Identification of ligands for human LOX-1 through fluorescence polarization-based high throughput screening.
- Author:
Tian-Tai ZHANG
1
;
Zhen-Tai HUANG
;
Ying DAI
;
Ai-Lin LIU
;
Ping ZHU
;
Guan-Hua DU
Author Information
- Publication Type:Journal Article
- MeSH: Binding, Competitive; Drug Evaluation, Preclinical; methods; Fluorescence Polarization; methods; Humans; Ligands; Lipoproteins, LDL; metabolism; Scavenger Receptors, Class E; metabolism
- From: Acta Pharmaceutica Sinica 2005;40(9):792-795
- CountryChina
- Language:Chinese
-
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).
METHODSSequential 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.
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 micromol x 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.