DNA Topoisomerases as Target of Action of Anticancer Drugs

DNA拓扑异构酶作为抗癌药物的作用靶点

• 批准号: 6433071
• 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/6433071
• 关键词: DNA topoisomerases; antineoplastics; apoptosis; camptothecin; cell growth regulation; cytotoxicity; drug design /synthesis /production; drug interactions; drug screening /evaluation; enzyme activity; enzyme inhibitors; enzyme mechanism; pharmacokinetics; point mutation; tissue /cell culture; tumor suppressor genes

DNA topoisomerases (top1 & top2) are important targets for anticancer therapeutics. The top2 inhibitors, etoposide and DNA intercalators (such as adriamycin and derivatives) are the most commonly used anticancer drugs today. Camptothecins are specific top1 poisons and have recently been approved by the FDA for the treatment of human carcinomas resistant to prior chemotherapy. 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 respond to topoisomerase-mediated DNA damage and contribute to the selectivity of topoisomerase inhibitors in cancer cells, and iii) discover novel topoisomerase inhibitors.Goal 1: To elucidate the molecular interactions between topoisomerase inhibitors and their target enzymes, we have set up a baculovirus expression system for high expression of recombinant top1. We have used this top1 enzyme with oligonucleotides containing a single polycyclic aromatic adduct that mimics a topoisomerase inhibitor, and found that intercalation at the site of top1 cleavage mimics the effect of camptothecin. Based on molecular modeling and crystal structure data, we proposed that polycyclic aromatics intercalate in the DNA and stabilize an intermediate in which a DNA base is flipped out of the DNA duplex. A second approach to elucidate the drug binding sites has been to identify top1 mutations that selectively confer camptothecin resistance. Analysis of camptothecin-resistant human prostate carcinoma cell lines (DU145/RC.1 & 1) demonstrated that amino acid residue 364 of top1 is important for camptothecin activity (interaction with top1?). In these cells, mutation of arginine 364 to histidine confers high resistance to camptothecin both with the mutated recombinant top1 enzyme and in cells.Goal 2: To elucidate the molecular pathways that respond to topoisomerase-mediated DNA damage, we have initiated studies with a newly discovered enzyme, tyrosyl-DNA-phosphodiesterase (TDP-1) that selectively removes the tyrosyl residue bound at the 3-end of the DNA. In collaboration with Dr. Grandas (University of Barcelona) and Dr. Nash (NIH), we found that the activity of TDP-1 is optimum when the top1 peptide is short and when it is linked to a long DNA oligonucleotide. This suggests that the catalytic site of TDP-1 interacts both with the DNA and a short peptide segment. These findings underline the potential importance of top1 proteolysis prior to TDP-1 action in cells.Goal 3: We have pursued our investigations for the discovery and molecular pharmacology investigations of novel topoisomerase I inhibitors. First, in the areas of camptothecins, we have identified novel camptothecins with enhanced stability in the bloodstream and which should be useful clinical candidates. We have also discovered in collaboration with Dr. Gamcsik (Duke University) and Dr. Wall (Research Triangle Institute) new camptothecin-peptide conjugates (glutathione bound to position 7 of camptothecin) that produce remarkably stable top1 cleavage complexes. These compounds have been patented because they can be used to specifically deliver drugs to the tumor cells. Secondly, we have continued our studies on the indenoisoquinolines that we discovered in collaboration with Drs Cushman. We now have more potent top1 poisons that are being investigated for pre-clinical development. Finally, we have started investigations on indolocarbazole derivatives that are a new class of top1 inhibitors that will be introduced in the clinic soon. We are currently determining: i) the drug molecular interactions with top1 using various camptothecin-resistant top1 mutants in cells and in biochemical assays, and ii) whether top1 is the only target of indolocarbazoles using cell lines with top1 alterations that should confer drug resistance.

DNA 拓扑异构酶（top1 和 top2）是抗癌治疗的重要靶标。 Top2 抑制剂、依托泊苷和 DNA 嵌入剂（如阿霉素及其衍生物）是当今最常用的抗癌药物。喜树碱是特定的顶级毒物，最近被 FDA 批准用于治疗对先前化疗耐药的人类癌症。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) 阐明响应拓扑异构酶介导的 DNA 损伤并有助于拓扑异构酶抑制剂在癌细胞中选择性的分子途径，以及iii) 发现新型拓扑异构酶抑制剂。目标 1：为了阐明拓扑异构酶抑制剂与其靶酶之间的分子相互作用，我们设定了建立了杆状病毒表达系统以高表达重组top1。我们将这种top1酶与含有模拟拓扑异构酶抑制剂的单个多环芳香加合物的寡核苷酸一起使用，并发现top1切割位点的插入模拟了喜树碱的作用。基于分子模型和晶体结构数据，我们提出多环芳族化合物插入 DNA 中并稳定中间体，其中 DNA 碱基从 DNA 双链体中翻转出来。阐明药物结合位点的第二种方法是识别选择性赋予喜树碱抗性的 top1 突变。对喜树碱抗性人前列腺癌细胞系 (DU145/RC.1 & 1) 的分析表明，top1 的氨基酸残基 364 对于喜树碱活性很重要（与 top1 相互作用？）。在这些细发现了酪氨酰-DNA-磷酸二酯酶 (TDP-1)，它可以选择性地去除结合在 DNA 3 端的酪氨酰残基。 我们与 Grandas 博士（巴塞罗那大学）和 Nash 博士（NIH）合作，发现当 top1 肽较短且与长 DNA 寡核苷酸连接时，TDP-1 的活性最佳。这表明 TDP-1 的催化位点与 DNA 和短肽片段相互作用。这些发现强调了细胞中 TDP-1 作用之前 top1 蛋白水解的潜在重要性。目标 3：我们一直致力于新型拓扑异构酶 I 抑制剂的发现和分子药理学研究。首先，在喜树碱领域，我们已经确定了在血液中稳定性增强的新型喜树碱，它们应该是有用的临床候选物。 我们还与 Gamcsik 博士（杜克大学）和 Wall 博士（三角研究所）合作发现了新的喜树碱-肽缀合物（与喜树碱的 7 位结合的谷胱甘肽），可产生非常稳定的 top1 裂解复合物。这些化合物已获得专利，因为它们可用于特异性地将药物递送至肿瘤细胞。其次，我们继续与库什曼博士合作发现的茚并异喹啉的研究。 我们现在拥有更有效的顶级毒药，正在研究其临床前开发。最后，我们已经开始对吲哚并咔唑衍生物进行研究，这是一类新型的 top1 抑制剂，即将引入临床。 我们目前正在确定：i) 在细胞和生化测定中使用各种喜树碱抗性 top1 突变体与 top1 的药物分子相互作用，以及 ii) 使用具有应赋予耐药性的 top1 改变的细胞系，top1 是否是吲哚并咔唑的唯一靶标。