Application of Dually Activated Michael Acceptor to the Rational Design of Reversible Covalent Inhibitor for Enterovirus 71 3C Protease

Targeted covalent inhibitors (TCIs) have attracted growing attention from the pharmaceutical industry in recent decades because they have potential advantages in terms of efficacy, selectivity, and safety. TCIs have recently evolved into a new version with reversibility that can be systematically modulated. This feature may diminish the risk of haptenization and help optimize the drug–target residence time as needed. The enteroviral 3C protease (3C^pro) is a valuable therapeutic target, but the development of 3C^pro inhibitors is far from satisfactory. Therefore, we aimed to apply a reversible TCI approach to the design of novel 3C^pro inhibitors. The introduction of various substituents onto the α-carbon of classical Michael acceptors yielded inhibitors bearing several classes of warheads. Using steady-state kinetics and biomolecular mass spectrometry, we confirmed the mode of reversible covalent inhibition and elucidated the mechanism by which the potency and reversibility were affected by electronic and steric factors. This research produced several potent inhibitors with good selectivity and suitable reversibility; moreover, it validated the reversible TCI approach in the field of viral infection, suggesting broader applications in the design of reversible covalent inhibitors for other proteases.