In this proposal, EpiCypher Inc. will develop the first commercially available assay to monitor nucleosome remodeling, a major current focus for rational drug discovery that is starved of HTS tools. Nucleosomesarethebasicunitsofchromatin,madeupof~147bpDNAtightlywrappedaroundanoctamerof histone proteins. Chromatin remodeling complexes utilize ATP to disrupt DNA-histone interactions and repositionnucleosomes,thusregulatingDNAaccess.Aberrantnucleosomeorganizationcanseverelydisrupt geneexpression,DNArepairandcellulardifferentiation,andisassociatedwithcanceranddisordersincluding schizophrenia,chronicinflammation,andintellectualdisability.Theprofoundroleofremodelingcomplexesin tumorigenesis and cancer progression represents a new target class for epigenetic drug discovery rapidly gatheringattentionfromourcommercialpartners.OfparticulartherapeuticinterestaretheSWI/SNFfamilyof chromatin remodeling complexes, with 20% of all human cancers harboring a subunit mutation. SWI/SNF complexes are comprised of a remodeling ATPase (SMARCA2 [BRM] or SMARCA4 [BRG1]), three core proteins, and an additional four to eight accessory proteins (varying by cell type) that direct genomic localization of the complex and stimulate catalysis. The catalytic ATPase domains of SWI/SNF complexes have been identified in multiple studies as rational targets for therapeutic development. However, chromatin remodeling assays amenable to high throughput screening are currently lacking, a problem EpiCypher will addressthroughthisproposal.Wewillassessthefeasibilityofusingrecombinantnucleosomes(rNucs) homogenously assembled on sliding DNA sequences as a high-throughput, biologically relevant screening platform to identify compounds that directly inhibit chromatin remodeling.
In Aim 1, EpiCypher will develop an innovative rNuc-based nucleosome sliding assay, which capitalizes on a unique DNA template that can be radiolabeled after SWI/SNF repositions the histone octamer to expose a Dam methylation site (GATC).
In Aim 2, we will validate the sliding rNuc substrates by assaying chromatin remodeleractivitywiththeisolatedSMARCA4enzyme.InPhaseII,wewillfocusoncommercialproductionof thishigh-throughputslidingrNucscreeningplatformfortheidentificationofinhibitorsthattargettheSWI/SNF chromatin remodeling family. This highly innovative research program will enable therapeutic development towardchromatinremodelingcomplexes,amajorclassofdrugtargetsthatcurrentlylackadequatescreening tools.
Chromatin remodeling, or the repositioning of nucleosomes, regulates DNA access and plays major rolesinhumandiseasefrominflammationandautoimmunitytocancer.However,theenzymesresponsiblefor modulatingnucleosomepositioningarerelativelyinaccessibletoHTSanddrugdevelopment,withnostandard approaches to interrogate their modeling activities. In this proposal, EpiCypher will develop the first high- throughput nucleosome sliding assay to accelerate the identification of therapeutics that target chromatin remodelingenzymes.