High-throughput fluorescence polarization method for identifying ligands of LOX-1.
OBJECTIVE
To develop and optimize a competitive fluorescence polarization (FP) method, and use it as a high-throughput screening (HTS) assay for drug discovery.


METHODS
Human lectin-like oxidized low-density lipoprotein receptor-1 (hLOX-1) and oxidized low-density lipoprotein (oxLDL) were used to establish a high-throughput fluorescence polarization assay to screen ligands of human LOX-1. A 96-well plate assay was performed with a fast plate reader. Three fluorescein isothiocyanate-labeled hLOX-1 concentrations (100, 200, and 400 nmol/L) were selected to be titrated by oxLDL (from 0.05 nmol/L to 100 micromol/L) in order to obtain optimal reactive concentrations. The concentration of Me2SO used (0%, 1%, 3%, 5%) and incubation time (15 min, 30 min, 1 h, 2 h) were optimized. The Z' factor was calculated to estimate the quality of FP-based HTS.


RESULTS
Concentrations of 200 nmol/L for human LOX-1 and 50 micromol/L for oxLDL were used in the actual assay. Concentrations of 0% to 5% Me2SO and different reaction times did not affect the FP-based HTS. The Z' value was 0.66. By using this detection and screening system, 12 700 compounds were screened and 3 ligands with an IC50 of less than 4.5 micromol/L were found.


CONCLUSIONS
The established competitive FP-based assay is sensitive, stable, highly reproducible and robust, and suitable for HTS for ligands of the hLOX-1 receptor.
National Centre for Pharmaceutical Screening, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, China.
Tian-Tai
TT
Zhang
Zhen-Tai
ZT
Huang
Ying
Y
Dai
Xiu-Ping
XP
Chen
Ping
P
Zhu
Guan-Hua
GH
Du
D001667:Binding, Competitive; 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; D051127:Scavenger Receptors, Class E; C099252:oxidized low density lipoprotein