五味子甲素是从中药五味子中提取出的联苯环辛烯型木脂素，具有神经保护活性，但是其作用靶标和作用机制尚未阐明。我们采用基于活性的蛋白质分析技术（activity-based protein profiling，ABPP）揭示五味子甲素的神经保护作用的靶标与机制，为后续相关研究提供参考。

首先基于五味子甲素的构效关系，我们设计并合成了4个五味子甲素光亲和性探针，通过生物活性评价和光标记能力测试后，获得一个生物活性较好且具有良好标记效率的探针P2。之后，我们从探针浓度、孵育时间、紫外光照时间三个方面优化了蛋白标记实验条件，并且通过竞争性实验验证了探针P2与五味子甲素作用于相同靶标。基于ABPP原理，我们利用探针P2在人神经母细胞瘤细胞（SH-SY5Y Human Neuroblastoma，SH-SY5Y）垂钓五味子甲素的直接作用靶蛋白，通过液质联用技术和非标定量法，鉴定到13个高可信度蛋白，结合生物信息学分析和文献调研，最终确定YKT6 v-SNARE 同系物（Synaptobrevin homolog YKT6，Ykt6）为五味子甲素的潜在靶蛋白。之后，通过Pull down/WB实验、免疫荧光共定位实验、细胞热迁移分析验证了五味子甲素与Ykt6的直接相互作用，利用表面等离子共振实验进一步测定二者之间的KD值为14.6 μM。五味子甲素是首次发现的作用于Ykt6的小分子。功能验证实验中，通过Ykt6敲低实验和自噬流实验，进一步证实Ykt6是五味子甲素发挥神经保护活性的重要靶点。五味子甲素通过作用于Ykt6调节自噬从而保护细胞免受过量谷氨酸诱导的细胞损伤。

本课题利用ABPP技术首次发现五味子甲素的直接作用靶标Ykt6，验证了二者之间的相互作用并初步探索五味子甲素通过Ykt6发挥神经保护活性的作用机制。这一发现为后续五味子甲素的神经保护作用研究提供参考，并可能为神经保护治疗提供新的策略。

Schisandrin A (Sch A) is a dibenzocyclooctene lignan extracted from traditional chinese medicine wuweizi, which has neuroprotective activity, however its target and mechanism of action have not been clarified. We revealed the target and mechanism of Sch A for neuroprotection by using activity-based protein profiling (ABPP) in order to provide reference for subsequent relevant studies.

Firstly, based on the structure activity relationship of Sch A, we designed and synthesized four photoaffinity probes. After biological activity evaluation and photoaffinity labeling ability test, probe P2 with good activity and labeling efficiency was obtained. After that, we optimized labeling experiment conditions from three aspects: probe concentration, incubation time and UV irradiation time, and verified that probe P2 acted on the same targets with Sch A through competitive experiments later. Based on ABPP principle, probe P2 was utilized to disclose the targets of Sch A in SH-SY5Y cells, and identified 13 high confidence proteins through LC-MS/MS with label free quantification method. Combined with bioinformatics analysis and literature research, Synaptobrevin homolog YKT6 (Ykt6) is identified as a potential target protein of Sch A. Pull down/WB assay, immunofluorescence experiment and cellular thermal shift assay validated the direct binding of Ykt6 and Sch A. The interaction between Sch A and Ykt6 was further confirmed by surface plasmon resonance assay, and the KD value was measured to be 14.6 μM. To the best of our knowledge, it was the first discovered small molecule that directly interacted with Ykt6. In functional verification experiments, Ykt6 knockdown experiment and autophagy flow experiment further confirmed that Ykt6 is an important target for Sch A to exert neuroprotective activity. Schisandrin A protected the cells against the injury induced by glutamic acid by regulating autophagy via Ykt6.

In this study, the direct target of Sch A, Ykt6, was discovered for the first time by using ABPP technology, and the interaction between them was verified. The mechanism of Sch A's neuroprotective activity through Ykt6 was preliminarily explored. These findings can provide reference for subsequent studies on the neuroprotective effects of Sch A, and may provide a new strategy for neuroprotective treatment.