A fluorescence-based, gain-of-signal, live cell system to evaluate SARS-CoV-2 main protease inhibition

Area of Science:

• Virology
• Drug Discovery
• Biochemistry

Background:

• The ongoing threat of SARS-CoV-2 (CoV2) and other coronaviruses necessitates the development of effective antiviral therapies.
• A significant gap exists in the availability of approved antivirals targeting coronaviruses.
• The Main 3C-like protease (Mpro) is a critical and conserved target for antiviral development against CoV2.

Purpose of the Study:

• To develop and validate a novel live-cell based assay for evaluating the intracellular activity of the SARS-CoV-2 Main 3C-like protease (Mpro).
• To enable high-throughput screening for potential Mpro inhibitors.
• To assess the efficacy of known and potential Mpro inhibitors in a cellular context.

Main Methods:

• Developed a live-cell assay utilizing native wild-type mature CoV2 Mpro expressed in cells.
• Quantified Mpro activity by measuring the cleavage of a fluorescent biosensor, leading to a loss of fluorescence.
• Assessed drug inhibition and recovery of Mpro function in real-time without cell harvesting.

Main Results:

• The assay successfully demonstrated inhibition of intracellular CoV2 Mpro by the pan-coronavirus inhibitor GC376 at low micromolar levels.
• GC376's inhibitory levels correlated with those required to inhibit CoV2 infection in human lung cells.
• Other tested compounds, bepridil and alverine, showed no significant Mpro inhibition in this assay.

Conclusions:

• The developed live-cell assay is a robust and effective system for screening coronavirus Mpro inhibitors.
• This assay facilitates the discovery of novel antivirals targeting Mpro for treating SARS-CoV-2 and other coronavirus infections.
• The system offers a valuable tool for high-throughput screening, accelerating antiviral drug development.