A potent inhibitor of B-Raf
CEP-32496 is a?highly potent and orally efficacious V600E mutant BRAF, WT BRAF and c-Raf inhibitor with Kd values of 14 nM, 36 nM and 39 nM, respectively.
In addition to BRAFV600E, CEP-32496 has exhibited high binding affinity for both wild-type BRAF and related CRAF, as well as certain receptor tyrosine kinases of known therapeutic utility, such as Abl-1, c-Kit, Ret, PDGFR-β, and VEGFR-2. However,?CEP-32496?proved selective for the RAF members of the MAPK signal transduction pathway, as no significant affinity was observed for other key kinases of the MAPK pathway, including MEK-1, MEK-2, ERK-1, and ERK-2. This suggests that the observed cellular activity was driven primarily through inhibition of BRAFV600E, which is further supported by the observation that CEP-32496?exhibited selective cytotoxicity for tumor cell lines expressing mutant BRAF versus those expressing wild-type BRAF [1].
Oral administration of?CEP-32496?to Colo-205 tumor xenograft-bearing mice resulted in significant inhibition of pMEK in tumor cell lysates [1].
参考文献:
[1] Rowbottom MW1,?Faraoni R,?Chao Q,?Campbell BT,?Lai AG,?Setti E,?Ezawa M,?Sprankle KG,?Abraham S,?Tran L,?Struss B,?Gibney M,?Armstrong RC,Gunawardane RN,?Nepomuceno RR,?Valenta I,?Hua H,?Gardner MF,?Cramer MD,?Gitnick D,?Insko DE,?Apuy JL,?Jones-Bolin S,?Ghose AK,?Herbertz T,?Ator MA,Dorsey BD,?Ruggeri B,?Williams M,?Bhagwat S,?James J,?Holladay MW. Identification of 1-(3-(6,7-dimethoxyquinazolin-4-yloxy)phenyl)-3-(5-(1,1,1-trifluoro-2 -methylpropan- 2-yl) isoxazol-3-yl) urea hydrochloride (CEP-32496), a highly potent and orally efficacious inhibitor of V-RAF murine sarcoma viral oncogene homologue B1 (BRAF) V600E. J Med Chem.?2012 Feb 9;55(3):1082-105. doi: 10.1021/jm2009925. Epub 2012 Jan 23.
Cell experiment: | A375 cells are seeded at 10,000 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. The test compounds (e.g., Agerafenib; 10 μM) are 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 h. 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 and are presented as mean values from experiments performed in duplicate[1]. |
Animal experiment: | Mice[1]Six to eight week old athymic nu/nu nude mice (20-25 g) are inoculated subcutaneously with Colo-205 tumor cells (1×106/mouse) in the right flank. Upon reaching an average tumor volume of 150-200 mm3 (10-12 days post implantation), animals are randomized into treatment groups (n=10 mice/group). Each group is dosed orally for 14 days with either vehicle only (22% HPβCD) or with Agerafenib at 10, 30, or 100 mg/kg twice daily (BID), and each dose of drug is given in a volume of 0.1 mL per 20 g of body weight, adjusted for the body weight of the animal. Tumor volumes are measured three times weekly using vernier calipers, and volumes are calculated[1]. |
参考文献: [1]. Rowbottom MW, et al. Identification of 1-(3-(6,7-dimethoxyquinazolin-4-yloxy)phenyl)-3-(5-(1,1,1-trifluoro-2-methylpropan-2-yl)isoxazol-3-yl)urea hydrochloride (CEP-32496), a highly potent and orally efficacious inhibitor of V-RAF murine sarcoma viral oncogene homologue B1 (BRAF) V600E. | |
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