Discovery of metabolic regulators of DNA topology and cellular responses to topoisomerase II inhibition

发现 DNA 拓扑代谢调节剂和细胞对拓扑异构酶 II 抑制的反应

• 批准号: 9766078
• 负责人: Joyce Lee
• 金额: $ 4.5万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-08-01 至 2020-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9766078
• 关键词: Affect; Alleles; Allosteric Site; Antineoplastic Agents; Binding; Binding Sites; Biochemical; Biological Models; Biological Process; Cell Cycle; Cell Proliferation; Cells; Chemicals; Chromosomes; Clinical; Crude Extracts; Cues; DNA; DNA Damage; DNA Structure; DNA biosynthesis; DNA strand break; Data; Development; Dominant-Negative Mutation; Drug Efflux; Drug resistance; Enzymes; Eukaryotic DNA Topoisomerases II; Event; Fractionation; Functional disorder; Gene Deletion; Gene Expression; Genes; Genetic; Genetic Screening; Genetic Transcription; Goals; Growth; Health; Homeostasis; Human; Knock-out; Lead; Libraries; Ligands; Malignant Neoplasms; Mass Spectrum Analysis; Metabolic; Metabolism; Morphologic artifacts; Natural Products; Ontology; Orphan; Outcome; Pathway interactions; Pharmaceutical Preparations; Pharmacogenetics; Physiological; Plants; Play; Process; Regulation; Regulatory Pathway; Research; Resistance; Resveratrol; Role; Saccharomyces cerevisiae; Sampling; Site; Structure; Superhelical DNA; Therapeutic; Topoisomerase; Topoisomerase II; Topoisomerase II inhibition; Topoisomerase Inhibitors; Topoisomerase-II Inhibitor; Toxic effect; Work; Yeasts; anti-cancer; base; cancer cell; clinical application; clinically relevant; combinatorial; cytotoxic; design; experimental study; improved; inhibitor/antagonist; insight; interest; knock-down; metabolomics; novel; novel strategies; novel therapeutics; preservation; response; screening; side effect; small molecule; synergism; targeted treatment; tumorigenic

Project Summary/Abstract The goal of my research is to understand the cellular mechanisms that regulate topoisomerase II (topo II) function and respond to topo II inhibition. Type II topoisomerases, such as topo II, are essential enzymes that manage DNA superhelical structure and decatenate entangled DNA strands to facilitate critical processes such as transcription, DNA replication, and chromosome segregation1. With respect to human health, topo II is a tremendously important cancer target, and a variety of chemotherapeutics are clinically used to exploit the ability to topo II to generate DNA damage and kill replicating cells. Biochemical and structural studies have provided valuable details about the “strand passage” mechanism of topo II and the means by which synthetic inhibitors affect this activity. However, the field lacks a clear understanding of how topo II activity is regulated in accordance with cell needs. To better understand how topo II is connected to regulatory pathways and DNA damage responses, I aim to use the S. cerevisiae model system to explore the role of cellular metabolism in regulating topo II function and perform an unbiased screen for genes involved in responding to topo II inhibition. While analyzing the evolutionary conservation of eukaryotic topo IIs, I discovered a highly conserved pocket on topo II that interacts with ICRF-187, a clinically approved, synthetic inhibitor that is used in chemotherapeutic regimens2. I later discovered that this site also engages resveratrol, a natural product found in red wine3. These observations suggest that this site may be an orphan allosteric site and inspired the hypothesis that small-molecule metabolites may allosterically regulate topo II function. Preliminary data indicate that components of yeast metabolite extracts are able to modulate topo II activity. Aim 1 outlines the natural product purification strategies and mass spectrometry-based metabolite ligand screening I will employ to identify and characterize endogenous small molecules that may have topo II-regulatory function. In addition to probing previously unexplored connections between cellular metabolism and DNA topology, I aim to uncover pathways involved in cellular responses to abnormal topo II activity. Thus, in Aim 2 I propose an unbiased pharmacogenetic screen with yeast deletion and hypomorph libraries to identify alleles that affect growth in the presence of clinically- relevant topo II inhibitors. Together, these aims have the potential to discover novel interactions between topo II and regulatory pathways. Such discoveries, in turn, could unveil new ways to allosterically modulate topo II function and new targets for combinatorial therapy in conjunction with anti-topoisomerase inhibitors to improve the efficacy and/or decrease toxicity of currently available treatments.

项目摘要/摘要 我的研究的目的是了解调节拓扑异构酶II的细胞机制（TOPO II） 功能并响应TOPO II抑制作用。 II型拓扑异构酶（例如Topo II）是必不可少的酶 管理DNA超螺旋结构和脱发纠缠的DNA链，以促进关键过程此类 作为转录，DNA复制和染色体分离1。关于人类健康，Topo II是 极其重要的癌症靶标，以及各种化学治疗剂临床用来利用 能够产生DNA损伤并杀死复制细胞的能力。生化和结构研究 提供了有关Topo II的“链”机制以及合成的手段的宝贵细节 抑制剂会影响这一活性。但是，该领域对Topo II活动的调节方式缺乏清晰的了解 因此，有细胞的需求。 为了更好地了解Topo II如何连接到监管途径和DNA损伤响应，我的目标是 使用S. cerevisiae模型系统探索细胞代谢在控制Topo II功能和 对涉及对Topo II抑制作用的基因进行无偏屏幕。在分析 真核托普II的进化保守性，我在Topo II上发现了一个高度保守的口袋，可以相互作用 与ICRF-187（一种经过临床批准的合成抑制剂，用于化学治疗方案2）。我后来 发现该站点还与红色葡萄酒中发现的天然产品互动3。这些观察 建议该地点可能是一个孤儿变构地点，并启发了小分子的假设 代谢产物可以变构调节TOPO II功能。初步数据，表明酵母的成分 代谢物提取物能够调节TOPO II活性。 AIM 1概述了天然产品纯化 策略和基于质谱的代谢物配体筛选我将采用我来识别和表征 可能具有TOPO II调控功能的内源性小分子。除了探测以前 细胞代谢与DNA拓扑之间的未开发连接，我的目的是发现涉及的途径 细胞对异常TOPO II活性的反应。在AIM 2我提出了一个公正的药物遗传筛查 通过酵母缺失和降压图，可以确定在临床上存在影响生长的等位基因 相关的TOPO II抑制剂。这些目标在一起有可能发现Topo之间的新型相互作用 II和监管途径。反过来，这样的发现可以揭示新的方法来调节托普II 与抗昆虫异构酶抑制剂结合使用的功能和新靶标 效率和/或降低当前可用治疗的毒性。