GW 5074 (Raf1 Kinase Inhibitor I)(IC50=9 nM) is an effective and specific c-Raf inhibitor. It has no effect on the activities of JNK1/2/3, MEK1, MKK6/7, CDK1/2, c-Src, p38 MAP, VEGFR2 or c-Fms.

C-Raf:9 nM

GW5074是一种强效且特异性的c-Raf抑制剂，其IC50为9 nM，但对MKK6、MKK7、p38 MAP激酶及cdks无影响。然而，在体外条件下，神经细胞培养处理中GW5074可促进c-Raf及B-Raf上激活修饰的积累。GW5074抑制LK引起的小脑颗粒神经元凋亡，并非依赖MEK-ERK。GW5074能延迟Akt活性下调，但通过Akt独立机制抑制凋亡。此外，GW5074对Ras、核因子-κB及c-jun有影响，并抑制由神经毒素引起的颗粒细胞及其他神经类型的细胞死亡。[1]

GW5074在亨廷顿病的活体实验模型中表现出保护作用。GW5074（5 mg/Kg）完全阻止了3-NP在小鼠中引发的广泛双侧纹状体损伤。[1] GW5074彻底消除了慢性吗啡在稳定表达人μ-阿片受体的CHO细胞中引起的AC超激活I。[2] 同时，GW5074还抑制了侧流烟雾引起的小鼠气道高反应性。[3]

Affinity determination: In general, in vitro kinase assays are performed using purified kinase and synthetic substrates under standard conditions using the Kinase Profiling service of Upstate Biotechnology. Briefly, for each assay 5–10 mU of purified kinase is used. For GSK3β, cdk1, cdk2, cdk3, cdk5, the kinase is incubated with 1 μM GW5074 in a buffer containing 8 mM MOPS, pH 7.2, 0.2 mM EDTA, 10 mM magnesium acetate and [c- 33P-ATP] for 40 min at room temperature. Kinase activity is quantified by measuring 33P incorporation by spotting an aliquot on P30 filters, washing in 50 mM phosphoric acid and scintillation counting. The buffer composition for c-Raf, JNK1, JNK2, JNK3, MEK1, MKK6, MKK7 is 50 mM Tris pH 7.5, 0.1 mM EGTA, 10 mM magnesium acetate and [c- 33P-ATP]. The peptide substrates used are as follows: For c-Raf, 0.66 mg/mL MBP; for cdks, 0.1 mg/mL histone H1; for JNKs, 3 μM ATF2; for MEK1, 1 μM MAPK2; for MKK6, 1 μM of SAPK2a and for MKK7, 2 μM JNK1α.

HCA is diluted from 100-fold concentrated solutions that are adjusted to pH 7.5. To evaluate the effects of GW5074 on HCA-induced cytotoxicity, GW5074 is added at the time cortical neurons are exposed to HCA. Viability is assessed 24 h later.(Only for Reference)