【作者】 卢艳；

【导师】 赵春燕；

【作者基本信息】 兰州大学 ， 药物化学， 2012， 硕士

【摘要】 鬼臼毒素(podophyllotoxin)最早是从盾叶鬼臼(P.peltatum)中提取分离得到的一类具有抗肿瘤活性的木脂素类化合物。尽管有很强的体外抗肿瘤活性,但由于其较大的细胞毒性,临床上主要外用治疗皮肤癌。因此研究学者致力于一系列以鬼臼为母核结构的半合成衍生物的研究,希望找到副作用小并且可口服的鬼臼衍生物。鬼臼毒素是通过抑制微管聚合而发挥抗癌活性。微管是一个关键的真核细胞的细胞骨架成分,参与了有丝分裂,细胞分裂等重要生理过程。α-微管蛋白和β-微管蛋白是组成微管的基本单位,鬼臼毒素与微管蛋白结合于秋水仙碱位点,抑制微管聚合,可以阻止细胞生长于M期(细胞周期的有丝分裂阶段),产生细胞毒作用从而导致细胞死亡。近些年发现鬼臼毒素类似物的另一种药用价值体现在杀虫作用方面,研究发现木脂素类化合物可以帮助植物抵御昆虫的侵袭,对昆虫主要表现为拒食作用,在一些特定情况下,他们也可以影响昆虫的生理功能。植物源杀虫剂由于其高效低残留等优点深受国内外学者青睐。首先,本研究针对19个具有杀虫活性的鬼臼类衍生物,为了阐明化合物结构与杀虫活性的关系,运用了比较分子场分析(CoMFA)和比较分子相似性指数分析法(CoMSIA)对19个化合物进行建模,得到了可信的模型,此外,我们还运用了分子对接方法,研究微管蛋白与化合物的结合模式。3D-QSAR模型结合对接方法,不仅可以更好地了解昆虫拒食活性与结构的关系,也有助于今后设计合成更高活性的杀虫剂。其次,我们对十个鬼臼类衍生物进行体外活性测定,将化合物作用于一系列人类癌细胞：人肺腺癌细胞株A549,人宫颈癌细胞株HeLa,人肝癌肝癌细胞株HepG2,人胃癌细胞株BGC-832,得到了这类化合物的IC50值,其中化合物6b和6g的活性最高。此外,为了证明这类化合物在体外可直接作用于微管蛋白,我们从羊脑中提取微管蛋白,测试抑制微管聚合的活性,得到了化合物6b和6g的微管抑制率IC50值,为了更好的探讨鬼臼类衍生物与微管蛋白的相互作用,开展了6b和6g的动力学模拟研究,在此基础上利用MMPBS/GBSA方法计算体系的结合自由能,探究分子间的相互作用,获得了结合位点处详细的结构和能量信息,这对于理解蛋白质的活性位点和设计类似的小分子抑制剂具有重要的意义。更多还原

【Abstract】 Podophyllotoxin is a natural medicine possessing an outstanding anti-tumour activity. It can be extracted from the rhizome of Podophillum peltatum (American Podophyllum). Due to its severe toxicity, it was mainly prescribed for the treatment of venereal warts. Prompted by the clinical successes of the podophyllotoxin, significant efforts have been focused on identifying new analogues that have a similar mechanism of action yet superior properties such as low or nil toxic side effects and better oral availability.Current evidence suggests that podophyllotoxin affect tubulin polymerization dynamics by binding to microtubules (MT) at colchicine pocket. Mircotubules, a major component of the cytoskeleton composed of a and β subunit, play a role in a variey of cellular functions including mitosis, intracellar transport, and the modulation of cell morphology. There was conclusive evidence that microtubule disruption affected by podophyllotoxin causes cell cycle arrest mainly at the G2/M phase, and subsequently induceed apoptosis, a cell suicide mechanism.Lignans and biogenetically-related secondary metabolites play a significant role in the defence of plants against insects. They act largely as regulators of insect feeding, but in a few cases they can influence also specific physiological functions of insects. Due to the high insecticidal activity and low toxicity of botanical pesticides, it attract great attention from in and abroad.Podophyllotoxin analogues were proved to be potential insecticides against the fifth-instar larvae of Brontispa longissima in vivo. To elucidate the structural properties of these compounds for insecticidal activity, comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA) were applied to a novel class of19pyrazolopyrimidine derivatives. Based upon the information derived from CoMFA and CoMSIA contour maps, we have identified some key structural features which explain the observed variance in the activities. In addition, a docking approach was used to map the3D-QSAR models to the active site of tubulin protein, which can provide us new insights into the protein-ligand interactions. The3D-QSAR model and docking study not only lead to a better understanding of structural requirements of antifeedant activities but also help us in the design of potent and selective compounds with enhanced activities.A series of ten podophyllotoxin derivatives were evaluated for biological activities. The anti-proliferative activities were tested against a panel of human cancer cell lines (human lung adenocarcinoma epithelial cell line A549, human carvical carcinoma cell line HeLa, human hepatocellular liver carcinoma cell line HepG2and human gastric carcinoma cell line BGC-832). Cytotoxic effects of the two lead molecules, namely6b and6g, were further confirmed and evaluated by conventional assays with tubulin including cellular microtubule distribution, and finally in vitro microtubule assembly with purified tubulin. More importantly, all experimental measurements were supported by molecular modeling and dynamic simulation study to understand the nature of these interactions. The information available from molecular modeling and dynamic simulation. These conclusions and methods used in this article will provide theoretical guide for further study, and help us to design and synthesize novel anticancer drugs.更多还原