Abstract: AimTo 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 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. ResultsThe 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 μmol·L^-1 from total 3 200 compounds. ConclusionThe results indicated that the fluorescence polarization assay is stable, sensitive, reproducible and well suited for high throughput screening efforts.