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

项目摘要/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。 并消除DNA缠结以支持关键过程，如转录、DNA复制和 染色体分离。Topo II依靠三磷酸腺苷捕获DNA片段并通过可逆的， TOPO-II介导的第二个DNA片段双链断裂。有许多经过临床证明的抗生素 以及针对这些酶的基本但有风险的活性的抗癌药物，这些酶对ATP的依赖 Topo II链传递反应已经建立；然而，Topo II如何利用ATP和局部DNA相互作用 支持单向链传递活性(确保基因组结被移除，而不是形成)尚不清楚。在……里面 与其他Topo II形成鲜明对比的是锥虫线粒体特异性IIA拓扑异构酶 (TxTopo IImt)据报道具有意想不到的ATP非依赖性链传递活性。此外, TxTopo IImt似乎在规范的Topo II活性(例如，杀死DNA)和相反的Topo II活动之间切换 连接DNA分子的活性。链通道方向的切换被认为是TxTopOIImt 使线粒体DNA网络永久化，这种网络被称为动丝泡体或kDNA，由数千个DNA组成 圆圈交织成一个巨大的结构，类似于中世纪的锁链邮件。独特的kDNA结构是 锥虫，引起三种被忽视的热带疾病的隐形寄生虫：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开发为治疗目标 了解和利用酶异常活性背后的基本机制。 目标1中概述的策略将机械地定义TxTopo IImt在体外的奇怪活动，这些活动是 现在，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，TxTopoIImt成为可能。 将评估TxTopOIImt的辅因子需求(使用体外拓扑异构酶去除和超级线圈 松弛分析)和DNA链通道方向性的分子决定因素(使用 各种组成拓扑的单链DNA底物)。目标2的目标是 鉴定纯化的TxTopo IImt的小分子抑制剂的机制(与体外鉴定) 临床已知的Topo II抑制剂和Johns Hopkins FDA批准的药物库的筛选)影响 酶活性，然后用杀灭试验验证这些抑制剂的抗寄生虫治疗潜力 血液形态的非洲锥虫培养物。这些目标加在一起，有可能提供新奇的 对一般Topo II功能的机械性洞察，建立对寄生锥虫的分子理解 独特的生物学，并支持未来开发治疗锥虫感染的新方法的研究工作。AS 这样，这个项目利用了约翰霍普金斯大学的合作学术环境。 医学提供基本的生物物理训练，注意临床应用的翻译机会。

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.