Structural basis for small molecule binding to the SARS-CoV-2 nsp10–nsp14 ExoN complex

Kozielski et al., Nucleic Acids Research, doi:10.1093/nar/gkaf753, Aug 2025

https://c19early.org/kozielski.html

In vitro and crystallographic study identifying 14 small molecule fragments that bind to novel sites in the SARS-CoV-2 nsp10-nsp14 ExoN complex, potentially inhibiting viral proofreading activity that causes resistance to nucleoside analogue drugs.

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Kozielski et al., 5 Aug 2025, peer-reviewed, 10 authors.

In Vitro studies are an important part of preclinical research, however results may be very different in vivo.

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Structural basis for small molecule binding to the SARS-CoV-2 nsp10-nsp14 ExoN complex

F R Ank Kozielski, Suzanne Zoë Fisher, Shumeng Ma, Fatma Al Busaidi, Ewa Krupinska, Maria Nyblom, Céleste Sele, Heather Mcduffie Sullivan, Tobias Krojer, Wolfg Ang Knec Ht

doi:10.1093/nar/gkaf753

Coronavirus outbreaks have occurred over the past 25 years with SARS-CoV-2 (severe acute respiratory syndrome coronavirus-2) causing a global pandemic. T he SAR S-CoV-2 non-str uct ural proteins 10 (nsp10) and 1 4 (nsp1 4) are considered as potential drug targets. Nsp10 stimulates the 3to-5 e x oribonuclease (Ex oN) activity of nsp14. The ExoN domain excises mis-incorporated nucleotides from the nascent RNA chain and therefore causes resistance to nucleoside analogue drugs. We cry stalliz ed the nsp10-nsp14 Ex oN comple x in distinct space groups, allowing us to describe conformational changes. In particular, the general base, His268, classifying the ExoN domain as a member of the DEDDh family, is trapped in the inactive and active orientations. By X-ray fragment screening, we identified five novel fragment binding sites in the nsp10-nsp14 interface, the hinge region connecting ExoN and N7 -methyltransf erase domains, and on nsp10. One new site in the nsp10-nsp14 interface accommodates nine str uct urally and chemically related hits, providing an initial str uct ure-activity relationship st udy. We could also identify enantiomers of one fragment selectively bound to two different binding sites. The binding affinities of fragment hits were estimated using microscale thermophoresis and the new sites were investigated for their potential to inhibit protein-protein interactions between nsp10 and nsp14. Our fragments represent no v el starting points for hit development by str uct ure-based design.

Supplementary data Supplementary data is available at NAR online. Conflict of interest None declared.

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