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A genetically encoded BRET-based SARS-CoV-2 Mpro protease activity sensor.

Anupriya M Geethakumari1, Wesam S Ahmed1, Saad Rasool2

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New bioluminescence resonance energy transfer (BRET) sensors detect SARS-CoV-2 main protease (Mpro) activity in cells and vitro. These tools aid drug discovery and functional genomics for SARS-CoV-2 Mpro.

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Virology

Background:

  • The SARS-CoV-2 main protease (Mpro) is essential for viral replication.
  • Targeting Mpro is a key strategy for developing antiviral therapies.
  • Existing methods for monitoring Mpro activity have limitations.

Purpose of the Study:

  • To develop novel, genetically encoded sensors for real-time detection of Mpro proteolytic activity.
  • To validate sensor performance in live cells and in vitro.
  • To utilize sensors for drug screening and functional genomics studies.

Main Methods:

  • Engineered BRET-based sensors incorporating Mpro cleavage sites between fluorescent proteins (mNeonGreen and NanoLuc).
  • Co-expression of sensors with wild-type or mutant Mpro in mammalian cells.
  • In vitro activity assays with purified sensors and Mpro under varying conditions (e.g., molecular crowding).
  • Assessment of sensor response to the Mpro inhibitor GC376.

Main Results:

  • BRET sensors successfully detected Mpro cleavage in live cells.
  • Cleavage was dependent on active Mpro, as demonstrated by C145A mutant studies.
  • Sensors accurately reflected Mpro inhibition by GC376.
  • In vitro assays showed molecular crowding enhances Mpro activity and reduces GC376 efficacy.

Conclusions:

  • Developed BRET sensors provide a robust tool for monitoring SARS-CoV-2 Mpro activity.
  • These sensors facilitate drug discovery efforts targeting Mpro.
  • The sensors are valuable for functional genomics to study Mpro sequence variations and their impact.