Cell-Based Luciferase Assay for Testing SARS-CoV-2 3CL Protease Inhibitors

Area of Science:

• Biochemistry
• Virology
• Drug Discovery

Background:

• Viral proteases are crucial targets for antiviral drug development.
• Existing screening methods may not fully replicate the cellular environment, impacting drug efficacy assessment.
• Developing robust cell-based assays is essential for identifying effective viral protease inhibitors.

Purpose of the Study:

• To establish a reliable cell-based screening system for viral protease inhibitors.
• To validate the system using the SARS-CoV-2 3CLpro (main protease) model.
• To assess the impact of cellular context on inhibitor activity.

Main Methods:

• Utilized firefly luciferase fragment complementation assay.
• Developed a bicistronic construct (pCAG-Luc-III-IRES-nsp4-6) for coordinated reporter and protease expression.
• Incorporated an extended viral polyprotein fragment (nsp4-nsp6) to maintain native protease context.
• Validated the system with known inhibitors (nirmatrelvir, GC376) and compared results to cell-free assays.

Main Results:

• An optimized luciferase variant (Luc III) retained 88% of native activity.
• The use of an extended viral polyprotein fragment was critical for system performance.
• IC50 values correlated with live-virus assays but differed from cell-free FRET assays, highlighting cellular barrier effects.
• The assay demonstrated high reproducibility and simplicity.

Conclusions:

• The developed cell-based assay is effective for screening viral protease inhibitors.
• It accurately reflects inhibitor activity within a cellular context, outperforming cell-free methods.
• This system is adaptable for high-throughput screening of various viral proteases in basic laboratory settings.