Identification of Pregnane X Receptor Ligands Using Time-Resolved Fluorescence Resonance Energy Transfer and Quantitative High-Throughput Screening
Identification of Pregnane X Receptor Ligands Using Time-Resolved Fluorescence Resonance Energy Transfer and Quantitative High-Throughput Screening
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DOI：
10.1089/adt.2009.193
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发表时间：
2009-04-01
影响因子：
1.8
通讯作者：
Auld, Douglas S.
中科院分区：
文献类型：
--
作者：
Shukla, Sunita J.;Nguyen, Dac-Trung;Auld, Douglas S.
人类的PXR是一种由外源性物质调节的受体，可被一系列不同的化学物质激活，包括抗生素、抗真菌药、糖皮质激素和草药提取物。由于细胞色素P450同工酶的诱导和大量处方药物的激活，PXR已被表征为外源性物质代谢中的重要受体。开发能够有效检测PXR配体的方法将在临床上有益于避免潜在的药物相互作用。为了促进PXR配体的鉴定，将时间分辨荧光共振能量转移（TR-FRET）测定小型化至1，536孔微量滴定板格式以采用定量高通量筛选（qHTS）。优化的1，536孔TR-FRET测定显示Z '因子>= 0.5。使用铽和荧光素发射数据生成8，280种化合物的7至15点浓度-响应曲线（CRC），从而生成241，664个数据点。qHTS方法允许我们回顾性检查单一浓度筛选数据集，以评估PXR测定在不同化合物筛选浓度下的灵敏度和选择性。此外，通过检查CRC的特定发射通道，确定了非特异性测定伪影，例如铽信号和化合物荧光的基于浓度的淬灭。CRC信息也用于定义与PXR配体相关的化学型。这项研究证明了以高通量形式使用TR-FRET测定分析数千种化合物对PXR的可行性。
The human pregnane X nuclear receptor (PXR) is a xenobiotic-regulated receptor that is activated by a range of diverse chemicals, including antibiotics, antifungals, glucocorticoids, and herbal extracts. PXR has been characterized as an important receptor in the metabolism of xenobiotics due to induction of cytochrome P450 isozymes and activation by a large number of prescribed medications. Developing methodologies that can efficiently detect PXR ligands will be clinically beneficial to avoid potential drug-drug interactions. To facilitate the identification of PXR ligands, a time-resolved fluorescence resonance energy transfer (TR-FRET) assay was miniaturized to a 1,536-well microtiter plate format to employ quantitative high-throughput screening (qHTS). The optimized 1,536-well TR-FRET assay showed Z'-factors of >= 0.5. Seven- to 15-point concentration-response curves (CRCs) were generated for 8,280 compounds using both terbium and fluorescein emission data, resulting in the generation of 241,664 data points. The qHTS method allowed us to retrospectively examine single concentration screening datasets to assess the sensitivity and selectivity of the PXR assay at different compound screening concentrations. Furthermore, nonspecific assay artifacts such as concentration-based quenching of the terbium signal and compound fluorescence were identified through the examination of CRCs for specific emission channels. The CRC information was also used to define chemotypes associated with PXR ligands. This study demonstrates the feasibility of profiling thousands of compounds against PXR using the TR-FRET assay in a high-throughput format.