Biochemistry/Molecular Biology - ARVO

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Biochemistry/Molecular Biologywas found in the membrane fraction, and was not co-localized withvisual pigment. The activity was extracted by a detergent.Conclusions: Substrate specificity for oxidation of alcohol was high,but that for reduction of aldehyde was low. Presumably, the bindingsite for alcohol is specific only for 11-cis and 9-cis form of retinol,but the site for aldehyde accepts a wide range of hydrophobicaldehydes. This AL-OL coupling activity seems to be localized in theCIS membrane. These findings suggest that in the AL-OL couplingreaction, not only all-trans retinal produced by visual pigment bleachbut also other hydrophobic aldehydes produced in the CIS could bethe substrate to oxidize 11-cis retinol.Commercial Relationships: Satoru Kawamura, None; ShinyaSato, None; Shuji Tachibanaki, None; Takashi Fukagawa, NoneSupport: JSPS KAKENHI Grant Number 23227002Program Number: 1702Presentation Time: 11:45 AM - 12:00 PMTargeting the STRA6/RBP4 Binding interaction to TreatMacular DegenerationKrysten M. Farjo, Gennadiy P. Moiseyev, Jian-Xing Ma. Physiology,Univ of Oklahoma Hlth Sciences, Oklahoma City, OK.Purpose: Macular degeneration is initiated and perpetuated by theaccumulation of toxic Vitamin A derivatives in the retinal pigmentepithelium (RPE). Pharmacological inhibition of Vitamin A deliveryor metabolism in the RPE can significantly slow and reduce visionloss in animal models of macular degeneration. Serum retinol bindingprotein (RBP4) transports Vitamin A, and delivers Vitamin A to RPEthrough binding to its receptor, STRA6. We sought to develop andcharacterize novel inhibitory peptides that target the STRA6/RBP4interaction to reduce Vitamin A delivery to RPE.Methods: We synthesized candidate inhibitory peptides based on theprotein domains of STRA6 that are implicated in binding to RBP4(including C43, C194-Q196, Y335-G341, and R461-N462). HEK-293 cells were used to generate a STRA6-stable cell line, whichserved as the basis for the development of a high throughputSTRA6/RBP4 competition binding assay in which alkalinephosphatase-tagged RBP4 (AP-RBP4) is incubated with STRA6-stable cells alone or in combination with increasing concentrations ofcandidate inhibitory peptides. Quantification of AP activity isproportional to the amount of AP-RBP4 bound to STRA6-stablecells. In addition, we have established a cellular Vitamin A uptakeassay to evaluate the efficacy of inhibitory peptides to reduceSTRA6-mediated uptake of Vitamin A in STRA6-stable cells.Results: We have identified four inhibitory peptides that significantlyreduce the STRA6/RBP4 binding interaction and/or inhibit STRA6-mediated Vitamin A uptake.Conclusions: Peptides derived from the STRA6 protein domainsimplicated in binding to RBP4 have inhibitory activity tosignificantly reduce STRA6/RBP4 binding and STRA6-mediatedVitamin A uptake in cell culture. Ongoing studies are evaluating theability of these peptides to inhibit Vitamin A uptake and visual cycleactivity in mouse eyecups. These inhibitory peptides could serve asthe basis for the development of a novel therapeutic to treat maculardegeneration.Commercial Relationships: Krysten M. Farjo, None; Gennadiy P.Moiseyev, Charlesson LLC (E); Jian-Xing Ma, NoneSupport: AHAF M2012057, OCAST HR10-150, FFS-GIA-10-015Program Number: 1703Presentation Time: 12:00 PM - 12:15 PMEvaluation of different classes of RBP4 antagonists as potentialtreatments for AMDNicoleta Dobri 1 , Qiong Qin 1 , Jian Kong 1 , Rando Allikmets 1, 2 , JanetR. Sparrow 1, 2 , Konstantin Petrukhin 1 . 1 OPHTHALMOLOGY,COLUMBIA UNIVERSITY, NEW YORK, NY; 2 PATHOLOGY,COLUMBIA UNIVERSITY, NEW YORK, NY.Purpose: Pharmacological inhibition of the retinol-inducedinteraction of Retinol-Binding Protein 4 (RBP4) with transthyretin(TTR) in the serum may decrease the uptake of serum retinol to theretina and reduce formation of lipofuscin bisretinoids. A1120 is nonretinoidRBP4 ligand capable of antagonizing retinol-induced RBP4-TTR interaction. Here we present characterization of A1120 andother classes of RBP4 agonists in a battery of in vivo and in vitroassays.Methods: RBP4 binding potency, ability to antagonize RBP4-TTRinteraction and compound specificity were compared for therepresentatives of different classes of RBP4 antagonists. Specificityof compounds was confirmed in in vitro assays probing thecompound effect on the activity of protein targets capable of bindingdifferent types of retinoids. The in vivo effect of compoundadministration on levels of serum RBP4, visual cycle retinoids,lipofuscin bisretinoids, and retinal visual function was evaluatedusing a combination of biochemical and electrophysiologicaltechniquesResults: We documented significant reduction of serum RBP4 inresponse to administration of RBP4 antagonists representing differentstructural classes. Compound administration induced partial depletionof visual cycle retinoids and significant inhibition of bisretinoidaccumulation in the mouse model of enhanced lipofuscinogenesiswhile no significant changes in kinetics of dark adaptation after thephotobleach were evident following long-term compound dosing.Conclusions: Medicinal chemistry optimization in selected structuralseries may yield drug candidates with optimized potency andimproved pharmacokinetic characteristics which could allow theiruse in treatment of dry AMD, Stargardt disease and other conditionscharacterized by excessive lipofuscin accumulation.Commercial Relationships: Nicoleta Dobri, None; Qiong Qin,None; Jian Kong, None; Rando Allikmets, None; Janet R.Sparrow, None; Konstantin Petrukhin, NoneSupport: NIH Grants R21 NS067594 (KP), U01 NS074476 (KP),R24 EY019861 (RA, JRS, KP), R01 EY012951 (JRS), P30EY019007 (Core Support for Vision Research), and unrestrictedfunds from Research to Prevent Blindness (New York, NY) to theDepartment of Ophthalmology, Columbia University, and gifts fromThe Burch Family Foundation, the Mary Jaharis-John CatsimatidisScholarship Fund, the Kaplen Foundation, and the Eye Surgery FundProgram Number: 1704Presentation Time: 12:15 PM - 12:30 PMBestrophinopathy Large Animal Model Shows AbnormalAccumulation of Lipofuscin in the Retinal Pigment EpitheliumNestor Mas Gomez 1 , Emily V. Dutrow 1 , Simone Iwabe 1 , Frank P.Stefano 2 , Kathleen Boesze-Battaglia 2 , Gustavo D. Aguirre 1 , KarinaE. Guziewicz 1 . 1 Department of Clinical Studies, University ofPennsylvania, Philadelphia, PA; 2 Department of Biochemistry,University of Pennsylvania, Philadelphia, PA.Purpose: BEST1 gene encodes for bestrophin-1, an integralmembrane protein localized basolaterally in the RPE. BEST1mutations are responsible for a group of inherited retinal disordersknown as bestrophinopathies. A major pathological hallmark of thesediseases is lipofuscin overload within the RPE cell monolayer. Themain lipofuscin component, fluorophore A2E, has been shown tocause accumulation of free and esterified cholesterol in RPE cells.The aim of this study was to investigate whether canine multifocalretinopathy 1 (cmr1), a large animal model for human©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. 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