Orthogonal dual reporter-based gain-of-signal assay for probing SARS-CoV-2 3CL protease activity in living cells: inhibitor identification and mutation investigation
The main protease (3-chymotrypsin-like protease, 3CLpro) of SARS-CoV-2 has become a focus of anti-coronavirus research. Despite efforts, drug development targeting 3CLpro has been hampered by limitations in the currently available activity assays. Additionally, the emergence of 3CLpro mutations in c...
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| Format: | Article |
| Language: | English |
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Taylor & Francis Group
2023-12-01
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| Series: | Emerging Microbes and Infections |
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| Online Access: | https://www.tandfonline.com/doi/10.1080/22221751.2023.2211688 |
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| author | Zhu-Chun Bei Huanhuan Yu Hong Wang Qingyun Li Baogang Wang Dongna Zhang Likun Xu Liangliang Zhao Shuwei Dong Yabin Song |
| author_facet | Zhu-Chun Bei Huanhuan Yu Hong Wang Qingyun Li Baogang Wang Dongna Zhang Likun Xu Liangliang Zhao Shuwei Dong Yabin Song |
| author_sort | Zhu-Chun Bei |
| collection | DOAJ |
| description | The main protease (3-chymotrypsin-like protease, 3CLpro) of SARS-CoV-2 has become a focus of anti-coronavirus research. Despite efforts, drug development targeting 3CLpro has been hampered by limitations in the currently available activity assays. Additionally, the emergence of 3CLpro mutations in circulating SARS-CoV-2 variants has raised concerns about potential resistance. Both emphasize the need for a more reliable, sensitive, and facile 3CLpro assay. Here, we report an orthogonal dual reporter-based gain-of-signal assay for measuring 3CLpro activity in living cells. It builds on the finding that 3CLpro induces cytotoxicity and reporter expression suppression, which can be rescued by its inhibitor or mutation. This assay circumvents most limitations in previously reported assays, especially false positives caused by nonspecific compounds and signal interference from test compounds. It is also convenient and robust for high throughput screening of compounds and comparing the drug susceptibilities of mutants. Using this assay, we screened 1789 compounds, including natural products and protease inhibitors, with 45 compounds that have been reported to inhibit SARS-CoV-2 3CLpro among them. Except for the approved drug PF-07321332, only five of these inhibit 3CLpro in our assays: GC376; PF-00835231; S-217622; Boceprevir; and Z-FA-FMK. The susceptibilities of seven 3CLpro mutants prevalent in circulating variants to PF-07321332, S-217622, and GC376 were also assessed. Three mutants were identified as being less susceptible to PF-07321322 (P132H) and S-217622 (G15S, T21I). This assay should greatly facilitate the development of novel 3CLpro-targeted drugs and the monitoring of the susceptibility of emerging SARS-CoV-2 variants to 3CLpro inhibitors. |
| format | Article |
| id | doaj-art-25113ab9400d4eaa9989cb77deafb417 |
| institution | DOAJ |
| issn | 2222-1751 |
| language | English |
| publishDate | 2023-12-01 |
| publisher | Taylor & Francis Group |
| record_format | Article |
| series | Emerging Microbes and Infections |
| spelling | doaj-art-25113ab9400d4eaa9989cb77deafb4172025-08-20T02:58:46ZengTaylor & Francis GroupEmerging Microbes and Infections2222-17512023-12-0112110.1080/22221751.2023.2211688Orthogonal dual reporter-based gain-of-signal assay for probing SARS-CoV-2 3CL protease activity in living cells: inhibitor identification and mutation investigationZhu-Chun Bei0Huanhuan Yu1Hong Wang2Qingyun Li3Baogang Wang4Dongna Zhang5Likun Xu6Liangliang Zhao7Shuwei Dong8Yabin Song9State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Beijing, People’s Republic of ChinaState Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Beijing, People’s Republic of ChinaState Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Beijing, People’s Republic of ChinaState Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Beijing, People’s Republic of ChinaState Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Beijing, People’s Republic of