High Throughput Assay Identifies Glafenine as a Corrector for the Folding Defect in Corneal Dystrophy–Causing Mutants of SLC4A11

Abstract

Citation: Chiu AM, Mandziuk JJ, Loganathan SK, Alka K, Casey JR. High throughput assay identifies glafenine as a corrector for the folding defect in corneal dystrophy-causing mutants of SLC4A11. Invest Ophthalmol Vis Sci. 2015;56:7739-7753. DOI:10.1167/ iovs.15-17802 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 EC 50 of 1.5 6 0.7 lM.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.

Ophthalmic Nonsteroidal Anti-Inflammatory Drugs as a Therapy for Corneal Dystrophies Caused by SLC4A11 Mutation

“…pAMC1 encoding N-terminally HA-tagged human SLC4A11 in pcDNA3.1 20 or wild-type (WT) Luc-SLC4A11 with N-terminal luciferase tag 12 were used as template for mutagenesis experiments to create point mutants.…”

“…SLC4A11 variants carried a double HA tag insertion in extracellular loop 3, following amino acid 564, and have been previously reported as normal in functional activity and cell surface localization. 20 One hour post transfection, cells were treated with 500 lL of drug to a final concentration as indicated in the Table in 0.2% DMSO (vol/ vol), or DMSO alone as control. Forty-eight hours later, cells were washed in PBS, fixed with 4% paraformaldehyde in PBS for 10 minutes, followed by a quenching step with 100 mM glycine in PBS for 5 minutes at 208C.…”

“…cDNA for SLC4A11 variants encoded a shortened version of splicing variant 2 of human SLC4A11 (891 amino acid, NCBI reference: NG_017072.1) with an N-terminal HA tag, as previously described previously. 20 One hour post transfection, cells were treated with the final concentration of drug in 0.2% DMSO (Table), and control cells received DMSO to a final concentration of 0.2% DMSO (vol/vol). Forty-eight hours later, coverslips were mounted in a 35-mm diameter Attofluor Cell Chamber (Molecular Probes, Ottawa, ON, Canada) and washed with PBS.…”

“…The tag was inserted into extracellular loop 3 of SLC4A11, following P564, and does not affect cell surface trafficking or water flux function of SLC4A11. 20 Representative CHED (E143K) and FECD (G709E) mutants were tested in this assay. Cells were treated with indicated ophthalmic NSAIDs in 0.2% DMSO (Table).…”

“…7,10,[14][15][16][17][18][19] Most SLC4A11 mutations lead to biosynthetic defects marked by accumulation in the ER, and protein degradation before reaching the plasma membrane. 12 A small-scale drug screen showed that Glafenine, a disused NSAID, was able to move some SLC4A11 mutants to the cell surface, 20 suggesting that other NSAIDs might also have therapeutic potential. We thus set out to screen NSAIDs available as eye drops for their ability to correct the cell-surfacing trafficking of SLC4A11 proteins, using a bioluminescence resonance energy transfer (BRET) assay that enabled rapid analysis of SLC4A11 cell surface abundance.…”