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

EAI045 potently inhibits EGFR Y1173 phosphorylation in H1975 cells (half maximal effective concentration (EC50)=2 nM), 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 10 μM[1]. 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[2].

Mouse pharmacokinetic studies with EAI045 reveals a maximal plasma concentration of 0.57 μM, a half-life of 2.15 h, and oral bioavailability of 26% after dosing at 20 mg/kg[1]. When combined with cetuximab that blocks EGFR dimerization, EAI045 markedly reduces tumor growth in a mouse model of L858R/T790M-mutant-driven lung cancer. The mice treated alone with EAI045 do not respond. EAI045 in combination with cetuximab also induces marked tumor shrinkage in the mouse model carrying L858R/T790M/C797S, a mutant known to be resistant to all third-generation EGFR TKIs. EAI045 and cetuximab exhibits mechanistic synergy[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)