Wang Li, Bao Yi, Yang Yuan, Wu Yexiang, Chen Xiaofang, Si Shuyi, Hong Bin
Key Laboratory of Biotechnology of Antibiotics of Ministry of Health, Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, China.
J Biomol Screen. 2010 Mar;15(3):239-50. doi: 10.1177/1087057109359686. Epub 2010 Feb 11.
CD36, a member of the class B scavenger receptor, is a high-affinity receptor for oxidatively modified low-density lipoprotein (oxLDL). Extensive evidence points to a significant role of CD36 in atherosclerosis and suggests that CD36 could be a potential target for treatment of atherosclerosis. Here, the extracellular domain of human CD36 (Gly(30)-Asn(439)) was expressed in Escherichia coli as His(6)-tagged soluble CD36 (sCD36), which could bind oxLDL specifically and effectively inhibit the uptake of oxLDL by murine macrophage RAW 264.7 cells. An enzyme-linked immunosorbent assay (ELISA)-like high-throughput screening (HTS) assay was developed for the discovery of CD36 antagonists, based on the competition of sCD36 binding to immobilized oxLDL and detection with a monoclonal antibody against His-tag. This assay was suitable for HTS in a 96-well format and was robust and reliable according to the evaluation parameter Z' value of 0.82. The developed HTS assay was applied to both pure chemical compounds and microbial secondary metabolite crude extracts to identify CD36 antagonists. Three active compounds-sodium danshensu (DSS), rosmarinic acid (RA), and salvianolic acid B (SAB)-were shown to be antagonistic to sCD36-oxLDL binding and further validated by their inhibition of oxLDL uptake in RAW 264.7 cells. These results suggest that the ELISA-like assay represents a promising screening for identifying bioactive molecules targeting atherosclerosis at the level of CD36-ligand binding.
CD36是B类清道夫受体家族的一员，是氧化修饰低密度脂蛋白（oxLDL）的高亲和力受体。大量证据表明CD36在动脉粥样硬化中起重要作用，并提示CD36可能是治疗动脉粥样硬化的潜在靶点。在此，人CD36的胞外域（Gly(30)-Asn(439)）在大肠杆菌中表达为His(6)标签的可溶性CD36（sCD36），其可特异性结合oxLDL并有效抑制鼠巨噬细胞RAW 264.7细胞对oxLDL的摄取。基于sCD36与固定化oxLDL结合的竞争性以及用抗His标签单克隆抗体进行检测，开发了一种类似酶联免疫吸附测定（ELISA）的高通量筛选（HTS）测定法以发现CD36拮抗剂。该测定法适用于96孔板形式的HTS，根据评估参数Z'值为0.82，其稳健且可靠。所开发的HTS测定法应用于纯化学化合物和微生物次级代谢产物粗提物以鉴定CD36拮抗剂。三种活性化合物——丹参素钠（DSS）、迷迭香酸（RA）和丹酚酸B（SAB）——被证明对sCD36-oxLDL结合具有拮抗作用，并通过它们对RAW 264.7细胞中oxLDL摄取的抑制作用进一步得到验证。这些结果表明，类似ELISA的测定法在识别靶向CD36-配体结合水平的动脉粥样硬化生物活性分子方面具有前景广阔的筛选作用。
