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Screening and Characterization of Drugs That Protect Corneal Endothelial Cells Against Unfolded Protein Response and
Eun Chul Kim1, Tetsuya Toyono2, Cynthia A Berlinicke3
1Wilmer Eye Institute, Johns Hopkins Medical Institutions, Baltimore, Maryland, United States 2Department of Ophthalmology & Visual Science, Catholic University of Korea, Seoul, Korea.
Purpose:
To screen for and characterize compounds that protect corneal endothelial cells against unfolded protein response (UPR) and oxidative stress.
Methods:
Bovine corneal endothelial cells (BCECs) were treated for 48 hours with 640 compounds from a Food and Drug Administration (FDA)-approved drug library and then challenged with thapsigargin or H2O2 to induce UPR or oxidative stress, respectively. Cell viability was measured using the CellTiter-Glo survival assay. Selected "hits" were subjected to further dose-response testing, and their ability to modulate expression of UPR and oxidative stress markers was assessed by RT-PCR, Western blot, and measurement of protein carbonyl and 8-hydroxydeoxyguanosine (8-OHdG) adducts in immortalized human corneal endothelial cells (iHCECs).
Results:
Forty-one drugs at 20 μM and 55 drugs at 100 μM increased survival of H2O2-challenged cells, and 8 drugs at 20 μM and 2 drugs at 100 μM increased survival of thapsigargin-challenged cells, compared with untreated control cells. Nicergoline, ergothioneine, nimesulide, oxotremorine, and mefenamic acid increased survival of both H2O2- and thapsigargin-challenged cells. Oxotremorine altered DNA damage inducible 3 (CHOP) gene expression, glucose-regulated protein 78 kDa (GRP78) and activating transcription factor 4 (ATF4) protein expression, and protein carbonyl and 8-OHdG levels. Mefenamic acid altered GRP78 protein expression and protein carbonyl and 8-OHdG levels.
Conclusions:
Oxotremorine and mefenamic acid are potential survival factors for corneal endothelial cells under UPR and oxidative stress. The described assay can be further expanded to screen additional drugs for potential therapeutic effect in corneal endothelial diseases such as Fuchs' endothelial corneal dystrophy.
Insights
This study identified oxotremorine and mefenamic acid as compounds that protect corneal endothelial cells from unfolded protein response and oxidative stress. These findings offer potential therapeutic avenues for corneal diseases.
Area of Science:
- Ophthalmology
- Cell Biology
- Pharmacology
Background:
- Corneal endothelial cells are vital for vision and susceptible to damage from unfolded protein response (UPR) and oxidative stress.
- Diseases like Fuchs' endothelial corneal dystrophy involve the loss of these critical cells.
- Identifying protective compounds is crucial for developing novel therapeutic strategies.
Purpose of the Study:
- To screen a library of Food and Drug Administration (FDA)-approved drugs for compounds that protect corneal endothelial cells.
- To characterize the protective mechanisms of identified compounds against UPR and oxidative stress.
- To evaluate the potential of these compounds for treating corneal endothelial diseases.
Main Methods:
- Screened 640 FDA-approved drugs using bovine corneal endothelial cells (BCECs).
- Induced UPR and oxidative stress using thapsigargin and H2O2, respectively.
- Assessed cell viability, gene expression (CHOP), protein levels (GRP78, ATF4), and oxidative damage markers (protein carbonyl, 8-OHdG).
Main Results:
- Forty-one and 55 drugs enhanced cell survival under oxidative stress at 20 μM and 100 μM, respectively.
- Eight and 2 drugs protected cells against UPR at 20 μM and 100 μM, respectively.
- Oxotremorine and mefenamic acid demonstrated protective effects against both stressors and modulated key UPR and oxidative stress markers.
Conclusions:
- Oxotremorine and mefenamic acid show promise as survival factors for corneal endothelial cells under stress.
- The developed screening assay can be utilized to discover additional therapeutic agents for corneal endothelial diseases.
- These findings may lead to new treatments for conditions like Fuchs' endothelial corneal dystrophy.
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