Over a broad dose range, BAM 15 can raise O2 consumption without raising ROS. The cellular O2 consumption rate (OCR) was found to be increased by BAM 15 at low doses (100 nM to 1 μM) in a manner similar to that of FCCP, despite the structural differences between the two compounds. However, BAM 15 was also able to sustain high rates of uncoupled respiration in a variety of cell lines at higher concentrations (1 μM to 50 μM). BAM 15 was able to increase mitochondrial respiration in myoblasts and hepatocytes in the presence of oligomycin, and it was able to do so across a wider concentration range than FCCP. Mitochondrial enlargement caused by BAM 15 indicates that it is a protonophore. When supplied at concentrations up to 50 μM, BAM15-treated cells were more viable than FCCP-treated cells [1].

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In Vitro: BAM15 (100 nM – 50 µM) increased oxygen consumption rate (OCR) in a broad concentration range across multiple cell types (L6 myoblasts, rat primary cardiomyocytes, mouse C2C12 myoblasts, normal murine liver cells, and human primary fibroblasts), maintaining uncoupled respiration at higher rates than FCCP at concentrations >1 µM [1].
BAM15 (0.1–10 µM) fully stimulated mitochondrial respiration in the presence of the ATP synthase inhibitor oligomycin, indicating genuine uncoupling activity [1].
BAM15 (1 µM and 10 µM) depolarized the mitochondrial membrane potential in L6 cells as measured by tetramethylrhodamine (TMRM) fluorescence, comparable to FCCP [1].
In isolated mouse liver mitochondria, BAM15 (1–64 µM) increased oxygen consumption on complex I substrates (pyruvate+malate) and complex II substrate (succinate+rotenone) with similar potency to FCCP [1].
BAM15 (5 µM) did not alter electron flow through complexes I, II, III, or IV in isolated mitochondria, as shown by sequential injection of rotenone, succinate, antimycin A, and TMPD/ascorbate [1].
BAM15 (10 µM) induced proton‑dependent mitochondrial swelling in isotonic potassium acetate buffer containing valinomycin, confirming its protonophore activity [1].
BAM15 (1 µM) increased respiration in permeabilized C2C12 cells even in the presence of the adenine nucleotide translocase (ANT) inhibitor carboxyatractyloside, indicating that its uncoupling action is ANT‑independent [1].
BAM15 (1 µM and 10 µM) did not alter plasma membrane electrophysiology: it caused no inward current under voltage clamp and no membrane depolarization under current clamp in L6 cells, in contrast to FCCP which induced significant changes [1].
BAM15 was less cytotoxic than FCCP across a broad concentration range up to 50 µM in NMuLi, L6, C2C12, and primary cardiomyocytes, as measured by MTT or crystal violet assays [1].
BAM15 did not increase reactive oxygen species (ROS) production in L6 cells at the screening concentration (0.5 ng/µL) as measured by CM‑H₂OCFDA fluorescence [1].

In mice administered BAM 15, there was protection against renal damage compared to those treated with a vehicle. Additionally, 24 and 48 hours after ischemia, the animals' plasma creatinine levels were lower, there was less obstruction and immune cell infiltration, less tubular necrosis, and less brush border villus depletion.
In Vivo: Male C57BL/6 mice (8‑week‑old) were pretreated with BAM15 (1 or 5 mg/kg, intraperitoneal) 1 hour before bilateral renal ischemia (26 min) followed by 24 h or 48 h reperfusion. BAM15 dose‑dependently protected against kidney injury: plasma creatinine levels were significantly reduced at 24 h and 48 h after reperfusion compared to vehicle‑treated controls [1].
Histological analysis at 48 h post‑reperfusion showed that BAM15 pretreatment decreased acute proximal tubular necrosis, reduced depletion of brush border villi, less obstruction of proximal tubules, and reduced leukocyte infiltration (CD45+ cells) into the kidney medulla [1].
Sham‑operated mice underwent identical surgery without renal pedicle clamping and showed normal kidney function [1].

