Development of a highly sensitive luciferase assay for intracellular evaluation of coronavirus Mpro activity
COVID-19, caused by SARS-CoV-2 virus, has emerged as a global threat to human health. The main protease (Mpro) of SARS-CoV-2 is an excellent target for the development of antiviral drugs against COVID-19, and various protease biosensors have been developed to evaluate anti-coronavirus drugs. However...
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| Format: | Article |
| Language: | English |
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Frontiers Media S.A.
2025-04-01
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| Series: | Frontiers in Microbiology |
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| Online Access: | https://www.frontiersin.org/articles/10.3389/fmicb.2025.1560251/full |
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| author | Bao Dong Bao Dong Yuehong Chen Xin Wang Jing Li Sen Zhang Xiaoping Kang Yuchang Li Biao Li Liangning Liao Zhengwei Zhang Jiaqi Xiong Lele Shao Shenghai Huang Ye Feng Tao Jiang Tao Jiang Tao Jiang Tao Jiang |
| author_facet | Bao Dong Bao Dong Yuehong Chen Xin Wang Jing Li Sen Zhang Xiaoping Kang Yuchang Li Biao Li Liangning Liao Zhengwei Zhang Jiaqi Xiong Lele Shao Shenghai Huang Ye Feng Tao Jiang Tao Jiang Tao Jiang Tao Jiang |
| author_sort | Bao Dong |
| collection | DOAJ |
| description | COVID-19, caused by SARS-CoV-2 virus, has emerged as a global threat to human health. The main protease (Mpro) of SARS-CoV-2 is an excellent target for the development of antiviral drugs against COVID-19, and various protease biosensors have been developed to evaluate anti-coronavirus drugs. However, the application of these protease biosensors was limited due to high background fluorescence, poor signal-to-noise ratios, and constraints in enzyme activity thresholds for accessing live viruses. In this study, we rationally designed a highly conserved Mpro cleavage site sequence among different coronaviruses (CoVs) with high proteolytic activity, and described an intracellular coronavirus Mpro proteolytic (ICMP) reporter system that takes advantage of virus-encoded Mpro expressed in infected cells to reform the NanoBiT fluorescent protein. The system can be used to visualize and identify cells infected with coronavirus, and demonstrated high compatibility with various Mpro proteins from 13 different mammalian coronaviruses (covering α, β, γ, and δ CoVs), exhibiting at least a 1,030-fold increase in luminescence. Stronger Nluc signals were detectable with CoV 229E virus infection at a MOI of 0.001. Additionally, the system proved suitable for evaluating and screening of antiviral compounds, including lufotrelvir, GC376, Nirmatrelvir, X77, MG-101, and the potential inhibitor Cynaroside. The ICMP system is not only an invaluable tool for the detection of live coronaviruses, but also for the discovery of antivirals against current and future pandemic coronaviruses. |
| format | Article |
| id | doaj-art-7b086559c83c434fbcf3e9c7b51cfdc0 |
| institution | OA Journals |
| issn | 1664-302X |
| language | English |
| publishDate | 2025-04-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Microbiology |
| spelling | doaj-art-7b086559c83c434fbcf3e9c7b51cfdc02025-08-20T01:55:37ZengFrontiers Media S.A.Frontiers in Microbiology1664-302X2025-04-011610.3389/fmicb.2025.15602511560251Development of a highly sensitive luciferase assay for intracellular evaluation of coronavirus Mpro activityBao Dong0Bao Dong1Yuehong Chen2Xin Wang3Jing Li4Sen Zhang5Xiaoping Kang6Yuchang Li7Biao Li8Liangning Liao9Zhengwei Zhang10Jiaqi Xiong11Lele Shao12Shenghai Huang13Ye Feng14Tao Jiang15Tao Jiang16Tao Jiang17Tao Jiang18School of Basic Medical Sciences, Anhui Medical University, Hefei, ChinaState Key Laboratory of Pathogen and Biosecurity, Academy of Military Medical Sciences, Beijing, ChinaState Key Laboratory of Pathogen and Biosecurity, Academy of Military Medical Sciences, Beijing, ChinaLaboratory of Advanced Biotechnology, Beijing Institute of Biotechnology, Beijing, ChinaState Key Laboratory of Pathogen and Biosecurity, Academy of Military Medical Sciences, Beijing, ChinaState Key Laboratory of Pathogen and Biosecurity, Academy of Military Medical