Identifying Small-Molecule Inhibitors of SARS-CoV-2 RNA-Dependent RNA Polymerase by Establishing a Fluorometric Assay
SARS-CoV-2 (severe acute respiratory syndrome coronavirus‐2), a member of the coronavirus family, appeared in 2019 and has caused the largest global public health and economic emergency in recent history, affecting almost all sectors of society. SARS-CoV-2 is a single-stranded positive-sense RNA vir...
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| Format: | Article |
| Language: | English |
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Frontiers Media S.A.
2022-04-01
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| Series: | Frontiers in Immunology |
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| Online Access: | https://www.frontiersin.org/articles/10.3389/fimmu.2022.844749/full |
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| author | Xiaoming Bai Xiaoming Bai Hongmin Sun Hongmin Sun Shuo Wu Shuo Wu Yuhuan Li Yuhuan Li Lifei Wang Lifei Wang Bin Hong Bin Hong |
| author_facet | Xiaoming Bai Xiaoming Bai Hongmin Sun Hongmin Sun Shuo Wu Shuo Wu Yuhuan Li Yuhuan Li Lifei Wang Lifei Wang Bin Hong Bin Hong |
| author_sort | Xiaoming Bai |
| collection | DOAJ |
| description | SARS-CoV-2 (severe acute respiratory syndrome coronavirus‐2), a member of the coronavirus family, appeared in 2019 and has caused the largest global public health and economic emergency in recent history, affecting almost all sectors of society. SARS-CoV-2 is a single-stranded positive-sense RNA virus that relies on RNA‐dependent RNA polymerase (RdRp) activity in viral transcription and replication. Due to its high sequence and structural conservation in coronavirus and new SARS-CoV-2 variants, RdRp has been recognized as the key therapeutic target to design novel antiviral strategies. Nucleotide analogs (NAs), such as remdesivir, is the most promising class of RdRp inhibitors to be used in the treatment of COVID-19. However, the presence of exonucleases in SARS-CoV-2 caused a great challenge to NAs; the excision of incorporated NAs will lead to viral resistance to this group of inhibitors. Here, we expressed active RdRp protein in both a eukaryotic expression system of baculovirus-infected insect cells and a prokaryotic expression system of Escherichia coli cells. Nsp7 and nsp8 of the functional RdRp holoenzyme were generated in E. coli. An in vitro RdRp activity assay has been established with a reconstituted nsp12/nsp7/nsp8 complex and biotin-labeled self-priming RNAs, and the activity of the RdRp complex was determined by detecting binding and extension of RNAs. Moreover, to meet the needs of high-throughput drug screening, we developed a fluorometric approach based on dsRNA quantification to assess the catalytic activity of the RdRp complex, which is also suitable for testing in 96-well plates. We demonstrated that the active triphosphate form of remdesivir (RTP) and several reported non-nucleotide analog viral polymerase inhibitors blocked the RdRp in the in vitro RdRp activity assay and high-throughput screening model. This high-throughput screening model has been applied to a custom synthetic chemical and natural product library of thousands of compounds for screening SARS-CoV-2 RdRp inhibitors. Our efficient RdRp inhibitor discovery system provides a powerful platform for the screening, validation, and evaluation of novel antiviral molecules targeting SARS-CoV-2 RdRp, particularly for non-nucleotide antivirals drugs (NNAs). |
| first_indexed | 2024-12-14T05:56:40Z |
| format | Article |
| id | doaj.art-cba72a9fb1d94692864239e728760c1a |
| institution | Directory Open Access Journal |
| issn | 1664-3224 |
| language | English |
| last_indexed | 2024-12-14T05:56:40Z |
| publishDate | 2022-04-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Immunology |
| spelling | doaj.art-cba72a9fb1d94692864239e728760c1a2022-12-21T23:14:33ZengFrontiers Media S.A.Frontiers in Immunology1664-32242022-04-011310.3389/fimmu.2022.844749844749Identifying Small-Molecule Inhibitors of SARS-CoV-2 RNA-Dependent RNA Polymerase by Establishing a Fluorometric AssayXiaoming Bai0Xiaoming Bai1Hongmin Sun2Hongmin Sun3Shuo Wu4Shuo Wu5Yuhuan Li6Yuhuan Li7Lifei Wang8Lifei Wang9Bin Hong10Bin Hong11National Health Commission of the People's Republic of China (NHC) Key Laboratory of Biotechnology of Antibiotics, Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, ChinaChinese Academy of Medical Sciences (CAMS) Key Laboratory of Synthetic Biology for Drug Innovation, Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, ChinaNational Health Commission of the People's Republic of China (NHC) Key Laboratory of Biotechnology of Antibiotics, Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, ChinaChinese Academy of Medical Sciences (CAMS) Key Laboratory of Synthetic Biology for Drug Innovation, Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, ChinaNational Health Commission of the People's Republic of China (NHC) Key Laboratory of Biotechnology of Antibiotics, Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, ChinaChinese Academy of Medical