Cell Assay: Non‑quantitative oxygen consumption assay: L6 myoblasts (500,000 cells/well) were seeded in 96‑well Oxygen Biosensor plates containing an O₂‑sensitive ruthenium dye. Cells were incubated with library compounds (5 µg/mL) or vehicle (DMSO), and fluorescence (ex 485 nm/em 630 nm) was recorded over 45–90 min at 37 °C. FCCP was used as positive control [1].
ROS production assay: L6 myoblasts were co‑incubated with 7.5 µM CM‑H₂OCFDA and 0.5 ng/µL test compound in Krebs‑Ringer phosphate buffer with 25 mM glucose for 1 h at 37 °C. After washing, fluorescence (ex 495 nm/em 530 nm) was measured. 100 mM H₂O₂ served as positive control. Compounds increasing ROS >20% were eliminated [1].
Seahorse XF24/96 analysis: Cells (NMuLi, C2C12, L6, primary cardiomyocytes, human fibroblasts) were seeded in XF plates and allowed to adhere for 24 h. Before assay, medium was changed to unbuffered DMEM (pH 7.4) containing pyruvate and glutamine. Compounds were injected, and OCR and ECAR were measured using 2‑min measurement periods. For isolated mitochondria, 5 µg mitochondrial protein in MAS buffer was loaded into XF24 plates, centrifuged at 2000×g for 15 min, then incubated with substrates and uncouplers [1].
Mitochondrial membrane potential in cells: L6 cells were loaded with 125 nM TMRM for 30 min, then treated with BAM15 or FCCP for 10 min before flow cytometry analysis (PE channel) [1].
Mitochondrial membrane potential in isolated mitochondria: Isolated liver mitochondria were incubated with 200 nM TMRM in MASₛᵣ₀ buffer (supplemented with 10 mM succinate, 1 µM rotenone, 1 µM oligomycin) for 20 min at 25 °C, then centrifuged and resuspended. After adding uncouplers and incubating for 20 min, mitochondria were centrifuged and supernatant TMRM fluorescence (ex 545/em 580) was measured [1].
Mitochondrial swelling assay: Isolated liver mitochondria (0.25 mg/mL) were added to isotonic acetate buffer (145 mM potassium acetate, 5 mM Tris‑HCl, 0.5 mM EDTA, 3 µM valinomycin, 1 µM rotenone, pH 7.4). Absorbance at 600 nm was measured for 60 s before and after addition of 10 µM uncoupler. Swelling indicated protonophore activity [1].
Plasma membrane electrophysiology: Whole‑cell voltage clamp and current clamp recordings were performed on L6 cells using borosilicate glass patch pipettes (3–5 MΩ) and an Axopatch 200B amplifier. Internal solution: 120 mM KCH₂SO₃, 4 mM NaCl, 1 mM MgCl₂, 0.5 mM CaCl₂, 10 mM HEPES, 10 mM EGTA, 3 mM ATP‑Mg, 0.3 mM GTP‑Tris (pH 7.2). Bath solution: 140 mM NaCl, 3 mM KCl, 2 mM MgCl₂, 2 mM CaCl₂, 10 mM HEPES, 10 mM glucose (pH 7.3). For voltage clamp, cells were held at −70 mV with a 750‑ms ramp from −150 mV to +80 mV applied every 10 s. Conductance was measured between −130 mV and −60 mV. For current clamp, cells were recorded at resting membrane potential [1].
Cytotoxicity assay: Cells (5000/well for NMuLi, L6, C2C12; 10,000/well for primary cardiomyocytes) were seeded in 96‑well plates and treated with compounds for 48 h. Viability was measured by MTT or crystal violet (0.5% w/v in 50% methanol) staining, and absorbance was read. Results expressed as percentage of DMSO‑treated controls [1].

