海洋真菌新颖生物碱代谢产物的发现及靶向Wnt/β-catenin信号通路的抗肿瘤作用研究

In our previous studies, we found that amino acid-directed strategy, that is feeding various amino acids, such as tryptophan, phenylalanine, cysteine, tyrosine and methionine into the culture media, can efficiently induce marine-derived fungi to produce an abundant of structurally interesting alkaloids. These alkaloids displayed potent antitumor activities and obvious inhibitory effect on the Wnt/β-catenin signaling pathway. Additionally, we had isolated a large number of fungal strains from the soft corals, sponges, ascidians, and sea stars from the South China Sea, and these fungi are stored in the refrigerator at -80 ℃ in our laboratory. The metabolites of most of these marine-derived fungi have not yet been investigated. Based on the previous success, in this project, we will continuously use the effective amino acid-directed strategy to explore the enormous alkaloid biosynthesis potential of marine-derived fungi and the prospect in medicine. Firstly, the marine-derived fungi which can produce a great number of bioactive alkaloids metabolites will be selected based on the structural novelty pre-screening by HPLC-MS and NMR experiments, and the preliminary antitumor activities screening targeting the Wnt/β-catenin signaling pathway. The culture conditions for producing novel active alkaloids with high yield will be optimized. Marine-derived fungi are cultured in the optimized culture condition on a large scale for the production and accumulation of metabolites. Then, we will develop efficient separation methods for active alkaloids metabolites by tracking the structure and activity. The chemical structures of the pure compounds will be determined by comprehensive spectral analysis. HCT116, SW620 and other colorectal cancer cell lines are selected to evaluate the inhibitory activities in vitro. As for the active alkaloids, the derivatives are prepared and the structure-activity relationships are studied. The inhibitory effect of active alkaloids in vivo Xenograft tumor models will also be determined by utilizing sensitive human colorectal cancer cell lines. Furthermore, the antitumor mechanisms of active alkaloids will be investigated by analysis their effects on the related genes and proteins in the Wnt/β-catenin signaling pathway. Overall, the results will discover a large number of novel alkaloid metabolites from marine-derived fungi and provide a solid foundation for the development of new antitumor drugs with the mechanism of action targeting the Wnt/β-catenin signaling pathway.

前期研究发现氨基酸导向策略，即培养基中添加色氨酸、苯丙氨酸、半胱氨酸等多种氨基酸，能促使海洋真菌产丰富的新结构生物碱，它们抗肿瘤活性强，对Wnt/β-catenin信号通路的抑制作用明确。研究组保藏了大批未研究过的海洋真菌，本项目将探究海洋真菌产生物碱的潜力和药用前景，继续发展氨基酸导向策略，以液质联用、核磁共振对结构新颖性检测和靶向Wnt/β-catenin信号通路的细胞抑制活性测试为指标，筛选并优化产新颖活性生物碱的培养条件，发展高效的活性生物碱跟踪分离技术。得到的纯的生物碱化合物选取HCT116、SW620等结肠癌肿瘤细胞系，开展体外细胞毒实验；活性显著的化合物进行衍生物制备和构效关系研究；选取敏感瘤株，测定活性生物碱化合物的体内抑瘤效果；开展作用机制研究，分析活性生物碱化合物对Wnt/β-catenin信号通路相关的基因或蛋白的影响，确定作用靶点，为新型靶向抗肿瘤药物研发奠定基础。

本项目首次运用氨基酸导向策略（Amino Acid-Directed Strategy），即在海洋真菌常规培养基中添加适量的色氨酸或色氨酸、苯丙氨酸、组氨酸、蛋氨酸、苏氨酸、赖氨酸、丝氨酸、缬氨酸等一种或多种天然氨基酸，以及邻氨基苯甲酸、烟酰胺等，深入开展了11株南海海洋真菌Pseudallescheria boydii、Aspergillus sp.、Fusarium sp.、Trichoderma erinaceum、Scedosporium apiospermum、Lecanicillium fusisporum、Dichotomomyces cejpii、Fusarium sp.、Trichoderma sp.、Exophiala oligosperma和Fusarium equiseti的活性代谢产物研究，从中发现100余个化合物，绝大部分属于生物碱类化合物，尤其是吲哚类生物碱明显占优势，其中38个新化合物，34个化合物显示了抗肿瘤、抗病毒等多种活性；推测了重要吲哚类生物碱代谢产物的生源合成途径；通过优化培养条件，显著提高了部分活性吲哚类生物碱的产量。研究结果表明：氨基酸导向策略能诱导海洋真菌多产、高产结构新颖的活性生物碱类代谢产物。目前正在开展部分抗肿瘤活性生物碱类代谢产物的体内药效评价和靶向Wnt/β-catenin信号通路的作用机制研究。相关研究结果在Journal of Natural Products, Phytochemistry，Bioorganic Chemistry,等国际著名期刊发表12篇文章，获得1件授权专利。