Assay Development and Validation for Innovative Antiviral Development Targeting the N-Terminal Autoprocessing of...

The SARS-CoV-2 main protease (M^pro) is initially synthesized as part of polyprotein precursors that undergo autoproteolysis to release the free mature M^pro. To investigate the autoprocessing mechanism in transfected mammalian cells, we examined several fusion precursors, with the mature SARS-CoV-2 M^pro along with the flanking amino acids (to keep the native substrate sequences) sandwiched between different tags. Our analyses revealed differential proteolysis kinetics at the N- and C-terminal cleavage sites. Particularly, N-terminal processing is differentially influenced by various upstream fusion tags (GST, sGST, CD63, and Nsp4) and amino acid variations at the N-terminal P1 position, suggesting that precursor catalysis is flexible and subject to complex regulation. Mutating Q to E at the N-terminal P1 position altered both precursor catalysis and the properties of the released M^pro. Interestingly, the wild-type precursors exhibited different enzymatic activities compared to those of the released M^pro, displaying much lower susceptibility to known inhibitors targeting the mature form. These findings suggest the precursors as alternative targets for antiviral development. Accordingly, we developed and validated a high-throughput screening (HTS)-compatible platform for functional screening of compounds targeting either the N-terminal processing of the SARS-CoV-2 M^pro precursor autoprocessing or the released mature M^pro through different mechanisms of action.