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Nicoleta Dobri, Xiaoming Xu, Konstantin Petrukhin; Identification of a New Class of Non-Retinoid RBP4 Antagonists. Invest. Ophthalmol. Vis. Sci. 2012;53(14):3349.
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Visual cycle inhibitors may reduce the formation of toxic bisretinoids and prolong RPE and photoreceptor survival in dry AMD. Rates of the visual cycle and A2E production depend on the influx of all-trans retinol from serum to the RPE. Formation of the tertiary retinol-binding protein 4 (RBP4)-transthyretin (TTR)-retinol complex in serum is required for retinol uptake from circulation to the RPE. Retinol-binding site on RBP4 is sterically proximal to the interface mediating the RBP4-TTR interaction. RBP4 antagonists that compete with serum retinol for binding to RBP4 while blocking the RBP4-TTR interaction would reduce serum retinol, slow down the visual cycle, and inhibit formation of cytotoxic bisretinoids. The purpose of this study is to identify new classes of RBP4 antagonists.
We developed and optimized the TR-FRET assay for compounds antagonizing retinol-dependent RBP4-TTR interaction using purified MBP-tagged RBP4 and apo-TTR directly labeled with Eu3+-cryptate. We used the assay to screen several commercial libraries of compounds with drug-like properties. Positive compounds were further evaluated using the developed competition binding assays which unutilized the scintillation proximity (SPA) format for probing the displacement of tritiated retinol from MBP-RPB4. Medicinal chemistry optimization of one positive compound was attempted in order to establish the SAR, structure-activity relationship, in this structural series.
Using the TR-FRET assay for the library screening, we identified a farnesoid derivative, a non-retinoid compound, as a low micromolar inhibitor of the retinol-dependent RBP4-TTR interaction. Using the competition binding assay, we established the compound’s ability to directly compete with all-trans retinol for binding to RBP4. We used the two assays to perform characterization of 9 newly synthesized compounds along with 12 commercially available structures in order to establish SAR in this structural series.
We identified a new class of non-retinoid RBP4 antagonists that may potentially be used in dry AMD treatment. Conducted experiments defined the path for further SAR optimization in this structural series.
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