中药枸骨中11β-HSD1选择性抑制剂18,19-secoursane皂苷的定向发现、结构优化及构效关系研究

Abnormal elevation of local tissue glucocorticoids (GC) can lead to insulin resistance, which is central to the onset of type 2 diabetes.11β-HSD1 is an important metabolic enzyme of GC, whose high activity will cause elevated GC levels in local tissues, and thus leading to insulin resistance. Therefore, the high selective inhibitor of 11β-HSD1 is expected to be a noval strategy for the treatment of type 2 diabetes. In the previous study, the applicant found that the plant Ilex cornuta metabolites a severely rare skeleton of 18,19-secoursane saponins, which showed significant selective inhibitory activity against 11β-HSD1, with IC50 value reaching 33 nM. Based on this, the project is to take Ilex cornuta as the research object, firstly using the approach named "precise separation" guided by biological activity and LC-MS to obtain 18,19-secoursane saponins and their analogues, following by the summary of preliminary structure-activity relationship. Then, molecular docking was performed on the active molecules and 11β-HSD1 to obtain the molecular/enzyme docking patterns, on which performing on the study of structural modification and structure-activity relationship were based. Finally, an effective inhibitor with high selectivity was obtained by the activity evaluation.

局部组织糖皮质激素（GC）水平的异常升高会导致胰岛素抵抗，而胰岛素抵抗是2型糖尿病发病的中心环节。11β-HSD1是GC的主要代谢调节酶，大量实验表明其异常高活性会引起局部组织GC水平的升高，进而导致胰岛素抵抗。因此，11β-HSD1的高选择性抑制剂有望成为治疗2型糖尿病的新策略。申请人在前期工作中发现中药枸骨代谢一种极为罕见的18,19-secoursane皂苷，并首次发现该类化合物对11β-HSD1具有显著的选择性抑制活性，IC50值达到33nM。基于此，本项目拟以枸骨为研究对象，首先利用生物活性和LC-MS导向的精准分离手段从枸骨中定向获得18,19-secoursane皂苷及其类似物，总结初步构效关系；然后，对活性分子与11β-HSD1进行分子对接研究，获取分子/酶结合模式，继而以此为基础进行结构修饰和构效关系研究，通过活性评价最终获得11β-HSD1的高选择性高效抑制。

局部组织糖皮质激素（GC）水平的异常升高会导致胰岛素抵抗，而胰岛素抵抗是2型糖尿病发病的中心环节。11beta-HSD1是GC的主要代谢酶，是一种氧化还原酶，在代谢旺盛的组织中主要起到还原的作用，催化无活性的皮质酮转变为有活性的皮质醇。大量实验表明，11beta-HSD1的异常升高会引起局部组织GC水平的升高，进而导致胰岛素抵抗，从而介导2型糖尿病的发生和发展，而11beta-HSD1的抑制剂可降低GC的水平，因而11beta-HSD1可作为治疗2型糖尿病的潜在靶点。项目负责人的前期实验表明，三萜类化合物作为糖皮质激素的结构类似物，具有显著抑制11beta-HSD1的活性，因而三萜类化合物被提上日程。本项目以中药枸骨的乙醇提取物为研究对象，借助现代先进的分离提取和结构鉴定技术，从中共分离天然产物71个，其中包括三萜及其皂苷类化合物63个；建立了基于LC-MS/MS的酶活筛选方法，该方法包含11beta-HSD1的表达纯化到筛选的整套体系；活性筛选表明，三萜类化合物大都具有显著的抑制活性，而三萜皂苷类化合物表现不良；18,19-开环乌苏烷型三萜皂苷，结构新颖，为中药枸骨的主要成分，经结构修饰得到46个衍生物，经活性测试，发现苷元及其酯化产物具有显著的抑制活性。本项目发现了一系列11beta-HSD1的抑制剂，为治疗2型糖尿病的新药研发提供了新的化学实体。