DNA Topoisomerases as Target of Action of Anticancer Drugs

DNA拓扑异构酶作为抗癌药物的作用靶点

• 批准号: 7965088
• 负责人: YVES POMMIER
• 金额: $ 110.4万
• 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/7965088
• 关键词: 2-butenal; 4-Nitroquinoline-1-oxide; ABCB1 gene; ABCC1 gene; ABCG2 gene; Antibodies; Antineoplastic Agents; Apoptosis; Apoptotic; Autoimmune Responses; Benzo(a)pyrene; Binding; Binding Sites; Biological Markers; Camptothecin; Carcinogens; Cell Nucleus; Cell Survival; Cells; Chemicals; Chickens; Chordata; Chromatin; Clinic; Clinical; Clinical Oncology; Clinical Trials; Collaborations; Colon; Complex; DNA; DNA Adducts; DNA Topoisomerases; DNA biosynthesis; Development; Down-Regulation; Doxorubicin; Enzymes; Ethanol Metabolism; Etoposide; Evolution; Family; Formaldehyde; Genes; Genome; Genomics; Genotype; Goals; Half-Life; Histones; Human; Intercalating Agents; Knockout Mice; Laboratories; Leishmania donovani; Maps; Mitochondria; Mitochondrial DNA; Molecular; Multi-Drug Resistance; Mus; Nuclear; Outcome; Ovarian Carcinoma; Paclitaxel; Pathway interactions; Pharmaceutical Preparations; Pharmacodynamics; Pharmacology; Phase; Phenotype; Physical condensation; Plants; Play; Poison; Rattus; Reporting; Role; Site; TOP1 gene; Time; Topoisomerase; Topoisomerase II; Topoisomerase Inhibitors; Tubulin; Type I DNA Topoisomerases; Universities; Vertebrates; Yeasts; Zebrafish; adduct; anti-cancer therapeutic; base; benzo(c)phenanthrene; cancer cell; chromatin protein; drug development; drug synthesis; efflux pump; genome sequencing; inhibitor/antagonist; mouse model; novel; response; vertebrate genome

We have pursued our discovery and molecular pharmacology of novel topoisomerase I (Top1) and topoisomerase II (Top2) inhibitors to alleviate the limitations of camptothecins, doxorubicin and etoposide while retaining their potent antitumor activity. The indenoisoquinolines have been discovered and pursued in collaboration with Dr. Cushman at Purdue University. 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). We have continued to discover and characterize novel derivatives to optimize the indenoisoquinolines. As a result, two indenoisoquinolines (NSC 725776 and 743400) have been selected for clinical development by the NCI. This drug development is a collaboration between several groups: LMP (our group and Dr. Bonner for gamma-H2AX biomarker), 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 have also studied and characterized novel non-camptothecin and non-indenoisoquinoline topoisomerase inhibitors that are in clinical trials and developments. Those inhibitors belong to different chemical families: the homocamptothecins and camptothecins keto derivatives against Top1, and Dp44mT against Top2. We are currently studying the molecular and cellular pharmacology of a novel non-camptothecin Top1 inhibitors from Genzyme Co., which is just starting clinical trials. We have extended our studies on the induction of Top1-DNA complexes by carcinogens and during apoptosis. We had previously reported that polycyclic aromatics (benzo[a]pyrene, benzo[c]phenanthrene), formaldehyde (a bioproduct generated in humans from alcohol metabolism) and 4-nitroquinoline-1-oxide (4-NQO) were potent inducers of Top1 cleavage complexes. We have now shown that another carcinogen, crotonaldehyde can also trap Top1 cleavage complexes both with purified Top1 and in cells. We have also shown for the first time that crotonaldehyde adducts can form Top1-DNA adducts independently of Top1 cleavage complexes. This result is the first proof of principle that crotonaldehyde adducts can form adducts between chromatin protein (here Top1) and DNA. Regarding the induction of topoisomerase cleavage complexes during apoptosis, we have now demonstrated that the formation of Top1 cleavage complexes is a conserved and ubiquitous feature of apoptosis induced by a variety of anticancer drugs including Top2 inhibitors (etoposide) and tubulin inhibitors (paclitaxel, vinblastin). We have also shown that the formation of Top1 cleavage complexes plays an active role in the execution of apoptosis since cells with Top1 down-regulation produce abnormal chromatin condensation and delayed formation of apoptotic bodies. This finding may be important since partial (incomplete apoptosis) allows the survival of cells with carcinogenic potential and can elicit autoimmune responses. The first and still the only specific mitochondrial topoisomerase, Top1mt, was discovered in our laboratory. Top1mt is encoded by a nuclear gene present in all vertebrate genomes sequenced: mouse, rat, chicken, and zebra fish. However, the gene is absent in non-vertebrate including yeast and plants. We have proposed that Top1mt arose by duplication of a common ancestral TOP1 gene (found today in simple chordates) during evolution of vertebrates. The other TOP1 gene encodes the previously known Top1 devoted to the nuclear genome. We have generated specific antibodies for Top1mt, which enabled us to demonstrate that Top1mt is absent from nuclei and concentrated in mitochondria. We have also found that Top1mt can be trapped by camptothecin and used this finding to map the Top1mt binding sites in mitochondrial DNA (mtDNA). Mapping of Top1mt sites in the regulatory D-loop region of mtDNA in mitochondria revealed the presence of an asymmetric cluster of Top1mt sites confined to a 150-bp segment downstream from, and adjacent to, the site at which replication is prematurely terminated, generating a ≈ 650-base (7S DNA) product that forms the mitochondrial D-loop. Moreover, we showed that inhibition of Top1mt by camptothecin reduces formation of the 7S DNA. Our results suggest novel roles for Top1mt in regulating mtDNA replication. We have also generated Top1mt knockout mice and are presently studying their phenotype and their genotype. In collaboration with Dr. Rafa Balana, we have studied the effects of Top1 inhibitors on the leishmania donovani Top1 and analyzed the functional role of key catalytic residues. One potential outcome will be the discovery of novel antiparasite drugs potentially related to indenoisoquinolines.

