High throughput screening for SARS-CoV-2 inhibitors targeting 5 helix bundle

Area of Science:

• Virology
• Drug Discovery
• Molecular Biology

Background:

• Viral entry into host cells, including SARS-CoV-2, involves receptor recognition and membrane fusion.
• The 5-Helix Bundle (5HB) protein is critical for this process by binding to viral spike heptad repeats (HR2).
• 5HB represents a potential druggable target for inhibiting virus entry.

Purpose of the Study:

• To develop and validate a 5-Helix Bundle (5HB) pentamer assay for high-throughput screening (HTS).
• To identify small molecule inhibitors that block SARS-CoV-2 virus entry by targeting the 5HB-HR2 interaction.
• To validate identified compounds in secondary assays, including cell-based virus entry and cytotoxicity.

Main Methods:

• Implementation and optimization of a 5HB pentamer assay in a 1536-well format.
• High-throughput screening of 635,262 compounds using the 5HB pentamer assay.
• Validation of hits using monomeric 5HB assay, dose titration, cell-based SARS-CoV-2 and Machupo virus entry assays, and cytotoxicity counterscreen.

Main Results:

• Successful identification of small molecule inhibitors targeting the 5HB-HR2 interaction in a pilot screen.
• Completion of a full HTS campaign, identifying 41 selective inhibitors of the 5HB pentamer assay.
• Selection of 5 potent compounds demonstrating efficacy in both pentamer and monomer assays, and further validated in a SARS pseudovirus assay.

Conclusions:

• The developed 5HB pentamer assay is effective for identifying inhibitors of SARS-CoV-2 entry.
• The study identified promising small molecules that interfere with viral fusion mechanisms.
• These findings support 5HB as a viable target for developing novel antiviral therapeutics.