Identification of pregnane X receptor ligands using time-resolved fluorescence resonance energy transfer and quantitative high-throughput screening - PubMed (original) (raw)

doi: 10.1089/adt.2009.193.

Dac-Trung Nguyen, Ryan Macarthur, Anton Simeonov, William J Frazee, Tina M Hallis, Bryan D Marks, Upinder Singh, Hildegard C Eliason, John Printen, Christopher P Austin, James Inglese, Douglas S Auld

Affiliations

Identification of pregnane X receptor ligands using time-resolved fluorescence resonance energy transfer and quantitative high-throughput screening

Sunita J Shukla et al. Assay Drug Dev Technol. 2009 Apr.

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.

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Figures

Scheme 1.

Scheme 1.

Example classification scheme for assignment of resulting curve-fitted data into CRC classes. The following classification criteria are used to establish SAR: Class 1 curves display two asymptotes, an inflection point, and r2 ≥ 0.9; subclasses 1a versus 1b are differentiated by full (>80%) versus partial (≤80%) response. Class 1a curves demonstrate high activity. Class 2 curves display a single left-hand asymptote and inflection point; subclasses 2a and 2b are differentiated by a maximum response and _r_2 with either >80% and >0.9 or <80% and <0.9, respectively. Class 3 curves have a single left-hand asymptote, no inflection point, and a response >3 standard deviations the mean activity of the sample field; hence they are often termed “inconclusive” and are lower confidence curves. Class 4 defines those samples showing no concentration–response relationship; thus they are inactive.

Fig. 1.

Fig. 1.

PXR TR-FRET assay overview. The TR-FRET assay principle relies on the proximity of two fluorophores, where excitation of a Tb3+-labeled antibody (donor molecule), bound to the GST-tagged portion of the nuclear receptor, at 340 nm results in fluorescent emission at 495 nm and energy transfer to a fluorescein-labeled acceptor ligand (Fluormone tracer) bound to the nuclear receptor. The transfer of energy to the acceptor molecule results in fluorescent emission at 520 nm and a high TR-FRET ratio (520 nm/495 nm). The TR-FRET assay reports on the ability of a test ligand to displace the Fluormone tracer molecule from the nuclear receptor, which results in a disruption of the energy transfer and loss of the TR-FRET signal.

Fig. 2.

Fig. 2.

qHTS Reproducibility of PXR qHTS. Intra-screen data from triplicate qHTS runs of the LOPAC library are shown. (A) Seven-point CRCs for 1,280 compounds, performed in triplicate, demonstrate concentration-dependent inhibition of the TR-FRET signal (blue), activation of the TR-FRET signal (red), or inactives (gray). (B) Log AC50 correlation plot of compounds with class 1a, 1b, and 2a curves from runs 1 and 2 (_r_2 = 0.89; n = 43). Runs 1 versus 3 and 2 versus 3 had _r_2 values of 0.86 and 0.84, respectively (all P < 10−4).

Fig. 3.

Fig. 3.

qHTS CRCs generated in the PXR assay. Ratiometric data are shown. (A) 134 CRCs for T0901317 (MSR = 2.25). (B) 134 CRCs for SR12813 (MSR = 3.25). (C) Seven- to 15-point qHTS data for all compounds tested from five different libraries where inhibitors of the TR-FRET signal (blue), activators (red), and inactives (gray) are shown. qHTS data shown do not include the LOPAC validation runs.

Fig. 4.

Fig. 4.

qHTS titration curves for CRC classes 1 and 2. CRCs for (A) ratiometric, (B) 520 nm, and (C) 495 nm channels are shown for the entire screen (excluding LOPAC validation runs).

Fig. 5.

Fig. 5.

qHTS curves for well-known PXR ligands: (A) hyperforin ratiometric CRC data taken from the Biomol collection and (B) rifampicin taken from the FDA 2 compound collection.

Fig. 6.

Fig. 6.

Intra-vendor and follow-up PXR qHTS curves for retinoic acid from various vendors: FDA (▪), LOPAC (▴), Tocris (⋄), Biomol (•), and follow-up sample (▾). The mean values taken from duplicate titration points are shown, and error bars represent standard error of the mean.

Fig. 7.

Fig. 7.

Fluorescent interference with Tb3+ fluorescence by rifampicin. (A) Response of rifampicin in the TR-FRET PXR confirmatory assay for the ratio (○), 495 nm (•), and 520 nm (□) emission channels. (B) Fluorescence excitation and emission spectra for 5 n_M_ Tb-anti-GST antibody used in the PXR assay. The positive spectra represent the fluorescent signal of Tb3+ alone or with increasing concentrations of rifampicin (concentration range from 0 to 65 μ_M_; gray shaded lines decreasing in fluorescent intensity). The negative spectrum represents the difference in intensity of the Tb3+ complex in the presence of 60 μ_M_ rifampicin minus the Tb3+ alone. The dotted line is represents the absorbance (Abs) spectrum of 16.7 μ_M_ rifampicin (scale shown on right y-axis). CPS, counts per second.

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