Development of a new experimental NMR strategy for covalent cysteine protease inhibitors screening: toward enhanced drug discovery.

In the development of antiviral drugs, proteases and polymerases are among the most important targets. Cysteine proteases, also known as thiol proteases, catalyze the degradation of proteins by cleaving peptide bonds using the nucleophilic thiol group of cysteine. As part of our research, we are examining how cysteine, an essential amino acid found in the active site of the main protease (M pro ) enzyme in SARS-CoV-2, interacts with electrophilic groups present in ethacrynic acid (EA) and compounds 4, 6, and 8 to form sulfur-carbon bonds. Nuclear magnetic resonance (NMR) spectroscopy was used to monitor the reaction rate between cysteine and Michael acceptors. We found that the inhibitory activity of these compounds towards M pro is correlated to their chemical reactivity toward cysteine. This approach may serve as a valuable tool in drug development for detecting potential covalent inhibitors of M pro and other cysteine proteases.