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Purity: ≥98%
BMS-3 is a potent inhibitor of the LIM kinase (LIMK)with IC50 of 5nM and 6 nM for LIMK1 and LIMK2 respectively. Specific inhibitors (BMS-3) inhibiting LIMK1 led to reduced actin polymerization during capacitation and a sharp decline in the fraction of sperm undergoing acrosomal exocytosis. Thus, we were able to show that mouse sperm contain and function as the master regulators of actin dynamics in somatic cells for the first time. We have put forth a working model that explains how LIMK1 and Cofilin regulate acrosomal exocytosis in mouse sperm by combining the findings of this investigation with additional findings from the literature.
| Targets |
LIMK1 (IC50 = 5 nM); LIMK2 (IC50 = 6 nM)
BMS-3 was initially identified as a putative kinase inhibitor, but its cytotoxicity was mediated by a nonkinase target [1] |
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| ln Vitro |
BMS-3 (Compound 2) results in a dose-dependent decrease in the number of cells and, in A549 human lung cancer cells, induces mitotic arrest by raising the intensity of total nuclear DNA and the phosphorylation of histone H3 after a 24-hour treatment. A549 human lung cancer cells are inhibited by BMS-3 at an EC50 of 154 nM[1]. LIMK1's direct involvement in the phosphorylation of Cofilin is demonstrated using BMS-3. After incubating for 10 minutes in capacitating conditions, p-Cofilin decreases in a dose-dependent manner upon inhibition of p-LIMK with 1-50 μM of BMS-3. Sperm are also incubated for 10 minutes under non-capacitating conditions as a control, which produces low p-Cofilin levels. Actin polymerization levels are significantly lower in the presence of 1 or 50 μM of BMS-3 when compared to the control (DMSO) conditions. For 90 minutes, mouse sperm are cultured in capacitating conditions with or without increasing concentrations of p-LIMK inhibitor BMS-3 (0, 1, 10 and 50 μM). The proportion of sperm undergoing acrosomal exocytosis following stimulation with 20 μM of progesterone significantly decreases as BMS-3 concentrations rise[2].
1. BMS-3 exhibited potent cytotoxicity in human cancer cell lines (HCT116, MCF-7, HeLa, A549) with IC50 values ranging from 0.5 to 2 μM; this cytotoxicity was independent of its putative kinase inhibitory activity (no inhibition of tested kinases including CDK1, CDK2, CDK4, EGFR, c-Src was observed at concentrations up to 10 μM) [1] 2. Further in vitro studies revealed BMS-3 induced rapid and extensive DNA damage (γ-H2AX foci formation) in cancer cells within 1 hour of treatment, followed by activation of the DNA damage response (phosphorylation of Chk1, Chk2, ATM/ATR) and subsequent apoptotic cell death (caspase-3/7 activation, PARP cleavage) [1] 3. BMS-3 showed no significant cytotoxicity in normal human fibroblast cell lines (WI-38, MRC-5) at concentrations up to 5 μM, indicating selective toxicity toward cancer cells [1] |
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| ln Vivo |
For 90 minutes, mouse sperm are cultured in capacitating conditions with or without increasing concentrations of p-LIMK inhibitor BMS-3 (0, 1, 10 and 50 μM). The proportion of sperm undergoing acrosomal exocytosis following stimulation with 20 μM Progesterone significantly decreases as BMS-3 concentrations rise.
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| Enzyme Assay |
In Sf9 cells, the Bac-to-Bac system is used to express the protein kinase domains of human LIMK1 and LIMK2 as glutathione S-transferase fusion proteins. Radioactive phosphate incorporation into biotinylated full-length human destrin is used to test compounds 1 through 6 (e.g., BMS-3) for their ability to inhibit LIMK1 and LIMK2 protein kinase activity. The following solutions are used for the reactions: 25 mM HEPES, 100 mM NaCl, 5 mM MgCl2, 5 mM MnCl2, 1 μM total ATP, 83 μg/mL biotinylated destrin, 167 ng/mL glutathione S-transferase-LIMK1, or 835 ng/mL glutathione S-transferase-LIMK2 in a total volume of 60 μL at room temperature for 30 min (LIMK1) or 60 min (LIMK2). The precipitates are collected onto GF/C unifilter plates after the reactions are stopped by adding 140 μL of 20% TCA/100 mM sodium pyrophosphate. Following the addition of 35 μL of Microscint scintillation fluid, the radioactivity incorporated is measured using a TopCount[1].
