Alectinib (RG-7853) is an ALK inhibitor (IC50=1.9 nM, Kd=2.4 nM) that is selective, orally active, and ATP-competitive. Alectinib has antitumor activity and is used in the treatment of non-small cell lung cancer.

ALK:1.9 nM, ALK (R1275Q):3.5 nM, ALK (F1174L):1 nM

方法：六种神经母细胞瘤 (NB) 细胞系用 Alectinib (0.001-50 µM) 处理 72 h，通过 CCK-8 assay 检测细胞活力。
结果：选择了六种 NB 细胞系，包括三个 ALK-WT 细胞系 (IMR-32、NB-19 和 SK-N-AS) 和三个 ALK 突变细胞系 (Kelly、SH-SY5Y 和 LA-N-6)。Alectinib 处理大大降低了细胞存活率，所测试细胞系的 IC50 范围为 3.181-9.6 µM。[1]
方法：NSCLCA 细胞 NCI-H2228 用 Alectinib (10-1000 nM) 处理 2 h，通过 Western Blot 检测靶点蛋白表达水平。
结果：Alectinib 可以防止表达 EML4-ALK 的 NCI-H2228 NSCLC 细胞中 ALK 的自磷酸化，并且 Alectinib 还导致 STAT3 和 AKT 的磷酸化受到实质性抑制，但 ERK1/2 的磷酸化没有受到实质性的抑制。[2]

方法：为检测体内抗肿瘤活性，将 Alectinib (25 mg/kg) 腹腔注射给 TH-MYCN 转基因小鼠，每两天一次，给药 11 次。
结果：用 Alectinib 治疗导致 TH-MYCN 转基因小鼠的肿瘤生长减少。Alectinib 在 TH-MYCN 肿瘤组织中诱导 PARP 和 Caspase 3 切割，并阻断 PI3K/Akt/mTOR 信号传导。与 DMSO 治疗相比，Alectinib 暴露延长了 TH-MYCN 转基因小鼠的存活时间。[1]

Kinase inhibitory assays in Vitro: The inhibitory ability against each kinase except for MEK1 and Raf-1 is evaluated by examining their ability to phosphorylate various substrate peptides in the presence of CH5424802 using time-resolved fluorescence resonance energy transfer (TR-FRET) assay or fluorescence polarization (FP) assay. The inhibitory activity against MEK1 is evaluated by quantitative analysis of the phosphorylation of a substrate peptide by a recombinant ERK2 protein in the presence of CH5424802. The inhibitory activity against Raf-1 is evaluated by examining the ability of the kinases to phosphorylate MEK1 in the presence of CH5424802.

Cells including NSCLC, A549 and HCC827 are seeded in 96-well plates overnight and incubated with various concentrations of CH5424802 for the indicated time. For spheroid cell growth inhibition assay, cells are seeded on spheroid plates, incubated overnight, and then treated with compound for the indicated times. The viable cells are measured by the Luminescent Cell Viability Assay. Caspase-3/7 assay is evaluated using the Caspase-Glo 3/7 Assay Kit.(Only for Reference)