Bax (IC50 = 3.3 μM)
BAX (proapoptotic BCL-2 family member) – binds to the BH3 trigger site (α1-α6 groove) with IC50 = 3.3 μM (competitive fluorescence polarization assay against FITC-BIM SAHB); resynthesized BAM7 showed IC50 = 4.4 μM. No binding detected to BCL-XLΔC, MCL-1ΔNΔC, BFL-1/A1ΔC, or BAKΔC at up to 50 μM dosing.

BAM7 directly binds the previously uncharacterized BH3-binding groove at the N-terminal face of BAX. At the N-terminal face of BAX, the BH3-binding groove is specifically recognized by BAM7. In fact, BAM7 directly interacts with BAX at the surface where the BIM BH3 helix activates BAX. Functional activation of BAX is the result of BAM7. In a dose- and time-responsive manner, BAM7 causes BAX to change from monomer to oligomer, with the kinetics approaching saturation at a dose ratio of 1:8 for BAX:BAM7. In vitro BAX oligomerization, BAX-mediated pore formation, and BAX-dependent cell death are all sparked by BAM7. BAM7 selectively induces BAX-mediated apoptosis by triggering the hallmark features of intracellular BAX activation. The biochemical and morphological characteristics of BAX-mediated apoptosis are only induced by BAM7 in the cell line that contains BAX. [1]

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BAM7 (10-40 μM) induced dose- and time-responsive conversion of monomeric BAX (5 μM) to oligomeric species as detected by size-exclusion chromatography; at a 1:8 BAX:BAM7 ratio, oligomerization approached saturation. The inactive analog ANA-BAM16 and BAXK21E mutant showed little to no effect. [1]
BAM7 (5-40 μM) triggered dose-responsive BAX-mediated release of entrapped fluorophore (ANTS/DPX) from liposomes, indicating functional pore formation; BAX alone, BAM7 alone, or combinations of BAX/ANA-BAM16 or BAXK21E/BAM7 had no effect. [1]
BAM7 (15 μM) induced dose-responsive exposure of the N-terminal BAX activation epitope (6A7 antibody) in Bak−/− MEFs, as detected by immunoprecipitation. [1]
BAM7 (15 μM) caused dose-responsive mitochondrial cytochrome c release in Bak−/− MEFs, as assessed by western blot of supernatant and pellet fractions. [1]
BAM7 (15 μM) induced BAX translocation from cytosol to mitochondria in Bax−/−Bak−/− MEFs reconstituted with EGFP-BAX, observed by confocal microscopy. [1]

In order to create direct binding curves, FITC-BIM SAHB (50 nM) is first incubated with serial dilutions of full-length BAX, BCL-XLΔC, MCL-1ΔNΔC, BFL-1/A1ΔC or BAKΔC, or BAKC, with fluorescence polarization being measured after 20 minutes on a SpectraMax M5 microplate reader. For competition assays, small molecules or acetylated BIM SAHB (Ac-BIM SAHB) are mixed with FITC-BIM SAHB (50 nM), and then recombinant protein is added at an ~EC75 concentration (BAX, BAKΔC: 500 nM; BCL-XLΔC, MCL-1ΔNΔC, BFL-1/A1ΔC: 200 nM) determined by the direct binding assay. IC50 values are determined by nonlinear regression analysis of competitive binding curves using Prism software after 20 minutes of fluorescence polarization measurement.

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Competitive fluorescence polarization binding assay: Recombinant BAX (50 nM) was incubated with FITC-labeled BIM SAHB (10 nM) and various concentrations of BAM7 (0.01–100 μM) in assay buffer (PBS, 0.01% Tween-20, pH 7.4). After equilibration, fluorescence polarization was measured at excitation 485 nm and emission 530 nm. IC50 values were calculated by nonlinear regression using a four-parameter logistic equation. Similar assays were performed for BCL-XLΔC, MCL-1ΔNΔC, BFL-1/A1ΔC, and BAKΔC using FITC-BIM SAHB and unlabeled competitors. [1]
NMR binding analysis: Uniformly 15N-labeled full-length BAX or BAXK21E (100 μM) was titrated with BAM7 (0–200 μM) in 20 mM phosphate buffer pH 6.5, 50 mM NaCl, 1 mM DTT, 10% D2O. 2D 1H-15N HSQC spectra were acquired at 25°C on a 600 MHz spectrometer. Chemical shift changes were plotted and compared to threshold values. [1]
Size-exclusion chromatography oligomerization assay: Monomeric BAX (5 μM) was incubated with BAM7 (10–40 μM) at 25°C for 0–16 h, then separated on a Superdex 200 column equilibrated with 20 mM HEPES pH 7.2, 150 mM NaCl. Eluted fractions were monitored by absorbance at 280 nm and western blot. [1]
Liposomal release assay: Liposomes composed of 50% POPC, 30% POPS, and 20% cholesterol were loaded with 50 mM ANTS and 20 mM DPX. BAX (1 μM) was pre-incubated with BAM7 (5–40 μM) for 30 min, then added to liposomes (final lipid concentration 50 μM). Fluorescence (ex 355 nm, em 520 nm) was monitored over time; maximal release determined by Triton X-100. [1]

