BAM7

Catalog No.S7105 Batch:S710501

Technical Data

Formula	C₂₁H₁₉N₅O₂S
Molecular Weight	405.47	CAS No.	331244-89-4
Solubility (25°C)*	In vitro	DMSO	2 mg/mL (4.93 mM)
		Water	Insoluble
		Ethanol	Insoluble
* <1 mg/ml means slightly soluble or insoluble. * Please note that Selleck tests the solubility of all compounds in-house, and the actual solubility may differ slightly from published values. This is normal and is due to slight batch-to-batch variations. * Room temperature shipping (Stability testing shows this product can be shipped without any cooling measures.)

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Biological Activity

Description

BAM 7 is a direct and selective activator of proapoptotic Bax with EC50 of 3.3 μM.

Targets

Bax ^[1]

3.3 μM

In vitro

BAM7 directly binds the previously uncharacterized BH3-binding groove at the N-terminal face of BAX. BAM7 is selective for the BH3-binding groove at the N-terminal face of BAX. BAM7 directly interacts with BAX at the very surface used by the BIM BH3 helix to trigger BAX activation. BAM7 results in functional BAX activation. BAM7 triggers the conversion of BAX from monomer to oligomer in a dose- and time-responsive manner, the kinetics of which approach saturation at a 1:8 dose ratio of BAX:BAM7. BAM7 triggers in vitro BAX oligomerization, BAX-mediated pore formation and BAX-dependent cell death. BAM7 selectively induces BAX-mediated apoptosis by triggering the hallmark features of intracellular BAX activation. BAM7 only kills the cell line that contains BAX, eliciting the biochemical and morphologic features of BAX-mediated apoptosis. ^[1]

Features

Does not interact with the BH3-binding pocket of antiapoptotic proteins or proapoptotic BAK and induces cell death in a BAX-dependent fashion.

Protocol (from reference)

Kinase Assay:^{^[1]}	Fluorescence polarization binding assays Direct binding curves are first generated by incubating FITC-BIM SAHB (50 nM) with serial dilutions of fulllength BAX, BCL-XLΔC, MCL-1ΔNΔC, BFL-1/A1ΔC or BAKΔC and fluorescence polarization measured at 20 minutes on a SpectraMax M5 microplate reader. For competition assays, a serial dilution of small molecule or acetylated BIM SAHB (Ac-BIM SAHB) is combined with FITC-BIM SAHB (50 nM), followed by the addition of recombinant protein at ~EC75 concentration, as determined by the direct binding assay (BAX, BAKΔC: 500 nM; BCL-XLΔC, MCL-1ΔNΔC, BFL-1/A1ΔC: 200 nM). Fluorescence polarization is measured at 20 minutes and IC50 values calculated by nonlinear regression analysis of competitive binding curves using Prism software.
Cell Assay:^{^[1]}	Cell lines MEFs Concentrations ~15 μM Incubation Time 24 h Method MEFs (2.5 × 10³ cells per well) are seeded in 96-well opaque plates for 18-24 h and then incubated with serial dilutions of BAM7, ANA-BAM16 or vehicle (0.15% (v/v) DMSO) in DMEM at 37 °C in a final volume of 100 μL. Cell viability is assayed at 24 h by addition of CellTiter-Glo reagent according to the manufacturer’s protocol, and luminescence is measured using a SpectraMax M5 microplate reader. Viability assays are performed in at least triplicate, and the data are normalized to vehicle-treated control wells.

Kinase Assay:^{^[1]}

Fluorescence polarization binding assays
Direct binding curves are first generated by incubating FITC-BIM SAHB (50 nM) with serial dilutions of fulllength BAX, BCL-XLΔC, MCL-1ΔNΔC, BFL-1/A1ΔC or BAKΔC and fluorescence polarization measured at 20 minutes on a SpectraMax M5 microplate reader. For competition assays, a serial dilution of small molecule or acetylated BIM SAHB (Ac-BIM SAHB) is combined with FITC-BIM SAHB (50 nM), followed by the addition of recombinant protein at ~EC75 concentration, as determined by the direct binding assay (BAX, BAKΔC: 500 nM; BCL-XLΔC, MCL-1ΔNΔC, BFL-1/A1ΔC: 200 nM). Fluorescence polarization is measured at 20 minutes and IC50 values calculated by nonlinear regression analysis of competitive binding curves using Prism software.

Cell Assay:^{^[1]}

Cell lines
MEFs
Concentrations
~15 μM
Incubation Time
24 h
Method
MEFs (2.5 × 10³ cells per well) are seeded in 96-well opaque plates for 18-24 h and then incubated with serial dilutions of BAM7, ANA-BAM16 or vehicle (0.15% (v/v) DMSO) in DMEM at 37 °C in a final volume of 100 μL. Cell viability is assayed at 24 h by addition of CellTiter-Glo reagent according to the manufacturer’s protocol, and luminescence is measured using a SpectraMax M5 microplate reader. Viability assays are performed in at least triplicate, and the data are normalized to vehicle-treated control wells.

References

[1] Gavathiotis E, et al. Nat Chem Biol, 2012, 8(7), 639-645.

Selleck's BAM7 has been cited by 2 publications

Small-molecule allosteric inhibitors of BAX. [ Nat Chem Biol, 2019, 15(4):322-330]	PubMed: 30718816
Survival of midbrain dopamine neurons depends on the Bcl2 factor Mcl1 [ Cell Death Discov, 2018, 4:107]	PubMed: 30479840

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SHIPPING AND STORAGE
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