Bax; Bak
MSN-125 targets Bax and Bak, two pro-apoptotic members of the Bcl-2 family that are essential for the intrinsic apoptosis pathway. Upon activation by apoptotic stimuli, Bax and Bak undergo a conformational change, oligomerize, and insert into the mitochondrial outer membrane, leading to MOMP, the release of cytochrome c, and activation of the caspase cascade. MSN-125 is a specific and potent inhibitor of Bax and Bak oligomerization. By inhibiting this oligomerization, MSN-125 prevents MOMP, thereby blocking the intrinsic apoptosis pathway. This mechanism of action underlies its neuroprotective effects and its ability to prevent cell death.

In a coordinated manner, MSN-125 concentration suppresses tBid/Bax-mediated MOMP[1]. MSN-50, MSN-125, and DAN004 Inhibit Bax- and Bak- Mediated MOMP. MSN-125 and MSN-50 Inhibit Apoptosis in Cells.MSN-125 Protects Primary Neurons against Glutamate Excitotoxicity. MSN-125 Inhibits Bax Oligomerization at Defined Dimer Interfaces[1].

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In vitro, MSN-125 is a potent inhibitor of Bax and Bak oligomerization that prevents MOMP with an IC50 of 4 μM. It potently inhibits Bax/Bak-mediated apoptosis in HCT-116, BMK Cells, and primary cortical neurons. Its activity is typically measured using cell-based assays that assess cell viability, apoptosis, and MOMP. These in vitro studies confirm MSN-125's mechanism of action as an inhibitor of Bax and Bak oligomerization and its neuroprotective effects.

In vivo, MSN-125 promotes neuroprotection. By inhibiting Bax and Bak oligomerization and preventing MOMP, MSN-125 blocks the intrinsic apoptosis pathway, which is a key mechanism of cell death in neurodegenerative diseases. This suggests potential in vivo applications in models of stroke, traumatic brain injury, and neurodegenerative disorders. However, specific in vivo protocols and results are not detailed in standard product descriptions. MSN-125 is a research compound used to study apoptosis and neuroprotection.

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.

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In vitro assays for MSN-125 measure its inhibition of Bax and Bak oligomerization and its effects on MOMP. Bax and Bak oligomerization can be assessed using crosslinking assays, co-immunoprecipitation, or fluorescence resonance energy transfer (FRET). MOMP can be assessed by measuring the release of cytochrome c from mitochondria or by using a mitochondrial membrane potential-sensitive dye. These assays confirm the compound's mechanism of action as an inhibitor of Bax and Bak oligomerization.

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.

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In vitro cell-based assays for MSN-125 are used to study its effects on apoptosis and cell survival. Cells (e.g., HCT-116, BMK cells, primary cortical neurons) are treated with apoptotic stimuli in the presence or absence of MSN-125. Cell viability is assessed using assays such as MTT or CellTiter-Glo. Apoptosis is measured using Annexin V staining or caspase-3/7 activation assays. MOMP is assessed by measuring the release of cytochrome c from mitochondria or by using a mitochondrial membrane potential-sensitive dye. These assays confirm the compound's anti-apoptotic activity.

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]

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In vivo animal experiments for MSN-125 are not extensively described in the available literature. As a research compound, its use in vivo would be determined by the specific research question being addressed. A typical protocol for studying a neuroprotective compound like MSN-125 would involve its administration to animal models of stroke, traumatic brain injury, or neurodegenerative diseases. The compound's ability to reduce neuronal cell death and improve functional outcomes would be assessed. However, specific protocols for MSN-125 are not detailed.

MSN-125 has a molecular weight of 688.61 g/mol and a molecular formula of C36H38BrN3O6. It has a CAS number of 1592908-16-1. It is a solid compound with a purity of 98.64%. For storage, it is recommended to keep the powder in a cool, dry place. Detailed pharmacokinetic properties such as absorption, distribution, metabolism, and excretion (ADME) have not been extensively characterized. As a research compound, its stability is maintained by proper storage as a dry powder.

Detailed toxicity data for MSN-125 is not provided in standard product descriptions. As a research compound, its toxicity profile has not been extensively characterized. MSN-125 is an inhibitor of Bax and Bak oligomerization, and its toxicity would be related to its effects on the apoptosis pathway in normal tissues. However, comprehensive toxicological studies have not been reported. As with all research chemicals, standard laboratory safety precautions should be followed when handling MSN-125. Its use is limited to research applications and it is not intended for human or veterinary use.

[1]. A Small-Molecule Inhibitor of Bax and Bak Oligomerization Prevents Genotoxic Cell Death and Promotes Neuroprotection. Cell Chem Biol. 2017 Apr 20;24(4):493-506.e5.

