苦木和臭椿中靶向Hsp90蛋白抑制肝癌的生物碱类成分的快速发现及作用机制研究

China is the country with the highest incidence and mortality rate of liver cancer in the world. Sorafenib, one of the most important clinical drug targeting liver cancer, can be suitable for patients with unresectable advanced liver cancer, but it is very susceptible to drug resistance. Therefore, it is of great significance to find new lead compounds targeted on liver cancer from natural products. Preliminary activity screening by liver cancer cell and animal experiments confirmed that the roots, stems and leaves of Picrasma quassioides as well as the roots, barks and leaves of Ailanthus altissima from Simarubaceae possessed good inhibitory activities on liver cancer. These active constituents were isolated and identified from the roots and stems of Picrasma quassioides and their inhibitory activity on liver cancer were investigated, leading to the exploration of three active constituents which were effective against sorafenib-resisting liver cancer cells. Subsequently, a network pharmacology model of Simarubaceae/liver cancer was established. Based on the prediction results of network pharmacology and verification of relevant pharmacological experiments, we proposed a scientific hypothesis of this project that the alkaloids from Picrasma quassioides and Ailanthus altissima might treat liver cancer by targeting at Hsp90 protein. Thus, based on the existing researches, this project was aimed at isolating alkaloids which were targeted at Hsp90 protein from the leaves of Picrasma quassioides and the roots, barks and leaves of Ailanthus altissima by using UF-LC-MS technology, and screening for their anti-hepatoma activities and investigating possible mechanism. Moreover, further study was performed to explore the subtypes and the active sites of subtypes which the compounds acted on when targeting at Hsp90 protein. The implementation of this project can lay a theoretical foundation for the research and development of anti-hepatoma effects from the plants of Simarubaceae.

中国是全球肝癌发病率最高和死亡人数最多的国家，对无法手术的晚期肝癌患者，索拉非尼是最重要的一种靶向治疗肝癌的药物，但其极易产生耐药，因此从天然产物中寻找新的靶向治疗肝癌的先导化合物意义重大。通过前期实验确定了苦木的枝、叶；臭椿的根皮、叶具有很好的肝癌抑制活性，并完成了苦木枝中活性成分的分离及抗肝癌药理实验，从中发现3个具有很好抗肝癌活性的生物碱，且对索拉非尼耐药的肝癌细胞有效。在此基础上建立了苦木科/肝癌的网络药理模型。基于网络药理的预测的靶点及相关药理学实验验证，我们提出了本项目的科学假说：苦木和臭椿中的生物碱，可能通过靶向调控Hsp90抑制肝癌。因此，本项目拟采用超滤液质技术导向分离苦木叶以及臭椿根皮、叶中靶向抑制Hsp90蛋白的生物碱，对其进行抗肝癌活性筛选及作用机制研究，并阐明其对Hsp90蛋白亚型的抑制选择性及作用位点。本项目的实施可为苦木科植物抗肝癌的研究和开发奠定理论基础。

原发性肝癌是我国乃至全世界常见的恶性肿瘤之一，中国是全球肝癌发病率最高和死亡人数最多的国家，开发新型治疗肝癌的药物十分迫切。我国的传统中药植物资源是一个蕴含大量潜在天然药物小分子的资源库，从中寻找治疗肝癌的先导化合物具有十分重要的意义。本项目对苦木科两种中药苦木（枝、叶）和臭椿（根皮、叶）中的抗肝癌活性成分进行了分离鉴定，得到了470个化合物，包括127个新化合物。对分离的化合物进行了肝癌细胞抑制活性筛选，优选出了先导化合物去氢苦木碱，并进行了体内外的抗肝癌药效学实验，发现其在体内外均表现出了很好的抗肝癌活性。药理机制研究结果显示去氢苦木碱通过影响线粒体复合物 I、III、IV的功能，使得两种肝癌细胞系（Hep3B，HepG2）的线粒体功能发生障碍，进而诱导了肝癌细胞发生凋亡，由此发挥抗肿瘤的作用。为了进一步将其开发成抗肝癌药物，我们对去氢苦木碱进行了全合成，解决了其药物资源问题。并在此基础上进行了结构优化，获得了20个去氢苦木碱的衍生物，对其进行了肝癌细胞抑制活性筛选，发现部分衍生物显示了更优的抗肝癌活性。本项目的研究结果显示去氢苦木碱及其衍生物有进一步开发成抗肝癌新药的潜能，相关研究成果为苦木科药用植物的活性成分及抗肝癌新药开发提供了重要的科学数据，具有很好的科学意义和应用价值。本项目执行过程中在Natural Product Reports、Journal of Natural Products、Phytotherapy Research、Phytochemistry等本领域权威杂志上发表标注本项目基金号的SCI论文26篇，申请专利3项，授权2项。培养毕业博士研究生1名，硕士研究生6名。