A dual-activity ExoN/MTase platform for SARS-CoV2 NSP14 drug development

Authors

• QM Hanson
• AV Zakharov
• S Pal
• K Recabo
• S Messing
• D Esposito
• NJ Martinez

Abstract

SARS-CoV-2 carries one of the largest RNA genomes (~30 kilobases) among all RNA virus families. The nonstructural protein 14 (NSP14) is a dual-function exonuclease (ExoN)/methyltransferase (MTase) protein responsible for preservation of the viral genome. NSP14 requires the accessory protein NSP10 for ExoN activity, but not for MTase activity. While the ExoN activity allows SARS-CoV-2 to edit faulty replication, the MTase activity performs the final step of viral RNA capping to prevent viral genome degradation by host cells. Both enzymatic domains are essential for successful viral replication, making NSP14 an appealing drug target. Our goal is to identify small molecules capable of impairing NSP14 activity. To this end, we developed high-throughput assays to measure both MTase and ExoN activity and interrogated these assays against a collection of ~20K approved, investigational, and pharmacologically active compounds. Our screened identified compounds active against the MTase activity of NSP14, compounds active against the ExoN activity, and compounds active against both domains. Hits were further validated using counter assays to parse out false positives. Selected hits were subsequently validated for target engagement using an activity-agnostic nano-differential scanning fluorimetry (nano-DSF) assay. Together, these assays constitute a platform amenable for NSP14 drug development, which can be deployed for screening larger and chemically diverse small molecule libraries to develop novel small molecule inhibitors of NSP14.

Scientific Focus Area: Chemical Biology

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