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1.
Viruses ; 13(9)2021 09 12.
Article in English | MEDLINE | ID: covidwho-1411086

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.


Subject(s)
Antiviral Agents/isolation & purification , Biosensing Techniques/methods , Coronavirus 3C Proteases/metabolism , SARS-CoV-2/physiology , Virus Replication , Animals , Antiviral Agents/pharmacology , Cell Line , Chlorocebus aethiops , Drug Discovery , Drug Evaluation, Preclinical , Enzyme Activation , HEK293 Cells , Humans , Luciferases, Firefly/metabolism , Nasal Mucosa/virology , Pyrazolones/pharmacology , Pyridones/pharmacology , SARS-CoV-2/metabolism , Vero Cells , Virus Internalization/drug effects , Virus Replication/drug effects
2.
Sci Rep ; 10(1): 10100, 2020 06 22.
Article in English | MEDLINE | ID: covidwho-832425

ABSTRACT

RNA viruses are responsible for a large variety of animal infections. Equine Arteritis Virus (EAV) is a positive single-stranded RNA virus member of the family Arteriviridae from the order Nidovirales like the Coronaviridae. EAV causes respiratory and reproductive diseases in equids. Although two vaccines are available, the vaccination coverage of the equine population is largely insufficient to prevent new EAV outbreaks around the world. In this study, we present a high-throughput in vitro assay suitable for testing candidate antiviral molecules on equine dermal cells infected by EAV. Using this assay, we identified three molecules that impair EAV infection in equine cells: the broad-spectrum antiviral and nucleoside analog ribavirin, and two compounds previously described as inhibitors of dihydroorotate dehydrogenase (DHODH), the fourth enzyme of the pyrimidine biosynthesis pathway. These molecules effectively suppressed cytopathic effects associated to EAV infection, and strongly inhibited viral replication and production of infectious particles. Since ribavirin is already approved in human and small animal, and that several DHODH inhibitors are in advanced clinical trials, our results open new perspectives for the management of EAV outbreaks.


Subject(s)
Arterivirus Infections/drug therapy , Equartevirus/metabolism , Ribavirin/pharmacology , Animals , Antiviral Agents/pharmacology , Arterivirus Infections/veterinary , Cell Line , Cytopathogenic Effect, Viral/drug effects , Horse Diseases/virology , Horses/genetics , Oxidoreductases Acting on CH-CH Group Donors/metabolism , Purines/antagonists & inhibitors , Purines/biosynthesis , Purines/pharmacology , Pyrimidines/antagonists & inhibitors , Pyrimidines/biosynthesis , Pyrimidines/pharmacology , RNA/pharmacology , Virus Replication/drug effects , Virus Replication/physiology
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