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

被引:0
作者
Huang, Liangqun [1 ]
Gish, Megan [1 ]
Boehlke, James [1 ]
Jeep, Ryan H. [1 ]
Chen, Chaoping [1 ]
机构
[1] Colorado State Univ, Dept Biochem & Mol Biol, Ft Collins, CO 80523 USA
来源
VIRUSES-BASEL | 2024年 / 16卷 / 08期
关键词
autoproteolysis; 3C-like protease; catalysis flexibility; glycosylation; main protease; Nsp5; precursor autoprocessing; protease precursor; SARS-CoV-2; HIV-1; PROTEASE; SARS; VIRUS; INHIBITORS; MECHANISM; CLEAVAGE; IDENTIFICATION; IMMUNOASSAY; PROTEINASE; KINETICS;
D O I
10.3390/v16081218
中图分类号
Q93 [微生物学];
学科分类号
071005 ; 100705 ;
摘要
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.
引用
收藏
页数:19
相关论文
共 76 条
[1]   Impact of Early Pandemic Stage Mutations on Molecular Dynamics of SARS-CoV-2 Mpro [J].
Amamuddy, Olivier Sheik ;
Verkhivker, Gennady M. ;
Bishop, Ozlem Tastan .
JOURNAL OF CHEMICAL INFORMATION AND MODELING, 2020, 60 (10) :5080-5102
[2]   3-Chymotrypsin-like Protease (3CLpro) of SARS-CoV-2: Validation as a Molecular Target, Proposal of a Novel Catalytic Mechanism, and Inhibitors in Preclinical and Clinical Trials [J].
Amorim, Vitor Martins de Freitas ;
Soares, Eduardo Pereira ;
Ferrari, Anielle Salviano de Almeida ;
Merighi, Davi Gabriel Salustiano ;
de Souza, Robson Francisco ;
Guzzo, Cristiane Rodrigues ;
de Souza, Anacleto Silva .
VIRUSES-BASEL, 2024, 16 (06)
[3]   Coronavirus main proteinase (3CLpro) structure:: Basis for design of anti-SARS drugs [J].
Anand, K ;
Ziebuhr, J ;
Wadhwani, P ;
Mesters, JR ;
Hilgenfeld, R .
SCIENCE, 2003, 300 (5626) :1763-1767
[4]   Insights into the mechanism of SARS-CoV-2 main protease autocatalytic maturation from model precursors [J].
Aniana, Annie ;
Nashed, Nashaat T. ;
Ghirlando, Rodolfo ;
Coates, Leighton ;
Kneller, Daniel W. ;
Kovalevsky, Andrey ;
Louis, John M. .
COMMUNICATIONS BIOLOGY, 2023, 6 (01)
[5]   N-Terminal Finger Stabilizes the S1 Pocket for the Reversible Feline Drug GC376 in the SARS-CoV-2 Mpro Dimer [J].
Arutyunova, Elena ;
Khan, Muhammad Bashir ;
Fischer, Conrad ;
Lu, Jimmy ;
Lamer, Tess ;
Vuong, Wayne ;
van Belkum, Marco J. ;
McKay, Ryan T. ;
Tyrrell, D. Lorne ;
Vederas, John C. ;
Young, Howard S. ;
Lemieux, M. Joanne .
JOURNAL OF MOLECULAR BIOLOGY, 2021, 433 (13)
[6]   The Design, Synthesis and Mechanism of Action of Paxlovid, a Protease Inhibitor Drug Combination for the Treatment of COVID-19 [J].
Bege, Miklos ;
Borbas, Aniko .
PHARMACEUTICS, 2024, 16 (02)
[7]   SARS-CoV-2 Main Protease Inhibitors That Leverage Unique Interactions with the Solvent Exposed S3 Site of the Enzyme [J].
Blankenship, Lauren R. ;
Yang, Kai S. ;
Vulupala, Veerabhadra R. ;
Alugubelli, Yugendar R. ;
Khatua, Kaustav ;
Coleman, Demonta ;
Ma, Xinyu R. ;
Sankaran, Banumathi ;
Cho, Chia-Chuan D. ;
Ma, Yuying ;
Neuman, Benjamin W. ;
Xu, Shiqing ;
Liu, Wenshe Ray .
ACS MEDICINAL CHEMISTRY LETTERS, 2024, 15 (06) :950-957
[8]   Structural and Evolutionary Analysis Indicate That the SARS-CoV-2 Mpro Is a Challenging Target for Small-Molecule Inhibitor Design [J].
Bzowka, Maria ;
Mitusinska, Karolina ;
Raczynska, Agata ;
Samol, Aleksandra ;
Tuszynski, Jack A. ;
Gora, Artur .
INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES, 2020, 21 (09)
[9]   Liberation of SARS-CoV main protease from the viral polyprotein: N-terminal autocleavage does not depend on the mature dimerization mode [J].
Chen, Shuai ;
Jonas, Felix ;
Shen, Can ;
Higenfeld, Rolf .
PROTEIN & CELL, 2010, 1 (01) :59-74
[10]   Characterization of human immunodeficiency virus type-1 (HIV-1) particles that express protease-reverse transcriptase fusion proteins [J].
Cherry, E ;
Liang, C ;
Rong, LW ;
Quan, YD ;
Inouye, P ;
Li, XG ;
Morin, N ;
Kotler, N ;
Wainberg, MA .
JOURNAL OF MOLECULAR BIOLOGY, 1998, 284 (01) :43-56