Ocotillol型活性小分子探针的化学构建及其抗心肌缺血/再灌注损伤的靶点探索

Myocardial ischemia-reperfusion injury (IRI) is known to significantly cause the myocardial damage in coronary heart disease. Ocotillol-type derivatives (ODs) have been shown the similarity in pharmacokinetics and transmembrane permeability, but only the 24R-ODs exert cardioprotective effects against IRI both in vivo and in vitro assay. It strongly suggested that ODs is stereospecificly recognized by the target of ODs in cardioprotection against IRI. Therefore, it’s necessary to further study the molecular mechanism and the target. Based on the results of proposer’s research team on the structure activity relationship study of ODs in cardioprotection against IRI, this project will apply the proposer’s experience and method in his successful study on detecting and studying the unknown target of bioactive metabolite by the small molecular probe, and chemically construct the active ODs molecular probes. The modular synthesis will be studied and be applied in the synthesis of ODs molecular probes. In the 24R-ODs fluorescence probe, the fitting linker between the ODs and fluorescence group will be examined to reduce the sterical effect of bulky fluorescence group on the activity of the ODs. In the compact molecular probe of 24R-ODs which is expected to have activity, the suitable reactive functionality will also be investigated to increase the efficiency of cross-linking between target protein and the compact molecular probe. These two kinds of developed molecular probes will be used in detecting the distribution of the target in living cell and exploring the target protein, respectively. The corresponding 24S-epimer will be used as ideal control because it has the similar chemical character with the 24R-ODs molecular probe, but it is expected to have no activity. Achievements of this project will strongly support the discovery of new target in cardioprotection against IRI and the research and development of the related drug innovation.

心肌缺血/再灌注损伤（IRI）是冠心病心肌损伤的重要原因。体内外实验证实ocotillol型人参皂苷（ODs）的药代和细胞透膜能力相似，但只有24R-ODs具有确切的抗大鼠IRI活性，提示其作用靶点能特异性识别ODs差向异构体，因此，其作用的靶点和分子机制值得深入研究。本项目利用申请人以往凭借活性小分子探针成功研究天然产物未知靶蛋白的方法和经验，在本课题组对ODs抗IRI构效关系研究的基础上，拟化学构建活性ODs探针分子。研究利用模块化合成手段，探讨在ODs荧光探针分子中引入恰当的连接分子，以隔开并缓和荧光基团对活性中心的影响；探讨在具有潜在活性的ODs紧凑型探针分子中使用合适的交联基团，提高其与靶蛋白的交联效率。由此获得的两类活性探针分子，以对应化学性质相似但无活性的差向异构体为理想对照，分别用于探索靶点在活细胞内的分布和靶蛋白发现。为抗心肌缺血新靶点的发现及其相关创新药物研究提供支持。

心肌缺血/再灌注损伤（IRI）是冠心病心肌损伤的重要原因。研究证实ocotillol型人参皂苷（ODs）有确切的抗大鼠IRI活性，且提示其作用靶点能特异识别24R-ODs。因此，开展其作用靶点和分子机制的研究具有重要意义。.本项目按照国家自然科学基金项目计划书内容进行，已完成全部研究计划，包括：1、构建系列含不同连接链的ODs荧光探针，利用体外IRI细胞模型，筛选出含柔性乙氧基链的24R-ODs荧光探针能保留ODs的生物活性；2、利用该系列活性荧光探针，基于活性差异法、共定位实验和竞争性实验等手段，首次发现了24R-ODs的活性靶点位于线粒体上；3、利用模块化合成手段，构建了系列含不同反应功能性基团的ODs紧凑型探针，通过H9C2心肌细胞模型实验，筛选出含光亲和基团的24R-ODs紧凑型探针分子能保留ODs的生物活性；4、利用活性24R-ODs紧凑型探针，初步探索发现了其活性靶蛋白的存在；5、在为紧凑型探针合成的糖苷化条件探索中，建立了ocotillol型皂苷元C3位羟基选择性糖苷化条件，因此而开发了一个从商品20(S)-原人参二醇出发三步反应合成拟人参皂苷HQ的简明高效合成线路；6、基于ODs抗IRI研究过程中发现其对P糖蛋白功能的调节能力，合成了系列ODs酰胺衍生物，通过构效关系等研究，在体内外活性试验和机制研究中证实了衍生物46c作为一种有效的P糖蛋白调节剂，在口服情况下，也能有效恢复多药耐药人源肿瘤对紫杉醇的敏感性。这些结果为ODs在抗IRI及P糖蛋白调节剂等方面的研究和应用奠定了基础。