Animal Protocol: Male C57BL/6 mice (8‑weeks‑old, from National Cancer Institute) were anesthetized with ketamine (120 mg/kg), xylazine (12 mg/kg), and atropine (0.324 mg/kg) intraperitoneally. Bilateral renal ischemia‑reperfusion injury was induced by clamping both renal pedicles for 26 min, followed by reperfusion for 24 h or 48 h. Core body temperature was maintained at 34–36 °C during surgery, and mice were housed in a warming incubator (30–32 °C) during recovery. BAM15 was prepared in 3% DMSO in 50% PEG400 and administered intraperitoneally at 1 or 5 mg/kg 1 hour before ischemia. Vehicle control mice received the same solution. Sham‑operated mice underwent identical surgery without clamping. Plasma creatinine was measured colorimetrically. For histology, kidneys were fixed in periodate‑lysine‑paraformaldehyde, embedded in paraffin, sectioned, and stained with H&E. Acute tubular necrosis was scored blinded based on percentage of outer medulla tubules with pink casts: 1 (<10%), 2 (10–25%), 3 (25–75%), 4 (>75%). Kidney leukocyte content was analyzed by flow cytometry using anti‑CD45 and other markers after enzymatic digestion [1].

Toxicity/Toxicokinetics: In vitro cytotoxicity: BAM15 was less cytotoxic than FCCP across a broad dose range up to 50 µM in NMuLi, L6, C2C12, and primary rat cardiomyocytes; cell viability remained higher with BAM15 treatment compared to FCCP at equivalent concentrations [1].
In vivo tolerability: No overt toxicity or adverse effects were reported for mice treated with BAM15 at 1 or 5 mg/kg (i.p.) in the renal ischemia‑reperfusion study; all mice survived the protocol and showed normal behavior [1].
Compound did not increase reactive oxygen species production in L6 cells at the screening concentration, indicating low oxidative stress liability [1].

[1]. Identification of a novel mitochondrial uncoupler that does not depolarize the plasma membrane. Mol Metab. 2013 Nov 28;3(2):114-23.

Additional Info: BAM15 is a structurally novel mitochondrial protonophore uncoupler unrelated to FCCP or DNP. It exhibits equal potency to FCCP but has a broader effective concentration range, does not depolarize the plasma membrane, and has lower cytotoxicity. Its uncoupling activity is independent of the adenine nucleotide translocase (ANT) and does not alter electron flow through complexes I‑IV. BAM15 shows in vivo efficacy in a mouse model of acute kidney ischemia‑reperfusion injury, reducing plasma creatinine, tubular necrosis, and immune cell infiltration. These properties make BAM15 a valuable tool for studying mitochondrial function without off‑target plasma membrane effects and a potential therapeutic candidate for diseases involving mitochondrial oxidative stress, such as ischemia‑reperfusion injury, obesity, and neurodegeneration [1].

C16H10F2N6O

340.29

340.088

210302-17-3

565708

Light yellow to yellow solid powder

1.5±0.1 g/cm3

421.7±55.0 °C at 760 mmHg

208.9±31.5 °C

0.0±1.0 mmHg at 25°C

1.728

4.19

2

9

4

25

409

0

OEGJBRZAJRPPHL-UHFFFAOYSA-N

InChI=1S/C16H10F2N6O/c17-9-5-1-3-7-11(9)19-13-14(20-12-8-4-2-6-10(12)18)22-16-15(21-13)23-25-24-16/h1-8H,(H,19,21,23)(H,20,22,24)

5-N,6-N-bis(2-fluorophenyl)-[1,2,5]oxadiazolo[3,4-b]pyrazine-5,6-diamine

BAM-15 BAM 15 BAM15

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 (~146.93 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (7.35 mM) (saturation unknown) in 10% DMSO + 40% PEG300 +5% Tween-80 + 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 25.0 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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 (Please use freshly prepared in vivo formulations for optimal results.)