Identification of ligands for human LOX-1 through fluorescence polarization-based high throughput screening / 药学学报

ABSTRACT

<p><b>AIM</b>To develop a fluorescence polarization-based high throughput screening and identify ligands for human Lectin-like oxidized low-density lipoprotein receptor-1 (hLOX-1).</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSION</b>The results indicated that the fluorescence polarization assay is stable, sensitive, reproducible and well suited for high throughput screening efforts.</p>