FABP4 (Ki < 2 nM); FABP3 (Ki = 250 nM); FABP5 (Ki = 350 nM)[1]

BMS30943刺激分化C2C12肌管的葡萄糖摄取、AMPK和p38磷酸化。[2]
BMS309403通过激活AMPK刺激C2C12肌管的葡萄糖摄取。[2]
BMS309403独立于FABP3激活AMPK 。[2]
AMPK未被BMS309403直接激活。[2]
BMS309403对线粒体膜电位去极化的影响。[2]
BMS-309403 治疗以时间和剂量依赖性方式显着降低 THP-1 巨噬细胞 MCP-1 的产生 [2]。

BMS-309403 钠（15 mg/kg；每天一次，持续六周；长期）可降低甘油三酯水平，增强内皮功能、磷酸化和总 eNOS，但对内皮非应激松弛影响不大 [3]。

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A-FABP在12周龄及以上的ApoE−/−小鼠的主动脉内皮中表达，但在8周龄或C57野生型小鼠的主动脉内皮中不表达。与年龄匹配的对照组相比，18周龄ApoE−/−小鼠主动脉中乙酰胆碱、UK14304(选择性α2-肾上腺素能受体激动剂)和A23187(钙离子载体)的内皮依赖性松弛减少，磷酸化eNOS和总eNOS的蛋白含量减少。在12周龄小鼠中，A- fabp抑制剂BMS309403治疗了6周，改善了内皮功能，磷酸化和总eNOS，降低了血浆甘油三酯水平，但不影响内皮非依赖性松弛。BMS309403对uk14304所致松弛的有益作用被百日咳毒素减弱。在培养的人微血管内皮细胞中，脂质诱导的A-FABP表达与磷酸化eNOS和NO生成的减少有关，并被BMS309403逆转。[3]

pCMV-3tag介导hFABP3在C2C12中的过表达[2]
cDNA编码全长人FABP3已市购。将hFABP3 cDNA连接到pcmv -3标签载体上，其C端带有3FLAG标签。该构建体经DNA测序验证，并用于细胞系的生成。用1.5 mg/mL G418选择构建pcmv - hfabp3 -3标签的稳定转染物或空pcmv -3标签载体，培养10天。克隆挑选稳定的转染物，转入分化培养基(2%马血清)，再培养7天(肌管)，然后用BMS30943处理。

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AMPK酶活性的体外测定[2]
在体外测定AMPK酶活性的方法已在前面介绍过。我们选择AMPKα2β1γ1作为活性形式，并通过将[γ-33P]掺入到SAMS肽中来评价其活性。在Wallac MicroBeta TriLus中通过液体闪烁计数来测定蛋白质中的放射性。

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腺嘌呤核苷酸的提取与测定[2]
在60 mm培养皿中培养C2C12肌管，用20µM BMS30943处理，PBS洗涤，胰蛋白酶化。细胞腺嘌呤核苷酸测量的样品按前面描述的方法制备和分析。

分化C2C12的葡萄糖摄取[2]
分化后的C2C12细胞在无血清培养基中饥饿2 h后，用BMS30943孵育。肌管用无糖KRPH缓冲液[140 mM NaCl, 5 mM KCl, 1 mM CaCl2, 1.2 mM KH2PO4, 2.5 mM MgSO4, 5 mM NaHCO3, 25 mM Hepes, pH 7.4, 0.2%脂肪酸游离牛血清白蛋白]洗涤2次，与0.5 ml不同浓度的BMS30943在KRPH缓冲液中孵育15分钟。切换到含有BMS30943的KRPH缓冲液中。5 mM d -葡萄糖和0.5µCi/孔的2-脱氧-d [3H]-葡萄糖持续15分钟或5分钟。然后用冰冷的PBS洗涤3次，用0.5 M NaOH和0.1% SDS裂解Myotubes。细胞裂解液用盐酸中和。用液体闪烁计数法测定放射性。

Animal/Disease Models: C57BL/6J mice (ApoE−/− mice) [3]
Doses: 15 mg/kg
Route of Administration: Chronic treatment; one time/day for 6 weeks
Experimental Results: 18weeks old ApoE−/− mice Phosphorylated eNOS (Ser1177) and total eNOS were Dramatically increased in arteries, but the ratio of phosphorylated to total eNOS was not increased.

