TMPRSS2 and SARS-CoV-2 SPIKE interaction assay for uHTS

Area of Science:

• Virology
• Biochemistry
• Drug Discovery

Background:

• Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) causes COVID-19, a global health crisis.
• Viral entry inhibitors targeting the Spike protein and host cell interactions are crucial for therapeutic development.
• The interaction between the SARS-CoV-2 Spike protein and host transmembrane serine protease 2 (TMPRSS2) is essential for viral entry.

Purpose of the Study:

• To develop and optimize a high-throughput screening assay for the SARS-CoV-2 Spike-TMPRSS2 interaction.
• To enable the discovery of small-molecule inhibitors targeting this critical viral-host protein-protein interaction.

Main Methods:

• Development of a time-resolved fluorescence/Förster resonance energy transfer (TR-FRET) assay.
• Optimization of the assay for 384-well and 1536-well ultrahigh-throughput screening (uHTS) formats.
• Utilizing cell lysate co-expressing TMPRSS2 and the viral Spike protein.

Main Results:

• A robust TR-FRET assay was established for monitoring the Spike-TMPRSS2 interaction.
• The assay was successfully miniaturized to a 1536-well uHTS format.
• A pilot screen demonstrated the assay's utility for large-scale compound screening.

Conclusions:

• The optimized TR-FRET uHTS assay is a valuable platform for discovering novel small-molecule inhibitors.
• This assay facilitates the identification of therapeutics targeting the initial step of SARS-CoV-2 infection.
• The developed assay supports expanded screening campaigns to combat COVID-19.