Sciences (CAMS) Key Laboratory of Antiviral Drug Research, Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, ChinaNational Health Commission of the People's Republic of China (NHC) Key Laboratory of Biotechnology of Antibiotics, Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, ChinaChinese Academy of Medical Sciences (CAMS) Key Laboratory of Antiviral Drug Research, Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, ChinaNational Health Commission of the People's Republic of China (NHC) Key Laboratory of Biotechnology of Antibiotics, Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, ChinaChinese Academy of Medical Sciences (CAMS) Key Laboratory of Synthetic Biology for Drug Innovation, Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, ChinaNational Health Commission of the People's Republic of China (NHC) Key Laboratory of Biotechnology of Antibiotics, Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, ChinaChinese Academy of Medical Sciences (CAMS) Key Laboratory of Synthetic Biology for Drug Innovation, Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, ChinaSARS-CoV-2 (severe acute respiratory syndrome coronavirus‐2), a member of the coronavirus family, appeared in 2019 and has caused the largest global public health and economic emergency in recent history, affecting almost all sectors of society. SARS-CoV-2 is a single-stranded positive-sense RNA virus that relies on RNA‐dependent RNA polymerase (RdRp) activity in viral transcription and replication. Due to its high sequence and structural conservation in coronavirus and new SARS-CoV-2 variants, RdRp has been recognized as the key therapeutic target to design novel antiviral strategies. Nucleotide analogs (NAs), such as remdesivir, is the most promising class of RdRp inhibitors to be used in the treatment of COVID-19. However, the presence of exonucleases in SARS-CoV-2 caused a great challenge to NAs; the excision of incorporated NAs will lead to viral resistance to this group of inhibitors. Here, we expressed active RdRp protein in both a eukaryotic expression system of baculovirus-infected insect cells and a prokaryotic expression system of Escherichia coli cells. Nsp7 and nsp8 of the functional RdRp holoenzyme were generated in E. coli. An in vitro RdRp activity assay has been established with a reconstituted nsp12/nsp7/nsp8 complex and biotin-labeled self-priming RNAs, and the activity of the RdRp complex was determined by detecting binding and extension of RNAs. Moreover, to meet the needs of high-throughput drug screening, we developed a fluorometric approach based on dsRNA quantification to assess the catalytic activity of the RdRp complex, which is also suitable for testing in 96-well plates. We demonstrated that the active triphosphate form of remdesivir (RTP) and several reported non-nucleotide analog viral polymerase inhibitors blocked the RdRp in the in vitro RdRp activity assay and high-throughput screening model. This high-throughput screening model has been applied to a custom synthetic chemical and natural product library of thousands of compounds for screening SARS-CoV-2 RdRp inhibitors. Our efficient RdRp inhibitor discovery system provides a powerful platform for the screening, validation, and evaluation of novel antiviral molecules targeting SARS-CoV-2 RdRp, particularly for non-nucleotide antivirals drugs (NNAs).https://www.frontiersin.org/articles/10.3389/fimmu.2022.844749/fullSARS-CoV-2RNA-dependent RNA polymerase (RdRp)non-nucleoside analog inhibitors (NNAIs)high-throughput screening (HTS)nucleoside analogs (NAs) |
| spellingShingle | Xiaoming Bai Xiaoming Bai Hongmin Sun Hongmin Sun Shuo Wu Shuo Wu Yuhuan Li Yuhuan Li Lifei Wang Lifei Wang Bin Hong Bin Hong Identifying Small-Molecule Inhibitors of SARS-CoV-2 RNA-Dependent RNA Polymerase by Establishing a Fluorometric Assay Frontiers in Immunology SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) non-nucleoside analog inhibitors (NNAIs) high-throughput screening (HTS) nucleoside analogs (NAs) |
| title | Identifying Small-Molecule Inhibitors of SARS-CoV-2 RNA-Dependent RNA Polymerase by Establishing a Fluorometric Assay |
| title_full | Identifying Small-Molecule Inhibitors of SARS-CoV-2 RNA-Dependent RNA Polymerase by Establishing a Fluorometric Assay |
| title_fullStr | Identifying Small-Molecule Inhibitors of SARS-CoV-2 RNA-Dependent RNA Polymerase by Establishing a Fluorometric Assay |
| title_full_unstemmed | Identifying Small-Molecule Inhibitors of SARS-CoV-2 RNA-Dependent RNA Polymerase by Establishing a Fluorometric Assay |
| title_short | Identifying Small-Molecule Inhibitors of SARS-CoV-2 RNA-Dependent RNA Polymerase by Establishing a Fluorometric Assay |
| title_sort | identifying small molecule inhibitors of sars cov 2 rna dependent rna polymerase by establishing a fluorometric assay |
| topic | SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) non-nucleoside analog inhibitors (NNAIs) high-throughput screening (HTS) nucleoside analogs (NAs) |
| url | https://www.frontiersin.org/articles/10.3389/fimmu.2022.844749/full |
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