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.
Physicochemical Properties
| Molecular Formula | C17H14CL2F2N4OS |
| Molecular Weight | 431.29 |
| Exact Mass | 430.023 |
| Elemental Analysis | C, 47.34; H, 3.27; Cl, 16.44; F, 8.81; N, 12.99; O, 3.71; S, 7.43 |
| CAS # | 1338247-35-0 |
| Related CAS # | 1338247-30-5 (BMS-3) |
| PubChem CID | 56965901 |
| Appearance | White to off-white solid powder |
| Density | 1.5±0.1 g/cm3 |
| Index of Refraction | 1.662 |
| LogP | 5.97 |
| Hydrogen Bond Donor Count | 1 |
| Hydrogen Bond Acceptor Count | 6 |
| Rotatable Bond Count | 5 |
| Heavy Atom Count | 27 |
| Complexity | 523 |
| Defined Atom Stereocenter Count | 0 |
| 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 |
| InChi Key | IVUGBSGLHRJSSP-UHFFFAOYSA-N |
| 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) |
| Chemical Name | N-[5-[2-(2,6-dichlorophenyl)-5-(difluoromethyl)pyrazol-3-yl]-1,3-thiazol-2-yl]-2-methylpropanamide |
| Synonyms | BMS-5; BMS 5; BMS5; LIMKI-3; LIMKI 3; LIMKI3 |
| HS Tariff Code | 2934.99.9001 |
| 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) |
Biological Activity
| Targets | LIMK1 (IC50 = 7 nM); LIMK2 (IC50 = 8 nM) |
| 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]. |
| 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]. |
| 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]. |
| 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]. |
| 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. |
| References |
[1]. Identification of a nonkinase target mediating cytotoxicity of novel kinase inhibitors. Mol Cancer Ther. 2008 Nov;7(11):3490-8. [2]. LIM Domain Kinases as Potential Therapeutic Targets for Neurofibromatosis Type 2.Oncogene. Oncogene. 2014 Jul 3;33(27):3571-82. [3]. Effects of Hippocampal LIMK Inhibition on Memory Acquisition, Consolidation, Retrieval, Reconsolidation, and Extinction. Mol Neurobiol. 2017 Jan 13. |
| 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. |
Solubility Data
| Solubility (In Vitro) |
|
|||
| 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. 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. 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 corn oil and mix evenly. (Please use freshly prepared in vivo formulations for optimal results.) |
| 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 |