ABSTRACT
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.