Investigating the Selectivity of Allosteric Inhibitors for Mutant T790M EGFR over Wild Type Using Molecular Dynamics and Binding Free Energy Calculations

The recent discovery of the fourth generation EAI045 allosteric inhibitor, which potently and selectively inhibits mutant EGFR, represents an important step forward for the treatment of non-small cell lung cancer. However, the structural determinants of EAI045 selectivity with respect to the wild type (wt) protein have not been fully investigated. To this aim, we performed a comparative analysis of long-scale molecular dynamics simulations and binding free energy calculations on wt and T790M EGFR in complexes with the EAI001 and EAI045 allosteric ligands. Unexpectedly, we found that the observed selectivity for T790M EGFR over wt is not due to more favorable interactions of the two ligands with the mutated gatekeeper residue, as previously suggested. Rather, the allosteric ligands were engaged in a direct hydrogen bond with the Asp855 residue of the DFG motif in mutant T790M but not in wt, in which the hydrogen bond was found to be water-mediated. Per-residue decomposition of binding free energies suggests that the loss of a direct interaction with Asp855 is the main cause of inhibitor selectivity. Moreover, the possibility that the allosteric ligands and adenosine triphosphate may have synergistic binding effects, as previously observed in MEK allosteric inhibitors, was investigated. Altogether, the results suggest that ligand selectivity arises from direct hydrogen bonds with the Asp855 side chain, and that the design of mutant-selective inhibitors should be focused on ligands that form direct hydrogen bonds with Asp855 in T790M EGFR but not in wt EGFR. These results may provide useful hints for future structural design of mutant-selective allosteric inhibitors that spare wt EGFR, which is a highly desirable goal.