13-(6-叔丁基吡啶-3-基)-甲基小檗碱（BD-10-4）抗真菌作用机制研究

The incidence of fungal infections worldwide has risen considerably in recent years. It is urgent to develop new classes of drugs for the treatment of fungal infection. Berberine, possessing weak antifungal activity, is attractive as a hit compound for the development of antifungal agents.We have carried out structure and antifungal activity relationship and molecular structure optimization study on berberine. Our results indicated that C-13 position was the key position for antifungal activity guided structure optimization. We first found that introduction of (substituted pyridin-3-yl)-methyl at C-13 position of berberine remarkably improved the antifungal activities. Among the 13-(substituted pyridin-3- yl)-methyl berberine derivatives, BD-10-4[13-(6-tert-butyl pyridin-3-yl)-methyl berberine] exhibited the most potent antifungal activities. Our previous mechanism study on antifungal activity of BD-10-4 indicated that it acted on the new antifungal target unknown to us. Based on the previous study, in this item we will continue to study the antifungal mechanism of BD-10-4 using Candida albicans, the most common pathogenic fungi,as model organism. Screening the interaction protein in Candida albicans with BD-10-4 by affinity chromatography and cellular colocalization technology is the first and key step in this item. Finally,we will combine the reults in this item with our previous results to find the antifungal target of BD-10-4.In addition, we will construct gene knockout mutation in Candida albicans to validate the target of BD-10-4. We aim to not only clarify the antifungal mechanism of BD-10-4 and find its target, but also provide new potential antifungal target for the new drug development.

目前真菌感染发生率逐年上升，已成为临床上棘手难题。本课题组前期以具有一定抗真菌活性的小檗碱为苗头化合物开展了系统性的构效关系及结构改造研究，发现C-13位取代的衍生物BD-10-4[13-(6-叔丁基吡啶-3-基)-甲基小檗碱]具有显著的抗真菌活性。BD-10-4是本课题组合成的新型抗真菌活性化合物，对临床分离耐药菌株亦具有较强活性，但作用机制尚未明确，前期研究表明BD-10-4作用于目前未知的新靶点。本项目拟以临床最常见病原真菌白念珠菌为研究对象，以利用连续、竞争亲和层析技术和细胞内共定位技术研究BD-10-4与真菌细胞内特异性相互作用蛋白为核心，并结合细胞水平作用机制研究和前期工作基础系统性的研究BD-10-4作用机制，以期明确其作用靶点，并通过基因敲除技术构建基因缺失菌对作用靶点进行验证，不仅为BD-10-4进一步研究开发提供理论依据，也为发现新型抗真菌药物作用靶点奠定工作基础。

目前真菌感染发生率逐年上升，已成为临床上棘手难题。本课题组前期以具有一定抗真菌活性的小檗碱为苗头化合物开展了系统性的构效关系及结构改造研究，发现C-13位取代的衍生物BD-10-4[13-(6-叔丁基吡啶-3-基)-甲基小檗碱]具有显著的抗真菌活性。BD-10-4是本课题组合成的新型抗真菌活性化合物，对临床分离耐药菌株亦具有较强活性，小檗碱衍生物BD-10-4通过靶向破坏细胞壁甘露糖蛋白成分发挥抗真菌作用，BD-10-4作用于白念珠菌后细胞壁松散不完整、甘露糖蛋白层缺失，不能覆盖内层的葡聚糖，细胞最内层的几丁质由于细胞壁受损而代偿性增生。BD-10-4作用于白念珠菌后能够显著抑制其对宿主口腔粘膜上皮细胞和肠粘膜上皮细胞的粘附和侵袭作用。我们采用蛋白质组学研究方法考察BD-10-4作用于白念珠菌后细胞壁蛋白的表达变化，并采用Label free方法进行定量，结果表明：很多文献报道的白念珠菌毒力因子（如：Rbt5p, Utr2p, Pga29p, Als1p, Sod5p, Ecm33p, Pib5p等）在BD-10-4处理后在细胞壁上的定位均显著降低。这一发现阐明了BD-10-4抑制白念珠菌粘附、侵袭宿主细胞的作用机制。BD-10-4作用白念珠菌细胞后，宿主天然免疫细胞能够增强对其免疫识别，免疫效应包括NF-κB和MAPK信号通路的激活、炎症细胞因子释放增加等。我们进一步通过生物素标记的BD-10-4，利用生物素-亲和素系统研究了BD-10-4与白念珠菌细胞的相互作用蛋白，综合进行生物信息学分析表明，小檗碱衍生物BD-10-4通过抑制GPI锚合成通路产生对细胞壁的破坏作用，从而发挥抗真菌活性作用。本项目研究不仅为小檗碱衍生物抗真菌药理作用进一步深入研究提供理论依据，也为发现新型抗真菌药物作用靶点奠定工作基础。