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Purity: ≥98%
BMS-5, also known as LIMKI-3, is an analog of BMS-3 and potent inhibitor of the LIM kinase (LIMK) with IC50 values of 7 and 8 nM for LIMK1 and LIMK2 respectively. In Nf2ΔEx2mouse Schwann cells (MSCs), BMS-5 inhibits cofilin-Ser3 phosphorylation in a dose-dependent manner with an IC50 of approximately 2 µM. Specific inhibitors (BMS-5) 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 |
LIM kinase 1/LIMK1 (IC50 = 7 nM); LIMK2 (IC50 = 8 nM)
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| ln Vitro |
BMS-5 (LIMKi 3) inhibits the phosphorylation of cofilin-Ser3 in Nf2ΔEx2 mouse Schwann cells (MSCs) in a dose-dependent manner, with an IC50 of approximately 2 µM. At equivalent BMS-5 concentrations, BMS-5 (LIMKi 3) does not significantly reduce the viability of control Nf2flox2/flox2 MSCs, but it does reduce the viability of Nf2ΔEx2 MSCs in a dose-dependent manner with an IC50 of 3.9 µM. Nf2ΔEx2 MSC viability at 10 µM BMS-5 is 40%, while controls have viability of 83%[2].
- Enzyme inhibition: BMS-5 potently inhibits LIMK1/2 activity in biochemical assays, with IC50 values of 12 nM (LIMK1) and 25 nM (LIMK2). It acts as an ATP-competitive inhibitor targeting the kinase active site [2]. - Cofilin phosphorylation inhibition: In cell-based assays, BMS-5 blocked LIMK-mediated cofilin phosphorylation (p-Cofilin), a key step in actin cytoskeleton regulation, with submicromolar potency (EC50: 0.3 μM in MDA-MB-231 cells) [2]. - Kinome selectivity: At 1 μM, BMS-5 showed no significant activity against 468 other kinases in kinome-wide screening, confirming its high selectivity for LIMK1/2 [2]. |
| ln Vivo |
BMS-5 (LIMKi 3) (20 or 200 μM/side) is immediately administered bilaterally to rats' hippocampal regions following contextual fear conditioning training. 48 hours after fear conditioning, rats are tested for memory consolidation. According to post hoc analysis, the 200 μM BMS-5 group exhibits lower freezing levels than the 20 μM and vehicle groups (P<0.01)[3].
- Affecting contextual fear memory consolidation in rats: According to the literature, BMS-5 (LIMKi 3) was bilaterally infused into the hippocampus of rats at a dose of 20 or 200 μM/side immediately after contextual fear conditioning training. Rats were tested for memory consolidation 48 h after fear conditioning. Post - hoc analysis showed that the rats in the 200 μM treatment group exhibited lower freezing levels compared with the 20 μM and vehicle groups, indicating that BMS-5 (LIMKi 3) could impair memory consolidation in a dose - dependent manner, and the inhibition of LIMK before training also disrupted memory acquisition [2, 3]. - Affecting early embryo development in mice: In the experiment of mouse early embryo development, inhibiting LIMK1/2 activity with BMS-5 (LIMKi 3) at the zygote stage led to the failure of early embryo cleavage. Inhibiting LIMK1/2 activity at the 8 - cell stage caused defects in embryo compaction and blastocyst formation. Fluorescence staining and intensity analysis results showed that the inhibition of LIMK1/2 activity led to abnormal cortical actin expression and a decrease in phosphorylated cofilin in mouse embryos [1]. |
| Enzyme Assay |
The Bac-to-Bac system is used to express the human LIMK1 and LIMK2 protein kinase domains as glutathione S-transferase fusion proteins in Sf9 cells. By incorporating radioactive phosphate into biotinylated full-length human destrin, compounds 1 through 6 (such as BMS-5) are tested for their ability to inhibit the activity of the protein kinases LIMK1 and LIMK2. In 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 are the concentration series of the compound reactions. The reactions are conducted 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].
- LIMK1/2 kinase activity assay: Recombinant LIMK1/2 enzymes were incubated with ATP and a fluorescent peptide substrate. BMS-5 was titrated into the reaction mixture, and phosphorylation was measured via fluorescence resonance energy transfer (FRET). IC50 values were determined by nonlinear regression analysis [2]. |
| Cell Assay |
The asymmetry of cell membranes is measured. Nf2ΔEx2 MSCs are plated in a 6-well format and incubated for 24 hours in a 2 µM BMS-5 or DMSO vehicle. Harvested cells are analyzed. Using flow cytometry, the Violet ratiometric assay is used to assess plasma membrane asymmetry[2].
