A Gaussia luciferase reporter assay for the evaluation of coronavirus Nsp5/3CLpro activity.
3CLpro
Coronaviruses
Gaussia reporter assay
Nsp5
Protease inhibitor
SARS-CoV-2
Journal
Scientific reports
ISSN: 2045-2322
Titre abrégé: Sci Rep
Pays: England
ID NLM: 101563288
Informations de publication
Date de publication:
05 Sep 2024
05 Sep 2024
Historique:
received:
03
05
2024
accepted:
27
08
2024
medline:
6
9
2024
pubmed:
6
9
2024
entrez:
5
9
2024
Statut:
epublish
Résumé
Human coronaviruses (hCoVs) infect millions of people every year. Among these, MERS, SARS-CoV-1, and SARS-CoV-2 caused significant morbidity and mortality and their emergence highlights the risk of possible future coronavirus outbreaks. Therefore, broadly-active anti-coronavirus drugs are needed. Pharmacological inhibition of the hCoV protease Nsp5 (3CLpro) is clinically beneficial as shown by the wide and effective use of Paxlovid (nirmatrelvir, ritonavir). However, further treatment options are required due to the risk of drug resistance. To facilitate the assessment of coronavirus protease function and its pharmacological inhibition, we developed an assay allowing rapid and reliable quantification of Nsp5 activity under biosafety level 1 conditions. It is based on an ACE2-Gal4 transcription factor fusion protein separated by a Nsp5 recognition site. Cleavage by Nsp5 releases the Gal4 transcription factor, which then induces the expression of Gaussia luciferase. Our assay is compatible with Nsp5 proteases from all hCoVs and allows simultaneous measurement of inhibitory and cytotoxic effects of the tested compounds. Proof-of-concept measurements confirmed that nirmatrelvir, GC376 and lopinavir inhibit SARS-CoV-2 Nsp5 function. Furthermore, the assay accurately predicted the impact of Nsp5 mutations on catalytic activity and inhibitor sensitivity. Overall, the reporter assay is suitable for evaluating viral protease activity.
Identifiants
pubmed: 39237598
doi: 10.1038/s41598-024-71305-6
pii: 10.1038/s41598-024-71305-6
doi:
Substances chimiques
Luciferases
EC 1.13.12.-
Coronavirus 3C Proteases
EC 3.4.22.28
Antiviral Agents
0
Types de publication
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Pagination
20697Subventions
Organisme : Deutsche Forschungsgemeinschaft
ID : CRC 1279
Organisme : European Commission
ID : 101062524
Organisme : Medizinische Fakultät, Universität Ulm
ID : L.SBN.0208
Organisme : Else Kröner-Fresenius-Stiftung
ID : 2022_EKEA.47
Informations de copyright
© 2024. The Author(s).
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