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Development of a Fluorescence-Based, High-Throughput SARS-CoV-2 3CL(pro) Reporter Assay

In late 2019, a human coronavirus, now known as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), emerged, likely from a zoonotic reservoir. This virus causes COVID-19, has infected millions of people, and has led to hundreds of thousands of deaths across the globe. While the best interv...

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Autores principales: Froggatt, Heather M., Heaton, Brook E., Heaton, Nicholas S.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: American Society for Microbiology 2020
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7592234/
https://www.ncbi.nlm.nih.gov/pubmed/32843534
http://dx.doi.org/10.1128/JVI.01265-20
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author Froggatt, Heather M.
Heaton, Brook E.
Heaton, Nicholas S.
author_facet Froggatt, Heather M.
Heaton, Brook E.
Heaton, Nicholas S.
author_sort Froggatt, Heather M.
collection PubMed
description In late 2019, a human coronavirus, now known as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), emerged, likely from a zoonotic reservoir. This virus causes COVID-19, has infected millions of people, and has led to hundreds of thousands of deaths across the globe. While the best interventions to control and ultimately stop the pandemic are prophylactic vaccines, antiviral therapeutics are important to limit morbidity and mortality in those already infected. At this time, only one FDA-approved anti-SARS-CoV-2 antiviral drug, remdesivir, is available, and unfortunately, its efficacy appears to be limited. Thus, the identification of new and efficacious antivirals is of the highest importance. In order to facilitate rapid drug discovery, flexible, sensitive, and high-throughput screening methods are required. With respect to drug targets, most attention is focused on either the viral RNA-dependent RNA polymerase or the main viral protease, 3CL(pro). 3CL(pro) is an attractive target for antiviral therapeutics, as it is essential for processing newly translated viral proteins and the viral life cycle cannot be completed without protease activity. In this work, we report a new assay to identify inhibitors of 3CL(pro). Our reporter is based on a green fluorescent protein (GFP)-derived protein that fluoresces only after cleavage by 3CL(pro). This experimentally optimized reporter assay allows for antiviral drug screening in human cell culture at biosafety level 2 (BSL2) with high-throughput compatible protocols. Using this screening approach in combination with existing drug libraries may lead to the rapid identification of novel antivirals to suppress SARS-CoV-2 replication and spread. IMPORTANCE The COVID-19 pandemic has already led to more than 700,000 deaths and innumerable changes to daily life worldwide. Along with development of a vaccine, identification of effective antivirals to treat infected patients is of the highest importance. However, rapid drug discovery requires efficient methods to identify novel compounds that can inhibit the virus. In this work, we present a method for identifying inhibitors of the SARS-CoV-2 main protease, 3CL(pro). This reporter-based assay allows for antiviral drug screening in human cell culture at biosafety level 2 (BSL2) with high-throughput compatible sample processing and analysis. This assay may help identify novel antivirals to control the COVID-19 pandemic.
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spelling pubmed-75922342020-11-06 Development of a Fluorescence-Based, High-Throughput SARS-CoV-2 3CL(pro) Reporter Assay Froggatt, Heather M. Heaton, Brook E. Heaton, Nicholas S. J Virol Vaccines and Antiviral Agents In late 2019, a human coronavirus, now known as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), emerged, likely from a zoonotic reservoir. This virus causes COVID-19, has infected millions of people, and has led to hundreds of thousands of deaths across the globe. While the best interventions to control and ultimately stop the pandemic are prophylactic vaccines, antiviral therapeutics are important to limit morbidity and mortality in those already infected. At this time, only one FDA-approved anti-SARS-CoV-2 antiviral drug, remdesivir, is available, and unfortunately, its efficacy appears to be limited. Thus, the identification of new and efficacious antivirals is of the highest importance. In order to facilitate rapid drug discovery, flexible, sensitive, and high-throughput screening methods are required. With respect to drug targets, most attention is focused on either the viral RNA-dependent RNA polymerase or the main viral protease, 3CL(pro). 3CL(pro) is an attractive target for antiviral therapeutics, as it is essential for processing newly translated viral proteins and the viral life cycle cannot be completed without protease activity. In this work, we report a new assay to identify inhibitors of 3CL(pro). Our reporter is based on a green fluorescent protein (GFP)-derived protein that fluoresces only after cleavage by 3CL(pro). This experimentally optimized reporter assay allows for antiviral drug screening in human cell culture at biosafety level 2 (BSL2) with high-throughput compatible protocols. Using this screening approach in combination with existing drug libraries may lead to the rapid identification of novel antivirals to suppress SARS-CoV-2 replication and spread. IMPORTANCE The COVID-19 pandemic has already led to more than 700,000 deaths and innumerable changes to daily life worldwide. Along with development of a vaccine, identification of effective antivirals to treat infected patients is of the highest importance. However, rapid drug discovery requires efficient methods to identify novel compounds that can inhibit the virus. In this work, we present a method for identifying inhibitors of the SARS-CoV-2 main protease, 3CL(pro). This reporter-based assay allows for antiviral drug screening in human cell culture at biosafety level 2 (BSL2) with high-throughput compatible sample processing and analysis. This assay may help identify novel antivirals to control the COVID-19 pandemic. American Society for Microbiology 2020-10-27 /pmc/articles/PMC7592234/ /pubmed/32843534 http://dx.doi.org/10.1128/JVI.01265-20 Text en Copyright © 2020 American Society for Microbiology. All Rights Reserved (https://doi.org/10.1128/ASMCopyrightv2) . https://doi.org/10.1128/ASMCopyrightv2 This article is made available via the PMC Open Access Subset for unrestricted noncommercial re-use and secondary analysis in any form or by any means with acknowledgement of the original source. These permissions are granted for the duration of the World Health Organization (WHO) declaration of COVID-19 as a global pandemic.
spellingShingle Vaccines and Antiviral Agents
Froggatt, Heather M.
Heaton, Brook E.
Heaton, Nicholas S.
Development of a Fluorescence-Based, High-Throughput SARS-CoV-2 3CL(pro) Reporter Assay
title Development of a Fluorescence-Based, High-Throughput SARS-CoV-2 3CL(pro) Reporter Assay
title_full Development of a Fluorescence-Based, High-Throughput SARS-CoV-2 3CL(pro) Reporter Assay
title_fullStr Development of a Fluorescence-Based, High-Throughput SARS-CoV-2 3CL(pro) Reporter Assay
title_full_unstemmed Development of a Fluorescence-Based, High-Throughput SARS-CoV-2 3CL(pro) Reporter Assay
title_short Development of a Fluorescence-Based, High-Throughput SARS-CoV-2 3CL(pro) Reporter Assay
title_sort development of a fluorescence-based, high-throughput sars-cov-2 3cl(pro) reporter assay
topic Vaccines and Antiviral Agents
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7592234/
https://www.ncbi.nlm.nih.gov/pubmed/32843534
http://dx.doi.org/10.1128/JVI.01265-20
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