Assay Development and Validation for Innovative Antiviral Development Targeting the N-Terminal Autoprocessing of SARS-CoV-2 Main Protease Precursors

Autor: Liangqun Huang, Megan Gish, James Boehlke, Ryan H. Jeep, Chaoping Chen
Jazyk: angličtina
Rok vydání: 2024
Předmět:
Zdroj: Viruses, Vol 16, Iss 8, p 1218 (2024)
Druh dokumentu: article
ISSN: 16081218
1999-4915
DOI: 10.3390/v16081218
Popis: The SARS-CoV-2 main protease (Mpro) is initially synthesized as part of polyprotein precursors that undergo autoproteolysis to release the free mature Mpro. To investigate the autoprocessing mechanism in transfected mammalian cells, we examined several fusion precursors, with the mature SARS-CoV-2 Mpro 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 Mpro. Interestingly, the wild-type precursors exhibited different enzymatic activities compared to those of the released Mpro, 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 Mpro precursor autoprocessing or the released mature Mpro through different mechanisms of action.
Databáze: Directory of Open Access Journals
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