DNA Topoisomerases as Target of Action of Anticancer Dru

DNA拓扑异构酶（top1和top2）是一些最有效的抗癌药物的靶点。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)阐明确定癌细胞对拓扑异构酶抑制剂反应的分子途径，iii)发现新的拓扑异构酶抑制剂，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，因为当拓扑异构酶形成其瞬时DNA切割复合物中间体时，这些药物通过在酶-DNA界面上结合来改变拓扑异构酶与DNA的结合。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药物发现的自然范式之一。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）而不是仅仅筛选阻止形成或解离蛋白质复合物的药物（过去和现在的方法）具有深远的意义。我们已经确定了几种拓扑异构酶I-DNA复合物的结构与单点突变导致喜树碱抗性。这些研究为耐药突变提供了理论依据。它们也为酶- dna结构的有效性提供了证据，可用于分子对接和合理的药物发现。为了进一步阐明拓扑异构酶抑制剂与其靶酶- 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裂解，例如模拟拓扑异构酶抑制剂的单个多环芳香族加合物。我们发现，在拓扑异构酶切割位点插入类似喜树碱对拓扑异构酶I的作用，以及在拓扑异构酶II的情况下插入抗癌药物的作用。我们还发现，在饮酒过程中容易形成的乙醛加合物可以增强喜树碱诱导的拓扑异构酶I-DNA复合物。目标2：药物外排增强是对治疗的常见耐药机制。喜树碱由半转运体ABCG2运输，也称为BCRP或MXR。我们已经评估了ABCG2对喜树碱活性的影响。我们发现新的喜树碱衍生物，同型喜树碱在abcg2过表达细胞系中比目前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。为了阐明响应拓扑异构酶介导的DNA损伤的分子途径，我们继续研究新发现的酶，酪氨酸-DNA-磷酸二酯酶（TDP-1），它选择性地去除结合在DNA 3'端的酪氨酸残基。我们已经证明Tdp1与XRCC1相关，XRCC1是BER（断裂诱导修复）途径中的支架蛋白，并且缺乏XRCC1的细胞对喜树碱选择性过敏。利用重组Tdp-1和修饰的寡核苷酸，我们正在寻找Tdp-1抑制剂来阻断拓扑异构酶i介导的DNA损伤下游的修复途径，以选择性地增强喜树碱在检查点缺陷细胞中的活性。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。吲哚异喹啉是与库什曼博士合作发现的。与喜树碱相比，吲哚异喹啉类化合物有几个潜在的优势：1、化学性质稳定；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，与喜树碱捕获的复合物不同；3/它们的细胞半衰期比喜树碱长得多，喜树碱的卵裂复合物比喜树碱捕获的复合物更稳定。我们最近获得了一种吲哚咔唑与拓扑异构酶I-DNA复合物结合的共晶体。我们现在有更多强效的top1毒药正在进行临床前开发研究。目标4：我们发现了人类线粒体拓扑异构酶I，这是一种由核基因编码的特定酶。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：小鼠、大鼠、鸡和斑马鱼。然而，该基因在非脊椎动物中不存在，包括肠虫、酵母和植物。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。

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.