title: Improved fluorescence assays to measure the defects associated with F508del-CFTR allow identification of new active compounds.
abstract: Cystic fibrosis (CF) is a debilitating disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which codes for a Cl-/HCO3- channel. F508del, the most common CF-associated mutation, causes both gating and biogenesis defects in the CFTR protein. This paper describes the optimization of two fluorescence assays, capable of measuring CFTR function and cellular localization, and their use in a pilot drug screen.
  
  
  
  HEK293 cells expressing YFP-F508del-CFTR, in which halide sensitive YFP is tagged to the N-terminal of CFTR, were used to screen a small library of compounds based on the VX-770 scaffold. Cells expressing F508del-CFTR-pHTomato, in which a pH sensor is tagged to the fourth extracellular loop of CFTR, were used to measure CFTR plasma membrane exposure following chronic treatment with the novel potentiators.
  
  
  
  Active compounds with efficacy ~50% of VX-770, micromolar potency, and structurally distinct from VX-770 were identified in the screen. The F508del-CFTR-pHTomato assay suggests that the hit compound MS131A, unlike VX-770, does not decrease membrane exposure of F508del-CFTR.
  
  
  
  Most known potentiators have a negative influence on F508del-CFTR biogenesis/stability, which means membrane exposure needs to be monitored early during the development of drugs targeting CFTR. The combined use of the two fluorescence assays described here provides a useful tool for the identification of improved potentiators and correctors. The assays could also prove useful for basic scientific investigations on F508del-CFTR, and other CF-causing mutations.
authors.affiliation: Research Department of Neuroscience, Physiology and Pharmacology, University College London, London, UK.
authors.forename: Emily
authors.identifier: 0000-0001-7183-7383
authors.initials: E
authors.lastname: Langron
authors.affiliation: Discipline of Chemistry, School of Environmental and Life Sciences, Priority Research Centre for Chemical Biology and Clinical Pharmacology, The University of Newcastle, Callaghan, NSW, Australia.
authors.forename: Michela I
authors.initials: MI
authors.lastname: Simone
authors.affiliation: Department of Chemistry and Biochemistry, University of Bern, Bern, Switzerland.
authors.forename: Clémence M S
authors.initials: CM
authors.lastname: Delalande
authors.affiliation: Department of Chemistry and Biochemistry, University of Bern, Bern, Switzerland.
authors.forename: Jean-Louis
authors.initials: JL
authors.lastname: Reymond
authors.affiliation: Wolfson Institute for Biomedical Research, University College London, London, UK.
authors.forename: David L
authors.initials: DL
authors.lastname: Selwood
authors.affiliation: Research Department of Neuroscience, Physiology and Pharmacology, University College London, London, UK.
authors.forename: Paola
authors.identifier: 0000-0002-5422-4662
authors.initials: P
authors.lastname: Vergani
mesh_terms: D000627:Aminophenols / Q000032:analysis* / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology*; D001426:Bacterial Proteins / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000235:genetics / Q000378:metabolism*; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D005453:Fluorescence; D057809:HEK293 Cells; D006801:Humans; D008164:Luminescent Proteins / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D015394:Molecular Structure; D015363:Quinolones / Q000032:analysis* / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology*; D054852:Small Molecule Libraries / Q000032:analysis* / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology*
chemical_list: D000627:Aminophenols; D001426:Bacterial Proteins; D008164:Luminescent Proteins; D015363:Quinolones; D054852:Small Molecule Libraries; C054562:yellow fluorescent protein, Bacteria; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; C545203:ivacaftor