title: Identification of pregnane X receptor ligands using time-resolved fluorescence resonance energy transfer and quantitative high-throughput screening.
abstract: 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 >or=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.
authors.affiliation: NIH Chemical Genomics Center, National Institutes of Health, Bethesda, Maryland, USA.
authors.forename: Sunita J
authors.initials: SJ
authors.lastname: Shukla
authors.forename: Dac-Trung
authors.initials: DT
authors.lastname: Nguyen
authors.forename: Ryan
authors.initials: R
authors.lastname: Macarthur
authors.forename: Anton
authors.initials: A
authors.lastname: Simeonov
authors.forename: William J
authors.initials: WJ
authors.lastname: Frazee
authors.forename: Tina M
authors.initials: TM
authors.lastname: Hallis
authors.forename: Bryan D
authors.initials: BD
authors.lastname: Marks
authors.forename: Upinder
authors.initials: U
authors.lastname: Singh
authors.forename: Hildegard C
authors.initials: HC
authors.lastname: Eliason
authors.forename: John
authors.initials: J
authors.lastname: Printen
authors.forename: Christopher P
authors.initials: CP
authors.lastname: Austin
authors.forename: James
authors.initials: J
authors.lastname: Inglese
authors.forename: Douglas S
authors.initials: DS
authors.lastname: Auld
mesh_terms: D004305:Dose-Response Relationship, Drug; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D006801:Humans; D008024:Ligands; D000077297:Pregnane X Receptor; D011987:Receptors, Steroid / Q000032:analysis / Q000378:metabolism*; D013329:Structure-Activity Relationship
chemical_list: D008024:Ligands; D000077297:Pregnane X Receptor; D011987:Receptors, Steroid