Defining the mechanisms of kinetoplast DNA assembly by trypanosomal topoisomerase II for therapeutic target development

定义锥虫拓扑异构酶 II 的动质体 DNA 组装机制，用于治疗靶点开发

基本信息

批准号：
10386849
负责人：
Arman Alam Siddiqui
金额：
$ 4.68万
依托单位：
JOHNS HOPKINS UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-04-01 至 2023-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10386849
关键词：
ATP phosphohydrolase ATPase Domain Adenylyl Imidodiphosphate Affect African African Trypanosomiasis Antibiotics Antineoplastic Agents Antiparasitic Agents Binding Biochemical Biological Assay Biology Biophysics Blood Circulation Catenated DNA Cells Chagas Disease Chemicals Chromosome Segregation Chromosomes Clinical DNA DNA Binding DNA Ligation DNA biosynthesis DNA topoisomerase II alpha Data Dependence Development Dimerization Disease Drug Targeting Ensure Environment Enzymes Eukaryota FDA approved Fluorescence Resonance Energy Transfer Fluoroquinolones Future Genetic Transcription Genomics Goals Health Homology Modeling Hydrolysis In Vitro Infection Infectious Agent Intervention Kinetoplast DNA Knowledge Label Leishmaniasis Libraries Measurement Measures Mediating Mitochondria Mitochondrial DNA Molecular Molecular Biology Molecular Machines Molecular Structure Monitor Nucleotides Outcome Parasites Persons Pharmaceutical Preparations Plasmids Population Process Property Protein Isoforms Public Health RNA Interference RNA primers Reaction Recombinants Recycling Reduce health disparities Relaxation Reporting Research Running Saccharomyces cerevisiae Sodium Chloride Source Structure Superhelical DNA Temperature Testing Therapeutic Therapeutic Intervention Topoisomerase Topoisomerase II Topoisomerase Inhibitors Topoisomerase-II Inhibitor Training Trypanosoma Trypanosoma brucei brucei Trypanosoma cruzi Work analog base clinical application cofactor deviant dimer divalent metal enzyme activity gel electrophoresis health disparity high throughput screening improved inhibitor inorganic phosphate insight interest medical schools mindfulness neglect neglected tropical diseases next generation novel overexpression pathogen preference small molecule small molecule inhibitor therapeutic development therapeutic target

项目摘要

Project Summary/Abstract Type IIA topoisomerases (topo II) are ubiquitous molecular machines that manage DNA superhelical structure and decatenate DNA entanglements to support critical processes such as transcription, DNA replication, and chromosome segregation. Topo II relies on ATP to capture and pass one DNA segment through a reversible, topo-II mediated, double-strand break in a second DNA segment. With numerous clinically proven antibiotics and anti-cancer drugs targeting the essential, yet risky, activities of these enzymes, the ATP-dependence of the topo II strand passage reaction is well-established; however, how topo IIs use ATP and local DNA interactions to favor unidirectional strand passage activity (ensuring genomic knots are removed, not formed) is unknown. In sharp contrast to other topo IIs, the mitochondrial-specific type IIA topoisomerase from trypanosomes (TxTopoIImt) is reported to possess an unexpected ATP-independent strand passage activity. In addition, TxTopoIImt appears to switch between canonical topo II activities (e.g., decatenating DNA) and the antithetical activity of catenating DNA molecules. Switching of strand passage directionality is believed to be how TxTopoIImt perpetuates networks of mitochondrial DNA, known as kinetoplast or kDNA, which comprise thousands of DNA circles interlocked into a giant structure akin to medieval chain mail. The unique kDNA structure is a hallmark of trypanosomatids, recondite parasites that cause three neglected tropical diseases: African sleeping sickness, Chagas disease, and leishmaniasis. The goal of this project is to develop TxTopoIImt as a therapeutic target by understanding and exploiting the fundamental mechanisms underlying the enzyme’s deviant activities. Strategies outlined in Aim 1 will mechanistically define the strange activities of TxTopoIImt in vitro, which are now possible due to a recent breakthrough in producing soluble TxTopoIImt purified from recombinant sources. Cofactor requirements for TxTopoIImt will be assessed (with in vitro topoisomerase decatenation and supercoil relaxation assays) and the molecular determinants of DNA strand passage directionality will be explored (using singly-catenated DNA substrates of various compositional topologies). The objectives of Aim 2 are to characterize the mechanisms by which small-molecule inhibitors of purified TxTopoIImt (identified with in vitro screens of both clinically known topo II inhibitors and the Johns Hopkins FDA-Approved Drug Library) affect enzymatic activities, and then validate the anti-parasitic therapeutic potential of these inhibitors with killing assays of bloodstream-form African trypanosome cultures. Together, these aims have the potential to offer novel mechanistic insights into general topo II function, establish a molecular understanding of parasitic trypanosomes’ peculiar biology, and support future research efforts to develop novel treatments for trypanosome infections. As such, this project draws upon the collaborative academic environment found at Johns Hopkins School of Medicine to provide basic biophysical training that is mindful of translational opportunities for clinical application.

