title: Development of filtration-based time-resolved fluorescence assay for the high-throughput screening of urotensin II receptor antagonist.
abstract: The time-resolved fluorescence (TRF) receptor binding assay has many advantages over the traditional radioligand binding assay in terms of sensitivity and reproducibility for the screening of receptor ligands. The TRF-based urotensin receptor (UT) binding assay with an automatic vacuum filtration system was developed and evaluated for the high-throughput screening of UT receptor antagonists. For this assay development, the human recombinant urotensin II (UII) was modified by labeling europium at its N-terminal position (Eu-UII) and used as a fluorescent tracer. The microsomal membrane fraction of UT receptor was prepared from HEK293 cells stably expressing the human UT receptor. The 50% inhibitory concentration (IC(50)) values of UII from competition binding assays with Eu-UII were 2.76 nM, which is very similar to that of fluorescence polarization (FP)-based UT receptor binding experiment (2.18 nM). Comparing with the FP-based receptor binding assay for UII (Z' factor, 0.36), the current TRF assay presented improved Z' factor (0.76) with a relatively higher signal-to-background ratio (1.5 and 2.1, respectively). The known high-affinity UT receptor antagonists, palosuran and SB657510, exhibited IC(50) values of 23.6 and 73.4 nM, respectively, which were consistent with the IC(50) values from FP-based receptor binding assay (30.6 and 78.7 nM, respectively). These results suggest that our filtration-based TRF UT receptor binding assay can achieve the desired sensitivity with higher reproducibility to adapt for the high-throughput screening of compound libraries.
authors.affiliation: Bio-Organic Science Division, Korea Research Institute of Chemical Technology Yuseong, Daejeon, Republic of Korea.
authors.forename: Kwang-Seok
authors.initials: KS
authors.lastname: Oh
authors.forename: Sunghou
authors.initials: S
authors.lastname: Lee
authors.forename: Byung Ho
authors.initials: BH
authors.lastname: Lee
mesh_terms: D000818:Animals; D001667:Binding, Competitive; D001681:Biological Assay; D055808:Drug Discovery; D004353:Drug Evaluation, Preclinical; D005063:Europium / Q000737:chemistry*; D005453:Fluorescence; D005454:Fluorescence Polarization / Q000379:methods*; D057809:HEK293 Cells; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D020128:Inhibitory Concentration 50; D008024:Ligands; D051379:Mice; D058990:Molecular Targeted Therapy; D011485:Protein Binding; D011868:Radioisotopes; D011869:Radioligand Assay; D051381:Rats; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013449:Sulfonamides / Q000032:analysis / Q000138:chemical synthesis / Q000494:pharmacology*; D013997:Time Factors; D014579:Urotensins / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism
chemical_list: D008024:Ligands; D011868:Radioisotopes; C553589:SB 657510; D013449:Sulfonamides; D014579:Urotensins; D005063:Europium; C029677:urotensin II