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.

Physicochemical Properties

Molecular Formula	C17H12CL2F2N4OS
Molecular Weight	429.27
Exact Mass	428.007
Elemental Analysis	C, 47.57; H, 2.82; Cl, 16.52; F, 8.85; N, 13.05; O, 3.73; S, 7.47
CAS #	1338247-30-5
Related CAS #	1338247-30-5
PubChem CID	73265272
Appearance	White to off-white solid powder
Density	1.7±0.1 g/cm3
Index of Refraction	1.733
LogP	5.46
Hydrogen Bond Donor Count	1
Hydrogen Bond Acceptor Count	6
Rotatable Bond Count	5
Heavy Atom Count	27
Complexity	549
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(C2([H])C([H])([H])C2([H])[H])=O)S1)Cl
InChi Key	YBGGBHCJSAEIAS-UHFFFAOYSA-N
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)
Chemical Name	N-[5-[2-(2,6-dichlorophenyl)-5-(difluoromethyl)pyrazol-3-yl]-1,3-thiazol-2-yl]cyclopropanecarboxamide
Synonyms	BMS-3; BMS3; BMS 3
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 = 5 nM); LIMK2 (IC50 = 6 nM)
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].
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.
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].
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.
Animal Protocol
References	[1]. Identification of a nonkinase target mediating cytotoxicity of novel kinase inhibitors. Mol Cancer Ther. 2008 Nov;7(11):3490-8. [2]. PKA-dependent phosphorylation of LIMK1 and Cofilin is essential for mouse sperm acrosomal exocytosis. Dev Biol. 2015 Sep 15;405(2):237-49.

Solubility Data

Solubility (In Vitro)

DMSO: ≥ 30 mg/mL Water: <1 mg/mL Ethanol: <1 mg/mL

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. 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. 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 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	2.3295 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.