藤黄酸肿瘤特异性靶蛋白谱动态变化的定量化学蛋白组分析与整体“靶-效”网络构建

Gambogic acid (GA) is a cytotoxic natural product discovered from the resin of Garcinia hanburyi Hook.f. (Clusiaceae). The antitumor activities and safety of GA have been well demonstrated by targeting multiple anti-tumor molecular pathway and thereby inhibiting the growth and progression of aggressive tumor. We have previously shown that GA is a natural Michael reaction acceptor molecule with extremely high electrophilicity and that it is prone to react with nucleophilic groups, such as thiol of cysteine residues on cellular proteins. Using mass spectrometry, in combination with biochemical methods, we have previously identified thioredoxins and thioredoxin reductase as the possible molecular targets of GA. It provides a plausible molecular basis of ROS toxicity and the downstream electrophilic cellular responses stimulated by GA in cancer cells, which shed light on the relationship between Michael reactivity to cellular proteins and antitumor activities of GA for the first time. Nonetheless, we cannot rule out the possibility that GA interacts with other proteins also with reactive thiol, especially knowing that most functional thiol containing cysteine residues in proteomes are hyper-reactive. To this end, here we aim to develop a novel quantitative chemoproteomic strategy to globally investigate target profile of GA. Afterwards, we will be able to discovery tumor-specific targets of GA in situ or in vivo and study the effects of exogenous or endogenous oxidative perturbations on GA’s target profile in cells. Moreover, we intend to apply iTRAQ-based quantitative proteomics to study changes of global protein expression and phosphorylation events induced by GA in cells. Finally, we will use the state-of-the-art bioinformatics to generate the first global “target-effector” network induced by GA in cancer cells. These studies are not only useful in dissection of the molecular base of the selective anti-tumor effects of GA, but also of value for new therapeutic strategy discovery in which GA is serving as a testing compound in a Phase II clinical trial. More generally, this project will provide a chemoproteomic platform for investigating target engagement of chemically synthesized or naturally occurring covalent inhibitors.

藤黄酸是一种提取自中药藤黄的天然抗肿瘤化合物。大量药理学研究表明藤黄酸能够通过多种途径抑制肿瘤发生与发展，提示藤黄酸可能存在多靶点作用。然而，基于“还原论”的研究思路与方法既难以全面鉴定藤黄酸的直接作用靶蛋白，也无法以建立其诱导的不同分子通路之间的联系，从而限制了人们对其抗肿瘤作用机制的全面认识。本项目拟采用定量化学蛋白组学技术系统分析藤黄酸的肿瘤特异性靶蛋白谱及其在肿瘤发展与干预过程中的动态变化，结合多种系统生物学研究手段探索藤黄酸的靶蛋白-效应蛋白之间以及效应蛋白-效应蛋白之间的关联性，最终全面揭示藤黄酸的整体“靶-效”关系网络与抗肿瘤作用机制。本项研究不仅将为正在开展的藤黄酸临床试验及其衍生物研发工作提供重要理论支持，而且对其它化学合成或天然迈克尔反应受体分子的靶点鉴定工作也有重要参考价值和示范作用。

天然产物是新药开发和化学生物学研究的重要来源。天然产物化学结构上常见一些亲电性基团如迈克尔反应受体、环氧。这类具有亲电性的天然产物能够通过共价修饰特定功能半胱氨酸残基上的亲核性巯基基团，从而影响其所在蛋白质的功能，并且最终发挥生物学效应。鉴定一个生物活性物质的蛋白靶点对于了解其分子作用机制至关重要。然而，天然产物往往化学结构复杂，合成困难，难以采用添加亲和基团或固载化的方式富集其在蛋白质组上的作用靶点。为此，本项目开发了一种新型巯基反应性多重定量技术，高通量地通过测定细胞巯基蛋白质组被巯基反应性探针标记的效率变化，从而间接识别哪些半胱氨酸残基位点能够被受试亲电性天然产物所修饰。该技术成功鉴定了八种含有α，β不饱和酮式结构的亲电性天然产物在肿瘤细胞中的作用位点。例如，本项目发现抗肿瘤天然产物藤黄酸能够通过共价修饰核转运蛋白Exportin-2上的842和939位半胱氨酸残基，抑制其对Importin-1α在细胞质-核间的转运能力，进而诱导肿瘤细胞死亡。值得一提的是，该化学蛋白质组学技术还能用于分析细胞巯基蛋白质组随氧化还原内稳态扰动的动态变化，或用于鉴定其它巯基反应性物质（包括氧化剂、化学毒剂、环境污染物、共价药物、反应性代谢物等）的分子靶点，因此具有广泛的应用前景。

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