Targeting Twist: Single Molecule Insights into the Topological Dependence of DNA - Topoisomerase Interactions

靶向扭曲：单分子洞察 DNA 拓扑依赖性 - 拓扑异构酶相互作用

• 批准号: 2066521
• 金额: --
• 依托单位: University College London
• 依托单位国家: 英国
• 项目类别: Studentship
• 财政年份: 2018
• 资助国家: 英国
• 起止时间: 2018 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=studentship-2066521
• 关键词: Targeting; Twist; Single; Molecule; Insights

Topoisomerase inhibitors are a vital class of therapeutics used in oncology. They target ubiquitous topoisomerase enzymes which are responsible for maintaining the state of cellular DNA. Topoisomerase-targeting therapeutics are currently challenged by off-target toxicity and the emergence of drug resistance, and a comprehensive understanding of their mechanism of action remains elusive. Furthermore, some topoisomerases remain untargeted despite a strong rational for their effectiveness in eliciting cell death. A complete description of the catalytic cycle of topoisomerases and the mechanics of their inhibition will aid in improving the selectivity, specificity, and safety of these therapeutics and facilitate an improved drug refinement and development pipeline.Underpinning this is a better understanding of the dynamics between the therapeutic target, topoisomerases, and cellular DNA. In particular, a consideration of how these enzymes interact with DNA under topological or supercoiling stress, has limited exploration.Topoisomerases relieve stress in DNA by cutting and rearranging DNA through nanometre conformational changes on the order of seconds within a complex cellular environment. Here, we perform single molecule in vitro studies to improve our understanding of how DNA structure varies under topological stress, and how this affects topoisomerase activity, using Atomic Force Microscopy. Atomic Force Microscopy scans a sharp tip over molecules immobilised on a surface in fluid, to 'feel' the contours of the molecule with nanometre precision and sub-second temporal resolution.The overarching aim of my project is to therefore study the topological dependence of DNAtopoisomerase/ topoisomerase inhibitors interactions to facilitate therapeutic refinement.We use small closed circular DNA molecules with controlled levels of superhelical stress to mimic the globally underwound state of genomic eukaryotic DNA and investigate changes in higher order structure in response to supercoiling. To this end, we have observed the onsetof defects in the double helical structure of closed circular DNA at physiological levels of superhelical stress. These defects increase the local flexibility of DNA resulting in increased conformational heterogeneity. Currently, we are using bacterial topoisomerases, in particularGyrase, to gain preliminary results about binding affinities and preferences of these enzymes to supercoiled DNA. We observe preferential binding of Gyrase, with the majority of binding events located at the outside of both open and kinked conformers inducing conformationalchanges in DNA proximal to the binding site. These observations provide insight into the spatial and energetic requirements of these enzymes, upon which we are able to optimise our imaging conditions further.Subsequently, we aim to optimise a protocol that allows us to achieve high-resolution dynamic imaging of eukaryotic topoisomerases (TOP1 and TOP2) to supercoiled DNA. This will allow us to gain information regarding topoisomerase structure but also the conformational changes that the enzymes undergo during catalysis. This will be followed by the addition of gold-standard topoisomerase inhibitors; the selection of which will be based on current practice guidelines such that our research is driven by clinical practice. In turn, these experiments will be complemented by the use of novel topoisomerase inhibitors, such as with the idenoisoquinolone drug class, to visually compare mechanistic inhibitory effects.

拓扑异构酶抑制剂是用于肿瘤学的重要类别的治疗剂。它们靶向负责维持细胞DNA状态的普遍存在的拓扑异构酶。拓扑异构酶靶向治疗目前受到脱靶毒性和耐药性的挑战，并且对其作用机制的全面理解仍然难以捉摸。此外，一些拓扑异构酶仍然是非靶向的，尽管它们在引发细胞死亡中的有效性有很强的合理性。拓扑异构酶的催化循环及其抑制机制的完整描述将有助于提高这些治疗药物的选择性，特异性和安全性，并促进改善药物的精制和开发pipeline. Understanding这是一个更好地理解治疗靶点，拓扑异构酶和细胞DNA之间的动力学。特别是，考虑到这些酶如何与DNA相互作用的拓扑或超螺旋应力，有有限的exploration.Topoisomerases减轻压力，通过切割和重排DNA通过纳米构象变化在一个复杂的细胞环境中的秒级。在这里，我们使用原子力显微镜进行单分子体外研究，以提高我们对DNA结构在拓扑应力下如何变化以及这如何影响拓扑异构酶活性的理解。原子力显微镜用一个尖端扫描固定在液体表面的分子，以纳米级的精度和亚秒级的时间分辨率来“感受”分子的轮廓。因此，我的项目的首要目标是研究DNA拓扑异构酶/拓扑异构酶抑制剂的相互作用，以促进治疗的完善。我们使用小的封闭环状DNA分子与控制水平的超螺旋应力，以模拟基因组真核DNA的全局欠缠绕状态，并研究响应于超螺旋的高阶结构的变化。为此，我们观察到在生理水平的超螺旋应力下闭合环状DNA双螺旋结构中的缺陷的发生。这些缺陷增加了DNA的局部柔性，导致构象异质性增加。目前，我们正在利用细菌拓扑异构酶，特别是Gyrase，获得有关这些酶与超螺旋DNA结合亲和力和偏好性的初步结果。我们观察到优先结合的促旋酶，与大多数的结合事件位于外部的开放和扭结的构象诱导conformalchanges在DNA近端的结合位点。这些观察提供了深入了解这些酶的空间和能量的要求，在此基础上，我们能够进一步优化我们的成像conditions.Subsequently，我们的目标是优化协议，使我们能够实现高分辨率的动态成像真核拓扑异构酶（TOP 1和TOP 2）超螺旋DNA。这将使我们能够获得有关拓扑异构酶结构的信息，也可以获得酶在催化过程中发生的构象变化。随后将添加金标准拓扑异构酶抑制剂;其选择将基于当前的实践指南，以便我们的研究由临床实践驱动。反过来，这些实验将通过使用新的拓扑异构酶抑制剂来补充，例如与异喹诺酮类药物一起，以视觉上比较机制抑制作用。