海绵共附生真菌Aspergillus flocculosus 16D-1中抗炎活性物质的发现及作用机制

Inflammation is associated with many diseases. Although the mechanism of actions of traditional SAIDs and NSAIDs are clear, their long term use would lead to drug resistance and adverse effects. New anti-inflammatory drugs are urgently needed in clinic. In previous study, the applicant screened out a genius strain Aspergillus flocculosus 16D-1, which is capable of producing strong anti-inflammatory substances, from many sponge-associated microorganisms. 23 new anti-inflammatory compounds, three of which have new skeletons, were found in the rice solid culture of this fungus. However, these compounds belong to only three structural types, suggesting that A. flocculosus 16D-1 still has great secondary metabolic potential that has not been exploited. This study intends to activate the silent gene clusters of this "genius" fungus by epigenome manipulation and OSMAC metabolic regulation; to track and isolate the anti-inflammatory compounds from A. flocculosus 16D-1 by combination of activity- and chemical-directed methods; to determine the structures of the isolated compounds by spectroscopic analysis, computational chemistry, and chemical modification; to evaluate the anti-inflammatory activity of these compounds and explore their structure-activity relationships; to study the mechanism of actions of the strong anti-inflammatory compounds from their effects on inflammation-related signaling pathways; to screen potential targets of the strong anti-inflammatory molecules via reverse molecular docking; and to validate the potential targets by SPR, enzyme inhibition tests in vitro, and Western blot. This project is plan to discover 1-2 new anti-inflammatory lead compounds.

炎症与多种疾病相关。传统作用机制抗炎药物SAIDs和NSAIDs长期使用多有耐药性和不良反应，临床迫切需求新型抗炎药物。申请者从海绵共附生微生物中筛选出一株高产抗炎活性化合物的天才菌株Aspergillus flocculosus 16D-1，仅在大米培养条件下便发现23个新抗炎活性分子，其中3个为全新骨架。但它们仅属3种结构类型，提示其尚有巨大的次生代谢潜力。本项目拟对该“天才”真菌进行表观遗传和OSMAC代谢调控，激活沉默基因簇；采用活性和化学导向相结合的方法，追踪分离其中的抗炎活性成分；运用NMR、计算化学及化学沟通等方法确定化合物的平面及立体结构；系统评价化合物的抗炎活性，探讨其构效关系；阐明强活性化合物对炎症相关信号通路的影响及其作用机制；通过反向分子对接筛选其潜在作用靶点；应用SPR、体外酶催化抑制实验和Western blot等技术进行靶点验证。获得1-2个新型抗炎先导分子。

炎症与多种疾病相关。传统作用机制SAIDs和NSAIDs抗炎药物的长期使用多有耐药性和不良。临床迫切需求新型抗炎药物。基于上述背景和思路，在基金的资助下，申请人对天才真菌Aspergillus flocculosus 16D-1进行了表观遗传和OSMAC代谢调控，并采用CBA抗炎活性和化学导向相结合的方法追踪分离其中的抗炎活性成分。共分离鉴定49个化合物，其中新化合物28个，获得2个抗炎活性显著的多羟基麦角甾醇化合物和1个抗炎活性显著的吡咯类生物碱。主要内容和结果如下：. 运用二代和三代测序完成了真菌A. flocculosus 16D-1基因组近完成图测序。同时对真菌A. flocculosus 16D-1进行OSMAC发酵条件优化，发现在简单葵花阳光大米的培养条件下最优，能够产生最丰富的LC-MS质谱峰和最强的抗炎活性。该研究结果表明大米固体培养仍是目前获取该真菌化学多样性最方便稳妥的培养条件。. 用葵花阳光大米培养基对真菌A. flocculosus 16D-1进行大量发酵（1000 × 2 L，40 天）。从其粗提物浸膏中共分离得到49个化合物（新化合物28个），包括10个多羟基麦角甾醇、2个环肽、14个吡喃酮类化合物、6个呋喃酮类化合物、2个吡咯类生物碱、1个异喹啉酮类生物碱、1个大环内酯类化合物、2个赭曲霉素类化合物、2个金属配合物类化合物及9个其它类化合物。该研究结果拓宽了各相关结构类型化合物的结构数量，同时进一步揭示了天才菌株A. flocculosus 16D-1的化学多样性和产新化合物的能力。对上述分离得到的单体化合物进行活性评价，最终获得2个抗炎活性显著的新骨架多羟基麦角甾醇化合物asperfloketal A和asperfloketal B和1个抗炎活性显著的已知吡咯类生物碱preussin G。. 上述研究结果发表基金标注SCI文章2篇，其中第一作者（共同第一排第二）1篇。以第三发明人授权发明专利1件。在基金资助下，协助培养了博士研究生1名，硕士研究生1名。通过本项目的实施，丰富了抗炎活性分子的分子来源，为充分了解真菌活性物质基础和抗炎活性小分子的发现提供了依据和参考。此外A. flocculosus 16D-1基因组近完成图的获取使将来开展其重要次生代谢分子的合成生物学研究成为可能。