滨海湿地真菌中Hedgehog信号通路抑制因子的发现及抗肿瘤机制研究

Microorganisms from unusual inches are important sources of new structural and active natural products. The coastal wetland of the Yellow River delta, characterized as salinization and alkalization, is rich in halotolerant microorganisms. The Hedgehog (HH) pathway is considered to be closely related to the initiation and progression of tumors. There have been inhibitors approved for market, but the emergence of severe drug resistance and side effects limits their application. In the preliminary study, halotolerant microorganisms were obtained from the coastal wetland of the Yellow River delta. A series of HH pathway inhibitors were isolated from Aspergillus flavipes RD-14-2. Based on the previous study, this project will use high salt stress strategy to activate the microorganism silenced genes, impelling the full expression of its metabolic and biosynthetic pathways. The transcriptional activity of GLI in the Hedgehog signaling pathway is used as an indicator to screen strains with HH pathway inhibitory activities further, which are used as target strains. Using the dual luciferase reporter gene model as bioassay-guided method, compounds with HH inhibitory activity are isolated and identified by modern chromatography technology and spectroscopy. Cytotoxicities on tumor cell lines of the inhibitors and the changes of the expression levels of Hedgehog downstream factors are analyzed to elucidate the preliminary anti-tumor mechanism of small molecule inhibitors. This project will provide scientific basis for the development of anti-tumor drugs and the utilization of special microorganisms.

特殊环境微生物是新结构活性天然产物发现的重要来源。黄河三角洲滨海湿地具有盐碱化的特征，富含耐盐特境微生物。Hedgehog（HH）信号通路与肿瘤的发生、发展密切相关，已上市的该通路抑制剂由于严重的耐药和毒副作用限制了其应用。本项目前期从黄河三角洲滨海湿地分离获得耐盐特境微生物，并从菌株Aspergillus flavipes RD-14-2代谢产物中获得系列HH通路抑制剂。本项目拟在此基础上，运用高盐胁迫策略，激活微生物体内沉默基因，使其代谢途径和生物合成途径充分表达。以HH通路中GLI的转录活性为指标，继续筛选具有该通路抑制活性的菌株作为目标菌株。运用双荧光素酶报告基因模型结合现代色谱、波谱技术追踪分离、鉴定具有HH通路抑制活性的化合物。通过检测化合物的抗肿瘤活性及HH通路下游因子表达量的变化，初步阐释小分子抑制剂的抗肿瘤作用机制。为抗肿瘤药物的研发和特境微生物的开发利用提供科学依据。

海洋天然产物是新药研发的重要源泉。本项目以黄河三角洲来源的真菌和海洋海绵Aaptos suberitoides为研究对象，对其进行化学成分、活性和机制研究。对采自黄河三角洲盐碱地的14个样品进行了真菌菌株的分离，鉴定菌株54株，对这些真菌资源进行了保藏和HPLC指纹图谱库构建，为后续开展药用先导化合物挖掘相关工作提供了资源微生物。选用4种培养基对菌株进行小试发酵，对216份发酵产物进行了抗肿瘤和抗菌活性的测定。选择其中5株真菌进行了次级代谢产物的研究，共分离鉴定化合物42个，新化合物6个。另外从海绵Aaptos suberitoides中分离得到化合物14个，其中新化合物5个。基于1D和2D核磁共振波谱、化学衍生、与Rh2(OCOCF3)4络合诱导的CD光谱、X-Ray（铜靶）等确定了化合物的绝对构型。对分离得到的化合物进行了Hedgehog信号通路抑制活性测定，遗憾的是所测单体化合物均无显著的Hedgehog信号通路抑制活性。为了充分挖掘化合物的活性，我们对化合物进行了抗肿瘤活性、抗炎活性、抗胆碱酯酶活性、CDK2激酶抑制活性的测定。发现抗肿瘤活性化合物9个，抗炎活性化合物10个，抗胆碱酯酶活性化合物6个，CDK2激酶抑制活性化合物3个。另外我们对其中一个新的萘啶类抗炎活性化合物进行了深入的机制研究，发现化合物通过抑制MAPK通路、激活Nrf2/HO-1信号通路而发挥抗炎作用。对海绵来源抗胆碱酯酶活性化合物aaptamine进行了深入的研究，发现aaptamine对乙酰胆碱酯酶和丁酰胆碱酯酶均具有显著的抑制活性，能够与胆碱酯酶的关键催化位点发生作用，并且能够显著改善斑马鱼模型的感知能力和运动功能，有望为抗阿尔茨海默症药物的研发提供新的模板分子。在本项目的资助下，以第一或通讯作者发表天然产物相关SCI论文7篇（5篇为第一标注），核心论文1篇（第一标注），申请专利4项，授权1项。