Thiophene-fused -lactams inhibit the SARS-CoV-2 main protease via reversible covalent acylation

Enzyme inhibitors working by O-acylation of nucleophilic serine residues are of immense medicinal importance, as exemplified by the beta-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 gamma-lactams can potently inhibit M-pro by reversible covalent reaction with Cys145 of M-pro. The results suggest that gamma-lactams have potential as electrophilic warheads for development of covalently reacting small-molecule inhibitors of M-pro and, by implication, other nucleophilic cysteine enzymes.