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 ( 18 ), an irreversible inhibitor of the L858R/T790M double-mutant EGFR (EGFR DM ) using α-chlorofluoroacetamide (CFA) as a novel warhead, has seven times the inhibition selectivity for EGFR DM over the wild type (EGFR WT ), as compared to clinically approved osimertinib ( 7 ). 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 18 over 7 . We find that EGFR DM undergoes significantly larger conformational changes than EGFR WT upon binding to 18 . The conformational stability of the diamine side chain and the CFA motif of 18 in the orthosteric site of EGFR DM is identified as key for the disparate binding mechanism and inhibitory prowess of 18 with respect to EGFR WT and EGFR DM and 18 's higher selectivity than 7 . The binding free energy of the 18 -bound complexes is -6.38 kcal/mol greater than that of the 7 -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 18 -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.