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Cytochrome P450 Gene Regulation: Reporter Assays to Assess Pregnane X Receptor (PXR, NR1I2) Activation

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Cytochrome P450

Part of the book series: Methods in Pharmacology and Toxicology ((MIPT))

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Abstract

Identifying new molecular entities (NMEs) that activate the pregnane X receptor (PXR, NR1I2) can provide critical information regarding their potential to produce DDIs. Experiments can be designed that recognize such entities by assessing receptor transactivation in cell-based systems and monitoring reporter gene activity. These in vitro assays have several advantages; activation of a specific nuclear receptor (NR) can be determined, there is a high degree of reproducibility between experiments, the assays are cost and time-efficient, and they allow for flexibility to adjust for medium to high throughput. The techniques described here involve direct assessment of PXR activation in cell-based assays employing full-length PXR from various species, including human, monkey, rat, murine, canine, and sheep. In addition to receptor activation, cytotoxicity can be assessed in the same well of a multi-well plate. In this manner, compounds that are toxic and receptor activators are identified in a single assay. Moreover, if six or more concentrations of NME are evaluated, and a plateau is achieved, it may be possible to construct a dose-response curve and derive EC50 and Emax values. Step-by-step procedures are provided to thaw, seed, and culture the cells in 96-well plates, treat cells with NMEs, and simultaneously assess cytotoxicity and reporter gene activity. Construction of dose-response curves and interpretation of results generated will also be discussed.

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Abbreviations

CTF:

Cell-Titer Fluor™ cell viability assay

DDIs:

Drug–drug interactions

DMSO:

Dimethyl sulfoxide

FBS:

Fetal bovine serum

FLU:

Fluorescent light units

NME:

New molecular entity

NR:

Nuclear receptor

PBS:

Phosphate buffered saline

PCN:

Pregnenolone 16-α carbonitrile

PXR:

Pregnane X receptor

PXRE:

Pregnane X receptor element

RIF:

Rifampicin

RIS:

Relative induction score

RLU:

Relative light units

SR12813:

Tetraethyl 2-(3,5-di-tert-butyl-4-hydroxyphenyl)ethenyl-1,1-bisphosphonate

XREM:

Xenobiotic-response enhancer module

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Author information

Authors and Affiliations

  1. Puracyp Inc., Carlsbad, CA, USA

    Marija Pinne & Judy L. Raucy

Authors
  1. Marija Pinne
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  2. Judy L. Raucy
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Corresponding author

Correspondence to Marija Pinne .

Editor information

Editors and Affiliations

  1. Genentech Inc., South San Francisco, CA, USA

    Zhengyin Yan

  2. Blue Bell, PA, USA

    Gary W. Caldwell

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Pinne, M., Raucy, J.L. (2021). Cytochrome P450 Gene Regulation: Reporter Assays to Assess Pregnane X Receptor (PXR, NR1I2) Activation. In: Yan, Z., Caldwell, G.W. (eds) Cytochrome P450. Methods in Pharmacology and Toxicology. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1542-3_9

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  • DOI: https://doi.org/10.1007/978-1-0716-1542-3_9

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-1541-6

  • Online ISBN: 978-1-0716-1542-3

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