Ma, S.; Patel, H.; Peeples, C. A.; Shen, J.

Targeted covalent inhibitor (TCI) design has become increasingly popular. Acrylamides are most commonly used warheads for targeting reactive cysteines; however, the reaction mechanisms of acrylamides in proteins remain controversial, particularly for those involving unreactive cysteines. Using combined semiempirical quantum mechanical (QM)/molecular mechanical (MM) simulations, we investigated the reaction between afatinib, the first TCI drug for cancer treatment, and the front-pocket cysteine Cys797 in EGFR, which has been suggested as unreactive in a recent study. Two base-assisted mechanisms were examined, in which either 1) the beta-dimethylaminomethyl substitution or 2) the nearby Asp800 serves as a general base/acid for the Michael addition between afatinib and Cys797 of EGFR. The calculated reaction free energy profiles suggested that while both mechanisms are stepwise, Mechanism 1 is more likely due to a lower free energy barrier for the rate-limiting nucleophilic attack, in close agreement with experimental measurement. Importantly, the simulations demonstrated that Asp800 plays an assisting role by stabilizing the thiolate-afatinib ion-pair reactant state as well as lowering the barrier of the nucleophilic attack. Our work elucidates an important structure-activity relationship of acrylamides that is generally applicable for cysteine-directed TCI designs.