Discovery of Antagonists for Human Scavenger Receptor CD36 via an ELISA-Like High-Throughput Screening Assay
Discovery of Antagonists for Human Scavenger Receptor CD36 via an ELISA-Like High-Throughput Screening Assay
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DOI：
10.1177/1087057109359686
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发表时间：
2010-03-01
影响因子：
--
通讯作者：
Hong, Bin
中科院分区：
文献类型：
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作者：
Wang, Li;Bao, Yi;Hong, Bin
CD36是B类清道夫受体的一员，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在动脉粥样硬化中的重要作用，并提示CD36可能是动脉粥样硬化治疗的潜在靶点。在这里，人CD36的胞外结构域(Gly（30)-Asn(439)）在大肠杆菌中表达为His(6)标记的可溶性CD36 (sCD36)，它可以特异性结合oxLDL并有效抑制小鼠巨噬细胞RAW 264.7细胞对oxLDL的摄取。基于sCD36与固定化oxLDL结合的竞争和针对His-tag的单克隆抗体检测，开发了一种类似于酶联免疫吸附试验（elisa）的高通量筛选（HTS）方法，用于发现CD36拮抗剂。该方法适用于96孔格式的HTS，评价参数Z′值为0.82，稳健可靠。开发的HTS法应用于纯化合物和微生物次生代谢物粗提取物，以鉴定CD36拮抗剂。丹参素钠（DSS）、迷迭香酸（RA）和丹酚酸B （SAB）三种活性化合物被证明对sCD36-oxLDL结合具有拮抗作用，并通过抑制RAW 264.7细胞对oxLDL的摄取进一步验证。这些结果表明，类似elisa的检测方法在cd36配体结合水平上代表了一种有希望的筛选靶向动脉粥样硬化的生物活性分子的方法。（Journal of biomolmolecular Screening 2010: 239-250）
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. (Journal of Biomolecular Screening 2010: 239-250)