BIX02189 is a selective inhibitor of MEK5 with IC50 of 1.5 nM.

MEK5:1.5 nM

用BIX02189预处理以剂量依赖性方式抑制山梨醇诱导的HeLa细胞中ERK5的磷酸化,并且对ERK1/2,p38和JNK1/2 MAPKs的磷酸化没有抑制活性。在HeLa或HEK293细胞中仅用BIX02189处理24小时不显示任何细胞毒性作用。BIX02189抑制HeLa和HEK293细胞中MEK5/ERK5/MEF2C驱动的萤光素酶表达,IC50分别为 0.53 μM和0.26 μM。BIX02189抑制MEK5和ERK5催化活性,IC50分别为1.5 nM和59 nM。BIX02189抑制CSF1R(FMS),IC50为46 nM,但对相关激酶MEK1,MEK2,ERK1,p38α,JNK2,EGFR和STK16无活性,IC50 >3.7 μM。BIX02189增强山梨醇诱导的NRCM细胞凋亡,证实了ERK5在心肌细胞中的保护作用。BIX02189（10 μM）抑制ERK5磷酸化,并降低由异丙肾上腺素刺激的新生大鼠心肌细胞（NRCMs）中的肌细胞增强因子2（MEF2）转录活性。

Catalytic 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.

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. (Only for Reference)