2 peptide is still obscure in amniotes compared with that in other vertebrates. Therefore, we performed genome database analyses in primates and reptiles to investigate the molecular evolution of the kiss2 gene in vertebrates. Because the mature kiss2 peptide has been identified only in amphibians, we further performed immunoaffinity purification and mass spectrometry to identify the mature endogenous kiss2 peptide in the brains of salmon and turtle that possessed the kiss2 gene. Here we provide the first evidence for the presence of a kiss2-like gene in the genome database of primates including humans. Synthetic amidated human KISS2 peptide activated human GPR54 expressed in COS7 cells, but nonamidated KISS2 peptide was inactive. The endogenous amidated kiss2 peptide may not be produced in primates because of the lack of an amidation signal in the precursor polypeptide. The kiss2-like gene may be nonfunctional in crocodilians because of premature stop codons. We identified the mature amidated kiss2 peptide in turtles and fish and analyzed the localization of kiss2 peptide mRNA expression in fish. The present study suggests that the kiss2 gene may have mutated in primates and crocodilians and been lost in birds during the course of evolution. In contrast, the kiss2 gene and mature kiss2 peptide are present in turtles and fish.

The Antiallergic Mast Cell Stabilizers Lodoxamide and Bufrolin as the First High and Equipotent Agonists of Human and Rat GPR35

Lack of high potency agonists has restricted analysis of the G protein-coupled receptor GPR35. Moreover, marked variation in potency and/or affinity of current ligands between human and rodent orthologs of GPR35 has limited their productive use in rodent models of physiology. Based on the reported modest potency of the antiasthma and antiallergic ligands cromolyn disodium and nedocromil sodium, we identified the related compounds lodoxamide and bufrolin as high potency agonists of human GPR35. Unlike previously identified high potency agonists that are highly selective for human GPR35, both lodoxamide and bufrolin displayed equivalent potency at rat GPR35. Further synthetic antiallergic ligands, either sharing features of the standard surrogate agonist zaprinast, or with lodoxamide and bufrolin, were also shown to display agonism at either human or rat GPR35. Because both lodoxamide and bufrolin are symmetric di-acids, their potential mode of binding was explored via mutagenesis based on swapping between the rat and human ortholog nonconserved arginine residues within proximity of a key conserved arginine at position 3.36. Computational modeling and ligand docking predicted the contributions of different arginine residues, other than at 3.36, in human GPR35 for these two ligands and were consistent with selective loss of potency of either bufrolin or lodoxamide at distinct arginine mutants. The computational models also suggested that bufrolin and lodoxamide would display reduced potency at a low-frequency human GPR35 single nucleotide polymorphism. This prediction was confirmed experimentally.

6-Bromo-8-(4-[3H]methoxybenzamido)-4-oxo-4H-chromene-2-carboxylic Acid: A Powerful Tool for Studying Orphan G Protein-Coupled Receptor GPR35

The potent and selective GPR35 agonist 6-bromo-8-(4-methoxybenzamido)-4-oxo-4H-chromene-2-carboxylic acid (12) was obtained in tritium-labeled form, designated [(3)H]PSB-13253, with a specific activity of 36 Ci (1.33 TBq)/mmol. Radiolabeling was achieved by methylation of ethyl 6-bromo-8-(4-((tert-butyldimethylsilyl)oxy)benzamido)-4-oxo-4H-chromene-2-carboxylate (19) with [(3)H]methyl tosylate followed by ester hydrolysis. The radioligand was characterized by kinetic, saturation, and competition assays at membrane preparations of Chinese hamster ovary cells recombinantly expressing the human GPR35. [(3)H]12 labeled the receptor with high affinity (KD = 5.27 nM). Binding was saturable (Bmax = 12.6 pmol/mg of protein) and reversible. Affinities of selected standard ligands and a library of amidochromen-4-one-2-carboxylates were determined. Binding data mostly correlated with potencies determined in β-arrestin assays. On the basis of the test results, several new fluorine-substituted 6-bromo-8-benzamidochromen-4-one-2-carboxylic acids were obtained, which represent the most potent GPR35 agonists known to date. 6-Bromo-8-(2,6-difluoro-4-methoxybenzamido)-4-oxo-4H-chromene-2-carboxylic acid (83; Ki = 0.589 nM, EC50 = 5.54 nM) showed the highest affinity with a Ki value in the subnanomolar range.

