Increasing the Endoplasmic Reticulum Pool of the F508del Allele of the Cystic Fibrosis Transmembrane Conductance Regulator Leads to Greater Folding Correction by Small Molecule Therapeutics.

W Joon Chung,Jennifer L Goeckeler-Fried,Annette Chiang,Lynn Rasmussen,Steven M Rowe,Jeong S Hong,Eric J Sorscher,Allan M Weissman,Gary A Piazza,Jeffrey L Brodsky,Marina Mazur,E Lucile White,Adam B Keeton,Viktoria Havasi,Zackery E Plyler,R Aldrin Denny,Estelle Cormet-Boyaka

doi:10.1371/journal.pone.0163615

Abstract

Small molecules that correct the folding defects and enhance surface localization of the F508del mutation in the Cystic Fibrosis Transmembrane conductance Regulator (CFTR) comprise an important therapeutic strategy for cystic fibrosis lung disease. However, compounds that rescue the F508del mutant protein to wild type (WT) levels have not been identified. In this report, we consider obstacles to obtaining robust and therapeutically relevant levels of F508del CFTR. For example, markedly diminished steady state amounts of F508del CFTR compared to WT CFTR are present in recombinant bronchial epithelial cell lines, even when much higher levels of mutant transcript are present. In human primary airway cells, the paucity of Band B F508del is even more pronounced, although F508del and WT mRNA concentrations are comparable. Therefore, to augment levels of “repairable” F508del CFTR and identify small molecules that then correct this pool, we developed compound library screening protocols based on automated protein detection. First, cell-based imaging measurements were used to semi-quantitatively estimate distribution of F508del CFTR by high content analysis of two-dimensional images. We evaluated ~2,000 known bioactive compounds from the NIH Roadmap Molecular Libraries Small Molecule Repository in a pilot screen and identified agents that increase the F508del protein pool. Second, we analyzed ~10,000 compounds representing diverse chemical scaffolds for effects on total CFTR expression using a multi-plate fluorescence protocol and describe compounds that promote F508del maturation. Together, our findings demonstrate proof of principle that agents identified in this fashion can augment the level of endoplasmic reticulum (ER) resident “Band B” F508del CFTR suitable for pharmacologic correction. As further evidence in support of this strategy, PYR-41—a compound that inhibits the E1 ubiquitin activating enzyme—was shown to synergistically enhance F508del rescue by C18, a small molecule corrector. Our combined results indicate that increasing the levels of ER-localized CFTR available for repair provides a novel route to correct F508del CFTR.

Highlights

Cystic fibrosis is a genetic disease caused by mutations in the gene encoding CFTR [1]
Our findings demonstrate proof of principle that agents identified in this fashion can augment the level of endoplasmic reticulum (ER) resident “Band B” F508del CFTR suitable for pharmacologic correction
Restoration of F508del CFTR function has been difficult to achieve as monotherapy, hundreds of thousands of discrete molecules have been analyzed for this purpose by unbiased high throughput screening [9, 10]

Summary

Introduction

Cystic fibrosis is a genetic disease caused by mutations in the gene encoding CFTR [1]. Correctors of F508del (e.g., C18 and Vertex-809, or lumacaftor) were identified by high throughput functional assays that monitor CFTRdependent anion transport [8]. Restoration of F508del CFTR function has been difficult to achieve as monotherapy, hundreds of thousands of discrete molecules have been analyzed for this purpose by unbiased high throughput screening [9, 10]. A combination of lumacaftor, which increases the activity of F508del CFTR to ~25% of WT levels in cell culture [8], only increases lung function by ~4% in F508del homozygotes when combined with a potentiator that helps to open the channel [11]

Methods

Results

Conclusion