基于极化荧光方法的人LOX-1配体高通量筛选  

Identification of ligands for human LOX-1 through fluorescence polarization-based high throughput screening

作　　者：张天泰[1] 黄镇太[1] 戴瑛[1] 刘艾林[1] 朱平[1] 杜冠华[1] 

机构地区：[1]中国医学科学院,中国协和医科大学药物研究所,北京100050

出　　处：《药学学报》2005年第9期792-795,共4页Acta Pharmaceutica Sinica

基　　金：国家高技术研究发展计划(863计划)资助项目(2002AA27343B);国家自然科学基金资助项目(30271504);博士后科学基金资助项目.

摘　　要：目的建立以极化荧光为检测方法的高通量筛选技术,通过大规模筛选发现氧化型低密度脂蛋白受体1(LOX1)的天然配体。方法密度梯度超速离心获得正常人血中低密度脂蛋白(LDL),然后用CuSO4(5μmol·L-1)修饰为氧化型低密度脂蛋白(oxLDL)。FITC标记hLOX1,以受体(LOX1)和配体(oxLDL)的相互作用为基础建立筛选模型,用极化荧光检测方法,在激发光485nm、发射光525nm,对3200个样品进行高通量筛选,并用Z′因子值评价实验。结果Z′因子值为0.75,根据建立的基于极化荧光的高通量筛选实验方法,发现3个化合物与hLOX1有较高的结合活性,IC50值小于31.6μmol·L-1。结论极化荧光检测方法适合于高通量筛选技术,具有较高的稳定性、灵敏性和可重复性。Aim To develop a fluorescence polarization-based high throughput screening and identify ligands for human Lectin-like oxidized low-density lipoprotein receptor-1 （hLOX-1）. Methods Sequential ultracentrifugation at 4 ℃ from normolipidemic fasting volunteers to obtain low density lipoprotein （LDL）, which was modified by CUSO4（5 μmol·L^-1） at 37℃ for 24 h. The assay was based on the interaction between receptor and ligand, and hLOX-1 was labeled by FITC and bound to its specific ligand, oxLDL. Different reaction time and DMSO concentration were optimized to determine the stability and tolerance of fluorescence polarization （FP） assay. 3 200 compounds were screened in black 384-well mieroplate by FP-based competitive displacement assay, at excitation filter of 485 nm and emission filter of 530 nm. Z′was used to assess the assay quality. Results The FP-based HTS was formatted in a 384-well mieroplate with a Z′ factor of 0. 75, and three active compounds for hLOX-1 were identified with IC50 below 40 μmol · L^-1 from total 3 200 compounds. Conclusion The results indicated that the fluorescence polarization assay is stable, sensitive, reproducible and well suited for high throughput screening efforts.

关 键 词：极化荧光 高通量筛选 氧化型低密度脂蛋白受体-1 动脉粥样硬化 

分 类 号：R965.1[医药卫生—药理学]