title: High Throughput Assay Identifies Glafenine as a Corrector for the Folding Defect in Corneal Dystrophy-Causing Mutants of SLC4A11.
abstract: OBJECTIVE
  Protein misfolding, causing retention of nascent protein in the endoplasmic reticulum (ER), is the most common molecular phenotype for disease alleles of membrane proteins. Strategies are needed to identify therapeutics able to correct such folding/trafficking defects. Mutations of SLC4A11, a plasma membrane transport protein of the human corneal endothelial cell layer, cause cases of congenital hereditary endothelial dystrophy, Harboyan syndrome, and Fuchs' endothelial corneal dystrophy. Most SLC4A11 mutations induce SLC4A11 misfolding and retention in the ER.
  
  
  METHODS
  An assay amenable to high-throughput screening was developed to quantify SLC4A11 at the plasma membrane, enabling a search for potential traffic-correcting small molecules. The assay was validated by comparing cell surface abundance of SLC4A11 mutants measured in the assay to observations from confocal immunofluorescence and values from cell surface biotinylation. Functionality of mutant proteins was assessed, using a confocal microscopic green fluorescent protein (GFP) water flux assay where relative rates of cell swelling are compared.
  
  
  RESULTS
  A small-scale screen revealed that the nonsteroidal anti-inflammatory drugs (NSAIDs), glafenine, ibuprofen, and acetylsalicylic acid dissolved in 0.2% dimethyl sulfoxide (DMSO), partially rescued the trafficking defect in some SLC4A11 mutants, expressed in HEK293 cells. These SLC4A11 mutants retained functional activity when rescued to the plasma membrane by glafenine treatment. Glafenine was effective with an EC50 of 1.5 ± 0.7 μM.
  
  
  CONCLUSIONS
  These data suggest that glafenine, and perhaps other NSAIDs, hold potential as therapeutics for misfolded membrane proteins, like SLC4A11. The high throughput approach described here can be modified to identify correctors of other misfolded plasma membrane proteins that cause eye disease.
authors.affiliation: Department of Physiology, University of Alberta, Edmonton, Alberta, Canada.
authors.forename: Anthony M
authors.initials: AM
authors.lastname: Chiu
authors.affiliation: Department of Physiology, University of Alberta, Edmonton, Alberta, Canada 2Department of Biochemistry, University of Alberta, Edmonton, Alberta, Canada.
authors.forename: Jake J
authors.initials: JJ
authors.lastname: Mandziuk
authors.affiliation: Department of Physiology, University of Alberta, Edmonton, Alberta, Canada 2Department of Biochemistry, University of Alberta, Edmonton, Alberta, Canada.
authors.forename: Sampath K
authors.initials: SK
authors.lastname: Loganathan
authors.affiliation: Department of Physiology, University of Alberta, Edmonton, Alberta, Canada 2Department of Biochemistry, University of Alberta, Edmonton, Alberta, Canada.
authors.forename: Kumari
authors.initials: K
authors.lastname: Alka
authors.affiliation: Department of Physiology, University of Alberta, Edmonton, Alberta, Canada 2Department of Biochemistry, University of Alberta, Edmonton, Alberta, Canada.
authors.forename: Joseph R
authors.initials: JR
authors.lastname: Casey
mesh_terms: D018712:Analgesics, Non-Narcotic / Q000494:pharmacology*; D027321:Anion Transport Proteins / Q000235:genetics / Q000378:metabolism*; D017920:Antiporters / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D003317:Corneal Dystrophies, Hereditary / Q000188:drug therapy / Q000235:genetics / Q000378:metabolism*; D005897:Glafenine / Q000494:pharmacology*; D057809:HEK293 Cells / Q000187:drug effects / Q000378:metabolism; D006319:Hearing Loss, Sensorineural / Q000378:metabolism; D006801:Humans; D020125:Mutation, Missense / Q000187:drug effects*; D017510:Protein Folding / Q000187:drug effects*; D021381:Protein Transport / Q000187:drug effects / Q000235:genetics
chemical_list: D018712:Analgesics, Non-Narcotic; D027321:Anion Transport Proteins; D017920:Antiporters; C425809:SLC4A11 protein, human; D005897:Glafenine