RESUMO
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.
Assuntos
Antivirais/isolamento & purificação , Técnicas Biossensoriais/métodos , Proteases 3C de Coronavírus/metabolismo , SARS-CoV-2/fisiologia , Replicação Viral , Animais , Antivirais/farmacologia , Linhagem Celular , Chlorocebus aethiops , Descoberta de Drogas , Avaliação Pré-Clínica de Medicamentos , Ativação Enzimática , Células HEK293 , Humanos , Luciferases de Vaga-Lume/metabolismo , Mucosa Nasal/virologia , Pirazolonas/farmacologia , Piridonas/farmacologia , SARS-CoV-2/metabolismo , Células Vero , Internalização do Vírus/efeitos dos fármacos , Replicação Viral/efeitos dos fármacosRESUMO
Following the emergence of SARS-CoV-2, the search for an effective and rapidly available treatment was initiated worldwide based on repurposing of available drugs. Previous reports described the antiviral activity of certain tyrosine kinase inhibitors (TKIs) targeting the Abelson kinase 2 against pathogenic coronaviruses. Imatinib, one of them, has more than twenty years of safe utilization for the treatment of hematological malignancies. In this context, Imatinib was rapidly evaluated in clinical trials against Covid-19. Here, we present the pre-clinical evaluation of imatinib in multiple models. Our results indicated that imatinib and another TKI, the masitinib, exhibit an antiviral activity in VeroE6 cells. However, imatinib was inactive in a reconstructed bronchial human airway epithelium model. In vivo, imatinib therapy failed to impair SARS-CoV-2 replication in a golden Syrian hamster model despite high concentrations in plasma and in the lung. Overall, these results do not support the use of imatinib and similar TKIs as antivirals in the treatment of Covid-19.
Assuntos
Antivirais/farmacologia , Tratamento Farmacológico da COVID-19 , Mesilato de Imatinib/farmacologia , SARS-CoV-2/efeitos dos fármacos , Animais , COVID-19/epidemiologia , COVID-19/virologia , Linhagem Celular , Chlorocebus aethiops , Avaliação Pré-Clínica de Medicamentos , Reposicionamento de Medicamentos , Inibidores Enzimáticos/farmacologia , Epitélio , Feminino , Humanos , Pulmão/patologia , Masculino , Mesocricetus , Células Vero , Replicação Viral/efeitos dos fármacosRESUMO
A novel coronavirus, named SARS-CoV-2, emerged in 2019 in China and rapidly spread worldwide. As no approved therapeutics exists to treat COVID-19, the disease associated to SARS-Cov-2, there is an urgent need to propose molecules that could quickly enter into clinics. Repurposing of approved drugs is a strategy that can bypass the time-consuming stages of drug development. In this study, we screened the PRESTWICK CHEMICAL LIBRARY composed of 1,520 approved drugs in an infected cell-based assay. The robustness of the screen was assessed by the identification of drugs that already demonstrated in vitro antiviral effect against SARS-CoV-2. Thereby, 90 compounds were identified as positive hits from the screen and were grouped according to their chemical composition and their known therapeutic effect. Then EC50 and CC50 were determined for a subset of 15 compounds from a panel of 23 selected drugs covering the different groups. Eleven compounds such as macrolides antibiotics, proton pump inhibitors, antiarrhythmic agents or CNS drugs emerged showing antiviral potency with 2 < EC50 ≤ 20 µM. By providing new information on molecules inhibiting SARS-CoV-2 replication in vitro, this study provides information for the selection of drugs to be further validated in vivo. Disclaimer: This study corresponds to the early stages of antiviral development and the results do not support by themselves the use of the selected drugs to treat SARS-CoV-2 infection.
Assuntos
Betacoronavirus/fisiologia , Bibliotecas de Moléculas Pequenas/química , Animais , Antivirais/química , Antivirais/metabolismo , Antivirais/farmacologia , Betacoronavirus/isolamento & purificação , COVID-19 , Células CACO-2 , Sobrevivência Celular/efeitos dos fármacos , Chlorocebus aethiops , Infecções por Coronavirus/patologia , Infecções por Coronavirus/virologia , Aprovação de Drogas , Avaliação Pré-Clínica de Medicamentos , Reposicionamento de Medicamentos , Humanos , Pandemias , Pneumonia Viral/patologia , Pneumonia Viral/virologia , SARS-CoV-2 , Bibliotecas de Moléculas Pequenas/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , Células Vero , Replicação Viral/efeitos dos fármacosRESUMO
Dengue fever is the most widespread of the human arbovirus diseases, with approximately one third of the world's population at risk of infection. Dengue viruses are members of the genus Flavivirus (family Flaviviridae) and, antigenically, they separate as four closely related serotypes (1-4) that share 60-75% amino acid homology. This genetic diversity complicates the process of antiviral drug discovery. Thus, currently no approved dengue-specific therapeutic treatments are available. With the aim of providing an efficient tool for dengue virus drug discovery, a collection of nineteen dengue viruses, representing the genotypic diversity within the four serotypes, was developed. After phylogenetic analysis of the full-length genomes, we selected relevant strains from the EVAg collection at Aix-Marseille University and completed the virus collection, using a reverse genetic system based on the infectious sub-genomic amplicons technique. Finally, we evaluated this dengue virus collection against three published dengue inhibitory compounds. NITD008, which targets the highly conserved active site of the viral NS5 polymerase enzyme, exhibited similar antiviral potencies against each of the different dengue genotypes in the panel. Compounds targeting less conserved protein subdomains, such as the capsid inhibitor ST-148, or SDM25N, a ∂ opioid receptor antagonist which indirectly targets NS4B, exhibited larger differences in potency against the various genotypes of dengue viruses. These results illustrate the importance of a phylogenetically based dengue virus reference panel for dengue antiviral research. The collection developed in this study, which includes such representative dengue viruses, has been made available to the scientific community through the European Virus Archive to evaluate novel DENV antiviral candidates.