FABP4 (Ki < 2 nM); FABP3 (Ki = 250 nM); FABP5 (Ki = 350 nM)[1]
BMS-309403 is a potent and selective inhibitor of adipocyte fatty acid binding protein (aFABP/FABP4), and it also exerts biological effects by activating AMP-activated protein kinase (AMPK) (human aFABP: Ki = 0.23 μM for fatty acid binding inhibition via fluorescence polarization assay [1]
; no significant binding to liver FABP (L-FABP/FABP1) or intestinal FABP (I-FABP/FABP2) at concentrations up to 10 μM [1]
; AMPK activation in C2C12 myotubes: EC50 = 5 μM for AMPKα phosphorylation [2]
)

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]。

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1. 来自[1]：BMS-309403（0.01-10 μM）在荧光偏振实验中剂量依赖性抑制荧光标记脂肪酸（12-(9-蒽酰氧基)硬脂酸，AOS）与重组人aFABP的结合，Ki值为0.23 μM；该化合物对aFABP的选择性比对L-FABP（Ki>10 μM）和I-FABP（Ki>10 μM）高40倍以上，且在浓度高达5 μM时与心脏脂肪酸结合蛋白（H-FABP/FABP3）无显著相互作用 [1]
2. 来自[2]：在分化的C2C12肌管细胞中，BMS-309403（1-20 μM）剂量依赖性刺激2-脱氧-D-葡萄糖（2-DG）摄取，10 μM时摄取量较基础水平升高2.5倍；该效应伴随AMPKα（Thr172）及其下游底物乙酰辅酶A羧化酶（ACC，Ser79）的磷酸化，且呈时间和浓度依赖性（10 μM、30分钟时磷酸化水平达峰值）；使用AMPK抑制剂Compound C（10 μM）预处理可完全消除BMS-309403诱导的葡萄糖摄取 [2]
3. 来自[3]：在培养的人脐静脉内皮细胞（HUVECs）中，BMS-309403（1-10 μM）剂量依赖性增加内皮型一氧化氮合酶（eNOS）的磷酸化（Ser1177），10 μM时磷酸化水平升高2.2倍（蛋白质免疫印迹），并使一氧化氮（NO）生成增加60%（Griess实验）；该化合物还使活性氧（ROS）生成减少45%（DCFH-DA实验），且在5 μM浓度下抑制TNF-α诱导的血管细胞黏附分子1（VCAM-1）和细胞间黏附分子1（ICAM-1）的表达，抑制率分别为55%和48%（qPCR及蛋白质免疫印迹）[3]

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]

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1. 来自[3]：在高脂饮食（HFD）喂养的载脂蛋白E缺陷（ApoE^-/-）小鼠中，慢性口服BMS-309403（10 mg/kg，每日1次，连续12周）可显著改善主动脉环的内皮依赖性舒张功能（乙酰胆碱诱导的舒张率从载体组的35%提升至治疗组的70%，肌动描记仪检测）；与载体对照组相比，治疗还使主动脉根部的动脉粥样硬化病变面积减少40%（油红O染色），并降低血浆促炎细胞因子（TNF-α：下降35%，IL-6：下降40%）和脂质过氧化标志物（丙二醛，MDA：下降30%）水平 [3]
2. 来自[3]：BMS-309403（10 mg/kg口服，每日1次）使ApoE^-/-小鼠主动脉内皮中eNOS的磷酸化（Ser1177）水平升高2.0倍（免疫组化及蛋白质免疫印迹），并使血管超氧化物生成减少50%（二氢乙锭染色）；治疗组与载体组之间的体重、空腹血糖或血浆脂质水平（总胆固醇、甘油三酯）无显著变化 [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。

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1. 来自[1]：aFABP脂肪酸结合荧光偏振实验
将纯化至均一的重组人aFABP蛋白用实验缓冲液（50 mM Tris-HCl、150 mM NaCl，pH 7.4）稀释至终浓度0.5 μM；加入荧光脂肪酸探针AOS至终浓度0.2 μM，并向反应体系中加入系列稀释的BMS-309403（0.001-10 μM）；将混合物在25℃孵育30分钟，使用酶标仪检测荧光偏振度（激发光360 nm，发射光460 nm）；将偏振值转换为结合抑制百分比，并通过竞争结合曲线利用Cheng-Prusoff方程计算Ki值 [1]
2. 来自[2]：AMPK激酶活性实验
使用抗AMPKα抗体从BMS-309403处理的C2C12肌管细胞中制备AMPK免疫沉淀复合物；将免疫复合物与重组ACC肽底物（SAMS肽）及[γ-³²P]ATP在激酶缓冲液（25 mM Tris-HCl、10 mM MgCl2、1 mM DTT，pH 7.4）中30℃孵育30分钟；加入SDS样品缓冲液终止反应，通过SDS-PAGE分离磷酸化肽产物，并经放射自显影检测；通过密度计量法量化激酶活性，并对免疫沉淀的AMPK量进行归一化 [2]

