海洋生物来源天然小分子生物碱抗HIV-1机制研究

Discovery of natural compounds with anti-HIV activities from marine biological sources has become a new hot area of anti-HIV drug research. From an established natural compound library from Beibu gulf marine biological sources, we screened the library and found that three types of small molecule alkaloids (zoanthoxanthin, quinoldione, cyclopeptide alkaloids, respectively) display strong anti-HIV-1 activities. Among them, zoanthoxanthin alkaloids are a new type of alkaloids with novel structure, and the other two have known structure. However, their anti-HIV-1 mechanism(s) are not yet clear. This project will reveal the anti-HIV-1 molecular mechanism(s) of these three types of alkaloids on the base of our previous results. Firstly, we will use a variety of HIV-1-cell infection models to clarify their cytotoxicity and anti-HIV-1 activities. And then TOA (time of addition) experiments will be employed to identify the certain process of HIV-1 replication aimed by three types of alkaloids to play their anti-HIV-1 roles, respectively. Furthermore, we will determine whether they inhibit HIV-1 through inhibiting the fusion or entry of HIV-1 into host cells, or suppressing the activities of HIV-1 critical enzymes. Finally, at the level of host genes, RT-PCR array will be used to screen the host factors or cytokines related to the host immune response, which are regulated by alkaloid treatment. The regulated host genes will be verified at transcriptional and protein levels. On this basis, overexpression/gene silence or pathway activation/inhibition will be used to further elucidate the anti-HIV-1 molecular mechanism(s) of the three types of alkaloids. The research is expected to clarify the similarities and differences of anti-HIV activities and mechanism(s) of these three types of alkaloids. The results will lay the foundation for the further development of new HIV-1 inhibitors with novel structure and new mechanism(s) from marine natural small molecule alkaloids.

从海洋生物中寻找抗人类免疫缺陷病毒（HIV）的天然活性成分是目前抗HIV药物研究的新热点之一。我们前期从已建立的北部湾海洋天然化合物库中筛选获得了三类生物碱：zoanthoxanthin类（新型结构）、quinoldione类及环肽类生物碱，均具有较强的抗HIV-1活性，但具体机制未明。本项目将在此基础上揭示三类生物碱的抗HIV-1机制。首先用不同的细胞感染模型确定其细胞毒性及抗HIV-1活性；然后通过TOA实验判断生物碱抑制HIV复制的作用环节，并进一步确定其抗HIV功能是否通过抑制病毒融合、进入或病毒关键酶活性等实现；最后在宿主基因水平，通过RT-PCR阵列筛选受生物碱调节的HIV感染及宿主应答相关的宿主因子，在转录和蛋白水平进行验证，用过表达/基因沉默或通路激活/抑制等探索生物碱抗HIV-1作用靶点，为进一步从海洋生物来源天然小分子中开发具有新结构和新机制的HIV-1抑制剂奠定基础。

从海洋生物及传统中药资源中挖掘抗HIV天然活性成分，是开发副作用小和药物依赖性低的新型抗HIV药物的有效途径。本项目利用前期建立的天然化合物库，采用三种细胞病毒感染模型明确海洋天然小分子化合物及中草药制剂/提取物的抗HIV-1活性，利用TOA实验、病毒关键酶活性实验、PCR阵列、分子对接等确定化合物抗HIV-1的作用靶点，并结合药物化学技术，制备小分子化合物系列衍生物，获得显效活性先导化合物。此外，针对中草药物药效成分复杂的特点，利用网络药理学研究主要药效成分及主要作用靶点，挖掘其潜在的抗病毒机制。本项目利用细胞病毒感染模型筛选了一批具有较强抗HIV-1活性的海洋天然小分子化合物，获得了zoanthoxanthin类、quinoldione类及环肽类三类小分子生物碱、聚醚类化合物K-41A及其双糖基衍生物K-41B、K-41Bm等具有显效抗HIV-1活性的先导化合物。三类生物碱在不同细胞病毒感染模型中的选择指数（SIs）均高于5，对HIV-1 IN和RT均具有抑制作用。聚醚类化合物K-41A的SI最高可达64.6，从9个衍生物中发现K-41Am、K-41B和K-41Bm具有更高的SI指数，该组聚醚类化合物对HIV-1的抑制作用涉及病毒多个靶点，对病毒进入、HIV-1 IN、RT等都具有抑制作用，同时双糖基衍生物K-41B和K-41Bm具有与前体物K-41A不同的抗病毒靶点。此外，本项目还发现了归芪补血口服液、双金花茶、余甘子提取物、芒果叶提取物、枸杞多糖、扶芳藤多糖等一批具有较强抗HIV作用的区域特色中药资源，并基于网络药理学研究发现上述传统中草药物主要通过调节免疫及炎症相关通路发挥免疫调节及炎症抑制作用从而抑制病毒复制。本项目促进了区域特色生物资源的研究与开发，为研制具有自主知识产权的原创抗HIV-1 新药提供了模式结构和先导化合物，对提高广西艾滋病领域应用基础研究水平具有重大意义。