BMS-3 is a potent LIMK inhibitor with IC50 values of 5 nM and 6 nM for LIMK1 and LIMK2, respectively.

LIMK1:5 nM, LIMK2:6 nM

BMS-3 (Compound 2) exhibits a dose-dependent decrease in A549 human lung cancer cell viability by inducing mitotic arrest, characterized by enhanced total nuclear DNA intensity and histone H3 phosphorylation following a 24-hour exposure. It effectively inhibits these cells with an EC50 value of 154 nM[1]. Additionally, BMS-3 elucidates the role of LIMK1 in Cofilin phosphorylation; inhibition of p-LIMK by BMS-3 (1-50 μM) leads to a notable reduction in p-Cofilin levels after a 10-minute incubation under capacitating conditions, with a marked decrease in actin polymerization levels compared to the DMSO controls. Furthermore, under capacitating conditions, mouse sperm exposure to escalating concentrations of BMS-3 (0, 1, 10, and 50 μM) for 90 minutes significantly diminishes the percentage of sperm undergoing acrosomal exocytosis upon Progesterone stimulation[2], highlighting BMS-3's potential in affecting sperm functionality.

The protein kinase domains of human LIMK1 and LIMK2 are expressed as glutathione S-transferase fusion proteins using the Bac-to-Bac system in Sf9 cells. Compounds 1 to 6 (e.g., BMS-3) are assayed for inhibition of LIMK1 and LIMK2 protein kinase activity by radioactive phosphate incorporation into biotinylated full-length human destrin. Reactions are done with a concentration series of compound 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 in a total volume of 60 μL at room temperature for 30 min (LIMK1) or 60 min (LIMK2). Reactions are terminated by addition of 140 μL of 20% TCA/100 mM sodium pyrophosphate, and the precipitates are harvested onto GF/C unifilter plates. The radioactivity incorporated is determined using a TopCount after addition of 35 μL Microscint scintillation fluid[1]