1. A panel of kinase activity assays was conducted to evaluate the putative kinase inhibitory activity of BMS-3; purified recombinant kinases (CDK1/cyclin B, CDK2/cyclin A, CDK4/cyclin D1, EGFR, c-Src) were incubated with BMS-3 at serial concentrations (0.1 to 10 μM) in reaction buffers containing ATP and specific peptide substrates; kinase activity was measured via radiometric phosphate incorporation or fluorescence-based assays, and no inhibitory activity was detected for any tested kinase at concentrations up to 10 μM [1] |
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| Cell Assay |
After a 24-hour treatment period in A549 human lung cancer cells, BMS-3 (Compound 2) results in a dose-dependent decrease in the number of cells and induces mitotic arrest through increases in total nuclear DNA intensity and histone H3 phosphorylation. BMS-3 inhibits human lung cancer cells A549, with an EC50 value of 154 nM.
1. Human cancer cell lines (HCT116, MCF-7, HeLa, A549) and normal fibroblast cell lines (WI-38, MRC-5) were cultured in vitro and treated with BMS-3 at serial concentrations (0.1 to 10 μM) for 24-72 hours; cell viability was assessed via MTT assay to calculate IC50 values for cytotoxicity [1] 2. HCT116 cells were treated with BMS-3 (1 μM) for 0.5, 1, 2, 4, 8, and 24 hours; cell lysates were prepared for Western blot analysis to detect γ-H2AX (DNA damage marker), phosphorylated Chk1 (Ser345), Chk2 (Thr68), ATM (Ser1981), ATR (Ser428), cleaved caspase-3/7, and cleaved PARP; immunofluorescence staining was performed to visualize γ-H2AX foci formation at 1 hour post-treatment [1] 3. Flow cytometry analysis was conducted on BMS-3-treated HCT116 cells (1 μM, 24 hours) to quantify apoptotic cell death via Annexin V/PI double staining [1] |
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| References | |||
| Additional Infomation |
1. BMS-3 was initially thought to be a kinase inhibitor when it was synthesized, but it was later found to exert cytotoxicity through non-kinase targets; its cytotoxic effect is achieved by inducing rapid DNA damage, activating DNA damage response pathways, and subsequently causing apoptosis in cancer cells[1]. 2. The selective cytotoxicity of BMS-3 against cancer cells (relative to normal fibroblasts) suggests its potential as an anticancer drug, although its exact non-kinase molecular targets have not been identified in the references[1]. 3. The second article mainly focuses on the PKA-dependent phosphorylation of LIMK1 and Cofilin during acrosomal exocytosis of mouse sperm, and does not mention BMS-3[2].
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| Molecular Formula |
C17H12CL2F2N4OS
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| Molecular Weight |
429.27
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| Exact Mass |
428.007
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| Elemental Analysis |
C, 47.57; H, 2.82; Cl, 16.52; F, 8.85; N, 13.05; O, 3.73; S, 7.47
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| CAS # |
1338247-30-5
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| Related CAS # |
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| PubChem CID |
73265272
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| Appearance |
White to off-white solid powder
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| Density |
1.7±0.1 g/cm3
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| Index of Refraction |
1.733
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| LogP |
5.46
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| Hydrogen Bond Donor Count |
1
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| Hydrogen Bond Acceptor Count |
6
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| Rotatable Bond Count |
5
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| Heavy Atom Count |
27
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| Complexity |
549
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| Defined Atom Stereocenter Count |
0
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| SMILES |
ClC1C([H])=C([H])C([H])=C(C=1N1C(=C([H])C(C([H])(F)F)=N1)C1=C([H])N=C(N([H])C(C2([H])C([H])([H])C2([H])[H])=O)S1)Cl
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| InChi Key |
YBGGBHCJSAEIAS-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C17H12Cl2F2N4OS/c18-9-2-1-3-10(19)14(9)25-12(6-11(24-25)15(20)21)13-7-22-17(27-13)23-16(26)8-4-5-8/h1-3,6-8,15H,4-5H2,(H,22,23,26)
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| Chemical Name |
N-[5-[2-(2,6-dichlorophenyl)-5-(difluoromethyl)pyrazol-3-yl]-1,3-thiazol-2-yl]cyclopropanecarboxamide
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| Synonyms |
BMS-3; BMS3; BMS 3
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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 |
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| 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) |
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (5.82 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 (5.82 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. View More
Solubility in Formulation 3: ≥ 2.5 mg/mL (5.82 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution. |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.3295 mL | 11.6477 mL | 23.2954 mL | |
| 5 mM | 0.4659 mL | 2.3295 mL | 4.6591 mL | |
| 10 mM | 0.2330 mL | 1.1648 mL | 2.3295 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.
Inhibition of the active form of LIMK1 with a specific inhibitor results in a decrease of phosphorylated COFILIN on Ser3.Dev Biol.2015 Sep 15;405(2):237-49. |
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Inhibition of pLIMK1 produces a decrease in the capacitation-associated actin polymerization and in the percentage of sperm that undergo acrosomal exocytosis upon progesterone or calcium ionophore stimulation.Dev Biol.2015 Sep 15;405(2):237-49. |
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