C6-Alkoxy substituted penicillins are potent non-covalently binding inhibitors of the SARS-CoV-2 main protease

Inhibition of the SARS-CoV-2 main protease (M^pro) by small-molecules is a validated strategy for COVID-19 treatment. There is a need for improved M^pro inhibitors, including because M^pro mutations can confer resistance to clinically used M^pro inhibitors. Previous work has revealed the potential of penicillin derivatives as covalently reacting M^pro inhibitors. Here we report studies on M^pro inhibition by C6-alkoxy substituted penicillin derivatives. The combined mass spectrometric and computational evidence imply most of the tested penicillin C6-alkoxy derivatives bind <i>via</i> non-covalent interactions at the M^pro active site, resulting in potent substrate-competitive inhibition. Some penicillin C6-alkoxy derivatives ((<i>R</i>)-, but not (<i>S</i>)-sulfoxides) manifest covalent reaction to different extents. Penicillins and related drugs are widely used antibiotics, acting <i>via</i> covalent reaction of their β-lactam with a nucleophilic serine in their transpeptidase targets to give an acyl-enzyme complex. The results imply penicillin derivatives can be developed to inhibit enzymes via mechanisms other than formation of stable acyl-enzyme complexes.