DNA TOPOISOMERASES AS TARGET OF ACTION OF ANTICANCER DRUGS

DNA拓扑异构酶(top1和top2)已被确定为抗癌研究的重要靶点。依托泊苷和DNA嵌入剂是Top2抑制剂，也是当今最常用的抗癌药物。喜树碱是一种特殊的TOP1毒药，最近已被引入临床。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)发现新的拓扑异构酶抑制剂。我们继续在喜树碱领域进行研究。首先，我们已经确定了新的喜树碱，它们在血液中更稳定，应该可以作为临床候选药物。其次，我们已经鉴定了一种人类前列腺癌细胞系，并发现了一种新的Top1突变，它赋予了喜树碱耐药性。有趣的是，这个残基与DNA密切接触，这与我们的模型一致，喜树碱堆积在top1裂解复合体DNA裂解位点的+1碱基上。在top2区域，我们确定top2的α-4区突变对DNA裂解活性和药物反应有深远影响。根据这些结果，我们推断这个α-4螺旋对于与在拓扑异构化反应中切割的DNA双链(G链)相互作用是关键的。这个区域也可能与药物相互作用，这与Top2毒素在酶-DNA界面上的结合是一致的。我们也一直在研究新的非喜树碱Top1抑制剂。首先，我们继续我们与Paull&Cushman博士合作发现的吲哚异喹啉的研究。我们现在有更多有效的TOP1毒药，正在进行临床前开发的研究。其次，我们发现Eecteinassidin 743是一种新的TOP1毒物。ECTEINASCIDIN 743是一种在临床试验中非常有效的抗癌药物，它在DNA小沟(鸟氨酸N_2)中形成加合物。我们最近的结果表明，ecteinassidin 743在细胞中形成蛋白质连接的DNA断裂，top1是该药物的靶点。与喜树碱不同的是，这些断裂位于基因组的特定区域，在药物移除后非常持久，这应该会使它们具有高度的细胞毒性。最后，我们已经开始了对吲哚咔唑类化合物的研究，这是一类即将引入临床的新型TOP1抑制剂。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耐药的top1突变体与top1的药物分子相互作用，以及ii)top1是否是使用具有top1改变的细胞系的吲哚卡巴索的唯一靶点，这应该会导致耐药性。-乳腺癌、细胞周期、化疗、结直肠癌、DNA结合蛋白、DNA修复、药理学、蛋白酶、

DNA topoisomerases (top1 & top2) have been identified as essential targets for anticancer research. Etoposide and DNA intercalators are top2 inhibitors and are the most commonly used anticancer drugs today. Camptothecins are specific top1 poisons and have recently been introduced in the clinic. The goals of this project are: i) to elucidate the molecular interactions between topoisomerase inhibitors and their target enzymes and ii) to discover novel topoisomerase inhibitors.We have continued our investigations in the areas of camptothecins. First, we have identified novel camptothecins that are more stable in the bloodstream and which should be useful as clinical candidates. Secondly, we have characterized a human prostate carcinoma cell line and found a novel top1 mutation that confers camptothecin resistance. Interestingly, this residue is in close contact with the DNA, which is consistent with our model that camptothecins stack against the +1 base in the DNA cleavage site of the top1 cleavage complex.In the area of top2, we have determined that mutations in the alpha-4 region of top2 have profound impact on DNA cleavage activity and response to drugs. From these results, we infer that this alpha-4 helix is critical for interacting with the DNA duplex that is cleaved during the topoisomerization reaction (G strand). This same region also probably interacts with drugs, which is consistent with the binding of top2 poison at the enzyme-DNA interface.We have also been studying novel non-camptothecin top1 inhibitors. First, we have continued our studies on the indenoisoquinolines that we discovered in collaboration with Drs. Paull & Cushman. We now have more potent top1 poisons that are being investigated for pre-clinical development. Secondly, we have found that ecteinascidin 743 is a novel top1 poison. Ecteinascidin 743 is an extremely potent anticancer drug in clinical trials, which forms adducts in the DNA minor groove (guanine N2). Our recent results indicate that ecteinascidin 743 forms protein-linked DNA breaks in cells and that top1 is a target of the drug. By contrast to camptothecins, these breaks are located in specific regions of the genome and are very persistent after drug removal, which should make them highly cytotoxic. 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 indolocarbaxoles using cell lines with top1 alterations that should confer drug resistance. - breast cancer, Cell Cycle, chemotherapy, colorectal cancer, DNA binding proteins, DNA repair, Pharmacology, proteases,