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Balendu Shekhar Jha, Madhu Lal, Ruchi Sharma, Fang Hua, Marc Ferrer, Kapil Bharti; A High-throughput Screen to Identify Candidate Drugs to Prevent Proteotoxic Stress-Induced Retinal Pigment Epithelium Cell Death. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):4207.
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Retinal pigment epithelium (RPE) degeneration in age-related macular degeneration (AMD) has been linked with accumulation of proteotoxic damage in RPE cells. The goal of this study is to perform high-throughput screening (HTS) assays using pharmacologically active drugs to identify compounds that can mitigate the proteotoxic stress on the RPE cells. These HTS assays can provide candidate drugs for AMD and other RPE-associated degenerative diseases.
RPE cells derived from iPS cells were used to create monolayer phenotype in 384-well plates. To identify compounds that mitigated the effects of proteotoxic stress, an endoplasmic reticulum (ER) stressor was optimized to obtain approximately 50% cell death in 48 hours. A library of pharmacologically active compounds (LOPAC) comprising of 1280 drugs was used at 9 µM and 46 µM to prevent stressor induced cell death. Both the ER stressor and the LOPAC candidates were added to RPE monolayers in 384-well plates at the same time and the plates were incubated for 48 hours. The efficacy of the LOPAC candidates in preventing proteotoxic stress induced cell death was measured using CellTiter-Glo, a luminescence based ATP-release assay for analysis of cell viability.
iPS cell-derived RPE cells form confluent RPE monolayers in 384-well plates. 10 µM of ER stressor leads to 50% RPE cell death within 48 hours. LOPAC candidates at 46 µM are toxic for RPE cells, but at 9 µM several LOPAC compounds prevent ER stressor induced RPE cell death. The primary HTS assay identified 40 LOPAC compounds that were able to prevent proteotoxic stressor induced cell death. Currently, we are testing these 40 compounds in follow-up assays to identify secondary hits, which will be tested in animal models.
The compounds that mitigate the effects of proteotoxic stressors can potentially be therapeutic drugs for RPE-associated degenerative diseases like AMD.
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