The Utility of Stable Cell Lines to Assess Species Differences in PXR Transactivation

Sinz, Michael; Pray, Devin; Raucy, Judy L.

doi:10.2174/187231207780363561

Cited by 11 publications

(17 citation statements)

References 17 publications

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“…The assays used were highly robust and allowed us to directly compare hPXR and rPXR activation at the cellular level. The use of stably transfected cell lines containing the PXR coding region and CYP3A4 promoter and enhancer elements to identify CYP3A4 inducers has been validated previously (Raucy et al, 2002;Yueh et al, 2005;Sinz et al, 2007). In a recent study, the use of in vitro cell-based methods that measure nuclear receptor activation has been demonstrated as a reliable method of identifying P450 inducers in a species-specific manner (Mueller et al, 2010).…”

Section: Discussionmentioning

confidence: 99%

“…The luciferase construct containing the CYP3A4 enhancer (upstream of the luciferase reporter gene) is necessary for detection of CYP3A4 transcription, as measured by luminescence. The rPXR cell line was generated through cotransfection with an expression vector containing the full-length rPXR and a luciferase construct into a rodent hepatoma cell line (Sinz et al, 2007). Cell culture and assay medium were purchased from Puracyp, and the cells were maintained at 37°C under a humidified atmosphere and 5% CO 2 .…”

Section: Methodsmentioning

confidence: 99%

“…SHP/SMRT transcription is blocked by ligand-activated PXR, and the ligand-PXR complex forms a heterodimer with RXR␣ before stabilization by SRC-1 and other coactivators. The coactivator-PXR-RXR␣ complex binds the xenobiotic responsive enhancer molecule/PXRE (XREM/PXRE) region containing an everted repeat with a spacer of six nucleotides (ER6) in the proximal promoter and direct repeats with a three-nucleotide spacing (DR3) in the distal XREM upstream of the CYP3A4 transcription start site (Stanley et al, 2006;Sinz et al, 2007). Bi, the hPXR/rPXR activation assay relies on a luciferase reporter gene (green), which is downstream of the PXRE (gray).…”

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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Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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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“…The most widely used are those that express hPXR [82][83][84] and those that determine human AhR activation [85][86][87][88]. Rodent PXR cell lines have also been developed [89] because of the species variability associated with PXR-activation. The rodent lines have proven useful in guiding both in vivo toxicology and pharmacokinetic studies.…”

Section: Transactivation In Stable Cell Linesmentioning

confidence: 99%

Current In Vitro High Throughput Screening Approaches to Assess Nuclear Receptor Activation

Raucy¹,

Lasker²

2010

CDM

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The screening of new drug candidates for nuclear receptor activation can identify agents with the potential to produce drug-drug interactions or elicit adverse drug effects. The nuclear receptors of interest are those that control the expression of drug metabolizing enzymes and drug transporters, and include the constitutive androstane receptor (CAR, NR1I3), the pregnane X receptor (PXR, NR1I2) and the aryl hydrocarbon receptor (AhR). This review will focus on the methods currently used to assess activation of these receptors. Assessment of nuclear receptor activation can be accomplished using direct or indirect approaches. Indirect methods quantify specific gene products that result from nuclear receptor activation while direct approaches measure either the binding of ligands to the receptors or the transcriptional events produced by ligand binding. Assays that directly quantify nuclear receptor activation are growing in popularity and, importantly, are amenable to high throughput screening (HTS). Several ligand binding assays are currently being utilized, including radioligand competition binding, where compounds compete with radiolabelled ligand for binding to PXR or CAR, such as the scintillation proximity binding assay that measures the reaction of ligands with receptor-coated beads. A fluorescence resonance energy transfer assay has also been developed, where the fluorescent signal is generated via the ligand-dependent interaction between the fluorescently-labeled ligand binding domain of a nuclear receptor and co-activator proteins. Other in vitro activation assays include transient- and stably-transfected cell lines incorporating an expression vector for PXR, CAR or AhR plus a reporter gene vector containing response elements. The methods focused on in this review will be limited to the more direct in vitro approaches that are amenable to high throughput screening.

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“…The luciferase construct containing the CYP3A4 enhancer (upstream of the luciferase reporter gene) is necessary for detection of CYP3A4 transcription, as measured by luminescence. The rPXR cell line was generated through co-transfection with an expression vector containing the full-length rPXR and a luciferase construct into a rodent hepatoma cell line (Sinz et al, 2007). Cell culture and assay medium were purchased from Puracyp (Carlsbad, CA) and the cells were maintained at 37ºC under a humidified atmosphere and 5% CO 2 .…”

Section: Cell Lines and Cell Culture Conditionsmentioning

confidence: 99%

Correction to “Identification of Clinically Utilized Drugs That Activate Pregnane X Receptors”

2011

Drug Metab Dispos

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The Utility of Stable Cell Lines to Assess Species Differences in PXR Transactivation

Cited by 11 publications

References 17 publications

Identification of Clinically Used Drugs That Activate Pregnane X Receptors

Identification of Clinically Used Drugs That Activate Pregnane X Receptors

Current In Vitro High Throughput Screening Approaches to Assess Nuclear Receptor Activation

Correction to “Identification of Clinically Utilized Drugs That Activate Pregnane X Receptors”

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