Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) main protease (Mpro) 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 Mpro. 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 WT glutamine at the P1 position with isoleucine retains Mpro 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 Mpro inhibition and the fraction of uncleaved precursor form of Mpro through separable fluorescent signals. We observed that inhibitory compounds preferentially block mature Mpro. Bofutrelvir and a novel compound designed in-house showed the lowest selectivity between precursor and mature Mpro, 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 precursor form of Mpro for inhibition or premature activation of Mpro.

Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences Prague Czech Republic

Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences Prague Czech Republic; Department of Biochemistry and Microbiology University of Chemistry and Technology Prague Prague Czech Republic

Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences Prague Czech Republic; Faculty of Science Department of Biochemistry Charles University Prague Prague Czech Republic

Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences Prague Czech Republic; Faculty of Science Department of Genetics and Microbiology Charles University Prague Prague Czech Republic

Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences Prague Czech Republic; Faculty of Science Department of Physical and Macromolecular Chemistry Charles University Prague Prague Czech Republic

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