Mapping the Fragment-Binding Landscape of SARS-CoV-2 nsp14 for Antiviral Drug Discovery

Non-structural protein 14 (nsp14) is a bifunctional coronavirus drug target, combining a 3'-to-5' exoribonuclease domain with an N7-methyltransferase domain. Proof-of-concept data have demonstrated beneficial effects in animal models upon inhibition. Using microscale thermophoresis and X-ray crystallography, we screened full-length nsp14 for fragment hits and developed SAR-by-catalogue analogues with five- to eight-fold improved affinity. Several scaffolds showed pronounced overlap between distinct pockets, complicating structure-based optimisation. Analysis of mutational frequencies across over 14 million SARS-CoV-2 genome sequences identified nsp14 residues with markedly high and very low mutation rates, highlighting sites best suited to pan-coronavirus inhibitor design. Combining these data with all 19 publicly known fragment- and ligand-binding regions on nsp14, most of which have never been directly compared, we introduce a systematic site-naming system and rank every site by its potential for future structure-based development. Weighing criteria include proof-of-principle inhibitor data, cross-coronavirus conservation, mutation frequency, the number of fragments bound per site, and susceptibility to crystallographic artefacts from symmetry mates. This ranked, unified binding-site atlas resolves which of the many previously reported nsp14 sites merit prioritisation, providing a practical resource for future coronavirus antiviral drug development.