Development And Optimization Of The Htrf Assay For Antagonists Of Retinol-dependent Rbp4-ttr Interaction

Abstract

Purpose: : Excessive accumulation of lipofuscin in the RPE cells is a key feature of the AMD retina. The major cytotoxic component of RPE lipofuscin is a bisretinoid A2E. A2E forms in the retina as a product of a properly functioning visual cycle. Rates of the visual cycle and A2E production depend on influx of retinol from serum to the RPE. Transportation of all-trans retinol from serum to the retina is mediated by complex formation between retinol binding protein (RBP4), transthyretinin (TTR) and retinol. Pharmacological down regulation of serum retinol may represent a treatment strategy for dry AMD. Our goal is to identify potent and selective non-retinoid antagonists of retinol binding to RBP4.

Methods: : We developed the HTRF (Homogeneous Time-Resolved Fluorescence) assay for RBP4 antagonists disrupting the retinol-induced RBP4-TTR interaction. The assay is based on disruption of retinol-dependent interaction between MBP-tagged RBP4 with Eu³⁺-cryptate labeled TTR in the presence of a detector reagent, anti-MBP Ab conjugated to d2. The assay sensitivity and dynamic range were first optimized in the agonist mode in respect to RBP4, TTR and detection reagent concentrations. In order to determine the optimum concentration of all-trans retinol stimulating the RBP4-TTR interaction we performed eight-point retinol titrations. We converted the assay to the antagonist mode by testing fixed concentration of retinol within 1-10 µM range and using the saturating concentration of antagonists (fenretinide and A1120). We used the optimized assay to screen two commercially available collections of pharmacologically active compounds with drug-like properties.

Results: : We identified three structurally unrelated non-retinoid compounds disrupting the retinol-induced RBP4-TTR interaction. Eight-point dose titration established compound potency in the lower micromolar range. Binding assay confirmed compound specificity as retinol antagonists.

Conclusions: : The HTRF assay for RBP4 antagonists disrupting retinol-induced RBP4-TTR interaction represents a sensitive and reliable tool for high throughput identification of potential visual cycle inhibitors capable of reducing accumulation of cytotoxic bisretinoids in the retina.

Keywords: age-related macular degeneration • enzymes/enzyme inhibitors • signal transduction: pharmacology/physiology