Identification of viral cap-methyltransferase inhibitors by fluorescence screening assay

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A smart assay for screening RNA methyl transferase inhibitors

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Severe acute respiratory syndrome (SARS) is a viral respiratory illness caused by a coronavirus called SARS-associated coronavirus (SARS-CoV). SARS typically begins with flu-like signs and symptoms but can lead to more serious forms of pneumonia causing acute respiratory distress (severe breathing difficulty) and sometimes death. The virus is believed to be of animal origin, recently crossing the species barrier to infect humans. Following the outbreak of SARS that began in 2002 in Asia, with secondary cases presumably spread elsewhere around the world by travelers, the World Health Organization (WHO) mobilized scientists worldwide to prevent an epidemic situation.The race for new antivirals was launched…In the complex world of virology, RNA methylation is a key biological process involved in RNA virus replication and propagation. It is a reversible post-translational modification of RNA that impacts numerous biological events epigenetically during the virus life cycle.In this article, Wahiba Aouadi and colleagues focused their efforts on the protein nsp14, involved in the RNA cap methylation of SARS-CoV. A high-throughput in-vitro assay was developed and used to screen libraries of compounds. Robustness of the assay in a 384-well plate format was assessed by determining the Z’ factor. Structure Activities Relationship studies were performed, leading to identification of compounds with very potent IC50 values.The threat of SARS and the risk of a more global epidemic will remain reality for some time. Pictures of people wearing nose-masks to prevent contamination strike everyone’s mind. Virologists are working diligently to find a treatment to cure coronavirus infections. We have no doubt they will succeed!

Abstract

Two highly pathogenic human coronaviruses associated with severe respiratory syndromes emerged since the beginning of the century. The severe acute respiratory syndrome SARS-coronavirus (CoV) spread first in southern China in 2003 with about 8000 infected cases in few months. Then in 2012, the Middle East respiratory syndrome (MERS-CoV) emerged from the Arabian Peninsula giving a still on-going epidemic associated to a high fatality rate. CoVs are thus considered a major health threat. This is especially true as no vaccine nor specific therapeutic are available against either SARS- or MERS-CoV. Therefore, new drugs need to be identified in order to develop antiviral treatments limiting CoV replication. In this study, we focus on the nsp14 protein, which plays a key role in virus replication as it methylates the RNA cap structure at the N7 position of the guanine. We developed a high-throughput N7-MTase assay based on Homogenous Time Resolved Fluorescence (HTRF®) and screened chemical libraries (2000 compounds) on the SARS-CoV nsp14. 20 compounds inhibiting the SARS-CoV nsp14 were further evaluated by IC50 determination and their specificity was assessed toward flavivirus- and human cap N7-MTases. Our results reveal three classes of compounds: 1) molecules inhibiting several MTases as well as the dengue virus polymerase activity unspecifically, 2) pan MTases inhibitors targeting both viral and cellular MTases, and 3) inhibitors targeting one viral MTase more specifically showing however activity against the human cap N7-MTase. These compounds provide a first basis towards the development of more specific inhibitors of viral methyltransferases.

Details

Antiviral Research, 2017 Aug;144:330-339.

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