项目概要/摘要 IIA 型拓扑异构酶 (topo II) 是普遍存在的管理 DNA 超螺旋结构的分子机器并连接 DNA 缠结以支持关键过程，例如转录、DNA 复制和 Topo II 依靠 ATP 捕获 DNA 片段并将其通过可逆的、 topo-II 介导的第二个 DNA 片段的双链断裂，具有多种经过临床验证的抗生素。和针对这些酶的基本但有风险的活性的抗癌药物，这些酶的 ATP 依赖性 topo II 链传代反应已得到充分证实；然而，topo II 如何利用 ATP 和局部 DNA 相互作用有利于单向链传递活性（确保基因组结被去除，而不是形成）尚不清楚。与其他拓扑异构酶 II 形成鲜明对比的是，来自锥虫的线粒体特异性 IIA 型拓扑异构酶据报道，(TxTopoIImt) 具有意想不到的 ATP 依赖性链传代活性。 TxTopoIImt 似乎在典型的拓扑 II 活性（例如，连接 DNA）和对立的拓扑活性之间切换连接 DNA 分子的活性被认为是 TxTopoIImt 的作用机制。维持线粒体 DNA 网络，称为动质体或 kDNA，由数千个 DNA 组成圆圈交织成一个类似于中世纪锁子甲的巨大结构，独特的 kDNA 结构是其标志。锥虫，一种神秘的寄生虫，会导致三种被忽视的热带疾病：非洲昏睡病，该项目的目标是开发 TxTopoIImt 作为治疗靶点。了解和利用酶异常活动的基本机制。目标 1 中概述的策略将机械地定义 TxTopoIImt 的体外奇怪活动，这些活动是由于最近在生产从重组来源纯化的可溶性 TxTopoIImt 方面取得了突破，现在成为可能。将评估 TxTopoIImt 的辅因子要求（通过体外拓扑异构酶串联和超螺旋弛豫测定）和 DNA 链通道方向性的分子决定因素将被探索（使用不同组成拓扑的单链 DNA 底物）目标 2 的目标是表征纯化 TxTopoIImt 的小分子抑制剂的机制（通过体外鉴定临床已知的拓扑 II 抑制剂和约翰·霍普金斯 FDA 批准的药物库的筛选）酶活性，然后通过杀灭试验验证这些抑制剂的抗寄生虫治疗潜力血流形式的非洲锥虫文化的共同作用，这些目标有可能提供新颖的。对一般拓扑 II 功能的机制见解，建立对寄生锥虫的分子理解独特的生物学，并支持未来开发锥虫感染新疗法的研究工作。因此，该项目利用了约翰·霍普金斯大学的协作学术环境医学提供基本的生物物理培训，注重临床应用的转化机会。