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Updated: Feb 28, 2026

A Fluorogenic Peptide Cleavage Assay to Screen for Proteolytic Activity: Applications for coronavirus spike protein activation
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Development of a Complementation Assay to Monitor Pan-Coronavirus 3C-like Protease Activity.

Akhil Chameettachal1, Alice Duchon1, Matthew A Brown1

  • 1Viral Recombination Section, HIV Dynamics and Replication Program, National Cancer Institute, Frederick, MD 21702, USA.

Viruses
|February 27, 2026
PubMed
Summary

A new assay effectively measures 3C-like protease (3CLpro) activity across multiple coronaviruses. This tool aids in developing pan-coronavirus inhibitors for pandemic preparedness.

Keywords:
3CLprocoronavirusesluciferase reporter assaypan-coronavirus inhibitor

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Area of Science:

  • Virology
  • Antiviral Drug Development
  • Molecular Biology

Background:

  • Coronaviruses present a significant pandemic risk, necessitating broad-spectrum antiviral strategies.
  • The 3C-like protease (3CLpro) is a critical enzyme for coronavirus replication and a prime target for antiviral therapies.
  • Existing assays are often specific to certain coronaviruses, limiting their utility for pandemic preparedness.

Purpose of the Study:

  • To adapt and validate a previously developed cell-based assay for monitoring 3C-like protease activity across diverse human coronaviruses.
  • To optimize the assay by evaluating different protease cleavage sites for enhanced signal-to-background ratios.
  • To demonstrate the assay's efficacy in identifying broad-spectrum 3CLpro inhibitors.

Main Methods:

  • Adapted a dual-luciferase reporter assay designed for SARS-CoV-2 3CLpro to evaluate activity in six other human coronaviruses (SARS-CoV, MERS-CoV, HCoV-NL63, HCoV-229E, HCoV-OC43, HCoV-HKU1).
  • Tested various protease cleavage sites, including Nsp4-Nsp5 and super-active substrate (SAS), to maximize assay dynamic range.
  • Validated the assay's performance using the known broad-spectrum 3CLpro inhibitor GC376.

Main Results:

  • The adapted assay successfully measured 3CLpro activity across multiple human coronavirus species.
  • The Nsp4-Nsp5 cleavage site and SAS substrate provided the largest dynamic range for most tested coronaviruses.
  • Increased reporter activity was observed upon treatment with GC376, confirming the assay's ability to detect 3CLpro inhibition.

Conclusions:

  • The improved cell-based assay is effective for assessing 3CLpro activity across a range of human coronaviruses.
  • This versatile assay platform can accelerate the discovery and development of novel pan-coronavirus inhibitors.
  • The assay facilitates preparedness for future coronavirus outbreaks by enabling rapid screening of potential antiviral compounds.