双功能分子探针的制备及在青蒿素作用靶点筛查中的应用

Artemisinins (Arts) hold great potentials in tumor chemotherapy. Some of the research has reached out clinical stages. To reveal their molecular mechanisms is of importance in accelerating the development of new kinds of Art-based anti-tumor drugs. However, up to date, the exact mechanism is still unknown, and there are different explanations from different research groups. Although drug conjugated molecular probes offer possibilities in discovering the mechanism details at molecular levels, those currently used are lack of functions to meet the requirement in mechanism study. For example, it is still a question that whether Arts have reversible target proteins, which is impossible to be answered by such single functional probes. In this program, we aim to design and synthesize new kinds of Arts conjugated molecular probes with multi-functionalities. Compared with the probes with single-function, our designed probes will have two reactive moieties that can ensure protein labeling and subsequent labeling with chemical markers. These probes can be applied to discover protein targets which reversibly bind to Arts , thus overcome the drawbacks of the probes currently used . The success in the development and application of such probes is great helpful to further reveal the anti-tumor mechanisms of Arts.

青蒿素（Artemisinin）类分子具有广泛的抗肿瘤活性，部分研究已经进入临床验证阶段。目前，学术界对其作用机理的理解尚存争议。青蒿素偶联的分子探针技术为作用机理研究提供有力的化学生物学工具。然而，目前已经发展起来的青蒿素分子探针技术不足以回答青蒿素机理争议中遇到的关键问题，如：青蒿素是否存在非共价结合靶点。本项目拟合成一类新的双功能青蒿素偶联分子探针,并利用此类探针对青蒿素结合蛋白进行筛查。与目前只含有单炔基的分子探针相比，该类探针含有两个反应基团，可以依次实现药物结合蛋白的可控偶联和化学标记。此类探针可以弥补单功能探针的缺陷，用于筛查新的青蒿素类药物的非共价结合靶点。最终为进一步揭示青蒿素类药物的抗肿瘤作用机理提供实验依据。

青蒿素及其衍生物具有较好的抗肿瘤活性，但是其作用机理尚未完全揭示。利用药物偶联的多功能分子探针可以实现药物结合靶点的发现，有望进一步揭示青蒿素抗肿瘤作用机理。本项目拟合成新的多功能探针，该探针同时搭载光活化标记基团和“点击化学”标记基团。与以往的青蒿素探针不同的是，该探针可用于发现青蒿素非共价结合蛋白。截至目前，项目组已经完成两个多功能探针的制备与结构表征，利用该类探针进行了青蒿素结合蛋白分析。结果没有发现新的可以与青蒿素非共价结合的蛋白，且两种不同的光活化基团的引入所得结论一致。结果提示青蒿素有可能不存在有意义的非共价结合蛋白，其主要作用机理在于蛋白的非特异性烷基化和ROS的影响。研究中我们发现，青蒿素在血红素催化下可以产生具有较强生物毒性的羟基自由基，且靶向线粒体可以进一步加强自由基的产生进而增强抗肿瘤活性。此外，本项目制备了三个内质网靶向青蒿素衍生物及两个化学寻靶探针，并通过转录组、蛋白组以及分子水平实验对青蒿素靶向内质网抗肿瘤机理进行了研究。结果表明靶向内质网可以加强青蒿素对肿瘤细胞的氧化胁迫以及内质网相关的钙信号通路的影响。项目的研究结果对进一步理解青蒿素的作用机理有重要参考价值，相关结论可以用于指导青蒿素的衍生化及发现更高效的具有抗肿瘤作用的青蒿素衍生物。