ChinaState Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Beijing, People’s Republic of ChinaState Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Beijing, People’s Republic of ChinaState Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Beijing, People’s Republic of ChinaThe Affiliated AnNing First Hospital, Faculty of Life Science and Technology, Kunming University of Science and Technology, Kunming, People’s Republic of ChinaState Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Beijing, People’s Republic of ChinaThe main protease (3-chymotrypsin-like protease, 3CLpro) of SARS-CoV-2 has become a focus of anti-coronavirus research. Despite efforts, drug development targeting 3CLpro has been hampered by limitations in the currently available activity assays. Additionally, the emergence of 3CLpro mutations in circulating SARS-CoV-2 variants has raised concerns about potential resistance. Both emphasize the need for a more reliable, sensitive, and facile 3CLpro assay. Here, we report an orthogonal dual reporter-based gain-of-signal assay for measuring 3CLpro activity in living cells. It builds on the finding that 3CLpro induces cytotoxicity and reporter expression suppression, which can be rescued by its inhibitor or mutation. This assay circumvents most limitations in previously reported assays, especially false positives caused by nonspecific compounds and signal interference from test compounds. It is also convenient and robust for high throughput screening of compounds and comparing the drug susceptibilities of mutants. Using this assay, we screened 1789 compounds, including natural products and protease inhibitors, with 45 compounds that have been reported to inhibit SARS-CoV-2 3CLpro among them. Except for the approved drug PF-07321332, only five of these inhibit 3CLpro in our assays: GC376; PF-00835231; S-217622; Boceprevir; and Z-FA-FMK. The susceptibilities of seven 3CLpro mutants prevalent in circulating variants to PF-07321332, S-217622, and GC376 were also assessed. Three mutants were identified as being less susceptible to PF-07321322 (P132H) and S-217622 (G15S, T21I). This assay should greatly facilitate the development of novel 3CLpro-targeted drugs and the monitoring of the susceptibility of emerging SARS-CoV-2 variants to 3CLpro inhibitors.https://www.tandfonline.com/doi/10.1080/22221751.2023.2211688SARS-CoV-23CL proteaseprotease inhibitororthogonal dual reporter-based gain-of-signal assaysusceptibilityGC376 |
| spellingShingle | Zhu-Chun Bei Huanhuan Yu Hong Wang Qingyun Li Baogang Wang Dongna Zhang Likun Xu Liangliang Zhao Shuwei Dong Yabin Song Orthogonal dual reporter-based gain-of-signal assay for probing SARS-CoV-2 3CL protease activity in living cells: inhibitor identification and mutation investigation Emerging Microbes and Infections SARS-CoV-2 3CL protease protease inhibitor orthogonal dual reporter-based gain-of-signal assay susceptibility GC376 |
| title | Orthogonal dual reporter-based gain-of-signal assay for probing SARS-CoV-2 3CL protease activity in living cells: inhibitor identification and mutation investigation |
| title_full | Orthogonal dual reporter-based gain-of-signal assay for probing SARS-CoV-2 3CL protease activity in living cells: inhibitor identification and mutation investigation |
| title_fullStr | Orthogonal dual reporter-based gain-of-signal assay for probing SARS-CoV-2 3CL protease activity in living cells: inhibitor identification and mutation investigation |
| title_full_unstemmed | Orthogonal dual reporter-based gain-of-signal assay for probing SARS-CoV-2 3CL protease activity in living cells: inhibitor identification and mutation investigation |
| title_short | Orthogonal dual reporter-based gain-of-signal assay for probing SARS-CoV-2 3CL protease activity in living cells: inhibitor identification and mutation investigation |
| title_sort | orthogonal dual reporter based gain of signal assay for probing sars cov 2 3cl protease activity in living cells inhibitor identification and mutation investigation |
| topic | SARS-CoV-2 3CL protease protease inhibitor orthogonal dual reporter-based gain-of-signal assay susceptibility GC376 |
| url | https://www.tandfonline.com/doi/10.1080/22221751.2023.2211688 |
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