Identification of Pregnane X Receptor Ligands Using Time-Resolved Fluorescence Resonance Energy Transfer and Quantitative High-Throughput Screening

Shukla, Sunita J.; Nguyễn, Ðắc-Trung; MacArthur, Ryan; Simeonov, Anton; Frazee, William J.; Hallis, Tina M.; Marks, Bryan D.; Singh, Ujjwal Kumar; Eliason, Hildegard C.; Printen, John A.; Austin, Christopher P.; Inglese, James; Auld, Douglas S.

doi:10.1089/adt.2009.193

Cited by 57 publications

(73 citation statements)

References 40 publications

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“…It is important to note that the TR-FRET assay was not applicable for rifampicin, because this compound quenched the fluorescence (unpublished data). This problem was also previously described by Shukla et al (2009) andLau et al (2012).…”

Section: Lanthascreen Tr-fret Pxr Competitive Binding Assaysupporting

confidence: 55%

Milk Thistle’s Active Components Silybin and Isosilybin: Novel Inhibitors of PXR-Mediated CYP3A4 Induction

et al. 2013

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Because cancer is often treated with combination therapy, unexpected pharmacological effects can occur because of drug-drug interactions. Several drugs are able to cause upregulation or downregulation of drug transporters or cytochrome P450 enzymes, particularly CYP3A4. Induction of CYP3A4 may result in decreased plasma levels and therapeutic efficacy of anticancer drugs. Since the pregnane X receptor (PXR) is one of the major transcriptional regulators of CYP3A4, PXR antagonists can possibly prevent CYP3A4 induction. Currently, a limited number of PXR antagonists are available. Some of these antagonists, such as sulphoraphane and coumestrol, belong to the so-called complementary and alternative medicines (CAM). Therefore, the aim was to determine the potential of selected CAM (b-carotene, Echinacea purpurea, garlic, Ginkgo biloba, ginseng, grape seed, green tea, milk thistle, saw palmetto, valerian, St. John's Wort, and vitamins B 6 , B 12 , and C) to inhibit PXR-mediated CYP3A4 induction at the transcriptional level, using a reporter gene assay and a real-time polymerase chain reaction assay in LS180 colon adenocarcinoma cells. Furthermore, computational molecular docking and a LanthaScreen time-resolved fluorescence resonance energy transfer (TR-FRET) PXR competitive binding assay were performed to explore whether the inhibiting CAM components interact with PXR. The results demonstrated that milk thistle is a strong inhibitor of PXR-mediated CYP3A4 induction. The components of milk thistle responsible for this effect were identified as silybin and isosilybin. Furthermore, computational molecular docking revealed a strong interaction between both silybin and isosilybin and PXR, which was confirmed in the TR-FRET PXR assay. In conclusion, silybin and isosilybin might be suitable candidates to design potent PXR antagonists to prevent drug-drug interactions via CYP3A4 in cancer patients.

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Section: Lanthascreen Tr-fret Pxr Competitive Binding Assaysupporting

confidence: 55%

Milk Thistle’s Active Components Silybin and Isosilybin: Novel Inhibitors of PXR-Mediated CYP3A4 Induction

et al. 2013

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“…The hPXR LBD assay ( Fig. 1Biii) was performed with LanthaScreen time-resolved fluorescence resonance energy transfer (TR-FRET)-based technology (Invitrogen, Carlsbad, CA) and is described in detail elsewhere (Shukla et al, 2009). In brief, the assay was performed using the LanthaScreen TR-FRET PXR Competitive Binding Assay Kit, which contains the TR-FRET PXR assay buffer, Fluormone PXR Green (fluorescein-labeled PXR ligand), human PXR LBD (GST) (amino acids 111-434), and LanthaScreen Tb-anti-GST antibody.…”

Section: Methodsmentioning

confidence: 99%

“…Furthermore, rats are the preferred models for drug metabolism and pharmacokinetic studies; hence, rat-human PXR activation differences are important to identify and quantify. We have previously reported the profiling of a structurally diverse collection of up to 17,000 compounds for PXR binding and P450 activity (Shukla et al, 2009;Veith et al, 2009) using quantitative high-throughput screening (qHTS) (Shukla et al, 2009). In this study, we used qHTS to profile more than 2800 clinically used drugs and bioactive compounds for their ability to activate hPXR and rat PXR (rPXR) and induce human CYP3A4 using cell-based in vitro assays.…”

