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.

项目概要/摘要 我的研究目标是了解调节拓扑异构酶 II (topo II) 的细胞机制 功能并对 II 型拓扑异构酶（例如拓扑异构酶 II）作出反应，是必需的酶。 管理 DNA 超螺旋结构并十连纠缠 DNA 链以促进关键过程，例如 就人类健康而言，拓扑 II 是一种转录、DNA 复制和染色体分离。 极其重要的癌症靶点，临床上使用多种化疗药物来开发 生化和结构研究表明，拓扑异构酶 II 具有产生 DNA 损伤和杀死复制细胞的能力。 提供了有关拓扑 II 的“链通道”机制以及合成方法的宝贵细节 然而，该领域对拓扑 II 活性的调节机制缺乏明确的了解。 根据细胞需要。 为了更好地了解 topo II 如何与调控途径和 DNA 损伤反应相关，我的目标是 利用酿酒酵母模型系统探索细胞代谢在调节topo II功能中的作用并 在分析拓扑 II 抑制时对参与响应的基因进行公正的筛选。 真核拓扑异构酶 II 的进化保守性，我发现拓扑异构酶 II 上有一个高度保守的口袋，它相互作用 ICRF-187 是一种临床批准的合成抑制剂，后来用于化疗方案2。 发现该网站还含有白藜芦醇，这是一种在红酒中发现的天然产物。 表明该位点可能是一个孤儿变构位点，并激发了小分子的假设 初步数据表明，酵母成分可能会变构调节拓扑 II 功能。 代谢物提取物能够调节拓扑 II 活性 目标 1 概述了天然产物的纯化。 我将采用策略和基于质谱的代谢物配体筛选来识别和表征 除了先前的探测外，还可能具有拓扑 II 调节功能的内源性小分子。 细胞代谢和 DNA 拓扑结构之间未经探索的联系，我的目标是揭示参与的途径 因此，在目标 2 中，我提出了一种公正的药物遗传学筛选。 与酵母缺失和亚型文库来识别在临床上存在影响生长的等位基因 这些目标共同有可能发现拓扑结构域之间的新相互作用。 反过来，这些发现可能会揭示变构调节拓扑 II 的新方法。 与抗拓扑异构酶抑制剂联合治疗以改善的功能和新靶点 当前可用治疗的功效和/或降低毒性。