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 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].

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1. From [1]: BMS-309403 (0.01-10 μM) dose-dependently inhibited the binding of fluorescently labeled fatty acid (12-(9-anthroyloxy)stearic acid, AOS) to recombinant human aFABP, with a Ki value of 0.23 μM in fluorescence polarization assays; the compound showed >40-fold selectivity for aFABP over L-FABP (Ki > 10 μM) and I-FABP (Ki > 10 μM), and no significant interaction with heart FABP (H-FABP/FABP3) at concentrations up to 5 μM [1]
2. From [2]: In differentiated C2C12 myotubes, BMS-309403 (1-20 μM) dose-dependently stimulated 2-deoxy-D-glucose (2-DG) uptake, with a maximal 2.5-fold increase at 10 μM (vs. basal uptake); this effect was accompanied by phosphorylation of AMPKα (Thr172) and its downstream substrate acetyl-CoA carboxylase (ACC, Ser79) in a time- and concentration-dependent manner (peak phosphorylation at 10 μM, 30 minutes); pretreatment with the AMPK inhibitor compound C (10 μM) completely abolished the glucose uptake induced by BMS-309403 [2]
3. From [3]: In cultured human umbilical vein endothelial cells (HUVECs), BMS-309403 (1-10 μM) dose-dependently increased endothelial nitric oxide synthase (eNOS) phosphorylation (Ser1177) by 2.2-fold at 10 μM (western blot), and enhanced nitric oxide (NO) production by 60% (Griess assay); the compound also reduced reactive oxygen species (ROS) generation by 45% (DCFH-DA assay) and inhibited TNF-α-induced expression of vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1) by 55% and 48% respectively (qPCR and western blot) at 5 μM [3]

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]

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1. From [3]: In apolipoprotein E-deficient (ApoE^-/-) mice fed a high-fat diet (HFD), chronic oral administration of BMS-309403 (10 mg/kg once daily for 12 weeks) significantly improved endothelium-dependent vasodilation of aortic rings (acetylcholine-induced relaxation increased from 35% (vehicle) to 70% (treated), assessed by myograph); the treatment also reduced atherosclerotic lesion area in the aortic root by 40% (Oil Red O staining) and decreased plasma levels of pro-inflammatory cytokines (TNF-α: -35%, IL-6: -40%) and lipid peroxidation markers (malondialdehyde, MDA: -30%) compared to vehicle controls [3]
2. From [3]: BMS-309403 (10 mg/kg PO qd) increased eNOS phosphorylation (Ser1177) in the aortic endothelium of ApoE^-/- mice by 2.0-fold (immunohistochemistry and western blot) and reduced vascular superoxide production by 50% (dihydroethidium staining); no significant changes in body weight, fasting blood glucose, or plasma lipid levels (total cholesterol, triglycerides) were observed between treated and vehicle groups [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.

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1. From [1]: aFABP fatty acid binding fluorescence polarization assay
Recombinant human aFABP protein (purified to homogeneity) was diluted in assay buffer (50 mM Tris-HCl, 150 mM NaCl, pH 7.4) to a final concentration of 0.5 μM; fluorescent fatty acid probe AOS was added to a final concentration of 0.2 μM, and serial dilutions of BMS-309403 (0.001-10 μM) were added to the reaction mixture; the mixture was incubated at 25°C for 30 minutes, and fluorescence polarization was measured using a plate reader (excitation 360 nm, emission 460 nm); polarization values were converted to binding inhibition percentages, and Ki values were calculated using the Cheng-Prusoff equation from competition binding curves [1]
2. From [2]: AMPK kinase activity assay
AMPK immunoprecipitates were prepared from BMS-309403-treated C2C12 myotubes using an anti-AMPKα antibody; the immunocomplexes were incubated with recombinant ACC peptide substrate (SAMS peptide) and [γ-³²P]ATP in kinase buffer (25 mM Tris-HCl, 10 mM MgCl2, 1 mM DTT, pH 7.4) at 30°C for 30 minutes; the reaction was terminated by adding SDS sample buffer, and phosphorylated peptide products were separated by SDS-PAGE and detected by autoradiography; kinase activity was quantified by densitometry and normalized to the amount of immunoprecipitated AMPK [2]

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.

