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

• 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
• 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

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本单元，由由两个副本组成的Hisstone Octamer组成 每个组蛋白亚基（H2A，H2B，H3和H4）的。染色质结构和功能的变化是 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，包括多种形式的癌症。 一些Hisstone PTM在精肠体组或系统中起作用以调节组蛋白的功能 甲基转移酶（HMTS），一个称为“组蛋白代码”的概念。例如，单素化 组蛋白H2b（H2BUB1）的赖氨酸120急剧增强了靶向组蛋白H3的HMT的酶促活性 包括dot1l和set1 -family（setd1a，setd1b，mll1，-2，-3，-4）。这些引人注目的观察 为有针对性的治疗发展提供了独特的机会。 PTM酶抑制剂分析当前使用 修改了Hisstone蛋白或碎片，因此在PTM的背景下未能筛选酶 组合，遗传到体内染色质调节。我们假设修改的半合成 带有特定PTM的核小体（即“设计器核小体”或“ dnucs”的核小体将提供一个 在染色质架构和 独特的表观遗传特征，使其成为PTM酶抑制剂分析的最佳底物。在这个 提案，Epicypher将开发创新的基于DNUC的HMT抑制剂筛选平台，该平台，该平台 利用HMTS和Hisstone泛素化之间的合作互动来识别特定于上下文的 癌症治疗的抑制剂。此外，我们将启用DNUCs作为高通量药物的底物 通过开发琥珀色抑制技术的商业潜力来筛选测定，以产生大型 高质量泛素化组蛋白的数量。我们将把研究重点放在DOT1L和 SETD1A，高度依赖于H2BUB1的酶，并在许多中进行了更新 癌症，包括白血病和大肠癌。虽然几种潜在的和特定的dot1l抑制剂具有 被鉴定出来，尚无setD1a的已知特异性抑制剂。那就是DOT1L提供独特的分子 途径可检验具有不同规格的抑制剂在我们的基于DNUC的测定中的功能，而 SETD1A提供了一个机会，可以识别具有即时无需临床的新抑制剂化合物 关联。此处提出的H2BUB1依赖性HTM抑制剂测定不仅是迈向的第一步 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。等待我们的成功 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。此外，我们将专注于优化DOT1L和 使用其他set1家族建立其他H2BUB1依赖性抑制剂测定法以及建立其他H2BUB1依赖性抑制剂分析 包括setD1b，mll1，-2，-3和-4的成员。