我们致力于新型拓扑异构酶 I (Top1) 和拓扑异构酶 II (Top2) 抑制剂的发现和分子药理学研究，以减轻喜树碱、阿霉素和依托泊苷的局限性，同时保留其有效的抗肿瘤活性。茚并异喹啉是与普渡大学的库什曼博士合作发现和研究的。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的底物（例如 ABCB1 (Pgp)、ABCG2 (Mrp/Bcrp) 和 ABCC1 (Mrp1)）。我们不断发现和表征新型衍生物以优化茚并异喹啉。因此， NCI 已选择两种茚并异喹啉（NSC 725776 和 743400）进行临床开发。多个小组之间的合作：LMP（我们小组和 Bonner 博士进行 gamma-H2AX 生物标志物）、临床肿瘤学分部（Doroshow 博士和 Shivaani Kummar 进行临床试验）、DTP 和 SAIC（Hollingshead 博士、Parchment 博士和 Kinders 博士）小鼠模型和药效生物标志物）和普渡大学（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 γ-H2AX 作为生物标志物的药物。我们还研究并表征了正在进行临床试验和开发的新型非喜树碱和非茚并异喹啉拓扑异构酶抑制剂。 这些抑制剂属于不同的化学家族：针对 Top1 的高喜树碱和喜树碱酮衍生物，针对 Top2 的 Dp44mT。我们目前正在研究Genzyme公司的一种新型非喜树碱Top1抑制剂的分子和细胞药理学，该抑制剂刚刚开始临床试验。我们扩展了致癌物诱导 Top1-DNA 复合物以及细胞凋亡过程中的研究。我们之前曾报道多环芳烃（苯并[a]芘、苯并[c]菲）、甲醛（人体酒精代谢产生的生物产物）和4-硝基喹啉-1-氧化物（4-NQO）是Top1的有效诱导剂裂解复合物。我们现在已经证明，另一种致癌物巴豆醛也可以与纯化的 Top1 和细胞内捕获 Top1 裂解复合物。我们还首次证明巴豆醛加合物可以独立于 Top1 裂解复合物形成 Top1-DNA 加合物。这一结果首次证明了巴豆醛加合物可以在染色质蛋白（此处为 Top1）和 DNA 之间形成加合物。 关于细胞凋亡过程中拓扑异构酶裂解复合物的诱导，我们现已证明Top1裂解复合物的形成是多种抗癌药物诱导的细胞凋亡的保守且普遍存在的特征，包括Top2抑制剂（依托泊苷）和微管蛋白抑制剂（紫杉醇、长春花素） 。我们还表明，Top1 裂解复合物的形成在细胞凋亡的执行中发挥着积极作用，因为 Top1 下调的细胞会产生异常的染色质浓缩并延迟凋亡小体的形成。这一发现可能很重要，因为部分（不完全凋亡）允许具有致癌潜力的细胞存活，并能引发自身免疫反应。第一个也是唯一一个特定的线粒体拓扑异构酶 Top1mt 是在我们的实验室发现的。 Top1mt 由所有已测序脊椎动物基因组中存在的核基因编码：小鼠、大鼠、鸡和斑马鱼。然而，该基因在包括酵母和植物在内的非脊椎动物中不存在。我们提出，Top1mt 是由脊椎动物进化过程中共同祖先 TOP1 基因（今天在简单脊索动物中发现）的复制而产生的。另一个 TOP1 基因编码先前已知的用于核基因组的 Top1。我们已经生成了 Top1mt 的特异性抗体，这使我们能够证明 Top1mt 不存在于细胞核中，而是集中在线粒体中。我们还发现 Top1mt 可以被喜树碱捕获，并利用这一发现绘制了线粒体 DNA (mtDNA) 中 Top1mt 结合位点的图谱。对线粒体 mtDNA 调节 D 环区域中 Top1mt 位点的作图揭示了 Top1mt 位点的不对称簇的存在，该簇局限于复制过早终止位点下游并邻近的 150 bp 片段，从而产生了≈形成线粒体 D 环的 650 个碱基 (7S DNA) 产物。此外，我们发现喜树碱抑制 Top1mt 会减少 7S DNA 的形成。我们的结果表明 Top1mt 在调节 mtDNA 复制中发挥新作用。我们还培育了 Top1mt 基因敲除小鼠，目前正在研究它们的表型和基因型。我们与 Rafa Balana 博士合作，研究了 Top1 抑制剂对杜氏利什曼原虫 Top1 的影响，并分析了关键催化残基的功能作用。一个潜在的结果将是发现可能与茚并异喹啉相关的新型抗寄生虫药物。