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John A Fuller, Karl Wahlin, Cynthia Berlinicke, Douglas Yasumura, Michael T Matthes, Ryan MacArthur, Patricia Dranchak, Matthew M LaVail, James Inglese, Donald J Zack; Quantitative High Throughput Screening for Small Molecules That Promote Photoreceptor Differentiation and Survival. Invest. Ophthalmol. Vis. Sci. 2014;55(13):1911. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Retinal degenerations are a heterogeneous group of diseases in which there is slow but progressive loss of photoreceptors. There are currently no approved therapies for treating retinal degenerations. In an effort to identify novel small molecules that are 1) neuroprotective and 2) promote photoreceptor differentiation, we have developed microscale (1536-well) high throughput assays using primary retinal neurons.
Primary cells from rhodopsin-GFP knock-in, QRX-EGFP transgenic, and C57BL/6 wild-type mice were dissociated, seeded into 1536-well plates, and treated with small molecule libraries in a concentration-dependent fashion. GFP positive cell generation is assessed by fixing cells after 7-14 days in culture and imaging with a microplate-based laser cytometer. Positive wells are verified with an automated microscopy platform. Cell viability of CD73 immunopanned photoreceptors is assayed following 96h in culture. Concentration-response curves are then generated to pharmacologically profile each small molecule. Putative actives from the primary screen are then verified by immunostaining and qRT-PCR analysis of an array of photoreceptor-associated genes.
We have developed an assay with a signal-to-background ratio of 3.2 and a coefficient of variation <20%. As an example of the potential of this screening paradigm, we have identified a small molecule that conferred a 4-fold increase in the number of differentiated rhodopsin-EGFP cells, and a 100% increase in the expression of QRX-GFP. Gene expression analysis of photoreceptor-associated genes showed a significant increase in expression of rhodopsin, NR2E3, NRL, Gnat1, and Pdc.
We have developed a primary cell-based high throughput screen to identify small molecules that influence photoreceptor development and survival. Our preliminary screening results demonstrate that our approach can successfully identify photoreceptor differentiation and survival promoting molecules, and ongoing screening will likely identify additional and more potent such molecules. It is hoped that this work will identify potential preclinical candidates, as well as molecular probes that will be useful for analysis of the molecular mechanisms that mediate photoreceptor differentiation and survival.
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