A high-throughput fluorescence polarization assay to discover inhibitors of arenavirus and coronavirus exoribonucleases

Viral exoribonucleases are uncommon in the world of RNA viruses. To date, this activity has been identified only in the Arenaviridae and the Coronaviridae families. These exoribonucleases play important but different roles in both families: for mammarenaviruses the exoribonuclease is involved in the suppression of the host immune response whereas for coronaviruses, exoribonuclease is both involved in a proofreading mechanism ensuring the genetic stability of viral genomes and participating to evasion of the host innate immunity. Because of their key roles, they constitute attractive targets for drug development. Here we present a high-throughput assay using fluorescence polarization to assess the viral exoribonuclease activity and its inhibition. We validate the assay using three different viral enzymes from SARS-CoV-2, lymphocytic choriomeningitis and Machupo viruses. The method is sensitive, robust, amenable to miniaturization (384 well plates) and allowed us to validate the proof-of-concept of the assay by screening a small focused compounds library (23 metal chelators). We also determined the IC50 of one inhibitor common to the three viruses. HighlightsO_LIArenaviridae and Coronaviridae viral families share an exoribonuclease activity of common evolutionary origin C_LIO_LIArenaviridae and Coronaviridae exoribonuclease is an attractive target for drug development C_LIO_LIWe present a high-throughput assay in 384 well-plates for the screening of inhibitors using fluorescence polarization C_LIO_LIWe validated the assay by screening of a focused library of 23 metal chelators against SARS-CoV-2, Lymphocytic Choriomeningitis virus and Machupo virus exoribonucleases C_LIO_LIWe determined the IC50 by fluorescence polarization of one inhibitor common to the three viruses. C_LI