SARS-CoV-2 Mpro inhibitor identification using a cellular gain-of-signal assay for high-throughput screening
Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2, SARS2) is responsible for the COVID-19 pandemic and infections that continue to affect the lives of millions of people worldwide, especially those who are older and/or immunocompromised. The SARS2 main protease enzyme, Mpro (also called 3C...
| Main Authors: | , , , , , , , , , , , , , , , , , , |
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| Format: | Journal Article |
| Language: | English |
| Published: |
2024
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| Online Access: | https://hdl.handle.net/10356/181907 |
| _version_ | 1826113752003510272 |
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| author | Delgado, Renee Vishwakarma, Jyoti Moghadasi, Seyed Arad Otsuka, Yuka Shumate, Justin Cuell, Ashley Tansiongco, Megan Cooley, Christina B. Chen, Yanjun Dabrowska, Agnieszka Basu, Rahul Anindita, Paulina Duhita Luo, Dahai Dosa, Peter I. Harki, Daniel A. Bannister, Thomas Scampavia, Louis Spicer, Timothy P. Harris, Reuben S. |
| author2 | Lee Kong Chian School of Medicine (LKCMedicine) |
| author_facet | Lee Kong Chian School of Medicine (LKCMedicine) Delgado, Renee Vishwakarma, Jyoti Moghadasi, Seyed Arad Otsuka, Yuka Shumate, Justin Cuell, Ashley Tansiongco, Megan Cooley, Christina B. Chen, Yanjun Dabrowska, Agnieszka Basu, Rahul Anindita, Paulina Duhita Luo, Dahai Dosa, Peter I. Harki, Daniel A. Bannister, Thomas Scampavia, Louis Spicer, Timothy P. Harris, Reuben S. |
| author_sort | Delgado, Renee |
| collection | NTU |
| description | Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2, SARS2) is responsible for the COVID-19 pandemic and infections that continue to affect the lives of millions of people worldwide, especially those who are older and/or immunocompromised. The SARS2 main protease enzyme, Mpro (also called 3C-like protease, 3CLpro), is a bona fide drug target as evidenced by potent inhibition with nirmatrelvir and ensitrelvir, the active components of the drugs Paxlovid and Xocova, respectively. However, the existence of nirmatrelvir and ensitrelvir-resistant isolates underscores the need to develop next-generation drugs with different resistance profiles and/or distinct mechanisms of action. Here, we report the results of a high-throughput screen of 649,568 compounds using a cellular gain-of-signal assay. In this assay, Mpro inhibits expression of a luciferase reporter, and 8,777 small molecules were considered hits by causing a gain in luciferase activity 3x SD above the sample field activity (6.8% gain-of-signal relative to 100 µM GC376). Single concentration and dose-response gain-of-signal experiments confirmed 3,522/8,762 compounds as candidate inhibitors. In parallel, all initial high-throughput screening hits were tested in a peptide cleavage assay with purified Mpro and only 39/8,762 showed inhibition. Importantly, 19/39 compounds (49%) re-tested positive in both SARS2 assays, including two previously reported Mpro inhibitors, demonstrating the efficacy of the overall screening strategy. This approach led to the rediscovery of known Mpro inhibitors such as calpain inhibitor II, as well as to the discovery of novel compounds that provide chemical information for future drug development efforts. |
| first_indexed | 2025-03-09T10:56:24Z |
| format | Journal Article |
| id | ntu-10356/181907 |
| institution | Nanyang Technological University |
| language | English |
| last_indexed | 2025-03-09T10:56:24Z |
| publishDate | 2024 |
| record_format | dspace |
