Bax; Bak

MSN-50 (0~10 μM) decreases baseline dye release in a concentration-dependent way. Actinomycin D and Star cytokine MSN-50 (5 μM; BMK cells) to inhibit tBid/Bax-mediated mitochondrial outer membrane permeabilization (MOMP) in a concentration-dependent manner. cell decontamination caused by STS [1][2].

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Nie et al. reported new five chemicals (molecular weight (MW) is ranging from 206–689) inhibiting the pore-forming activity of Bax. These chemicals were selected from a group of 87 compounds that have binding affinity to Mcl-1, an anti-apoptotic member of the Bcl-2 family of proteins that shares Bcl-2 homology (BH) domains with Bax. The names of these compounds are BJ-1, BJ-1-BP, MSN-50, MSN-125, and DAN004. Compounds were initially screened via liposome dye release assay to determine whether they could inhibit Bax-mediated membrane permeabilization. IC50 values from the liposome dye release assay were found to be 9, 6, 6, 4, and 0.7 µM respectively. MSN-50, MSN-125, and DAN004 were found to be successful in inhibiting MOMP (mitochondrial outer membrane permeabilization) induced by Bax using isolated mitochondria in vitro. Since these inhibitors inhibited MOMP of bax−/−bak+/+ cells in addition to Bax proficient cells, the authors proposed that these inhibitors are Bax/Bak dual inhibitors. MSN-50 (5 µM) and MSN-125 (10 µM) inhibited apoptosis induced by actinomycin D and staurosporin (STS) in BMK (baby mouse kidney) cells. However, these chemicals were cytotoxic at concentrations of 20 µM and higher in the culture medium. In cultured primary embryonic mouse brain cortical neurons, MSN-125 (at 5 µM) also successfully reduced cell death if added immediately after glutamate excitotoxicity was induced (25 or 100 mM glutamate for 30 min). The authors propose that these new small molecules may be useful in many applications, but protection from traumatic brain injury was emphasized since these chemicals protected primary cultured neurons. The authors also discussed the unexpected off-target effects and issues surrounding the unknown mechanisms of Bax and Bak, which remain problems[2].

To measure mitochondrial permeabilization for constitutively active Bak protein synthesized in SP6 RNA polymerase/reticulocyte lysate-based transcription and translation coupled system, 40 μM MSN-50 was added to 2 μl Bak prior to the addition of mitochondria and cytochrome c release was assayed by ELISA (Zhang et al., 2016). As a control, inhibition was measured after addition of 2 μl Bcl-XL synthesized in vitro instead of MSN-50[1].

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Small Molecule Bax Inhibitor Screen[1]
A collection of benzofuran-based flavonoid-inspired, and several tetrahydroquinoline alkaloid-inspired compounds were screened for inhibition of tBid/Bax-mediated dye release in a MOMP-mimicking liposome permeabilization assay. In the first round 86 compounds were individually added to liposomes and then purified Bax and tBid were added to induce permeabilization of liposomes. Small molecules that inhibited liposome permeabilization with aZ-score >2 were re-assayed manually. The top two compounds were used to select additional compounds for a second round of screening.

Long Term Survival after Replating[1]
For experiments with MSN-125 andMSN-50 male HCT-116 (Wang and Youle, 2012) and BMK cells (Mathew et al., 2008) of unspecified sex were seeded at a cell density of 3000 cells/well in a 96 well plate. For experiments with DAN004 BMK cells were seeded at 750 cells/well in 384 well plates. Cells were plated in in DMEM containing 10% FBS and after adhering, cells were treated with the indicated concentration of the inhibitors for 3–4 hours followed by addition of actinomycin D (ActD) or staurosporine (STS) for another 4 hours. The ActD/STS (+ compound) containing media was then replaced with drug-free media (containing inhibitor) and cells were grown for 4 days (to confluency for DMSO controls) and re-plated into 24 or 96 well plates for MSN and DAN004 inhibitors, respectively. Surviving cells were allowed to grow until the DMSO controls on the same plate just reached confluency and then the plate was stained with crystal violet. Images of the plates were obtained using a flat-bed scanner. For 96 well plates, quantification of surviving cells was done by absorbance measurements at 600 nm as described previously (Brahmbhatt et al., 2016).

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Immunofluorescence[1]
HCT-116 cells were pre-treated with 10 μM MSN-125 for three hours, followed by the addition of actinomycin D (50 ng/ml final) for 24 hours. Cells were fixed using paraformaldehyde and analyzed by immunofluorescence by double staining with primary sheep anti-cytochrome c antibody and mouse anti-Bax 6A7 monoclonal antibodies. Secondary donkey anti-sheep-Alexa Fluor 488 and goat anti-mouse-Alexa Fluor 555 antibodies were used for microscopy. The nuclei of HCT-116 cells were stained with DAPI according to the manufacturer’s instructions. Cells were imaged using a Zeiss LSM710 confocal microscope and associated software.

