Novel Topoisomerase I Inhibitors

描述(申请人提供)：该项目的长期目标是设计、合成和评估用于癌症治疗的新型拓扑异构酶I(Top1)抑制剂。临床研究将使临床结果揭示的未来问题能够立即和有效地得到解决。这可能包括结构修改，以解决药物毒性、耐药性、不利的药代动力学和缺乏效力所导致的潜在问题。Top1的设计策略将包括通过药物化学、计算机图形学分子建模、分子力学、从头算量子力学和生物化学研究来评估稳定抑制剂/酶/DNA三元复合体的基本力。此外，新的Top1抑制剂的设计将借助晶体学抑制剂/酶/DNA三元络合物，这将有助于基于结构的药物设计。新的Top I抑制剂的合成将采用多种合成方法，包括吲哚异喹啉-喜树碱杂化化合物，芳硫菌素的氮类似物，以及氮杂异喹啉类化合物。所产生的抗癌剂将通过附着低分子寻的配体而靶向于癌细胞和实体肿瘤，这些配体在选择性地吸收到癌细胞后将被代谢除去，而不是正常细胞。这些结合物将通过测试释放机制、监测细胞增殖抑制、阻断归巢配体与癌细胞的附着以帮助阐明作用机制以及确定癌细胞培养的NCI小组的选择性来进行评估。在停滞的裂解复合体中，连接Top I的Tyr723和DNA的3‘-磷酸的磷酸二酯键被酪氨酰-DNA-磷酸二酯酶I(Tdp1)水解。由于Tdp1抑制剂抵消Top1抑制剂的作用，Tdp1抑制剂可能与Top1抑制剂协同作用。该项目的一个目标是将Top1和Tdp1抑制活性整合到相同的抗癌药物中，这两种药物预计比缺乏Tdp1抑制活性的Top I抑制剂的效力要强得多。这将利用几种吲哚异喹啉中Tdp1抑制活性的独特发现。这项研究产生的Top1抑制剂将通过各种检测方法进行评估，包括：1)Top1介导的DNA切割反应；2)Top1-DNA连接和切割复合体的可逆性；3)切割复合体形成和逆转的动力学；4)DNA解离以监测嵌入；5)抑制Top1介导的DNA松弛；6)抑制剂在哺乳动物细胞中诱导的蛋白质链断裂；7)癌细胞培养中的细胞毒性检测，包括喜树碱耐药细胞系；8)中空纤维研究和异种移植试验；9)抗生素活性与非洲锥虫的对比。

DESCRIPTION (provided by applicant): The long-term goals of this project are to design, synthesize and evaluate novel topoisomerase I (Top1) inhibitors for the treatment of cancer. The clinical studies will allow future problems revealed by the clinical results to be addressed immediately and effectively. This could include structural modification to address potential problems resulting from drug toxicity, resistance, unfavorable pharmacokinetics, and lack of potency. The Top1 design strategy will involve an evaluation of the fundamental forces stabilizing the inhibitor/enzyme/DNA ternary complexes through medicinal chemistry, computer graphics molecular modeling, molecular mechanics, ab initio quantum mechanics, and biochemical studies. In addition, the design of new Top1 inhibitors will be aided by crystallography inhibitor/enzyme/DNA of ternary complexes, which will facilitate structure-based drug design. A variety of synthetic methods will be employed in the syntheses of new Top I inhibitors, including indenoisoquinoline-camptothecin hybrids termed "aromathecins", nitrogen analogues of the aromathecins, and azaindenoisoquinolines. The resulting anticancer agents will be targeted to cancer cells and solid tumors through attachment of low molecular weight homing ligands that will be removed metabolically after selective uptake into cancer cells as opposed to normal cells. The conjugates will be evaluated by testing the release mechanism, monitoring inhibition of cell proliferation, blocking the attachment of the homing ligand to cancer cells to help elucidate mechanism of action, and determining selectivities in the NCI panel of cancer cell cultures. The phosphodiester bond linking Tyr723 of Top I to the 3'-phosphate of DNA in stalled cleavage complexes is hydrolyzed by tyrosyl-DNA-phosphodiesterase I (Tdp1). Since Tdp1 inhibitors counteract the action of Top1 inhibitors, Tdp1 inhibitors might interact synergistically with Top1 inhibitors. A goal of this project is to incorporate both Top1 and Tdp1 inhibitory activities into the same anticancer agents, which are expected be significantly more potent than those of Top I inhibitors lacking Tdp1 inhibitory activity. This will exploit a unique discovery of Tdp1 inhibitory activity in several indenoisoquinolines. The Top1 inhibitors resulting from this study will be evaluated in a variety of assays including those involving: 1) Top1-mediated DNA cleavage reactions; 2) Top1-DNA linkage and reversibility of cleavage complexes; 3) kinetics of cleavage complex formation and reversal; 4) DNA unwinding to monitor intercalation; 5) inhibition of Top1-mediated DNA relaxation; 6) protein-linked strand breaks induced by inhibitors in mammalian cells; 7) cytotoxicity assays in cancer cell cultures, including camptothecin-resistant cells lines; 8) hollow fiber studies and xenograft testing; 9) antibiotic activity vs. African trypanosomes.