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

拓扑异构酶抑制剂是肿瘤学中一类重要的治疗药物。它们的靶标是普遍存在的负责维持细胞DNA状态的拓扑异构酶。拓扑异构酶靶向治疗目前面临靶外毒性和耐药性出现的挑战，对其作用机制的全面了解仍然难以捉摸。此外，一些拓扑异构酶仍然没有靶向，尽管它们在诱导细胞死亡方面具有很强的合理性。完整描述拓扑异构酶的催化循环及其抑制机制将有助于提高这些治疗药物的选择性、特异性和安全性，并有助于改进药物精制和开发流水线。了解这一点将有助于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，考虑这些酶如何在拓扑或超螺旋压力下与DNA相互作用，探索有限。在复杂的细胞环境中，拓扑异构酶通过切割和重新排列DNA，通过几秒钟量级的纳米构象变化来缓解DNA中的压力。在这里，我们进行单分子的体外研究，以提高我们对DNA结构在拓扑应力下的变化，以及这如何影响拓扑异构酶活性的理解，使用原子力显微镜。原子力显微镜扫描流体中固定在表面上的分子的尖端，以纳米级的精度和亚秒级的时间分辨率‘感觉’分子的轮廓。因此，我项目的首要目标是研究DNA拓扑异构酶/拓扑异构酶抑制剂相互作用的拓扑依赖关系，以促进治疗的改进。我们使用具有可控超螺旋应力水平的小的闭合环形DNA分子来模拟真核基因组DNA的整体缠绕状态，并研究响应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的双螺旋结构中出现了缺陷。这些缺陷增加了DNA的局部灵活性，导致了构象异质性的增加。目前，我们正在使用细菌拓扑异构酶，特别是Gyrase，来获得关于这些酶与超螺旋DNA的结合亲和力和偏好的初步结果。我们观察到旋转酶的优先结合，大多数结合事件都位于开放构象和扭结构象的外部，导致结合部位附近的DNA构象变化。这些观察提供了对这些酶的空间和能量需求的洞察，在此基础上，我们能够进一步优化我们的成像条件。因此，我们的目标是优化一种方案，使我们能够实现真核细胞拓扑异构酶(TOP1和TOP2)到超螺旋DNA的高分辨率动态成像。这将使我们能够获得关于拓扑异构酶结构的信息，也可以获得酶在催化过程中经历的构象变化。紧随其后的是添加金标准拓扑异构酶抑制剂；这些药物的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，例如使用相同的异喹诺酮类药物，以直观地比较机制抑制效果。

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.