All animal procedures were performed in accordance with the local standards for animal care and were approved by the animal care committee at Sunnybrook Research Institute. To establish primary neuron cultures of mixed sex, cerebral cortices from male and female mouse embryos (embryonic day E14.5–15) were dissected and cultured for up to 10 days to ensure maturation of neurons as described (Mergenthaler et al., 2012). Neurons from one embryo were considered as one independent “n”. A maximum of 3 embryos of the same litter were used. Briefly, neurons were cultured in Neurobasal-A medium (Life Technologies) supplemented with B-27 (Life Technolgies), 0.5 mM L-glutamine and 25 μM glutamate. The medium was partially replaced on day 6 in culture with Neuro- basal-A supplemented with B-27 and L-glutamine. On day 9, neurons were treated with either 25 μM or 100 μM glutamate in BSS0 (116 mM NaCl, 5.4 mM KCl, 0.8 mM MgSO4, 1 mM NaH2PO4, 26.2 mM NaHCO3, 10 μM glycine, 1.8 mM CaCl2, 10 mM HEPES pH 7.4) for 30 minutes (37°C, 5% CO2) after washing the cultures with PBS. Medium was pooled and added to cultures after the incubation period with or without addition of 5 μM MSN-125. Cell death was analyzed 20–24 hours after glutamate treatment by measuring lactate dehydrogenase release as previously described (Mergenthaler et al., 2012). Briefly, lactate dehydrogenase concentration in medium was analyzed by measuring NADH to NAD+ turnover (absorbance at 340 nm) in a coupled spectrophotometric assay on aTecan M1000 microplate reader at 37°C and normalized to total lactate dehydrogenase levels after volume correction. For each measurement, 200 μl LDH buffer (33 mM KH2PO4, 66 mM K2HPO4) containing 210 μM β-NADH, pH 7.4 were added to 50 μl media supernatant in a 96 well plate. Immediately before starting the measurements, 25 μl LDH buffer containing 22.7 mM sodium pyruvate were added. Total lactate dehydrogenase release was measured after incubating neuronal cultures with 0.5% TritonX-100 for 30 minutes (37° C). All measurements were normalized to control reactions containing 500 units/l l-lactic dehydrogenase (Sigma). Statistical analysis was performed in Prism 5.0 or SPSS 23 (IBM). ANOVA was performed after normality testing.[1]

[1]. A Small-Molecule Inhibitor of Bax and Bak Oligomerization Prevents Genotoxic Cell Death and Promotes Neuroprotection. Cell Chem Biol. 2017;24(4):493-506.e5.

[2]. Pharmacological inhibition of Bax-induced cell death: Bax-inhibiting peptides and small compounds inhibiting Bax. Exp Biol Med (Maywood). 2019;244(8):621-629.

Aberrant apoptosis can lead to acute or chronic degenerative diseases. Mitochondrial outer membrane permeabilization (MOMP) triggered by the oligomerization of the Bcl-2 family proteins Bax/Bak is an irreversible step leading to execution of apoptosis. Here, we describe the discovery of small-molecule inhibitors of Bax/Bak oligomerization that prevent MOMP. We demonstrate that these molecules disrupt multiple, but not all, interactions between Bax dimer interfaces thereby interfering with the formation of higher-order oligomers in the MOM, but not recruitment of Bax to the MOM. Small-molecule inhibition of Bax/Bak oligomerization allowed cells to evade apoptotic stimuli and rescued neurons from death after excitotoxicity, demonstrating that oligomerization of Bax is essential for MOMP. Our discovery of small-molecule Bax/Bak inhibitors provides novel tools for the investigation of the mechanisms leading to MOMP and will ultimately facilitate development of compounds inhibiting Bax/Bak in acute and chronic degenerative diseases.[1]

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Bax induces mitochondria-dependent programed cell death. While cytotoxic drugs activating Bax have been developed for cancer treatment, clinically effective therapeutics suppressing Bax-induced cell death rescuing essential cells have not been developed. This mini-review will summarize previously reported Bax inhibitors including peptides, small compounds, and antibodies. We will discuss potential applications and the future direction of these Bax inhibitors.[2]

C36H38BRN3O6

688.607429027557

687.194

C, 62.79; H, 5.56; Br, 11.60; N, 6.10; O, 13.94

1592908-75-2

1592908-75-2;

154730935

White to off-white solid powder

5.1

2

7

15

46

923

3

BrC1C=CC(=CC=1)CN(C([C@H](CC1C=CC=CC=1)NC(C1C=CC=CC=1)=O)=O)C[C@@H]1[C@H](C2C=CC(=CC=2O1)OCOCCOC)N

QRQAGDGLLUPMLV-IGOOQNSHSA-N

InChI=1S/C36H38BrN3O6/c1-43-18-19-44-24-45-29-16-17-30-32(21-29)46-33(34(30)38)23-40(22-26-12-14-28(37)15-13-26)36(42)31(20-25-8-4-2-5-9-25)39-35(41)27-10-6-3-7-11-27/h2-17,21,31,33-34H,18-20,22-24,38H2,1H3,(H,39,41)/t31-,33+,34-/m0/s1

N-{1(S)-[[3(S)-Amino-6-(2-methoxy-ethoxymethoxy)-2,3-dihydro-benzofuran-2(R)-ylmethyl]-(4-bromo-benzyl)-carbamoyl]-2-phenyl-ethyl}-benzamide

MSN-50; MSN 50; MSN-50; 1592908-75-2; CHEMBL5278460; N-[(2S)-1-[[(2R,3S)-3-amino-6-(2-methoxyethoxymethoxy)-2,3-dihydro-1-benzofuran-2-yl]methyl-[(4-bromophenyl)methyl]amino]-1-oxo-3-phenylpropan-2-yl]benzamide; N-((S)-1-((((2R,3S)-3-Amino-6-((2-methoxyethoxy)methoxy)-2,3-dihydrobenzofuran-2-yl)methyl)(4-bromobenzyl)amino)-1-oxo-3-phenylpropan-2-yl)benzamide; N-{1(S)-[[3(S)-Amino-6-(2-methoxy-ethoxymethoxy)-2,3-dihydro-benzofuran-2(R)-ylmethyl]-(4-bromo-benzyl)-carbamoyl]-2-phenyl-ethyl}-benzamide; BDBM50610529; MSN50.

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~200 mg/mL (~290.44 mM)

Solubility in Formulation 1: 5 mg/mL (7.26 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 50.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 2: ≥ 5 mg/mL (7.26 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 50.0 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.)