Rectify Pharmaceuticals LLC — Department of Health and Human Services STTR Phase I: NEI

Description

ABCAis an ABC transporter found in retinal photoreceptor cellsMutations in ABCAhave been implicated in retinopathies such as autosomal recessive Stargardt macular degenerationcone rod dystrophyretinitis pigmentosa and age related macular degenerationABCAfunctions as an importerandflippasemoving N retinylidene phosphatidylethanolamineN retinylidene PEfrom the lumen to the cytoplasmic leaflet of disc membranesPrevious studies indicate that mutations in ABCAprofoundly influence this flippase activityresulting in a buildup of toxic retinoid substrates in the lumenA potential approach for treatment of Stargardt Disease is to correct these functional defects with small molecule transport modulatorsThis approach is analogous to the use of small moleculepotentiatorssuch as KalydecoVertex Pharmaceuticalsto successfully correct channel gating defects in the GD CFTR mutant in Cystic FibrosisThe discovery of ABCAdirected therapeutics to treat retinal disease requires development of novel assays to measure restoration of transporter function for ABCAdisease mutantsMoreoverthese assays must be scalable to screen largee gK to andgtMsmall molecule compound librariesOur specific aims are toestablish proof ofconcept for a robust high throughput screeningHTSplatform for ABCAdirected drug discoveryFirsta biochemical platform must be developed for assessment of the functional and structural consequences of common disease variants of ABCAMammalian cell expression protocols will be established for wild type ABCAand a panel of ABCAdisease mutantsPurification and reconstitution of ABCAinto liposomes will then be optimized for functional assay developmentExisting literature lipid transport assays measure active transport of ABCAsubstratessuch as radiolabeled Nretinylidene PEor fluorescent lipidsfrom the membrane inner leaflet to the outer leafletThese assay protocols are unsuitable for high throughput screeningHTSof small molecule librariesas they rely either on a cumbersome centrifugation step to separate donor proteoliposomes from acceptor liposomesor multiple quenching steps and detergent additionwhich would introduce significant error and impose technical constraints in a high throughput settingTo address these limitationswe will use a simple phospholipid tagging strategy to allow separation of donor proteoliposomes from acceptor liposomes in a high throughput formatWe will establish protocols in which biotinylated lipids are incorporated into acceptor liposomesallowing these liposomes to be tethered to streptavidin coated substrates on multi well filter plates commonly used in HTSAfter a wash step to remove the proteoliposomesthe acceptor can then be measured by either scintillation proximity methods with a radiolabeled substrateor total fluorescence for a fluorescent phospholipid substrate Project Narrative This research describes a novel approach for identifying small molecule drugs for treatment of Stargardt Disease and related retinopathiesIf successfulthis strategy may provide a platform for discovery of new therapies for treatment of additional rare diseases caused by functional defects in ABC transporter proteins