苔藓植物内生真菌中靶向微管的抗肿瘤活性代谢产物的研究

Microtubules are key components of the cytoskeleton that are essential in all eukaryotic cells and are the therapeutic target of many important anticancer drugs. The discovery of many promising new antitubulin agents with novel properties from nature makes microtubule-targeting natural products a dynamic field of new anticancer drug discovery. Endophytic fungi from bryophytes (mosses and liverworts) have been recognized as a new source of bioactive natural compounds. However, there are few reports on the chemical investigation of them. In our previous study of the chemistry and pharmacology of the metabolites from bryophyte endophytes, a series of diterpenoids have been obtained and among them, sphaeropsidin A showed potent antiproliferative activity on several tumor cell lines by inhibiting the polymerization of tubulin. Based on the above results, the aims of the present project are listed as follows: i) To further enrich as much as possible of sphaeropsidin A and isolate as many as possible of its diterpene analogs from the sphaeropsidin-producing strains we have found in preliminary study. ii) To rapidly obtain microtubule inhibitors of other structral types through a bioactivity-guided isolation from othe strains of fungi. iii) To design and prepare a series of sphaeropsidin A derivatives, get the structure-activity relationship of microtubule inhibition, and find the most potent compound. iv) To investigate the anticancer michanism of action of the active componds, determine their binding domain to tubulin, elucidate their mechanism on microtubule dynamics alteration, uncover their way of changing the expression of tubulins and microtubule-assiciated proteins, and effects on related signaling pathway. The results of this project will make basis for the discovery of new anticancer drugs.

微管是真核细胞中重要的骨架蛋白成分，因此是许多重要的抗肿瘤药物的作用靶点，以微管为靶点的天然产物是抗肿瘤新药研究中一个重要且充满活力的领域。苔藓植物内生真菌是目前研究较少的活性化合物的新资源，本项目拟基于前期工作中对其次生代谢产物的化学和药理活性的研究积累，以我们发现的具有微管抑制活性的二萜化合物sphaeropsidin A及其同系物为目标分子，对产生菌进行深入研究，进一步富集活性目标分子并尽可能获得其它微量同系物。同时，应用活性筛选为指导，从另外几株活性真菌的提取物中快速分离其他结构的微管抑制剂。此外，在上述研究的基础上，针对sphaeropsidin A等化合物进行衍生物设计与合成，研究其抑制微管的构效关系，以获得强活性的目标分子，并探索其抗肿瘤作用机制，确证其与微管结合位点、揭示其对微管动态、微管蛋白和微管相关蛋白表达及信号通路影响的机制，为新型抗肿瘤药物的发现奠定基础。

本项目完成了6株苔藓或地衣内生真菌(Sphaeropsis sapinea、Aspergillus fumigatus、Aspergillus niger、Penicillium sp. SD1617、Tolypocladium cylindrosporum和Aspergillus versicolor)和两种药用植物(鸡毛松和罗汉松)中活性成分的系统研究工作，共分离获得各类化合物153个，其中包括新化合物37个，新骨架化合物4个，具有较强抗肿瘤活性的化合物多个，此外还发现部分化合物具有一定的抗氧化活性。采用NaBH4还原、催化氢化、酯化、醚化、环丙烷化、成酰胺及生物转化等反应，对从内生真菌中获得了二萜化合物sphaeropsidin A及从罗汉松中获得的二萜化合物podomacrohyllin A、inumakoic acid、4beta-carboxy-19-nor-totarol、19-hydroxytotarol和inumakiol A进行了结构修饰，共制备衍生物42个，采用多株肿瘤细胞，对其抑制肿瘤细胞增殖的活性进行了筛选评价，并初步总结了其构效关系。对活性化合物J5抗肿瘤和抑制微管的作用机制进行了研究，发现该化合物可通过抑制肿瘤细胞微管的形成，引起细胞周期抑制和诱导细胞凋亡。以上研究的完成，对阐明这几种内生真菌或药用植物的活性成分和新型抗肿瘤药物的发现具有重要意义。