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Y. Han, T.V. Bui, N.L. Mata; A Novel Screening Method for Compounds Which Reduce Serum Retinol and Arrest the Accumulation of Lipofuscin Fluorophores in the RPE . Invest. Ophthalmol. Vis. Sci. 2006;47(13):2020.
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© ARVO (1962-2015); The Authors (2016-present)
Dietary retinol is delivered to extra–hepatic target tissues as a complex with retinol binding protein (RBP) and transthyretin (TTR). Binding of TTR with RBP–retinol is necessary to maintain high steady–state concentrations of retinol in serum. N–(4–hydroxyphenyl)retinamide (HPR) binds competitively to RBP and prevents TTR binding leading to reduced levels of retinol in serum. Importantly, chronic HPR treatment reduced ocular retinoid concentrations and arrested the accumulation of retinal fluorophores in ABCA4–/– mice. The success of this approach has provided the impetus to develop a high throughput assay to screen for non–retinoid chemical entities which achieve a similar therapeutic effect.
The developed 384–well plate assay relies on the principles of fluorescence resonance energy transfer (FRET). Briefly, upon binding of RBP–retinol to TTR, the emission from excited RBP–retinol (donor fluorophore) is transferred to probe–labeled TTR (acceptor fluorophore). The emission intensity of the acceptor fluorophore is then used to determine the extent of RBP–retinol–TTR binding. Compounds which bind either RBP or TTR may disrupt RBP–retinol–TTR interaction and reduce the emission intensity of the acceptor fluorophore. Several retinoids were used as test compounds to validate this FRET assay, including HPR, all–trans retinoic acid and all–trans retinal.
Control assays containing only RBP–retinol and TTR demonstrated the remarkable sensitivity of this assay to detect complexation of RBP–retinol to TTR at physiological concentrations (0.5 – 2 µM). The presence of HPR (0.1 µM – 8 µM) reduced the fluorescence of both donor and acceptor fluorophores in a dose–dependent manner. These findings were consistent with data obtained from analyses of the effects of HPR on RBP–retinol–TTR binding using other less sensitive, more costly and more time consuming techniques (e.g., gel–filtration chromatography, tryptophan quenching and retinol anisotropy).
The sensitivity, versatility and quantitative nature of this assay make it a powerful tool to screen for modulators which disrupt formation of the RBP–retinol–TTR complex. The assay is currently being used to screen large chemical libraries for non–retinoid chemical entities which may ultimately be utilized as therapies for retinal degenerations characterized by excessive lipofuscin accumulation.
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