Non-natural Nucleoside Inhibitors of DNA Polymerases

DNA 聚合酶的非天然核苷抑制剂

• 批准号: 6983500
• 负责人: ANTHONY J BERDIS
• 金额: $ 24.17万
• 依托单位: CASE WESTERN RESERVE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-06-01 至 2010-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6983500
• 关键词: DNA directed DNA polymerase; DNA repair; DNA replication; SDS polyacrylamide gel electrophoresis; cell growth regulation; cyclin dependent kinase; cytotoxicity; enzyme inhibitors; high performance liquid chromatography; neoplastic cell culture for noncancer research; nuclear magnetic resonance spectroscopy; nucleic acid inhibitor; nucleic acid metabolism; nucleoside analog; nucleoside inhibitor; site directed mutagenesis; synthetic nucleic acid; western blottings

DESCRIPTION (provided by applicant): The inhibition of nucleic acid metabolism has long been recognized as a powerful chemotherapeutic target having one significant pitfall - non-selective killing of both diseased and healthy cells. We propose a unique strategy to potentiate the effects of existing chemotherapeutic agents by using nucleoside analogs that display preferential insertion opposite damaged DNA versus their unmodified counterparts. By targeting promutagenic DNA synthesis, these analogs will hinder the development of drug resistance and other diseases caused by inappropriate replication of damaged DNA. Aim 1 will use our experience in medicinal chemistry and enzymology to further develop a series of novel nucleoside analogs that are preferentially inserted opposite this DNA lesion. We shall employ a kinetic approach to generate a structure/function relationship for each non-natural analog to correlate the catalytic efficiency of insertion with the unique electronic signature of the substituted analog. Completion of this Aim will provide evidence for rationally targeting DNA lesions, the ability to selectively inhibit DNA polymerases, and define the contributions of p-electron interactions in DNA polymerization. We hypothesize that these non-natural nucleosides will selectively inhibit polymerases involved in DNA repair and/or error-prone synthesis. They are therefore predicted to potentiate the effects of chemotherapeutic agents that induce DNA damage. Aim 2 will test the functionality of these analogs by quantitatively evaluating the efficacy of each analog at the cellular level. The anti-proliferative effects of our nucleoside analogs will be addressed through a series of cell cytotoxicity studies using a T-lymphoblast cancer cell line (CEM-7) as our initial model. After defining the potency and selectivity of each non-natural nucleoside analog, we shall characterize the cellular pathways accounting for the induction of cell death and/or anti-proliferative behavior to describe their mechanism of action. Finally, the potentiating effects of these analogs will be evaluated by measuring the cytotoxic effect of the nucleosides used in conjunction with DNA damaging agents. Completion of this Aim will provide a new paradigm for treatment. Cancer is frequently characterized by cell cycle deregulation involving altered activity of cyclin-dependent kinases. Aim 3 will test the ability of the non-natural analogs to inhibit cell-cycle progression regulated by this class of kinase. In vitro experiments will delineate the mechanism of action. Results generated from this Aim will provide a greater understanding of the enzymes involved in regulating the cell cycle. This knowledge will facilitate the development of new drugs as the repertoire of targets becomes more differentiated.

描述（由申请人提供）： 长期以来，对核酸代谢的抑制作用已被公认为是一个有明显陷阱的强大化学治疗靶标 - 对患病和健康细胞的非选择性杀害。我们提出了一种独特的策略，通过使用核苷类似物来增强现有化学治疗剂的影响，这些核苷类似物与未修饰的DNA相对损坏的DNA相反。通过靶向正当的DNA合成，这些类似物将阻碍耐药性和其他疾病的发展，原因是受损的DNA不当复制引起的疾病。 AIM 1将利用我们在药物化学和酶学方面的经验进一步开发一系列新型的核苷类似物，这些类似物优先插入该DNA病变。我们将采用动力学方法来为每个非天然类似物生成结构/功能关系，以将插入的催化效率与取代类似物的唯一电子特征相关联。该目标的完成将为理性靶向DNA病变，选择性抑制DNA聚合酶的能力以及定义P电子相互作用在DNA聚合中的贡献。 我们假设这些非天然核苷将有选择地抑制参与DNA修复和/或易折叠合成的聚合酶。因此，预测它们会增强诱导DNA损伤的化学治疗剂的作用。 AIM 2将通过定量评估每个类似物在细胞水平上的疗效来测试这些类似物的功能。我们的核苷类似物的抗增殖作用将通过使用T-浓细胞细胞癌细胞系（CEM-7）作为我们的初始模型来解决一系列细胞细胞毒性研究。在定义了每个非天然核苷类似物的效力和选择性之后，我们将表征细胞死亡和/或抗增殖行为的细胞途径，以描述其作用机理。最后，这些类似物的增强作用将通过测量与DNA损害剂结合使用的核苷的细胞毒性作用来评估。此目标的完成将为治疗提供新的范式。癌症通常以细胞周期消耗为特征，涉及细胞周期蛋白依赖性激酶的活性改变。 AIM 3将测试非天然类似物抑制该类激酶调节的细胞周期进展的能力。体外实验将描述作用机理。从这个目标产生的结果将对调节细胞周期涉及的酶有更多的了解。随着目标的曲目变得更加区分，这些知识将促进新药的发展。