PT - JOURNAL ARTICLE AU - Stella Prins AU - Emily Langron AU - Cato Hastings AU - Emily J. Hill AU - Andra C. Stefan AU - Lewis D. Griffin AU - Paola Vergani TI - High-content assay for precision medicine discovery in cystic fibrosis AID - 10.1101/631614 DP - 2019 Jan 01 TA - bioRxiv PG - 631614 4099 - http://biorxiv.org/content/early/2019/08/08/631614.short 4100 - http://biorxiv.org/content/early/2019/08/08/631614.full AB - Background and Purpose Cystic fibrosis (CF) is a life-limiting disease caused by mutations in the human CFTR gene, encoding an anion-selective channel. Because CF-causing mutations affect both CFTR permeation/gating and biogenesis, multi-assay approaches have been implemented in drug development, sequentially screening for channel function and membrane density. Here we present the first assay capable of simultaneous assessment of both CFTR characteristics.Experimental approach Images of live HEK293 cells co-expressing a soluble and a CFTR-tagged fluorescent protein are automatically acquired and analysed to quantify both CFTR membrane density and ion channel function. We monitor F508del-CFTR, the most common disease-causing mutant. Furthermore we characterize a panel of 62 CF-causing mutations and profile effects of acute treatment with approved drug VX-770, mapping potentiation on CFTR structures.Key Results We validate our assay by confirming F508del-CFTR rescue by incubation at low temperature, treatment with CFTR-targeting drugs and introduction of second-site revertant mutation R1070W. Measurements using the rare mutations panel also correlate well with published results.Conclusions and Implications Mapping of VX-770 potentiation of mutants suggests that by increasing flexibility around the gate, the drug allows an alternative protein conformation at domain interfaces around site 1.The assay is a powerful tool for investigation of CFTR ion channel biophysics, allowing more accurate inferences on gating/permeation properties than can be obtained by measuring cellular conductance alone. Finally, by providing a two-dimensional molecular characterization of individual mutant CFTR proteins, our assay can better inform development of single-drug and combination therapies addressing the root cause of CF disease.What is already knownCFTR is an anion-selective channel, normally present in the plasma membrane of epithelial cells.Hundreds of different mutations affect CFTR biogenesis and/or function causing cystic fibrosis (CF).What this study addsWe present and validate an assay that simultaneously measures CFTR biogenesis and function.Profiling a panel of CF-causing mutants suggests hypotheses on how approved drug VX-770 works.Clinical SignificanceThe integrated assay boosts potential for discovery of more effective therapies, simultaneously repairing both defects.ABCATP-binding cassetteCFCystic FibrosisCFTRCystic Fibrosis Transmembrane Conductance RegulatorFYFP membraneaverage normalized YFP fluorescence intensity within the membrane zoneFmCherry cellaverage normalized mCherry fluorescence over the entire cellGCFTRCFTR conductanceGtranstransient anion conductanceIRESinternal ribosome entry siteNBDnucleotide binding domainPDLpoly-D-lysinePOopen probabilityσCFTR membrane densitySSRsum of squared residualsτtranstime constant of the transient anion conductanceVMmembrane potentialWTwild typeYFPyellow fluorescent protein