GPR119

MBX-2982 (1 μM) sheared cells in "Chronic Elution/Wash" resulted in a significant increase in cAMP accumulation as measured by IBMX inclusions when compared to control experimental cells (P<0.01; ANOVA; n=3-6). With pEC50s of 8.67±0.11 and 8.93±0.17, respectively, AR-231,453 produced sustained responses (1.82-fold change) across the concentration range observed with acute stimulation. Similar to this, MBX-2982 showed a greater but less severe change in the concentration response (57.54-fold), with acute and sustained pEC50 values of 8.79±0.12 and 7.03±0.13, respectively [1].

In order to investigate the physiological relevance of the findings in GLUTag and primary enterocytes, 10 mg/kg of the GPR119 agonist MBX-2982 was administered to C57BL/6 mice. In this experiment, DPP-IV was used to preserve active GLP-1 in blood samples; however, DPP-IV twins were not combined in order to examine direct GPR119 effects. Licenseed MBX-2982 embryos showed an increase in GLP-1 levels without an excess of oxidant, suggesting that GPR119-mediated GLP-1 is no longer Dock-dependent [2].

The GloSensor 22F plasmid is transfected into HEK-GPR119 cells, and 24–30 hours later, the cells are used to measure dynamiccAMP. PBS washing, Accutase treatment, and then resuspension in culture media are the steps involved in making cell suspensions. After two centrifugations (300 g, 5 min) to pellet the cells, they are resuspended in the assay buffer (Hank's Balanced Salt Solution, pH 7.4), which is supplemented with 20 mM HEPES and 0.01% fatty acid-free BSA. The cells are then counted and diluted to 600,000 cells/mL in buffer. Next, 2% v/v of GloSensor cAMP reagent is added, and the cells and reagent are allowed to equilibrate for two hours at 20°C with periodic mixing. In triplicate, 50 µl/well of cells are added to white-bottomed 384 well plates (30,000 cells/well), and an Envision plate reader is used to measure the baseline luminescence. To achieve the stated final concentration, 5 μL of MBX-2982 is manually added to the assay wells after being severely diluted in DMSO and then diluted 1:100 in assay buffer to obtain ×10 concentrated solution. To find dynamic cAMP changes over time within the same wells, plates are incubated at 20°C and luminescence is measured at regular intervals. The cAMP responses are expressed as fold over control (cells treated with vehicles) at each time point. [1]

The process of making cell suspensions involves dislodging HEK-GPR119 cells using PBS wash and accutase treatment, then resuspension in culture media. The cells are grown to confluency in flasks. After that, cells are twice washed by pelleting through centrifugation (227g, 7 min, 20°C), and then resuspension in warm assay buffer (Hank's Balanced Salt Solution, pH 7.4, supplemented with 20 mM HEPES and 0.01% fatty acid free BSA). The second wash is followed by a 5-minute incubation at 37°C. Following cell counting, the cells are diluted in warm assay buffer to 200,000 cells/mL[1].

Mice: Male C57BL/6 mice are employed. Male 10-week-old mice (n = 20 per group) are fasted for the entire night and given either MBX-2982 at 10 mg/kg or vehicle (15% polyethylene glycol 400+85% of 23.5% hydroxypropyl-β-cyclodextrin) orally. Thirty minutes after compound dosing, half of the animals (n = 10 per group) are killed by CO2 asphyxiation, and blood is extracted by cardiac puncture. In order to maintain active GLP-1, a DPP-IV inhibitor is pre-added to the blood collection tubes (10 µL per 1 mL of blood), and the syringe walls are rinsed with the inhibitor prior to the cardiac puncture. The other half of the animals (n = 10 per group) were put to death for blood collection 10 minutes after the glucose load and received an oral glucose bolus (3 g/kg) 30 minutes after compound dosing. The active GLP-1 (ver 2) kit is used to measure GLP-1 levels in plasma samples.

[1]. Sustained wash-resistant receptor activation responses of GPR119 agonists. Eur J Pharmacol. 2015 Sep 5;762:430-42.

[2]. Agonists at GPR119 mediate secretion of GLP-1 from mouse enteroendocrine cells through glucose-independent pathways. Br J Pharmacol. 2012 Apr;165(8):2799-807.

[3]. GPR119: a promising target for nonalcoholic fatty liver disease. FASEB J. 2016 Jan;30(1):324-35.

