基于HPLC-MS-SPE-NMR导向的三株赤芍内生真菌抑制CDC25A/B磷酸酶活性成分的发现

The cell division cycle 25 (CDC25) family of proteins have been identified as a group of highly conserved dual-specificity phosphatases, regulating key transitions between cell cycle phases during normal cell division through activation of cyclin dependan kinases and acting as mediators of the checkpoint response in the event of DNA damage. Overexpression of CDC25s has been frequently documented in multiple cancer cell lines, which is highly associated with clinical outcome. CDC25 phosphatases have been emerged as attractive chemotherapeutic targets in oncology. Secondary metabolites of endophytic fungi in medicinal plants, which possess varied structural types and diversity biological activities, are well-known resources for identifying natural anticancer drugs. In our ongoing search for new bioactive secondary metabolites from natural products, three strains of endophytic fungus with potent CDC25A/B phosphatase inhibitory activities were screened from healthy tissues of Peaonia lactiflora Pall which indicating the potential for discovering of novel CDC25A/B phosphatase inhibitors. On the basis of the above findings, a systematic and in-depth investigation of the secondary metabolites of the three candidate strains will be carried out. Key technologies of HPLC-HRMS-SPE-NMR and the CDC25A/B phosphatase target-based bioassay guide will be integrated and applied to discover compounds with novel structures and potent activities. Their accurate structures and pharmacological activities will be illuminated. Furthermore, chemical conversion and biotransformation methods will also be explored to supply the optimization of leading compounds of CDC25A/B phosphatase inhibitors. This study is hopefully to provide scientific basis for exploiting endophytic fungi resources of P. lactiflora Pall and lay foundation for development of new targeting anti-tumor drugs with independent intellectual property rights. Meanwhile, this project is also of great importance for protecting the wild resources of P. lactiflora Pall.

CDC25磷酸酶在细胞周期中具有重要调控作用，并且在肿瘤细胞中过量表达，已成为当今肿瘤靶向治疗药物研发的热点领域之一，而植物内生真菌次生代谢产物是天然产物创新药物的重要来源。本课题组前期发现，中药赤芍中的三株内生真菌发酵提取物显示出显著的抑制CDC25A/B磷酸酶活性，表明其可能存在显著抑制CDC25A/B磷酸酶活性的新型成分。本项目拟在此基础上，集成应用高效液相-质谱-固相萃取-核磁共振谱联用（HPLC-MS-SPE-NMR）和CDC25A/B磷酸酶靶标活性筛选导向关键技术，深入系统研究三株候选菌株的活性次生代谢产物，发现新颖结构类型的强活性化合物，阐明其精确结构、药理活性，探索转化合成的制备方法，优选出新型CDC25A/B磷酸酶抑制活性先导化合物，为赤芍内生真菌资源的开发应用及新的自主知识产权靶向抗肿瘤药物的研制奠定基础，同时对赤芍野生资源的保护具有重要意义。

CDC25磷酸酶在细胞周期中具有重要调控作用，并且在肿瘤细胞中过量表达，已成为当今肿瘤靶向治疗药物研发的热点领域之一，而植物内生真菌次生代谢产物是天然产物创新药物的重要来源。本课题以前期从中药赤芍中分离得到的内生真菌为研究对象，集成应用高效液相-质谱-固相萃取-核磁共振谱联用（HPLC-MS-SPE-NMR）和CDC25A/B磷酸酶靶标活性筛选导向关键技术，深入系统研究三株候选菌株的活性次生代谢产物。同时利用已建立的“基于HPLC-MS-SPE-NMR的真菌新颖活性次级代谢产物快速识别和获取技术”，对另外一株赤芍内生真菌和一株结香内生真菌的活性次级代谢产物进行了系统研究。共分离获得85个真菌次生代谢产物，其中新化合物30个，包括1个具有新颖骨架的螺环倍半萜类化合物，并通过多种波谱学技术及计算NMR、计算ECD、计算CPL、化学降解、X-ray单晶衍射等方法确定了它们的精确结构。通过探索转化合成方法，转化和富集微量成分。最终优选出新型CDC25A/B磷酸酶抑制活性先导化合物1个。为赤芍内生真菌资源的开发应用及新的自主知识产权靶向抗肿瘤药物的研制奠定基础。