Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, China.
Anal Biochem. 2010 May 15;400(2):207-12. doi: 10.1016/j.ab.2010.02.003. Epub 2010 Feb 10.
CD36, a class B scavenger receptor, is an integral membrane protein that mediates the endocytosis of modified lipoproteins. The functions of CD36 are complex and have been associated with atherosclerosis. In the current study, we developed a high-throughput screening (HTS) assay to identify small molecule antagonists by expressing human CD36 using a Bac-to-Bac baculovirus expression system in Spodoptera frugiperda (Sf9) cells. Uptake of 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate-labeled acetylated low-density lipoprotein (DiI-AcLDL) revealed that the IC(50) values for the CD36 ligands oxidatively modified LDL (Ox-LDL), Ac-LDL, and high-density lipoprotein (HDL) were 0.039, 0.019, and 0.010 microg/ml, respectively. Using the HTS assay, two novel compounds, 2016481B and 2038751B, were found to inhibit DiI-AcLDL uptake in insect cells and exhibited IC(50) values of 17.4 and 23.7 microM, respectively. These two novel compounds also inhibited DiI-AcLDL uptake in cultured Chinese hamster ovary (CHO) cells permanently expressing human CD36. Furthermore, these two compounds inhibited lipid accumulation in RAW 264.7 murine macrophage cells in foam cell assays. This HTS assay represents a potential method for identifying more effective macrophage scavenger receptor antagonists, which may serve as starting points for the development of novel anti-atherosclerotic agents.
CD36，一种 B 型清道夫受体，是一种完整的膜蛋白，介导修饰脂蛋白的内吞作用。CD36 的功能复杂，与动脉粥样硬化有关。在本研究中，我们开发了一种高通量筛选（HTS）测定法，通过在 Spodoptera frugiperda（Sf9）细胞中使用 Bac-to-Bac 杆状病毒表达系统表达人 CD36 来鉴定小分子拮抗剂。摄取 1,1'-二辛基-3,3,3',3'-四甲基吲哚羰花青高氯酸盐标记的乙酰化低密度脂蛋白（DiI-AcLDL）表明 CD36 配体氧化修饰的低密度脂蛋白（Ox-LDL）、Ac-LDL 和高密度脂蛋白（HDL）的 IC50 值分别为 0.039、0.019 和 0.010 μg/ml。使用 HTS 测定法，发现两种新型化合物 2016481B 和 2038751B 可抑制昆虫细胞中 DiI-AcLDL 的摄取，IC50 值分别为 17.4 和 23.7 μM。这两种新型化合物也抑制了永久表达人 CD36 的中国仓鼠卵巢（CHO）细胞中 DiI-AcLDL 的摄取。此外，这两种化合物还抑制了泡沫细胞测定中 RAW 264.7 鼠巨噬细胞中脂质的积累。这种 HTS 测定法代表了一种潜在的方法，可以鉴定更有效的巨噬细胞清道夫受体拮抗剂，这可能成为开发新型抗动脉粥样硬化药物的起点。
