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S. Zimov, C. Mandavia, K. Petrukhin; Comparison of Corepressor Peptides in the Development of a High Throughput Screening Assay for Small Molecule Agonists of the Retinal Nuclear Receptor, NR2E3. Invest. Ophthalmol. Vis. Sci. 2008;49(13):1370. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
The orphan nuclear receptor, retina-specific nuclear receptor (NR2E3/RNR/PNR) is expressed exclusively in vertebrate photoreceptors, including humans. NR2E3 is involved in the development and maintenance of adult photoreceptors and mutations in NR2E3 are associated with photoreceptor degeneration. Through the identification of NR2E3 agonists, it may be possible to slow the blinding progress of disease by prolonging photoreceptor survival in patients with age-related macular degeneration. Here we developed a homogenous plate based HTRF® assay that will enable the analysis of compound libraries for potential small molecule NR2E3 agonists.
The assay is performed in 96-well microtiter plates in a total volume of 80 µL. We analyzed the molecular proximity of purified NR2E3 and its corepressors, NCOR and Ret-CoR . NR2E3-GST fusion protein and biotinylated NCOR, and two Ret-CoR peptides (Ret-CoR1 and Ret-CoR2) were used in concert with HTRF® detection agents: anti-GST-XL665 and Streptavidin-Europium Cryptate. FRET from the donor (Europium Cryptate) to the acceptor (XL665) was measured by computing the ratio: (emission at 665/ emission at 620) * 10,000. A dual-monochromator, SpectraMax M5e, multi-detection microplate reader was used to read time-resolved fluorescence.
Optimal concentrations for NR2E3 fusion protein and peptides were determined by matrix analysis. The concentration of NR2E3-GST fusion protein varied from 0 - 500 nM and the concentration of the NCOR, Ret-CoR1 and Ret-CoR2 peptides were varied from 0 - 1500 nM. NR2E3 and Ret-CoR1 yielded the highest ratio. Using optimal concentrations of NR2E3 and Ret-CoR1 resulted in a ratio that was consistently 4 times greater than detection agent control. Non-fusion GST protein yielded a ratio half that of NR2E3-GST fusion protein. Upon addition of agonist, we expect disruption of FRET by agonist-induced release of corepressor from the NR2E3-corepressor complex.
We developed an assay in a format compatible with high-throughput screening of large chemical libraries. The strategy employed in this study should allow the screening of a compound collection at one of the NIH MLSCN screening centers and potentially identify original agents for the treatment of macular degeneration.
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