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

• 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
• 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

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.

项目摘要/摘要 IIA型拓扑异构酶（Topo II）是无处不在的分子机器，可以管理DNA超螺旋结构 和脱发DNA纠缠，以支持转录，DNA复制和 染色体分离。 TOPO II依靠ATP捕获并通过一个可逆的DNA段， TOPO-II在第二个DNA段中介导的双链断裂。具有大量临床证明的抗生素 和抗癌药物针对这些酶的基本但有风险的活性，ATP依赖性 Topo II链条通过反应已建立了良好的。但是，TOPO IIS如何使用ATP和局部DNA相互作用 为了有利于单向链传递活性（确保去除基因组结，而不是形成）是未知的。 与其他TOPO IIS形成鲜明对比，锥虫的线粒体特异性IIA型tososomerase 据报道（TXTOPOIIMT）假设存在意外的非ATP链条传递活性。此外， txtopoiimt似乎在规范的Topo II活动（例如脱发DNA）和对立之间切换 arnat依的DNA分子的活性。据信，链通道的切换是txtopoiimt的方式 永久性线粒体DNA的网络，称为kinetoplast或kDNA，该网络包含数千个DNA 圈子互锁到类似于中世纪链邮件的巨大结构。独特的kDNA结构是 锥形剂，培育寄生虫会引起三种被忽视的热带疾病：非洲昏昏欲睡的疾病， 查加斯病和利什曼病。该项目的目的是通过 理解和利用酶偏差活动的基本机制。 AIM 1中概述的策略将在体外机械地定义TxTopoiimt的奇怪活动， 现在，由于最近产生从重组来源纯化的固体txtopoiimt的突破而可能。 将评估TXTOPOIIMT的辅助因子要求（使用体外拓扑异构酶衰减和超二元 将探索放松测定）和DNA链通道方向的分子决定剂（使用 各种组成拓扑的单一诱导的DNA底物）。目标2的目标是 表征了纯化txtopoiimt的小分子抑制剂的机制（并在体外鉴定 临床上已知的Topo II抑制剂和约翰·霍普金斯FDA批准的药物库的筛选） 酶促活性，然后通过杀伤测定法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培养物。这些目标在一起有可能提供小说 对一般TOPO II功能的机械洞察力，建立对寄生锥虫的分子理解 奇特的生物学，并支持未来的研究工作，以开发锥虫感染的新型治疗方法。作为 这样的项目借鉴了约翰·霍普金斯学校的协作学术环境 医学提供基本的生物物理培训，这些培训注意到临床应用的转化机会。