Cell experiment:

Proliferation of H727 and BON cells after treatment with ZM336372 is measured using a 3,4-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide. Cells are trypsinized and plated in triplicate to 24-well plates and allowed to adhere overnight. Then, cells are treated with either 100 μM ZM336372 or DMSO (2%) and incubated. Media are changed every 2 days with new treatment. At each time point, cell growth rates are analyzed after the addition of 3,4-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reagent to the cultured cells. Absorbance is determined using spectrophotometer at a wavelength of 540 nm[2].

ZM336372 is a potent inhibitor of C-Raf with IC50 value of 70 nM [1]. It is also reported that ZM336372 inhibits SAPK2b/p38β2 with IC50 value of 2 μM.

C-Raf is a member of the Raf kinase family of serine/threonine-specific protein kinases and plays an important role in the ERK1/2 pathway. It has been reported that abnormal expression of C-Raf correlates with human diseases and also involves in a variety of cancers.

ZM336372 is a potent C-Raf inhibitor and has a different selectivity with the reported C-Raf inhibitor PD98059. When tested with primary neurons, ZM3363372 treatment decreased low potassium-induced apoptosis percent by inhibiting C-Raf activation [2]. In tumor spheroids pretreated with H2O2 and ZM336372 for 24 hours totally abolished the ROS-induced eNOS up-regulation via mediating ERK1/2 signaling pathway [3].

References:
1. Hall-Jackson, C.A., et al., Paradoxical activation of Raf by a novel Raf inhibitor. Chem Biol, 1999. 6(8): p. 559-68.
2. Burgess, S. and V. Echeverria, Raf inhibitors as therapeutic agents against neurodegenerative diseases. CNS Neurol Disord Drug Targets, 2010. 9(1): p. 120-7.
3. Wartenberg, M., et al., Reactive oxygen species-mediated regulation of eNOS and iNOS expression in multicellular prostate tumor spheroids. Int J Cancer, 2003. 104(3): p. 274-82.