Development of a High throughput screening method based on fluorescence to discover inhibitors of viral exoribonucleases

The exoribonuclease activity (ExoN) is quite bizarre in the world of RNA viruses, as it is present uniquely in the Arenaviridae and the Coronaviridae families. ExoN plays important but different roles in both families : for arenaviruses the ExoN is involved in the suppression of the host immune response whereas for coronaviruses, ExoN is likely involved in the proofreading mechanism for the viral genome's replication. Because of their key roles, they are attractive targets for drug development, however, the most common current available technique to measure the ExoN activity and inhibition is the use of radiolabeled gel assays, which is not suitable for the screening of compounds libraries. Here we developed a method using fluorescence polarization to assess the ExoN activity and inhibition and we validated the method on three different viral enzymes (SARS-CoV-2, lymphocytic choriomeningitis virus and Machupo Virus. The method is very sensitive, robust, amenable to miniaturization (384 well plates) and allow us to screen a small compounds library (24). We are confident that this method is a method of choice for screening large libraries and will become a commonly used HTS screening method.