DNA Topoisomerases as Target of Action of Anticancer Dru

DNA拓扑异构酶作为抗癌药物的作用靶点

• 批准号: 7048145
• 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/7048145
• 关键词: DNA damage; DNA repair; DNA topoisomerases; active sites; adduct; antineoplastics; camptothecin; cell growth regulation; drug discovery /isolation; drug resistance; drug screening /evaluation; enzyme activity; enzyme complex; enzyme inhibitors; enzyme mechanism; enzyme structure; intermolecular interaction; mitochondria; molecular oncology; neoplasm /cancer chemotherapy; neoplasm /cancer pharmacology; oligonucleotides; pharmacokinetics; point mutation; tissue /cell culture

DNA topoisomerases (top1 and top2) are the targets for some of the most effective 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 determine the response to topoisomerase inhibitors in cancer cells, iii) discover novel topoisomerase inhibitors, and iv) elucidate the function of mitochondrial topoisomerase I.Goal 1: Our recent studies demonstrate that topoisomerase inhibitors are a paradigm for drug discovery as these drugs alter the binding of topoisomerases to DNA by binding at the enzyme-DNA interface when the topoisomerases form their transient DNA cleavage complex intermediates. We refer to this type of inhibition as "interfacial inhibition" and propose this type of inhibition to be one of Nature's paradigms for drug discovery. This concept has profound implication for the discovery of inhibitors of macromolecular complexes that stabilize protein complexes (novel approach) rather than screening only for drugs that prevent the formation or dissociate protein complexes (past and current approach). We have determined the structures of several topoisomerase I-DNA complexes with single point mutations resulting in camptothecin resistance. These studies provide a rationale for the drug-resistance mutations. They also provide evidence for the validity of the enzyme-DNA structures to be used for molecular docking and rational drug discovery. To further elucidate the molecular interactions between topoisomerase inhibitors and their target enzyme-DNA complexes, we have studied topoisomerase-mediated cleavage of oligonucleotides containing site-specific modification, such as a single polycyclic aromatic adduct that mimics a topoisomerase inhibitor. We found that intercalation at the sites of topoisomerase cleavage mimics the effect of camptothecin with topoisomerase I and of intercalating anticancer drugs in the case of topoisomerase II. We have also found that acetaldehyde adducts, which form readily during alcohol consumption can enhance camptothecin-induced topoisomerase I-DNA complexes.Goal 2: Enhanced drug efflux is a common resistance mechanism to therapy. Camptothecins are transported by the half transporter ABCG2, otherwise named BCRP or MXR. We have evaluated the implication of ABCG2 in the activity of camptothecins. We found the new camptothecin derivatives, the homocamptothecins to be more active than the camptothecins presently in clinical trials in ABCG2-overexpressing cell lines. We have also looked at differential expression of the ABC transporter in camptothecin-resistant cells. To elucidate the molecular pathways that respond to topoisomerase-mediated DNA damage, we have continued our studies with the newly discovered enzyme, tyrosyl-DNA-phosphodiesterase (TDP-1) that selectively removes the tyrosyl residue bound at the 3'-end of the DNA. We have demonstrated that Tdp1 is associated with XRCC1, the scaffolding protein in the BER (Break-Induced Repair) pathway, and that cells deficient for XRCC1 are selectively hypersensitive to camptothecin. Using recombinant Tdp-1 and modified oligonucleotides, we are looking for TDP-1 inhibitors to block the repair pathways downstream from topoisomerase I-mediated DNA damage in order to selectively enhance the activity of camptothecins in checkpoint-deficient cells.Goal 3: We have pursued our investigations for the discovery and molecular pharmacology investigations of novel topoisomerase I inhibitors. The indenoisoquinolines were discovered in collaboration with Dr. Cushman. The indenoisoquinolines have several potential advantages over camptothecins: 1/ they are chemically stable; 2/ they trap topoisomerase I cleavage complexes at specific genomic sites that differ from those trapped by camptothecins; 3/ their cellular half-life is much longer than camptothecins with cleavage complexes that are more stable than those trapped by camptothecins. We recently obtained co-crystals of one of the indolocarbazoles bound to the topoisomerase I-DNA complex. We now have more potent top1 poisons that are being investigated for pre-clinical development.Goal 4: We discovered human mitochondrial topoisomerase I, a specific enzyme encoded by a nuclear gene. We have now found the presence of homologs in all vertebrate genomes sequenced: mouse, rat, chicken, and zebra fish. However, the gene is absent in non-vertebrate including the Ciona intestinalis, yeast and plants. We are currently looking for mitochondrial DNA damage that may be induced by mitochondrial topoisomerase I, and attempting to knock out the gene in mice.

