Journal
INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE
Volume 56, Issue 13, Pages 7739-7753Publisher
ASSOC RESEARCH VISION OPHTHALMOLOGY INC
DOI: 10.1167/iovs.15-17802
Keywords
SLC4A11; corneal dystrophy; drug screening; CHED; Fuchs' endothelial corneal dystrophy
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Funding
- Canadian Institutes of Health Research (Ottawa, ON, Canada)
- Alberta Innovates Health Solutions
- Women's and Children's Health Research Institute (Edmonton, AB, Canada)
- International Research Training Group in Membrane Biology - National Sciences and Engineering Research Council (Ottawa, ON, Canada)
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PURPOSE. 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 mu M. 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.
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