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，是当今最常用的抗癌药物。喜树碱类药物是一种特异性最强的毒物，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。首先，我们已经确定了新的喜树碱，在血液中更稳定，这应该是有用的临床候选人。其次，我们已经确定了人前列腺癌细胞系的特征，并发现了一个新的top1突变，赋予喜树碱耐药。有趣的是，该残基与DNA紧密接触，这与我们的模型一致，即喜树碱与top1切割复合物的DNA切割位点中的+1碱基堆叠。在top2区域，我们已经确定了top2 α-4区域的突变对DNA切割活性和对药物的反应具有深远的影响。从这些结果中，我们推断，这种α-4螺旋对于与在拓扑异构化反应期间被切割的DNA双链体（G链）相互作用是至关重要的。这个区域也可能与药物相互作用，这与top2毒素在酶-DNA界面的结合是一致的。首先，我们继续研究我们与Paull和Cushman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。其次，我们发现海鞘素743是一种新的top1毒素。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（鸟嘌呤N2）中形成加合物。我们最近的研究结果表明，海鞘素743在细胞中形成蛋白质连接的DNA断裂，并且top1是药物的靶点。与喜树碱相反，这些断裂位于基因组的特定区域，并且在药物去除后非常持久，这应该使它们具有高度细胞毒性。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。- 乳腺癌，细胞周期，化疗，结肠直肠癌，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,