DNA Topoisomerases as nuclear and mitochondrial targets of Anticancer Drugs

人类编码6种拓扑异构酶：IB型有2种：Top1和Top1mt， IIA型有2种：Top2A和Top2B， IA型有2种：Top3A和Top3B。拓扑异构酶对于避免包括超级线圈、结和链链烷在内的DNA解开至关重要，并且是包括转录和复制在内的所有DNA交易所必需的。最近发现，Top3B可以解决RNA解结问题，对神经元的转录至关重要。TOP3B突变与神经缺陷和神经退行性疾病有关。Top1是伊立替康和拓扑替康的靶点，这两种喜树碱衍生物有效地用于治疗卵巢癌、结肠癌和肺癌以及血液和儿科恶性肿瘤。然而，喜树碱具有明确的局限性，包括化学不稳定性（由于其α -羟内酯结构），ABCG2和ABCB1质膜转运蛋白的药物外排，以及剂量限制的胃肠道和骨髓毒性。为了减轻这些限制，我们一直在探索新的top1靶向抗癌药物的发现和分子药理学。与普渡大学的Cushman博士和NCI药物开发计划（DTP）合作，发现了吲哚异喹啉类药物，并获得了专利。我们现在已经确定，与喜树碱相比，吲哚异喹啉具有以下几个优点：1 .它们化学稳定，易于合成和化学优化；2/它们在特定的基因组位点捕获与喜树碱捕获的不同的Top1切割复合物；3/其细胞半衰期比喜树碱长得多；4/它们产生的Top1切割复合物比喜树碱捕获的更稳定，表明Top1- dna切割复合物紧密配合；它们不是多药耐药外排泵(如ABCB1 （Pgp）、ABCG2 （Mrp/Bcrp）和ABCC1 （Mrp1）的底物。两种吲哚异喹啉（NSC 725776—indiitecan和743400—indotecan）正在NCI进行临床试验。这种药物的开发是LMP（我们的团队和Bonner博士研究γ - h2ax生物标志物）、临床肿瘤学分支（Doroshow博士和Shivaani Kummar博士研究临床试验）、DTP和SAIC （Hollingshead博士、Parchment博士和Kinders博士研究小鼠模型和药效学生物标志物）以及普渡大学（Mark Cushman博士研究药物合成）之间的合作。我们的目标是使吲哚异喹啉类药物成为第一个以组蛋白γ - h2ax作为生物标志物的nci在0/I期管道中发现的药物。我们也在继续开发第二代吲哚异喹啉衍生物。新的系列比目前在临床试验中的化合物更有效。此外，我们正在启动一个项目，在递送载体中配制吲哚异喹啉，以增加其在肿瘤中的浓度，同时保留正常组织。关于拓扑异构酶的基本生物学，我们发现，当Top1与错误结合的核糖核苷酸结合到DNA底物上时，糖的2 ‘端羟基通过在最初由Top1产生的断裂的3 ’端形成2 ‘,3 ’端的环核苷酸来消除Top1， Top1cc自发地转化为单链断裂。这一发现之所以重要，有两个原因：首先，我们的合作者之一Thomas Kunkel和他的团队最近表明，核糖核苷酸在正常复制过程中很容易错误结合，其次，这些错误结合的位点会以依赖于top1的方式产生短核苷酸缺失和插入。总之，这些新的结果增加了我们之前的发现，显示了Top1的重组和潜在的诱变特性。我们一直在研究Top1与转录的关系。首先，我们展示了Top1与转录停止点之间的关键关系，这些转录停止点与转录复核后出现的负超螺旋DNA片段中替代DNA结构（鸟苷四重奏和r环）的形成有关。Top1的缺乏促进了这种负超卷曲，在正常情况下，Top1的作用是消除转录复合物移动后产生的负超卷曲。我们还证明，Top1靶向药物稳定Top1（以及异常DNA结构，见上文）会诱导异常剪接，尤其是编码剪接因子的基因。最后，通过微阵列分析，我们能够证明喜树碱捕获Top1切割复合物选择性地阻断了长转录本和内含子-外显子连接处的转录，这与Top1缓解转录诱导的超卷曲和剪接的作用是一致的。我们实验室发现了线粒体IB型拓扑异构酶Top1mt。Top1mt是由一个存在于所有脊椎动物体内的核基因编码的，这个核基因可能是由一个共同的祖先TOP1基因的复制而产生的（在今天的简单脊索动物中发现，在更远的酵母和植物中发现）。虽然TOP1mt敲除小鼠是存活的，但我们发现它们对阿霉素的心脏毒性过敏。这是因为被阿霉素损伤的线粒体DNA需要Top1mt来再生。我们还发现，Top1mt是维持线粒体DNA正常超卷曲所必需的，并在整个线粒体基因组中发挥作用，其优先位点位于调控区和复制起始区。我们在实验室产生的TOP1mt敲除小鼠的生存能力促使我们确定哪些其他拓扑异构酶可以补充缺乏TOP1mt。我们发现Top2A（拓扑异构酶II α）和Top2B（拓扑异构酶II β）都存在于线粒体中并起作用。这一发现不仅解释了我们的Top1mt基因敲除小鼠的温和表型，而且还解释了阿霉素的心脏毒性，因为阿霉素捕获了线粒体拓扑异构酶II。我们已经开始将我们的Top1mt基因敲除小鼠与其他携带线粒体基因改变的转基因小鼠杂交。

Humans encode 6 topoisomerases: Two type IB: Top1 and Top1mt, two type IIA: Top2A and Top2B, and two type IA: Top3A and Top3B. Topoisomerase enzymes are critical for avoiding DNA untanglements including supercoils, knots and catenanes, and are required for all DNA transactions including transcription and replication. Top3B was recently discovered to resolve RNA untanglements and to be critical for transcription in neurons. TOP3B mutations have been associated with neurological defects and neurodegenerative diseases. Top1 is the target of irinotecan and topotecan, which are camptothecin derivatives efficiently used to treat ovarian, colon and lung cancers as well as hematologic and pediatric malignancies. However, camptothecins have well-defined limitations including chemical instability (due to their alpha-hydroxylactone structure), drug efflux by the ABCG2 and ABCB1 plasma membrane transporters, and dose-limiting gastro-intestinal and bone marrow toxicity. We have pursued our discovery and molecular pharmacology of novel Top1-targeted anticancer agents to alleviate these limitations. The indenoisoquinolines have been discovered, patented and pursued in collaboration with Dr. Cushman at Purdue University and the NCI Drug Development Program (DTP). We have now established that the indenoisoquinolines have several advantages over camptothecins: 1/ they are chemically stable and easy to synthesize and chemically optimize; 2/ they trap Top1 cleavage complexes at specific genomic sites that differ from those trapped by camptothecins; 3/ their cellular half-life is much longer than camptothecins; 4/ the Top1 cleavage complexes they produce are more stable than those trapped by camptothecins indicating a tight fit in the Top1-DNA cleavage complexes; 5/ they are not substrates for the multidrug resistance efflux pumps (such as ABCB1 (Pgp), ABCG2 (Mrp/Bcrp) and ABCC1 (Mrp1). Two indenoisoquinolines (NSC 725776 -- indimitecan and 743400--indotecan) are in clinical trials