DIYNENE ANTIBIOTICS AND THEIR DNA CLEAVAGE CHEMISTRY

二炔抗生素及其 DNA 裂解化学

• 批准号: 2414220
• 负责人: CRAIG ARTHUR TOWNSEND
• 金额: $ 17.04万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-05-10 至 1999-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2414220
• 关键词: DNA damage; RNA; adduct; aminoglycoside antibiotics; antineoplastic antibiotics; cell cycle; chemical cleavage; chemical kinetics; cyclization; cytotoxicity; deuterium; gel electrophoresis; nuclear magnetic resonance spectroscopy; nucleic acid sequence; nucleosomes; radiotracer

Calicheamicin gamma1I (CLM), the neocarzinostatin chromophore, esperamicin A1, dynemicin, kedarcidin, c-1027 and maduropeptin are members of the structurally unprecedented and growing family of diynene antitumor antibiotics. Reductive activation by thiols and/or simple thermal rearrangement generates diradical species by an electrocyclization process that is characteristic of this class. When bound in the minor groove of DNA these radicals initiate helix cleavage by hydrogen abstraction from one or both strands. The resulting DNA radicals react with molecular oxygen and fragmentation ensues. While single-strand breaks are typically the major cleavage event observed, CLM is both notably sequence-selective and very largely a double-strand cutter. Using a diverse array of experiments outlined in this renewal proposal we seek to understand the origins of these properties and how CLM can be used as a probe of protein/nucleic acid structure and in the design of useful tools in molecular biology. The presumed lethality of DNA damage, the induction of apoptosis and the demonstrated cytotoxicity and cell cycle effects of these compounds have animated hope that, through preferential uptake or selective delivery to cancer cells, effective therapies might be possible. Clinical trials testing the latter principle are underway. Investigation of the low kinetic isotope effects associated with CLM- induced hydrogen abstraction from DNA and the apparent absence of isotope-induced branching will be completed. Whether these two events occur in a simultaneous or stepwise manner will be distinguished. The unusual behavior of A-tracts will be examined for kinetic acceleration as a function of minor groove narrowing. The efficiencies of hydrogen abstraction will be monitored and the distribution of cuts in the cleavage cascade will be evaluated as a function of temperature. The interaction of CLM with supercoiled plasmid DNA will be investigated as a function of inserted A/T-rich segments causing varying degrees of curvature. Extensive experiments are planned with nucleosomes to study the occurrence of "hot spots" to understand whether CLM cleavage is associated with physical location in the nucleosome or features of particular DNA sequences. Automated methods will be used to evaluate large amounts of data generated in mixed sequence nucleosomes. A degradation product of CLM having essentially no sequence selectivity but retaining double-strand cutting properties will be linked to homeodomains to achieve specific binding for high efficiency cleavage of DNA. Success in this effort will be evaluated in tests with DNA sequences already in hand and will lay the groundwork for eventual experiments to be undertaken for gene identification e.g. in Drosophila. Finally, CLM reaction with transfer-, ribosomal- and messenger-RNA will be investigated. In eukaryotic cells, RNA is a more accessible target than DNA. These experiments will monitor the susceptibility of RNA to reaction with CLM and particularly with rRNA in the ribosome for comparison to nucleosomes and chromatin.

Calicheamicin Gamma1I(CLM)，新卡西诺抑素发色团， 埃司帕米星A1、达尼米星、克达西丁、c-1027和马多肽素 结构上史无前例且不断增长的二芳烃家族的成员 抗肿瘤抗生素。硫醇和/或简单的还原活化 热重排生成双自由基物种通过一种 电环化过程是这一类的特征。什么时候 这些自由基结合在DNA的小沟中，启动了螺旋切割 通过从一条或两条链中提取氢。由此产生的DNA 自由基与分子氧反应，随后发生裂解。而当 单链断裂通常是观察到的主要切割事件 既是明显的序列选择性，又是非常大的双链 卡特。使用本次更新中概述的一系列不同实验 我们试图了解这些属性的起源以及如何 激光共聚焦显微镜可作为蛋白质/核酸结构的探针，并可用于蛋白质/核酸结构的研究 分子生物学中有用工具的设计。推定的致命性 DNA损伤、诱导细胞凋亡及其细胞毒性 这些化合物对细胞周期的影响激发了人们的希望， 通过优先摄取或选择性地输送到癌细胞， 有效的治疗方法或许是可能的。测试后者的临床试验 原则正在进行中。 与CLM相关的低动能同位素效应的研究 诱导从DNA中抽氢和明显缺乏 同位素诱导的分支将完成。这两件事是否 同时发生的或逐步发生的将被区分开来。这个 A形束的异常行为将被检查动加速度 作为小凹槽变窄的函数。氢气的效率 将监测抽象化和削减在 解理级联将作为温度的函数进行评估。这个 CLM与超螺旋DNA的相互作用将被研究如下 插入的富A/T段的功能导致不同程度的 曲率。计划对核小体进行广泛的实验以进行研究 了解CLM卵裂是否 与核小体中的物理位置或特征有关 特定的DNA序列。将使用自动化方法来评估 在混合序列核小体中产生的大量数据。一个 CLM的降解产物基本上没有序列选择性，但 保留双链切割特性将与同源结构域相连 以实现高效切割DNA的特异性结合。成功 这一努力将在DNA序列测试中进行评估 并将为最终的实验奠定基础 用于基因鉴定的，例如在果蝇身上。最后，CLM 与转移、核糖体和信使RNA的反应将是 调查过了。在真核细胞中，rna是一个比 DNA这些实验将监测rna对反应的敏感性。 与CLM，特别是与核糖体中的rRNA进行比较 核小体和染色质。