Molecular Mechanism of Labelling Functional Cysteines by Heterocyclic Thiones

Heterocyclic thiones have recently been identified as reversible covalent warheads, consistent with their mild electrophilic nature. Little is known so far about their mechanism of action in labelling nucleophilic sidechains, especially cysteines. The vast number of tractable cysteines promotes a wide range of target proteins to examine; however, our focus was put on functional cysteines. We chose the main protease of SARS‐CoV‐2 harboring Cys145 at the active site that is a structurally characterized and clinically validated target of covalent inhibitors. We screened an in‐house, cysteine‐targeting covalent inhibitor library which resulted in several covalent fragment hits with benzoxazole, benzothiazole and benzimidazole cores. Thione derivatives and Michael acceptors were selected for further investigations with the objective of exploring the mechanism of inhibition of the thiones and using the thoroughly characterized Michael acceptors for benchmarking our studies. Classical and hybrid quantum mechanical/molecular mechanical (QM/MM) molecular dynamics simulations were carried out that revealed a new mechanism of covalent cysteine labelling by thione derivatives, which was supported by QM and free energy calculations and by a wide range of experimental results. Our study shows that the molecular recognition step plays a crucial role in the overall binding of both sets of molecules. A reversible mechanism of functional cysteine labelling by heterocyclic thiones is presented. The mechanism is supported by both experimental and computational results – including quantum mechanical (QM), molecular mechanical (MM) and hybrid QM/MM calculations.