DNA拓扑异构酶（top1和top2）是一些最有效的抗癌疗法的靶点。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。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）阐明决定癌细胞中对拓扑异构酶抑制剂的反应的分子途径，iii）发现新型拓扑异构酶抑制剂，以及iv）阐明线粒体拓扑异构酶I的功能。我们最近的研究表明，拓扑异构酶抑制剂是药物发现的一个范例，因为这些药物通过结合在拓扑异构酶与DNA的结合位点来改变拓扑异构酶与DNA的结合。当拓扑异构酶形成其瞬时DNA切割复合物中间体时，DNA界面。我们将这种类型的抑制称为“界面抑制”，并提出这种类型的抑制是自然界的药物发现范例之一。这一概念对于发现稳定蛋白质复合物的大分子复合物抑制剂（新方法）而不是仅筛选阻止蛋白质复合物形成或解离的药物（过去和当前的方法）具有深远的意义。我们已经确定了几个拓扑异构酶I-DNA复合物的结构与单点突变导致喜树碱耐药。这些研究为耐药突变提供了理论依据。它们还为酶-DNA结构用于分子对接和合理药物发现的有效性提供了证据。为了进一步阐明拓扑异构酶抑制剂和它们的靶酶-DNA复合物之间的分子相互作用，我们研究了拓扑异构酶介导的含有位点特异性修饰的寡核苷酸的切割，例如模仿拓扑异构酶抑制剂的单个多环芳香族加合物。我们发现，在拓扑异构酶裂解位点的嵌入模拟了喜树碱与拓扑异构酶I和在拓扑异构酶II的情况下嵌入抗癌药物的效果。我们还发现，乙醛加合物，这在酒精消费过程中容易形成可以增强喜树碱诱导的拓扑异构酶I-DNA complexes.Goal 2：增强药物外排是一种常见的耐药机制治疗。喜树碱由半转运蛋白ABCG 2转运，也称为BCRP或MXR。我们已经评估了ABCG 2在喜树碱活性中的意义。我们发现新的喜树碱衍生物，高喜树碱比目前在ABCG 2过表达细胞系的临床试验中的喜树碱更有活性。我们还研究了喜树碱耐药细胞中ABC转运蛋白的差异表达。为了阐明拓扑异构酶介导的DNA损伤的分子途径，我们继续研究新发现的酶，酪氨酰-DNA-磷酸二酯酶（TDP-1），选择性地去除结合在DNA的3 '端的酪氨酰残基。我们已经证明Tdp 1与BER（断裂诱导修复）途径中的支架蛋白XRCC 1相关，并且XRCC 1缺陷的细胞对喜树碱选择性超敏感。使用重组TDP-1和修饰的寡核苷酸，我们正在寻找TDP-1抑制剂，以阻止修复途径下游的拓扑异构酶I介导的DNA损伤，以选择性地提高喜树碱的活性在checkpoint-deficient cells.Goal 3：我们已经追求我们的调查，发现和分子药理学调查的新型拓扑异构酶I抑制剂。茚并异喹啉是与库什曼博士合作发现的。与喜树碱相比，茚并异喹啉具有几个潜在的优点：1/它们是化学稳定的; 2/它们在特定的基因组位点捕获拓扑异构酶I切割复合物，这些位点不同于喜树碱捕获的位点; 3/它们的细胞半衰期比喜树碱长得多，切割复合物比喜树碱捕获的切割复合物更稳定。我们最近获得了共晶体的吲哚并咔唑绑定到拓扑异构酶I-DNA复合物之一。目标4：我们发现了人类线粒体拓扑异构酶I，一种由核基因编码的特异性酶。我们现在已经在所有脊椎动物基因组测序中发现了同源物的存在：小鼠，大鼠，鸡和斑马鱼。然而，该基因在包括玻璃海鞘、酵母和植物在内的非脊椎动物中不存在。我们目前正在寻找可能由线粒体拓扑异构酶I诱导的线粒体DNA损伤，并试图在小鼠中敲除该基因。