藤黄科三种植物中靶向抑制CYP1B1酶天然香豆素化合物的发现及其作用机制研究

Cytochrome P450 1B1 (CYP1B1) is a heme-thiolate monooxygenase involved in epoxidating and hydroxylating of estradiol, procacinogens and some clinical first-line anticancer agents. So it participates in the occurrence and development of tumor and causes tumor cells to develop drug resistance. And it shows high expression in many kinds of tumor cells. So CYP1B1 enzyme has gained considerable attention. Most of the existing CYP1B1 enzyme inhibitors have poor selectivity to CYP1B1 enzyme, and can further lead to the high expression of CYP1B1 enzyme by activating the aryl hydrocarbon receptor (AhR). In our previous study, a series of coumarin compounds were found to showed inhibitory activity against CYP1B1 enzyme. The inhibitory activities were better than the positive control resveratrol, among them some compounds showed remarkable inhibitory activity. In this project, with an aim to further systematically acquire biological coumarins, especially 4-phenylcoumarin, continuing researches on the three Guttiferae medicinal plants will be studied. Combined our previous studies, a small molecular natural compound library will be built, which comprises 4-phenylcoumarin, simple coumarin, furocoumarin and pyranocoumarin. CYP1B1/CYP1A1/CYP1A2 enzyme inhibition assay, yeast AhR-signaling assay and NADPH-dependency assay in vitro of isolated coumarins will be studied. It is hopeful to find novel targeted tumor preventive, therapeutic and reversible drug resistant active compounds based on CYP1B1 enzyme inhibition without activating AhR. And to promote the further utilization and protection of the Guttiferae resources in China.

CYP1B1酶作为一种亚铁血红素-硫醇盐单加氧酶，可催化雌二醇、前致癌物质和部分临床一线抗癌药物的环氧化及羟基化反应，参与肿瘤的发生发展及使肿瘤细胞产生耐药性，并在多种肿瘤细胞有高表达，受到广泛关注。现有的CYP1B1酶抑制剂，大多对CYP1B1酶选择性差，且可通过激活芳香烃受体（AhR），进一步导致CYP1B1酶的高表达。在前期研究中，发现一系列对CYP1B1酶抑制率高于阳性对照白藜芦醇的香豆素化合物，其中部分化合物具有显著抑酶活性。本项目拟结合前期研究结果，对三种藤黄科植物进行系统的化学成分研究，分离植物中独特的4-苯代香豆素类成分，组成小分子天然香豆素化合物库，并对其进行体外CYP1B1/CYP1A1/CYP1A2酶抑制试验、酵母AhR-信号试验和NADPH依赖性试验，有望发现靶向抑制CYP1B1酶而不激活AhR的新型肿瘤预防、治疗和可逆转肿瘤耐药活性化合物。

细胞色素P450 1B1(CYP1B1)是一种催化底物环氧化及羟基化的亚铁血红素-硫醇盐单加氧酶，研究表明，CYP1B1酶正成为肿瘤预防、治疗和克服耐药研究中的一个新靶标，CYP1B1酶抑制剂的筛选将成为新型肿瘤药物发现的一个新途径。. 基于此，本项目采用硅胶柱色谱、Sephadex LH-20柱色谱、开放ODS柱色谱、制备薄层色谱、半制备高效液相色谱等分离手段对三种藤黄科植物滇南红厚壳、薄叶红厚壳和铁力木进行系统的化学成分研究，并利用1H-NMR、13C-NMR 2D-NMR、HR ESI-MS、UV、IR、CD等波谱技术和计算机计算辅助分析手段对分离得到化合物进行结构鉴定。从三种植物中获得99个化合物，新化合物为17个，99个化合物结构类型为香豆素类、苯并吡酮类、黄酮及黄酮苷类、萜类、甾醇类，其中30个为香豆素类化合物，35个苯并吡酮类化合物，16个黄酮及黄酮苷类化合物。香豆素类化合物的结构特点是C-4位均有取代，其中24个化合物C-4位为苯基取代。体外CYP1B1/CYP1A1/CYP1A2酶活性抑制试验的研究结果显示，8个香豆素类化合物和5个苯并吡酮类化合物对CYP1B1酶具有抑制活性，其中5个香豆素类化合物对CYP1B1酶具有靶向抑制作用；7个4-苯代香豆素类化合物和5个苯并吡酮类化合物对CYP1A1酶具有抑制活性，3个苯并吡酮类化合物对CYP1A2酶具有抑制活性，没有发现对CYP1A2酶具有抑制活性的香豆素化合物。7个具有CYP1B1酶抑制活性的香豆素化合物的IC50值测定实验、酵母AhR-信号试验和NADPH依赖性试验的结果显示，7个香豆素化合物抑制CYP1B1酶活性都需要NADPH，但是活性化合物都能显著激活AhR受体。. 该项目为获得基于CYP1B1 酶抑制的靶向肿瘤预防、治疗和可逆转肿瘤耐药活性化合物，及推动藤黄科植物资源的深层次利用和保护奠定了基础。