A nucleosome sliding assay platform to screen inhibitors of SWI/SNF chromatin remodeling complexes

筛选 SWI/SNF 染色质重塑复合物抑制剂的核小体滑动分析平台

基本信息

批准号：
9346171
负责人：
Michael-Christopher Keogh
金额：
$ 22.46万
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：
2017-06-01 至 2018-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9346171
关键词：
ATP phosphohydrolase ATPase Domain Address Affinity Attention Autoimmunity Biological Biological Assay Biomedical Research Cardiovascular Diseases Catalysis Cell Death Chromatin Chromatin Remodeling Factor Chronic Complex Core Protein DNA DNA Repair DNA Sequence Defect Disease Drug Targeting Engineering Enzymes Family Feasibility Studies Fluorescence Resonance Energy Transfer Gene Expression Genome Genomics Goals Histones Human Inflammation Institutes Intellectual functioning disability Label Lead Letters Location Malignant Neoplasms Mediating Methylation Methyltransferase Modeling Monitor Mutate Mutation Normal Cell Nucleosomes Pharmaceutical Preparations Pharmacologic Substance Phase Play Positioning Attribute Production Property Proteins Pump Radiolabeled Reagent Recombinants Recurrence Research Role SMARCA2 gene SMARCA4 gene SWI/SNF Family Complex Schizophrenia Site Slide Somatic Mutation Specificity Stretching Testing Therapeutic assay development base cancer cell cell type chromatin remodeling commercialization design drug development drug discovery epigenetic drug flexibility high throughput screening human disease industry partner inhibitor/antagonist innovation interest novel programs screening success targeted treatment therapeutic development therapeutic target tool tumor progression tumorigenesis

项目摘要

PROJECT SUMMARY In this proposal, EpiCypher Inc. will develop the first commercially available assay to monitor nucleosome remodeling, a major current focus for rational drug discovery that is starved of HTS tools. Nucleosomes are the basic units of chromatin, made up of ~147 bp DNA tightly wrapped around an octamer of histone proteins. Chromatin remodeling complexes utilize ATP to disrupt DNA-histone interactions and reposition nucleosomes, thus regulating DNA access. Aberrant nucleosome organization can severely disrupt gene expression, DNA repair and cellular differentiation, and is associated with cancer and disorders including schizophrenia, chronic inflammation, and intellectual disability. The profound role of remodeling complexes in tumorigenesis and cancer progression represents a new target class for epigenetic drug discovery rapidly gathering attention from our commercial partners. Of particular therapeutic interest are the SWI/SNF family of chromatin remodeling complexes, with 20% of all human cancers harboring a subunit mutation. SWI/SNF complexes are comprised of a remodeling ATPase (SMARCA2 [BRM] or SMARCA4 [BRG1]), three core proteins, and an additional four to eight accessory proteins (varying by cell type) that direct genomic localization of the complex and stimulate catalysis. The catalytic ATPase domains of SWI/SNF complexes have been identified in multiple studies as rational targets for therapeutic development. However, chromatin remodeling assays amenable to high throughput screening are currently lacking, a problem EpiCypher will address through this proposal. We will assess the feasibility of using recombinant nucleosomes (rNucs) homogenously assembled on sliding DNA sequences as a high-throughput, biologically relevant screening platform to identify compounds that directly inhibit chromatin remodeling. In Aim 1, EpiCypher will develop an innovative rNuc-based nucleosome sliding assay, which capitalizes on a unique DNA template that can be radiolabeled after SWI/SNF repositions the histone octamer to expose a Dam methylation site (GATC). In Aim 2, we will validate the sliding rNuc substrates by assaying chromatin remodeler activity with the isolated SMARCA4 enzyme. In Phase II, we will focus on commercial production of this high-throughput sliding rNuc screening platform for the identification of inhibitors that target the SWI/SNF chromatin remodeling family. This highly innovative research program will enable therapeutic development toward chromatin remodeling complexes, a major class of drug targets that currently lack adequate screening tools.

项目概要在本提案中，EpiCypher Inc. 将开发第一个可商业化的监测检测方法。核小体重塑是目前合理药物发现的主要焦点，但缺乏高温超导工具。核小体是染色质的基本单位，由紧密包裹在八聚体周围的约 147 bp DNA 组成组蛋白蛋白质。染色质重塑复合物利用 ATP 破坏 DNA-组蛋白相互作用并重新定位核小体，从而调节 DNA 访问。异常的核小体组织可能会严重破坏基因表达、DNA 修复和细胞分化，与癌症和疾病相关，包括重塑复合物在精神分裂症、慢性炎症和智力障碍中的深远作用。肿瘤发生和癌症进展代表了表观遗传药物快速发现的新靶标类别 SWI/SNF 家族引起了我们商业合作伙伴的特别关注。染色质重塑复合物，20% 的人类癌症含有 SWI/SNF 亚基突变。复合物由重塑 ATP 酶（SMARCA2 [BRM] 或 SMARCA4 [BRG1]）、三个核心组成蛋白质，以及另外四到八个指导基因组的辅助蛋白质（因细胞类型而异）复合物的定位并刺激催化作用。然而，多项研究已将染色质确定为治疗开发的合理靶点。目前缺乏适合高通量筛选的重塑测定，EpiCypher 将面临这个问题我们将通过该提案评估使用重组核小体（rNucs）的可行性。作为高通量、生物学相关的同质组装在滑动 DNA 序列上筛选平台来鉴定直接抑制染色质重塑的化合物。在目标 1 中， EpiCypher 将开发一种基于 rNuc 的创新核小体滑动测定法，该测定法利用了独特的 SWI/SNF 重新定位组蛋白八聚体以暴露 Dam 后可进行放射性标记的 DNA 模板在目标 2 中，我们将通过检测染色质来验证滑动 rNuc 底物。使用分离的 SMARCA4 酶进行重塑活性。在第二阶段，我们将专注于商业生产该高通量滑动 rNuc 筛选平台用于鉴定针对 SWI/SNF 的抑制剂这个高度创新的研究计划将促进治疗的发展。染色质重塑复合物是目前缺乏充分筛选的一类主要药物靶点。工具。