Novel Indenoisoquinoline Topoisomerase I Inhibitors

描述(由申请人提供)： 拓扑异构酶I是开发治疗人类癌症的新型化疗药物的重要靶点。尽管喜树碱类拓扑异构酶I抑制剂的一些成员目前在临床上用作抗癌药物，但喜树碱存在一些固有的局限性，包括内酯开环导致的化学和代谢不稳定，以及拓扑异构酶I抑制在药物去除后的快速可逆性。由于这些限制，有效的喜树碱化疗需要长时间的静脉输液和长期持续的暴露。 我们研究组最近的研究与其他人合作，合成了一类新的拓扑异构酶I抑制剂，吲哚异喹啉。酶抑制研究、体外细胞毒性结果和体内动物研究的结合提供了令人信服的证据，证明吲哚异喹啉类化合物将克服喜树碱的一些局限性。目前提出的研究计划的主要目标之一将是设计和合成更有效的吲哚异喹啉作为拓扑异构酶I抑制剂，在人类癌症治疗中具有潜在的临床应用价值。 可以假设，吲哚异喹啉拓扑异构酶I抑制剂的有效性可以通过利用稳定DNA、酶和抑制剂形成的三元络合物所涉及的各种化学力来最大化。负责药物与三元络合物中DNA碱基键合的作用力包括电荷转移络合物的形成、静电引力和伦敦弥散力。除了这些作用力外，三元络合物还通过吲哚异喹啉与酶的一些附近氨基酸残基的氢键来稳定。这项研究项目的主要目标之一将是利用我们对药物-酶-DNA三元复合体的晶体结构的知识，在一系列新的药物分子中最大化这些不同的力量。我们目前已经确定了两个吲哚异喹啉形成的配合物的晶体结构，我们预计通过未来的努力将有更多的晶体结构出现。吲哚异喹啉的结构也将被操纵，以便将它们靶向于癌细胞，最大化良好的溶解性，并增加它们的生物利用度。 在这项研究中制备的新化合物将在一些生化和生物测试中进行评估。这些将包括在抑制剂存在的情况下分析拓扑异构酶I诱导的DNA裂解模式，分析三元复合体的稳定性，检测插入的DNA解离研究，抑制拓扑异构酶I介导的DNA松弛，检测细胞系统中的DNA链断裂，以及分析喜树碱耐药突变细胞系的酶抑制活性。吲哚异喹啉类化合物在癌细胞培养中的细胞毒性将被确定，并将在各种动物模型系统中研究有前景的抗癌活性候选者。将详细研究吲哚异喹啉的作用机制，并将利用这些信息来最大限度地发挥其作为抗癌药物的治疗潜力。

DESCRIPTION (provided by applicant): Topoisomerase I is an important target for the development of new chemotherapeutic agents for the treatment of cancer in humans. Although some members of the camptothecin class of topoisomerase I inhibitors are presently in clinical use as anticancer agents, the camptothecins suffer from a number of inherent limitations, including chemical and metabolic instability due to lactone ring opening, and rapid reversibility of topoisomerase I inhibition upon drug removal. Because of these limitations, effective chemotherapy with the camptothecins requires long I.V. infusions and prolonged and continuous exposure. Recent studies in our research group, in collaboration with others, have resulted in the synthesis of a new class of topoisomerase I inhibitors, the indenoisoquinolines. A combination of enzyme inhibition studies, in vitro cytotoxicity results in human cancer cell cultures, and in vivo animal studies have provided compelling evidence that the indenoisoquinolines will overcome some of the limitations of the camptothecins. One of the main goals of the presently proposed research program will be to design and synthesize more effective indenoisoquinolines as topoisomerase I inhibitors with potential clinical application in the treatment of cancer in humans. It can be assumed that the efficacy of the indenoisoquinoline topoisomerase I inhibitors can be maximized by taking advantage of the various chemical forces that are involved in stabilization of the ternary complexes formed from DNA, the enzyme, and the inhibitors. The forces responsible for the bonding of the drug to DNA bases in the ternary complex include charge-transfer complex formation, electrostatic attraction, and London dispersion forces. In addition to these forces, the ternary complexes are also stabilized through hydrogen bonding of the indenoisoquinolines to some of the nearby amino acid residues of the enzyme. One of the main goals of this research project will be to maximize each of these various forces in an array of new drug molecules using our knowledge of the crystal structures of the drug-enzyme-DNA ternary complexes. We presently have determined the crystal structures of complexes formed from two indenoisoquinolines, and we expect that additional crystal structures will become available through future efforts. The structures of the indenoisoquinolines will also be manipulated in order to target them to cancer cells, maximize favorable solubility properties, and increase their bioavailabilities. The new compounds prepared in this study will be evaluated in a number of biochemical and biological assays. These will include analysis of DNA cleavage patterns induced by topoisomerase I in the presence of the inhibitors, analysis of the stabilities of the ternary complexes, DNA unwinding studies to detect intercalation, inhibition of topoisomerase I-mediated DNA relaxation, detection of DNA strand breaks in cellular systems, and analysis of enzyme-inhibitory activities in camptothecin resistant mutant cell lines. The cytotoxicities of the indenoisoquinolines in cancer cell cultures will be determined, and promising candidates will be investigated for anticancer activity in a variety of animal model systems. The mechanism of action of the indenoisoquinolines will be studied in detail, and the information will be used to maximize their therapeutic potential as anticancer agents.