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

BMS30943 Stimulates Glucose Uptake, AMPK and p38 Phosphorylation in Differentiated C2C12 Myotubes.[2]
BMS309403 Stimulates Glucose Uptake in C2C12 Myotubes Via AMPK Activation.[2]
BMS309403 Activates AMPK Independent of FABP3.[2]
AMPK is not Directly Activated by BMS309403 in vitro.[2]
BMS309403 Depolarizes Mitochondrial Membrane Potential and Increases Cytosolic AMP/ATP Ratio.[2]
Treatment with BMS-309403 dramatically decreased THP-1 macrophages' production of MCP-1 in a time- and dose-dependent manner [2].

BMS-309403 sodium (15 mg/kg; once daily for six weeks; long-term) decreases triglyceride levels, enhances endothelial function, phosphorylation, and total eNOS, but has little effect on endothelial non-stress relaxation [3].

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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 α2-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]

pCMV-3tag Mediated Overexpression of hFABP3 in C2C12[2]
cDNA encoding full-length human FABP3 was purchased commercially. The hFABP3 cDNA was ligated into pCMV-3tag vector with the 3FLAG tag in the C terminus. The construct was verified by DNA sequencing and used for generation of cell lines. Stable transfectants with pCMV-hFABP3-3tag construct or empty pCMV-3tag vector were selected with 1.5 mg/mL G418 for 10 days. The stable transfectants were clonally picked and switched to differentiation medium (2% horse serum), and cultured for an additional 7 days (myotube) before treatment with BMS30943.

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Measurement of AMPK Enzymatic Activity in vitro[2]
The method to measure AMPK enzymatic activity in vitro was described previously. We chose AMPKα2β1γ1 as the active form and its activity was evaluated by the incorporation of [γ-33P] into the SAMS peptide. Radioactivity that had been incorporated in the proteins was determined by liquid scintillation counting in a Wallac MicroBeta TriLus.

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Adenine Nucleotide Extraction and Measurement[2]
C2C12 myotubes cultured in 60 mm dishes were treated with 20 µM BMS30943, washed with PBS and trypsinized. The samples for cellular adenine nucleotides measurements were prepared and analyzed as previously described.

Glucose Uptake in Differentiated C2C12[2]
Differentiated C2C12 cells were starved in serum free-medium for 2 h before incubation with BMS30943. Myotubes were washed twice with glucose free KRPH buffer [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% fatty acid free bovine serum albumin], incubated with 0.5 ml of BMS30943 of various concentrations in KRPH buffer for 15 min. The medium was switched to KRPH buffer containing BMS30943, 5 mM D-glucose and 0.5 µCi/well of 2-deoxy-D [3H]-glucose for the last 15 min or 5 min. Myotubes were then washed three times with ice-cold PBS and lysed with 0.5 M NaOH and 0.1% SDS. Cell lysates were neutralized with HCl. Radioactivity was measured by liquid scintillation counting.

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

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

Solubility in Formulation 1: ≥ 2.08 mg/mL (4.38 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (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 20.8 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.08 mg/mL (4.38 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 20.8 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.)