BIX02188 is a specific MEK5 inhibitor (IC50: 4.3 nM), also inhibits ERK5 catalytic activity (IC50: 810 nM), and does not inhibit closely related kinases MEK1/2, JNK2, and ERK2.

ERK5:810 nM, MEK5:4.3 nM

BIX02188显著阻断MEK5的催化活性，IC50值为4.3 nM，且抑制ERK5的催化活性，IC50值达0.83 μM。对于与之密切相关的激酶MEK1、MEK2、ERK1、p38α、JNK2、TGFβR1、EGFR和STK16则无活性，其中TGFβR1、p38α的IC50值分别为1.8 μM和3.9 μM，其他激酶的IC50值>6.3 μM。BIX02188预处理能以剂量依赖的方式抑制甘露醇诱导的HeLa细胞中ERK5的磷酸化，但对ERK1/2、p38和JNK1/2 MAPKs的磷酸化无抑制作用。单独处理BIX02188 24小时，对HeLa或HEK293细胞无细胞毒作用。BIX02188通过抑制MEK5/ERK5信号传导途径，抑制MEK5/ERK5/MEF2C驱动的荧光素酶表达系统中MEF2C的转录激活，其IC50值在HeLa和HEK293细胞中分别为1.15 μM和0.82 μM。通过阻断MEK5信号途径，BIX02188还能以剂量依赖的方式抑制用300 μM Water2刺激的牛肺微血管内皮细胞(BLMECs)中BMK1的磷酸化，IC50为0.8 μM。BIX02188完全逆转了TNF介导的JNK激活的抑制效果，在BMK1抑制上有类似效应，表明它通过激活MEK5-BMK1信号途径抑制TNF信号。

Catalytic assay: MEK5 protein isolated from a 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 BIX02188. The kinase reaction mixture is incubated for 90 minutes at room temperature followed by addition of 10 μL of an 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. BIX02188 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. A 20 μl sample is run on SDS–PAGE 10% Tris-glycine gels and transferred to nitrocellulose. Western blotting is done with appropriate antibodies.(Only for Reference)