Identification and characterisation of candidate inhibitors of the SARS-CoV-2 nsp14 3-prime - 5-prime 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 an exoribonuclease (ExoN) activity possessed by 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 synergised with mutagenic nucleoside analogues. Next-generation sequencing analysis revealed an increased rate of mutation in the presence of these compounds, confirming their mode of action as inhibition of ExoN.