Sciences, Beijing, ChinaState Key Laboratory of Pathogen and Biosecurity, Academy of Military Medical Sciences, Beijing, ChinaState Key Laboratory of Pathogen and Biosecurity, Academy of Military Medical Sciences, Beijing, ChinaSchool of Basic Medical Sciences, Anhui Medical University, Hefei, ChinaSchool of Public Health, Mudanjiang Medical University, Mudanjiang, ChinaSchool of Basic Medical Sciences, Anhui Medical University, Hefei, ChinaSchool of Public Health, Mudanjiang Medical University, Mudanjiang, ChinaCollege of Veterinary Medicine, Yangzhou University, Yangzhou, ChinaSchool of Basic Medical Sciences, Anhui Medical University, Hefei, ChinaState Key Laboratory of Pathogen and Biosecurity, Academy of Military Medical Sciences, Beijing, ChinaSchool of Basic Medical Sciences, Anhui Medical University, Hefei, ChinaState Key Laboratory of Pathogen and Biosecurity, Academy of Military Medical Sciences, Beijing, ChinaSchool of Public Health, Mudanjiang Medical University, Mudanjiang, ChinaCollege of Veterinary Medicine, Yangzhou University, Yangzhou, ChinaCOVID-19, caused by SARS-CoV-2 virus, has emerged as a global threat to human health. The main protease (Mpro) of SARS-CoV-2 is an excellent target for the development of antiviral drugs against COVID-19, and various protease biosensors have been developed to evaluate anti-coronavirus drugs. However, the application of these protease biosensors was limited due to high background fluorescence, poor signal-to-noise ratios, and constraints in enzyme activity thresholds for accessing live viruses. In this study, we rationally designed a highly conserved Mpro cleavage site sequence among different coronaviruses (CoVs) with high proteolytic activity, and described an intracellular coronavirus Mpro proteolytic (ICMP) reporter system that takes advantage of virus-encoded Mpro expressed in infected cells to reform the NanoBiT fluorescent protein. The system can be used to visualize and identify cells infected with coronavirus, and demonstrated high compatibility with various Mpro proteins from 13 different mammalian coronaviruses (covering α, β, γ, and δ CoVs), exhibiting at least a 1,030-fold increase in luminescence. Stronger Nluc signals were detectable with CoV 229E virus infection at a MOI of 0.001. Additionally, the system proved suitable for evaluating and screening of antiviral compounds, including lufotrelvir, GC376, Nirmatrelvir, X77, MG-101, and the potential inhibitor Cynaroside. The ICMP system is not only an invaluable tool for the detection of live coronaviruses, but also for the discovery of antivirals against current and future pandemic coronaviruses.https://www.frontiersin.org/articles/10.3389/fmicb.2025.1560251/fullcoronavirusmain proteaseprotease activityreporter systemNanoBitprotease inhibitor |
| spellingShingle | Bao Dong Bao Dong Yuehong Chen Xin Wang Jing Li Sen Zhang Xiaoping Kang Yuchang Li Biao Li Liangning Liao Zhengwei Zhang Jiaqi Xiong Lele Shao Shenghai Huang Ye Feng Tao Jiang Tao Jiang Tao Jiang Tao Jiang Development of a highly sensitive luciferase assay for intracellular evaluation of coronavirus Mpro activity Frontiers in Microbiology coronavirus main protease protease activity reporter system NanoBit protease inhibitor |
| title | Development of a highly sensitive luciferase assay for intracellular evaluation of coronavirus Mpro activity |
| title_full | Development of a highly sensitive luciferase assay for intracellular evaluation of coronavirus Mpro activity |
| title_fullStr | Development of a highly sensitive luciferase assay for intracellular evaluation of coronavirus Mpro activity |
| title_full_unstemmed | Development of a highly sensitive luciferase assay for intracellular evaluation of coronavirus Mpro activity |
| title_short | Development of a highly sensitive luciferase assay for intracellular evaluation of coronavirus Mpro activity |
| title_sort | development of a highly sensitive luciferase assay for intracellular evaluation of coronavirus mpro activity |
| topic | coronavirus main protease protease activity reporter system NanoBit protease inhibitor |
| url | https://www.frontiersin.org/articles/10.3389/fmicb.2025.1560251/full |
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