Oh Kwang-Seok, Lee Sunghou, Lee Byung Ho
Bio-Organic Science Division, Korea Research Institute of Chemical Technology Yuseong, Daejeon, Republic of Korea.
Assay Drug Dev Technol. 2011 Oct;9(5):514-21. doi: 10.1089/adt.2010.0353. Epub 2011 May 11.
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.
在筛选受体配体时，时间分辨荧光（TRF）受体结合测定法相较于传统放射性配体结合测定法，在灵敏度和重现性方面具有诸多优势。我们开发了基于TRF的尿紧张素受体（UT）结合测定法，并配备自动真空过滤系统，用于高通量筛选UT受体拮抗剂。在该测定法的开发过程中，对人重组尿紧张素II（UII）进行了修饰，在其N端标记铕（Eu-UII），用作荧光示踪剂。从稳定表达人UT受体的HEK293细胞中制备UT受体的微粒体膜部分。在与Eu-UII的竞争结合试验中，UII的50%抑制浓度（IC(50)）值为2.76 nM，这与基于荧光偏振（FP）的UT受体结合实验（2.18 nM）非常相似。与基于FP的UII受体结合测定法（Z'因子为0.36）相比，当前的TRF测定法显示出更高的Z'因子（0.76），且信号与背景比值相对更高（分别为1.5和2.1）。已知的高亲和力UT受体拮抗剂，帕洛苏兰和SB657510，其IC(50)值分别为23.6和73.4 nM，这与基于FP的受体结合测定法得到的IC(50)值（分别为30.6和78.7 nM）一致。这些结果表明，我们基于过滤的TRF UT受体结合测定法能够实现所需的灵敏度，且具有更高的重现性，适用于化合物库的高通量筛选。
