Anti-topoisomerase drug action in yeast

描述（由申请人提供）：拓扑异构酶II（Top2）是多种活性抗癌剂的靶标。靶向Top2的药物是广泛使用的活性药物，但对它们如何对抗酶的详细了解已被证明是难以捉摸的。临床活性Top2靶向剂导致酶介导的DNA损伤，并且被称为Top2毒物。关于Top2毒物作用的详细信息有可能导致更安全和更有效的抗癌药物。酵母是理解DNA拓扑异构酶的生物学和生物化学性质的一个重要模型系统。这种生物体中可用的遗传工具也使其成为研究靶向拓扑异构酶的抗癌药物作用机制的强大系统。酵母Top2也是真核Top2结构研究的首选酶。该项目的总体目标是应用酵母系统可用的所有工具，以详细了解Top2毒物如何与酶相互作用，以及Top2药物相互作用如何导致Top2介导的DNA损伤。我们提出了一个遗传，生物化学和结构的方法相结合，以了解其目标酶的Top2毒药的行动。遗传学研究将允许分离和表征Top2的新型药物超敏突变体。这些研究将增加我们对Top2毒物如何与酶结合并产生酶介导的DNA损伤的理解。用于评估药物作用的测定，包括药物亲和力和酶介导的DNA切割的测量，将用于测试突变蛋白中如何发生药物超敏反应。我们还计划研究一种新的人类Top2突变体，它在没有药物的情况下模仿Top2毒药的作用。该突变体的研究将提供Top2介导的DNA损伤在整个反应周期的背景下的生物化学的理解。最后，药物过敏突变体将为结构研究提供必要的试剂，这些研究将试图解决药物：蛋白质：DNA三元复合物的结构。该实验为Top2靶向药物的设计提供了理论依据，也为Top2靶向药物的临床应用提供了新的思路。 公共卫生相关性：拓扑异构酶II（Top2）是常用的活性抗癌药物的靶点。虽然这些药物已经使用多年，但对它们如何对Top2起作用的详细了解已被证明是难以捉摸的。拟议的研究将提供Top2靶向药物如何与酶相互作用的详细信息，并可能导致设计更安全，更有效的靶向这种酶的药物所需的工具。

DESCRIPTION (provided by applicant): Topoisomerase II (Top2) is the target of a wide range of active anti-cancer agents. Drugs targeting Top2 are widely used and active agents, but a detailed understanding of how they act against the enzyme has proven elusive. Clinically active Top2 targeting agents lead to enzyme mediated DNA damage, and are referred to as Top2 poisons. Detailed information about the action of Top2 poisons has the potential for leading to safer and more active anti-cancer drugs. An important model system for understanding the biological and biochemical properties of DNA topoisomerases is the yeast Saccharomyces cerevisiae. The genetic tools available in this organism also make it a powerful system for studying mechanisms of action of anti-cancer drugs targeting topoisomerases. Yeast Top2 has also been the enzyme of choice for structural studies of eukaryotic Top2. The overall goal of this project is to apply all of the tools available with the yeast system to obtain a detailed understanding of how Top2 poisons interact with the enzyme, and how Top2 drug interactions lead to Top2 mediated DNA damage. We propose a combination of genetic, biochemical and structural approaches to understand the action of Top2 poisons on their target enzymes. Genetic studies will allow the isolation and characterization of novel drug hypersensitive mutants of Top2. These studies will increase our understanding of how Top2 poisons bind to the enzyme and generate enzyme-mediated DNA damage. Assays for assessing drug action, including measurements of drug affinity and enzyme mediated DNA cleavage will be applied to test how drug hypersensitivity occurs in the mutant proteins. We also plan to study a novel mutant of human Top2 that mimics the action of Top2 poisons in the absence of drug. Studies with this mutant will provide a biochemical understanding of Top2 mediated DNA damage in the context of the overall reaction cycle. Finally, the drug hypersensitive mutants will provide necessary reagents for structural studies that will attempt to solve the structure of drug:protein:DNA ternary complexes. The proposed experiments will be useful in the design of new agent targeting Top2, and may suggest more effective use of Top2 targeting drugs that are in clinical use. PUBLIC HEALTH RELEVANCE: Topoisomerase II (Top2) is the target of active anti-cancer agents that in commonly use. While these drugs have been in use for many years, a detailed understanding of how they act against the Top2 has proven elusive. The proposed studies will provide a detailed picture of how Top2 targeting drugs interact with the enzyme, and may lead to tools needed for designing safer and more effective drugs that target this enzyme.