三株西沙海绵来源真菌中抗手足口病致病病毒EV71活性代谢产物的发现

In recent years, the hand, food and mouth disease (HFMD) is an acute infectious disease caused by enterovirus family, in which enterovirus 71 (EV71) is the most common pathogen. This diease spreads through a variety of ways, and occurs all around the world. Pre-school children especially children aged 5 years and below have the highest disease incidence. Serious cases could lead to neurological symptoms and even death. Currently, clinically effective antivirals for use in the treatment of enteroviral infection do not exist. Thus, there is a tremendous clinical need to develop novel classes of antiviral agents for the treatment of enterovirus infection. Marine microorganisms with a high degree of species diversity can produce structurally novel secondary metabolites, which are an important resource for discovery of new leading compounds. Our aim was to identify antiviral metabolites from sponge-derived fungi. Twenty-one strains of marine fungi were isolated from the marine sponge, Phakellia fusca Thiele, collected from the Xisha Islands of China. The previous research has screened the effective extracts of different fermentation broth of sponge-derived fungi for treating the EV71 and for the first time to separate five 4-hydroxy-2-pyridone alkaloids, in which there are four new compounds. At the same time, arthpyrone D could significantly inhibit EV71 virus multiplication on Vero cells. Optimizations of fermentation condition for these strains were systematically studied. This research is on multi-discipling cooperation of marine biology, microbiology, and marine natural products. Guided by antiviral activity against the enterovirus 71, we will investigate the chemical components from the secondary metabolites of three sponge-derived fungi. Furthermore, we will study structure-activity relationships for some of the more promising compounds, and optimize the lead compounds. Above studies would provid theoretical basis to further research for obtaining antiviral agents from marine fungi.

由肠道病毒(EV71)引发手足口病(HFMD)的全球大流行严重威胁到人类尤其是儿童的健康。HFMD至今尚无有效药物和安全疫苗可用。研发结构新颖、活性显著的抗EV71药物刻不容缓。目前国内外学术界对微生物来源的抗EV71活性成分研究却罕见报道。因此，申请者系统筛选了21株分离自西沙海绵真菌发酵产物的抗EV71活性，并进一步从中首次发现抑制活性与Ribavirin相当的抗EV71活性生物碱Arthpyrone D，前期研究成果开拓了抗EV71先导化合物发现的新来源，提供了近年来预防HFMD的新思路。本项目以抗EV71活性为导向，运用化学和光谱学方法，拟从三株前期筛选具有显著抗病毒活性的海绵真菌中快速分离并结构确定抗EV71活性的化学分子，优化发酵条件，富集活性代谢产物，发现、衍生新的抗EV71先导结构，揭示其构效关系，探明其潜在的药效基团，为研制真菌来源的新型抗病毒药物提供科学依据和物质基础。

针对目前没有安全有效的小分子药物治疗由肠道病毒EV-71引起的手足口病(hand, food and mouth disease, HFMD)，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真菌来源的抗肠道病毒EV-71活性代谢产物。共计从海绵共附生真菌中分离鉴定单体化合物107个，其中新单体化合物31个，新天然产物1个；基于圆二色谱（CD）、计算ECD、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；系统研究了来自于棕色扁海绵（Phakellia fusca Thiele）和美丽属海绵（Callyspongia sp.）的六株海绵共附生真菌的次生代谢产物。基于OSMAC策略，通过改变微生物培养条件，将富营养液体摇床培养改为大米固体静置发酵培养，二次发酵节菱孢菌Arthrinium arundinis ZSDS1-F3，从中分离鉴定10个完全不同于液体发酵的聚酮产物，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肠道病毒EV-71活性的单体化合物4个，为发现高效、低毒的抗肠道病毒药物提供了科学依据。上述研究结果整理发表SCI论文7篇（均已标注），申报国家发明专利2项，圆满完成了预期的研究目标。