基于极化荧光方法的人LOX-1配体高通量筛选

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(LOX-1)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(5 μmol·L^-1)修饰为氧化型低密度脂蛋白(oxLDL)。FITC标记hLOX-1，以受体(LOX-1)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485 nm、发射光525 nm,对3 200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hLOX-1有较高的结合活性，IC₅₀值小于31.6 μmol·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。

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

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 degrees C from normolipidemic fasting volunteers to obtain low density lipoprotein (LDL), which was modified by CuSO4 (5 micromol x L(-1)) at 37 degrees C 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 microplate 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 microplate with a Z' factor of 0. 75, and three active compounds for hLOX-1 were identified with IC50 below 40 micromol x 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.