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Gennadiy P Moiseyev, Jian-Xing Ma, Konstantin Petrukhin; Identification and in vitro Characterization of Novel Non-retinoid RPE65 Inhibitors. Invest. Ophthalmol. Vis. Sci. 2014;55(13):3477.
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Accumulation of A2E, a toxic non-degradable bisretinoid, a byproduct of the visual cycle, in the retinal pigment epithelium (RPE) plays a major role in pathogenesis of age-related macular degeneration (AMD) and Stargardt disease (STGD), causing dysfunction and death of RPE cells. Slowing the visual cycle by inhibition of the rate-limiting step catalyzed by RPE65 is a promising strategy to decrease the accumulation of A2E. The aim of this study was the identification of new non-retinoid compounds capable of inhibiting RPE65 and slowing the visual cycle.
Using the available crystallographic model of bovine RPE65 we generated a docking model of its ligand-binding pocket and conducted the in silico screen of 350,000 compounds. Compounds with the highest QFIT score were characterized experimentally. All-trans-[3H]-retinol was used as a substrate to measure the activities of lecithin:retinol acyltransferase (LRAT) and isomerohydrolase in bovine RPE microsomes. The generated retinoids were analyzed by HPLC. To determine the inhibition mode of the compounds chicken RPE65 was used in a liposome isomerohydrolase assay.
A total of 65 compounds with the highest QFIT scores were synthesized for the experimental verification. Four compounds identified in the in silico docking completely inhibited bovine RPE65 when tested at 200 μM. IC50 for these compounds were determined from the concentration-dependent inhibition of RPE65 activity measured in bovine microsomes. IC50 for the most potent inhibitor CU239 was found to be 6 μM. LRAT activity was not affected by these inhibitors at a concentration as high as 200 μM. To analyze the inhibition type, all-trans retinyl palmitate was incorporated in liposomes and used as a substrate for chicken RPE65. The concentration dependence of RPE65 reaction was measured in the absence and presence of CU239. The Lineweaver-Burk graph demonstrated two lines intersected at Y-axis suggesting that CU239 inhibits the isomerohydrolase reaction in a competitive manner.
We identified CU239, a small molecule compound, which potently and selectively inhibited conversion of all-trans-retinyl ester to 11-cis-retinol catalyzed by RPE65 isomerohydrolase. The competitive mode of the inhibition suggests that this inhibitor is likely bound to the RPE65 active site. This inhibitor may be used to slow down the visual cycle and to prevent accumulation of A2E in STGD and AMD.
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