海蛇蛇毒选择性TNFR1拮抗肽Hydrostatin-SN10的分子结构及其抗炎机制研究

The anti-inflammatory bioactive components of sea snake venom and its mechanism of action is unknown. We previously obtained a TNFR1-selective antagonist peptide Hydrostatin-SN10 from Hydrophis cyanocinctus, which has significant anti-inflammatory activity in rheumatoid arthritis and related diseases. However, the molecular structure of Hydrostatin-SN10 and its anti-inflammatory mechanism remain unclear. This project aims to study the crystal structure of TNFR1-SN10 complex via structural biological methods, identify the binding sites and interacting properties of Hydrostatin-SN10 with TNFR1, and elucidate its molecular mechanism of selective antagonism towards TNFR1; analyze the effects of the peptide on the pro-inflammatory of TNF-α and associated signaling pathways, and finally evaluate the in vivo anti-inflammatory and immunoregulatory effects of Hydrostatin-SN10 in animal models of arthritis. Our project will provide a direct structural biological evidence to clarify the anti-inflammatory mechanism of TNFR1-selective antagonist peptide Hydrostatin-SN10, and can also lay a solid foundation for Hydrostatin-SN10 to develop into a new class of marine-derived TNFR-inhibitory peptide drugs with proprietary intellectual property rights.

海蛇蛇毒中的抗炎活性成分及其作用机理未明。本课题组前期从青环海蛇中获得了一条选择性TNFR1拮抗肽Hydrostatin-SN10，靶点明确，对类风关等疾病具有显著的抗炎活性。然而，Hydrostatin-SN10的分子结构及其抗炎机制尚不明确。本课题拟通过结构生物学方法研究Hydrostatin-SN10与TNFR1复合物晶体结构，明确Hydrostatin-SN10与TNFR1的结合位点、方式，阐明其选择性拮抗TNFR1的分子机制；分析对TNF-α促炎活性及相关信号通路的影响，最终在关节炎动物模型上评价体内抗炎和免疫调控效应。本课题的完成将给选择性TNFR1拮抗肽Hydrostatin-SN10抗炎机理的揭示提供最直接的结构生物学证据，为研发Hydrostatin-SN10成为具有自主知识产权的、靶点明确的海洋生物来源新型选择性TNF-α受体抑制剂多肽药物奠定最坚实的基础。

课题组前期从青环海蛇中获得了一条选择性TNFR1拮抗肽Hydrostatin-SN10，本课题在此工作基础上探讨了SN10的分子结构、抗炎效应及其作用机制：1、获得了多肽SN10与靶蛋白TNFR1的复合物晶体，解析了SN10的分子结构和SN10-TNFR1的复合物三维结构；结合分子对接、蛋白截短与突变等方法，分析并验证了SN10与TNFR1的结合位点与拮抗机制：E8（第8位氨基酸—谷氨酸）为SN10结合TNFR1的关键作用氨基酸，SN10通过E8占据TNFR1与TNF-α结合的关键位点，直接竞争性拮抗TNFR1与TNF-α的结合，从而抑制TNFR1-TNF三聚体复合物的形成及下游信号的激活。2、SN10的体外抗炎作用及机制：SN10能够明显抑制TNF-α介导的L929细胞毒效应，抑制TNF-α刺激后HUVEC内皮细胞和RAW264.7巨噬细胞的ICAM-1、VCAM-1、IL-8、IL-6、IL-1β等炎症因子的表达；利用TNFR1-/-和TNFR2-/- BMDM炎症细胞模型，证明了SN10对TNFR1靶标具有选择性；SN10可通过抑制TNF-α诱导的NF-κB及MAPKs炎症信号通路的激活来拮抗TNF-α的促炎活性。3、利用类风湿关节炎和结肠炎动物模型评价了SN10的整体抗炎效应及机制，其通过调节Th1/Th2以及Treg/Th17比例失衡发挥抗炎和免疫调控作用，具有显著的体内抗炎效应。此外，SN10的适应症炎症性肠病（IBD）也已完成临床前研究的所有内容，正在申报临床试验批件。总之，本课题系统阐明了SN10选择性拮抗TNFR1的结构生物学基础、体内外抗炎效应与分子作用机制，为研发选择性TNFR1拮抗肽Hydrostatin-SN10成为具有自主知识产权的、First-in-class海洋来源的1类新药奠定了坚实的基础。