A Mechanistic Study of a Potent and Selective Epidermal Growth Factor Receptor Inhibitor against the L858R/T790M Resistance Mutation

Covalent targeting is a promising strategy for increasing the potency and selectivity of potential drug candidates. This therapeutic approach was recently reported for the epidermal growth factor receptor (EGFR), wherein a covalent binder, 20g [N-(347-[2-methoxy-4-(4-methylpiperazin-1-yl)phenylamino]-3,4-dihydro-3-isopropy1-2,4- dioxopyrimido[4,S-d]pyrimidin-1(2H)-yllpheny1)- acrylamide], demonstrated significant selectivity and inhibitory activity toward the EGFR L858R/T790M double mutant (EGFR(DM)) relative to the EGFR wild-type form (EGFR(WT)). The enhanced therapeutic potency of 20g against EGFR(DM) is 263 times greater than that against EGFR(WT), which necessitates a rational explanation for the underlying selective and inhibitory mechanisms. In this work, we investigate the differential binding modes of 20g with EGFR(WT) and EGFR(DM) using molecular dynamics simulations coupled with free energy calculations and further identify key residues involved in the selective targeting, binding, and inhibitory mechanisms mediated by 20g. We find that systematic orientational and conformational changes in the alpha-loop, p-loop, active loop, and alpha C-helix are responsible for the disparate binding mechanisms and inhibitory prowess of 20g with respect to EGFR(WT) and EGFR(DM). The calculated binding free energies show good correlation with the experimental biological activity. The total binding free energy difference between EGFR(WT)-20g and EGFR(DM)-20g is -11.47 kcal/mol, implying that 20g binds more strongly to EGFR(DM). This enhanced binding affinity of 20g for EGFR(DM) is a result of a large increase in the van der Waals and electrostatic interactions with three critical residues (Met790, G1n791, and Met793) that are chiefly responsible for the high-affinity interactions mediated by 20g with EGFR(DM) relative to EGFR(WT).