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.

项目概要：核小体是染色质的基本单位，由两个拷贝组成的组蛋白八聚体组成每个组蛋白亚基（H2A、H2B、H3 和 H4）的染色质结构和功能发生变化。通过组蛋白翻译后修饰（PTM）介导，例如甲基化和泛素化。特定 PTM 的改变与人类疾病高度相关，包括多种癌症。一些组蛋白 PTM 在核小体内基团或系统中发挥作用，调节组蛋白的功能甲基转移酶（HMT），一个被称为“组蛋白密码”的概念，例如，单泛素化。组蛋白 H2B (H2Bub1) 的赖氨酸 120 显着增强靶向组蛋白 H3 的 HMT 酶活性，包括 DOT1L 和 SET1 家族（SETD1A、SETD1B、MLL1、-2、-3、-4）这些惊人的观察结果。为目前使用的靶向治疗开发提供了独特的机会。修饰组蛋白或片段，因此无法在 PTM 背景下筛选酶组合，这是体内染色质调节所固有的，我们勇敢地进行了改良的半合成。携带特定 PTM 的核小体（即“设计核小体”或“dNucs”）将提供用于在染色质结构和染色质结构的背景下鉴定抑制剂的生物学相关底物独特的表观遗传特征，使其成为 PTM 抑制剂酶测定的最佳底物。根据提案，EpiCypher 将开发一个基于 dNuc 的创新 HMT 抑制剂筛选平台，该平台利用 HMT 和组蛋白泛素化之间的合作相互作用来识别特定环境此外，我们将启用 dNucs 作为高通量药物的底物。利用琥珀抑制技术的商业潜力开发的筛选测定法可产生大量我们将重点研究 DOT1L 和 HMT 的活性。 SETD1A，高度依赖于 H2Bub1 存在的酶，并且在许多细胞中上调癌症，包括白血病和结直肠癌，而几种有效且特异性的 DOT1L 抑制剂具有作用。已被鉴定，没有已知的 SETD1A 特异性抑制剂，因此，DOT1L 提供了一种独特的分子。途径来检查具有不同特异性的已知抑制剂如何在我们基于 dNuc 的测定中发挥作用，而 SETD1A 提供了一个机会来识别具有立即且未满足的临床效果的新抑制剂化合物这里提出的 H2Bub1 依赖性 HTM 抑制剂测定不仅代表了第一步。一个新的研究平台，也是一个潜在的强大的新型药物筛选工具，有待我们的成功。第一阶段的努力，第二阶段计划将加速 H2Bub1 修饰的商业化生产此外，我们将重点优化 DOT1L 和核小体的高通量检测开发。 SETD1A 以及使用其他 SET1 家族建立额外的 H2Bub1 依赖性抑制剂测定成员包括 SETD1B、MLL1、-2、-3 和 -4。