DNA Topoisomerases as nuclear and mitochondrial targets of Anticancer Drugs

DNA 拓扑异构酶作为抗癌药物的核和线粒体靶标

• 批准号: 8937651
• 负责人: YVES POMMIER
• 金额: $ 94.67万
• 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/8937651
• 关键词: ABCB1 gene; ABCC1 gene; ABCG2 gene; Antineoplastic Agents; Binding; Biological Markers; Biology; Bone Marrow; Camptothecin; Cardiotoxicity; Catenanes; Cell Nucleus; Cell membrane; Chemicals; Chordata; Clinic; Clinical Oncology; Clinical Trials; Collaborations; Colon Carcinoma; Complement; Complex; Cyclic Nucleotides; DNA; DNA Damage; DNA Structure; DNA Topoisomerases; DNA topoisomerase II alpha; Defect; Dose-Limiting; Doxorubicin; Drug Efflux; Drug Targeting; Enzymes; Etoposide; Exons; Gene Mutation; Generations; Genes; Genetic Transcription; Genomics; Goals; Guanosine; Half-Life; Hematologic Neoplasms; Histones; Human; Intercalating Agents; Intestines; Introns; Knockout Mice; Laboratories; Legal patent; Malignant Childhood Neoplasm; Malignant neoplasm of lung; Malignant neoplasm of ovary; Medicine; Membrane Transport Proteins; Microarray Analysis; Mitochondria; Mitochondrial DNA; Molecular; Multi-Drug Resistance; Mus; Mutation; Natural regeneration; Neurodegenerative Disorders; Neurologic; Neurons; Normal tissue morphology; Nuclear; Nucleotides; Ovarian Carcinoma; Pathway interactions; Patients; Pharmaceutical Preparations; Pharmacodynamics; Pharmacology; Phase; Phenotype; Plants; Poison; Program Development; Property; RNA; RNA Splicing; Replication Origin; Ribonucleotides; Role; Series; Site; Stratification; Structure; Superhelical DNA; TOP1 gene; Topoisomerase; Topoisomerase II; Topoisomerase Inhibitors; Topotecan; Toxic effect; Transcript; Universities; Vertebrates; Yeasts; anti-cancer therapeutic; cancer cell; drug development; drug synthesis; efflux pump; hydroxyl group; inhibitor/antagonist; insertion/deletion mutation; irinotecan; lung Carcinoma; mitochondrial genome; mouse model; novel; programs; repaired; response; sugar; tumor; vector

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.

