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. Epub 2025 Jan 9.

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.

MeSH terms

  • Antiviral Agents* / chemistry
  • Antiviral Agents* / pharmacology
  • COVID-19 / virology
  • COVID-19 Drug Treatment
  • Enzyme Inhibitors* / chemistry
  • Enzyme Inhibitors* / pharmacology
  • Exoribonucleases / antagonists & inhibitors
  • Exoribonucleases / chemistry
  • Exoribonucleases / metabolism
  • High-Throughput Screening Assays* / methods
  • Humans
  • Methyltransferases* / antagonists & inhibitors
  • Methyltransferases* / chemistry
  • Methyltransferases* / metabolism
  • SARS-CoV-2* / drug effects
  • SARS-CoV-2* / enzymology
  • Viral Nonstructural Proteins* / antagonists & inhibitors
  • Viral Nonstructural Proteins* / chemistry
  • Viral Nonstructural Proteins* / metabolism

Substances

  • NSP14 protein, SARS-CoV-2
  • Viral Nonstructural Proteins
  • Antiviral Agents
  • Enzyme Inhibitors
  • Methyltransferases
  • Exoribonucleases