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Qing Chang, Siquan Chen, Bogaard Joseph, Dingcai Cao, Michael Grassi; Cytochrome P450 2C9 Is a Target for Photoreceptor Neuroprotection. Invest. Ophthalmol. Vis. Sci. 2013;54(15):1268.
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Age-related macular degeneration (AMD) represents the most common cause of irreversible vision loss in the elderly. Currently, there is no available treatment for the atrophic form of this condition. Visual impairment is caused by photoreceptor death which represents a major histopathological endpoint of AMD. Light is known to play a precipitating role in the injury of photoreceptors and has long been used as a disease relevant model to study photoreceptor death. We sought to identify novel neuroprotective mechanisms for photoreceptors in a chemical screen of a cellular phototoxicity model.
A cell-based high throughput screen of the FDA approved Prestwick library of 1200 small molecules was conducted using murine-derived cone photoreceptors (661W cells) cultured with 9-cis retinal and test compounds on 384-well plates. 661W cells were then exposed to 11,000 lux of bright white light for four hours. Informatic analysis of top hit compounds (>2 SD) was performed. Confirmatory validation studies were conducted. Apoptosis, necrosis, reactive oxygen species, mitochondrial stress and intracellular calcium were detected using fluorescent affinity labeling. Expression was determined by western blot. Gene targeted knockdown was performed with siRNA.
Sulfaphenazole, a selective inhibitor of cytochrome P450 (CYP) 2C9, was identified as the top hit (>2.5 SD) of the chemical screen. Sulfaphenazole inhibited both necrosis and mitochondria-initiated, caspase-mediated apoptosis. It prevented superoxide anion generation and blocked light-activated calcium influx. Expression of CYP2C9 was inducible by light and unaffected by sulfaphenazole. Targeted RNAi directed knockdown of CYP2C9 improved cell viability after light exposure. Treatment of 661W cells with CYP2C9-derived synthetic cis-epoxyeicosatrienoic acids potentiated the canonical TRPC and L-type calcium channels.
Sulfaphenazole, a selective inhibitor of cytochrome P450 2C9, protects photoreceptors from photochemical stress-mediated cell death. CYP2C9 plays a direct and causal role in the necrotic and apoptotic death of photoreceptors. CYP2C9-derived cis-epoxyeicosatrienoic acids promote extracellular calcium influx. This study establishes CYP2C9 of the cytochrome P450 mono-oxygenase system as a target for photoreceptor neuroprotection in AMD and other retinal degenerative diseases.
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