July 4th 2024

Published in Antiviral Research: A new fully automated dual-reporter HTS antiviral assay to facilitate drug discovery against SARS-CoV-2

Published in Antiviral Research: A new fully automated dual-reporter HTS antiviral assay to facilitate drug discovery against SARS-CoV-2.

CARE partner KU Leuven developed a dual-reporter high-throughput screening (HTS) assay to facilitate antiviral drug discovery against SARS-CoV-2 using a fully automated, high-containment robot system. 

For this purpose, by clonal selection, KU Leuven successfully obtained an A549 cell clone that expresses a strong fluorescent mCherry signal in the nucleus (for cytotoxicity evaluation) and that efficiently supports replication of a SARS-CoV-2 reporter virus that expresses the NeonGreen fluorescent reporter protein after infection (for antiviral potential), and this without being affected by virus-induced cytopathogenic effects. This dual-reporter system makes any staining steps redundant, and thus offers a convenient and robust strategy for phenotypic HTS. KU Leuven chose the A549 cell line, overexpressing the SARS-CoV-2 angiotensin converting enzyme 2 (ACE2) receptor and the spike priming protease transmembrane serine protease 2 (TMPRSS2) to develop this assay as this is a human airway epithelial cell line, a more relevant model than the Vero6 cell line, which is a monkey kidney-derived cell, often used for HTS campaigns against SARS-CoV-2.  

Image acquisition and analysis processes were automated using the Caps-It research infrastructure, a fully automated robotic system enclosed in an isolator, which facilitates HTS on high biosafety organisms, as developed by KU Leuven. An in-house algorithm was created for cell counting, segmentation, and validation for calculation of the percentage of infected cells. 

Two reference compounds with known antiviral activity against SARS-CoV-2, GS-441524 and PF-00835231, were tested in this assay and both resulted in antiviral activity against SARS-CoV-2 with similar EC50 and CC50 values reported in other publications, thus validating this novel assay.  

This newly developed high-content imaging-based HTS assay, which is automated, scalable, and robust, is suitable for screening of large compound libraries.   

To learn more, click here: Development of a robust and convenient dual-reporter high-throughput screening assay for SARS-CoV-2 antiviral drug discovery