阿霉素与紫杉醇生物素化探针的合成及初步应用

发布时间：2018-03-04 13:33

本文选题：多药耐药　切入点：生物素亲和素系统　出处：《江南大学》2017年硕士论文　论文类型：学位论文

【摘要】：已上市药物的药理学解释的关键涉及药物作用靶点的确认,研究方法主要是设计和获得保留药物作用的探针分子,进行生物材料中分析相应药物靶点。本研究以临床治疗肿瘤一线药物阿霉素(ADM)和紫杉醇(PTX)为研究材料,设计生物素化药物分子,通过亲和素组合形成药物探针分子,从乳腺导管上皮癌MCF-7细胞系的耐ADM细胞(MCF-7/ADM)、PTX细胞(MCF-7/PTX)中获得相应的药物结合蛋白,探讨MCF-7细胞的耐药机制。主要结果如下:1、计算机辅助设计保持药物识别功能的生物素-C6-药物的连接结构,在筛选中找到简便的一步法合成中间体生物素-氨基己酸(产物结构经核磁图谱确证,纯度为94.65%),并采取DCC,DMAP催化法合成获得生物素化ADM与生物素化PTX两种药物类似物,经1H NMR和HPLC确认结构及纯度后,测试药物类似物对细胞生长抑制情况及其在细胞中的分布并与原药对比:生物素化ADM和生物素化PTX干预MCF-7、MCF-7/ADM以及MCF-7/PTX的IC50没有明显的改变:当作用于MCF-7/W细胞,生物素化ADM的IC50值为8.554μM,ADM的IC50值为8.656μM,对于MCF-7/ADM细胞,生物素化ADM的IC50值为127.2μM,ADM的IC50值为104.5μM;当作用于MCF-7/W细胞,生物素化PTX的IC50值为10.17μM,PTX的IC50值为12.01μM;对于MCF-7/PTX细胞,生物素化PTX的IC50值为82.16μM,PTX的IC50值为93.26μM;激光共聚焦成像亦验证了其在细胞内的分布和作用方式没有明确的变化,说明生物素碳链结构的引入不会干扰ADM或PTX的药物作用。2、以生物素化ADM和生物素化PTX药物类似物为探针,与包被链霉亲和素的磁珠组建可快速分离药物探针分子,以MCF-7/ADM与MCF-7/PTX细胞裂解液为研究材料,分别采用生物素化ADM和生物素化PTX药物类似物-亲和素磁珠分析相应的结合蛋白。经电泳分离,Q TOF-MS检测蛋白序列并与数据库对比,获得对ADM耐药性状相关的蛋白Junction plakoglobin和Hyaluronidase,以及与PTX耐药性状相关的蛋白Glutathione S-transferase P,Peroxiredoxin-1等二十余种蛋白。本研究构建了具有一定通量的分析药物结合蛋白的方法,为筛选和确认药物作用靶点提供了新的可行的工具,有助于解析相应药物的作用机制和耐药机理。
[Abstract]:The key to the pharmacological interpretation of marketed drugs involves the identification of drug action targets, primarily by designing and obtaining probe molecules that retain the effects of drugs. Using adriamycin adriamycin (adriamycin) and paclitaxel (PTX) as the research materials, the biotin drug molecules were designed, and the drug probe molecules were formed by the combination of avidin, adriamycin (adriamycin) and paclitaxel (PTX). The corresponding drug-binding protein was obtained from MCF-7 / ADMN-PTX cell line of breast ductal carcinoma MCF-7 cell line, which was resistant to ADM, and the corresponding drug binding protein was obtained from MCF-7 / PTX cell line. To study the mechanism of drug resistance in MCF-7 cells. The main results are as follows: 1. Computer aided design of the connective structure of biotin -C6- drugs, which maintains drug recognition function, A simple one-step method was found for the synthesis of biotinylhexanoic acid (the structure of the product was confirmed by NMR spectra, the purity was 94.65g), and the biotinylated ADM and biotinylated PTX analogues were synthesized by DCC-DMAP catalytic method. The structure and purity were confirmed by 1H NMR and HPLC. The inhibition of drug analogues on cell growth and their distribution in cells were measured and compared with the original drug. Biotin ADM and biotinylated PTX did not change significantly in MCF-7 / 7 / ADM and MCF-7/PTX IC50: they were used in MCF-7/W cells. The IC50 value of biotinylated ADM was 8.554 渭 M, the IC50 value of biotin ADM was 8.656 渭 M, the IC50 value of biotin ADM was 127.2 渭 M, the IC50 value of biotinylated PTX was 104.5 渭 M, the IC50 value of biotinylated PTX was 12.01 渭 M for MCF-7/W cells, and the IC50 value for MCF-7/PTX cells was 10.17 渭 m. The IC50 value of biotinylated PTX was 82.16 渭 MPTX and the IC50 value of biotinylated PTX was 93.26 渭 M. Laser confocal imaging also proved that there was no clear change in the distribution and mode of action in the cells. It was suggested that the introduction of biotinylated carbon chain structure would not interfere with the drug action of ADM or PTX. Using biotinylated ADM and biotinylated PTX analogues as probes and magnetic beads coated with streptavidin, the drug probe molecules could be rapidly separated. Using MCF-7/ADM and MCF-7/PTX cell lysate as materials, the binding proteins were analyzed by biotin ADM and avidin magnetic beads respectively. The protein sequences were detected by electrophoresis and compared with the database. More than 20 proteins, such as Junction plakoglobin and Hyaluronidase associated with drug resistance to ADM, and Glutathione S-transferase P#en0# peroxiredoxin-1 were obtained. It provides a new and feasible tool for screening and confirming drug action targets, and helps to analyze the mechanism of drug resistance and action.
【学位授予单位】：江南大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R96

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