Abstract
Purpose :
Mutations of the membrane transport protein, SLC4A11, cause genetic endothelial corneal dystrophies: congenital hereditary endothelial corneal dystrophy (CHED), Harboyan syndrome (HS), and Fuchs endothelial corneal dystrophy (FECD). Out of 58 SLC4A11 point mutants, 36 were identified as candidates for folding correction therapy based on their endoplasmic reticulum (ER)-retained phenotype. Glafenine, which is a non-steroidal anti-inflammatory drug, was earlier found to be promising in rescuing cell surface functional activity of ER-retained mutants of SLC4A11. Here, we tested topical FDA- approved ophthalmic NSAIDs for their efficacy in moving ER-retained SLC4A11 mutants to the cell surface to restore functional activity.
Methods :
A bioluminescence resonance energy transfer (BRET)-based assay was established to identify SLC4A11 mutants amenable to folding correction therapy. With this assay FDA-approved ophthalmological non-steroidal anti-inflammatory drugs (NSAIDs) were screened to identify those enabling diseased SLC4A11 to target to the plasma membrane. Confocal immunofluorescence was also used to measure cell-surface localization of mutant SLC4A11. Functional activity of these mutants, upon drug treatment, was measured by monitoring rates of cell swelling in hypo-osmotically challenged transfected HEK293 cells.
Results :
The BRET assay enabled rapid, sensitive and accurate screening of ophthalmological NSAIDs. Ketorolac tromethamine, nepafenac, bromfenac, diclofenac, and flurbiprofen were tested for their ability to correct SLC4A11 ER-retained mutants (E143K and G709E). Nepafenac and diclofenac treatment gave rise to statistically significant increases in cell surface abundance of SLC4A11 mutants (BRET assay), which was confirmed by an increased cell surface abundance measured by confocal immunofluorescence. Ophthalmic NSAID-rescued mutants also retained the water flux function. The ability of nepafenac and diclofenac to rescue ER-retained SLC4A11 point mutants establishes the therapeutic potential of these drugs, when directed to individuals with particular SLC4A11 lesions.
Conclusions :
The BRET assay measured total cell surface SLC4A11 abundance. Nepafenac and diclofenac rescued the folding defect of E143K and G709E SLC4A11. These drugs were tested for their ability to correct the folding of the 36 identified ER-retained mutants of SLC4A11.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.