p97-Npl4复合物调控抗病毒免疫应答的信号机制与靶向药物筛选

Viral infections pose increasing threats to public health throughout the world. Innate immunity serves as the first line of defense in the response against the pathogenic invasion of hosts by viruses. RIG-I-MAVS and cGAS-STING signaling pathways have been uncovered recently to be crucial for host defense against a range of RNA and DNA viruses. However, the regulatory mechanisms involved in these two pathways remain to be elucidated. In previous studies, we have identified p97-Npl4 complex as a negative regulator of RIG-I-mediated antiviral responses. Structural and structure-directed mutational studies indicate that an intact state of the p97-Npl4 complex is essential for the regulation of antiviral signaling. Consistently, administration of compounds targeting p97 ATPase activity is shown to enhance antiviral response and protect mice from vesicular stomatitis virus infection. Taken together, these observations reveal the p97 complex as a potential drug target in antiviral therapy. Meanwhile, our preliminary findings suggest that p97 complex may also regulate cGAS-STING signaling pathway upon DNA virus infection. Here, we focus on p97-Npl4 complex-mediated regulation of cGAS-STING signaling pathway, intending to identify the key proteins associated with p97 or Npl4 and implore the novel mechanism about antiviral immune responses. Besides, we attempt to develop novel inhibitors targeting the p97-Npl4 interface through high-throughput screening, trying to find the lead compounds that interfere with the interaction between p97 and Npl4. Taken together, our research will contribute to further clarify the regulatory mechanism of antiviral signaling transduction and provide new strategies for antiviral therapy.

以RIG-I和STING等信号通路为代表的抗病毒应答是国际免疫学研究前沿。然而其关键调控机制仍有待阐明和完善。我们在前期的工作中部分探究了ATP酶p97在天然免疫中的功能与机制，发现p97与Npl4形成的复合物能够通过介导与K63泛素化相关联的K48泛素化来负调控RIG-I信号通路。进一步体内实验揭示破坏p97复合物的组装能够增强小鼠的抗病毒免疫反应。此外，初步研究表明p97复合物很可能也调控了STING信号通路。因此，本项目拟在上述工作基础上，一方面研究p97复合物对STING通路的潜在调控作用与机制，发现新靶点；另一方面靶向p97复合物进行抗病毒药物筛选，从分子水平深入探讨先导化合物对p97复合物组装的影响。最后，综合利用细胞和小鼠模型对p97调控STING通路的功能机制及先导化合物进行评估、验证与完善。本项目研究将进一步完善抗病毒免疫应答理论知识，并为相关药物研发及临床治疗提供基础。

病毒感染是威胁人类健康和生存的最危险的现实因素之一，其不断变异的特性以及新型未知病毒的出现是现代医学和生命科学所面临的巨大挑战。目前，关于抗病毒免疫应答作用机理与调控机制的研究已成为前沿领域。而p97-Npl4 复合物是抗病毒信号通路中的一个重要调控因子。.我们前期已经证实p97-Npl4复合物是RIG-I-MAVS介导的抗病毒信号通路的一个负调控因子。而通过本项目的研究，我们发现p97-Npl4复合物对cGAS-STING介导的抗DNA病毒信号通路的同样是起到了负调控的效果。进一步地，我们通过AlphaScreen这一筛选手段，寻找可以打破p97-Npl4相互作用的小分子化合物。并通过细胞内报告基因实验，检测对其相互作用抑制率>30%的40个化合物在抗病毒信号通路中的作用，确认其中三个化合物对于报告基因IFNβ以及ISRE的激活效果>30%，筛选出了能够有效阻断p97与Npl4的相互作用的抑制剂。.以上结果提示，p97复合物是一个抗病毒治疗的药物靶点，在药物筛选及药物研发方面有广阔的应用前景。同时，筛选获得的小分子化合物有望为抗病毒相关治疗及药物研发提供新的思路和方法。