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: 7732907
• 负责人: YVES POMMIER
• 金额: $ 79.97万
• 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/7732907
• 关键词: 2-butenal; 4-Nitroquinoline-1-oxide; ABCB1 gene; ABCC1 gene; ABCG2 gene; Active Sites; 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 Structure; DNA Topoisomerases; DNA biosynthesis; Development; Developmental Therapeutics Program; Distal; Docking; Down-Regulation; Doxorubicin; Doxorubicin/Etoposide; Enzymes; Ethanol Metabolism; Etoposide; Evolution; Family; Formaldehyde; Genes; Genome; Genomics; Genotype; Genus Cola; Goals; Half-Life; Histones; Human; Intercalating Agents; Knockout Mice; Laboratories; Leishmania donovani; Macromolecular Complexes; Maps; Mitochondria; Mitochondrial DNA; Molecular; Multi-Drug Resistance; Multidrug Resistance Associated Protein 1; Mus; Nature; Nuclear; Outcome; Ovarian Carcinoma; P-Glycoprotein; Paclitaxel; Pathway interactions; Pharmaceutical Preparations; Pharmacodynamics; Pharmacology; Phase; Phenotype; Physical condensation; Plants; Play; Point Mutation; Poison; Proteins; Rattus; Reporting; Resistance; Role; Screening procedure; Site; Structure; TOP1 gene; Time; Today; Topoisomerase; Topoisomerase II; Topoisomerase Inhibitors; Topotecan; Tryptophan; Tubulin; Type I DNA Topoisomerases; Universities; Vertebrates; Yeasts; Zebrafish; adduct; anti-cancer therapeutic; base; batracylin; benzo(c)phenanthrene; cancer cell; chromatin protein; concept; drug development; drug discovery; efflux pump; genome sequencing; human TOP1 protein; inhibitor/antagonist; interfacial; mouse model; mutant; novel; novel strategies; prevent; programs; response

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, three indenoisoquinolines (NSC 706744, 725776 and 724998) 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). 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, the camptothecins keto derivatives, and batracylin. We have provided further evidence that Top1 inhibitors are a paradigm for interfacial inhibitors. Crystal structure studies have now established that 5 different Top1 inhibitors (topotecan, natural camptothecin, an indenoisoquinoline, a norindenoisoquinoline and an indolocarbazole) all bind at the Top1-DNA interface when the Top1 forms its 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 Natures paradigms for drug discovery (TIPS). 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 Top1-DNA complexes with single point mutations resulting in camptothecin resistance. These studies provide molecular examples of structural alterations propagated from distal point mutants to enzyme active sites. They also provide evidence for the validity of the enzyme-DNA structures to be used for molecular docking and rational drug discovery, which we are applying to our indenoisoquinoline program. In the course of these structural studies we found that Top1 structure is stabilized a yet unrecognized motif made of tryptophan residues that tie the N- and C-terminus motif of Top1. 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 Cushman 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，三种茚并异喹啉（NSC 706744、725776 和 724998）已被 NCI 选择进行临床开发。该药物开发是多个团队之间的合作：LMP（我们团队和 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）。 我们的目标是使茚并异喹啉成为 NCI 发现的第一个处于 0/I 期管道的药物，并以组蛋白 gamma-H2AX 作为生物标志物。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： 高喜树碱、喜树碱酮衍生物和batracylin。我们提供了进一步的证据表明 Top1 抑制剂是界面抑制剂的范例。 晶体结构研究现已确定 5 种不同的 Top1 抑制剂（托泊替康、天然喜树碱、茚并异喹啉、去甲茚并异喹啉和吲哚咔唑）均 当 Top1 形成其瞬时 DNA 裂解复合物中间体时，结合在 Top1-DNA 界面上。我们将这种类型的抑制称为界面抑制，并提议将这种类型的抑制作为自然药物发现（TIPS）范例之一。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）具有深远的意义，而不仅仅是筛选药物 防止蛋白质复合物的形成或解离（过去和当前的方法）。我们已经确定了几种具有导致喜树碱抗性的单点突变的 Top1-DNA 复合物的结构。这些研究提供了从远端点突变体传播到酶活性位点的结构改变的分子例子。它们还为用于分子对接和合理药物发现的酶-DNA 结构的有效性提供了证据，我们对此 正在申请我们的茚并异喹啉项目。在这些结构研究过程中，我们发现 Top1 结构是稳定的，是一个由色氨酸残基组成的尚未识别的基序，该基序与 Top1 的 N 和 C 末端基序相连。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。我们之前曾报道过多环芳烃 （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 裂解复合物的有效诱导剂。我们现在已经证明，另一种致癌物巴豆醛也可以捕获 Top1 裂解复合物，无论是纯化的还是 Top1 和单元格中。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，我们现在已经证明 Top1 裂解复合物的形成是多种抗癌药物（包括 Top2 抑制剂（依托泊苷）和微管蛋白抑制剂（紫杉醇、长春花碱））诱导的细胞凋亡的保守且普遍存在的特征。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 下调的细胞产生 染色质浓缩异常和凋亡小体形成延迟。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 由所有脊椎动物中存在的核基因编码 基因组测序：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。我们已经产生了特异性抗体 Top1mt，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。线粒体 mtDNA 调节 D 环区域中 Top1mt 位点的定位揭示了这种现象的存在 Top1mt 位点的不对称簇局限于复制过早终止位点下游和邻近的 150 bp 片段，生成形成线粒体 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。我们的结果表明了新的角色 Top1mt 调节 mtDNA 复制。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相关的新型抗寄生虫药物 至茚并异喹啉类。

