MBX-2982 (SAR260093)

GPR119

在“慢性洗脱/清洗”中用MBX-2982 (1 μM)剪切细胞，与对照实验细胞比较，IBMX包涵体捕获的cAMP积累显着增加（P<0.01；ANOVA；n=3-6） AR-231，453在与急性刺激观察到的浓度范围内产生持续反应（1.82倍变化），pEC50分别为8.67±0.11和8.93±0.17。同样，观察到MBX-2982的浓度反应发生较大但不太严重的变化（57.54倍），持续和急性pEC50分别为7.03±0.13和8.79±0.12[1]。

为了检查 GLUTag 和原代肠细胞中的观察结果是否具有生理相关性，用 10 mg/kg 的 GPR119 激动剂 MBX-2982 处理 C57BL/6 小鼠。请注意，为了检查直接的 GPR119 效应，本实验中没有联合使用DPP-IV 双胞胎，而是使用DPP-IV 来保存血液样本中的活性GLP-1。 许可证MBX-2982 的胚胎的GLP-1 水平在没有氧化剂过量的情况下增加，表明GPR119介导的GLP-1不再依赖于码头[2]。

将 GloSensor 22F 质粒转染至 HEK-GPR119 细胞中，24-30 小时后，使用细胞测量动态 cAMP。制备细胞悬浮液的步骤包括 PBS 洗涤、Accutase 处理，然后重悬于培养基中。两次离心（300 g，5 分钟）沉淀细胞后，将它们重悬于测定缓冲液（Hank 平衡盐溶液，pH 7.4）中，其中补充有 20 mM HEPES 和 0.01% 不含脂肪酸的 BSA。然后对细胞进行计数并在缓冲液中稀释至 600,000 个细胞/mL。接下来，添加 2% v/v 的 GloSensor cAMP 试剂，并让细胞和试剂在 20°C 下平衡两小时，并定期混合。一式三份，将 50 µl/孔的细胞添加到白底 384 孔板（30,000 个细胞/孔）中，并使用 Envision 读板器测量基线发光。为了达到规定的最终浓度，将 5 μL MBX-2982 在 DMSO 中严重稀释后手动添加到测定孔中，然后在测定缓冲液中按 1:100 稀释以获得 ×10 浓缩溶液。为了发现同一孔内 cAMP 随时间的动态变化，将板在 20°C 下孵育，并定期测量发光。 cAMP 反应表示为每个时间点对照（用媒介物处理的细胞）的倍数。 [1]

制作细胞悬浮液的过程包括使用 PBS 洗涤和 Accutase 处理去除 HEK-GPR119 细胞，然后重新悬浮在培养基中。细胞在烧瓶中生长至汇合。之后，通过离心沉淀（227g，7分钟，20°C）将细胞洗涤两次，然后重悬于温热的测定缓冲液（Hank's平衡盐溶液，pH 7.4，补充有20 mM HEPES和0.01%不含脂肪酸的BSA）中）。第二次洗涤后在 37°C 下孵育 5 分钟。细胞计数后，将细胞在温热的测定缓冲液中稀释至 200,000 个细胞/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

122.98

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)

配方 1 中的溶解度: ≥ 2.75 mg/mL (6.13 mM) (饱和度未知) in 10% DMSO + 90% Corn Oil (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将100 μL 27.5 mg/mL 澄清 DMSO 储备液加入到 900 μL 玉米油中并混合均匀。

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请根据您的实验动物和给药方式选择适当的溶解配方/方案：
1、请先配制澄清的储备液(如：用DMSO配置50 或 100 mg/mL母液(储备液));
2、取适量母液，按从左到右的顺序依次添加助溶剂，澄清后再加入下一助溶剂。以 下列配方为例说明 (注意此配方只用于说明，并不一定代表此产品 的实际溶解配方):
10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
假设最终工作液的体积为 1 mL, 浓度为5 mg/mL: 取 100 μL 50 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀/澄清；向上述体系中加入50 μL Tween-80，混合均匀/澄清；然后继续加入450 μL ddH2O (或 saline)定容至 1 mL；
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7、 以上所有助溶剂都可在 Invivochem.cn网站购买。