A Modular APEX Biosensor Enables Sensitive and Visualized Monitoring of Viral Protease Activity

Area of Science:

• Biochemistry
• Molecular Biology
• Drug Discovery

Background:

• Viral proteases are crucial targets for broad-spectrum antiviral drug development.
• Existing screening methods like FRET assays have limitations in reflecting intracellular drug effects.
• A need exists for robust cellular screening platforms to assess protease inhibitor efficacy.

Purpose of the Study:

• To develop and validate novel modular biosensors for intracellular viral protease activity screening.
• To overcome limitations of traditional assays susceptible to compound interference.
• To facilitate the discovery of potent antiviral therapeutics.

Main Methods:

• Constructed two modular biosensors (TS3AR and C3SIR) utilizing engineered ascorbate peroxidase (APEX).
• Biosensor mechanism relies on protease-specific substrate cleavage, APEX fragment reassembly, and signal generation via cascade amplification.
• Validated using coronavirus main protease (Mpro) and tested against known inhibitors.

Main Results:

• TS3AR sensor achieved a 1500-fold signal-to-noise ratio for enzyme activity detection.
• C3SIR sensor accurately identified Mpro inhibitors (e.g., Enstrelvir, Nirmatrelvir) and avoided false positives.
• Biosensors demonstrated adaptability for various coronaviruses and other viral proteases.

Conclusions:

• The developed APEX-based biosensor platform offers a reliable and specific intracellular screening tool.
• This platform accelerates the development of broad-spectrum therapeutics against viral threats.
• Modular design allows flexible adaptation for diverse viral protease targets.