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ApoE−/− mice[3]
C57BL/6J mice (wild-type strain; ApoE+/+ mice) and ApoE−/− mice were studied. Mice homozygous for the Apoetm1Unc mutation were provided by the Jackson Laboratory. The breeding line was maintained by directly pairing male and female homozygous mutated Apoetm1Unc mice. The mice were maintained under pathogen-free conditions in filter-topped cages in an air-conditioned room at constant temperature (23 ± 1°C), fed a standard laboratory diet and given water ad libitum. To study endothelial function, ApoE−/− mice 8 to 18 weeks old, and age-matched wild-type mice were compared. To determine the effects of pharmacological inhibition of the actions of A-FABP, either the A-FABP inhibitor BMS30943(15 mg·kg−1·day−1) (Furuhashi et al., 2007) or vehicle (4% Tween 80) were administered chronically by daily oral gavage for 6 weeks in ApoE−/− mice (starting at weeks 12 of age). Mice were anaesthetized with a bolus injection of pentobarbitone sodium (230 mg·kg−1) and their aorta removed and dissected for ex vivo studies.
Blood samples from mice with or without BMS30943 treatment were collected at the time of death by direct puncture of the heart. They were centrifuged at 1500× g for 15 min at 15°C and the plasma was collected. The triglyceride concentration was determined with 20 µL plasma using a commercially available measurement kit (WAKO, Osaka, Japan). Plasma levels of LDL and high density lipoprotein (HDL) cholesterol were determined using another commercially available HDL and LDL/VLDL Cholesterol Quantification Kit

[1]. Potent and selective biphenyl azole inhibitors of adipocyte fatty acid binding protein (aFABP). Bioorg Med Chem Lett. 2007 Jun 15;17(12):3511-5.

[2]. BMS309403 stimulates glucose uptake in myotubes through activation of AMP-activated protein kinase. PLoS One. 2012;7(8):e44570.

[3]. Chronic administration of BMS309403 improves endothelial function in apolipoprotein E-deficient mice and in cultured human endothelial cells. Br J Pharmacol. 2011 Apr;162(7):1564-76.

Herein we report the first disclosure of biphenyl azoles that are nanomolar binders of adipocyte fatty acid binding protein (aFABP or aP2) with up to thousand-fold selectivity against muscle fatty acid binding protein and epidermal fatty acid binding protein. In addition a new radio-ligand to determine binding against the three fatty acid binding proteins was also synthesized.[1]

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BMS309403 is a biphenyl azole inhibitor against fatty acid binding protein 4 (FABP4) and regarded as a lead compound for effective treatment of obesity related cardio-metabolic diseases. Here we discovered an off-target activity of BMS309403 in that it stimulates glucose uptake in C2C12 myotubes in a temporal and dose dependent manner via activation of AMP-activated protein kinase (AMPK) signaling pathway but independent of FABPs. Further analysis indicated that BMS309403 activates AMPK through increasing the ratio of intracellular AMP:ATP while decreasing mitochondrial membrane potential. These findings provide mechanistic insights on the action of BMS309403.[2]