| spelling | ntu-10356/1819072025-01-05T15:39:29Z SARS-CoV-2 Mpro inhibitor identification using a cellular gain-of-signal assay for high-throughput screening Delgado, Renee Vishwakarma, Jyoti Moghadasi, Seyed Arad Otsuka, Yuka Shumate, Justin Cuell, Ashley Tansiongco, Megan Cooley, Christina B. Chen, Yanjun Dabrowska, Agnieszka Basu, Rahul Anindita, Paulina Duhita Luo, Dahai Dosa, Peter I. Harki, Daniel A. Bannister, Thomas Scampavia, Louis Spicer, Timothy P. Harris, Reuben S. Lee Kong Chian School of Medicine (LKCMedicine) Institute of Structural Biology Medicine, Health and Life Sciences Antiviral drugs Cell-based ultra-high throughput screening Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2, SARS2) is responsible for the COVID-19 pandemic and infections that continue to affect the lives of millions of people worldwide, especially those who are older and/or immunocompromised. The SARS2 main protease enzyme, Mpro (also called 3C-like protease, 3CLpro), is a bona fide drug target as evidenced by potent inhibition with nirmatrelvir and ensitrelvir, the active components of the drugs Paxlovid and Xocova, respectively. However, the existence of nirmatrelvir and ensitrelvir-resistant isolates underscores the need to develop next-generation drugs with different resistance profiles and/or distinct mechanisms of action. Here, we report the results of a high-throughput screen of 649,568 compounds using a cellular gain-of-signal assay. In this assay, Mpro inhibits expression of a luciferase reporter, and 8,777 small molecules were considered hits by causing a gain in luciferase activity 3x SD above the sample field activity (6.8% gain-of-signal relative to 100 µM GC376). Single concentration and dose-response gain-of-signal experiments confirmed 3,522/8,762 compounds as candidate inhibitors. In parallel, all initial high-throughput screening hits were tested in a peptide cleavage assay with purified Mpro and only 39/8,762 showed inhibition. Importantly, 19/39 compounds (49%) re-tested positive in both SARS2 assays, including two previously reported Mpro inhibitors, demonstrating the efficacy of the overall screening strategy. This approach led to the rediscovery of known Mpro inhibitors such as calpain inhibitor II, as well as to the discovery of novel compounds that provide chemical information for future drug development efforts. Published version This work was supported by National Institute of Allergy and Infectious Disease grant U19-AI171954. 2024-12-30T07:27:49Z 2024-12-30T07:27:49Z 2024 Journal Article Delgado, R., Vishwakarma, J., Moghadasi, S. A., Otsuka, Y., Shumate, J., Cuell, A., Tansiongco, M., Cooley, C. B., Chen, Y., Dabrowska, A., Basu, R., Anindita, P. D., Luo, D., Dosa, P. I., Harki, D. A., Bannister, T., Scampavia, L., Spicer, T. P. & Harris, R. S. (2024). SARS-CoV-2 Mpro inhibitor identification using a cellular gain-of-signal assay for high-throughput screening. SLAS Discovery, 29(6), 100181-. https://dx.doi.org/10.1016/j.slasd.2024.100181 2472-5552 https://hdl.handle.net/10356/181907 10.1016/j.slasd.2024.100181 39173830 2-s2.0-85202508542 6 29 100181 en SLAS Discovery © 2024 The Authors. Published by Elsevier Inc. on behalf of Society for Laboratory Automation and Screening. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). application/pdf |
| spellingShingle | Medicine, Health and Life Sciences Antiviral drugs Cell-based ultra-high throughput screening Delgado, Renee Vishwakarma, Jyoti Moghadasi, Seyed Arad Otsuka, Yuka Shumate, Justin Cuell, Ashley Tansiongco, Megan Cooley, Christina B. Chen, Yanjun Dabrowska, Agnieszka Basu, Rahul Anindita, Paulina Duhita Luo, Dahai Dosa, Peter I. Harki, Daniel A. Bannister, Thomas Scampavia, Louis Spicer, Timothy P. Harris, Reuben S. SARS-CoV-2 Mpro inhibitor identification using a cellular gain-of-signal assay for high-throughput screening |
| title | SARS-CoV-2 Mpro inhibitor identification using a cellular gain-of-signal assay for high-throughput screening |
| title_full | SARS-CoV-2 Mpro inhibitor identification using a cellular gain-of-signal assay for high-throughput screening |
| title_fullStr | SARS-CoV-2 Mpro inhibitor identification using a cellular gain-of-signal assay for high-throughput screening |
| title_full_unstemmed | SARS-CoV-2 Mpro inhibitor identification using a cellular gain-of-signal assay for high-throughput screening |
| title_short | SARS-CoV-2 Mpro inhibitor identification using a cellular gain-of-signal assay for high-throughput screening |
| title_sort | sars cov 2 mpro inhibitor identification using a cellular gain of signal assay for high throughput screening |
| topic | Medicine, Health and Life Sciences Antiviral drugs Cell-based ultra-high throughput screening |
| url | https://hdl.handle.net/10356/181907 |
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