RAF265 (CHIR-265) (CHIR-265) is a potent selective inhibitor of C-Raf/B-Raf/B-Raf V600E with IC50 of 3-60 nM, and exhibits potent inhibition on VEGFR2 phosphorylation with EC50 of 30 nM. Phase 2.

C-Raf:3-60 nM, B-Raf:3-60 nM, VEGFR2:30 nM(EC50)

RAF265抑制C-Raf、野生型B-Raf以及突变型(V600E)B-Raf。它有效阻断了Raf下游底物MEK和ERK在细胞中的磷酸化，并能杀死携带B-Raf突变的黑色素瘤和结直肠癌细胞株，这一作用与PTEN突变状态无关。在携带突变B-Raf的黑色素瘤细胞株中，RAF265通过抑制Raf激酶活性导致细胞周期阻断并诱导凋亡，这模仿了Raf RNAi在这些细胞中的效果。此外，RAF265还强效抑制VEGFR2的磷酸化和VEGF刺激下的hMVEC增殖。[1]在HT29和MDAMB231细胞中，RAF265显示出IC20为1到3μM和IC50为5到10μM的抑制活性。RAF265显著降低了所有测试细胞株的集落形成生存率，说明RAF265对集落形成生存率产生了显著的影响。在HCT116细胞中添加RAF265可以中度降低AKT、S6蛋白和4EBP1的磷酸化。[2]RAF265显著降低了Bcl-2的蛋白水平，在CM-和NCI-H727细胞中具有很强的抑制作用，但对BON1和GOT1细胞的TRAIL敏感性没有影响。[3]蛋白激酶D3 (PRKD3)的敲除能增强RAF265对A2058黑色素瘤细胞的杀伤作用，阻止MAPK信号通路的重新激活，诱导PARP切割，增加caspase活性，干扰细胞周期进展，并抑制集落形成。[4]

RAF265在12 mg/kg剂量下对HCT116异种移植瘤显示出71%至72%的TVI%（肿瘤体积抑制百分比）。而RAF265与RAD001的联合使用则显示出增强的抗肿瘤活性，表现在T10（达到初始肿瘤体积10倍的相对肿瘤体积的时间）增长和肿瘤生长延迟上。此外，RAD001与RAF265的组合显著增强了在HCT116和MDAMB231中的caspase-3激活，但在A549异种移植瘤中没有此效果。[2] RAF265能通过口服100 mg/kg剂量降低A375M异种移植瘤中FDG（2-脱氧-2-[18F]氟代葡萄糖）的积累并减少肿瘤体积。[5]

Assay Protocol: Raf and Mek are combined at 2 × final concentrations in assay buffer (50 mM Tris, pH 7.5, 15 mM MgCl2. 0.1 mM EDTA and 1 mM DTT) and dispensed 15 μL per well in polypropylene assay plates. Background levels are determined in wells containing Mek and DMSO without Raf. To the Raf/Mek containing wells is added 3 μL of 10 × of RAF265 diluted in 100% DMSO. The raf kinase activity reaction is started by the addition of 12 μL per well of 2.5 × 33P-ATP diluted in assay buffer. After 45-60 minutes, the reactions are stopped with the addition of 70 μL of stop reagent (30 mM EDTA). Filtration plates are pre-wetted for 5 min with 70% ethanol, and then rinsed by filtration with wash buffer. Samples (90 μL) from the reaction wells are then transferred to the filtration plates. The filtration plates are washed 6 × with wash buffer using Millipore filtration apparatus. The plates are dried and 100 μL per well of scintillation fluid is added. The CPM is then determined using a Wallac Microbeta 1450 reader.

The MTT assay and Bliss additivism model are used to assess the effect of RAF265 on cell viability. In each well of a 96-well plate, 1 × 104 cells are grown in 200 μL of medium. After 24 hours, RAF265 is added to achieve a final concentration of 0.1 to 10 μM. After 48 hours of treatment, 20 μL of 5 mg/mL MTT solution in PBS is added to each well. After 4 hours, supernatant is removed and formazan crystals are discarded in 200 μL of DMSO. Absorbance is then measured at 595 nm using an absorbance plate reader. Data are expressed as the percentage of viable cells.(Only for Reference)