Novel enzyme inhibitor screening platform using modified designer nucleosomes

使用改良设计核小体的新型酶抑制剂筛选平台

基本信息

批准号：
9253050
负责人：
Zu-Wen Sun
金额：
$ 22.49万
依托单位：
EPICYPHER, INC.
依托单位国家：
美国
项目类别：
财政年份：
2017
资助国家：
美国
起止时间：
2017-03-01 至 2018-02-28
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9253050
关键词：
Amber Architecture Biochemical Biological Biological Assay Biology Boston Cells Chemistry Chromatin Chromatin Structure Chromosomes Collaborations Colorectal Cancer Complex DNA Development Employee Strikes Enzyme Inhibitor Drugs Enzymes Epigenetic Process Family Family member Gene Expression Generations Gray unit of radiation dose Histone Code Histone H2B Histone H3 Histones Hypermethylation Link Lysine MLL gene Malignant Neoplasms Mediating Methods Methylation Modification Molecular Monoubiquitination Nucleosomes Phase Post-Translational Protein Processing Preclinical Drug Evaluation Process Proteins Quality Control Ramp Regulation Regulator Genes Research Specificity Structural Protein System Tail Technology Testing Ubiquitin Ubiquitination Validation Weight Work assay development base cancer therapy clinically relevant high throughput screening histone methyltransferase histone modification human disease in vivo inhibitor/antagonist innovation leukemia monomer novel novel therapeutics programs screening success targeted treatment therapeutic development tool

项目摘要

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 (H2A, H2B, H3, and H4). 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 120 of histone H2B (H2Bub1) dramatically enhances enzymatic activity of HMTs that target histone H3, including DOT1L and the SET1-family (SETD1A, SETD1B, MLL1, -2, -3, -4). 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 nucleosomes' or `dNucs' 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 DOT1L and SETD1A, enzymes that are highly dependent on the presence of H2Bub1 and are upregulated in numerous cancers, including leukemia and colorectal cancer. While several potent and specific DOT1L inhibitors have been identified, there are no known specific inhibitors of SETD1A. Thus, DOT1L provides a unique molecular avenue to examine how known inhibitors with varying specificities function in our dNuc-based assay, whereas SETD1A provides an opportunity to identify new inhibitor compounds with immediate and unmet clinical relevance. The H2Bub1-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 H2Bub1-modified nucleosomes. In addition, we will focus on optimizing high-throughput assay development for DOT1L and SETD1A as well as establishing additional H2Bub1-dependent inhibitor assays using other SET1 family members including SETD1B, MLL1, -2, -3, and -4.

项目摘要：核小体是染色质的基础单位，由两个副本组成的组蛋白八聚体的组成每个组蛋白亚基（H2A，H2B，H3和H4）的变化是介导的雷霆组蛋白后翻译后修饰（PTM），例如甲基化和泛素化。特定PTM的改变与人类疾病高度相关，倾向于多种形式的癌症。一些组蛋白PTM在核体内组或系统中起作用以调节组蛋白的功能甲基转移酶（HMT），例如。组蛋白H2b（H2BUB1）的赖氨酸120急剧增强了HMTS靶向组蛋白H3的酶活性，包含dot1l和set1 -family（setd1a，setd1b，mll1，-2，-3，-4）为有针对性的治疗发展提供独特的机会。修饰的组蛋白蛋白，片段或片段，因此在PTM的背景下无法筛选酶组合，是体内染色质调节的固有的。携带特定PTM的核小体（即“设计器核成像”或“ dnucs'forcs'）将提供一个与生物学相关的底物，用于在染色质架构和独特的表观签名，使PTM酶遗传测定法提案，具有开发基于DNUC的HMT抑制剂筛查平台的Epicypher，该平台利用HMT和组蛋白泛素化之间的合作互动，以识别特定于上下文癌症治疗的抑制剂。通过开发琥珀色支持技术的商业潜力来筛查测定法，以产生大型高质量的泛素化组蛋白的数量。 setD1a，酶高度取决于H2BUB1的存在，并在许多中上登记癌症，包括白血病和大肠癌。已识别，有setD1a的特异性抑制剂。途径检查如何在基于DNUC的测定中授予具有不同特异性功能的抑制剂，而 setD1a提供了一个机会，可以立即鉴定新的抑制剂化合物相关性。一个新的研究平台，也是一种潜在强大的新型药物筛查工具。第一阶段的努力，第二阶段计划将加强H2BUB1修改的商业制造核小组。 setD1a以及使用其他set1家族建立其他H2BUB1依赖性抑制剂测定包括setD1b，mll1，-2，-3和-4的成员。