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1. From [2]: C2C12 myotube glucose uptake assay
C2C12 myoblasts were seeded in 24-well plates and differentiated into myotubes by culturing in DMEM containing 2% horse serum for 7 days; the cells were serum-starved for 4 hours, then treated with serial dilutions of BMS-309403 (1-20 μM) for 1 hour; 2-deoxy-D-[³H]glucose (1 μCi/well) was added, and the cells were incubated for 10 minutes at 37°C; the reaction was stopped by washing with cold PBS, and intracellular radioactivity was measured by liquid scintillation counting; non-specific uptake was determined in the presence of cytochalasin B (10 μM), and specific glucose uptake was calculated by subtracting non-specific from total uptake [2]
2. From [2]: AMPK and ACC phosphorylation western blot assay
C2C12 myotubes were treated with BMS-309403 (1-20 μM) for 15-60 minutes; whole-cell lysates were prepared using RIPA buffer, separated by SDS-PAGE, and transferred to PVDF membranes; the membranes were probed with primary antibodies against phospho-AMPKα (Thr172), total AMPKα, phospho-ACC (Ser79), and total ACC, followed by horseradish peroxidase (HRP)-conjugated secondary antibodies; chemiluminescence signals were detected, and band intensities were quantified by densitometry and normalized to total protein levels [2]
3. From [3]: HUVEC eNOS activation and ROS detection assay
HUVECs were seeded in 6-well plates and cultured to confluency; the cells were treated with BMS-309403 (1-10 μM) for 2 hours, with or without TNF-α (10 ng/mL) stimulation for 4 hours; for eNOS phosphorylation analysis, cell lysates were prepared for western blot using anti-phospho-eNOS (Ser1177) and total eNOS antibodies; for ROS measurement, cells were loaded with DCFH-DA (10 μM) for 30 minutes, and fluorescence intensity was measured by flow cytometry (excitation 488 nm, emission 525 nm); NO production was determined by measuring nitrite levels in the culture supernatant using the Griess reaction [3]
4. From [3]: HUVEC adhesion molecule expression qPCR assay
Total RNA was extracted from BMS-309403 and TNF-α-treated HUVECs using an RNA isolation kit; cDNA was synthesized by reverse transcription, and qPCR was performed with gene-specific primers for VCAM-1, ICAM-1, and GAPDH (housekeeping gene); relative gene expression levels were calculated using the 2^-ΔΔCt method and normalized to GAPDH [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, food intake, or organ weight (liver, kidney, heart) were observed compared with the vector control group; serum liver function indicators (ALT, AST) and kidney function indicators (BUN, creatinine) levels were within the normal range, and no evidence of hepatotoxicity or nephrotoxicity was observed [3]. 2. According to [2]: After treatment with BMS-309403 (concentration up to 20 μM) for 24 hours, no significant cytotoxicity was observed in C2C12 myotube cells, and cell viability was >90% as detected by MTT assay [2]. 3. According to [3]: BMS-309403 (concentration up to 10 μM) After incubation with μM for 24 hours, it did not affect HUVEC cell viability (MTT assay) or induce apoptosis (Annexin V/PI staining) [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.

This article reports for the first time a class of bifonazole compounds that can bind to adipocyte fatty acid-binding proteins (aFABP or aP2) with nanomolar binding forces and exhibit selectivity of up to a thousand-fold for muscle fatty acid-binding proteins and epidermal fatty acid-binding proteins. In addition, we synthesized a new radioligand to determine its binding to these three fatty acid-binding proteins. [1]

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BMS309403 is a bifonazole compound that inhibits fatty acid-binding protein 4 (FABP4) and is considered a lead compound for the treatment of obesity-related cardiovascular and metabolic diseases. We found that BMS309403 has an off-target activity, namely, it can stimulate C2C12 myotube cells to take up glucose in a time- and dose-dependent manner by activating the AMP-activated protein kinase (AMPK) signaling pathway, but this process is independent of FABP. Further analysis showed that BMS309403 activates AMPK by increasing the intracellular AMP/ATP ratio and decreasing the mitochondrial membrane potential. These findings provide insights into the mechanism of action of BMS309403. [2]

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Adipocyte fatty acid-binding protein (A-FABP) is upregulated in regenerating endothelial cells and modulates the inflammatory response of macrophages. Hyperlipidemia accelerates endothelial dysfunction associated with regeneration. This study aimed to investigate the role of A-FABP in the pathogenesis of endothelial dysfunction in the aorta of apolipoprotein E-deficient (ApoE-/-) mice and cultured human endothelial cells. Experimental methods: The expression of A-FABP in the aorta of ApoE-/- mice and human endothelial cells was detected by RT-PCR, immunostaining, and Western blotting. The total protein and phosphorylated protein of endothelial nitric oxide synthase (eNOS) were detected by Western blotting. Changes in isometric tension were measured in the aortic ring of mice. Main results: A-FABP was expressed in the aortic endothelium of ApoE(-/-) mice aged 12 weeks and older, but not in 8-week-old mice or C57 wild-type mice. Compared with age-matched controls, 18-week-old ApoE(-/-) mice showed reduced endothelial-dependent vasodilation in the aorta in response to acetylcholine, UK14304 (a selective α2-adrenergic receptor agonist), and A23187 (a calcium ionocarrier), as well as decreased levels of phosphorylated and total eNOS proteins. In 12-week-old mice, treatment with the A-FABP inhibitor BMS309403 for 6 weeks improved endothelial function, increased phosphorylated and total eNOS levels, and decreased plasma triglyceride levels, but had no effect on endothelial-independent vasodilation. The beneficial effect of BMS309403 on UK14304-induced vasodilation 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, which BMS309403 reversed. [3]

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1. From [1]: BMS-309403 is a potent and selective bifenazole aFABP (FABP4) inhibitor that was discovered by targeting the fatty acid binding pocket of aFABP through a structure-based drug design approach; it is one of the most potent aFABP inhibitors reported in this series and has high selectivity for other FABP subtypes. [1]
2. According to [2], the mechanism by which BMS-309403 stimulates glucose uptake in myotubes involves the activation of the AMPK signaling pathway, which is independent of the insulin signaling pathway (without effect on IRS-1 or Akt phosphorylation); the compound increases AMPK phosphorylation by increasing the intracellular AMP/ATP ratio, which may be achieved by blocking aFABP to inhibit fatty acid metabolism [2]
3. According to [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, reduce oxidative stress (ROS), and inhibit the expression of pro-inflammatory adhesion molecules, thereby alleviating endothelial dysfunction and the development of atherosclerotic lesions [3]
4. BMS-309403 has potential applications in the treatment of metabolic diseases (such as type 2 diabetes and insulin resistance) and cardiovascular diseases (such as atherosclerosis) due to its dual effects of inhibiting aFABP and activating AMPK; it is currently still in the preclinical research stage and has not yet been submitted for FDA approval or entered the clinical trial stage [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

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.)