Identification of potent inhibitors of SARS-CoV-2 exoribonuclease by fluorescence polarization assay

The emergence of SARS-CoV-2 has triggered a pandemic with devastating consequences to the world. One of the proteins essential to the virus life cycle is nsp14, which is a bifunctional protein that encodes a 3'to 5' exoribonuclease activity in its N-terminus, and a methyl transferase activity in its C-terminus. Nsp14 in complex with the accessory protein nsp10 is involved in a proofreading mechanism that ensures the genetic stability of its massive viral genome, and is associated to the resistance against nucleotide analogs targeting the polymerase nsp12. Because of its key role, nsp14-nsp10 complex constitutes an attractive target for antiviral development. Here we present a fluorescence polarization (FP) assay development to measure the exoribonuclease activity and its inhibition in vitro. The FP method is sensitive, robust, amenable to miniaturization and offers confirmation by visualizing the degradation of the fluorescent RNA in acrylamide gels. We performed a screening of a focused library of 113 metal chelators at 20 and 5 μM compound concentration and IC50 measurement of 9 hits showing efficiency at micromolar level. We also tested the focused library in SARS-CoV-2 infected Vero cells and we confirmed 3 hits previously detected in the in vitro screening out of 6 promising inhibitors. In conclusion the FP method proposed is a reliable tool to discover inhibitors of the SARS-CoV-2 exoribonuclease activity and will help to find new antivirals to be used in combination with nucleoside analogs.

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.