人类编码 6 种拓扑异构酶：两种 IB 型：Top1 和 Top1mt，两种 IIA 型：Top2A 和 Top2B，以及两种 IA 型：Top3A 和 Top3B。拓扑异构酶对于避免 DNA 解缠（包括超螺旋、结和索链）至关重要，并且是所有 DNA 事务（包括转录和复制）所必需的。最近发现 Top3B 可以解决 RNA 解缠结问题，并且对神经元转录至关重要。 TOP3B 突变与神经缺陷和神经退行性疾病有关。 Top1 是伊立替康和托泊替康的靶标，这两种药物是喜树碱衍生物，可有效用于治疗卵巢癌、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-羟基内酯结构）、ABCG2 和 ABCB1 质膜转运蛋白的药物流出以及剂量限制性胃肠道和骨髓毒性。我们一直致力于新型 Top1 靶向抗癌药物的发现和分子药理学研究，以缓解这些限制。茚并异喹啉是与普渡大学的 Cushman 博士和 NCI 药物开发计划 (DTP) 合作发现、申请专利并进行开发的。我们现在已经确定，茚并异喹啉比喜树碱有几个优点： 1/它们化学稳定，易于合成和化学优化； 2/ 它们在特定基因组位点捕获 Top1 裂解复合物，该位点与喜树碱捕获的位点不同； 3/它们的细胞半衰期比喜树碱长得多； 4/ 他们产生的 Top1 裂解复合物比喜树碱捕获的那些更稳定，表明与 Top1-DNA 裂解复合物紧密配合； 5/ 它们不是多药耐药外排泵（例如 ABCB1 (Pgp)、ABCG2 (Mrp/Bcrp) 和 ABCC1 (Mrp1)）的底物。两种茚并异喹啉（NSC 725776 - indimitecan 和 743400 -indotecan）正在 NCI 进行临床试验。该药物开发是 LMP（我们的 小组和 Bonner 博士负责 γ-H2AX 生物标志物）、临床肿瘤学分部（Doroshow 博士和 Shivaani Kummar 博士负责临床试验）、DTP 和 SAIC（Hollingshead 博士、Parchment 博士和 Kinders 博士负责小鼠模型和药效生物标志物）以及普渡大学（Mark Cushman 博士负责药物合成）。我们的目标是使茚并异喹啉成为第一个 NCI 在 0/I 期管道中发现了以组蛋白 gamma-H2AX 作为生物标志物的药物。我们还继续开发第二代茚并异喹啉衍生物。新系列比目前临床试验中的化合物更有效。此外，我们正在启动一个项目，在递送载体中配制茚并异喹啉，以增加其在肿瘤中的浓度，同时避免 正常组织。关于拓扑异构酶的基本生物学，我们发现，当Top1与错误掺入的核糖核苷酸的DNA底物结合时，糖的2-prime-羟基通过在最初产生的断裂的3-prime末端形成2-prime，3-prime-环状核苷酸消除Top1，Top1cc自发地转化为单链断裂 通过 Top1。这一发现很重要，原因有两个：首先，因为 Thomas Kunkel 和他的团队（我们的合作者之一）最近表明，核糖核苷酸在正常复制过程中很容易发生错误掺入，其次是因为这些错误掺入位点以依赖于 Top1 的方式导致短核苷酸缺失和插入。这些新结果共同补充了我们之前的发现，显示了重组和潜在的诱变特性 顶部1。我们一直在进行有关 Top1 与转录的研究。首先，我们展示了 Top1 和转录终止点之间的关键关系，这些转录终止点与负超螺旋 DNA 片段中替代 DNA 结构（鸟苷四联体和 R 环）的形成相关，而负超螺旋 DNA 片段往往在转录复合物之后出现。 Top1 的缺陷促进了这种负超螺旋，在正常情况下，Top1 的作用是消除负超螺旋 在移动转录复合物之后产生。我们还证明，Top1 靶向药物（以及异常 DNA 结构；见上文）对 Top1 的稳定会诱导异常剪接，尤其是在编码剪接因子的基因中。最后，使用微阵列分析，我们能够证明喜树碱对 Top1 裂解复合物的捕获可选择性地阻断长转录本和长转录本中的转录。 内含子-外显子连接，这与 Top1 减轻转录诱导的超螺旋和剪接的作用一致。线粒体 IB 型拓扑异构酶 Top1mt 是我们实验室发现的。 Top1mt 由存在于所有脊椎动物中的核基因编码，该核基因可能是由共同祖先 TOP1 基因的复制引起的（今天在简单的脊索动物中发现，更遥远的在 酵母和植物）。尽管 TOP1mt 敲除小鼠是可行的，但我们发现它们对阿霉素的心脏毒性过敏。这是因为在阿霉素损伤后需要 Top1mt 来再生线粒体 DNA。我们还发现 Top1mt 是维持线粒体 DNA 正常超螺旋所必需的，并通过优先位点在整个线粒体基因组中发挥作用 在调节和复制起点区域。我们在实验室培育的 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 TOP1mt 的缺乏。我们发现 Top2A（拓扑异构酶 II α）和 Top2B（拓扑异构酶 II β）均存在于线粒体中并发挥作用。这一发现不仅解释了轻度表型 我们的 Top1mt 基因敲除小鼠也发现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 Top1mt 敲除小鼠与其他携带线粒体基因改变的转基因小鼠进行杂交。