Abnormal apoptosis can lead to acute or chronic degenerative diseases. Mitochondrial outer membrane permeability (MOMP) triggered by the oligomerization of Bcl-2 family proteins Bax/Bak is an irreversible step leading to apoptosis. This article describes the small molecule inhibitors of Bax/Bak oligomerization that we have discovered that can prevent MOMP. We demonstrate that these molecules can disrupt a variety of interactions (but not all) between Bax dimer interfaces, thereby interfering with the formation of higher oligomers in the MOM, without affecting the recruitment of Bax to the MOM. The small molecule inhibitors of Bax/Bak oligomerization enable cells to escape apoptotic stimuli and rescue neurons after excitotoxic damage, indicating that Bax oligomerization is essential for MOMP. Our discovery of small molecule Bax/Bak inhibitors provides new tools for studying the mechanisms leading to mitochondrial outer membrane permeability (MOMP) and will ultimately facilitate the development of Bax/Bak inhibitors for the treatment of acute and chronic degenerative diseases. [1]

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Nie et al. reported five new compounds (molecular weight (MW) ranging from 206 to 689) that inhibit the pore-forming activity of Bax. These compounds were screened from 87 compounds with binding affinity for Mcl-1. Mcl-1 is an anti-apoptotic member of the Bcl-2 protein family, sharing a Bcl-2 homology (BH) domain with Bax. The compounds were named BJ-1, BJ-1-BP, MSN-50, MSN-125, and DAN004. These compounds were initially screened using a liposomal dye release assay to determine their ability to inhibit Bax-mediated membrane permeability. The IC50 values for the liposomal dye release assay were 9, 6, 6, 4, and 0.7 µM, respectively. In vitro mitochondrial isolation experiments showed that MSN-50, MSN-125, and DAN004 effectively inhibited Bax-induced mitochondrial outer membrane permeability (MOMP). Since these inhibitors inhibited MOMP not only in Bax-functional cells but also in Bax−/−bak+/+ cells, the authors considered these inhibitors to be dual Bax/Bak inhibitors. MSN-50 (5 µM) and MSN-125 (10 µM) inhibited actinomycin D and STS-induced apoptosis in BMK (mouse kidney) cells. However, these compounds were cytotoxic at concentrations of 20 µM and above in the culture medium. In cultured primary mouse embryonic cortical neurons, the immediate addition of MSN-125 (5 µM) after inducing glutamate excitotoxicity (25 or 100 mM glutamate, lasting 30 minutes) also effectively reduced cell death. The authors suggest that these novel small molecules may play a role in many applications, but their application in the prevention of traumatic brain injury is highlighted due to their ability to protect primary cultured neurons. The authors also discuss the unintended off-target effects of Bax and Bak and related questions about their unknown mechanisms, which remain unresolved [Exp Biol Med (Maywood). 2019;244(8):621-629.].

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MSN-125 is a research compound and is not approved for any clinical or therapeutic use. It is a specific and potent inhibitor of the intrinsic apoptosis pathway. MSN-125 is a potent Bax and Bak oligomerization inhibitor that prevents MOMP with an IC50 of 4 μM. It potently inhibits Bax/Bak-mediated apoptosis in HCT-116, BMK Cells, and primary cortical neurons. MSN-125 is a valuable research tool for studying apoptosis and neuroprotection. Its mechanism of action involves inhibiting Bax and Bak oligomerization, thereby blocking the intrinsic apoptosis pathway.

C36H38BRN3O6

688.607429027557

687.194

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

1592908-16-1

1592908-16-1;

146014431

White to off-white solid powder

5.1

2

7

15

46

923

3

COCCOCOC1=CC2=C(C=C1)[C@@H]([C@H](O2)CN)N(CC3=CC=C(C=C3)Br)C(=O)[C@H](CC4=CC=CC=C4)NC(=O)C5=CC=CC=C5

NUPXNNVRTSEHSR-IGOOQNSHSA-N

InChI=1S/C36H38BrN3O6/c1-43-18-19-44-24-45-29-16-17-30-32(21-29)46-33(22-38)34(30)40(23-26-12-14-28(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)-[[2(R)-Aminomethyl-6-(2-methoxy-ethoxymethoxy)-2,3-dihydro-benzofuran-3(S)-yl]-(4-bromo-benzyl)-carbamoyl]-2-phenyl-ethyl}-benzamide InChi Key

MSN-125; MSN 125; MSN-125; 1592908-16-1; CHEMBL5271207; N-((S)-1-(((2R,3S)-2-(Aminomethyl)-6-((2-methoxyethoxy)methoxy)-2,3-dihydrobenzofuran-3-yl)(4-bromobenzyl)amino)-1-oxo-3-phenylpropan-2-yl)benzamide; N-[(2S)-1-[[(2R,3S)-2-(aminomethyl)-6-(2-methoxyethoxymethoxy)-2,3-dihydro-1-benzofuran-3-yl]-[(4-bromophenyl)methyl]amino]-1-oxo-3-phenylpropan-2-yl]benzamide; BDBM50610530; MSN125.

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: 4.5 mg/mL (6.53 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% 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 45.0 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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Solubility in Formulation 2: 4.5 mg/mL (6.53 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 ultrasonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 45.0 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 3: ≥ 4.5 mg/mL (6.53 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 45.0 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.)