MEFs (2.5 × 103 cells per well) are seeded in 96-well opaque plates for 18–24 h, after which they are incubated at 37 °C in a final volume of 100 L with serial dilutions of BAM7, ANA-BAM16, or vehicle (0.15% (v/v) DMSO). According to the manufacturer's instructions, CellTiter-Glo reagent is added to assay cell viability at 24 hours, and luminescence is measured using a SpectraMax M5 microplate reader. At least three replicates of the viability assays are carried out, and the data are then normalized to the vehicle-treated control wells.

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Cell viability assay: Bak−/−, Bax−/−, and Bax−/−Bak−/− MEFs were seeded in 96-well plates (2.5×10³ cells/well) for 18–24 h, then treated with serial dilutions of BAM7 (0–50 μM) or ANA-BAM16 (0–50 μM) in DMEM with 0.15% DMSO for 24 h. Viability was measured by CellTiter-Glo luminescent assay. BAM7 selectively impaired viability of Bak−/− MEFs (dose-dependent up to ~15 μM), with no effect on Bax−/− or Bax−/−Bak−/− MEFs. ANA-BAM16 had no effect. In Bax−/−Bak−/− MEFs reconstituted with wild-type BAX or BAXK21E, BAM7 killed only wild-type BAX-expressing cells. [1]
BAX conformational change (6A7 immunoprecipitation): Bak−/− MEFs (5×10⁵ cells) were treated with BAM7 (0–15 μM) for 6 h, lysed in CHAPS buffer, and immunoprecipitated with 6A7 antibody. Western blot with N20 anti-BAX antibody detected activated BAX. [1]
Cytochrome c release assay: Bak−/− MEFs treated with BAM7 (0–15 μM) for 15 h were digitonin-permeabilized, separated into supernatant (cytosolic) and pellet (mitochondrial) fractions, and analyzed by western blot for cytochrome c. [1]
BAX translocation assay: Bax−/−Bak−/− MEFs were transfected with EGFP-BAX (60% positive), treated with BAM7 (0–15 μM) for 24 h, stained with MitoTracker, and imaged by confocal microscopy. Percentage of cells with mitochondrial BAX localization was quantified. [1]
Live-cell morphology: Bak−/− MEFs were treated with BAM7 (15 μM) and time-lapse images captured to document apoptotic morphology (shrinkage, blebbing). [1]

[1]. Direct and selective small-molecule activation of proapoptotic BAX. Nat Chem Biol. 2012 Jul;8(7):639-45.

BAM7 is a first-in-class small-molecule direct activator of proapoptotic BAX. Its mechanism involves binding to a novel BH3 trigger site on BAX (distinct from the canonical BH3-binding groove of antiapoptotic proteins), inducing allosteric changes including α1-α2 loop displacement, 6A7 epitope exposure, BH3 domain mobilization, and C-terminal α9 helix rearrangement, ultimately leading to BAX oligomerization and mitochondrial outer membrane permeabilization. BAM7 is highly selective for BAX over BAK and antiapoptotic BCL-2 members. The compound serves as a chemical tool to study direct BAX activation in apoptosis and has potential therapeutic implications for cancers with apoptotic blocks. Negative control reagents include ANA-BAM16 (a structural analog with meta-chloro substitution that does not bind BAX) and the BAXK21E point mutant. [1]

C21H19N5O2S

405.476

405.125

C, 62.21; H, 4.72; N, 17.27; O, 7.89; S, 7.91

331244-89-4

331244-89-4

3101542

Pink to red solid powder

1.3±0.1 g/cm3

567.3±52.0 °C at 760 mmHg

296.9±30.7 °C

0.0±1.6 mmHg at 25°C

1.685

3.18

1

7

6

29

649

0

CCOC1=CC=CC=C1N/N=C2C(C)=NN(C3=NC(C4=CC=CC=C4)=CS3)C\2=O

WRLVHADVOGFZOZ-UHFFFAOYSA-N

InChI=1S/C21H19N5O2S/c1-3-28-18-12-8-7-11-16(18)23-24-19-14(2)25-26(20(19)27)21-22-17(13-29-21)15-9-5-4-6-10-15/h4-13,25H,3H2,1-2H3

4-[(2-ethoxyphenyl)diazenyl]-5-methyl-2-(4-phenyl-1,3-thiazol-2-yl)-1H-pyrazol-3-one

BAM 7; BAM7; BAM-7

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: 2~5 mg/mL (4.9~12.3 mM)

Solubility in Formulation 1: ≥ 0.5 mg/mL (1.23 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 5.0 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.)