Abstract
Enzyme inhibitors working by O-acylation of nucleophilic serine residues are of immense medicinal importance, as exemplified by the β-lactam antibiotics. By contrast, inhibition of nucleophilic cysteine enzymes by S-acylation has not been widely exploited for medicinal applications. The SARS-CoV-2 main protease (M(pro)) is a nucleophilic cysteine protease and a validated therapeutic target for COVID-19 treatment using small-molecule inhibitors. The clinically used M(pro) inhibitors nirmatrelvir and simnotrelvir work via reversible covalent reaction of their electrophilic nitrile with the M(pro) nucleophilic cysteine (Cys145). We report combined structure activity relationship and mass spectrometric studies revealing that appropriately functionalized γ-lactams can potently inhibit M(pro) by reversible covalent reaction with Cys145 of M(pro). The results suggest that γ-lactams have potential as electrophilic warheads for development of covalently reacting small-molecule inhibitors of M(pro) and, by implication, other nucleophilic cysteine enzymes.