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 trials studying the treatment of Diabetes.

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G protein-coupled receptor 119 (GPR119) is involved in regulating metabolic homoeostasis, with GPR119 agonists targeted for the treatment of type-2 diabetes and obesity. Using the endogenous agonist oleoylethanolamide and a number of small molecule synthetic agonists we have investigated the temporal dynamics of receptor signalling. Using both a dynamic luminescence biosensor-based assay and an endpoint cAMP accumulation assay we show that agonist-driven desensitization is not a major regulatory mechanism for GPR119 despite robust activation responses, regardless of the agonist used. Temporal analysis of the cAMP responses demonstrated sustained signalling resistant to washout for some, but not all of the agonists tested. Further analysis indicated that the sustained effects of one synthetic agonist AR-231,453 were consistent with a role for slow dissociation kinetics. In contrast, the sustained responses to MBX-2982 and AZ1 appeared to involve membrane deposition. We also detect wash-resistant responses to AR-231,453 at the level of physiologically relevant responses in an endogenous expression system (GLP-1 secretion in GLUTag cells). In conclusion, our findings indicate that in a recombinant expression system GPR119 activation is sustained, with little evidence of pronounced receptor desensitization, and for some ligands persistent agonist responses continue despite removal of excess agonist. This provides novel understanding of the temporal responses profiles of potential drug candidates targetting GPR119, and highlights the importance of carefully examining the the mechanisms through which GPCRs generate sustained responses.[1]

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Background and purpose: The 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 is not clear whether or not GPR119-mediated GLP-1 secretion similarly requires glucose. This study was designed to address the glucose-dependence of GPR119-mediated GLP-1 secretion, and to explore the cellular mechanisms of hormone secretion in L cells versus those in β cells. Experimental approach: GLP-1 secretion in response to GPR119 agonists and ion channel modulators, with and without glucose, was analysed in the intestinal L cell line GLUTag, in primary intestinal cell cultures and in vivo. Insulin secretion from Min6 cells, a pancreatic β cell line, was analysed for comparison. Key results: In GLUTag cells, GPR119 agonists stimulated GLP-1 secretion both in the presence and in the absence of glucose. In primary mouse colon cultures, GPR119 agonists stimulated GLP-1 secretion under glucose-free conditions. Moreover, a GPR119 agonist increased plasma GLP-1 in mice without a glucose load. However, in Min6 cells, GPR119-mediated insulin secretion was glucose-dependent. Among the pharmacological agents tested in this study, nitrendipine, an L-type voltage-dependent calcium channel blocker, dose-dependently reduced GLP-1 secretion from GLUTag cells, but had no effect in Min6 cells in the absence of glucose. Conclusions and implications: Unlike that in pancreatic β cells, GPR119-mediated GLP-1 secretion from intestinal L cells was glucose-independent in vitro and in vivo, probably because of a higher basal calcium tone in the L cells. [2]

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Nonalcoholic fatty liver disease is associated with metabolic syndrome and has the unique characteristic of excess lipid accumulation in liver. G-protein-coupled receptor 119 (GPR119) is a promising target for type 2 diabetes. However, the role of GPR119 activation in hepatic steatosis and its precise mechanism has not been investigated. In primary cultured hepatocytes from wild-type and GPR119 knockout (KO) mice, expression of lipogenic enzymes was elevated in GPR119 KO hepatocytes. Treatment of hepatocytes and HepG2 cells with GPR119 agonists in phase 2 clinical trials (MBX-2982 [MBX] and GSK1292263) inhibited protein expression of both nuclear and total sterol regulatory element binding protein (SREBP)-1, a key lipogenesis transcription factor. Oral administration of MBX in mice fed a high-fat diet potently inhibited hepatic lipid accumulation and expression levels of SREBP-1 and lipogenesis-related genes, whereas the hepatic antilipogenesis effects of MBX were abolished in GPR119 KO mice. MBX activated AMPK and increased Ser-372 phosphorylation of SREBP-1c, an inhibitory form of SREBP-1c. Moreover, inhibition of AMPK recovered MBX-induced down-regulation of SREBP-1. These findings demonstrate for the first time that the GPR119 ligand alleviates 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.)