Chemical Approaches to DNA Topoisomerase Inhibition and Function

描述（由申请人提供）：DNA拓扑异构酶（Topo）是在DNA复制、转录和染色体分离过程中维持基因组DNA超螺旋拓扑结构所必需的酶。所有拓扑异构酶都是通过使用活性位点酪氨酸攻击DNA主干，形成动态共价磷酸酪氨酸连锁和柔性链缺口的机制特征而结合在一起的，这在DNA代谢和药理学中具有非常重要的意义。拓扑异构酶长期以来一直是结合和稳定共价复合物的药物的靶标，但在二十多年来尚未发现机制新颖的化合物类别。这种竞争性更新旨在阐明酶DNA共价复合物的动力学和移动性如何促进酶催化的各种DNA转化，包括药物结合。我们还致力于开发一个化学平台，以发现抑制或毒害人类，细菌和寄生虫I型Topo的新型小分子配体。这项工作的意义在于将这些酶的基本动态特征与生物学功能和药物作用联系起来。目的是：(i)了解拓扑异构酶IB DNA复合物的动态迁移如何导致超级线圈松弛和药物抑制。Topo IB的DNA松弛的自由链旋转模型要求DNA片段3'与共价连接暂时从其与酶的非共价相互作用中释放出来，以允许DNA绕超螺旋轴旋转。使用19F NMR松弛方法，我们将研究共价结合DNA的动力学，使用在DNA复合物的刚性和动态区域特异性标记19F的新型底物。（ii）了解拓扑异构酶IB DNA复合物的动态迁移如何导致重组DNA链交换反应。由Topo促进的易链交换反应也需要在裂解位点附近的DNA双链的动力学和热力学不稳定。我们将探索这些关键的机制方面使用新的核磁共振微量质子交换测量，并停止流动动力学和热力学测量。（三）发展化学工具以快速分析拓扑异构酶的活性，并发现调节拓扑功能的新配体。一个完整的高通量拓扑异构酶筛选库将被开发，以促进发现新的小分子拓扑异构酶抑制剂或毒药。这些方法将为临床样品的拓扑异构酶活性分析提供新的分析工具。

DESCRIPTION (provided by applicant): DNA topoisomerases (Topo) are essential enzymes required to maintain the superhelical topology of genomic DNA during the processes of DNA replication, transcription and chromosome segregation. All topoisomerases are united by the mechanistic feature of using an active site tyrosine to attack the DNA backbone and form a dynamic covalent phosphotyrosine linkage and flexible strand nick that is of extraordinary importance in DNA metabolism and pharmacology. Topoisomerases have long been targets for drugs that bind to and stabilize the covalent complex, but mechanistically novel classes of compounds have not been discovered in more than two decades. This competitive renewal seeks to elucidate how the dynamics and mobility of the enzyme DNA covalent complex facilitate the various DNA transformations catalyzed by the enzyme, including drug binding. We also aim to develop a chemical platform to discover new classes of small molecule ligands that inhibit or poison human, bacterial and parasite type I Topo's. The significance of this work is linking the essential dynamic features of these enzymes to biological function and drug action. The aims are to: (i) Understand how the dynamic mobility of the topoisomerase IB DNA complex leads to supercoil relaxation and drug inhibition. The free strand rotation model for DNA relaxation by Topo IB requires that the DNA segment 3' to the covalent attachment is transiently released from its noncovalent interactions with the enzyme to allow rotation of the DNA around the superhelical axis. Using 19F NMR relaxation methods, we will investigate the dynamics of the covalently bound DNA using novel substrates that are specifically labeled with 19F in rigid and dynamic regions of the DNA complex. (ii) Understand how the dynamic mobility of the topoisomerase IB DNA complex leads to recombinogenic DNA strand exchange reactions. The facile strand exchange reactions promoted by Topo also necessitate dynamic and thermodynamic destabilization of the DNA duplex near to the cleavage site. We will explore these key mechanistic aspects using novel NMR imino proton exchange measurements, and stopped flow kinetic and thermodynamic measurements. (iii) Develop chemical tools to rapidly profile the activities of topoisomerases and discover new ligands that modulate Topo function. A complete repertoire of high throughput topoisomerase screens will be developed to facilitate the discovery of novel small molecule topoisomerase inhibitors or poisons. These methods will provide new profiling tools for topoisomerase activity in clinical samples.