DNA Topoisomerases as Target of Action of Anticancer Drugs

我们致力于新的拓扑异构酶I(Top1)和拓扑异构酶II(TOP2)抑制剂的发现和分子药理学研究，以缓解喜树碱、阿霉素和依托泊苷的局限性，同时保持其强大的抗肿瘤活性。吲哚异喹啉是与普渡大学的库什曼博士合作发现并研究的。我们现在已经确定吲哚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/它们在不同于喜树碱捕捉的特定基因组位置捕获Top1裂解复合体；3/它们的细胞半衰期比喜树碱长得多；4/它们产生的Top1裂解复合体比喜树碱捕捉的那些更稳定，表明它们与Top1-DNA裂解复合体紧密结合；5/它们不是多药耐药外排泵(如CB1(PGP)、ABCG2(MRP/BCRP)和CC1(MRp1))的底物。我们一直在不断地发现和表征新的衍生物，以优化吲哚异喹啉。因此，两种吲哚异喹啉(NSC 725776和743400)已被美国国家药品监督管理局选择用于临床开发。这项药物开发是几个小组之间的合作：LMP(我们小组和Bonner博士研究伽马-H2AX生物标记物)，临床肿瘤学分部(Doroshow和Shivaani Kummar博士研究临床试验)，DTP和SAIC(Hollinghead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)。我们的目标是使吲哚异喹啉成为NCI在0/I期流水线中发现的第一种药物，组蛋白伽马-H2AX作为生物标记物。我们还研究和表征了正在进行临床试验和开发的新型非喜树碱和非吲哚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类化合物，以及针对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，该药物刚刚开始临床试验。我们扩展了我们对致癌物诱导Top1-DNA复合体和在细胞凋亡过程中的研究。我们曾报道多环芳烃(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裂解复合体的有效诱导剂。我们现在已经证明，另一种致癌物质巴豆醛也可以捕获Top1裂解复合物与纯化的Top1和在细胞中。我们还首次证明了巴豆醛加合物可以独立于Top1裂解复合体形成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(这里是Top1)和DNA之间形成加合物。关于在细胞凋亡过程中拓扑异构酶裂解复合体的诱导，我们已经证明Top1裂解复合体的形成是多种抗癌药物诱导的一种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)。我们还发现Top1裂解复合体的形成在执行细胞凋亡中起着积极的作用，因为Top1下调的细胞会产生异常的染色质凝聚和凋亡小体的延迟形成。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。我们实验室发现了第一个也是唯一一个特异的线粒体拓扑异构酶Top1mt。Top1mt是由所有脊椎动物基因组中存在的核基因编码的，这些脊椎动物基因组包括小鼠、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缺失。我们提出，在脊椎动物的进化过程中，Top1mt是通过复制共同的祖先TOP1基因(今天在简单的脊索动物中发现)而产生的。另一个TOP1基因编码先前已知的专用于核基因组的Top1。我们已经产生了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，并利用这一发现定位了线粒体DNA(MtDNA)中的Top1mt结合位点。线粒体线粒体DNA调控D-环区的Top1mt位点图谱显示，存在一个不对称的Top1mt位点簇，限制在复制过早终止的位点下游和邻近的150个碱基片段上，产生形成线粒体D-环的650碱基(7S DNA)产物。此外，我们还发现喜树碱对Top1mt的抑制减少了7S DNA的形成。我们的结果表明Top1mt在调节mtDNA复制中扮演了新的角色。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。与Rafa Balana博士合作，我们研究了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吲哚异喹啉相关的新型抗寄生虫药物。

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.