Novel approaches for studying topoisomerase 2 targeting anti-cancer drugs

研究靶向抗癌药物的拓扑异构酶 2 的新方法

• 批准号: 9306402
• 负责人: JOHN L NITISS
• 金额: $ 8万
• 依托单位: UNIVERSITY OF ILLINOIS AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-04-01 至 2019-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9306402
• 关键词: ATP phosphohydrolase; ATPase Domain; Adopted; Affect; Affinity; Alleles; Antineoplastic Agents; Binding; Biochemical; Biochemistry; Biological Assay; C-terminal; Cell Death; Cells; Characteristics; Clinical; Collaborations; Complex; Crystallization; Cytotoxic agent; DNA; DNA Binding; DNA Damage; DNA Double Strand Break; DNA Repair; DNA Structure; DNA Topoisomerases; DNA strand break; Dimerization; Doxorubicin; Drug Hypersensitivity; Drug Interactions; Drug Targeting; Drug effect disorder; Drug resistance; Ectopic Expression; Enzymes; Etoposide; Exhibits; Generations; Genetic Transcription; Goals; Human; Hydrolysis; Hypersensitivity; Lead; Lesion; Location; Mediating; Mitoxantrone; Modeling; Molecular; Molecular Conformation; Mutation; Names; Pharmaceutical Preparations; Plasmids; Poison; Poisoning; Process; Protein Isoforms; Proteins; RAD52 gene; Reaction; Reagent; Research; Resolution; Resources; Site; Testing; Topoisomerase; Topoisomerase II; Transducers; Universities; Work; Yeasts; base; cancer cell; cell type; chemotherapeutic agent; cytotoxicity; dimer; drug sensitivity; enzyme activity; experimental study; insight; interest; killings; mutant; neurotensin mimic 2; novel; novel strategies; overexpression; phosphodiester; prevent; programs; repaired; response; screening; small molecule; targeted agent; tumor

The long-term goal of our research program is to provide a biochemical and molecular blueprint for the action of anti-cancer drugs that target DNA topoisomerase 2 (Top2). Top2 carries out changes in DNA structure needed for efficient transcription, replication, and DNA repair. These enzymes introduce transient double strand breaks in DNA through a protein/DNA covalent intermediate termed the cleavage complex. This intermediate allows cells to catalyze changes in DNA conformation without the dangers of frank DNA double strand breaks. Anti-cancer drugs such as etoposide and doxorubicin generate elevated levels of cleavage complexes, leading to cytotoxicity. The biochemical details of trapping Top2 covalent complexes remain poorly understood. We devised a variety of approaches for developing novel reagents for studying Top2/drug interactions. One key strategy is to isolate and characterize mutants in eukaryotic Top2 with altered drug sensitivity. Classically, studying drug/protein interactions is facilitated by the identification of drug resistant mutant proteins. However, this approach has proven unsatisfactory for topoisomerases, since any mutations that reduce enzyme activity typically confer high levels of drug resistance. We have adopted two alternate approaches. First, we devised screens for the identification of drug hypersensitive alleles of Top2. This approach used ectopic expression of human Top2 enzymes (both Top2α and Top2β isoforms have been functionally expressed in yeast) with screening for alleles that conferred elevated levels of sensitivity to etoposide. We identified a large number of mutants with greatly elevated levels of sensitivity to etoposide. In our proposed experiments, we will carry out biochemical analyses of the hypersensitive alleles. One class of mutant alleles that is of great interest is mutants in the Top2 ATPase domain. Current models suggest that etoposide interacts with the protein at the protein/DNA interface. Therefore, the mutants we have identified in the ATPase domain likely regulate progression through the catalytic cycle rather than directly affect drug/protein interactions. How the enzyme regulates progression through the catalytic cycle, and how this progression affects drug binding and action remains poorly understood. A second approach is based on recently identified mutants of yeast Top2 that require cells be proficient in DNA repair for viability. Biochemical analysis of the purified mutant proteins demonstrated elevated levels of drug independent DNA cleavage. Therefore, the mutant Top2 mimics the action of Top2 poisons and are termed self-poisoning Top2 mutants. We expanded this observation to human Top2, and have identified several self-poisoning mutations in human Top2α. We hypothesize that these mutants will also have elevated levels of drug independent DNA cleavage. Biochemical and structural analysis of these mutants will lend unique insights into the processes of DNA cleavage and religation by human Top2 proteins. Taken together, our work will provide unique reagents and insights into the action of anti-cancer drugs that target Top2.

我们研究计划的长期目标是为行动提供生物化学和分子蓝图 靶向DNA拓扑异构酶2（Top2）的抗癌药物。Top2进行DNA结构的改变 这是高效转录、复制和DNA修复所必需的。这些酶将瞬时双链 通过称为切割复合物的蛋白质/DNA共价中间体的DNA链断裂。这 中间体允许细胞催化DNA构象的变化，而没有坦率的DNA加倍的危险。 链断裂。抗癌药物，如依托泊苷和阿霉素产生高水平的切割 复合物，导致细胞毒性。捕获Top2共价复合物的生物化学细节仍然很差 明白我们设计了多种方法来开发用于研究Top2/药物的新型试剂。 交互.一个关键的策略是分离和表征真核Top2中的突变体， 灵敏度传统上，研究药物/蛋白质相互作用是通过鉴定耐药蛋白来促进的。 突变蛋白然而，这种方法已被证明对拓扑异构酶是不令人满意的，因为任何突变 降低酶活性通常赋予高水平抗药性。我们采用了两个候补 接近。首先，我们设计了用于鉴定Top2的药物过敏等位基因的筛选。这 该方法使用人Top2酶的异位表达（Top2α和Top2β同种型都已被发现）。 在酵母中功能性表达），并筛选赋予高水平敏感性的等位基因， 依托泊苷我们鉴定了大量的突变体，其对依托泊苷的敏感性水平大大提高。在 在我们提议的实验中，我们将对过敏等位基因进行生化分析。一类 非常感兴趣的突变等位基因是Top2 ATP酶结构域中的突变体。目前的模型表明， 依托泊苷在蛋白质/DNA界面与蛋白质相互作用。因此，我们已经确定的突变体， ATP酶结构域可能通过催化循环调节进程，而不是直接影响 药物/蛋白质相互作用。酶如何通过催化循环调节进展，以及这是如何 进展影响药物结合，作用仍然知之甚少。第二种方法是基于 最近鉴定出酵母Top2的突变体，其需要细胞精通DNA修复以保持活力。生化 对纯化的突变蛋白的分析表明非药物依赖性DNA切割水平升高。 因此，突变体Top2模拟Top2毒物的作用，并被称为自毒Top2突变体。 我们将这一观察扩展到人类Top2，并在人类中发现了几个自毒突变。 Top2α.我们假设这些突变体也将具有升高的药物非依赖性DNA切割水平。 对这些突变体的生化和结构分析将为DNA的过程提供独特的见解。 通过人Top2蛋白的切割和再连接。总之，我们的工作将提供独特的试剂， 深入了解靶向Top2的抗癌药物的作用。