逆转肿瘤耐药性是肿瘤治疗的重要策略之一，基于膜转运蛋白活性抑制机制开发的拓扑异构酶2（Top2）毒剂肿瘤耐药逆转剂因毒副作用较大，临床应用受到了限制。因此，寻找高效、低毒的新型逆转剂成为肿瘤耐药研究的重要内容。在利用固定化酶进行药物筛选的前期工作基础上，本项目拟以TDP2为靶分子，以5种常用的抗肿瘤中药为研究对象，通过制备毛细管整体固定化TDP2亲和柱，构建二维整体亲和色谱-nLC-MS/MS系统筛选中药中TDP2抑制活性成分，从分子水平和细胞水平验证筛选出的活性成分的活性及其对化疗耐药细胞的增敏效果，根据酶学原理探讨其抑制机理，采用分子对接的方法研究活性成分与酶结合的位点。本研究首次制备固定化TDP2亲和柱，建立高效、快速的TDP2酶抑制剂筛选方法，如果课题成功实施和完成，将为中药TDP2抑制剂的筛选提供新技术，也为Top2毒剂肿瘤耐药逆转剂的开发提供新思路。

To reverse multidrug resistances(MDR) has been one of the important strategies in tumor treatment. The existing reversal agents for Top2 poisons designed by inhibiting the activity of membrane transporter protein are limited by severe side effects in clinic. Hence, it is urgent to search for safe and effective resistance reversal agents. On the basis of previous work on preparation of immobilized enzyme affinity column, this project aims to screen active constituents for TDP2 inhibition from five commonly used herbs by preparing capillary monolithic TDP2 affinity column and constructing two dimensional monolithic affinity chromatography(MAC)-nanoLC-MS/MS system. The activities of the screened compounds are verified by enzyme activity assay and tumor cell proliferation inhibition and chemotherapy sensitizer test. The inhibition mechanism is discovered by Lineweaver-Burk principle and binding sites are studied by molecular docking technology. In this project, the stable and robust monolithic affinity column is firstly designed to construct the on-line second dimensional MAC-nanoLC-MS/MS system, which provide a new approach for TDP2 inhibitors screening and new ideas for developing new tumor resistance reversal agents for Top2 poisons.