Application of a High-Content Screening Assay Utilizing Primary Human Lung Fibroblasts to Identify Antifibrotic Drugs for Rapid Repurposing in COVID-19 Patients

Abstract

Lung imaging and autopsy reports among COVID-19 patients show elevated lung scarring (fibrosis). Early data from COVID-19 patients as well as previous studies from severe acute respiratory syndrome, Middle East respiratory syndrome, and other respiratory disorders show that the extent of lung fibrosis is associated with a higher mortality, prolonged ventilator dependence, and poorer long-term health prognosis. Current treatments to halt or reverse lung fibrosis are limited; thus, the rapid development of effective antifibrotic therapies is a major global medical need that will continue far beyond the current COVID-19 pandemic. Reproducible fibrosis screening assays with high signal-to-noise ratios and disease-relevant readouts such as extracellular matrix (ECM) deposition (the hallmark of fibrosis) are integral to any antifibrotic therapeutic development. Therefore, we have established an automated high-throughput and high-content primary screening assay measuring transforming growth factor-β (TGFβ)-induced ECM deposition from primary human lung fibroblasts in a 384-well format. This assay combines longitudinal live cell imaging with multiparametric high-content analysis of ECM deposition. Using this assay, we have screened a library of 2743 small molecules representing approved drugs and late-stage clinical candidates. Confirmed hits were subsequently profiled through a suite of secondary lung fibroblast phenotypic screening assays quantifying cell differentiation, proliferation, migration, and apoptosis. In silico target prediction and pathway network analysis were applied to the confirmed hits. We anticipate this suite of assays and data analysis tools will aid the identification of new treatments to mitigate against lung fibrosis associated with COVID-19 and other fibrotic diseases.

A Novel High-Content Phenotypic Screen To Identify Inhibitors of Mitochondrial DNA Maintenance in Trypanosomes

“…All compounds were screened at a final concentration of 10 μM in 0.1% (vol/vol) DMSO in a 384-well format, where the first four columns had alternating positive (EtBr) and negative (DMSO) controls. Additionally, the PKE and Prestwick Chemical libraries were also screened at a lower final concentration of 1 μM, because both libraries have been reported to lead to the identification of potent inhibitors in different phenotypic screening assays at this lower dose, which may better reflect on-target activity rather than the off-target activity observed at higher doses ( 35 , 36 ). The screens were performed in five batches (48 plates in total), with a “robust” Z′ assay performance score ( 33 ) ranging from 0.63 to 0.9 between batches.…”

Advances in High-Content Imaging and Informatics: A Joint Special Collection with Society for Biomolecular Imaging and Informatics and SLAS

“…With the abundance of COVID-associated lung injuries, the need to identify new drugs and/or different mechanisms contributing to pulmonary fibrosis is great. In this Special Collection of SLAS Discovery , Marwick et al 1 describe their effort to develop a phenotypic-based drug screen using fibrotic readouts from primary human pulmonary fibroblast in a high-content screen. The authors describe a screen of 2743 compounds that yielded hits inhibiting fibrogenesis in a model of pulmonary fibrosis.…”