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，核酸代谢的抑制一直被认为是一种强大的化疗靶点，但有一个重大缺陷——非选择性杀死患病细胞和健康细胞。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疗药物的效果，这些核苷类似物与未修饰的对应物相比，会优先插入受损的 DNA 对面。通过靶向促突变 DNA 合成，这些类似物将阻碍耐药性的发展以及因受损 DNA 不适当复制而引起的其他疾病。 目标 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核苷类似物，这些类似物优先插入到该 DNA 损伤对面。我们将采用动力学方法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的类似物的独特电子特征相关联。该目标的完成将为合理靶向 DNA 损伤、选择性抑制 DNA 聚合酶的能力以及定义 DNA 聚合中 p 电子相互作用的贡献提供证据。 我们假设这些非天然核苷将选择性地抑制参与 DNA 修复和/或易错合成的聚合酶。因此，预计它们会增强诱导 DNA 损伤的化疗药物的效果。 目标 2 将通过定量评估每种类似物在细胞水平上的功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 淋巴细胞癌细胞系 (CEM-7) 作为我们的初始模型进行一系列细胞毒性研究来解决。在定义了每种非天然核苷类似物的效力和选择性后，我们将描述诱导细胞死亡和/或抗增殖行为的细胞途径，以描述其作用机制。最后，将通过测量与 DNA 损伤剂结合使用的核苷的细胞毒性作用来评估这些类似物的增强作用。这一目标的完成将为治疗提供新的范例。癌症通常以细胞周期失调为特征，涉及细胞周期蛋白依赖性激酶活性的改变。 目标 3 将测试非天然类似物抑制此类激酶调节的细胞周期进程的能力。体外实验将描述作用机制。该目标产生的结果将有助于更好地了解参与调节细胞周期的酶。随着靶点变得更加分化，这些知识将促进新药的开发。