The zymogenic form of SARS-CoV-2 main protease: A discrete target for drug discovery.

Abstract

SARS-CoV-2 main protease (M^pro) autocatalytically releases itself out of the viral polyprotein to form a fully active mature dimer in a manner that is not fully understood. Here, we introduce several tools to help elucidate differences between cis (intramolecular) and trans (intermolecular) proteolytic processing and to evaluate inhibition of precursor M^pro. We found that many mutations at the P1 position of the N-terminal autoprocessing site do not block cis autoprocessing but do inhibit trans processing. Notably, substituting the wild-type glutamine at the P1 position with isoleucine retains M^pro in an unprocessed precursor form that can be purified and further studied. We also developed a cell-based reporter assay suitable for compound library screening and evaluation in HEK293T cells. This assay can detect both overall M^pro inhibition and the fraction of uncleaved preM^pro through separable fluorescent signals. We observed that inhibitory compounds preferentially block mature M^pro. Bofutrelvir and a novel compound designed in-house showed the lowest selectivity between precursor and mature M^pro, indicating that inhibition of both forms may be possible. Additionally, we observed positive modulation of precursor activity at low concentrations of inhibitors. Our findings help expand understanding of the SARS-CoV-2 viral life cycle and may facilitate development of strategies to target preM^pro for inhibition or premature activation of M^pro.