title: Protein traffic disorders: an effective high-throughput fluorescence microscopy pipeline for drug discovery.
abstract: Plasma membrane proteins are essential molecules in the cell which mediate interactions with the exterior milieu, thus representing key drug targets for present pharma. Not surprisingly, protein traffic disorders include a large range of diseases sharing the common mechanism of failure in the respective protein to reach the plasma membrane. However, specific therapies for these diseases are remarkably lacking. Herein, we report a robust platform for drug discovery applied to a paradigmatic genetic disorder affecting intracellular trafficking - Cystic Fibrosis. This platform includes (i) two original respiratory epithelial cellular models incorporating an inducible double-tagged traffic reporter; (ii) a plasma membrane protein traffic assay for high-throughput microscopy screening; and (iii) open-source image analysis software to quantify plasma membrane protein traffic. By allowing direct scoring of compounds rescuing the basic traffic defect, this platform enables an effective drug development pipeline, which can be promptly adapted to any traffic disorder-associated protein and leverage therapy development efforts.
authors.affiliation: 1] University of Lisboa, Faculty of Sciences, BioISI - Biosystems &Integrative Sciences Institute, Campo Grande, C8, 1749-016 Lisboa, Portugal [2] Cell Biology and Biophysics Unit, European Molecular Biology Laboratory (EMBL), Meyerhofstraβe 1, 69117 Heidelberg, Germany [3] Advanced Light Microscopy Facility, European Molecular Biology Laboratory (EMBL), Meyerhofstraße 1, 69117 Heidelberg, Germany.
authors.forename: Hugo M
authors.initials: HM
authors.lastname: Botelho
authors.affiliation: 1] University of Lisboa, Faculty of Sciences, BioISI - Biosystems &Integrative Sciences Institute, Campo Grande, C8, 1749-016 Lisboa, Portugal [2] Cell Biology and Biophysics Unit, European Molecular Biology Laboratory (EMBL), Meyerhofstraβe 1, 69117 Heidelberg, Germany [3] Advanced Light Microscopy Facility, European Molecular Biology Laboratory (EMBL), Meyerhofstraße 1, 69117 Heidelberg, Germany.
authors.forename: Inna
authors.initials: I
authors.lastname: Uliyakina
authors.affiliation: University of Lisboa, Faculty of Sciences, BioISI - Biosystems &Integrative Sciences Institute, Campo Grande, C8, 1749-016 Lisboa, Portugal.
authors.forename: Nikhil T
authors.initials: NT
authors.lastname: Awatade
authors.affiliation: University of Lisboa, Faculty of Sciences, BioISI - Biosystems &Integrative Sciences Institute, Campo Grande, C8, 1749-016 Lisboa, Portugal.
authors.forename: Maria C
authors.initials: MC
authors.lastname: Proença
authors.affiliation: 1] Cell Biology and Biophysics Unit, European Molecular Biology Laboratory (EMBL), Meyerhofstraβe 1, 69117 Heidelberg, Germany [2] Advanced Light Microscopy Facility, European Molecular Biology Laboratory (EMBL), Meyerhofstraße 1, 69117 Heidelberg, Germany.
authors.forename: Christian
authors.initials: C
authors.lastname: Tischer
authors.affiliation: Department of Physiology, University of Regensburg, Universitätsstraße 31, 93053 Regensburg, Germany.
authors.forename: Lalida
authors.initials: L
authors.lastname: Sirianant
authors.affiliation: Department of Physiology, University of Regensburg, Universitätsstraße 31, 93053 Regensburg, Germany.
authors.forename: Karl
authors.initials: K
authors.lastname: Kunzelmann
authors.affiliation: 1] Cell Biology and Biophysics Unit, European Molecular Biology Laboratory (EMBL), Meyerhofstraβe 1, 69117 Heidelberg, Germany [2] Advanced Light Microscopy Facility, European Molecular Biology Laboratory (EMBL), Meyerhofstraße 1, 69117 Heidelberg, Germany.
authors.forename: Rainer
authors.initials: R
authors.lastname: Pepperkok
authors.affiliation: University of Lisboa, Faculty of Sciences, BioISI - Biosystems &Integrative Sciences Institute, Campo Grande, C8, 1749-016 Lisboa, Portugal.
authors.forename: Margarida D
authors.initials: MD
authors.lastname: Amaral
mesh_terms: D002460:Cell Line; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000235:genetics / Q000378:metabolism; D055808:Drug Discovery / Q000379:methods*; D015870:Gene Expression; D015723:Gene Library; D017930:Genes, Reporter; D057166:High-Throughput Screening Assays; D006801:Humans; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D008856:Microscopy, Fluorescence; D009154:Mutation; D018408:Patch-Clamp Techniques; D021381:Protein Transport; D034741:RNA, Small Interfering; D054852:Small Molecule Libraries
chemical_list: D008565:Membrane Proteins; D034741:RNA, Small Interfering; D054852:Small Molecule Libraries; D019005:Cystic Fibrosis Transmembrane Conductance Regulator