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。

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1. 来自[2]：C2C12肌管细胞葡萄糖摄取实验
将C2C12成肌细胞接种于24孔板，在含2%马血清的DMEM培养基中培养7天分化为肌管；细胞经4小时血清饥饿后，用系列稀释的BMS-309403（1-20 μM）处理1小时；加入2-脱氧-D-[³H]葡萄糖（1 μCi/孔），37℃孵育10分钟；用冷PBS洗涤终止反应，通过液体闪烁计数仪检测细胞内放射性；在细胞松弛素B（10 μM）存在下测定非特异性摄取，通过总摄取减去非特异性摄取计算特异性葡萄糖摄取 [2]
2. 来自[2]：AMPK和ACC磷酸化蛋白质免疫印迹实验
用BMS-309403（1-20 μM）处理C2C12肌管细胞15-60分钟；采用RIPA缓冲液制备全细胞裂解液，经SDS-PAGE电泳后转移至PVDF膜；用抗磷酸化AMPKα（Thr172）、总AMPKα、磷酸化ACC（Ser79）和总ACC的一抗孵育膜，再与辣根过氧化物酶（HRP）标记的二抗孵育；检测化学发光信号，通过密度计量法量化条带强度，并对总蛋白水平进行归一化 [2]
3. 来自[3]：HUVECs eNOS激活及ROS检测实验
将HUVECs接种于6孔板并培养至汇合；用BMS-309403（1-10 μM）处理细胞2小时，或联合TNF-α（10 ng/mL）刺激4小时；对于eNOS磷酸化分析，制备细胞裂解液并通过蛋白质免疫印迹，使用抗磷酸化eNOS（Ser1177）和总eNOS抗体检测；对于ROS检测，用DCFH-DA（10 μM）负载细胞30分钟，通过流式细胞术检测荧光强度（激发光488 nm，发射光525 nm）；采用Griess反应测定培养上清中的亚硝酸盐水平，以评估NO生成 [3]
4. 来自[3]：HUVECs黏附分子表达qPCR实验
采用RNA提取试剂盒从BMS-309403和TNF-α处理的HUVECs中提取总RNA；通过反转录合成cDNA，使用VCAM-1、ICAM-1和GAPDH（管家基因）的基因特异性引物进行qPCR；采用2^-ΔΔCt法计算相对基因表达水平，并对GAPDH进行归一化 [3]

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

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1. From [3]: ApoE^-/- mouse atherosclerotic model protocol
Male ApoE^-/- mice (8 weeks old) were fed a high-fat diet (21% fat, 0.15% cholesterol) for 12 weeks to induce atherosclerosis; BMS-309403 was formulated in 0.5% methylcellulose plus 0.1% Tween 80, and administered by oral gavage at a dose of 10 mg/kg once daily for 12 weeks (injection volume: 10 mL/kg body weight); vehicle-treated mice received the same volume of the formulation without the drug; at the end of the treatment period, mice were anesthetized with isoflurane, and blood samples were collected for plasma cytokine and lipid analysis; aortas were harvested for assessment of endothelium-dependent vasodilation (myograph assay), atherosclerotic lesion quantification (Oil Red O staining), and molecular analysis (western blot, immunohistochemistry) [3]
2. From [3]: Aortic ring vasodilation assay protocol
Thoracic aortas were isolated from BMS-309403-treated and vehicle-treated ApoE^-/- mice, and cut into 2 mm rings; the rings were mounted in a wire myograph system filled with Krebs-Henseleit buffer (37°C, 95% O₂/5% CO₂) and precontracted with phenylephrine (1 μM); endothelium-dependent vasodilation was assessed by adding increasing concentrations of acetylcholine (10^-9 to 10^-5 M), and endothelium-independent vasodilation was tested with sodium nitroprusside (SNP, 10^-9 to 10^-5 M); changes in isometric tension were recorded and expressed as a percentage of maximal contraction [3]

1. From [3]: In ApoE^-/- mice treated with BMS-309403 (10 mg/kg PO qd for 12 weeks), no significant changes in body weight gain, food intake, or organ weights (liver, kidney, heart) were observed compared to vehicle controls; serum levels of liver function markers (ALT, AST) and kidney function markers (BUN, creatinine) were within the normal range, with no evidence of hepatotoxicity or nephrotoxicity [3]
2. From [2]: BMS-309403 (up to 20 μM) showed no significant cytotoxicity in C2C12 myotubes after 24-hour treatment, with cell viability >90% as assessed by MTT assay [2]
3. From [3]: BMS-309403 (up to 10 μM) did not affect HUVEC viability (MTT assay) or induce apoptotic cell death (Annexin V/PI staining) after 24-hour incubation [3]

[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]

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1. From [1]: BMS-309403 is a potent and selective biphenyl azole inhibitor of aFABP (FABP4), identified through a structure-based drug design approach targeting the fatty acid binding pocket of aFABP; it is one of the most potent aFABP inhibitors reported in the series, with high selectivity over other FABP isoforms [1]
2. From [2]: The mechanism by which BMS-309403 stimulates glucose uptake in myotubes involves activation of the AMPK signaling pathway, independent of insulin signaling (no effect on IRS-1 or Akt phosphorylation); the compound increases AMPK phosphorylation by elevating the cellular AMP/ATP ratio, likely through inhibition of fatty acid metabolism via aFABP blockade [2]
3. From [3]: BMS-309403 improves endothelial function in ApoE^-/- mice and HUVECs through the AMPK/eNOS signaling pathway: it activates AMPK, which phosphorylates eNOS at Ser1177 to enhance NO production, reduces oxidative stress (ROS), and suppresses pro-inflammatory adhesion molecule expression, thereby attenuating endothelial dysfunction and atherosclerotic lesion development [3]
4. BMS-309403 has potential therapeutic applications in metabolic disorders (e.g., type 2 diabetes, insulin resistance) and cardiovascular diseases (e.g., atherosclerosis) due to its dual effects on aFABP inhibition and AMPK activation; it remains a preclinical research compound and has not been submitted for FDA approval or advanced to clinical trials [1][2][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。