High-Throughput Surface Liquid Absorption and Secretion Assays to Identify F508del CFTR Correctors Using Patient Primary Airway Epithelial Cultures

Abstract

High-throughput screening for drug discovery is increasingly utilizing cellular systems of high physiological relevance, such as patient primary cells and organoid cultures. We used 3D-cultured cystic fibrosis patient bronchial epithelial cells to screen for new small-molecule correctors of the disease-causing F508del mutation in CFTR. Impaired mucociliary clearance due to insufficient airway hydration is a hallmark of cystic fibrosis and we used a simple measure of surface liquid levels to quantify F508del CFTR correction in cultured bronchial epithelial cells. Two robust assay formats were configured and used to screen more than 100,000 compounds as mixtures or individual compounds in 96-well format. The corrector discovery success rate, as measured by the number of hits confirmed by an electrophysiology assay on patient primary bronchial epithelial cells, was superior to screens in cell lines expressing recombinant F508del CFTR. Several novel corrector scaffolds were discovered that when combined with the clinical corrector VX-809 delivered combination responses greater than double that of VX-809 alone. This work exemplifies the advantages of a disease-relevant readout and 3D-cultured patient primary cells for the discovery of new drug candidates.

Rescue of common exon skipping mutations in Cystic Fibrosis with modified U1 snRNAs

“…In general, this alternative ASO based approach for rescuing exon skipping defects is based on the identification of appropriate splicing regulatory elements and thus more time consuming and expensive and not easy testable on several splicing defects. Similarly, a pharmacological approach to correct splicing requires high-throughput screening platforms and may lack of specificity (Berg et al, 2019;Giuliano et al, 2018;Liang et al, 2017;Merkert et al, 2019;Pereira et al, 2019). The positive effect we observed here on different CFTR mutations, along with those previously reported for other diseases in several cellular and mouse models (Dario Balestra et al, 2020;Dal Mas, Fortugno, et al, 2015;Donadon et al, 2018;Tajnik et al, 2016), indicates a general applicability and translatability of the ExSpeU1 approach in rescuing exon skipping defects.…”

CFTR Modulators: The Changing Face of Cystic Fibrosis in the Era of Precision Medicine

“…Numerous libraries of compounds have been screened by distinct high-throughput screening (HTS) methods and using several cell models. These experimental approaches have been contributing to the identification of small-molecules from different chemical series (Pedemonte et al, 2005a;Van Goor et al, 2009;Van Goor et al, 2011;Phuan et al, 2014;Phuan et al, 2015;Liang et al, 2017;Giuliano et al, 2018;Van der Plas et al, 2018;Veit et al, 2018;Wang et al, 2018;Berg et al, 2019;De Wilde et al, 2019;Merket et al, 2019) (Figure 6). Notably, as cell background may significantly influence the pharmacological rescue of CFTR mutants (Pedemonte et al, 2010), most promising hits should be further validated in well-differentiated cells in order to identify non-cytotoxic and effective compounds that might result in optimal therapeutic benefits in the clinical scenario.…”

Intestinal organoids for Cystic Fibrosis research

“…This has led to the emergence of other epithelial-derived organoids, like hIPSC-derived pancreas [39] or liver [40] organoids. In addition, ASC-derived airway organoids are of special interest as CF mortality is mainly a result of pulmonary failure and enable the assessment of airway-specific fluid transport, mucus viscosity and patient-specific drug responses [41] in the context of airway cell environment. Culturing of airway-derived cells in matrigel results in the formation of spheroids that allow for CFTR function measurement in a comparable fashion to intestinal organoids, via FIS [42] .…”