at the NCI. This drug development is a collaboration between LMP (our group and Dr. Bonner for gamma-H2AX biomarker), the Clinical Oncology Branch (Dr. Doroshow and Shivaani Kummar for clinical trials), DTP and SAIC (Dr. Hollingshead, Dr. Parchment and Dr. Kinders for mouse models and pharmacodynamic biomarkers), and Purdue University (Dr. Mark Cushman for drug synthesis). Our goal is to make the indenoisoquinolines the first NCI-discovered drugs in the Phase 0/I pipeline with histone gamma-H2AX as a biomarker. We are also continuing to develop indenoisoquinoline derivatives as second generation. The new series is even more potent than the compounds in presently in clinical trials. Moreover, we are initiating a project to formulate the indenoisoquinolines in delivery vectors to increase their concentration in tumors while sparing normal tissues. Regarding the basic biology of topoisomerases, we discovered that, when Top1 binds to a DNA substrate with a misincorporated ribonucleotide, the Top1cc is spontaneously converted into a single-strand break after the 2-prime-hydroxyl group of the sugar eliminate Top1 by forming a 2-prime,3-prime-cyclic nucleotide at the 3-prime-end of the break that was initially made by Top1. This finding is important for two reasons: first, because Thomas Kunkel and his group, one of our collaborators, have recently shown that ribonucleotides are readily misincorporated during normal replication, and second because those misincorporation sites give rise to short nucleotide deletions and insertion in a Top1-dependent manner. Together these new results add to our previous findings showing the recombinogenic and potentially mutagenic properties of Top1. We have pursued our studies relating Top1 to transcription. First, we showed the critical relationship between Top1 and transcription stop points that are associated with the formation of alternative DNA structures (guanosine quartets and R-loops) in the negatively supercoiled DNA segments that tend to arise in the wake of transcription complexes. Such negative supercoiling is facilitated by deficiency in Top1, which under normal conditions functions to eliminate the negative supercoiling generated in the wake of moving transcription complexes. We also demonstrated that Top1 stabilization by Top1-targeted drugs (and abnormal DNA structures; see above) induces abnormal splicing, especially in genes that encode splicing factors. Finally, using microarray analysis, we were able to show that the trapping of Top1 cleavage complexes by camptothecins blocks transcription selectively in long transcripts and at intron-exon junctions, which is consistent with the role of Top1 to relieve transcription-induced supercoiling and in splicing. Mitochondrial type IB topoisomerase, Top1mt, was discovered in our laboratory. Top1mt is encoded by a nuclear gene present in all vertebrates, which probably arose by duplication of a common ancestral TOP1 gene (found today in simple chordates and more distantly in yeast and plants). Although TOP1mt knockout mice are viable, we found that they are hypersensitive to the cardiotoxicity of doxorubicin. This is because Top1mt is required to regenerate mitochondrial DNA upon damage by doxorubicin. We also found that Top1mt is required to maintain the normal supercoiling of mitochondrial DNA and acts throughout the mitochondrial genome with preferential sites in the regulatory and replication origin regions. The viability of the TOP1mt knockout mice, which we generated in our laboratory prompted us to determine which other topoisomerase could complement for lack of TOP1mt. We found that both Top2A (topoisomerase II alpha) and Top2B (topoisomerase II beta) are present and functional in mitochondria. This finding not only explains the mild phenotype of our Top1mt knockout mice but also the cardiotoxicity of doxorubicin because of the trapping of mitochondrial topoisomerase II by doxorubicin. We have begun crossing our Top1mt knockout mice with other genetically altered mice bearing mitochondrial gene alterations.