Epicypher, Inc. — Department of Health and Human Services SBIR Phase I: 300

Description

PROJECT SUMMARYEnzymes that addwritersor removeerasershistone post translational modificationsPTMsare high value drug targets with the potential to treat diverse human pathologiese gcancerneurodegeneration or inflammationModified histone peptides are widely used as in vitro substrates due to their short development cycle and low costHowevernucleosomesNucsrepresent the physiological substrate for chromatin writereraser enzymes and are thus preferable for the development of target specific therapeuticsRecent advances in the commercial manufacturing of PTM containing recombinant Nucsakadesigner Nucleosomes or dNucshave realized these substrates for epigenetic HTS assay developmentThere is a great unmet need for a universal HTS platform compatible with diverse epigenetic substratese ghistone peptides or Nucsand capable of quantifying the activity of writer and eraser enzymesIndeedmany current HTS assayse gFRET and FPare incompatible with Nuc based substrates due to their large size vshistone peptidesMoreovereraser assays provide a formidable challenge for the leading antibody based HTS platformse gAlphaLISA and TRFas antibodies that recognize specific unmodified histone residues are few and far betweenThis has hampered drug development programs to epigenetic erasersdespite the overwhelming evidence implicating these enzymes in disease initiationprogressionIn this proposalwe will develop EpiBeaconTMa novel non radioactiveno wash epigenetic HTS platformThis innovative HTS platform willbe compatible with both peptideand Nuc based substratesmitigate the costly infrastructure and reagents required by existing epigenetic HTS systemsandprovide access to difficult drug targetse gerasersThe cornerstone of our technology is the development of specialized RNA aptamerswhich will be used as `no washandaposdetection reagentsThese aptamer beacons will be engineered to distinguish the precise status of specific histone residuese gH KvsH Kmeand fluoresce only upon target bindingThe innovation of this project is a unique selection strategy allied with the novel incorporation of hydrophobic residues to generate aptamer beacons that will bind PTMstatus targets with exquisite specificity while being agnostic to substrate typei epeptides or NucsIn Phase Iwe will develop aptamer beacons to unmodified H KH Kmeand H KmeAimand validate these reagents by optimizing enzymatic assays for two high interest drug targetsSETDwriterand LSD neraserAimIn Phase IIwe will synthesizeoptimize new histone targeting aptamers as well as miniaturizewell formatSETDand LSD n assays for pilot screen validationPROJECT NARRATIVEChromatin targeting enzymes that addwritersand removeerasershistone modifications have emerged as promising therapeutic targetsand are subject to a growing number of preclinicalclinical inhibitor development programsHowever a large number of these targets remain challengingparticularly eraser enzymes due to a lack of appropriate high throughput screeningHTStoolsHerewe will develop a novel HTS platform using novel turn on fluorescent `aptamer beaconsandaposas no wash detection reagentsThis innovative technology will deliver a powerfullow cost screening platform and give access to currently challenging or inaccessible epigenetic targetse geraser enzymes