DNA Topoisomerases as Target of Action of Anticancer Drugs

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插入剂（如阿霉素及其衍生物）是当今最常用的抗癌药物。喜树碱是一种特殊的top1毒药，最近被FDA批准用于治疗对化疗有抗药性的人类癌症。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损伤的分子途径，并有助于拓扑异构酶抑制剂在癌细胞中的选择性；iii)发现新的拓扑异构酶抑制剂。目的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，我们建立了重组top1的杆状病毒表达系统。我们将这种top1酶与含有单一多环芳香族加合物的寡核苷酸一起使用，这种加合物可以模拟拓扑异构酶抑制剂，并发现在top1裂解位点插入类似喜树碱的作用。基于分子模型和晶体结构数据，我们提出了多环芳烃插入DNA并稳定DNA碱基从DNA双链中翻转出来的中间体。第二种阐明药物结合位点的方法是鉴定选择性赋予喜树碱耐药性的top1突变。喜树碱耐药人前列腺癌细胞株DU145/RC的分析。1 & 1)证明了top1的氨基酸残基364对喜树碱活性（与top1的相互作用）很重要。在这些细胞中，精氨酸364突变为组氨酸，在突变的重组top1酶和细胞中都对喜树碱具有高抗性。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，我们已经开始了一种新发现的酶的研究，酪氨酸-DNA-磷酸二酯酶（TDP-1）选择性地去除结合在DNA 3端的酪氨酸残基。在与Grandas博士（巴塞罗那大学）和Nash博士（美国国立卫生研究院）的合作中，我们发现TDP-1的活性在top1肽较短且与较长的DNA寡核苷酸连接时是最佳的。这表明TDP-1的催化位点与DNA和短肽段相互作用。这些发现强调了在细胞中TDP-1作用之前，top1蛋白水解的潜在重要性。目标3：我们对新型拓扑异构酶I抑制剂的发现和分子药理学研究进行了研究。首先，在喜树碱领域，我们已经确定了新的喜树碱，它在血液中具有更高的稳定性，应该是有用的临床候选药物。我们还与杜克大学的Gamcsik博士和三角研究所的Wall博士合作发现了新的喜树碱肽偶联物（谷胱甘肽结合到喜树碱的第7位），可以产生非常稳定的top1切割复合物。这些化合物已经获得了专利，因为它们可以用来专门向肿瘤细胞输送药物。其次，我们继续对我们与库什曼博士合作发现的吲哚异喹啉进行研究。我们现在有更多强效的top1毒药正在进行临床前开发研究。最后，我们已经开始研究吲哚咔唑衍生物，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)在细胞和生化分析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，ii) top1是否是吲哚咔唑的唯一靶标，使用top1突变的细胞系应该赋予耐药性。

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.