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.

项目总结： 核小体是染色质的基本单位，由一个由两个拷贝组成的组蛋白八聚体组成 每个组蛋白亚基(H_2A、H_2B、H_3和H_4)。染色质结构和功能的变化是 通过组蛋白翻译后修饰(PTM)，如甲基化和泛素化。 特定的PTM的改变与人类疾病高度相关，包括多种形式的癌症。 一些组蛋白PTM在核小体内的群或系统中工作，以调节组蛋白的功能 甲基转移酶(HMTs)，一个被称为“组蛋白密码”的概念。例如，单一泛素化在 组蛋白H_2B(H_2Bub1)的赖氨酸120显著增强以组蛋白H3为靶标的HMT的酶活性， 包括DOT1L和SET1-家族(SETD1A、SETD1B、MLL1、-2、-3、-4)。这些惊人的观察结果 为有针对性的治疗开发提供了独特的机会。目前使用的PTM酶抑制剂检测方法 修饰的组蛋白蛋白或片段，因此不能在PTM的背景下筛选酶 结合，这是体内染色质调节所固有的。我们假设改良型半合成 携带特定PTM的核小体(简写为“设计型核小体”或“dNucs”)将提供 用于在染色质结构和环境中鉴定抑制剂的生物相关底物 独特的表观遗传特征，使它们成为PTM酶抑制剂检测的最佳底物。在这 提议，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于上下文的 癌症治疗的抑制剂。此外，我们将允许将dNucs用作高通量药物的底物。 通过开发琥珀抑制技术的商业潜力来筛选检测产生大量 大量的高质量泛素化的组蛋白。我们将重点研究DOT1L和DOT1L的HMT活性 SETD1A，高度依赖于H_2Bub1的存在，并在许多 癌症，包括白血病和结直肠癌。而几种有效和特异的DOT1L抑制剂 目前，尚无已知的SETD1A的特异性抑制剂。因此，DOT1L提供了一种独特的分子 在我们基于dNuc的测试中，检查具有不同特异性的已知抑制剂如何发挥作用，而 SETD1A提供了一个机会，以立即和未达到临床要求的方式识别新的抑制剂化合物 关联性。这里提出的依赖于H_2Bub1的HTM抑制剂试验代表着不仅是朝着 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。我们的成功待定 第一阶段的努力，第二阶段的计划将加强H_2Bub1-改进型的商业生产 核小体。此外，我们将专注于优化DOT1L和DOT1L的高通量分析开发 SETD1a以及使用其他SET1家族建立额外的H2 Bub1依赖的抑制物检测 成员包括SETD1B、MLL1、-2、-3和-4。