一种基于液质联用与免疫沉淀技术的芳香化酶抑制剂的靶向发现及抑制剂结构与活性关系的研究

Aromatase is a key target for therapy of diseases caused of hyperestrogenism. Currently, Screening for aromatase inhibitors is using the methods by which aromatase interacts directly with compounds in biochemical reagent. These have differences with interaction in organism, and are laborious and time-consuming resulting in few naturally occurring aromatase inhibitors was discovered. In our previous study, many compounds capable of inhibiting aromatase from Mulberry Leaf and Glycyrrhiza uralensis Fisch were researched utilizing HPLC-MS coupling Ultrafiltration. The results indicated some isoflavone performed good aromatase inhibiting activity. However above method might lead to false positive results, and there are as yet little researches about isoflavone focused on its aromatase inhibiting activity, so it is difficult to elucidate the relationship of its structure and aromatase inhibiting activity. Therefore, this project is mainly focused on three aspects: 1) To establish an aromatase inhibitors screening method based on HPLC-MS and immunoprecipitation techniques, by which the interaction of aromatase and compounds is occurring in MCF-7 cell. 2) To discover and characterize all of isoflavones from Glycyrrhiza plants utilizing established method. 3) To elucidate isoflavone's relationship of chemical structure and aromatase activity, utilizing molecular mimicry computing. In summary, this project can supply a novel and reliable method and a good chemical source for the discovery of aromatase inhibitors for treatment of estrogen-related diseases.

芳香化酶(AR)是药物治疗高雌激素水平相关疾病的关键靶点。目前对于芳香化酶抑制剂(AIs)的筛选主要采用化学模拟酶催化反应的方式，与生物体内的作用存在较大差别，且操作繁琐、耗时导致从天然产物中发现的AIs极其有限。我们前期的工作通过LC-MS偶联超滤离心的方法，对桑叶、甘草等植物中具有AR抑制活性的成分进行了研究，发现一些甘草中的异黄酮类化合物(IFs)具有较好的AR抑制活性。但是LC-MS结合超滤离心的方法容易造成假阳性；且目前关于IFs在AR抑制活性方面的研究报道甚少，不足以对其结构与活性之间的关系进行分析。因此，本项目在工作基础上，①拟建立一种基于LC-MS结合免疫沉淀技术，从成活细胞中快速发现AIs的筛选方法；②筛选甘草属植物中具有AR抑制活性的IFs；③阐明IFs类芳香化酶抑制活性的构效关系。本项目为AIs的发现提供了新的方法及其物质来源。

芳香化酶（AR）是药物治疗高雌激素水平相关疾病的关键靶点。前期研究工作发现某些异黄酮类化合物具有很好的AR抑制活性。鉴于以往芳香化酶抑制剂（AIs）筛选都采用体外化学反应的方法，本项目建立了一种基于细胞内的AIs筛选模型。利用上述方法发现了甘草二氯甲烷提取物中含有一些AIs，并从中分离了6个含有异戊烯基取代的异黄酮，经测定具有较好的AR抑制活性。研究报道苗药蔓性千斤拔中含有大量异戊烯基取代的异黄酮，对蔓性千斤拔二氯甲烷提取物进行AR抑制活性测定发现其IC50值为10.0 μg/mL。利用UPLC-TOF/MS技术鉴定了蔓性千斤拔二氯甲烷提取物中20个异黄酮化合物，4个异戊烯基取代的异黄酮经测定其抑制AR活性的IC50为2.98-9.99 μM。酶动力学研究表明这些异戊烯基取代的异黄酮可以可逆地竞争性抑制AR活性，抑制能力最强的化合物6,8-diprenylorobol其抑制常数Ki值仅为1.42 μM。实验进一步对活性化合物在提取物中的含量进行了半定量分析，其中6,8-diprenylorobol在提取物中的含量为3.53%（w/w），含量相对较高。研究还表明蔓性千斤拔提取物中尚含有大量其它类似结构的AIs有待继续开发，蔓性千斤拔是一个很好的芳香化酶抑制剂来源。