In vitro activity: BIX02189 is a novel, potent and selective inhibitor of MEK5 with IC50 value of 1.5 nM, it also inhibits ERK5 catalytic activity with IC50 of 59 nM in cell-free assays; but it does not inhibit closely related kinases MEK1, MEK2, ERK2, and JNK2. BIX02189 was reported to inhibit catalytic function of purified, MEK5 enzyme. BIX02189 inhibited transcriptional activation of MEF2C, a downstream substrate of the MEK5/ERK5 signaling cascade, in a cellular trans-reporter assay system. BIX02189 may offer novel pharmacological tools to better characterize the role of the MEK5/ERK5 pathway in various biological systems. BIX02189 blocks MEK5 and ERK5 catalytic activity with IC50 values of 1.5 nM and 59 nM, respectively. They are more potent than the effect caused by BIX02188 with IC50 values of 4.3 nM and 810 nM, respectively. BIX02189 shows inhibitory activity against CSF1R (FMS) with IC50 of 46 nM but displays no activity against related kinases MEK1, MEK2, ERK1, p38α, JNK2, EGFR, and STK16 with IC50 values of>3.7 μM. Pretreatment with BIX02189 inhibits sorbitol-induced phosphorylation of ERK5 in HeLa cells in a dose dependent manner, and displays no inhibitory activity against the phosphorylation of ERK1/2, p38, and JNK1/2 MAPKs. Treatment with only BIX02189 for 24 hours in HeLa or HEK293 cells does not show any cytotoxic effect. BIX02189 inhibits MEK5/ERK5/MEF2C-driven luciferase expression in HeLa and HEK293 cells with IC50 values of 0.53 μM and 0.26 μM, respectively. This is a more significant than the effect caused by BIX02188. BIX02189 inhibits the activation of ERK5, and suppresses C-terminus of Hsc70-interacting protein (CHIP) mediated p53 ubiquitination, leading to the reverse of the protective effect caused by laminar flow (L-flow) in human umbilical vein endothelial cells (HUVECs) exposed to 15d-PGJ2. BIX02189 (10 uM) inhibits ERK5 phosphorylation, and reduces myocyte enhancer factor 2 (MEF2) transcriptional activity in neonatal rat cardiomyocytes (NRCMs) stimulated by isoproterenol. BIX02189 enhances the sorbitol induced apoptosis in NRCMs, confirming the protective role of ERK5 in cardiomyocytes.

Kinase Assay: MEK5 protein isolated from the baculovirus expression system is used to measure kinase activity utilizing PKLight ATP Detection Reagent. The assay is performed using 15 nM GST-MEK5 and 0.75 μM ATP in assay buffer consisting of 25 mM Hepes, pH 7.5, 10 mM MgCl2, 50 mM KCl, 0.2% BSA, 0.01% CHAPS, 100 μM Na3VO4, 0.5 mM DTT and 1% DMSO in the presence of varying concentrations of BIX02189. The kinase reaction mixture is incubated for 90 minutes at room temperature followed by addition of 10 μL of ATP detection reagent for 15 minutes. The relative light unit (RLU) signal is measured and the RLU signals are converted to percent of control (POC) values for the determination of IC50 value.

Cell Assay: The cells are serum starved for 20 hours prior to stimulation with sorbitol at a final concentration of 0.4 M for 20 minutes at 37 °C. BIX02189 is added 1.5 hours prior to the addition of sorbitol. The cells are harvested and lysed in 50 μL RIPA buffer containing Halt protease and phosphate inhibitors at 4 °C for 5-10 minutes. The lysates are centrifuged for 10 minutes at 14,000 rpm and 50 μL lysate is added to 50 μl 2× sample buffer and boiled for 4 minutes at 95 °C. Twenty microliters sample is run on SDS–PAGE 10% Tris-glycine gels and transferred to nitrocellulose. Western blotting is done with appropriate antibodies.