项目成果

Apoptotic susceptibility of cancer cells selected for camptothecin resistance: gene expression profiling, functional analysis, and molecular interaction mapping.

选择喜树碱抗性的癌细胞的凋亡易感性：基因表达谱、功能分析和分子相互作用图谱。

• 发表时间: 2003
• 期刊: Cancer research
• 影响因子: 11.2
• 作者: Reinhold,WilliamC;Kouros-Mehr,Hosein;Kohn,KurtW;Maunakea,AlikaK;Lababidi,Samir;Roschke,Anna;Stover,Kristen;Alexander,Jes;Pantazis,Panayotis;Miller,Lance;Liu,Edison;Kirsch,IlanR;Urasaki,Yoshimasa;Pommier,Yves;Weinstein,John
• 通讯作者: Weinstein,John

Non-camptothecin DNA topoisomerase I inhibitors in cancer therapy.

非喜树碱 DNA 拓扑异构酶 I 抑制剂在癌症治疗中的应用。

• DOI: 10.2174/1568026033452546
• 发表时间: 2003
• 期刊: Current topics in medicinal chemistry
• 影响因子: 3.4
• 作者: Meng,Ling-Hua;Liao,Zhi-Yong;Pommier,Yves
• 通讯作者: Pommier,Yves

Interfacial inhibitors of protein-nucleic acid interactions.

蛋白质-核酸相互作用的界面抑制剂。

• DOI: 10.2174/1568011054222337
• 发表时间: 2005
• 期刊: Current medicinal chemistry. Anti-cancer agents
• 作者: Pommier,Yves;Marchand,Christophe
• 通讯作者: Marchand,Christophe

Analysis of human topoisomerase I inhibition and interaction with the cleavage site +1 deoxyguanosine, via in vitro experiments and molecular modeling studies.

• DOI: 10.1016/j.bmc.2004.06.046
• 发表时间: 2004-10
• 期刊: Bioorganic & medicinal chemistry
• 影响因子: 3.5
• 作者: G. Laco;W. Du;G. Kohlhagen;J. M. Sayer;D. Jerina;T. Burke;D. Curran;Y. Pommier