Dysregulation of protein kinases is linked to cancers and other human diseases. Covalent kinase inhibitor design has attracted growing attention since the regulatory approval of cysteine-targeted kinase inhibitors as therapeutic agents for cancer patients. Understanding kinase covalent inhibition mechanisms is relevant for the design of novel inhibitors or improvement of the existing inhibitors to counter drug resistance. The epidermal growth factor receptor (EGFR) is a tyrosine kinase that catalyzes protein phosphorylation reactions to trigger signaling networks. Afatinib is a FDA approved covalent inhibitor of EGFR for the treatment of metastatic non-small cell lung cancer. X-ray structure demonstrates that afatinib forms a Michael adduct with Cys797 of EGFR; however, molecular dynamics based pKa calculation on apo EGFR suggested that Cys797 is protonated and hence not nucleophilic. Prompted by this controversy, we performed combined quantum mechanics/molecular mechanics (QM/MM) simulations to calculate the free energy profiles of the thiol-Michael addition reaction of afatinib with EGFR. Our simulations suggested that the Michael addition reaction follows a base-catalyzed mechanism in which a terminal tertiary amine group attached to afatinib’s acrylamide warhead serves as a general base to deprotonate Cys797 prior to the nucleophilic attack. Our data supports the notion that the electrophilic warheads in the covalent inhibitors can be properly designed to target unreactive but ligandable cysteines. Our study also demonstrates the utility of QM/MM simulations in covalent drug design.

Mechanism of the thiol-Michael addition reaction of afatinib with EGFR: A combined QM/MM study

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