Novel enzyme inhibitor screening platform using modified designer nucleosomes

Project Summary Nucleosomes are the basic units of chromatin comprised of a histone octamer made up of two copies of each of the histone subunits H A H B H and H Changes in chromatin structure and function are mediated through histone post translational modifications PTMs such as methylation and ubiquitination Alterations of specific PTMs are highly associated with human diseases including numerous forms of cancer Some histone PTMs work in intranucleosomal groups or systems to regulate the function of histone methyltransferases HMTs a concept referred to as the histone code For instance mono ubiquitination at lysine of histone H B H Bub dramatically enhances enzymatic activity of HMTs that target histone H including DOT L and the SET family SETD A SETD B MLL These striking observations provide a unique opportunity for targeted therapeutic development PTM enzyme inhibitor assays currently use modified histone proteins or fragments and therefore fail to screen enzymes in the context of PTM combinations which are inherent to chromatin regulation in vivo We hypothesize that modified semi synthetic nucleosomes carrying specific PTMs i e `designer nucleosomesandapos or `dNucsandapos for brevity will provide a biologically relevant substrate for identification of inhibitors in the context of both chromatin architecture and unique epigenetic signatures making them optimal substrates for PTM enzyme inhibitor assays In this proposal EpiCypher will develop an innovative dNuc based HMT inhibitor screening platform which capitalizes on cooperative interactions between HMTs and histone ubiquitination to identify context specific inhibitors for cancer therapy Further we will enable the use of dNucs as substrates in high throughput drug screening assays by developing the commercial potential of Amber suppression technology to generate large quantities of high quality ubiquitinated histones We will focus our study on the HMT activity of DOT L and SETD A enzymes that are highly dependent on the presence of H Bub and are upregulated in numerous cancers including leukemia and colorectal cancer While several potent and specific DOT L inhibitors have been identified there are no known specific inhibitors of SETD A Thus DOT L provides a unique molecular avenue to examine how known inhibitors with varying specificities function in our dNuc based assay whereas SETD A provides an opportunity to identify new inhibitor compounds with immediate and unmet clinical relevance The H Bub dependent HTM inhibitor assay proposed here represents a first step toward not only a new research platform but also a potentially powerful novel drug screening tool Pending success of our Phase I efforts the Phase II program will ramp up commercial manufacturing of H Bub modified nucleosomes In addition we will focus on optimizing high throughput assay development for DOT L and SETD A as well as establishing additional H Bub dependent inhibitor assays using other SET family members including SETD B MLL and Project Narrative The progression and onset of numerous of cancers have been linked to modifications of histones the structural proteins of chromosomes that serve as modification dependent regulators of gene expression Understanding how one histone modification relates to the presence absence or function of another is challenging given the dozens of potential modifications that can appear on any of the histone subunits found in a mature nucleosome complex Here we propose a yet undeveloped and novel designer nucleosome based platform comprising specifically modified histones to screen for context dependent inhibitors of histone modifying enzymes