ZM 336372 is a potent and selective c-Raf inhibitor.

C-Raf:70 nM

1 μM ZM 336372消除过氧化氢处理后eNOS的上调。ZM 336372在HepG2中以剂量依赖性方式诱导抑制增殖,抑制激素分泌和上调细胞周期抑制剂。ZM 336372作用于C-Raf 的选择性是作用于B-Raf的10倍。ZM 336372抑制嗜铬细胞瘤细胞的增殖,并抑制NE血管活性肽产生。ZM 336372微弱抑制SAPK2a/p38α和SAPK2b/p38β,IC50 为2 μM,即使在高达50 μM浓度下,对C-Raf的选择性也高于包括PKA,PKC,AMPK,p42 MAPK,MKK1,SAPK1/JNK和CDK1等17种其他蛋白激酶。ZM 336372不会阻止生长因子或佛波酯诱导的MKKl或p42 MAPK/ERK2激活。通过抑制MAPK级联反应,蛋白激酶C或磷脂酰肌醇3-激酶不能阻止ZM 336372诱导的c-Raf的激活。ZM 336372处理诱导c-Raf和B-Raf异构体> 100的活化,但它不引发MKKI或p42MAPK/ERKP的任何活化或诱导Ras的GTP加载的任何增加,表明反馈控制环Raf异构体抑制其自身激活,从而抑制总是通过再激活抵消。ZM 336372在胰腺癌细胞系中,也会通过Ser 9上GSK-3β的磷酸化作用抑制糖原合成酶激酶-3β,从而诱导细胞凋亡。

In vitro kinase assay: c-Raf kinase activity is assayed directly in Sl9 cell lysates. Human c-Raf is activated in Sf9 cells by cotransfection from baculovirus vectors containing DNA encoding v-Ras and Lck in the absence of ZM 336372. The cell lysates are then assayed for c-Raf activity in the presence of increasing concentrations of ZM 336372.

Cells are exposed to various concentrations of ZM 336372 for 48 and 72 hours. After incubation, the medium is removed and cells are trypsinized. Cells are incubated on ice, and 2.5 μg/mL propidium iodide is added 5 minutes before flow cytometry. Data is acquired using a FACSCalibur benchtop flow cytometer using CellQuest acquisition and analysis software. Cytotoxicity is done using Cell Titer Glo Assay. Cell proliferation is measured using MTT assay.(Only for Reference)