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.

Indole‐2‐carboxamide Derivatives as Potent Urotensin‐II Receptor (UT) Antagonists

“…The binding affinities of the substituted indole‐2‐carboxamide derivatives 2–8 to the human UT receptor expressed in membranes of HEK293 cells were determined using a competitive binding assay with Eu‐labeled U‐II and a time‐resolved fluorometric (TRF) assay . The initial SAR study focused on exploration of the effects of several substituents of the indole‐2‐carboxamide at 1‐position.…”

1,3,4‐Thiadiazol‐2‐amine Derivatives as Urotensin‐II Receptor (UT) Antagonists

“…The binding affinities of the 1,3,4‐thiadiazol‐2‐amine derivatives 2 to membranes of HEK293 cells expressing the human UT receptor were determined by conducting a competitive binding assay with Eu‐labeled U‐II and a time‐resolved fluorometric (TRF) assay . The initial SAR study focused on an exploration of substances containing a variety of substituents on the aryloxymethyl group at C‐5 of the 1,3,4‐thiadiazol‐2‐amine moiety.…”