Abstract
Aim:
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 μmol/L) in order to obtain optimal reactive concentrations. The concentration of Me2SO used (0%, 1%, 3%, 5%) and incubation time (15 min, 30 min, 1h, 2h) 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 μmol/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 μmol/L were found.
Conclusion:
The established competitive FP-based assay is sensitive, stable, highly reproducible and robust, and suitable for HTS for ligands of the hLOX-1 receptor.
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References
Hertzberg RP, Pope AJ . High-throughput screening: new technology for the 21st century. Curr Opin Chem Biol 2000; 4: 445–51.
Silverman L, Campbell R, Broach JR . New drug assay technologies for high throughput screening. Curr Opin Chem Biol 1998; 2: 397–403.
Banks P, Harvey M . Consideration for using fluorescence polarization in the screening of G protein-coupled receptor. J Biomol Screen 2002; 7: 111–7.
Rodiger M, Haupts U, Moore KJ . Single-molecule detection technologies in miniaturized high throughput screening: binding as says for G protein-coupled receptors using fluorescence intensity distribution analysis and fluorescence anisotropy. J Biomol Screen 2001; 6: 29–37.
Banks P, Gosselin M, Prystay L . Impact of a red-shifted dye label for high throughput fluorescence polarization assays of G protein-coupled receptors. J Biomol Screen 2000; 5: 329–34.
Sawamura T, Kume N, Aoyama T, Moriwakl H, Hoshikawa H, Alba Y, et al. An endothelial receptor for oxidized low-density lipoprotein. Nature 1997; 386: 73–7.
Masaki T . Endothelial dysfunction and LOX-1: forty years from muscle to endothelium. Circ Res 2003; 92: 819–20.
Cominacini L, Garbin U, Davoli A, Micciolo R, Bosello O, Gaviraghi G, et al. A simple test for predisposition to LDL oxidation based on the fluorescence development during copper-catalyzed oxidative modification. J Lipid Res 1991; 32: 349–58.
Bradford MM . Rapid and sensitive method for quantitation of microgram quantities of protein utilizing principle of protein-dye binding. Anal Biochem 1976; 72: 248–54.
Zouhair FS, Yurachek EC . Rapid fluorometric assay of LDL receptor activity by Dil-labeled LDL. J Lipid Res 1993; 34: 325–30.
Takefumi N, Yasuda T, Hoshikawa H, Shimizu M, Kakinuma T, Chen MY, et al. LOX1-1 expressed in culture rat chondrocytes mediates oxidized LDL-induced cell death: possible role of de-phosphorylation of Akt. Biochem Biophys Res Commun 2002; 299: 91–7.
Huang ZT, Zhang TT, Yang JL, Zhu P, Du GH, Chen KD . Cloning and expression of human lectin-like oxidized low density lipoprotein receptor-1 in Pichia pastoris. Biotech Lett 2005; 27: 49–52.
Li DY, Liu L, Chen HJ, Sawamura T, Ranganathan S, Mehta JL . LOX-1 mediates oxidized low-density lipoprotein-induced expression of matrix metalloproteinases in human coronary artery endothelial cells. Circulation 2003; 107: 612–7.
Jameson DM, Seifried SE . Quantification of protein-protein interaction using fluorescence polarization. Methods 1999; 19: 222–33.
Zhang JH, Chung T, Oldenburg K . A simple statistical parameter for use in evaluation of high throughput screening assays. J Biomol Screen 1999; 4: 67–73.
Thum T, Borlak J . Mechanistic role of cytochrome P450 monooxygenases in oxidized low-density lipoprotein-induced vascular injury: therapy through LOX-1 receptor antagonism? Circ Res 2004; 94: e1–e13.
Jameson DM, Croney JC . Fluorescence polarization: past, present and future. Comb Chem High Throughput Screen 2003; 6: 167–76.
Kakehi K, Oda Y, Kinoshita M . Fluorescence polarization: analysis of carbohydrate-protein interactions. Anal Biochem 2001; 297: 111–6.
Banks P, Gosselin M, Prystay L . Fluorescence polarization as says for high throughput screening of G protein-coupled receptors. J Biomol Screen 2000; 5: 159–67.
Heyduk T, Ma Y, Tang H, Ebright RH . Fluorescence anisotropy: rapid, quantitative assay for protein-DNA and protein-protein interaction. Methods Enzymol 1996; 274: 492–503.
Fisher BM, Ha JH, Raines RT . Coulombic forces in protein-RNA interactions: binding and cleavage by ribonuclease A and variants at Lys7, Arg10 and Lys66. Biochemistry 1998; 37: 12121–32.
Qian J, Voorbach MJ, Huth JR, Coen ML, Zhang H, Ng SC, et al. Discovery of novel inhibitors of Bcl-xL using multiple high-through put screening platforms. Anal Biochem 2004; 328: 131–8.
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Project supported by the High Technology Research and Development Program of China (No 2002AA27343B), the National Natural Science Foundation of China (No 30271504) and the China Postdoctoral Science Foundation.
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Zhang, Tt., Huang, Zt., Dai, Y. et al. High-throughput fluorescence polarization method for identifying ligands of LOX-1. Acta Pharmacol Sin 27, 447–452 (2006). https://doi.org/10.1111/j.1745-7254.2006.00281.x
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DOI: https://doi.org/10.1111/j.1745-7254.2006.00281.x
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