基于亲和整体柱的药物选择性杀伤MDR肿瘤细胞靶标蛋白的垂钓研究

Multidrug resistance (MDR) represents a core issue in chemotherapy of cancer research. Collateral sensitivity (CS) compounds can kill MDR cells selectively over its parental cells. Insights into CS may offer an alternative strategy for the clinical resolution of MDR. However, the complex mechanisms by which CS agents exert selective killing of MDR cells have not been elucidated so far. It is of great interest to find MDR-selective compounds (CS agents), identify their target proteins and elucidate a CS mechanism. Our preliminary studies showed that a derivative of 23-hydroxybetulinic acid (23-HBA), 23-hydroxy-28-bi-piperidine betulinic acid (B5G1) can selectively kill MDR tumor cell lines over the drug sensitive parent cell line. In this project, both the CS agent B5G1 and non-CS agent 23-HBA will be utilized as molecular probes, for the serial preparation of novel monolithic capillary affinity columns through a two-step or single-step co-polymerization of these two molecular probes with glycidyl methacrylate and ethylene dimethacrylate. These affinity monolithic columns combined with nano-HPLC-LTQ-Orbitrap MS will be used to seek out and identify the drug binding proteins from the cell lysate of both the MDR tumor cell line and parent cell line. By combining bioinformatics, gene Silencing etc, the detected proteins will be further analyzed and verified in order to explore the CS target proteins of B5G1. This research provides not only a valuable tool for the profiling of binding proteins of small molecular drugs as well as bioactive compounds, but also a theoretical foundation for elucidating the mechanisms by which CS agents exert selective killing of MDR cells.

如何克服肿瘤多药耐药（MDR）是当前肿瘤治疗研究中的核心问题之一。近年研究发现某些药物能选择性杀伤MDR细胞而对母体肿瘤细胞无效，这为攻克MDR问题提供了一条新的思路，但相关作用机制和作用靶点还不明确。我们前期研究表明，23-羟基白桦酸（23-HBA）衍生物23-羟基-28-双哌啶白桦酸（B5G1）在人肝癌多柔比星耐药细胞株上的增殖抑制作用明显优于其相对应的敏感细胞株。本项目拟选取具有选择性杀伤MDR细胞活性的B5G1以及不具活性的23-HBA为小分子探针化合物，开发新型毛细管亲和整体色谱柱，通过色谱法从肿瘤细胞的敏感株以及耐药株裂解液中分别垂钓特异性结合的高亲和蛋白，利用LTQ-Orbitrap质谱技术对蛋白进行识别与鉴定，结合生物信息学、基因沉默等技术探索研究B5G1选择性杀伤耐药性肿瘤细胞作用的相关靶标蛋白。本课题的开展将为进一步揭示药物选择性杀伤肿瘤细胞的机理研究提供重要理论依据。

克服肿瘤多药耐药（multidrug resistance, MDR）是当前肿瘤治疗研究中的重要问题。23-羟基白桦酸（23-HBA）衍生物23-羟基-28-双哌啶白桦酸（B5G1）在人肝癌多柔比星耐药细胞株上的增殖抑制作用明显优于其相对应的敏感细胞株，提示其能选择性杀伤MDR细胞而对母体肿瘤细胞无效，为攻克MDR问题提供了一条新的思路。为了进一步认清相关作用机制和作用靶点，本研究首先以代表性化合物B5G1为基本结构，合成了一系列衍生物，结构经MS、HR-MS、NMR等确证，通过MTT方法对药物单体衍生物的肿瘤细胞杀伤活性进行考察，初步阐明了构效关系：C-3、C-23及C-28位取代基的相互影响；然后，通过对活性小分子的固定化，制备成相应的小分子亲和色谱整体柱，对细胞裂解液中靶蛋白的垂钓，并通过SDS-PAGE凝胶电泳对靶蛋白进行分析；最后，利用Sybyl 8.1软件中的Sulflex-Dock模块，以维拉帕米作为对照，对优势衍生物进行了系统的研究。本研究成果成功总结了固定化药物分子有机聚合物整体柱的制备方法，同时为进一步揭示药物选择性杀伤肿瘤细胞的机理研究提供重要理论依据。