A High-Throughput Screening Pipeline to Identify Methyltransferase and Exonuclease Inhibitors of SARS-CoV-2 NSP14

  • Biochemistry. 2025 Jan 21;64(2):419-431. doi: 10.1021/acs.biochem.4c00490.
Quinlin Hanson  1 ,  Xin Hu  1 ,  Sourav Pal  1 ,  Katlin Recabo  1 ,  Lin Ye  1 ,  Ivy Poon  2 ,  John-Paul Denson  2 ,  Simon Messing  2 ,  Min Shen  1 ,  Kelli M Wilson  1 ,  Alexey Zakharov  1 ,  Dominic Esposito  2 ,  Natalia J Martinez  1
Affiliations
  • 1. National Center for Advancing Translational Sciences, National Institutes of Health, Rockville, Maryland 20850, United States.
  • 2. Protein Expression Laboratory, Cancer Research Technology Program, Frederick National Laboratory for Cancer Research, Frederick, Maryland 21701, United States.
PMID: 39789725 DOI: 10.1021/acs.biochem.4c00490
Abstract

SARS-CoV-2 infections led to a worldwide pandemic in 2020. As of 2024, therapeutics against SARS-CoV-2 have continued to be desirable. NSP14 is a dual-function methyltransferase (MTase) and exonuclease (ExoN) with key roles in SARS-CoV-2 genome propagation and host immune system evasion. In this work, we developed high-throughput screening (HTS) assays for NSP14 MTase and ExoN activities. We screened both activities against a collection of 40,664 compounds. A total of 1677 initial hit compounds were identified, cherrypicked, counterscreened for assay interference, and screened for off-target selectivity. We identified 396 and 174 high-quality hits against the MTase and ExoN activities, respectively. Along with inhibitors for individual activities, we identified dual-activity inhibitors, including a novel inhibitor that is not competitive with any substrate and interacts with a putative allosteric binding site. This study represents the largest published screen of SARS-CoV-2 NSP14 MTase and ExoN activities to date and culminates in a pipeline for the NSP14 drug discovery.

Products