苦参碱化学小分子探针的构建及其抗特发性肺纤维化候选靶标的探寻

Idiopathic pulmonary fibrosis (IPF) is a diffuse lung disease, with rapid progress, short survival time and high mortality rate, still lack effective treatments. Matrine is a component of the traditional Chinese medical herb Sophora flavescens Ait. Previous studies have found it can reduce the extent of fibers in bleomycin-induced pulmonary fibrosis in mice. But its molecular targets and regulation mechanisms have not yet been fully clarified. This project will carry out a systematic research on molecular targets, utilizing small molecules probes technology: 1) Study on structural modification and structure-activity relationship of matrine, screening the activity compounds for probe synthesis. 2) Design and synthesis of three types of small molecular probes and screening through the biological activity tests to obtain the activity molecular probes. 3) Using affinity chromatography, gel electrophoresis and mass spectrometry technology to probe the candidate targets of matrine. The objective of this work will obtain the candidate targets of matrine for the treatment of idiopathic pulmonary fibrosis for the first time, and provide the foundation for revealing the antifibrotic activity targets. It can also give the direction for the matrine-based antifibrotic drugs research and development, and provide new research ideas and methods for development of antifibrotic drugs with independent intellectual property right.

特发性肺纤维化(idiopathic pulmonary fibrosis，IPF)是一种进展迅速、中位生存期短、死亡率高的弥漫性肺疾病，至今仍缺乏有效的治疗药物。苦参碱是我国传统中药苦参的主要成分，课题组前期研究发现，它能减轻博来霉素诱导的小鼠肺组织纤维化程度，具有很好的抗IPF作用。但目前其抗IPF的作用靶标和调节机制尚不清楚。本项目拟采用小分子探针技术，以寻找苦参碱抗IPF作用候选靶标为核心开展系统研究：1）对苦参碱进行结构修饰，结合体外生物活性筛选结果总结构效关系，确定其抗IPF的活性基团；2）合成3种苦参碱类小分子探针，通过生物活性评价筛选出活性探针分子；3）运用亲和层析、凝胶电泳及质谱分析技术探寻候选靶标。本项目的开展将首次获取苦参碱抗IPF作用的候选靶标，为揭示其真实作用靶标奠定基础，为苦参碱类抗IPF药物的研发指明方向，为开发具有自主知识产权的抗IPF药物提供新的研究思路。

前期研究发现苦参碱能减轻博来霉素诱导的小鼠肺组织纤维化程度，具有很好的抗特发性肺纤维化(idiopathic pulmonary fibrosis，IPF)作用，但其抗IPF的作用靶标和调节机制尚不清楚。为明确其靶标，本项通过对苦参碱及其类似物进行结构修饰，设计并合成了苦参碱衍生物147个；建立了天狼星红染色法检测TGF-β1诱导人胚肺成纤维细胞Mrc-5细胞外基质胶原沉积量的体外抗IPF筛选模型；通过活性筛选，获得了体外具有良好抗IPF活性的苦参碱衍生物并总结了构效关系；通过已有构效关系，结合以化合物为中心的化学蛋白质组学技术设计并合成了3类共4个新型苦参碱类小分子探针，并通过体外活性评价筛选出活性探针分子；选取具有荧光功能的光亲和标记型小分子探针，运用原位"Click-chemistry"反应策略，通过凝胶电泳等方法进行候选靶标探寻。结果显示小分子探针的蛋白条带与阴性对照的蛋白条带并无明显差异，提示我们小分子探针与靶点可能并未发生特异性结合，或者其作用靶点可能为低丰度蛋白。