Histone phosphorylation-dependent screening platform for identification of inhibitors to treat neuroblastoma

组蛋白磷酸化依赖性筛选平台，用于鉴定治疗神经母细胞瘤的抑制剂

基本信息

批准号：
9201485
负责人：
Zu-Wen Sun
金额：
$ 22.43万
依托单位：
EPICYPHER, INC.
依托单位国家：
美国
项目类别：
财政年份：
2016
资助国家：
美国
起止时间：
2016-09-01 至 2017-09-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9201485
关键词：
Acetylation Affect Age Architecture Biochemical Biological Assay Cell Death Child Chromatin Chromatin Structure DNA Data Development Diagnosis Disease EP300 gene Engineering Enzymes Epigenetic Process Excision FDA approved Gene Activation Gene Expression Goals Histone Acetylation Histone Code Histone H3 Histones Human Malignant Neoplasms Marketing Mediating Methods Methylation Neuroblastoma Nucleosomes Phase Phosphorylation Phosphoserine Post-Translational Protein Processing Preclinical Drug Evaluation Process Production Protein Fragment Proteins Reaction Recombinants Regulation Research Serine Signal Transduction System Technology Time United States assay development base cancer cell commercialization drug discovery head-to-head comparison high throughput analysis high throughput screening histone acetyltransferase human disease in vivo inhibitor/antagonist innovation manufacturing process monomer novel therapeutics screening success therapy development tool tumor progression

项目摘要

Project Summary: In this proposal, we will develop a synthetic nucleosome-based drug-screening platform to identify novel therapeutics to treat neuroblastoma. Histone acetylation is associated with gene activation and is catalyzed by histone acetyltransferase enzymes (HATs). Histone hyperacetylation is a key driver of neuroblastoma. Several recent studies demonstrate that inhibition of HAT activity dramatically slows cancer progression in vivo. However, there are no FDA approved HAT inhibitors for human use. New HAT inhibitors therefore, are greatly needed to develop therapies for hyperacetylation-based diseases such as neuroblastoma. Nucleosomes, composed of the core histone proteins and DNA, are the fundamental repeating units of chromatin. Chromatin structure and function are altered upon the addition or removal of histone post- translational modifications (PTMs), such as methylation, acetylation, and phosphorylation by histone modifying enzymes. The “histone code hypothesis” stipulates that nucleosomal PTMs function in interdependent networks to regulate downstream gene expression. Current histone modifying assays typically use modified histone proteins/fragments as substrates, which poorly mimic native chromatin architecture. By contrast, synthetic nucleosomes carrying specific PTMs (termed “designer nucleosomes” or “dNucs”) provide a superior substrate for the study of histone modifying enzymes by better replicating chromatin structure. EpiCypher is a world leader in recombinant nucleosome synthesis and is pioneering the development of dNuc-based technologies for drug discovery applications. Phosphorylation of histone H3 at serine 10 (H3S10ph) is strongly associated with gene activation and acts as an epigenetic signaling hub that significantly enhances the activity of multiple HATs. In this Phase I proposal, we will leverage this unique feature of the histone code to develop an innovative screening platform to identify HAT inhibitors. We hypothesize that screening HAT enzymes in the presence of H3S10ph will recapitulate in vivo activity and reveal context-dependent inhibitors, providing a robust and powerful assay platform for drug discovery. We will develop for the first time methods to synthesize high quality H3S10ph- modified nucleosomes at commercial-grade and -scale. We will then use these dNucs as biochemical substrates to establish HAT activity assays using H3S10ph-dependent enzymes. Finally, we will demonstrate feasibility that this assay platform can be used for drug discovery, by examining phosphorylation context- dependent HAT activity following treatment of H3S10ph-modified nucleosomes with tool HAT inhibitors. In Phase II, we will further optimize commercialization of H3S10ph-modified nucleosomes to support high throughput assay development. We will also develop additional H3S10ph-dependent HAT activity/inhibitor assays, which we will market as stand-alone inhibitor kits to both industrial and academic research customers. The innovative drug discovery platform described herein will accelerate the identification HAT inhibitors to treat devastating human diseases such as neuroblastoma.

项目概要：在本提案中，我们将开发一个基于合成核小体的药物筛选平台，以识别治疗神经母细胞瘤的新疗法与基因激活有关。由组蛋白乙酰转移酶 (HAT) 催化，是组蛋白过度乙酰化的关键驱动因素。最近的几项研究表明，抑制 HAT 活性可显着减缓癌症的发生。然而，FDA 尚未批准新的 HAT 抑制剂用于人体。因此，非常需要开发针对基于过度乙酰化的疾病的治疗方法，例如成神经细胞瘤。核小体由核心组蛋白和 DNA 组成，是细胞的基本重复单位。染色质的结构和功能随着组蛋白的添加或去除而改变。翻译修饰 (PTM)，例如组蛋白修饰的甲基化、乙酰化和磷酸化 “组蛋白密码假说”规定核小体 PTM 相互依赖地发挥作用。目前的组蛋白修饰检测通常使用修饰的网络。组蛋白/片段作为底物，其模仿天然染色质结构的能力较差。携带特定 PTM 的合成核小体（称为“设计核小体”或“dNucs”）提供了优越的通过更好地复制染色质结构来研究组蛋白修饰酶的底物是一种。重组核小体合成领域的世界领先者，并引领基于 dNuc 的开发药物发现应用技术。组蛋白 H3 丝氨酸 10 (H3S10ph) 的磷酸化与基因激活密切相关在第一阶段中，它作为表观遗传信号中枢显着增强了多个 HAT 的活性。建议，我们将利用组蛋白代码的这一独特功能来开发创新的筛选平台我们发现，在 H3S10ph 存在的情况下筛选 HAT 酶会产生影响。重现体内活性并揭示背景依赖性抑制剂，提供稳健且强大的检测我们将首次开发合成高质量 H3S10ph- 的方法。然后我们将使用这些 dNucs 作为生物化学物质。最后，我们将证明使用 H3S10ph 依赖性酶来确定 HAT 测定活性。通过检查磷酸化背景，该检测平台可用于药物发现的可行性用工具 HAT 抑制剂处理 H3S10ph 修饰的核小体后依赖的 HAT 活性。第二阶段，我们将进一步优化 H3S10ph 修饰核小体的商业化，以支持高我们还将开发额外的 H3S10ph 依赖性 HAT 活性/抑制剂。我们将作为独立的抑制剂试剂盒向工业和学术研究客户销售。本文描述的创新药物发现平台将加速识别 HAT 抑制剂来治疗毁灭性的人类疾病，例如神经母细胞瘤。