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

项目总结： 在这项提案中，我们将开发一个基于人工核小体的药物筛选平台来识别 治疗神经母细胞瘤的新疗法。组蛋白乙酰化与基因激活有关 由组蛋白乙酰转移酶(HATS)催化。组蛋白的超乙酰化是 神经母细胞瘤。最近的几项研究表明，抑制HAT活性可以显著延缓癌症。 在体内的进展。然而，目前还没有FDA批准的人类使用的HAT抑制剂。新型HAT抑制剂 因此，非常需要开发基于高乙酰化的疾病的治疗方法，如 神经母细胞瘤。 核小体由核心组蛋白和DNA组成，是组蛋白的基本重复单位。 染色质。染色质的结构和功能在组蛋白的添加或去除后发生改变。 翻译修饰(PTM)，如组蛋白修饰的甲基化、乙酰化和磷酸化 酵素。“组蛋白密码假说”认为，核小体PTMS的功能是相互依赖的。 调控下游基因表达的网络。目前的组蛋白修饰分析通常使用修饰后的 组蛋白蛋白质/片段作为底物，不能很好地模仿天然的染色质结构。相反， 携带特定PTM的合成核小体(称为“设计核小体”或“dNucs”)提供了一种更好的 通过更好地复制染色质结构，研究组蛋白修饰酶的底物。EpiCypher是一种 重组核小体合成的世界领先者，并率先开发基于dNuc的 用于药物发现应用的技术。 组蛋白H3丝氨酸10(H3S10ph)的磷酸化与基因激活和 作为表观遗传信号枢纽，显著增强多个HAT的活性。在此阶段I 建议，我们将利用组蛋白编码的这一独特功能来开发创新的筛查平台 以确定HAT抑制剂。我们假设在H3S10ph存在的情况下筛选HAT酶将 概述了体内活性并揭示了上下文依赖的抑制物，提供了一种强有力的分析方法 药物发现的平台。我们将首次开发合成高质量H3S10ph的方法- 商业化和规模化的修饰核小体。然后我们将使用这些dNucs作为生化 用H3S10ph依赖酶建立HAT活性测定的底物。最后，我们将演示 通过检测磷酸化背景，该分析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。在……里面 第二阶段，我们将进一步优化H3S10ph修饰的核小体的商业化，以支持高 吞吐量测定的发展。我们还将开发更多依赖于H3S10ph的HAT活性/抑制剂 分析，我们将作为独立的抑制剂试剂盒销售给工业和学术研究客户。 本文所述的创新药物发现平台将加速识别HAT抑制剂进行治疗 毁灭性的人类疾病，如神经母细胞瘤。

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.