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| 25mg |
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| Targets |
RIP1 (Kd = 5.3 μM); Bcl-2; Bcl-xL; Bim; Bak
BH3I-1 targets the Bcl-2 family of proteins, with primary binding to the BH3-binding groove of anti-apoptotic proteins Bcl-xL and Bcl-2. It inhibits the interaction between the Bak BH3 peptide and Bcl-xL with a Ki of 2.4 μM in a fluorescence polarization (FP) binding assay. BH3I-1 also binds to the p53/MDM2 interaction with a Kd of 5.3 μM. Additionally, it shows significant inhibition of the p300/Hif-1α interaction. By disrupting these critical protein-protein interactions, BH3I-1 promotes the release of pro-apoptotic factors, leading to the induction of apoptosis in cancer cells that are dependent on Bcl-2 family proteins for survival. |
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| ln Vitro |
In vitro, BH3I-1 prevents Bcl-xL heterodimerization. It also causes the release of cytochrome c[1]. While inhibiting its reported targets Bcl-2/Bim and Bcl-xL/Bim, BH3I-1 also significantly inhibits the interactions between p53/hDM2 and p300/Hif-1a[2].
In vitro, BH3I-1 exhibits potent inhibition of Bcl-xL-BH3 domain interactions with a Ki of 2.4 μM in fluorescence polarization assays. It demonstrates significant inhibition of both the p53/hDM2 and p300/Hif-1α interactions. BH3I-1 induces cytochrome c release from mitochondria and promotes apoptosis in Bcl-xL-expressing cells. The compound is cell-permeable and effectively disrupts Bcl-2 family protein-protein interactions in cellular contexts. In cell-based assays, BH3I-1 treatment leads to the activation of the intrinsic apoptotic pathway, characterized by mitochondrial outer membrane permeabilization, caspase activation, and eventual cell death. |
| ln Vivo |
In vivo activity data for BH3I-1 are limited, as the compound is primarily used as a research tool in in vitro studies. However, based on its mechanism of action as a Bcl-2 family antagonist and its ability to induce apoptosis in cancer cells in vitro, BH3I-1 has the potential to exhibit antitumor activity in vivo. For in vivo administration, BH3I-1 can be formulated as a homogeneous suspension using CMC-Na at concentrations of ≥5 mg/mL. The compound's ability to inhibit Bcl-2 family proteins suggests that it could be effective in animal models of Bcl-2-dependent cancers, although comprehensive in vivo efficacy and pharmacokinetic studies are needed.
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| Enzyme Assay |
In vitro enzyme/receptor binding assays for BH3I-1 are typically conducted using fluorescence polarization (FP) to measure the inhibition of BH3 peptide binding to Bcl-xL. In this assay, a fluorescently labeled BH3 peptide (e.g., Bak BH3) is incubated with recombinant Bcl-xL protein and varying concentrations of BH3I-1. The displacement of the labeled peptide by BH3I-1 results in a decrease in fluorescence polarization, from which the Ki value is calculated. The p53/MDM2 binding inhibition can be assessed using similar FP or AlphaScreen assays, with a reported Kd of 5.3 μM. Surface plasmon resonance (SPR) or isothermal titration calorimetry (ITC) can also be used to directly measure binding affinity and thermodynamic parameters.
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| Cell Assay |
Cells (5×104 cells per well) are seeded into white 96-well plates (Costar) and treated with various concentrations of the compounds for 48 h. Cells are preincubated with 100 µM zVAD-FMK for 1 hour prior to the addition of chemicals for zVAD-FMK protection experiments. An MTS assay is used to gauge cell viability. Cells are expanded in 24-well plates for PI staining experiments before being incubated with 2 g ml/L PI. FACS analysis is used to identify cell death.
In vitro cell-based assays for BH3I-1 evaluate its ability to induce apoptosis in Bcl-xL-expressing cancer cell lines. Cells are cultured in appropriate media and treated with serial dilutions of BH3I-1 for 24-72 hours. Cell viability is assessed using MTT, CellTiter-Glo, or trypan blue exclusion assays. Apoptosis is quantified by measuring caspase-3/7 activity, Annexin V staining, or detection of cytochrome c release from mitochondria. The compound's specificity can be assessed by comparing its effects in Bcl-2 family-dependent versus -independent cell lines. The IC₅₀ for apoptosis induction is typically calculated from dose-response curves. |
| Animal Protocol |
In vivo animal experiments for BH3I-1 have not been extensively reported in the literature. For potential in vivo studies, BH3I-1 can be administered orally or via intraperitoneal injection in mouse xenograft models of cancer. A homogeneous suspension can be prepared using CMC-Na at concentrations of ≥5 mg/mL for oral administration. Tumor-bearing mice would be treated with BH3I-1 at various doses, and tumor growth inhibition would be monitored by caliper measurements. Pharmacodynamic endpoints would include assessment of apoptosis in tumor tissues by TUNEL staining or immunohistochemistry for cleaved caspase-3. Comprehensive in vivo studies are needed to establish efficacy, optimal dosing, and safety.
