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-(3-{7-[2-methoxy-4-(4-methylpiperazin-1-yl)phenylamino]-3,4-dihydro-3-isopropyl-2,4-dioxopyrimido[4,5-d]pyrimidin-1(2H)-yl}phenyl)acrylamide], demonstrated significant selectivity and inhibitory activity toward the EGFR L858R/T790M double mutant (EGFRDM) relative to the EGFR wild-type form (EGFRWT). The enhanced therapeutic potency of 20g against EGFRDM is 263 times greater than that against EGFRWT, which necessitates a rational explanation for the underlying selective and inhibitory mechanisms. In this work, we investigate the differential binding modes of 20g with EGFRWT and EGFRDM 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 α-loop, p-loop, active loop, and αC-helix are responsible for the disparate binding mechanisms and inhibitory prowess of 20g with respect to EGFRWT and EGFRDM. The calculated binding free energies show good correlation with the experimental biological activity. The total binding free energy difference between EGFRWT-20g and EGFRDM-20g is -11.47 kcal/mol, implying that 20g binds more strongly to EGFRDM. This enhanced binding affinity of 20g for EGFRDM is a result of a large increase in the van der Waals and electrostatic interactions with three critical residues (Met790, Gln791, and Met793) that are chiefly responsible for the high-affinity interactions mediated by 20g with EGFRDM relative to EGFRWT.