酸浆属清热解毒中药抗肿瘤睡茄内酯的发现及其作用靶点研究

The genus Physalis (Solanaceae) are widely distributed are widely distributed throughout tropical and subtropical regions of the world. Some of them are used as common Chinese herbs with heat-clearing and detoxifying effects. Withanolides, a group of naturally occurring C28 steroids, are responsible for these functions. Recently, the researchers throw their sight to the withanolides due to their diverse structures and significant bioactivities. In this project, we will applied an established reactivity-based screening (RBS) method, for effectively screening the cytotoxic withanolides with the 2,3-unsaturated ketone moiety in ring A and the 5,6-epoxy group in ring B from Physalis herbals in China. These withanolides will be isolated and elucidated using our established techniques including specific detection, fast separation and structure identification based on the spectroscopic and chemical methods. A biotin-withanolide affinity probe will be synthesized using the optimal withanolide as a substrate. The probe will be applied to fish the proteins from lysate of cancer cells. After the proteins were identified by MS analysis, pull-down, isothermal titration calorimetry (ITC), surface plasmon resonance (SPR) and molecule dynamic experiments will be performed to further verify the interactions between the withanolide and target proteins. Moreover, site-specific mutagenesis, enzyme activity detection and in vivo experiments also will be carried out to validate the protein as a antitumor target of the withanolide and investigate its mechanism as an anticancer agent. This project will provide a new strategy to effectively find the target of bioactive compounds from TCM sources for the first time. It will illuminate the material basis and mechanism of Chinese herbals from Physalis exhibiting heat-clearing and detoxifying effects, and also promote the application of the withonaloids as promising therapeutic agents for use in cancer treatments.

酸浆属中药具“清热解毒”功效，其中富含特征性成分——睡茄内酯。近年来，睡茄内酯因结构变化多样、活性显著而倍受关注。本项目拟采用已建立基于药效团的化学反应筛选法（RBS）从酸浆属中药快速发现细胞毒活性的睡茄内酯，并对目标化合物进行分离、制备、结构测定和活性评价。继而以显著活性化合物为底物，合成生物素-睡茄内酯亲和探针，结合质谱分析开展从肿瘤细胞裂解液中垂钓和鉴定靶蛋白的研究。并利用Pull down、等温滴定量热法（ITC）、表面等离子共振法（SPR）和分子动力学实验进一步验证目标化合物与靶蛋白的结合，进而通过定点突变、酶活检测、体内实验等方法对靶点验证。从而确认睡茄内酯抗肿瘤作用靶点、探究其作用机制，最终阐明酸浆属中药“清热解毒”的分子生物学原理，为从中药物质基础研究提供新的思路。

睡茄内酯是酸浆属清热解毒中药的特征性成分，也是主要的活性成分。为了阐释酸浆属中药清热解毒的分子生物学基础，本项目对酸浆、苦蘵、毛苦蘵、龙珠等的睡茄内酯类成分开展了定向分离和结构测定，从中共分离得到61个睡茄内酯成分，包括23个新化合物。并对分离获得的睡茄内酯化合物进行了多种肿瘤细胞的细胞毒活性和结构修饰，获得25个衍生物，优选了GLS1抑制剂化合物7。通过WA小分子探针结合蛋白质谱分析，发现WA能够直接结合结肠癌细胞中PHGDH蛋白。进一步研究显示WA能够共价结合到PHGDH的Cys295位点，阻断了PHGDH的底物结合域并产生变构作用，进而抑制了PHGDH的酶活。在此基础上，通过细胞内的“click”反应结合SILAC-ABPP技术发现PRDX6是WA在非小细胞肺癌中的直接作用靶点。质谱鉴定与点突变实验证明WA是通过共价结合到PRDX6的Cys46位点，抑制了PRDX6的活性，而发挥抑制非小细胞肺癌增殖的作用。此外，还发现睡茄内酯Tubocapsenolide A（TA-1）能够直接与SHP-2结合，抑制其降解，升高酪氨酸磷酸酶活性，抑制STAT3的磷酸化并发挥抑制肿瘤细胞增殖的作用。TA-1还能够共价结合HSP90的C端Cys521位点，抑制HSP90与CDC37的相互作用，进一步抑制了肿瘤细胞的增殖。.本项目对酸浆属中药中睡茄内酯成分及其抗肿瘤作用研究，阐释了酸浆属中药“清热解毒”的物质基础和药效机制，为酸浆属中药的临床应用与开发的提供了科学依据。本项目研究已发表SCI论文12篇（其中影响因子大于5的11篇，大于10的3篇），获专利授权2项。