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Mark Anthony Fields, Huey Cai, Jie Gong, laura abriola, Denton Hoyer, Lucian V Del Priore, Scott Noggle, Daniel Paull; High-throughput Screening Identifies Compounds that Protect RPE from Physiological Stressors Present in AMD. Invest. Ophthalmol. Vis. Sci. 2019;60(9):2990.
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Dysfunction and eventual loss of retinal pigment epithelial (RPE) cells is a hallmark of age-related macular degeneration (AMD), and linked to oxidative and nitrosative damage. Here, we use high-throughput screening (HTS) to identify compounds that protect human RPE cells from oxidative damage. We then verify the ability of identified compounds to protect RPE cells (primary adult human RPE and induced pluripotent stem cell-derived (iPSC)-derived RPE cells generated from AMD patients): against (1) oxidative damage, (2) nitrosative damage to the basement membrane, and (3) ultraviolet-B (UV-B) light damage.
Human ARPE-19 cells were seeded in 384 well format, exposed to oxidative damage using tert-butyl hydroperoxide (TBHP), and screened using compound libraries containing 5,065 compounds with known bioactivity, safety, bioavailability, and history of Food and Drug Administration (FDA) approval. Identified compound (s) were tested in models of RPE stress; primary human RPE and iPSC-derived RPE cells were also used to verify activity. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) and a Seahorse XF96 analyzer measured gene expression and mitochondrial function, respectively.
Twelve compounds passed the activity threshold for a hit rate of 0.3%. All 12 compounds were retested in TBHP-treated RPE cells in dose response. Of the 12 compounds tested, 7 were active upon retest. After chemical structure review, compound 33746 and its derivatives were identified as potent inhibitors of oxidative damage to RPE. Compound 33746 increased cell viability in ARPE-19 cells treated with TBHP (compound 33746 with TBHP vs. TBHP-only, 0.874 ± 0.028 vs. 0.521 ± 0.021, p < 0.01) or UV-B light (0.78 ± 0.02 vs. 0.51 ± 0.04, p < 0.01). Treatment with TBHP altered expression of genes related to oxidative stress and apoptosis which was reversed treatment with compound 33746. Compound 33746 improved mitochondrial function in TBHP-exposed ARPE-19 and improved cell survival on damaged basement membrane by (0.802 vs. 0.185, p < 0.001). Compound 33746 protected primary human RPE and iPSC-derived RPE cells from the oxidative stress, UV-B light induced damage, or damaged basement membrane.
This strategy of HTS screening can be used to identify small molecules that protects RPE cells exposed to various stressors associated with disease progression of AMD.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
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