Section: Introductionmentioning

confidence: 99%

“…In this study, we used qHTS to profile more than 2800 clinically used drugs and bioactive compounds for their ability to activate hPXR and rat PXR (rPXR) and induce human CYP3A4 using cell-based in vitro assays. The activities of these drugs at the cellular level (human) were further compared with their ability to bind the human PXR LBD at the protein level (Shukla et al, 2009) to identify compounds that directly bind to the LBD and those that potentially bind outside this region along the full-length PXR. Finally, we identified compounds that induced human CYP3A4 with an additional cell-based assay.…”

Section: Introductionmentioning

confidence: 99%

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Identification of Clinically Used Drugs That Activate Pregnane X Receptors

et al. 2010

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ABSTRACT:The pregnane X receptor (PXR) binds xenobiotics and regulates the expression of several drug-metabolizing enzymes and transporters. Human PXR (hPXR) activation and CYP3A4 induction can be involved in drug-drug interactions, resulting in reduced efficacy or increased toxicity. However, there are known species-specific differences with regard to PXR activation that should be taken into account when animal PXR data are extrapolated to humans. We profiled 2816 clinically used drugs from the National Institutes of Health Chemical Genomics Center Pharmaceutical Collection for their ability to activate hPXR and rat PXR (rPXR) at the cellular level, induce human CYP3A4 at the cellular level, and bind human PXR at the protein level. From 6 to 11% of drugs were identified as active across the four assays, which included assay-specific and pan-active compounds. The lowest concordance was observed between the hPXR and rPXR assays, and many compounds active in both assays nonetheless demonstrated significant potency differences between species. Analysis based on clustering potency values demonstrated the greatest activity correlation between the hPXR activation and CYP3A4 induction assays. Structure-activity relationship analysis identified chemical scaffolds that were panactive (e.g., dihydropyridine calcium channel blockers) and others that were uniquely active in individual assays (e.g., steroids and fatty acids). These results provide important information on PXR activation by clinically used drugs, highlight the species specificity of PXR activation by xenobiotics, and provide a means of prioritizing compounds for follow-up studies and optimization efforts.

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“…Recently, homogeneous and versatile TR-FRET-based nuclear receptor co-activator assays have been designed for a wide selection of nuclear receptors (Table 1) (Brown et al, 1999), stimulated coactivator recruitment and GW9662, PPARγ antagonist (Davies et al, 2001), inhibited coactivator recruitment in a dose-dependent manner. Shukla et al (2009) screened over 80,000 compounds from various libraries for their ability to bind PXR using a homogeneous PXR TR-FRET assay containing fluorescein-labelled PXR ligands, GST-tagged PXR, and Tb-labelled anti-GST antibodies. The authors also took advantage of the qHTS in generating multiple concentration-response curves to probe for assay artefacts caused by compound autofluorescence, signal quenching, aggregate formation, or diffusion-enhanced FRET.…”

Section: Nuclear Receptor Assaysmentioning

confidence: 99%

Advances in high-throughput screening technology for toxicology

Hsu

Huang

Attene‐Ramos

et al. 2017

IJRAM

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Abstract:The US Tox21 collaboration utilises a quantitative high-throughput screening (qHTS) platform to efficiently profile a large number of environmental chemicals. qHTS combined with informatics facilitates chemical prioritisation for further in-depth toxicity testing and development of computational models to predict chemical toxicity. The NIH Chemical Genomics Center (NCGC), now part of the National Center for Advancing Translational Sciences (NCATS), is a key contributor during all phases of the Tox21 collaboration, from assay development and compound screening to data analysis and model building. Since 2011, the Tox21/NCGC has been profiling the phase 2 Tox21 library of approximately 10,000 (10K) environmental chemicals and drugs. The advances in HTS assays and qHTS screens against the Tox21's and other chemical libraries are described in this review.

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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 xenobioticregulated receptor that is activated by a range of diverse chemicals, including antibiotics, antifungals, glucocorticoids, and

Cited by 57 publications

References 40 publications

Milk Thistle’s Active Components Silybin and Isosilybin: Novel Inhibitors of PXR-Mediated CYP3A4 Induction

Milk Thistle’s Active Components Silybin and Isosilybin: Novel Inhibitors of PXR-Mediated CYP3A4 Induction

Identification of Clinically Used Drugs That Activate Pregnane X Receptors

Advances in high-throughput screening technology for toxicology

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