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Yuanyuan Chen, Krzysztof Palczewski, Xujie Liu; Small molecules selectively reduce the misfolded rhodopsin that causes retinitis pigmentosa. Invest. Ophthalmol. Vis. Sci. 2019;60(9):43.
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Rhodopsin mutations cause autosomal dominant retinitis pigmentosa (adRP). Many of these mutations lead to rhodopsin misfolding, which in turn result in rod death. This study is to discover novel small molecules which selectively clear the misfolded rhodopsin to alleviate rod stress and protect vision for rhodopsin associated adRP.
Using a luciferase reporter assay quantifying the amount of the misfolded P23H rhodopsin, we performed a cell-based high-throughput screening testing a total of 69,989 small molecules. The hit compounds were then counter screened using Hek293 cells expressing the WT rhodopsin that is conjugated with a luciferase reporter. The lead compounds with selective activities reducing only the P23H rhodopsin vs. the WT rhodopsin are further confirmed by immunostaining and immunoblots. Using a non-radioactive labeled pulse-chase assay, we followed the degradation of the nascent P23H rhodopsin mutant with or without treatment of the lead compounds to test if these compounds reduce the misfolded rhodopsin via accelerating misfolded protein degradation or reducing protein biosynthesis.
We identified a total of nine small molecule compounds which selectively clear the P23H rhodopsin at micromolar or submicromolar potencies. Among these molecules, five are documented with known pharmacological activities, and the rest four are not known with any activities. The pulse-chase assay showed that co-treated with an inhibitor of protein synthesis, cycloheximide and a lead compound that is known to interfere DNA and amino acid synthesis, the degradation of the nascent P23H rhodopsin does not change, compared to cycloheximide treatment alone, suggesting the selective clearance of P23H rhodopsin is not due to accelerated protein degradation, but its inhibition of rhodopsin biosynthesis.
We discovered a group of novel small molecules which selectively clear the P23H rhodopsin mutant without affecting the WT rhodopsin in vitro. Our discovery of a lead compound that reduces protein synthesis reveals a novel strategy to clear out the misfolded rhodopsin. A slower rate of rhodopsin production reduces the load of the protein quality control system in the cells that in turn leads to a more efficient degradation of the misfolded rhodopsin. This novel mechanism of action suggests that this compound can be used to clear other misfolded proteins.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
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