A fluorescence-based high throughput-screening assay for the SARS-CoV RNA synthesis complex

• Published in: Journal of virological methods 2021-02, Vol.288, p.114013-114013, Article 114013
• Main Authors: Eydoux, Cecilia, Fattorini, Veronique, Shannon, Ashleigh, Le, Thi-Tuyet-Nhung, Didier, Bruno, Canard, Bruno, Guillemot, Jean-Claude
• Format: Article
• Language: English
• Subjects: Antiviral Agents - pharmacology; Coronavirus; Dose-Response Relationship, Drug; Drug Evaluation, Preclinical; Enzyme Activation; Fluorescent Dyes; High-Throughput Screening Assays - methods; High-Throughput Screening Assays - standards; Humans; Inhibitory Concentration 50; Life Sciences; Replication complex; RNA, Messenger - genetics; RNA, Viral; RNA-Dependent RNA Polymerase - metabolism; SARS Virus - genetics; Screening; Severe Acute Respiratory Syndrome - diagnosis; Severe Acute Respiratory Syndrome - genetics; Small molecule; Templates, Genetic
• ISSN: 0166-0934; 1879-0984
• DOI: 10.1016/j.jviromet.2020.114013

Principle of SARS-CoV RNA synthesis detection by a fluorescence-based High Throughput Screening assay. During the enzymatic step, the RNA template was incubated with the SARS-CoV RTC, the nucleotides triphosphate and the compound to be tested (putative inhibitor). The polymerase activity released a double stranded RNA which was detected by adding a fluorescent intercalant agent (Picogreen®, ex: 480 nM, em: 530 nm). In presence of an inhibitor, the fluorescence signal decreased and is quantified. NTP: Nucleotides Tri-Phosphate. [Display omitted] •A new non-radioactive SARS-CoV RNA polymerase assay was developed.•The robotized assay is suitable to identify RdRp inhibitors based on HTS.•A total of 1520 FDA-approved compounds were tested using the SARS-CoV-RTC assay. The Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) emergence in 2003 introduced the first serious human coronavirus pathogen to an unprepared world. To control emerging viruses, existing successful anti(retro)viral therapies can inspire antiviral strategies, as conserved viral enzymes (eg., viral proteases and RNA-dependent RNA polymerases) represent targets of choice. Since 2003, much effort has been expended in the characterization of the SARS-CoV replication/transcription machinery. Until recently, a pure and highly active preparation of SARS-CoV recombinant RNA synthesis machinery was not available, impeding target-based high throughput screening of drug candidates against this viral family. The current Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) pandemic revealed a new pathogen whose RNA synthesis machinery is highly (>96 % aa identity) homologous to SARS-CoV. This phylogenetic relatedness highlights the potential use of conserved replication enzymes to discover inhibitors against this significant pathogen, which in turn, contributes to scientific preparedness against emerging viruses. Here, we report the use of a purified and highly active SARS-CoV replication/transcription complex (RTC) to set-up a high-throughput screening of Coronavirus RNA synthesis inhibitors. The screening of a small (1520 compounds) chemical library of FDA-approved drugs demonstrates the robustness of our assay and will allow to speed-up drug discovery against the SARS-CoV-2.