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Fragment-Based Development of NSP14 Exonuclease Inhibitors Confounded by Batch-to-Batch Variability
Jesse A Coker1,2, Rong Sun3,4, Paul M Polzer1
1Cleveland Clinic Center for Therapeutics Discovery, Cleveland Clinic Research, Cleveland, Ohio 44106, United States.
ACS Chemical Biology
|February 16, 2026
Summary
Researchers explored inhibiting the nonstructural protein 14 (NSP14) exonuclease (ExoN) site for antiviral strategies. Initial pyrazole compounds showed promise but were false positives due to divalent cations, highlighting challenges in targeting NSP14 ExoN.
Area of Science:
- Virology
- Drug Discovery
- Biochemistry
Background:
- The exonuclease (ExoN) activity of nonstructural protein 14 (NSP14) is crucial for betacoronavirus replication, including SARS-CoV-2.
- Inhibiting NSP14's ExoN function presents a potential antiviral strategy against these viruses.
Purpose of the Study:
- To identify novel compounds that inhibit the ExoN activity of NSP14.
- To investigate the feasibility of targeting the NSP14 ExoN site using fragment-based drug design.
Main Methods:
- Utilized crystal structures of fragments bound to the NSP14 ExoN site to guide compound design.
- Synthesized and tested a series of 3,5-disubstituted pyrazoles for NSP14 ExoN inhibition.
- Re-synthesized and re-evaluated putative inhibitory compounds.
Main Results:
- A series of 3,5-disubstituted pyrazoles were initially identified as binders and inhibitors of NSP14 ExoN.
- Upon re-synthesis, these compounds were found to be false positives.
- Divalent cations were identified as potent inhibitors of NSP14 ExoN, likely responsible for the false-positive results.
Conclusions:
- Directly targeting the NSP14 ExoN site with fragment-merging approaches faces significant challenges.
- The high sensitivity of NSP14 ExoN to divalent cations must be carefully considered in future antiviral drug discovery efforts targeting this site.
