biostudies-literatureGayatriWellcome TrustBiotechnology and Biological Sciences Research Council7667-7678https://www.ebi.ac.uk/biostudies/studies/S-EPMC11110133biostudies-literatureUnknown15(20)Enzyme inhibitors working by <i>O</i>-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 <i>S</i>-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 <i>via</i> 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.Chemical scienceThiophene-fused γ-lactams inhibit the SARS-CoV-2 main protease <i>via</i> reversible covalent acylation.PMC11110133106244/Z/14/ZBB/R000344/1BB/J003018/1Salah EBasak SIbbotson LBrewitz LChoudhry HGayatriSchofield CJfalseThiophene-fused γ-lactams inhibit the SARS-CoV-2 main protease <i>via</i> reversible covalent acylation.Enzyme inhibitors working by <i>O</i>-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 <i>S</i>-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 <i>via</i> 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.2024-01-01T00:00:00Z2024 May2026-06-03T04:26:08.065Z2026-04-24T03:09:32.137ZS-EPMC111101333878472910.1039/d4sc01027b