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| ADME/Pharmacokinetics |
Pharmacokinetic (PK) data for BH3I-1 are not well-characterized in the available literature. The compound has a molecular weight of 400.31 g/mol and is soluble in DMSO but insoluble in water. For in vivo formulation, BH3I-1 can be prepared as a homogeneous suspension in CMC-Na at ≥5 mg/mL for oral administration. The compound's poor aqueous solubility may limit its oral bioavailability and could present challenges for in vivo administration. Comprehensive ADME (absorption, distribution, metabolism, excretion) studies are needed to fully characterize the pharmacokinetic profile of BH3I-1. For in vitro studies, stock solutions are typically prepared in DMSO at 64 mg/mL.
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| Toxicity/Toxicokinetics |
The toxicity profile of BH3I-1 has not been extensively characterized in preclinical studies. As a Bcl-2 family antagonist that induces apoptosis, the compound may have off-target effects on normal tissues that depend on Bcl-2 family proteins for survival. For laboratory handling, BH3I-1 should be treated as a hazardous chemical. Appropriate personal protective equipment should be used when handling the compound. The compound is for research use only and is not intended for human or veterinary use. Comprehensive toxicological studies are needed to fully characterize the safety profile of BH3I-1.
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| References | |
| Additional Infomation |
2-[5-[(4-bromophenyl)methylene]-4-oxo-2-thiomethylene-3-thiazolyl]-3-methylbutyric acid is a derivative of valine.
BH3I-1 (BHI1) is a small-molecule Bcl-2 antagonist that inhibits the interaction of Bak BH3 and Bcl-xL with a Ki of 2.4 μM in an FP binding assay. It is a cell-permeable inhibitor that targets Bcl-2 family proteins and induces apoptosis of Bcl-xL-expressing cells. BH3I-1 also shows significant inhibition of the p53/hDM2 and p300/Hif-1α interactions. The compound has a molecular weight of 400.31 g/mol and is soluble in DMSO but insoluble in water. BH3I-1 is a valuable research tool for studying apoptosis and the role of Bcl-2 family proteins in cancer biology. |
| Molecular Formula |
C15H14BRNO3S2
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|---|---|
| Molecular Weight |
400.3106
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| Exact Mass |
398.96
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| Elemental Analysis |
C, 45.01; H, 3.53; Br, 19.96; N, 3.50; O, 11.99; S, 16.02
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| CAS # |
300817-68-9
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| Related CAS # |
300817-68-9
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| PubChem CID |
5720188
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| Appearance |
Light yellow to yellow solid powder
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| LogP |
3.697
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| Hydrogen Bond Donor Count |
1
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| Hydrogen Bond Acceptor Count |
5
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| Rotatable Bond Count |
4
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| Heavy Atom Count |
22
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| Complexity |
513
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| Defined Atom Stereocenter Count |
0
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| SMILES |
BrC1C([H])=C([H])C(=C([H])C=1[H])C([H])=C1C(N(C(=S)S1)C([H])(C(=O)O[H])C([H])(C([H])([H])[H])C([H])([H])[H])=O
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| InChi Key |
COHIEJLWRGREHV-YRNVUSSQSA-N
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| InChi Code |
InChI=1S/C15H14BrNO3S2/c1-8(2)12(14(19)20)17-13(18)11(22-15(17)21)7-9-3-5-10(16)6-4-9/h3-8,12H,1-2H3,(H,19,20)/b11-7+
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| Chemical Name |
2-[(5E)-5-[(4-bromophenyl)methylidene]-4-oxo-2-sulfanylidene-1,3-thiazolidin-3-yl]-3-methylbutanoic acid
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| Synonyms |
BH3I1; BH 3I1; BH-3I1; BH3I-1; BH 3I-1; BH-3I-1
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| HS Tariff Code |
2934.99.9001
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| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month |
| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vitro) |
DMSO: 64~100 mg/mL (159.9~249.8 mM)
Ethanol: ~13 mg/mL (~32.5 mM) |
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (6.25 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 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. Solubility in Formulation 2: ≥ 2.5 mg/mL (6.25 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in 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 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. (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.4981 mL | 12.4903 mL | 24.9806 mL | |
| 5 mM | 0.4996 mL | 2.4981 mL | 4.9961 mL | |
| 10 mM | 0.2498 mL | 1.2490 mL | 2.4981 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
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