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Joseph Bogaard, Qing Chang, Evgeny A Berdyshev, Siquan Chen, Bhargava Karumudi, Yueting Wang, Tom G Driver, Sam Bettis, Gregory R. J Thatcher, Michael A Grassi, Retina Cell Biology; Cytochrome P450 2C Inhibitors Protect Photoreceptors from Light Induced Cell Death. Invest. Ophthalmol. Vis. Sci. 2014;55(13):4387.
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© ARVO (1962-2015); The Authors (2016-present)
Photoreceptor cell death is the primary pathologic endpoint in many retinal degenerative diseases and ultimately results in vision loss and blindness in affected individuals. Previously, we conducted a high throughput chemical screen using a cell based phototoxicity assay. Our top hit from the screen was sulfaphenazole, a selective cytochrome P450 2C inhibitor that is a novel target for neuroprotection. In this study we further investigated the neuroprotection of CYP2C inhibitors.
661W cells were seeded onto 96 well plates and given 9-cis retinal overnight to sensitize them to light. Cells were then given serum free media with/without a compound of interest. Then cells were exposed to 11,000 lux of light to inflict cell death. Media fractions and lipid extracts were obtained following light exposure to perform lipid profiling using LC/MS. Controls were media only wells and untreated wells on the plate and an identical plate that was unexposed to light. Liver microsome experiments were performed to evaluate the IC50 value of compounds by mass spectrometry with known CYP2C metabolites.
Over 40 sulfaphenazole analogs were tested using our light induced cell death model and with liver microsomes. Several of the top analogs demonstrated improved protection over sulfaphenazole over multiple doses. Of the compounds tested, more than half had increased activity compared to sulfaphenazole. Our top two hits, KB-2-1 and KB-2-3 demonstrated greater than a 3-fold increase (p < .0001) in viable cells compared to neighboring control wells. Lipid studies revealed that two metabolites matching the mass/transition ion pairs of 8,9- and 14,15-EETs were significantly increased (>2-fold) following exposure to light after normalization to total lipid phosphates. Treatment with also sulfaphenazole increased the quantities of these metabolites. These molecules had slightly different (<0.1 min) retention time compared to the deuterated standards indicating they are not authentic EETs but have the same molecular mass and can create the same transition ions.
Sulfaphenazole analogs continue to validate our original hit as novel therapeutic agent. Downstream lipid analysis investigations have revealed that arachidonic acid metabolites are putative players in the neuroprotection of sulfaphenazole. Testing with sulfaphenazole and our top analogs in animal models of retinal degeneration is currently underway.
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