Fragment-Based Development of NSP14 Exonuclease Inhibitors Confounded by Batch-to-Batch Variability

TL;DR

Researchers tried to develop inhibitors for a key SARS-CoV-2 enzyme but found their initial results were unreliable due to contamination.

Contribution

Highlights the challenges of fragment-based drug design for NSP14 ExoN due to batch variability and cation interference.

Findings

3,5-disubstituted pyrazoles initially appeared to inhibit NSP14 ExoN but were later found to be false positives.

Divalent cations, possibly from contamination, strongly inhibit NSP14 ExoN activity.

The study warns about the sensitivity of NSP14 ExoN to environmental factors during drug development.

Abstract

Point mutations in the exonuclease (ExoN) site of nonstructural
protein 14 (NSP14) compromise the fitness of betacoronaviruses such
as SARS-CoV-2, implicating NSP14 ExoN inhibition as an antiviral strategy.
However, there are no advanced compounds that inhibit NSP14’s
ExoN activity. Building upon the reported crystal structures of two
fragments bound to NSP14’s ExoN site, we identified a series
of 3,5-disubsituted pyrazoles that bound to and inhibited NSP14 ExoN.
However, upon resynthesis, we discovered that these putative leads
were false positives, perhaps due to contaminating divalent cations,
which potently inhibit NSP14 ExoN. Our results provide a cautionary
tale to the field about the sensitivity of NSP14 to divalent cations
and illustrate the challenges associated with directly targeting the
NSP14 ExoN site via fragment merging.

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