MBX-2982 has been used in clinical trials for diabetes treatment. G protein-coupled receptor 119 (GPR119) is involved in the regulation of metabolic homeostasis, and GPR119 agonists are targets for the treatment of type 2 diabetes and obesity. We investigated the temporal dynamics of receptor signaling using the endogenous agonist oleoylethanolamine and several small-molecule synthetic agonists. Through detection using a dynamic luminescent biosensor and endpoint cAMP accumulation assays, we found that although GPR119 exhibits a strong activation response regardless of the agonist used, agonist-driven desensitization is not its primary regulatory mechanism. Temporal analysis of the cAMP response showed that some (but not all) of the tested agonists produced sustained signaling that was unaffected by drug elution. Further analysis showed that the sustained effect of a synthetic agonist, AR-231,453, was consistent with slow dissociation kinetics. In contrast, the sustained responses of MBX-2982 and AZ1 appeared to be related to membrane deposition. We also detected a elution-resistant response of AR-231,453 in the endogenous expression system (GLP-1 secretion in GLUTag cells), with levels that correlated with physiological parameters. In summary, our results indicate that GPR119 activation is persistent in the recombinant expression system with little apparent sign of receptor desensitization, and that persistent agonist response persists for some ligands even after removal of excess agonists. This provides new insights into the time-response profile of potential drug candidates targeting GPR119 and underscores the importance of carefully studying the mechanisms by which GPCRs generate persistent responses. [1]

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Background and Objectives: G protein-coupled receptor 119 (GPR119) mediates insulin secretion from pancreatic β cells and glucagon-like peptide-1 (GLP-1) release from intestinal L cells. While GPR119-mediated insulin secretion is glucose-dependent, it remains unclear whether GPR119-mediated GLP-1 secretion is similarly glucose-dependent. This study aimed to investigate whether GPR119-mediated GLP-1 secretion is glucose-dependent and to compare the cellular mechanisms of hormone secretion in L cells and β cells. Methods: The effects of GPR119 agonists and ion channel modulators on GLP-1 secretion in the presence and absence of glucose were analyzed in the intestinal L cell line GLUTag, primary intestinal cell cultures, and in vivo. Insulin secretion in the pancreatic β cell line Min6 was analyzed as a control. Main Results: In GLUTag cells, GPR119 agonists stimulated GLP-1 secretion regardless of the presence of glucose. In mouse primary colon cell cultures, GPR119 agonists also stimulated GLP-1 secretion in the absence of glucose. Furthermore, GPR119 agonists increased plasma GLP-1 levels in glucose-naïve mice. However, in Min6 cells, GPR119-mediated insulin secretion was glucose-dependent. In the pharmacological reagents tested in this study, nifedipine (an L-type voltage-dependent calcium channel blocker) dose-dependently reduced GLP-1 secretion in GLUTag cells, but had no effect on Min6 cells under glucose-free conditions. Conclusion and significance: Unlike pancreatic β cells, GPR119-mediated GLP-1 secretion in intestinal L cells is glucose-independent both in vitro and in vivo, which may be due to the higher basal calcium tension in L cells. [2]

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Nonalcoholic fatty liver disease is associated with metabolic syndrome and is characterized by excessive lipid accumulation in the liver. G protein-coupled receptor 119 (GPR119) is a promising therapeutic target for type 2 diabetes. However, the role of GPR119 activation in hepatic steatosis and its exact mechanism have not been investigated. In primary cultured hepatocytes of wild-type and GPR119 knockout (KO) mice, the expression of lipogenesis enzyme was increased in GPR119 KO hepatocytes. In a phase II clinical trial, treatment of hepatocytes and HepG2 cells with GPR119 agonists (MBX-2982 [MBX] and GSK1292263) suppressed the protein expression of nuclear and total sterol regulatory element binding protein (SREBP)-1, a key transcription factor for lipogenesis. In mice fed a high-fat diet, oral administration of MBX significantly suppressed hepatic lipid accumulation and the expression levels of SREBP-1 and lipogenesis-related genes, while the anti-lipogenesis effect of MBX in the liver disappeared in GPR119 KO mice. MBX activated AMPK and increased Ser-372 phosphorylation of SREBP-1c, an inhibitory SREBP-1c. Furthermore, inhibition of AMPK reversed MBX-induced downregulation of SREBP-1. These findings demonstrate for the first time that GPR119 ligands alleviate hepatic steatosis by inhibiting SREBP-1-mediated lipogenesis in hepatocytes. [3]

C22H24N8OS

448.54396

448.179

C, 58.91; H, 5.39; N, 24.98; O, 3.57; S, 7.15

1037792-44-1

25025505

White to off-white solid powder

1.4±0.1 g/cm3

683.6±65.0 °C at 760 mmHg

367.3±34.3 °C

0.0±2.1 mmHg at 25°C

1.739

3.88

0

9

7

32

564

0

CCC1=CN=C(N2CCC(C3=NC(COC4=CC=C(N5N=NN=C5)C=C4)=CS3)CC2)N=C1

NFTMKHWBOINJGM-UHFFFAOYSA-N

InChI=1S/C22H24N8OS/c1-2-16-11-23-22(24-12-16)29-9-7-17(8-10-29)21-26-18(14-32-21)13-31-20-5-3-19(4-6-20)30-15-25-27-28-30/h3-6,11-12,14-15,17H,2,7-10,13H2,1H3

2-[1-(5-ethylpyrimidin-2-yl)piperidin-4-yl]-4-[[4-(tetrazol-1-yl)phenoxy]methyl]-1,3-thiazole

SAR-260093; SAR 260093; SAR260093; MBX-2982; MBX2982; MBX 2982

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO: ~50 mg/mL (~111.5 mM)

Solubility in Formulation 1: ≥ 2.75 mg/mL (6.13 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 27.5 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)