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Publication Detail
High-content assay for CFTR monitoring: simultaneous quantification of channel function and biogenesis
  • Publication Type:
    Working discussion paper
  • Authors:
    Prins S, Langron E, Hastings C, Hill E, Stefan A, Griffin L, Vergani P
  • Publication date:
  • Status:
Abstract 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. Images of live HEK293 cells co-expressing a soluble and a CFTR-tagged fluorescent protein are 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. 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-mutation panel also correlate well with published results. Mapping the potentiation profile on CFTR structures raises mechanistic hypotheses on drug action, suggesting that VX-770 might allow an open-channel conformation with an alternative arrangement of domain interfaces around site 1. The assay is a powerful tool for investigation of CFTR ion channel biophysics, allowing rapid and accurate inferences on gating/permeation properties and on how these are affected by mutations and compounds. By providing a two-dimensional molecular characterization of mutant CFTR proteins, our assay can better inform development of therapies tailored for individual CFTR variants. Finally, the integrated assay boosts the potential for discovery of dual-acting compounds, simultaneously repairing both biogenesis and function. Summary CFTR mutations cause cystic fibrosis by affecting how many channels reach the membrane, and/or how well they work. JGP study presents an assay that simultaneously measures CFTR biogenesis and function. A screen of 62 disease-causing mutations provides clues on how approved drug VX-770 works. Graphical Summary
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