基于甜菊苷和冬凌草甲素复杂母核结构高内涵化合物库的构建和抗肿瘤活性评价

The structural modification of natural products with unique scaffolds and excellent bioactivities is an important approach to attain new antitumor drugs. Among the natural compounds, terpenoids have been the focus of research for a long time. In the year of 2015, oridonin derivative HAO472 was advanced into human clinical trial, which raised the attention of this kind of compounds once again. In the previous study, the applicant has completed parts of structural modification work of oridonin and accumulated some research experiences. In this project, sustainable, commercially available ent-kaurane diterpenoid compounds oridonin and stevioside will be used as the leads. Based on the prior established ring systems and chiral centers in the structures, and the analysis of the relationships of biogenesis and structures between different kinds of diterpenoids, high content compound library of natural derivatives with novel structures and excellent bioactivities will be constructed. The antitumor effects of the derivatives in the library will be evaluated and the structure-activity relationships will be concluded. By applying human apoptosis and cell cycle protein array kits, chemical probes, drug affinity responsive target stability assay and classical pharmacological methods, the antitumor molecular mechanisms will be well studied to reveal the pathways and targets of drug action. It is hoped that we will have a better understanding of the pharmaceutical properties of these compounds, and finally obtain novel and valuable drug candidates through the research work in this project.

对结构独特、活性优良的天然产物进行结构改造是获得抗肿瘤新药的重要途径之一，其中萜类一直是研究的热点。2015年，以冬凌草甲素为先导的新药HAO472进入临床研究，再一次提高了对此类化合物的关注度。前期工作中，完成了部分对冬凌草甲素的结构改造工作，积累了研究经验。本项目将以具有共同ent-贝壳杉烷二萜母核结构的、可持续获得的、市售的冬凌草甲素和甜菊苷为先导，利用其结构中已具有的环系和手性中心等因素，在分析不同类型二萜生源关系和结构联系的基础上，构建包含结构新颖、活性优良的天然产物衍生物的高内涵化合物库。对库中衍生物进行抗肿瘤活性评价，获得构效关系。通过凋亡和细胞周期蛋白相关试剂盒、探针类衍生物辅助和DARTS靶点垂钓等有效的方法和经典的药理手段，深入研究抗肿瘤分子机制，确定其作用通路和靶标。希望通过该项目的研究对这些类型化合物的药学特性有更深入的认识，最终获得全新的、有价值的抗肿瘤候选药物。

本项目通过化学合成解决了十余类结构新颖、活性优良，但天然含量低、提取分离困难的二萜衍生物的来源问题。获得具有新颖结构的百余个衍生物，初步构建了高内涵化合物库。完成了体内外活性评价，总结构效关系，并寻找每种结构类型的优选化合物的作用通路和靶标。得到可持续性获得、药效优良的抗肿瘤候选化合物。如氨基酸修饰衍生物YMC-HX-014对人肝癌细胞Bel-7402的IC50值为0.70 μM，对人正常肝细胞L-02的IC50值大于50 μM，表现出很好的抗肿瘤细胞与正常细胞增殖选择性。初步的机制研究显示，YMC-HX-014能诱导Bel-7402细胞发生凋亡，阻滞细胞周期于G1期，并使线粒体膜电位下降。Western blot实验结果显示，YMC-HX-014处理的Bel-7402细胞中，Bcl-2、Bcl-xL和pro-caspase-3的表达水平下调，Bax和caspase-3的表达水平上调。氮芥类衍生物YMC-GX-016对Bel-7402细胞的IC50值达到0.77 μM，对耐5-Fu的Bel-7402细胞IC50值为2.07 μM，而对人正常肝细胞几乎没有抗增殖活性，IC50值大于100 μM，选择性指数SI大于130。YMC-GX-016处理Bel-7402细胞，线粒体膜电位下降，细胞发生凋亡，阻滞细胞周期于G1期。上调Bax/Bcl-2的比例，以及cytochrome c、p53、caspase-3和-9的表达水平。硫化氢供体型衍生物DLC-LHN-146对人髓性白血病K562细胞、人肝癌HepG2细胞、人结肠癌HCT-116细胞三种肿瘤细胞系的IC50值分别为0.95、2.57和5.81 μM，对人正常肝细胞L-02和人外周血细胞PBMC的IC50值分别为17.59 μM和大于100 μM。运用Human Apoptosis Array试剂盒测试用DLC-LHN-146处理后K562细胞35种凋亡相关蛋白的变化趋势。发现procaspase-3、c-IAP1、c-IAP2、Survivin、XIAP和Livin等蛋白的表达量下降，Bax、DR4和DR5的表达量上升。表明DLC-LHN-146对K562细胞的抗增殖作用，可能通过内源性和外源性凋亡的共同作用。相关成果已发表通讯作者第一标注SCI论文8篇，授权发明专利9项。培养博士研究生2名，硕士研究生5名。

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