A Bioluminescent 3CL^Pro Activity Assay to Monitor SARS-CoV-2 Replication and Identify Inhibitors

Area of Science:

• Virology
• Drug Discovery
• Biochemistry

Background:

• Limited antiviral therapies necessitate new treatments for emerging coronaviruses like SARS-CoV-2.
• High-throughput cellular assays are crucial for identifying effective antiviral compounds.
• Developing novel therapeutic strategies requires understanding viral metabolic vulnerabilities.

Purpose of the Study:

• To develop a novel cell-based assay for quantifying SARS-CoV-2 replication.
• To screen a metabolism-oriented compound library for potential SARS-CoV-2 inhibitors.
• To identify and validate new antiviral compounds for therapeutic development.

Main Methods:

• Generated cell lines expressing a firefly luciferase biosensor activated by the viral 3CL protease.
• Screened a library of 492 metabolism-focused compounds using the developed assay.
• Validated antiviral activity of hit compounds in ACE2-expressing pulmonary and primary nasal epithelial cells.

Main Results:

• The cell-based assay successfully detected SARS-CoV-2 infection and viral protease activity.
• Inhibitors of pyrimidine biosynthesis demonstrated efficacy in preventing SARS-CoV-2 replication.
• The NADPH oxidase (NOX) inhibitor Setanaxib showed significant anti-SARS-CoV-2 activity in multiple cell types.

Conclusions:

• The developed cell-based functional assay is a validated tool for SARS-CoV-2 antiviral screening.
• Screening diverse compound libraries can identify novel therapeutic targets and repurposable drugs.
• Setanaxib and pyrimidine biosynthesis inhibitors represent promising leads for developing new anti-coronavirus therapies.