基于药物亲和诱导蛋白稳定性差异鉴定大戟/甘遂毒性二萜成分的靶蛋白研究

Knowledge about the protein targets of therapeutic agents is critical for understanding drug mode of action. But identifying the molecular targets for the beneficial or detrimental effects of small-molecule drugs is currently challenged. Only very few protein targets were confirmed for natural compounds. The present study was conducted to investigate the protein targets of cytotoxic diterpenes from Euphorbia Pekinensis/Kansui, using the drug affinity responsive target stability difference (DRTSD), proteomics and bioassay. Based on the realization of the TCMs properties and our previous work, we hypothesized that the diterpenes with similar chemical structure may interact with the similar protein targets and then produce the similar intestinal toxic reactions. To find the protein targets meditating the intestinal toxicity of diterpenes from Euphorbia Pekinensis/Kansui, the research pathways were as follows:（1）Toxic ingenane and casbane type diterpenes were isolated and identified from the Euphorbia Pekinensis/Kansui. The intestinal toxicities were evaluated in vivo and in vitro. The experimental diarrhea induced by these diterpenes was observed in mice, and the contractive function was recorded in the isolated intestinal issue. Moreover, the contents of intracellular Ca2+, Cl- and active oxygen species (ROS) labeled with different fluorescent probes were determined in human epithelial colorectal adenocarcinoma cells (Caco-2), to reflect the intestinal toxicities induced by diterpenes from Euphorbia Pekinensis/Kansui. (2) A new technology, drug affinity responsive target stability difference (DRTSD), was used to discover the direct binding targets (and off targets) of toxic ingenane and casbane type diterpenes on a proteome scale from Caco-2. Such an approach could potentially identify any protein targets of small molecules with no limitations posed by chemistry or mechanism of action. We also selected a diterpene without any intestinal toxicity to identify its possible target(s) at the same way, which should not be the targets for toxic ingenane and casbane type diterpenes. (3) We determined whether activation/inhibition of the targets can change the intestinal toxicity induced by the toxic diterpenes, using agonist/antagonist and siRNAs of the targets. In addition, we explore the direct interactions between toxic ingenane/casbane type diterpenes and target proteins, by liquid chromatography mass spectrometry, spectroscopy and computer modeling. In summary, the main objective of the present study was to find the target proteins for cytotoxic diterpenes from Euphorbia Pekinensis/Kansui by DRTSD on a proteome scale, trying to provide the evidence for the antidote research, rational use and new drug development.

本项目基于对大戟/甘遂药性的认识和课题组前期工作基础，采用药物亲和诱导蛋白稳定性差异（DRTSD）方式，结合蛋白组学及生物效应评价，研究大戟类药材毒性二萜成分的靶蛋白。认为结构相近而毒性不同的二萜结合至近似的靶蛋白从而产生肠道毒性。工作路径：（1）在整体/离体/细胞水平评价西松烷和巨大戟烷型二萜及代表性化合物对肠道刺激毒性；（2）采用蛋白脉冲消化（DRTSD方法）和液相-质谱技术，从Caco-2细胞总蛋白中鉴定代表性毒性二萜成分的结合蛋白，并从中排除无毒性二萜成分的结合蛋白，以发现关键结合蛋白；（3）使用蛋白激动/抑制剂和RNA干扰等手段对靶蛋白介导肠道毒性进行验证；并进一步分析关键靶蛋白与毒性二萜类成分的相互作用。从而揭示大戟类药材毒性发生的机理和毒性物质的构-毒关系，为其解毒药物寻找、配伍禁忌和新药开发提供研究依据。

中药活性成分的靶蛋白鉴定是研究难点。本项目应用药物亲和诱导蛋白稳定性改变（DARTS）方法，结合代谢组学和蛋白组学及传统生物效应评价，在不同层面研究大戟类药材芫花毒性二萜成分yuanhuacine的毒性标志物和靶蛋白。1）采用UPLC-QTOF鉴定出的化合芫花酯戊、genkwanine L、芫花酯丙、芫花烯、芫花酯丁、芫花酯庚、芫花酯乙、芫花酯己、芫花酯甲等二萜类成分；2）研究发现芫花酯甲和异大戟素具有显著的细胞毒性作用，Hoechst 33342荧光染色显示细胞明显发生凋亡，10uM浓度下48h细胞凋亡率为21.13%和25.98%。细胞上清液中AST、ALT 含量显著增加3-4倍。近一步研究显示细胞凋亡发生于细胞内活性ROS上升有密切关系。而且芫花酯甲和异大戟素显著诱导PGE2和PGF2前列腺素类炎性成分的释放，PGE2和PGF2通过经典MAPK途径加剧细胞凋亡。3）采用UPLC-QTOF-MS/MS分析异大戟素诱导肝细胞损伤的代谢组学变化。运用PLS-DA模型区别空白对照组和给药组L02细胞代谢物差异，鉴定出10个与细胞毒性高度相关的细胞代谢物。包括，磷脂类、脂肪酸类等。涉及甘油磷脂代谢、脂肪酸过氧化、花生四烯酸代谢、MAPK等细胞凋亡相关信号通路。4）采用色谱共流出技术（离子交换），从细胞L02的裂解液中，鉴定发现了芫花酯甲的直接蛋白结合部位。对芫花酯甲的蛋白结合部位，经蛋白脉冲消化处理，使用NanoLC-LTQ-Orbitrap从L02蛋白部位Fra.4中，鉴定Yuanhuacine的结合蛋白为Enolase, Ran GTPase-activating protein 1，Histidine triad nucleotide-binding protein 1（H1NT1） 和PARK7。该研究在分子水平揭示了芫花酯甲等二萜成分细胞毒、致炎性作用，特别是使用代谢组学及蛋白脉冲消化蛋白组学技术，在代谢物和蛋白组层面深入鉴定了芫花酯甲等二萜成分毒性标志物和靶蛋白，为揭示揭示大戟类药材毒性发生机理提供研究依据。