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.

项目总结/摘要 IIA型拓扑异构酶（topoII）是一种普遍存在的控制DNA超螺旋结构的分子机器 和十链DNA缠结，以支持关键过程，如转录，DNA复制， 染色体分离Topo II依赖于ATP捕获并通过一个可逆的DNA片段， topo-II介导的第二DNA片段中的双链断裂。具有多种临床证明的抗生素 以及针对这些酶的基本但有风险的活性的抗癌药物， topo II链通过反应是公认的;然而，topo II如何使用ATP和局部DNA相互作用 以有利于单向链通过活性（确保基因组结被去除，而不是形成）是未知的。在 与其他拓扑异构酶II形成鲜明对比的是，锥虫特异性IIA型拓扑异构酶 （TxTopoIImt）具有出乎意料的ATP非依赖性链通过活性。此外，本发明还提供了一种方法， TxTopoIImt似乎在典型的拓扑II活动之间切换（例如，decatenating DNA）和对立的 链化DNA分子的活性。链通道方向性的转换被认为是TxTopolimt 使线粒体DNA网络永久化，称为动质体或kDNA，它包含数千个DNA 圆圈联锁成一个巨大的结构，类似于中世纪的锁子甲。独特的kDNA结构是 锥虫是一种隐蔽的寄生虫，可引起三种被忽视的热带疾病：非洲昏睡病， 恰加斯病和利什曼病。该项目的目标是通过以下方式将TxTopoIImt开发为治疗靶点： 理解和利用酶的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在体外的奇怪活性，其为 由于最近在生产从重组来源纯化的可溶性TxTopoIImt方面的突破， 将评估TxTopoIImt的辅因子要求（体外拓扑异构酶脱链和超螺旋 松弛试验）和DNA链通过方向性的分子决定因素将被探索（使用 各种组成拓扑结构的单链DNA底物）。目标2的目标是 表征纯化的TxTopoIImt的小分子抑制剂（用体外鉴定）的机制， 临床已知的拓扑异构酶II抑制剂和约翰霍普金斯FDA批准的药物库的筛选）影响 酶活性，然后用杀伤试验验证这些抑制剂的抗寄生虫治疗潜力 非洲锥虫的血液流培养。总之，这些目标有可能提供新的 一般拓扑结构II功能的机制见解，建立寄生锥虫的分子理解 独特的生物学，并支持未来的研究工作，以开发新的治疗锥虫感染。作为 因此，该项目借鉴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，注意临床应用的转化机会。