Correlation of High-Throughput Pregnane X Receptor (PXR) Transactivation and Binding Assays

Zhu, Zhengrong; Kim, Sean; Chen, Taosheng; Lin, Jun-Hsiang; Bell, Aneka; Bryson, James; Dubaquié, Yves; Yan, Ning; Yanchunas, Joseph; Xie, Dan; Stoffel, Robert H.; Sinz, Michael; Dickinson, Kenneth E.J.

doi:10.1177/1087057104264902

Cited by 67 publications

(68 citation statements)

References 21 publications

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“…Unlike most steroid hormone receptors that have dissociation constants in the nanomolar range, and activate gene expression when steroids are present at 1-100 nM, the Kd for allopregnanolone binding to PXR is in the micromolar range, and requires 10-50 μM allopregnanolone for activation of gene expression in cell transfection assays (Lamba et al, 2004). PXR is activated by a wide range of structurally dissimilar compounds, consistent with its function in drug/xenobiotic sensing (Dixit et al, 2005;Ekins and Erickson, 2002;Kliewer et al, 2002;Poso and Honkakoski, 2006;Zhu et al, 2004), bile acid metabolism (Dussault et al, 2003;Goodwin et al, 2003), owing to its large ligand binding pocket (Ekins and Schuetz, 2002;Orans et al, 2005;Schuster and Langer, 2005;Watkins et al, 2002;Watkins et al, 2001). …”

Section: Allopregnanolone Effects In Developmentmentioning

confidence: 89%

Neurosteroid regulation of central nervous system development

Mellon

2007

Pharmacology & Therapeutics

189

117

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Neurosteroids are a relatively new class of neuroactive compounds, brought to prominence in the past two decades. Despite knowing of their presence in the nervous system of various species for over twenty years and knowing of their functions as GABA A and NMDA ligands, new and unexpected functions of these compounds are continuously being identified. Absence or reduced concentrations of neurosteroids during development and in adults may be associated with neurodevelopmental, psychiatric, or behavioral disorders. Treatment with physiologic or pharmacologic concentrations of these compounds may also promote neurogenesis, neuronal survival, myelination, increased memory, and reduced neurotoxicity. This review highlights what is currently known about the neurodevelopmental functions and mechanisms of action of four distinct neurosteroids -pregnenolone, progesterone, allopregnanolone and dehydroepiandrosterone.

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Section: Allopregnanolone Effects In Developmentmentioning

confidence: 89%

Neurosteroid regulation of central nervous system development

Mellon

2007

Pharmacology & Therapeutics

189

117

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“…Here, increased luminescence is typically measured as an indication of enzyme induction potential and EC 50 and E max values are determined. In general, there is a good correlation between EC 50 and IC 50 values in PXR transactivation and binding assays, respectively (16). However, there are examples where significant binding to the PXR receptor does not lead to transactivation or enzyme induction.…”

Section: Nuclear Hormone Receptors-transcription Factorsmentioning

confidence: 94%

“…Ligand binding assays generally consist of genetically expressed and isolated receptors incubated with test compound and a radiolabeled ligand. Competition of the radiolabeled ligand with test compound is measured and an IC 50 determined (16). These assays are reasonably straightforward to conduct, however their simplicity can also be a deficit in some situations.…”

Section: Nuclear Hormone Receptors-transcription Factorsmentioning

confidence: 99%

Current Industrial Practices in Assessing CYP450 Enzyme Induction: Preclinical and Clinical

Sinz

Wallace

Sahi³

2008

AAPS J

Self Cite

114

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Abstract. Induction of drug metabolizing enzymes, such as the cytochromes P450 (CYP) is known to cause drug-drug interactions due to increased elimination of co-administered drugs. This increased elimination may lead to significant reduction or complete loss of efficacy of the co-administered drug. Due to the significance of such drug interactions, many pharmaceutical companies employ screening and characterization models which predict CYP enzyme induction to avoid or attenuate the potential for drug interactions with new drug candidates. The most common mechanism of CYP induction is transcriptional gene activation. Activation is mediated by nuclear receptors, such as AhR, CAR, and PXR that function as transcription factors. Early high throughput screening models utilize these nuclear hormone receptors in ligand binding or cell-based transactivation/reporter assays. In addition, immortalized hepatocyte cell lines can be used to assess enzyme induction of specific drug metabolizing enzymes. Cultured primary human hepatocytes, the best established in vitro model for predicting enzyme induction and most accepted by regulatory agencies, is the predominant assay used to evaluate induction of a wide variety of drug metabolizing enzymes. These in vitro models are able to appropriately predict enzyme induction in patients when compared to clinical drug-drug interactions. Finally, transgenic animal models and the cynomolgus monkey have also been shown to recapitulate human enzyme induction and may be appropriate in vivo animal models for predicting human drug interactions.

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“…The assay was carried out in 96-well white plates in a total volume of 100 ml per well as described previously (Zhu et al, 2004). Curve fitting software, Grafit 5.0 (Erithacus Software Ltd, Surrey, UK) was used to generate IC 50 values (see Information for details under Materials and methods).…”

Section: Rna Preparation and Northern Blot Analysismentioning

confidence: 99%

Inhibition of drug metabolism by blocking the activation of nuclear receptors by ketoconazole

et al. 2006

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Individual variation in drug metabolism is a major cause of unpredictable side effects during therapy. Drug metabolism is controlled by a class of orphan nuclear receptors (NRs), which regulate expression of genes such as CYP (cytochrome)3A4 and MDR-1 (multi-drug resistance-1), that are involved in this process. We have found that xenobiotic-mediated induction of CYP3A4 and MDR-1 gene transcription was inhibited by ketoconazole, a commonly used antifungal drug. Ketoconazole mediated its effect by inhibiting the activation of NRs, human pregnenolone X receptor and constitutive androstene receptor, involved in regulation of CYP3A4 and MDR-1. The effect of ketoconazole was specific to the group of NRs that control xenobiotic metabolism. Ketoconazole disrupted the interaction of the xenobiotic receptor PXR with the co-activator steroid receptor co-activator-1. Ketoconazole treatment resulted in delayed metabolism of tribromoethanol anesthetic in mice, which was correlated to the inhibition of PXR activation and downmodulation of cyp3a11 and mdr-1 genes and proteins. These studies demonstrate for the first time that ketoconazole represses the coordinated activation of genes involved in drug metabolism, by blocking activation of a specific subset of NRs. Our results suggest that ketoconazole can be used as a pan-antagonist of NRs involved in xenobiotic metabolism in vivo, which may lead to novel strategies that improve drug effect and tolerance.

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Correlation of High-Throughput Pregnane X Receptor (PXR) Transactivation and Binding Assays

Cited by 67 publications

References 21 publications

Neurosteroid regulation of central nervous system development

Neurosteroid regulation of central nervous system development

Current Industrial Practices in Assessing CYP450 Enzyme Induction: Preclinical and Clinical

Inhibition of drug metabolism by blocking the activation of nuclear receptors by ketoconazole

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