蛋白泛素化在NF-κB信号通路的活化中起关键性调控作用。我们在前期研究中发现一种从药用植物旋覆花中分离的倍半萜成分IJ-5能直接与UbcH5和Ubc13等泛素结合酶(E2)结合并抑制其酶促活性，抑制TNF-α刺激的293细胞内IKK复合物的调控亚基NEMO的泛素化，阻断NF-κB通路，从而发挥抗炎效应。本项目拟对从同一中药部位分离的IJ-5结构类似物展开系列研究，首先采用TNF-α或IL-1刺激的293细胞为体外模型，分析该类化合物对NEMO等多种与NF-κB通路相关的信号分子泛素化的影响；进而比较它们与相应E2酶的直接相互作用以及对各种E2酶活性的影响，探明其抑制NF-κB通路的分子靶点；最后在多种炎症细胞模型和胶原诱导的关节炎动物模型上评价它们的体内外抗炎效应。本项目的完成将有望发现一类靶点明确的抗炎候选药物，为新型NF-κB通路抑制剂的进一步结构优化、分子设计和的新药研发奠定基础。

Recent evidence supports the key role of ubiquitination in regulating the NF-κB signaling cascades, one of the most important immune and inflammatory pathways. It is now clear that ubiquitination not only controls the degradation of inhibitory proteins of κB family (IκBs) by the proteasome, but also regulates the activation of IκB kinase complex (IKK) through proteasome-independent mechanisms. In our previous study, we have found that IJ-5, a sesquiterpene compound isolated from medical plants of genus Inula, directly bound to the ubiquitin-conjugating E2 enzymes such as UbcH5 and Ubc13, and inhibited their activities, thus reduced the ubiquitination of NEMO, the essential regulatory subunit of IKK, in TNF-α-stimulated 293 cells, and subsequently blocked NF-κB pathway via inhibition the activation of IKK, and exerted anti-inflammatory effects. In this proposed research project, we will develop a series of research on the structural analogues isolates from the same fraction of the plants. We will at first use TNF-α- or IL-1-stimulated 293 cells as a cellular model to analyze the effects of these analogues on the ubiquitination of diverse signal proteins related to NF-κB pathway, including NEMO, RIP1, IRAK1, TRAF2 and TRAF6. Furthermore, we will compare their direct interactions with the relevant E2 enzymes, including Ubc13、UbcH5、UbcH7 and E2-25K, and study the effects of these compounds on the activities of the aforementioned E2 enzymes. Through these studies, we attempt to identify the molecular target of these compounds and clarify their mechanisms by which they block NF-κB pathway. Finally, we will evaluate the anti-inflammatory effects of these compounds on various inflammatory cellular models, including TNF-α- or IL-1-stimulaed RAW264.7 macrophages, bEnd.3 mouse endothelial cells, and synoviocytes. Their in vivo anti-inflammatory efficacy will also be evaluated on the collagen-induces arthritis murine model. The accomplishment of this project holds the promise for discovery of a new class of drug candidates for anti-inflammatory therapy and will provide basis for further structural optimization, molecular design, and drug development of novel NF-κB antagonists.

本项目旨在探明倍半萜内酯类化合物IJ-5及其结构类似物调控蛋白泛素化而阻断NF-κB 通路的作用机制，并评价其在细胞和动物整体水平上的抗炎效应，为研发抗炎新药提供候选化合物。迄今，所有的计划研究内容均已完成，研究成果包括：（1）阐明了IJ-5共价结合于E2酶UbcH5c活性中心的Cys85位点而抑制其活性，抑制TNF-α诱导细胞内RIP1和NEMO泛素化，抑制NF-κB通路激活的作用机制；（2）对IJ-5的结构类似物的UbcH5c抑制活性进行筛选，通过对20余种不同结构类型的天然倍半萜内酯类化合物的筛选，发现多种结构类型的倍半萜类酯化合物具有不同的UbcH5c抑制活性；进而以α-山道年为起始原料合成十余种结构类型的倍半萜内酯化合物，发现其中3个衍生物具有一定的UbcH5c抑制活性；进一步以这3个化合物为前体进行结构修饰得到3个系列、共73个结构衍生物，从中筛选得到了14个具有显著增强的抑制活性；对上述活性化合物阻断细胞内NF-κB通路的活性进行筛选，发现其中6个化合物能抑制TNF-α诱导的NF-κB通路活化；进一步检测确定了这些化合物抑制UbcH5c作用的量效依赖关系以及与UbcH5c的结合动力学，发现化合物6d与UbcH5c的亲和力最强；此外，从植物中高丰度的天然倍半萜内酯类化合物中筛选发现土木香内酯对UbcH5c有较强的抑制作用，土木香总内酯对UbcH5c也具有显著的抑制活性。（3）分析了IJ-5和6d与UbcH5c的结合模式，进一步明确了IJ-5和6d在UbcH5c蛋白分子上的作用位点。（4）证明了上述UbcH5c抑制剂确能抑制TNF-α诱导的细胞内NEMO或RIP1等信号分子的泛素化。（5）发现IJ-5及其具有UbcH5c抑制活性的结构类似物对TNF-α诱导的细胞内NF-κB通路的活化具有抑制作用，并发现它们对NF-κB调控的多种炎症因子基因的表达亦具有显著的抑制作用。（6）采用多种炎症动物模型研究发现IJ-5 对胶原诱导的小鼠关节炎具有显著的治疗作用，并且可以减轻 TNF-α 联合 D-GaLN 诱导的小鼠急性肝损伤； 6d对佐剂性大鼠关节炎具有显著的治疗作用；土木香总内酯无论是治疗性给药还是预防性给药均能减轻胶原诱导的大鼠关节炎，而且土木香总内酯皮肤给药能够有效的防治DNCB诱导的小鼠湿疹。