影响PI3K/Akt通路的西南沙海绵真菌来源功能分子的发现及其机理研究

The PI3K/Akt signaling pathway plays essential roles in cell growth, survival, apoptosis, and autophagy, and the excessive activation of PI3K/Akt is closely related to tumorigenesis. The specific PI3K/Akt inhibitor is an important strategy of the treatment of hepatoma cell. The fungus SCSIO 41051 (Arthrinium arundinis) was isolated from a marine sponge sample, Phakellia fusca Thiele, collected from the Xisha Islands of China. From this strain, three new 4-hydroxy-2-pyridone alkaloids, arthpyrones A–C, together with one known pyridone alkaloid derivative, N-hydroxyapiosporamide, were isolated. Noteworthly, N-hydroxyapiosporamide could obviously induce apoptosisi of hepatoma cell HepG2 targeting the Akt/Caspase-9 pathway. Considering these novel structures and the significant bioactivity, we have obtained patent protection about these arthpyrones together with their producing strain SCSIO 41051. On the basis of huge microorgamisn resources of the RNAM Center for Marine Microbiology, all 131 sponge-derived fungi, collected from the Xisha and Nansha Islands of China, were selected and subjected to small-scale fermentation. Their crude extracts were tested for anti-HepG2 activity and analysed for chemical compositions as well. Recently, two fungi strains SCSIO 41107 and 41134 were found to have significant in vitro cytotoxicities against hepatoma cell and diverse components. Optimizations of fermentation condition for these two strains were systematically studied to discover effective natural PI3K/Akt inhibitors. The most bioactive compounds will be accumulated by 300L large-scale fermentation under optimized media and conditions. Then, the hit compounds will be study their effects on anti-HepG2 mechanism including proliferation, survival, and apoptosis by using the cellular and molecular biology methods. The complishment of this research proposal will establish the base for the marine microbial drug innovation in our country with entire intellectual property rights.

PI3K/Akt通路在细胞生长、存活、凋亡、自噬等过程中发挥重要作用，其过度激活与肿瘤发生密切相关。特异性PI3K/Akt抑制剂是肝癌治疗的重要策略。真菌SCSIO41051分离自西沙海域的海绵，从该菌中获得一类新骨架吡啶酮，能够特异性地促进Akt/Casp9介导的人肝癌细胞凋亡反应。目前该菌及相关化合物已获专利授权。前期选取中科院海洋微生物研究中心保藏的131株西南沙海绵真菌，集成UPLC/DAD/MS化学与生物活性筛选，发现SCSIO41107和41134发酵产物丰富且具有显著的抗肿瘤活性。因此本项目拟以该两株海绵真菌为研究对象，筛选发现西南沙海绵真菌来源代谢产物中骨架新颖的抗人肝癌细胞活性的功能分子，基于培养调控策略富集目标活性化合物，深入研究影响PI3K/Akt通路的特异性目标功能分子对人肝癌细胞凋亡的诱导途径及其作用机制，为我国拥有完全自主知识产权的海洋微生物创新药物奠定基础。

为了获得更多高效低毒有效的靶向抗肿瘤先导化合物，本项目集成化学和生物活性筛选策略，运用多种化学和光谱学手段，筛选和发现我国海绵微生物来源的抗肿瘤活性代谢产物。共计从海绵共附生微生物中分离鉴定单体化合物191个，其中新单体化合物61个；基于圆二色谱（CD）、计算ECD、计算NMR核磁碳谱、X-Ray单晶衍射（铜靶）等手段精确阐明了大部分新化合物的立体绝对构型；利用化学衍生手段（Mosher′s Method）确定部分新化合物中含有仲醇碳的手性构型；系统研究了七株海绵共附生微生物的次生代谢产物。基于微生物培养调控策略，通过改变微生物培养条件，二次发酵节菱孢菌Arthrinium sp. SCSIO41051，从中分离鉴定4个不同于以前发酵的吡啶酮生物碱类化合物，丰富了海绵真菌代谢产物的结构类型；研究发现具有较强抗肿瘤活性的单体化合物22个，包括显著影响PI3K/Akt、AR等信号通路的先导化合物4个，为发现高效、低毒的抗肿瘤候选药物提供了科学依据和物质基础。上述研究结果整理发表SCI论文18篇（均已标注），申报国家发明专利3项，圆满完成了预期的研究目标。