G-protein coupled receptor 35 activation mediates human vascular smooth muscle migration

Introduction: GPR35 is a poorly characterised G protein-coupled receptor endogenously activated by kynurenic acid and selectively coupled to Gα13, a G protein involved in regulation of cellular motility and proliferation via the Rho kinase pathway. The role of GPR35 in cardiovascular disease (CVD) is poorly explored, though it has been proposed to regulate blood pressure in rodents. Ligands which activate GPR35 are orthologue selective however, we have identified potent novel human GPR35 specific ligands; agonist pamoic acid and antagonists, CID-2745687 and ML145 for further dissection of GPR35 pharmacology. Based on GPR35's coupling to Gα13, here we investigated the role of GPR35 in primary human saphenous vein vascular smooth muscle cell (HSVSMC) migration and proliferation; a pathological process integral to the development of vein graft failure and restenosis. Methods: Human selective GPR35 ligands were pharmacologically characterised in β-arrestin translocation assays via bioluminenscence resonence energy transfer (BRET) in cells stably expressing FLAG-hGPR35-eYFP (Flp-In TREx 293). Endogenous expression of GPR35 was quantified in HSVSMC via QRT-PCR and migration was measured in HSVSMCs stimulated with 10-500nM pamoic acid, +/- CID-2745687 and ML145 (100nM) or ROCK1/2 inhibitor Y-27632 (10μM) in scratch wound assays using ImageJ. HSVSMC proliferation was assessed via MTS assay. Changes in HSVSMC morphology and actin-cytoskeletal structure were assessed via tetramethyl rhodamine isothiocyanate (TRITC) actin phalloidin staining. Results: BRET assays in FLAG-hGPR35-eYFP cells demonstrated that pamoic acid was a potent, human selective GPR35 agonist (pEC50 7.28±0.007) while CID-2745687 (pIC50 7.16±0.12) and ML145 (pIC50 8.08±0.12) were potent antagonists. QRT-PCR demonstrated HSVSMC endogenously express GPR35 (ΔCT=19.97±0.23, normalised to 18s). Analysis of the actin cytoskeleton revealed GPR35 activation induced a rearrangement of actin filaments consistent with HSVSMC migration. Quantification of HSVSMC migration revealed the GPR35 agonist pamoic acid induced a 30% increase in migration (p 0.05 vs -serum control). GPR35 activation did not affect HSVSMC proliferation. Conclusion: Here, we have described a novel role for GPR35 activation in the migratory capacity of HSVSMC via its downstream effector ROCK1/2. This suggests GPR35 might be a therapeutic target in the setting of vein graft failure and restenosis.

Novel GPR119 agonist HD0471042 attenuated type 2 diabetes mellitus

In type 2 diabetes mellitus (T2DM) patients, the gradual loss of pancreatic β-cell function is a characteristic feature of disease progression that is associated with sustained hyperglycemia. Recently, G protein-coupled receptor 119 (GPR119) has been identified as a promising anti-diabetic therapeutic target. It is predominantly expressed in pancreatic β-cells, directly promotes glucose stimulated insulin secretion and indirectly increases glucagon-like peptide 1 (GLP-1) levels reducing appetite and food intake. Activation of GPR119 leads to insulin release in β-cells by increasing intracellular cAMP. Here, we identified a novel structural class of small-molecule GPR119 agonists, HD0471042, consisting of substituted a 3-isopropyl-1,2,4-oxadiazol-piperidine derivative with promising potential for the treatment of T2DM. The GPR119 agonist, HD0471042 increased intracellular cAMP levels in stably human GPR119 expressing CHO cell lines and HIT-T15 cell lines, hamster β-cell line expressing endogenously GPR119. HD0471042, significantly elevated insulin release in INS-1 cells of rat pancreatic β-cell line. In in vivo experiments, a single dose of HD0471042 improved glucose tolerance. Insulin and GLP-1 level were increased in a dose-dependent manner. Treatment with HD0471042 for 6 weeks in diet induced obesity mice and for 4 weeks in ob/ob and db/db mice improved glycemic control and also reduced weight gain in a dose-dependent manner. These data demonstrate that the novel GPR119 agonist, HD0471042, not only effectively controlled glucose levels, but also had an anti-obesity effect, a feature observed with GLP-1. We therefore suggest that HD0471042 represents a new type of anti-diabetes agent with anti-obesity potential for the effective treatment of type 2 diabetes.

