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: 8157199
• 负责人: YVES POMMIER
• 金额: $ 121.57万
• 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/8157199
• 关键词: Antineoplastic Agents; DNA Topoisomerases

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. Two indenoisoquinolines (NSC 725776 and 743400) have begun clinical trial at the NCI with histone gamma-H2AX as biomarker in tumor and normal tissues. This translational drug development has been made possible by a close collaboration between the LMP (our group and Dr. Bonner for gamma-H2AX biomarker), the Clinical Oncology Branch (Dr. Doroshow and Shivaani Kummar for clinical trials), the NCI-DTP-DCTD (Dr. Hollingshead, Dr. Parchment and Dr. Kinders for mouse models and pharmacodynamic biomarkers), and Purdue University (Dr. Mark Cushman for drug synthesis). 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 also studied and characterized additional novel topoisomerase inhibitors. Those inhibitors belong to different chemical families: the homocamptothecins, camptothecins keto derivatives and aromathecins against Top1, and Dp44mT and epoxy-anthraquinones against Top2. We have also studied the molecular and cellular pharmacology of novel non-camptothecin Top1 inhibitors from Genzyme Co., which is just starting clinical trials. Using Top1-depleted human cells, we have shown the critical role of Top1 in genomic stability. Depletion of top1 produces replication damage. We have also shown for the first time that the trapping of Top1 (by drugs or potentially DNA damage) produces transcription-associated DNA double-strand breaks, as a result of R-loop formation. We have extended our studies on the repair of topoisomerase-associated DNA damage and focused on tyrosyl-DNA-phosphodiesterase (Tdp1). Our studies are focused on the cellular regulation of Tdp1 and the discovery of inhibitors that could be used in combination with camptothecins or indenoisoquinolines for the treatment of cancer. 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 to date. 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 an almost equal to 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. In collaboration we Dr. Fritz Boege in Germany, we have shown that targeting (nuclear) Top1 is toxic, which demonstrate the different functions of Top1mt and Top1 in spite of their high degree of homology. We have also generated the Top1mt knockout mice and are studying their phenotype.

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。两种茚并异喹啉（NSC 725776 和 743400）已在 NCI 开始临床试验，以组蛋白 γ-H2AX 作为肿瘤和正常组织的生物标志物。这种转化药物的开发是通过 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 负责临床试验）、NCI-DTP- DCTD（Hollingshead 博士、Parchment 博士和 Kinders 博士负责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）。与喜树碱相比，茚并异喹啉有几个优点： 1/化学性质稳定，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)）。我们还研究并表征了其他新型拓扑异构酶抑制剂。这些抑制剂属于不同的化学家族：针对 Top1 和 Dp44mT 的高喜树碱、喜树碱酮衍生物和芳香皂素我们还研究了 Genzyme Co. 的新型非喜树碱 Top1 抑制剂的分子和细胞药理学，该抑制剂刚刚开始使用 Top1 耗尽的人体细胞，我们已经证明了 Top1 在治疗中的关键作用。我们还首次表明，top1 的捕获（通过药物或潜在的 DNA 损伤）会产生转录相关的 DNA 双链断裂。 R 环的形成。我们扩展了对拓扑异构酶相关 DNA 损伤修复的研究，并将重点放在酪氨酰-DNA-磷酸二酯酶 (Tdp1) 上。我们的研究重点是 Tdp1 的细胞调节以及可与喜树碱或茚并异喹啉类药物联合使用来治疗癌症的抑制剂的发现。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 由迄今为止已测序的所有脊椎动物基因组中存在的核基因编码。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，从而产生了几乎等于形成线粒体 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。我们与德国的 Fritz Boege 博士合作，证明了靶向（核）Top1 是有毒的，这表明尽管 Top1mt 和 Top1 具有高度同源性，但它们的功能不同。我们还培育了 Top1mt 基因敲除小鼠并正在研究它们的表型。