AZ628 is a new pan-Raf inhibitor for BRAF, BRAFV600E, and c-Raf-1 with IC50 of 105 nM, 34 nM and 29 nM, also inhibits VEGFR2, DDR2, Lyn, Flt1, FMS, etc.

B-Raf (V600E):34 nM, BRAF:105 nM, C-Raf1:29 nM

AZ628可阻止包括VEGFR2、DDR2、Lyn、Flt1、FMS等在内的多种酪氨酸蛋白激酶的活化。它抑制锚定依赖性和独立性生长，引起细胞周期停滞，并在携带B-RafV600E突变的结肠和黑色素瘤细胞系中诱导凋亡。AZ628可能具有抗血管生成性。[1]相较于亲本细胞系的IC50为0.1μM，AZ628抗性克隆细胞对AZ628的抗性约增加100倍，显示出大约10μM的IC50。在M14亲本细胞系中，随着AZ628浓度的增加，有效抑制了p-ERK1/2水平。AZ628抗性克隆细胞表达上调CRAF，上调CRAF表达可能是持续暴露于AZ628后获得抗性的潜在机制，导致ERK1/2持续激活。在这三种AZ628不敏感细胞系之一（Wm1552C）中，p-ERK1/2活性未被AZ628暴露显著抑制。与在AZ628抗性M14细胞中AZ628未能抑制ERK激活不同，AZ628治疗在NRAS突变黑色素瘤细胞中有效减弱了ERK激活。[2]

Approximately 0.5-2.5 × 105 M14 cells are seeded in 12 or 24 - well plates, respectively, in medium supplemented with 5% FBS. After overnight incubation, the cells are treated with various concentrations of AZ628. Fresh medium and drug is replaced every 2 days until the untreated control wells reached confluence. At this time-point, the media is removed and the cells are fixed in 4% formaldehyde in PBS for 20 minutes at room temperature. Cells are then washed twice with PBS and stained with a 1:5000 solution of the fluorescent nucleic acid stain Syto60. Quantitation of fluorescent signal intensity is carried out at 700 nm, using an Odyssey Infrared Imager. Each experiment is performed in quadruplicate and the results shown represent the average of the four values compared to untreated wells. Error bars represent standard deviation of the 4 values from the mean. High-throughput cell growth/viability assays are performed.(Only for Reference)