利用活性筛选-质谱特征引导的双示踪法快速发现海绵环肽类抗肿瘤先导化合物

Due to the novel chemical structure (rich in rare amino acids), significant bioactivity and potential medicinal value, the cyclic peptides originated from marine sponge have been attracted the world wide attention. Based on the successful experience in searching cyclic peptides from Phakellia fusca, a rapid, sensitive and integrated method using LC-QTRAP-MS/MS has been established by the applicant to monitor the cyclic peptide skeleton in 24 diversified marine sponges. After combination with bioactivity-guided fractionation, 10 cyclic peptides with antitumor activity have been separated and identified preliminarily from two sponge specimens, including Theonella swinhoei ad Reniochalina stalagmitis collected off Xisha islands in our previous study.. Therefore, we expected to systematically investigate the chemical diversities and antitumor activities of active peptides in the above mentioned two sponge specimens by using double tracer method: integrated bioactivity screening and LC-QTRAP-MS/MS monitoring. As expected results, more cyclic peptides with novel structures and potent antitumor activities will be discovered, the action mechanism and structure-activity relationship of which will be investigated as well. It can not only provide a rapid and orientation tracking method for monitoring trace concentration cyclic peptides in complex system, but also have a great significance for the rational exploration of the distant-sea biological resources in the future.

海绵中的环肽类化合物因其结构新颖（富含非常见氨基酸）、活性显著且成药性强而备受关注。申请者基于从棕色扁海绵中获得的抗肿瘤环肽系列结构，运用LC-QTRAP-MS/MS的PI扫描结合IDA触发的EPI扫描技术，建立了选择性识别粗提物中环肽类特征结构的快速定位方法，并结合活性筛选，从24种西沙海绵中筛选出两种抗肿瘤活性显著且富含环肽的海绵：隋氏蒂壳海绵和肾指海绵，成功获得10种细胞毒活性的环肽类化合物。. 本课题拟采用活性筛选-质谱特征引导的双示踪法，结合先进的中压液相和高效液相质谱引导制备技术，继续追踪两种海绵中微量环肽类细胞毒活性成分；运用化学和波谱学方法确定其平面及立体结构；对活性单体进行体内外抗肿瘤活性评价和构效关系分析，探讨强活性化合物作用机制，获得1-2种结构特异的环肽类抗肿瘤先导化合物。构建环肽类微量复杂结构的快速识别、定向追踪的技术方法，为新型海洋抗肿瘤药物研究奠定基础。

本课题基于前期从西沙棕色扁海绵Phakellia fusca 获得的抗肿瘤环肽系列结构的基础上，结合环肽类化合物的主要质谱特征，建立活性筛选-质谱特征引导的双示踪法。利用该方法发现肾指海绵Reniochalina stalagmitis、棕色扁海绵Phakellia fusca和棕色扁海绵共附生真菌稻黑孢菌Nigrospora oryzae PF18粗提物中富含微量环肽类成分；进而结合质谱导向的程序性分离方法（开放柱-中压制备-质谱引导自动纯化系统）对其中环肽类成分进行追踪分离。最终，共从肾指海绵Reniochalina stalagmitis的活性部位中发现了16个环肽类化合物，其中12个新化合物；从棕色扁海绵Phakellia fusca中发现了24个环肽类化合物，其中包括6个新化合物；从棕色扁海绵共附生真菌稻黑孢菌Nigrospora oryzae PF18中发现了6个化合物，其中5个为新化合物。利用HRESI和NMR等现代波谱学方法确定了这些化合物的平面结构，采用2D NMR配合Q-TOF MS/MS技术确定氨基酸残基的连接顺序，通过铜靶X-ray单晶衍射法和高级Marfey法分析确定环肽的绝对构型。对所分到的环肽化合物进行了抗肿瘤活性筛选，发现了2个结构新颖且对肺癌干细胞具有显著活性的分子。在该项目的资助下，已经发表SCI论文12篇，培养博士研究生1名，硕士研究生2名，基本完成了项目的预期目标。构建环肽类微量复杂结构的快速识别、定向追踪的技术方法，为新型海洋抗肿瘤药物研究奠定基础。