A nucleosome sliding assay platform to screen inhibitors of SWI/SNF chromatin remodeling complexes

PROJECT SUMMARYNearlyof all cancers contain mutations in subunits from the SWI SNF family of NucleosomeNucremodeling complexesThe SWI SNF family regulates local genome access by pumping DNA around histone octamersthusslidingor remodeling Nucs within chromatinRecurrent somatic mutations in SWI SNF subunits are observed in multiple cancerssupporting a driver role in tumorigenesisThe mutated complexes are desirable therapeutic targetssince removing their ATPase activity promotes cancer cell death but spares normal cellsThis phenomenon is known as synthetic lethality and identifying inhibitors to exploit it may lead to drugs with cancer specificityIn Phase IEpiCypher developed EpiDyneTMrecombinant nucleosome remodeling substrates for SWI SNF family enzymesOur assay design utilized a GATC motif within a defined Nucleosome positioning sequenceNPSwhere the positioned histone octamer blocks access to the motifUpon nucleosome remodelingthe GATC motif is exposed and can be subsequently cleaved or labeled as an assay readoutThe primary goals of our Phase II research program are to evolve EpiDyne toward non radioactive HTS readouts as we begin to scale up and validate our SWI SNF inhibitor screening platform for commercial releaseIn Aimwe will develop novel specialized EpiDyne nucleosomesepiNucscompatible with multiple non radioactive HTS readoutse gFluorescence polarizationFPor AlphaIn additionwe will engineer epiNucs containing acetylation on histone Ha post translational modificationPTMthat interacts with a bromodomain on SMARCAthe SWI SNF complex ATPase subunitand enhances remodeling activity in vitroin vivoThe use of physiological substrates is preferable for biochemical inhibitor assays as these are more likely to identify compounds with in vivo activityThuswe hypothesize that acetylated epiNucs may not only enhance enzyme activityi eincrease assay windowbut may lead to the identification of more target specific inhibitorsvsunmodified epiNucsIn Aimwe will develop methods for the manufacturing of SMARCAand SMARCAcontaining SWI SNF remodeling complexesnot commercially availableUsing these remodeling complexeswe will optimize HTS assays using Alpha or FP readoutsthe development of orthogonal HTS platforms will allow users to perform both primary screens and hit counterscreeningIn Aimwe will scale up manufacturing of epiNucsunmodified and acetylatedFinallywe will perform a series of pilot screenscompound libraryin collaboration with Jim Bruenig at BeanTown Biotechusing our optimized SMARCAor SMARCAHTS assaysGiven the remarkable disease prevalence of dysfunctional nucleosome remodelingthe HTS platform swe develop will have a profound impact on the identification of new therapeutics for diverse human diseasesmost notably cancers with poor prognosis PROJECT NARRATIVEChromatin remodelingor the repositioning of nucleosomesregulates DNA access and plays major roles in human disease from inflammation and autoimmunity to cancerHoweverthe enzymes that mediate nucleosome positioning are relatively inaccessible to HTSdrug developmentwith no standard approaches to interrogate their remodeling activitiesHereEpiCypher will develop the first high throughput nucleosome remodeling assay platform to accelerate therapeutic development for chromatin remodeling enzymes