Deciphering the Mechanism of Binding Selectivity of Chlorofluoroacetamide-Based Covalent Inhibitors toward L858R/T790M Resistance Mutation.

Covalent modification of the oncogenic mutant epidermal growth factor receptor (EGFR) by small molecules is an efficient strategy for achieving an enhanced and sustained pharmacological effect in the treatment of non-small-cell lung cancer. NSP-037 (), an irreversible inhibitor of the L858R/T790M double-mutant EGFR (EGFR) using α-chlorofluoroacetamide (CFA) as a novel warhead, has seven times the inhibition selectivity for EGFR over the wild type (EGFR), as compared to clinically approved osimertinib (). Here, we employ multiple computational approaches to elucidate the mechanism underlining this improved selectivity, as well as the effect of CFA on the selectivity enhancement of inhibitor over . We find that EGFR undergoes significantly larger conformational changes than EGFR upon binding to . The conformational stability of the diamine side chain and the CFA motif of in the orthosteric site of EGFR is identified as key for the disparate binding mechanism and inhibitory prowess of with respect to EGFR and EGFR and 's higher selectivity than . The binding free energy of the -bound complexes is -6.38 kcal/mol greater than that of the -bound complexes, explaining the difference in selectivity of these inhibitors. Further, free energy decomposition analysis indicates that the electrostatic contribution of key residues plays an important role in the -bound complexes. QM/MM calculations show that the most favored mechanism for the Cys797 alkylation reaction is the direct displacement mechanism through a CFA-based inhibitor, producing a reaction with the lowest energy barrier and most stable product.