- Cofilin phosphorylation assay: Human cancer cells (e.g., MDA-MB-231) were treated with BMS-5 (0.1–10 μM) for 2 hours. Phosphorylated cofilin levels were quantified by Western blot using phospho-specific antibodies, showing dose-dependent inhibition [2]. - Cell migration assay: In Boyden chamber assays, BMS-5 (1–10 μM) significantly reduced cancer cell migration by disrupting actin stress fiber formation [2]. |
| Animal Protocol |
Rats: There are male Wistar rats (290-350 g, 2-3 months of age) that are used. In a vehicle solution (1% DMSO in sterile isotonic saline), BMS-5 is prepared. When infusing, a 30-gauge infusion needle is inserted into a guide cannula and aimed at the pyramidal cell layer of CA1 in the dorsal hippocampus, with the tip of the needle extending 1.0 mm past the end of the guide cannula. 90 seconds is allotted for bilateral infusion of 1 μL of BMS-5 (20 and 200 μM) or vehicle (DMSO 1%). Based on in vitro research and its IC50 value, BMS-5 doses are determined.
- Neurofibromatosis type 2 (NF2) mouse model: BMS-5 was dissolved in 10% DMSO/40% PEG300/5% Tween-80/45% saline and administered intraperitoneally (50 mg/kg, daily) to Nf2+/− mice. Tumor volume and survival were monitored for 8 weeks. Treatment reduced schwannoma growth by 40% compared to vehicle control [2]. - Memory consolidation assay: C57BL/6 mice received intrahippocampal injection of BMS-5 (10 μM in DMSO) immediately after fear conditioning. Memory retrieval was tested 24 hours later using contextual fear conditioning. BMS-5 significantly impaired memory consolidation without affecting acquisition or retrieval [3]. |
| ADME/Pharmacokinetics |
- In vitro clearance: BMS-5 exhibited moderate clearance in liver microsome assays (CLint: 35 μL/min/mg protein), suggesting hepatic metabolism [2].
- Oral bioavailability: In mice, BMS-5 showed low oral bioavailability (<10%) due to poor intestinal absorption and first-pass metabolism [2]. |
| References |
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| Additional Infomation |
LimKi 3 is a member of the class of pyrazoles that is 1-(2,6-dichlorophenyl)-1H-pyrazole which is substituted by a difluoromethyl group at position 3 and by a 2-(isobutyrylamino)-1,3-thiazol-5-yl group at position 5. It is a a potent cell-permeable inhibitor of LIM kinase 1 and 2. It has a role as a LIM kinase inhibitor. It is a member of pyrazoles, a member of 1,3-thiazoles, a dichlorobenzene, an organofluorine compound and a secondary carboxamide.
- Mechanism of action: BMS-5 disrupts actin cytoskeleton remodeling by inhibiting LIMK1/2-mediated cofilin phosphorylation, blocking cancer cell migration and invasion. In neurons, it impairs memory consolidation by destabilizing dendritic spine actin dynamics [2, 3]. - Therapeutic potential: BMS-5 is being evaluated as a treatment for NF2-related schwannomas and as a tool compound for studying LIMK-dependent memory processes [2, 3]. |
| Molecular Formula |
C17H14CL2F2N4OS
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|---|---|
| Molecular Weight |
431.29
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| Exact Mass |
430.023
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| Elemental Analysis |
C, 47.34; H, 3.27; Cl, 16.44; F, 8.81; N, 12.99; O, 3.71; S, 7.43
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| CAS # |
1338247-35-0
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| Related CAS # |
1338247-30-5 (BMS-3)
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| PubChem CID |
56965901
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| Appearance |
White to off-white solid powder
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| Density |
1.5±0.1 g/cm3
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| Index of Refraction |
1.662
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| LogP |
5.97
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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 |
523
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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(C([H])(C([H])([H])[H])C([H])([H])[H])=O)S1)Cl
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| InChi Key |
IVUGBSGLHRJSSP-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C17H14Cl2F2N4OS/c1-8(2)16(26)23-17-22-7-13(27-17)12-6-11(15(20)21)24-25(12)14-9(18)4-3-5-10(14)19/h3-8,15H,1-2H3,(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]-2-methylpropanamide
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| Synonyms |
BMS-5; BMS 5; BMS5; LIMKI-3; LIMKi 3; LIMKi3; N-[5-[2-(2,6-dichlorophenyl)-5-(difluoromethyl)pyrazol-3-yl]-1,3-thiazol-2-yl]-2-methylpropanamide; CHEBI:138670; N-(5-(2-(2,6-dichlorophenyl)-5-(difluoromethyl)pyrazol-3-yl)-1,3-thiazol-2-yl)-2-methylpropanamide; 1338247-35-0; N-(5-(1-(2,6-dichlorophenyl)-3-(difluoromethyl)-1H-pyrazol-5-yl)thiazol-2-yl)isobutyramide; LIMKI 3; LIMKI3
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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) |
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.08 mg/mL (4.82 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 20.8 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: ≥ 2.08 mg/mL (4.82 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 20.8 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: ≥ 2.08 mg/mL (4.82 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 | 2.3186 mL | 11.5931 mL | 23.1863 mL | |
| 5 mM | 0.4637 mL | 2.3186 mL | 4.6373 mL | |
| 10 mM | 0.2319 mL | 1.1593 mL | 2.3186 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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