[Identification of ligands for human LOX-1 through fluorescence polarization-based high throughput screening].
OBJECTIVE
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.


CONCLUSIONS
The results indicated that the fluorescence polarization assay is stable, sensitive, reproducible and well suited for high throughput screening efforts.
Zhang
Tian-Tai
TT
Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, China.
Huang
Zhen-Tai
ZT
Dai
Ying
Y
Liu
Ai-Lin
AL
Zhu
Ping
P
Du
Guan-Hua
GH
D001667:Binding, Competitive; D004353:Drug Evaluation, Preclinical / Q000379:methods; D005454:Fluorescence Polarization / Q000379:methods*; D006801:Humans; D008024:Ligands; D008077:Lipoproteins, LDL / Q000378:metabolism*; D051127:Scavenger Receptors, Class E / Q000378:metabolism*
D008024:Ligands; D008077:Lipoproteins, LDL; C495137:OLR1 protein, human; D051127:Scavenger Receptors, Class E; C099252:oxidized low density lipoprotein