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Adipocyte fatty acid-binding protein (A-FABP) is up-regulated in regenerated endothelial cells and modulates inflammatory responses in macrophages. Endothelial dysfunction accompanying regeneration is accelerated by hyperlipidaemia. Here, we investigate the contribution of A-FABP to the pathogenesis of endothelial dysfunction in the aorta of apolipoprotein E-deficient (ApoE(-/-) ) mice and in cultured human endothelial cells. Experimental approach: A-FABP was measured in aortae of ApoE(-/-) mice and human endothelial cells by RT-PCR, immunostaining and immunoblotting. Total and phosphorylated forms of endothelial nitric oxide synthase (eNOS) were measured by immunoblotting. Changes in isometric tension were measured in rings of mice aortae Key results: A-FABP was expressed in aortic endothelium of ApoE(-/-) mice aged 12 weeks and older, but not at 8 weeks or in C57 wild-type mice. Reduced endothelium-dependent relaxations to acetylcholine, UK14304 (selective α(2) -adrenoceptor agonist) and A23187 (calcium ionophore) and decreased protein presence of phosphorylated and total eNOS were observed in aortae of 18 week-old ApoE(-/-) mice compared with age-matched controls. A 6 week treatment with the A-FABP inhibitor, BMS309403, started in 12 week-old mice, improved endothelial function, phosphorylated and total eNOS and reduced plasma triglyceride levels but did not affect endothelium-independent relaxations. The beneficial effect of BMS309403 on UK14304-induced relaxations was attenuated by Pertussis toxin. In cultured human microvascular endothelial cells, lipid-induced A-FABP expression was associated with reduced phosphorylated eNOS and NO production and was reversed by BMS309403.[3]

C31H26N2O3

474.5497

474.194

C, 78.46; H, 5.52; N, 5.90; O, 10.11

300657-03-8

BMS-309403 sodium;2802523-05-1

16122583

White to off-white solid powder

1.2±0.1 g/cm3

657.5±55.0 °C at 760 mmHg

351.4±31.5 °C

0.0±2.1 mmHg at 25°C

1.623

7.69

64.35

1

4

8

36

689

0

SJRVJRYZAQYCEE-UHFFFAOYSA-N

InChI=1S/C31H26N2O3/c1-2-27-30(22-12-5-3-6-13-22)31(23-14-7-4-8-15-23)32-33(27)28-19-10-9-18-26(28)24-16-11-17-25(20-24)36-21-29(34)35/h3-20H,2,21H2,1H3,(H,34,35)

((2'-(5-Ethyl-3,4-diphenyl-1H-pyrazol-1-yl)-1,1'-biphenyl-3-yl)oxy)acetic acid

BMS-309403; BMS309403; 2-((2'-(5-Ethyl-3,4-diphenyl-1H-pyrazol-1-yl)-[1,1'-biphenyl]-3-yl)oxy)acetic acid; FABP4 Inhibitor; [2'-(5-Ethyl-3,4-diphenyl-pyrazol-1-yl)-biphenyl-3-yloxy]acetic acid; ((2'-(5-Ethyl-3,4-diphenyl-1H-pyrazol-1-yl)-1,1'-biphenyl-3-yl)oxy)acetic acid; BMS 309403.

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 : ~100 mg/mL (~210.73 mM)
H2O : < 0.1 mg/mL

配方 1 中的溶解度: ≥ 2.08 mg/mL (4.38 mM) (饱和度未知) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (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 20.8 mg/mL澄清DMSO储备液加入400 μL PEG300中，混匀；然后向上述溶液中加入50 μL Tween-80，混匀；加入450 μL生理盐水定容至1 mL。
*生理盐水的制备：将 0.9 g 氯化钠溶解在 100 mL ddH₂O中，得到澄清溶液。

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配方 2 中的溶解度: ≥ 2.08 mg/mL (4.38 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 20.8 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；
3、溶剂前显示的百分比是指该溶剂在最终溶液/工作液中的体积所占比例;
4、 如产品在配制过程中出现沉淀/析出，可通过加热(≤50℃)或超声的方式助溶;
5、为保证最佳实验结果，工作液请现配现用！
6、如不确定怎么将母液配置成体内动物实验的工作液，请查看说明书或联系我们;
7、 以上所有助溶剂都可在 Invivochem.cn网站购买。