鬼伞属真菌中抗肿瘤guanacastane类二萜及其作用机制研究

Coprinus are a class of edible and medicinal facultative fungi. However, the pharmaceutical components of Coprinus, especially the anticancer small molecules, have yet to be further investigated. In our previous work, we isolated and identified a series of novel guanacastane-type diterpenoids from two fungal strains of Coprinus species. Specifically, a new guanacastane-type diterpene lactone named plicatilisin A, was found to have strong inhibitory activities against the human colorectal cancer cell lines HCT 116 mainly by inducing cell cycle arrests, but its targets and specific mechanism of action is unclear. Based on these results, we intend to explore more antitumor guanacastane-type diterpenoids from the two fungal strains mentioned above by using the OSMAC strategy, to characterize and validate the targeted proteins of plicatilisin A by using “Tag-free” chemical proteomic strategies, bioinformatical analysis and immunoblotting methods. Subsequently, we will build the possible signaling pathway of plicatilisin A based on the validated targets and the mechanism of colorectal cancer, which would lay the foundation for elucidating the antitumor mechanisms of guanacastane-type diterpenoids.

鬼伞属真菌是一类药食两用兼性真菌，但目前有关其抗癌活性的小分子化学成分的研究尚未深入开展。前期研究中，我们从2株鬼伞属真菌中分到了一系列结构新颖的guanacastane类二萜，并发现其中的一个二萜内酯—plicatilisin A对人大肠癌细胞HCT 116具有很强的体外增殖抑制活性，且主要通过引起HCT 116细胞发生细胞周期停滞从而抑制它的增殖，但其作用靶点和具体作用机制尚不清楚。在此基础上，我们拟采用OSMAC策略进一步发掘上述两株真菌中新的抗肿瘤活性guanacastane类二萜；运用“免亲和标签”化学蛋白质组学方法并结合生物信息学、免疫印迹分析等方法，发现、鉴定和验证plicatilisin A作用的靶标蛋白；随后基于验证的作用靶点，结合大肠癌发生机理，构建可能的plicatilisin A作用信号通路，为阐明guanacastane类二萜抗肿瘤作用机制奠定基础。

曲张链丝菌素（Streptovaricins）是一类对结核分枝杆菌等具有广谱抗菌作用的重要抗生素，结构分类上属于典型的I型聚酮类安莎霉素类化合物，但目前对其生物合成机制特别是聚酮骨架形成后发生的羟基化、甲基化、乙酰化和氧化还原等后修饰反应步骤仍不清楚。前期研究中，我们从一株土壤放线菌Streptomyces spectabilis CCTCC M2017417分离得到了一系列Streptovaricins类化合物，生测结果显示它们具有很强的抗MRSA活性；基于对该菌株的全基因组测序和生物信息学分析，我们进一步推测了Streptovaricins可能的生物合成途径，并定位了可能的5个P450羟化修饰基因stvP1-P5。在此基础上，我们拟通过基因敲除及回补等体内遗传学手段证明stvP1-P5基因的功能；随后在体外异源表达、纯化StvP1-P5蛋白以及发酵突变株制备反应底物dehydroxyl Streptovaricins，通过体外生物化学手段验证StvP1-P5的功能，从而在分子水平上阐明Streptovaricins萘环羟化及氧化还原修饰等步骤的发生次序，为全面解析Streptovaricins生物合成途径奠定基础；同时基于突变株代谢产物的分离，发现和鉴定新结构Streptovaricins中间代谢前体并考察其抗MRSA活性。