基于靶标鉴定研究黄芩素抗帕金森病作用的分子机制

Parkinson's disease (PD), the second neurodegenerative disease in the world, is characterized by a combination of motor and non-motor symptoms. Currently, clinical treatment for PD is symptomatic and there is no effective treatment able to restore neuronal degeneration. Research and development in novel drugs to prevent or treat PD has been a crucial subject, and some novel candidates are in the development. .Baicalein, a major bioactive flavone constituent isolated from Scutellaria baicalensis Georgi., has been shown to be neuroprotective in several PD animal models. In our previous study, we verified the anti-PD effect of baicalein in rotenone-induced models, and investigated its possible mechanism. The previous results suggested that baicalein might be effective in the prevention or treatment of PD. However, the action mechanism of baicalein, especially the target, is largely unknown..In this project, we aim to fundamentally elucidate the molecular mechanism of baicalein against PD based on its targets identification. First, we identify the direct binding protein of baicalein through chemical biology techniques. Combined with gene editing technology, we verify the functional targets of baicalein. Based on the identified targets, the modulatory mechanism of baicalein on the target and the signal pathway involved in its anti-PD effect will been further investigated. Through this study, we will provide crucial clues for the specific indications of baicalein in PD treatment and its further structural optimization.

帕金森病（Parkinson's disease, PD）是全球第二大神经退行性疾病，现有药物因疗效限制性及副作用明显，不能持久有效缓解PD症状，因此亟需深入开展抗PD药物的开发研究。传统中药黄芩的主要活性成分黄芩素在抗PD药物研发中，能够明显改善多种PD模型动物的运动功能障碍，申请人更明确了黄芩素对鱼藤酮致PD模型的治疗作用，探究了其对线粒体及神经炎症通路的调控机制，这些结果提示黄芩素可能是新型抗PD候选药物。然而黄芩素的作用机制不清，作用靶标尚未明确。本项目旨在基于靶标鉴定研究黄芩素抗PD作用的分子机制。采用化学生物学手段高通量鉴定黄芩素抗PD的直接结合蛋白；同时利用基因编辑技术鉴定黄芩素抗PD的功能靶蛋白；最后深入研究黄芩素与其功能靶标的结合位点及调控机制。本项目有望从分子水平阐明黄芩素抗PD的机制，为全面认识其对PD的治疗特点及其优化方向提供理论支持，具有重要的科学意义和社会价值。

帕金森病（Parkinson’s disease, PD）是全球第二大神经退行性疾病，现有药物因疗效限制性及副作用明显，不能持久有效缓解PD症状。传统中药黄芩的主要活性成分黄芩素可明显改善多种PD模型动物的运动功能障碍，然而黄芩素抗PD的作用靶标仍有待阐明。本研究采用药物亲和反应靶点稳定性（Drug affinity responsive target stability, DARTS）联合蛋白质谱技术筛选了黄芩素直接结合蛋白。采用微量热泳动技术（microscale thermophoresis, MST）以及细胞热转变分析（Cellular Thermal Shift Assay, CETSA）验证了黄芩素与磷脂酰乙醇胺结合蛋白1（Phosphatidylethanolamine-binding protein 1，PEBP1）在PD体内外模型上直接结合。进一步构建PEBP1突变蛋白，利用MST实验明确了黄芩素直接与PEBP1的C端结合，同时分子对接实验结果发现了黄芩素在PEBP1结合口袋中的关键位点。通过基因编辑技术CRISPR/Cas9建立了PEBP1敲除细胞，发现黄芩素依赖PEBP1发挥药理活性。调控机制上，黄芩素靶向PEBP1后抑制Raf1/ERK通路和GPX4蛋白而保护神经细胞抵抗铁死亡，改善铁死亡神经细胞模型中的自噬水平；并且发现了黄芩素不影响正常神经细胞中PEBP1水平，而是调控PD模型细胞内PEBP1的磷酸化从而干预下游信号通路，这与黄芩素和PEBP1结合位点也是相关的。综上所述，本项目发现了黄芩素靶向PEBP1调控Raf1/ERK通路而抑制神经细胞铁死亡，从而发挥抗PD作用。