五环三萜类化合物抑制流感病毒的构效关系及其作用机制的研究

In the past few years, frequent outbreaks of influenza epidemics have severely endangered health and life of humans and animals. Antiviral treatment is one of indispensable strategies for preventing influenza epidemics. Unfortunately, the extreme variability of the virus makes drugs which inhibit viruses by targeting viral antigens, easy to induce resistance and become inefficacy. The large aggregation of studies has shown that the inhibition on activation of NF-κB signaling pathway induced by influenza virus infection not only reduces the excretion of pro-inflammatory cytokines and the recruitment of inflammatory cells but also inhibits the replication of progeny viruses without tendency to induce resistance. Hence, NF-κB signaling pathway becomes a novel promising antiviral target. In preliminary studies, we have discovered that saikosaponins (SSs) and other pentacyclic triterpenes posses potent antiviral activities against influenza virus both in vitro and in vivo. In addition, we showed that SSs blunts the activation of NF-κB signaling pathway and thereby reduces the production of pro-inflammatory cytokines and inhibits virus replication by suppressing nuclear export of vRNP. Based on the previous studies, this project will further isolate related medicine materials and get more than 50 pentacyclic triterpene compounds as well as evaluate their inhibitory activities on influenza viruses and NF-κB signaling pathway. Besides, we will also focus on analysis of structure-activity relationships and elucidation of the underlying mechanism of action. Our study will provide the scientific basis for the development of novel antiviral lead compounds with high efficiacy, low toxicity and low tendency of resistance induction.

近年来，流感疫情频繁爆发，严重危害人类和动物生命健康。药物防治是防控流感的重要手段，但流感病毒的高度变异性使得以病毒蛋白为作用靶点的药物容易诱导耐药性而失效。大量研究表明，NF-κB信号通路是机体对病毒感染免疫应答的重要枢纽，对炎症进程起决定性作用。抑制流感病毒感染诱导激活的NF-κB通路，可有效抑制肺部炎症及病毒的复制，且不易诱导耐药性。NF-κB通路成为极具前景的新型流感病毒药物靶标。前期我们发现，柴胡皂苷等五环三萜类化合物具有显著的抗流感病毒作用；初步的作用机制研究表明，柴胡皂苷通过抑制NF-κB通路的活化而减少肺部炎症，并通过抑制病毒核糖核蛋白出核而抑制病毒复制。本项目拟在前期工作基础上，分离获得更多五环三萜类化合物（≥50种），评价和研究该类成分对流感病毒及NF-κB通路的抑制活性、构效关系及作用机制，为研究和开发高效广谱、不易诱导耐药的新型抗流感病毒药物提供科学依据。

近年来，流感疫情频繁爆发，严重危害人类和动物生命健康。药物防治是防控流感的重要手段，但现有流感防治药物M2通道抑制剂（金刚烷胺、金刚乙胺）已有严重耐药性，NA抑制剂（奥司他韦、帕拉米韦等）的耐药性报道也日益增多。研究开发具有新作用机制的抗流感病毒(IAV)新药的需求十分迫切。中药及天然产物蕴含大量具有显著药理活性的化合物，是开发抗病毒药物的重要资源。本项目从中药、天然产物及其衍生物中发现多个具有显著抗IAV活性的化合物，包括：(1) 以五环三萜类天然产物齐墩果酸(OA)和熊果酸(UA)为前体化合物，分别合成了11个OA衍生物和12个UA衍生物，其中10个OA衍生物(EC50≤14.0μM)和10个UA衍生物(EC50≤5.06μM)具有显著抗IAV活性，分析总结了该类衍生物抗IAV的构效关系，并探明了代表性活性物OA-10通过抑制病毒膜融合过程抑制IAV复制的作用机制；(2)发现柴胡皂苷a(SSa)对多种亚型IAV具有显著抑制作用，25mg/kg皮下注射给药对感染PR8(H1N1)小鼠的保护率达90%（对照组10%存活），并揭示其通过抑制NF-κB信号通路、vRNP出核过程及小鼠肺病理炎症损伤的抗IAV作用机制；(3)发现胆汁中的主要活性成分鹅去氧胆酸(CDCA)通过抑制vRNP出核抑制IAV的复制，提示在动物的消化道CDCA发挥抗IAV作用；(4)发现人参皂苷G-rk1和G-rg5通过抑制病毒的吸附过程而发挥显著的抗IAV活性(EC50≤7.0μM)，并保护感染IAV小鼠(70% VS 10%)；⑤发现临床上抗疟药物青蒿琥酯(AS)及双氢青蒿素对IAV具有显著抑制作用，对感染H1N1(pan2009)和高致病性H5N1 IAV小鼠均有显著保护作用，40mg/kg剂量的AS对感染H5N1 IAV小鼠的保护率为70%（对照组20%存活），AS与帕拉米韦联用对感染小鼠的保护率达100%，预示其在抗流感临床上具有潜在的应用价值。