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

基本信息

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
项目类别：
财政年份：
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

项目摘要

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.

我们追求了新型拓扑异构酶I（TOP1）和拓扑异构酶II（TOP2）抑制剂的发现和分子药理学，以减轻凸甲状腺素，阿霉素和依托泊苷的局限性，同时保持其有效的抗肿瘤活性。 Indenoisoquinolines已与普渡大学的Cushman博士合作发现并追求。我们现在已经确定，indenoisoquinolines比camptothecins具有多个优点：1/它们在化学上稳定且易于合成和化学优化； 2/它们在特定的基因组部位捕获了与被kptothecins捕获的基因组部位的裂解复合物； 3/他们的细胞半衰期比camptothecins长得多； 4/它们产生的Top1裂解复合物比被camptothecins捕获的配合物更稳定，表明在Top1-DNA裂解复合物中紧密拟合。 5/它们不是多药抗性外排泵（例如ABCB1（PGP），ABCG2（MRP/ BCRP）和ABCC1（MRP1）。我们继续发现并表征新颖的衍生物以优化以优化Indenoisoquinolines。结果，三个IndenoisoIsoquinolines。 NCI已选择了724998）。我们的目标是将Indenoisoquinolines作为生物标志物在0/I期中的第一批NCI下发现的药物。这些抑制剂属于不同的化学家族：同质膜片，Camptothecins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，当Top1-DNA界面形成瞬时DNA Cleavage IntermedMediatedes时，5种不同的TOP1抑制剂（TOPOTECAN，TOPOTECAN，天然Camptothecin，一种Norindenoisoquinoline和intolocarbazole）都在Top1-DNA界面上结合。我们将这种抑制作用称为界面抑制作用，并提出这种类型的抑制作用是药物发现的自然范式之一（TIPS）。这个概念对发现稳定蛋白质复合物（新方法）的大分子复合物的抑制剂具有深远的影响，而不是仅用于防止形成或解离蛋白质复合物（过去和当前方法）的药物。我们已经确定了具有单点突变的几种Top1-DNA复合物的结构，从而导致了Camptothecin抗性。这些研究提供了从远端突变体传播到活性位点的结构改变的分子例子。它们还提供了证据证明用于分子对接和合理药物发现的酶-DNA结构的有效性，我们将其应用于indenoisoquinoline程序。在这些结构研究的过程中，我们发现TOP1结构稳定在TOP1的N-和C末端基序中稳定了一个未识别的基序。我们扩展了有关致癌物和凋亡期间诱导Top1-DNA复合物的研究。我们先前曾报道过多环芳烃（苯并[a] pyrene，苯并[C]稳定），甲醛（来自酒精代谢的人类在人类中产生的生物产生）和4-硝基喹啉素-1-氧化物（4-NQO）是Top1裂解复合物的有效诱导剂。现在，我们已经表明，另一种致癌物，克罗托醛醛还可以捕获带有纯化的top1和细胞中的Top1裂解复合物。我们还首次表明，双醛加合物可以独立于Top1乳化络合物形成Top1-DNA加合物。该结果是原理的第一个证明，即硬甲醛加合物可以在染色质蛋白（此处TOP1）和DNA之间形成加合物。关于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由多种抗癌药物（包括TOP2抑制剂（Etoposide））和大型抑制剂抑制剂（Paclitaxel，Vinblitaxel，vinblastin，vinblastin）所诱导的凋亡的保守和普遍特征。我们还表明，TOP1裂解复合物的形成在凋亡的执行中起着积极作用，因为具有TOP1下调的细胞会产生异常的染色质凝结和凋亡体的延迟形成。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，并且可以引起自身免疫反应。在我们的实验室中发现了第一个也是仍然是唯一的特定线粒体拓扑酶Top1mt。 TOP1MT由所有脊椎动物基因组中存在的核基因编码：小鼠，大鼠，鸡肉和斑马鱼。然而，在包括酵母和植物在内的非魔术中不存在该基因。我们已经提出，在脊椎动物进化过程中，复制一个共同的祖先TOP1基因（如今在简单的弦弦中）出现了TOP1MT。另一个TOP1基因编码了专用于核基因组的先前已知的TOP1。我们已经生成了TOP1MT的特异性抗体，这使我们能够证明TOP1MT是从核中不存在的，并将其浓缩在线粒体中。我们还发现，Top1MT可以被凸轮皮蛋白捕获，并使用此发现来绘制线粒体DNA（mtDNA）中的Top1mt结合位点。线粒体中MTDNA调节的D-Loop区域的TOP1MT位点的映射揭示了存在不对称的TOP1MT位点的群集，仅限于从下游的150 bp段，并毗邻与该站点的重复位置过早地终止，并产生650 Base DNA-DNA-DNA-DNA-DNA-DNA dna-dna-dna-dna-dna-dna-dna-dna-dna-dna-dna-dna-dna-dna-dna-dna-dna dnna形式。此外，我们表明，甲虫对TOP1MT的抑制会减少7S DNA的形成。我们的结果表明，TOP1MT在调节mtDNA复制中的新作用。我们还产生了TOP1MT敲除小鼠，目前正在研究其表型和基因型。与Rafa Balana博士合作，我们研究了TOP1抑制剂对Leishmania Donovani 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与indenoisoquinolines可能相关的新型抗寄生虫药物。

项目成果

期刊论文数量（16）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

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

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。

DOI：
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

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
通讯作者：
G. Laco;W. Du;G. Kohlhagen;J. M. Sayer;D. Jerina;T. Burke;D. Curran;Y. Pommier

Interfacial inhibitors of protein-nucleic acid interactions.

蛋白质-核酸相互作用的界面抑制剂。