Biochemical and genomic tools to study histone ADP ribosylation signaling

PROJECT SUMMARY Histone ADP-ribosylation (ADPr) is a complex post-translational modification (PTM) with strong disease associations. ADPr occurs on all five histone proteins in diverse states (e.g., mono-, di-, tri-, poly-ADPr) that are dynamically regulated to control various cellular functions. The proteins that add, recognize, and remove histone ADPr are rapidly emerging as high-value therapeutic targets. For example, one class of enzymes that catalyze histone poly-ADPr (e.g., PARPs) is the target of many FDA-approved therapeutics for cancer and part of many on-going clinical trials for other disease indications. Despite this, an understanding of the biological roles and therapeutic potential of histone ADPr and histone ADPr-interacting enzymes remains largely unrealized due to a dearth of molecular tools to study ADPr in vivo and in vitro. The goal of this Direct-to-Phase-II proposal is to develop a novel molecular toolkit to enable the study of histone ADPr, advancing our knowledge of this important PTM and providing access to new therapeutic targets in an emerging segment of histone biology. EpiCypher® is pioneering the development of fully-defined designer nucleosomes (dNucs), which we and others have shown to be the ideal physiological substrates and controls for diverse epigenetic assays. The power of EpiCypher’s fully-defined dNuc platform stems from its broad chemical diversity, containing >100 unique dNucs carrying the most widely studied PTM classes (e.g., lysine methylation and acetylation, serine phosphorylation). Our team has successfully leveraged the diversity of this flagship technology for a range of high value applications including: I) high-throughput biochemical assays for drug discovery, inhibitor screening, and antibody profiling (EpiDyne® and dCypher® assays) and II) ultra-sensitive genomic mapping (CUTANA® CUT&RUN / CUT&Tag assays). The central innovations of this proposal are I) the development of a novel chemoenzymatic manufacturing approach to generate the first fully-defined histone ADPr dNucs (ADPr-dNucs) at commercial scale and II) leveraging these novel tools to develop cutting-edge assays to enable new avenues of histone ADPr research and access new drug targets. In Phase I Equivalent feasibility studies, we partnered with ADPr expert Dr. Glen Liszczak of the University of Texas Southwestern to develop a novel two-step chemoenzymatic ADPr-dNuc manufacturing method and generate a focused panel of defined ADPr-dNucs, whose functional importance was validated in a chromatin remodeling assay. Here, we will expand our catalog of ADPr-dNucs (Aim 1) and use these reagents to develop robust biochemical (Aim 2) and genomics (Aim 3) assays to accelerate histone ADPr basic and drug development research. Together, this work will lead to the development and commercialization of a first-in-class molecular toolkit for the study of histone ADPr, enabling multiple high-value applications to drive novel research into currently inaccessible therapeutic targets.