海洋微生物来源新型抗流感病毒先导分子的发现与作用机制研究

Influenza virus induced serious harm to human health, while the virus mutation led to failure treatment for clinical drugs. Thus, it is imminent to discover new anti-influenza virus agents which possess strong efficiency and low toxicity as well as the action with new mechanistic pathway which may be used for the remedy of drug-resistant viral species. Marine derived microorganisms inhabited under ecological stresses and possessed chemical defense capabilities, they are thus considered as the new and promising source provided for the finding of new anti-influenza virus lead compounds. Based on our previous achievement to obtain a series of marine microorganisms with potential inhibition against influenza virus strains and handled several hundreds of marine microorganism-derived new natural products and the results of their antivirus assay, this project aimed to discover structurally novel natural products from marine microorganisms with potential anti-influenza virus activity which are able to be developed as lead compounds. A protocol is designed using viral enzyme-oriented UF-LC-MSn method and other related methods for efficiently obtaining active molecules from microorganism library toward the key enzymes (HA, DNA polymerase, etc.) which related to influenza virus entry and replication. Additional anti-virus pharmacological evaluation was performed to confirm the anti-influenza virus effects of the targeted-bonded small molecules. In order to accumulate the minor active compounds and to find the unexpressed natural products from the bioactive microorganisms, gene-mining and optimizing methods were applied including OSMAC and silent gene regulation. Particularly those containing synthetic terpene-PKS gene cluster and synthetic alkaloid hybrid gene cluster will be selected for activation to mine new structures with potential anti-influenza virus. In addition, the bioactive compounds with four different molecular scaffolds which had proved to possess anti-flu virus effects are selected for further investigation of their mechanistic pathways related to the targets of influenza virus entry, replication and release. The molecules with anti-drug resistant flu virus species and without resistant action with flu virus such as diketopiperazines and biphenyl esters will be examined in detail to find the new mechanism of drug resistant inhibition. Moreover, the potent antiviral molecules as the molecular probes will be selected for further investigation with proteomic and genomic methods to find new virus/host cell targets, which may insigne new antiviral pathway. Conclusively, following the research project, we intend to provide the marine microorganisms as a new source for the discovery of new antiviral agents and to overcome the resistant influenza virus which is unable to be treated by the drugs currently used for clinic trail.

流感病毒严重危害人类健康，病毒变异导致临床药物失效。发现新作用机制、高效低毒且不易耐药抗流感病毒天然药源分子迫在眉睫。海洋微生物具备抗病毒胁迫和化学防御功能，是抗流感病毒新型药源分子最有潜力的生物资源。课题基于前期从海洋微生物库中发现具有显著抑制流感病毒菌株及其抗病毒成分的研究基础，设计以病毒重要靶标为导向的谱效相关等方法，从分子库中高效获得对流感病毒进入和复制关键靶酶有显著靶向作用的活性分子，并进行抗病毒药效评价，获得新型抗流感病毒药源分子。基于抗流感病毒活性菌种的合成基因簇等生物信息，应用合成生物学等技术挖掘全新结构抗流感病毒分子。此外，对已明确抗流感病毒作用的天然分子进行深入抗病毒作用机制研究，阐明活性分子对关键靶蛋白的调控作用；并对其中具有抗临床耐药毒株且对病毒低耐药的化合物开展抗耐药作用分子机制研究，揭示抗耐药靶向因子。课题研究将为抗流感病毒药物研究提供全新药源分子。

课题建立以流感病毒关键靶酶为导向的筛选技术，深入开展以病毒进入和复制/转录过程关键靶标为核心的抗流感病毒分子机制研究，以获得若干新颖结构抗病毒海洋天然产物。课题建立了LC-MSn-GNPS和谱-效相关双导向及抗病毒筛选技术体系，从自建的海洋生物库中确定10种海洋生物有抑制流感病毒H1N1活性。通过化学与生物功能双导向技术，对海洋生物开展了抗病毒活性化合物的分子发掘与作用机制相关研究，取得的主要成果包括：1）应用表观遗传操作激活5种海洋真菌萜类等合成基因簇，结合化学和生物功能双导向以及DNPS分析网络排重方法，色谱制备与波谱确定获得450种结构各异海洋天然产物，其中160种为结构新颖化合物，并评价了抗多种病毒功能；2）发现非核苷类新颖结构共13种化合物包括多氯代联苯醚，哌嗪生物碱和链肽在细胞水平抗H1N1和H3N2流感病毒功效强于达菲等临床药物，多氯代联苯醚和哌嗪生物碱对多种临床耐药流感病毒皆有抑制作用，且为低细胞毒化合物，多周期病毒复制对活性化合物不产生耐药作用，与临床药物易耐药不同；3）多氯联苯醚的抗病毒机制为同时作用于病毒的进入与病毒复制过程，并作用于病毒血凝素HA的亚型HA1，阻断与宿主sialic acid受体结合，并抑制病毒基因复制RNP复合体，协同抑制病毒进入和复制。哌嗪生物碱作用于病毒HA以阻断病毒与sialic acid受体结合，对多种耐药流感病毒有抑制作用，但不产生病毒耐药性；4）发现8种新颖结构链肽在体内外皆具有显著抗流感病毒作用，强于利巴韦林，并对HCoV-229E有显著抑制作用，体内活性表明明显减低肺病毒滴度。该课题提供了3类非核苷高活性低耐药性抗流感新颖结构海洋先导化合物，并获得抗艾滋病，抗HBV，HCV和抗Zika等病毒活性化合物，为开发海洋生物抗病毒潜力奠定了化学与药理学基础，并揭示海洋天然产物作为抗病毒候选药物具有巨大潜力。课题发表SCI论文38篇，申请发明专利13件.