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 (3Cpro) is
a valuable therapeutic target, but the development of 3Cpro inhibitors is far from satisfactory. Therefore, we aimed to apply
a reversible TCI approach to the design of novel 3Cpro 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.

近年来，目标共价抑制剂（TCIs）因其效能、选择性和安全性方面的潜在优势，受到了制药行业的广泛关注。此类抑制剂近期发展出一种可逆的新版本，其可系统调节的特性可能降低半抗原化的风险，并有助于优化药物与靶点之间的停留时间。肠病毒3C蛋白酶（3Cpro）是一个具有重要治疗价值的靶点，但其抑制剂的研发尚不尽人意。因此，本研究旨在将可逆的TCI方法应用于新型3Cpro抑制剂的研发。通过对经典迈克尔受体α-碳引入多种取代基，我们得到了携带多种弹头类抑制剂的化合物。通过稳态动力学和生物分子质谱分析，我们确认了可逆共价抑制的机制，并阐明了电子和立体因素如何影响其效力和可逆性。本研究产出了数种具有良好选择性和适宜可逆性的强效抑制剂；此外，它还验证了可逆TCI方法在病毒感染领域的应用，为其他蛋白酶的可逆共价抑制剂设计提供了更广泛的应用前景。