苦豆子中TopoⅠ/Ⅱ酶抑制剂的靶向筛选及其抗肿瘤机制研究

Sophora alopecuroides L. belongs to leguminous Sophora plant，which has a variety of effects such as detoxification, expelling wind and dampness, relieving pain and insecticide. It is used to obtain better curative effect in the treatment of a variety of cancers in the clinic. It was found that several quinolone alkaloids has a strong inhibitory activity for topoisomerase Topo in Chinese medicine Sophora alopecuroides L., and their inhibitory activity is far stronger than that of the positive drug etoposide in our past work. But due to the limited sensitivity of separation technology, low content active compounds of Sophora alopecuroides failed to be separated. Therefore, the proper bio-targets TopoⅠ and Topo II would be used to prepare new affinity materials base on the target protein hybrid nano-flowers which could be used to capture the new active ingredients from Sophora alopecuroides. And these compounds would be isolated and identified combined with UPLC-QTOF-MS technology. Cytotoxic test and report gene technology would be used to confirm the antitumor activity of compounds. Then, 1-2 lead compounds are selected for further study. The inhibitory activity and inhibiting TopoⅠ / II mechanism of lead compounds would be studied by DNA relaxation, DNA fragmentation detection model, and in vivo tumor-bearing animal model. It is expected that a clear mechanism and good lead compounds are obtained from Sophora alopecuroides L.

中药苦豆子具有清热解毒、祛风燥湿、止痛杀虫等功效，临床用于治疗多种癌症，疗效确切。前期研究发现苦豆子中多个喹诺里西啶类生物碱抑制拓扑异构酶Topo，抗肿瘤活性远强于阳性药物依托泊苷，极具应用前景；同时发现苦豆子还存在微量强活性成分，限于常规分离技术的灵敏度未能分离成功。为此，拟利用课题组专利纳米固定化成熟技术，以商品化TopoⅠ和TopoⅡ为靶标，制备靶蛋白杂化纳米花亲和材料，从苦豆子中靶向捕获强活性成分，并结合UPLC-QTOF-MS技术高效分离鉴定化合物，并利用细胞毒实验模型、报告基因技术确证化合物抗肿瘤活性，优选1-2个极具前景的活性化合物深入研究。建立DNA松弛、DNA断裂检测模型及整体动物荷瘤抑制模型，研究化合物的抑制活性、抑制类型及方式，明确活性化合物通过抑制TopoⅠ/II酶活性发挥抗肿瘤作用机理，有望从苦豆子中获得机制清楚、成药性好的先导化合物，为创新药物开发奠定基础。

前期在国家自然科学基金资助下，多年研究发现特色中药苦豆子中存在多个喹诺里西啶生物碱具有抑制拓扑异构酶TopoI，抗肿瘤活性强，但限于分离筛选技术未能成功分离且作用机制不清。为了快速精准筛选其这些目标成分，本研究以TopoI酶作为靶标，以Cu离子作为固定支架，通过无机-有机蛋白杂化成功制备了高特异性和高载量的亲和多维纳米材料，从苦豆子中靶向捕获强结合活性成分，并结合UPLC-QTOF-MS技术高效分离鉴定化合物，获得了6个活性化合物，它们是苦参碱、氧化苦参碱、槐定碱、氧化槐果碱，金雀花碱和槐胺碱，并利用细胞毒实验模型、报告基因技术等初筛、复筛确证3个活性较强的TopoI酶抑制剂，它们是苦参碱、金雀花碱和槐定碱，并进一步对其作用机制进行系统研究，经TopoI介导的DNA裂解实验、凝胶阻滞实验、DNA嵌入实验等证明了它们抑制TopoI的双重作用机制：(1)稳定TopoI-DNA裂解复合物；(2)抑制或阻断TopoI与底物DNA的结合。本项目所建立地从复杂体系中靶向筛选活性成分的新方法，能有效地保持了靶蛋白的原有活性，且在有机溶剂环境中稳定性更好。依据锁钥理论，特异性铺获目标化合物，结合UPLC-QTOF-MS的分离和鉴定优势，简化了分离鉴定和活性验证步骤，大大提高复杂体系中靶蛋白抑制剂识别和分离的效率，同时兼具了耐用性，制备简单的特点。这种靶向捕获技术为实现复杂体系中活性化合物的快速筛选分离提供了新思路和新手段，在活性成分筛选分离方面拥有很大的应用潜能。首次阐明了苦参碱、金雀花碱和槐定碱抑制TopoI酶的作用机制，为后续组合抗肿瘤药物的开发奠定基础。