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A Fluorogenic Peptide Cleavage Assay to Screen for Proteolytic Activity: Applications for coronavirus spike protein activation
Published on: January 9, 2019
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A genetically encoded BRET-based SARS-CoV-2 Mpro protease activity sensor
Anupriya M Geethakumari1, Wesam S Ahmed1, Saad Rasool2
1Division of Biological and Biomedical Sciences, College of Health & Life Sciences, Hamad Bin Khalifa University, Education City, Qatar Foundation, Doha, 34110 Qatar.
Communications Chemistry
|October 3, 2022
Summary
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.
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.
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