Stimulation of Proglucagon Gene Expression by Human GPR119 in Enteroendocrine L-cell Line GLUTag

GPR119 is a G protein-coupled receptor expressed on enteroendocrine L-cells that synthesize and secrete the incretin hormone glucagon-like peptide-1 (GLP-1). Although GPR119 agonists stimulate L-cell GLP-1 secretion, there is uncertainty concerning whether GLP-1 biosynthesis is under the control of GPR119. Here we report that GPR119 is functionally coupled to increased proglucagon (PG) gene expression that constitutes an essential first step in GLP-1 biosynthesis. Using a mouse L-cell line (GLUTag) that expresses endogenous GPR119, we demonstrate that PG gene promoter activity is stimulated by GPR119 agonist AS1269574. Surprisingly, transfection of GLUTag cells with recombinant human GPR119 (hGPR119) results in a constitutive and apparently ligand-independent increase of PG gene promoter activity and PG mRNA content. These constitutive actions of hGPR119 are mediated by cAMP-dependent protein kinase (PKA) but not cAMP sensor Epac2. Thus, the constitutive action of hGPR119 to stimulate PG gene promoter activity is diminished by: 1) a dominant-negative Gαs protein, 2) a dominant-negative PKA regulatory subunit, and 3) a dominant-negative A-CREB. Interestingly, PG gene promoter activity is stimulated by 6-Bn-cAMP-AM, a cAMP analog that selectively activates α and β isoforms of type II, but not type I PKA regulatory subunits expressed in GLUTag cells. Finally, our analysis reveals that a specific inhibitor of Epac2 activation (ESI-05) fails to block the stimulatory action of 6-Bn-cAMP-AM at the PG gene promoter, nor is PG gene promoter activity stimulated by: 1) a constitutively active Epac2, or 2) cAMP analogs that selectively activate Epac proteins. Such findings are discussed within the context of ongoing controversies concerning the relative contributions of PKA and Epac2 to the control of PG gene expression.

8-Benzamidochromen-4-one-2-carboxylic Acids: Potent and Selective Agonists for the Orphan G Protein-Coupled Receptor GPR35

8-Amido-chromen-4-one-2-carboxylic acid derivatives were identified as novel agonists at the G protein-coupled orphan receptor GPR35. They were characterized by a β-arrestin recruitment assay and optimized to obtain agonists with nanomolar potency for the human GPR35. The compounds were found to exhibit high selectivity versus the related GPR55. The most potent agonists were 6-bromo-8-(4-methoxybenzamido)-4-oxo-4H-chromene-2-carboxylic acid (85, EC50 12.1 nM) and 6-bromo-8-(2-chloro-4-methoxybenzamido)-4-oxo-4H-chromene-2-carboxylic acid (90, EC50 11.1 nM), both of which were >1700-fold selective versus GPR55. Most compounds were considerably less potent at rat and mouse than at human GPR35. 6-Bromo-8-(2-methoxybenzamido)-4-oxo-4H-chromene-2-carboxylic acid (87) was the only derivative that activated GPR35 of all three species at similar, low micromolar concentration. Compounds 85 and 90 are the most potent agonists at the human GPR35 known to date and might thus serve as powerful pharmacological tools to further elucidate the receptor's (patho)physiological role and its potential as a future drug target.

Pharmacological characterization of GPR120 ligands

Omega‐3 fatty acids (ω‐3 FAs) and other long chain free fatty acids activate GPR120, which has been shown to mediate some of the anti‐inflammatory effects of ω‐3 FAs. Loss‐of‐function mutation in human GPR120 confers increased risk for obesity and GPR120 deficient mice develop diet induced obesity, glucose intolerance and fatty liver disease. The goal of the current study was to understand GPR120 ligand selectivity by testing agonist activity of various fatty acids in cell based assays to measure G‐protein activated second messengers, β‐arrestin recruitment and inhibition of cytokine production. HEK 293 cells over‐expressing GPR120 and Gα15 were used to detect agonist mediated IP‐1 production and measure intracellular Ca2+. β‐arrestin recruitment was measured using DiscoveRx enzyme complementation technology. GPR120 agonist mediated inhibition of LPS‐induced cytokine production was measured in the RAW 264.7 cells. We confirmed the previously reported agonist activity of ω‐3 FAs including DHA, EPA, α‐linolenic acid and other polyunsaturated fatty acids such as palmitoleate and oleic acid. However, in contrast to earlier reports ω‐6 FAs linoleic acid, arachidonic acid and γ‐linolenic acid were found to robustly activate GPR120 signaling. Further characterization of GPR120 ligands will help understand its physiological role and determine its potential as a therapeutic target.