A Bioluminescent 3CLPro Activity Assay to Monitor SARS-CoV-2 Replication and Identify Inhibitors.

Cyrille Mathieu,Branka Horvat,Manon Brailly,Pierre-Olivier Vidalain,Aymeric Hans,Vincent Lotteau,Yves L Janin,Franck Touret,Xavier De Lamballerie,Clémence Jacquemin,José-Carlos Valle-Casuso,Patrice André,Antoine Nougairède,Magalie Mazelier,Virginie Vasseur,Didier Décimo,Mustapha Si-Tahar

doi:10.3390/v13091814

Abstract

Our therapeutic arsenal against viruses is very limited and the current pandemic of SARS-CoV-2 highlights the critical need for effective antivirals against emerging coronaviruses. Cellular assays allowing a precise quantification of viral replication in high-throughput experimental settings are essential to the screening of chemical libraries and the selection of best antiviral chemical structures. To develop a reporting system for SARS-CoV-2 infection, we generated cell lines expressing a firefly luciferase maintained in an inactive form by a consensus cleavage site for the viral protease 3CLPro of coronaviruses, so that the luminescent biosensor is turned on upon 3CLPro expression or SARS-CoV-2 infection. This cellular assay was used to screen a metabolism-oriented library of 492 compounds to identify metabolic vulnerabilities of coronaviruses for developing innovative therapeutic strategies. In agreement with recent reports, inhibitors of pyrimidine biosynthesis were found to prevent SARS-CoV-2 replication. Among the top hits, we also identified the NADPH oxidase (NOX) inhibitor Setanaxib. The anti-SARS-CoV-2 activity of Setanaxib was further confirmed using ACE2-expressing human pulmonary cells Beas2B as well as human primary nasal epithelial cells. Altogether, these results validate our cell-based functional assay and the interest of screening libraries of different origins to identify inhibitors of SARS-CoV-2 for drug repurposing or development.

Highlights

In January 2020, SARS-CoV-2 was identified as the etiological agent of the currentCovid-19 pandemic
To develop a bioluminescence-based biosensor of the proteolytic activity of SARSCoV-2 3C-like protease (3CLPro), we took advantage of a previously reported construct based on circularly permuted N- and C-terminal fragments of firefly luciferase linked by a protease cleavage site (Figure 1B) [18]
We have developed and validated an in vitro luminescent assay relying on the detection of 3CLPro -mediated proteolysis as a reporter for SARS-CoV-2 infection

Summary

Introduction

In January 2020, SARS-CoV-2 was identified as the etiological agent of the current. Covid-19 is a complex respiratory disease with symptoms ranging from none to severe illness that can lead to death. Thanks to unprecedented worldwide efforts, this virus and the associated diseases are being characterized in great detail and vaccines have been developed and approved in less than a year. Covid-19 are urgently needed, but the current therapeutic arsenal is extremely limited despite research endeavors. Many molecules have been evaluated in clinical trials for either treating symptoms of the infection or blocking the virus replication. The anti-inflammatory corticosteroid dexamethasone and the anti-IL6 antibody tocilizumab have been shown to contain the cytokine storm associated with severe Covid and to improve patients’

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Conclusion