河朔荛花中激活潜伏HIV二萜的发现、构效关系及其作用机制研究

HIV latency is the major obstacle to the cure of ADIS. The discovery of the highly efficient latency reactivation agent and new activation mechanism is one of the important strategies to solve the problem. We previously found five diterpenoids with HIV latency reactivation activities in which they showed significant difference between each other from the buds of Wikstroemia chamaedaphne. One diterpenoid exhibited excellent reactivation activity which was 400 times as much as the positive control Prostratin, and also effective in tests with ADIS patients' blood peripheral mononuclear cells. However, the structure-activity relationship and mechanisms of these molecules are not yet clear. On the basis of the highly effective latent reactivation compounds found, this project is intended to: 1) extensively carry on isolation and enrichment of these highly efficient latency reactivation agents from Wikstroemia chamaedaphne, combined with latent HIV reactivation tests, deeply analyze the structure-activity relationships, and obtain lead compounds. 2) with the membrane and nuclear separation technology, chemical genetics, western blot and other methods together, discover whether the reactivation function of these agents is dependent on the activation of PKCs-NF-κB signaling pathway. 3) explore the targets of these agents at the level of proteome with chemical biological methods. This project will provide scientific basis for the discovery of highly efficient and safe HIV latent reactivation agents with clear mechanisms as candidate drugs.

HIV潜伏是艾滋病治愈的主要障碍，高效潜伏激活剂和新激活机制的发现是解决该障碍的一个关键。申请人前期从河朔荛花中发现5个二萜具有激活潜伏HIV的作用，并且它们的活性显著不同。其中一个二萜的体外激活效果不仅是阳性药物Prostratin的400倍，还对艾滋病人的外周血单核细胞显示出病毒激活作用。文献表明这类分子发挥激活作用的构效关系及作用机制尚不清楚。本项目拟在已发现的高效潜伏激活剂基础上继续开展：1）对河朔荛花中这类高效激活剂进行大量的分离、富集，结合潜伏HIV激活活性测试，深入分析构效关系，获得先导化合物；2）通过膜质、核质分离技术、化学遗传学以及免疫印迹等技术相结合，确证这类激活剂的作用是否依赖于激活PKCs-NF-κB信号通路所实现；3）运用化学生物学研究手段在蛋白组层面探索这类分子的作用靶点。本项目的开展可为发现高效、安全、作用机制清晰的HIV潜伏激活候选药物提供科学依据。

HIV潜伏是艾滋病治愈的主要障碍，高效潜伏激活剂和新激活机制的发现是解决该障碍的一个关键。天然产物一直是高效潜伏激活剂发现的重要源泉。本项目以山西特色植物河朔荛花为研究对象，对其具有激活潜伏HIV的作用成分特别是瑞香烷型二萜和惕各烷型二萜及作用机制进行了详细研究，试图发现高效新颖的HIV潜伏激活剂和评价构效关系。. 研究结果表明我们从河朔荛花中获得了78个成分，其中新化合物14个，二萜26个。通过4种细胞模型（J-Lat A2, 2D10和HeLa-NH2, NH1）完成二萜化合物激活潜伏HIV的评价，发现8个具有高效激活潜伏HIV作用的二萜，特别是Wikstroelide E的激活效果，是阳性对照prostratin的500倍。构效关系研究表明：大环结构、A环的完整性和20位羟基是瑞香完型二萜发挥激活潜伏HIV作用的重要活性基团。1）瑞香完型二萜中大环是必须基团；2）A环五元C环结构是必须的；3）20位羟基若被大基团取代活性会显著降低。. 通过转录组学、蛋白质组学、核质分离技术和细胞生物学等技术开展了Wikstroelide E激活潜伏HIV的机制研究。结果显示Wikstroelide E的激活潜伏HIV是通过激活NF-κB信号通路实现，且依赖于PKC的激活。通过DARTS联合蛋白组学技术对Wikstroelide E的作用靶点进行了研究，发现Wikstroelide E能够结合的靶蛋白约45个。. 此外，我们首次发现了Wikstroelide E等化合物具有激活STAT1的作用，这是一类新颖的STAT1激活剂，具有重要应用前景。同时，本项目还完成了河朔荛花花蕾中总二萜和总黄酮提取物的制备工艺研究。二萜提取物显示出了高效激活潜伏HIV作用，为未来二萜提取物治疗抗艾滋病药物研发提供重要基础。整个项目发表论文5篇，包括2篇SCI论文，授权专利2项。