Agerafenib (CEP32496) is a highly potent inhibitor of BRAF.

PDGFRβ:2 nM(Kd), c-Kit:2 nM(Kd), Abl-1:3 nM(Kd), RET:2 nM(Kd), Lck:2 nM(Kd)

在Colo-205异种移植小鼠模型中CEP-32496（30 mg/kg,口服,BID）显示肿瘤停滞和部分肿瘤消退（PR）的发生率为40％,而100 mg/kg剂量组显示肿瘤停滞和PR的发生率为80％.每天两次口服30 mg/kg CEP-32496处理肿瘤裂解物,在给药后2小时和6小时,分别抑制50％和75％归一化的pMEK,而给携带Colo-205移植瘤的小鼠模型用55 mg/kg CEP-32496处理2到10小时,抑制57％到75％pMEK.CEP-32496在多种临床前物种中口服生物可利用（大鼠,狗和猴子> 95％）.100 mg/kg CEP-32496处理导致裸鼠中的BRAF（V600E）结肠癌异种移植物中的pMEK和pERK的抑制和持续的肿瘤停滞和退化.CEP-32496在小鼠,狗,猴和人肝微粒体制剂中表现出良好的稳定性,在所有测定中测得的固有清除率值<23（μL/分钟）/ mg和t1/2> 60分钟.

CEP-32496抑制人类黑素瘤和结直肠癌细胞系中的MAPK/MEK磷酸化,IC50为78 nM和60 nM。CEP-32496抑制A375细胞(BRAFV600E) 增殖,EC50为78 nM。CEP-32496作用于肿瘤细胞系,对表达突变型BRAF的A375,SK-MEL-28,Colo-205,Colo-679和HT-144细胞比表达野生型BRAF的HCT116,Hs578T,LNCaP,DU145和PC-3细胞具有更敏感的细胞毒性作用。

Binding assay: Kinases are produced displayed on T7 phage or by expression in HEK-293 cells and tagged with DNA. Binding reactions are performed at room temperature for 1 hour, and the fraction of kinase not bound to test compound is determined by capture with an immobilized affinity ligand and quantitation by quantitative PCR. Each kinase is tested individually against CEP-32496. Kd values are determined using eleven serial 3-fold dilutions and presented as mean values from experiments performed in duplicate. Variability between individual values is less than 2-fold.

Cells are seeded at 104 cells per well in DMEM with 10% fetal calf serum and allowed to attach. The cells are washed with PBS and switched to DMEM with 0.5% of serum and incubated overnight. CEP-32496 is then added at various concentrations with a final DMSO concentration of 0.5% and incubated for 72 h. At the end of incubation, a Cell Titer Blue is added per instructions, and incubation is continued for 3 hours. Remaining viable cells are quantified by measuring the strength of the fluorescence signal using SoftMax Pro (excitation at 560 nm and emission at 590 nm). IC50 values are derived using a 9-point curve fitted with Igor Pro and are presented as mean values from experiments performed in duplicate. Variability between individual values is less than 2-fold.(Only for Reference)