In vitro activity: EAI045 potently inhibits EGFR Y1173 phosphorylation in H1975 cells (half maximal effective concentration (EC50)=2nM), but not in HaCaT cells, a keratinocyte cell line with wild-type EGFR. Despite potent inhibition of mutant EGFR, EAI045 shows no anti-proliferative effect in the H1975 and H3255 cell lines with concentrations as high as 10μM. EAI045 inhibits L858R/T790M mutant with an IC50 of 3 nM. However, EAI045 is not able to completely abolish EGFR autophosphorylation in H1975 NSCLC cell line harboring the L858R/T790M mutant. Dimerization-defective/independent mutants are markedly more sensitive to EAI045. Since EGFR dimerization is required for kinase enzyme activation, EAI045 may be active against one subunit of an EGFR heterodimer/asymmetric dimer.

Kinase Assay: EAI045 is an allosteric inhibitor of mutant EGFR with IC50s of 1.9, 0.019, 0.19 and 0.002 μM for EGFR, EGFRL858R, EGFRT790M and EGFRL858R/T790M at 10 μM ATP, respectively.

Cell Assay: 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.