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点。 Top2抑制剂依托泊苷和DNA嵌入剂（如阿霉素及其衍生物）是目前最常用的抗癌药物。喜树碱类药物是一种特异性最强的药物，最近FDA批准其用于治疗对化疗耐药的人类癌症。本项目的目标是：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的选择性的分子途径，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，我们建立了一个高效表达重组top1的杆状病毒表达系统。我们已经使用了这种top1酶与寡核苷酸含有一个多环芳香族加合物，模拟拓扑异构酶抑制剂，并发现在网站的top1切割嵌入模拟喜树碱的效果。基于分子模型和晶体结构数据，我们提出多环芳烃插入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鉴定选择性赋予喜树碱抗性的top1突变。喜树碱耐药的人前列腺癌细胞系（DU145/RC. 1和1）的分析表明，氨基酸残基364的top1是重要的喜树碱活性（与top1？）。在这些细胞中，精氨酸364突变为组氨酸，在突变的重组top1酶和细胞中都赋予了对喜树碱的高抗性。目标2：为了阐明响应拓扑异构酶介导的DNA损伤的分子途径，我们启动了对新发现的酶酪氨酰-DNA-磷酸二酯酶（TDP-1）的研究，它选择性地去除结合在DNA 3-末端的酪氨酰残基。 在与Grandas博士（巴塞罗那大学）和Nash博士（NIH）的合作中，我们发现当top1肽较短并且与长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和短肽段相互作用。这些研究结果强调了潜在的重要性top1蛋白水解之前TDP-1的行动在cells.Goal 3：我们一直在追求我们的调查发现和分子药理学研究的新型拓扑异构酶I抑制剂。首先，在喜树碱领域，我们已经鉴定了在血流中具有增强的稳定性并且应该是有用的临床候选物的新型喜树碱。 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（谷胱甘肽结合到喜树碱的7位），其产生非常稳定的top1切割复合物。这些化合物已经获得专利，因为它们可以用于将药物特异性地递送到肿瘤细胞。其次，我们继续研究我们与库什曼博士合作发现的茚并异喹啉。 我们现在有更强的顶级毒药，正在研究临床前的发展。最后，我们已经开始了对吲哚并咔唑衍生物的研究，这是一类新的top1抑制剂，将很快被引入临床。 我们目前正在确定：i）在细胞和生物化学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，和ii）使用具有应赋予药物抗性的Top1改变的细胞系，Top1是否是吲哚并咔唑的唯一靶标。