Identification and characterization of candidate inhibitors of the SARS-CoV-2 nsp14 3′-5′ exoribonuclease

Coronaviruses such as SARS-CoV-2 possess the largest positive-sense RNA virus genomes (30 kb). This poses a fidelity problem as the inherent lack of proof-reading capacity of the viral RNA-dependent RNA polymerase results in a high level of mutation. To overcome this issue, coronaviruses encode a 3'-5' exoribonuclease (ExoN) proof-reading activity, which is a property of a complex of two non-structural proteins nsp14 and nsp10. Inactivating ExoN mutants in SARS-CoV-2 are lethal, indicating the importance of this enzymatic activity for virus replication and raising the possibility that small-molecule inhibitors of ExoN activity could be potential antiviral agents. To evaluate this, we used structure-based drug design approaches to identify potential ExoN inhibitors and tested these for activity against infectious SARS-CoV-2. Two compounds had low micromolar EC₅₀ activity and synergized with mutagenic nucleoside analogues. Next-generation sequencing analysis revealed an increased rate of mutation in the presence of these compounds, which is consistent with their mode of action being inhibition of ExoN enzymatic activity.