两面针碱通过靶向Hsp90治疗实验性自身免疫性脑脊髓炎的作用机制研究

Multiple sclerosis (MS) is the one of the medical unsolved problem. Our previous study found that nitidine could mitigate experimental autoimmune encephalomyelitis (EAE), a current internationally recognized MS animal model. Further mechanism study found that nitidine could promote the activated dendritic cells and macrophages to secrete IL-10 in EAE treatment. Affinity chromatography results suggested that nitidine is able to bind and interact with Hsp90 in dendritic cells and macrophages (KD=1 uM). So we put forward a hypothesis, "Hsp90 is a potential target of nitidine in EAE treatment". This topic goal is to confirm the interaction between nitidine and Hsp90 to verify the above hypothesis. The main research contents include molecules dynamics, thermodynamics parameters assays; Hsp90 activity assay; molecules mode and binding site confirmation, etc. This subject will reveal the molecular mechanism of action of nitidine to promote IL-10 secretion in EAE treatment for MS and will provide important scientific foundation and information for drug design and development to study novel structure and mechanism anti-inflammatory and immune-modulating agents.

多发性硬化症（MS）是当今医学界的未解难题之一。本课题组采用实验性自身免疫性脑脊髓膜炎（EAE）这一目前国际公认的MS动物模型，发现两面针碱具有潜在的MS治疗活性。前期机制研究发现，两面针碱治疗EAE的关键在于促进活化的树突状细胞和巨噬细胞分泌IL-10；亲和色谱及其验证实验提示树突状细胞中的Hsp90可与两面针碱结合并发生相互作用(KD=1.0 uM)。因此我们提出了"Hsp90是两面针碱治疗EAE的潜在分子靶标"的假说。本课题的目标是确证两面针碱与Hsp90的相互作用，验证上述假说。主要研究内容包括：两面针碱与Hsp90直接相互作用的结构生物学验证；两者分子结合模式及结合位点的确认；两面针碱对Hsp90自身功能及其伴侣蛋白功能的影响等。本课题的完成将有助于从分子靶标层面揭示两面针碱促进IL-10分泌、发挥EAE治疗作用的机理，并为MS治疗药物研发提供结构新颖、机制明确的化学小分子模版。

本课题的目标是确证两面针碱与Hsp90的相互作用，主要研究内容包括：分子结合动力学、热力学参数的测量；两面针碱对Hsp90自身及其伴侣蛋白功能的影响；两者分子结合模式及结合位点的确认等。本课题的完成，将有助于从分子靶标层面揭示两面针碱促进IL-10分泌、发挥治疗EAE作用的机理，为MS治疗药物的研发提供结构新颖、机制明确的化学小分子模板。研究结果：①通过pulldown实验和western blot实验证明两面针碱可与HSP90相互作用；②SPR和ITC等实验表明HSP90与两面针碱确实存在相互作用(KD=1.0 uM)。③两面针碱通过与HSP90结合，调控HSP90与某伴侣蛋白的相互作用，从而诱导下游通路提高IL-10的分泌，并且进一步产生防治EAE的作用。