A dual-acceptor time-resolved Foster resonance energy transfer assay for simultaneous determination of thyroid hormone regulation of corepressor and coactivator binding to the thyroid hormone receptor: Mimicking the cellular context of thyroid hormone action
A dual-acceptor time-resolved Foster resonance energy transfer assay for simultaneous determination of thyroid hormone regulation of corepressor and coactivator binding to the thyroid hormone receptor: Mimicking the cellular context of thyroid hormone action
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DOI：
10.1016/j.ab.2008.11.039
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发表时间：
2009-03-01
影响因子：
2.9
通讯作者：
Katzenellenbogen, John A.
中科院分区：
文献类型：
--
作者：
Jeyakumar, M.;Katzenellenbogen, John A.
此前，我们报道了两种基于时间分辨福斯特共振能量转移(TRRET)的体外检测方法，用于评估不同的甲状腺激素受体(TR)配体在调节辅调节因子招募方面的效力和有效性。我们可以在不同的检测中独立地测量SRC3(类固醇受体辅活化子3，转录辅活化子)的募集和NCoR(核受体辅抑制子，转录辅阻遏子)从与DR+4甲状腺激素反应元件(TRE)结合的TR中心点维甲酸X受体(RXR)异二聚体的解离。在这里，通过使用Tb3+的不同发射峰，供体荧光团用于标记Tre结合的TrR中心点RXR异源二聚体，并选择两个不同的受体荧光素和花菁5来分别标记NCoR和SRC3，我们将前面的两种检测模式整合到一个单一的检测中。因此，我们可以同时测量TR配体对NCoR解离和SRC3募集活动的效力在一个模拟TRR作用的细胞环境的许多特征的系统中，这种双重测定的性能用已知的高度有效的生理学TRR配体三碘甲状腺原氨酸(TA)和合成的TRR拮抗剂NH-3进行测试。从这两种双重测定中测得的这两种受体配体的效价和效率与从两种独立的测定中获得的结果高度相似。因此，这种双受体tr-fret分析进一步简化了配体调节的tr-辅调节因子相互作用的测量，并应提高tr药物发现计划筛选过程的整体效率。(C)2008 Elsevier Inc.保留所有权利。
Previously, we reported the development of two in vitro time-resolved Foster resonance energy transfer (tr-FRET)-based assays for evaluating the potency and efficacy of different ligands of thyroid hormone receptor (TR) for regulating the recruitment of coregulators. We could measure independently, in separate assays, both the recruitment of SRC3 (steroid receptor coactivator 3, a transcriptional coactivator) and the dissociation of NCoR (nuclear receptor corepressor, a transcriptional corepressor) from a TR center dot retinoid X receptor (RXR) heterodimer bound to a DR+4 thyroid hormone response element (TRE). Here, by using the distinct emission peaks of Tb3+, the donor fluorophore used to label the TRE-bound TR center dot RXR heterodimers, and selecting two distinct acceptor fluorophores, fluorescein and cyanine 5, to label of NCoR and SRC3, respectively, we have integrated our previous two assay formats into a single assay. Thus, we can measure the potency of TR ligands simultaneously for NCoR dissociation and SRC3 recruitment activities in a system that mimics many features of the cellular context of TR action, The performance of this dual assay was tested with a known, highly potent physiological TR ligand, triiodothyronine (TA and with a synthetic TR antagonist, NH-3. Measured potencies and efficacies of these two TR ligands from this dual assay are highly comparable to those obtained from the two independent assays. Thus, this dual-acceptor tr-FRET assay further simplifies the measurement of ligand-modulated TR-coregulator interactions and Should improve the overall efficiency of the screening process of TR drug discovery programs. (c) 2008 Elsevier Inc. All rights reserved.