EAI045, an allosteric inhibitor, targets towards drug-resistant EGFR mutants but avoids the wild-type receptor.

EGFR (L858R):0.002 μM, EGFR (L858R):0.019 μM, EGFR:1.9 μM, EGFR (T790M):0.19 μM

EAI045在H1975细胞中有效抑制EGFR Y1173的磷酸化（半最大有效浓度（EC50）=2 nM），但在具有野生型EGFR的HaCaT细胞（一种角质形成细胞系）中则无此效果。尽管EAI045对突变型EGFR具有强效抑制作用，但在浓度高达10 μM的情况下，对H1975和H3255细胞系没有抗增殖效果[1]。EAI045对L858R/T790M突变体的抑制作用表现在IC50为3 nM。然而，EAI045无法完全消除携带L858R/T790M突变的H1975 NSCLC细胞系中EGFR的自磷酸化。对于二聚体缺陷/独立突变体，EAI045表现出明显更高的敏感性。由于EGFR二聚体化对于激酶酶活性的激活是必需的，EAI045可能针对EGFR异二聚体/非对称二聚体的一个亚单位发挥作用[2]。

在小鼠药代动力学研究中，EAI045的最大血浆浓度为0.57 μM，半衰期为2.15小时，20 mg/kg剂量下的口服生物利用度为26%[1]。结合阻碍EGFR二聚化的cetuximab时，EAI045显著减缓了L858R/T790M突变驱动的小鼠肺癌模型中的肿瘤生长。单独使用EAI045治疗的小鼠未显示出反应。EAI045与cetuximab联合使用在携带L858R/T790M/C797S突变（一种对所有第三代EGFR TKIs均呈耐药性的突变体）的小鼠模型中也引起了明显的肿瘤缩小。EAI045和cetuximab表现出机制上的协同作用[2]。

ERK dimerization assay: Compound screening is performed in HEK293T cells treated with the potential inhibitors (10 μM) for 30 min before EGF stimulation. Cellular lysates are tested for ERK dimerization by native PAGE and p-ERK evaluation of the potential positives. In vitro ERK dimerization is assayed using GST-MEK1 ΔN EE purified from bacteria, bound to glutathione sepharose beads, and incubated with 25 μg/ml of purified His-ERK2 plus increasing concentrations of DEL-22379. Western blotting, kinase assays, and luciferase assays are also performed. In silico docking of the DEL-22379 compound is carried out with the modeling tools provided by the OpenEye package (v. 2.1).

H1975, H3255 and HaCaT cell lines are plated in solid white 384-well plates at 500 cells per well in 10% FBS RPMI penicillin/streptomycin media. Using a Pin Tool, 50 nl of serial diluted compounds are transferred to the cells. After 3?days, cell viability is measured.(Only for Reference)