title: Simple image-based no-wash method for quantitative detection of surface expressed CFTR.
abstract: Cystic fibrosis (CF) is the most common lethal genetic disease among Caucasians. It is caused by mutations in the CF Transmembrane Conductance Regulator (CFTR) gene, which encodes an apical membrane anion channel that is required for regulating the volume and composition of epithelial secretions. The most common CFTR mutation, present on at least one allele in >90% of CF patients, deletes phenylalanine at position 508 (F508del), which causes the protein to misfold. Endoplasmic reticulum (ER) quality control elicits the degradation of mutant CFTR, compromising its trafficking to the epithelial cell apical membrane. The absence of functional CFTR leads to depletion of airway surface liquid, impaired clearance of mucus and bacteria from the lung, and predisposes to recurrent infections. Ultimately, respiratory failure results from inflammation and bronchiectasis. Although high throughput screening has identified small molecules that can restore the anion transport function of F508del CFTR, they correct less than 15% of WT CFTR activity, yielding insufficient clinical benefit. To date, most primary CF drug discovery assays have employed measurements of CFTR's anion transport function, a method that depends on the recruitment of a functional CFTR to the cell surface, involves multiple wash steps, and relies on a signal that saturates rapidly. Screening efforts have also included assays for detection of extracellularly HA-tagged or HRP-tagged CFTR, which require multiple washing steps. We have recently developed tools and cell lines that report the correction of mutant CFTR trafficking by currently available small molecules, and have extended this assay to the 96-well format. This new and simple no-wash assay of F508del CFTR at the cell surface may permit the discovery of more efficacious drugs, and hopefully thereby prevent the catastrophic effects of this disease. In addition, the modular design of this platform should make it useful for other diseases where loss-of-function results from folding and/or trafficking defects in membrane proteins.
authors.affiliation: Department of Cell Biology and Physiology, Center for Biologic Imaging, University of Pittsburgh School of Medicine, 3500 Terrace Street, S233 BST, Pittsburgh, PA 15261, USA. Electronic address: mbl6@pitt.edu.
authors.forename: Mads Breum
authors.initials: MB
authors.lastname: Larsen
authors.affiliation: Department of Cell Biology and Physiology, Center for Biologic Imaging, University of Pittsburgh School of Medicine, 3500 Terrace Street, S233 BST, Pittsburgh, PA 15261, USA. Electronic address: hu.jennifer@medstudent.pitt.edu.
authors.forename: Jennifer
authors.initials: J
authors.lastname: Hu
authors.affiliation: Department of Cell Biology and Physiology, University of Pittsburgh School of Medicine, 7116 Rangos Research Center, 4401 Penn Avenue, Pittsburgh, PA 15224, USA. Electronic address: frizzell@pitt.edu.
authors.forename: Raymond A
authors.initials: RA
authors.lastname: Frizzell
authors.affiliation: Department of Cell Biology and Physiology, Center for Biologic Imaging, University of Pittsburgh School of Medicine, 3500 Terrace Street, S233 BST, Pittsburgh, PA 15261, USA. Electronic address: swatkins@pitt.edu.
authors.forename: Simon C
authors.initials: SC
authors.lastname: Watkins
mesh_terms: D000631:Aminopyridines / Q000494:pharmacology*; D052117:Benzodioxoles / Q000494:pharmacology*; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000819:agonists* / Q000235:genetics / Q000378:metabolism; D005456:Fluorescent Dyes / Q000737:chemistry; D015870:Gene Expression; D057809:HEK293 Cells; D057166:High-Throughput Screening Assays; D006801:Humans; D057054:Molecular Imaging; D009154:Mutation; D010957:Plasmids / Q000737:chemistry / Q000378:metabolism; D015202:Protein Engineering; D011993:Recombinant Fusion Proteins / Q000235:genetics* / Q000378:metabolism; D012394:Rosaniline Dyes / Q000737:chemistry; D057127:Single-Chain Antibodies / Q000737:chemistry; D054852:Small Molecule Libraries / Q000494:pharmacology*; D014162:Transfection
chemical_list: D000631:Aminopyridines; D052117:Benzodioxoles; C505032:CFTR protein, human; D005456:Fluorescent Dyes; D011993:Recombinant Fusion Proteins; D012394:Rosaniline Dyes; D057127:Single-Chain Antibodies; D054852:Small Molecule Libraries; C005095:malachite green; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; C569105:lumacaftor
keywords: Corrector; F508del CFTR; Fluorogen activating protein tag; High throughput screening; Surface expression