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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

基本信息

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
项目类别：
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

项目摘要

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这种行为提供生化和分子蓝图