Mutagenesis-Guided Target Identification Reveals the Protein-Binding Domain of Nsp14 in Coronaviruses as the Target of a Labdane-Oxindole Compound.

The non-structural protein (nsp) 14 of coronaviruses plays an important role in maintaining the genomic stability of the virus during viral replication. This had garnered significant interest towards the identification and development of inhibitors against nsp14, specifically its exoribonuclease (ExoN) domain. However, no inhibitors have been successfully developed to date. The bioactivity of the nsp14-ExoN is governed through a complex formation with its co-factor nsp10. This provides opportunities to target the protein assembly as an antiviral modality. In this study, a labdane-oxindole compound (OX18) was identified as a promising new antiviral agent against coronaviruses. Through a combination of FRET- and BRET-based approaches, OX18 was found to target the nsp10-binding domain of nsp14. A key escape mutation to OX18 in nsp14 was also identified in our study, albeit compromising its exoribonuclease activity. To our knowledge, OX18 is the first small molecule to target the nsp14/10 protein assembly. As such, our work paves the way for the development of future inhibitors of the nsp14-ExoN with increased potency and complexity.