DNA Topoisomerases as Target of Action of Anticancer Dru

DNA拓扑异构酶作为抗癌药物的作用靶点

• 批准号: 7337933
• 负责人: YVES POMMIER
• 金额: --
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7337933
• 关键词: DNA; Topoisomerases; Target; Action; Anticancer

DNA topoisomerases (Top1 & Top2) are the targets for some of the most effective anticancer therapeutics. The Top2 inhibitors, etoposide and DNA intercalators (such as adriamycin and derivatives) are commonly used anticancer drugs. Camptothecins are specific Top1 poisons and have recently been approved by the FDA for the treatment of colon and ovarian carcinomas.The goals of this project are: i) to elucidate the molecular interactions between topoisomerase inhibitors and their target enzymes, ii) to elucidate the molecular pathways that determine the response to topoisomerase inhibitors in cancer cells, iii) discover novel topoisomerase inhibitors, and iv) elucidate the function of mitochondrial topoisomerase I (Top1mt).We have provided further evidence that topoisomerase I (Top1) inhibitors are a paradigm for interfacial inhibitors. Crystal structure studies have now established that 5 different Top1 inhibitors (topotecan, natural camptothecin, an indenoisoquinoline, a norindenoisoquinoline and an indolocarbazole) all bind at the Top1-DNA interface when the Top1 forms its transient DNA cleavage complex intermediates. We refer to this type of inhibition as "interfacial inhibition" and propose this type of inhibition to be one of Nature's paradigms for drug discovery. This concept has profound implication for the discovery of inhibitors of macromolecular complexes that stabilize protein complexes (novel approach) rather than screening only for drugs that prevent the formation or dissociate protein complexes (past and current approach). We have determined the structures of several Top1-DNA complexes with single point mutations resulting in camptothecin resistance. These studies provide molecular examples of structural alterations propagated from distal point mutants to enzyme active sites. They also provide evidence for the validity of the enzyme-DNA structures to be used for molecular docking and rational drug discovery.We have pursued our investigations to discover novel Top1 inhibitors to alleviate the limitations of camptothecins while retaining their potent antitumor activity. The indenoisoquinolines were discovered in collaboration with Dr. Cushman. The indenoisoquinolines have several potential advantages over camptothecins: 1/ they are chemically stable; 2/ they trap topoisomerase I cleavage complexes at specific genomic sites that differ from those trapped by camptothecins; 3/ their cellular half-life is much longer than camptothecins with cleavage complexes that are more stable than those trapped by camptothecins. The indenoisoquinolines have been selected for clinical development by the NCI. Four derivatives are in the development pipeline of DTP with the goal of making the indenoisoquinolines the first NCI-discovered drugs in the Phase 0 pipeline.In spite of the same induction of Top1 cleavage complexes in cancer and normal tissues, Top1 inhibitors exhibit some selectivity for cancer tissues. One of our goal is to determine the molecular determinants that account for this selectivity. We aim to translate this knowledge to the clinic by developing novel therapeutic approaches that increase the selectivity of Top1 inhibitors for cancer tissues, and by providing molecular biomarkers to direct therapeutic choices and follow therapeutic responses to Top1 inhibitors. Our first approach is to study step by step ("dissect") the molecular pathways activated by camptothecins and Top1 inhibitors. Our recent studies have implicated phosphorylation of BLM (on threonine 99) in association with phosphorylation of histone H2AX (gamma-H2AX). We are also finding phosphorylation of Chk2, which is conditional for presence of Mre11-Rad50-Nbs1 complexes. We are investigating the functional relevance of these molecular pathways in cells that are deficient for these pathways.

DNA 拓扑异构酶（Top1 和 Top2）是一些最有效的抗癌疗法的靶标。 Top2抑制剂、依托泊苷和DNA嵌入剂（如阿霉素及其衍生物）是常用的抗癌药物。喜树碱是特定的 Top1 毒物，最近已被 FDA 批准用于治疗结肠癌和卵巢癌。该项目的目标是：i) 阐明拓扑异构酶抑制剂与其靶酶之间的分子相互作用，ii) 阐明拓扑异构酶抑制剂与其靶酶之间的分子相互作用。决定癌细胞对拓扑异构酶抑制剂反应的途径，iii) 发现新型拓扑异构酶抑制剂，iv) 阐明线粒体的功能拓扑异构酶 I (Top1mt)。我们提供了进一步的证据表明拓扑异构酶 I (Top1) 抑制剂是界面抑制剂的范例。晶体结构研究现已确定，当 Top1 形成其瞬时 DNA 裂解复合物中间体时，5 种不同的 Top1 抑制剂（托泊替康、天然喜树碱、茚并异喹啉、去甲茚并异喹啉和吲哚咔唑）均结合在 Top1-DNA 界面上。我们将这种类型的抑制称为“界面抑制”，并提议将这种类型的抑制作为《自然》药物发现的范例之一。这一概念对于发现稳定蛋白质复合物的大分子复合物抑制剂（新方法）具有深远的意义，而不是仅筛选阻止形成或解离蛋白质复合物的药物（过去和当前的方法）。我们已经确定了几种具有导致喜树碱抗性的单点突变的 Top1-DNA 复合物的结构。这些研究提供了从远端点突变体传播到酶活性位点的结构改变的分子例子。它们还为用于分子对接和合理药物发现的酶-DNA 结构的有效性提供了证据。我们一直致力于发现新型 Top1 抑制剂，以减轻喜树碱的局限性，同时保留其有效的抗肿瘤活性。茚并异喹啉是与 Cushman 博士合作发现的。与喜树碱相比，茚并异喹啉有几个潜在的优点： 1/ 化学性质稳定； 2/ 它们在特定基因组位点捕获拓扑异构酶 I 裂解复合物，该位点与喜树碱捕获的位置不同； 3/ 它们的细胞半衰期比喜树碱长得多，其裂解复合物比喜树碱捕获的裂解复合物更稳定。 NCI 已选择茚并异喹啉进行临床开发。 DTP 正在开发四种衍生物，目标是使茚并异喹啉成为第 0 期 NCI 发现的药物。尽管在癌症和正常组织中对 Top1 裂解复合物的诱导相同，但 Top1 抑制剂对癌组织。我们的目标之一是确定导致这种选择性的分子决定因素。我们的目标是通过开发新的治疗方法来将这些知识转化为临床，以提高 Top1 抑制剂对癌症组织的选择性，并提供分子生物标志物来指导治疗选择并跟踪对 Top1 抑制剂的治疗反应。我们的第一个方法是逐步研究（“剖析”）喜树碱和 Top1 抑制剂激活的分子途径。我们最近的研究表明 BLM（苏氨酸 99）的磷酸化与组蛋白 H2AX (gamma-H2AX) 的磷酸化相关。我们还发现 Chk2 的磷酸化，这是 Mre11-Rad50-Nbs1 复合物存在的条件。我们正在研究这些分子途径在缺乏这些途径的细胞中的功能相关性。