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A highly sensitive cell-based luciferase assay for high-throughput automated screening of SARS-CoV-2 nsp5/3CLpro inhibitors.
Chen, K Y; Krischuns, T; Varga, L Ortega; Harigua-Souiai, E; Paisant, S; Zettor, A; Chiaravalli, J; Delpal, A; Courtney, D; O'Brien, A; Baker, S C; Decroly, E; Isel, C; Agou, F; Jacob, Y; Blondel, A; Naffakh, N.
  • Chen KY; Institut Pasteur, Université Paris Cité, CNRS UMR3569, Unité Biologie des ARN et Virus Influenza, F-75015 Paris, France.
  • Krischuns T; Institut Pasteur, Université Paris Cité, CNRS UMR3569, Unité Biologie des ARN et Virus Influenza, F-75015 Paris, France.
  • Varga LO; Institut Pasteur, Université Paris Cité, CNRS UMR3528, Unité de Bioinformatique Structurale, F-75015 Paris, France.
  • Harigua-Souiai E; Laboratory of Molecular Epidemiology and Experimental Pathology - LR16IPT04, Institut Pasteur de Tunis, Université de Tunis El Manar, Tunis, Tunisia.
  • Paisant S; Institut Pasteur, Université Paris Cité, CNRS UMR3569, Unité Biologie des ARN et Virus Influenza, F-75015 Paris, France.
  • Zettor A; Institut Pasteur, Université Paris Cité, CNRS UMR3523, Plateforme de Criblage Chémogénomique et Biologique, F-75015 Paris, France.
  • Chiaravalli J; Institut Pasteur, Université Paris Cité, CNRS UMR3523, Plateforme de Criblage Chémogénomique et Biologique, F-75015 Paris, France.
  • Delpal A; Architecture et Fonction des Macromolécules Biologiques, Aix-Marseille Université, CNRS UMR7257, F-13009 Marseille, France.
  • Courtney D; Institut Pasteur, Université Paris Cité, CNRS UMR3569, Unité Biologie des ARN et Virus Influenza, F-75015 Paris, France.
  • O'Brien A; Department of Microbiology and Immunology, Loyola University Chicago, Stritch School of Medicine, Maywood, IL, USA.
  • Baker SC; Department of Microbiology and Immunology, Loyola University Chicago, Stritch School of Medicine, Maywood, IL, USA.
  • Decroly E; Architecture et Fonction des Macromolécules Biologiques, Aix-Marseille Université, CNRS UMR7257, F-13009 Marseille, France.
  • Isel C; Institut Pasteur, Université Paris Cité, CNRS UMR3569, Unité Biologie des ARN et Virus Influenza, F-75015 Paris, France.
  • Agou F; Institut Pasteur, Université Paris Cité, CNRS UMR3523, Plateforme de Criblage Chémogénomique et Biologique, F-75015 Paris, France.
  • Jacob Y; Institut Pasteur, Université Paris Cité, CNRS UMR3569, Unité Génétique Moléculaire des Virus à ARN, F-75015 Paris, France.
  • Blondel A; Institut Pasteur, Université Paris Cité, CNRS UMR3528, Unité de Bioinformatique Structurale, F-75015 Paris, France.
  • Naffakh N; Institut Pasteur, Université Paris Cité, CNRS UMR3569, Unité Biologie des ARN et Virus Influenza, F-75015 Paris, France. Electronic address: nadia.naffakh@pasteur.fr.
Antiviral Res ; 201: 105272, 2022 05.
Article in English | MEDLINE | ID: covidwho-1729532
ABSTRACT
Effective drugs against SARS-CoV-2 are urgently needed to treat severe cases of infection and for prophylactic use. The main viral protease (nsp5 or 3CLpro) represents an attractive and possibly broad-spectrum target for drug development as it is essential to the virus life cycle and highly conserved among betacoronaviruses. Sensitive and efficient high-throughput screening methods are key for drug discovery. Here we report the development of a gain-of-signal, highly sensitive cell-based luciferase assay to monitor SARS-CoV-2 nsp5 activity and show that it is suitable for the screening of compounds in a 384-well format. A benefit of miniaturisation and automation is that screening can be performed in parallel on a wild-type and a catalytically inactive nsp5, which improves the selectivity of the assay. We performed molecular docking-based screening on a set of 14,468 compounds from an in-house chemical database, selected 359 candidate nsp5 inhibitors and tested them experimentally. We identified two molecules which show anti-nsp5 activity, both in our cell-based assay and in vitro on purified nsp5 protein, and inhibit SARS-CoV-2 replication in A549-ACE2 cells with EC50 values in the 4-8 µM range. The here described high-throughput-compatible assay will allow the screening of large-scale compound libraries for SARS-CoV-2 nsp5 inhibitors. Moreover, we provide evidence that this assay can be adapted to other coronaviruses and viruses which rely on a viral protease.
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Full text: Available Collection: International databases Database: MEDLINE Main subject: SARS-CoV-2 / COVID-19 Drug Treatment Limits: Humans Language: English Journal: Antiviral Res Year: 2022 Document Type: Article Affiliation country: J.antiviral.2022.105272

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Full text: Available Collection: International databases Database: MEDLINE Main subject: SARS-CoV-2 / COVID-19 Drug Treatment Limits: Humans Language: English Journal: Antiviral Res Year: 2022 Document Type: Article Affiliation country: J.antiviral.2022.105272