Altered Human CYP3A4 Activity Caused by Antley-Bixler Syndrome-Related Variants of NADPH-Cytochrome P450 Oxidoreductase Measured in a Robust In Vitro System

Daniela Moutinho, Christopher C Marohnic, Satya Prakash Panda, J. Rueff, Bettie Sue S.S.S. Masters, Michel Kranendonk

Research output: Contribution to journalArticle

19 Citations (Scopus)

Abstract

NADPH-cytochrome P450 oxidoreductase (CYPOR) variants have been described in patients with perturbed steroidogenesis and sexual differentiation, related to Antley-Bixler syndrome (ABS). It is important to determine the effect of these variants on CYP3A4, the major drug-metabolizing cytochrome P450 (P450) in humans. In this study, 12 CYPOR_ABS variants were separately coexpressed with CYP3A4 in a robust in vitro system to evaluate the effects of these variants on CYP3A4 activity in a milieu that recapitulates the stoichiometry of the mammalian systems. Full-length CYPOR variants were coexpressed with CYP3A4, resulting in relative expression levels comparable to those found in hepatic tissue. Dibenzylfluorescein (DBF), a CYP3A-specific reporter substrate (Biopharm Drug Dispos 24:375-384, 2003), was used to compare the variants and wild-type (WT) CYPOR activities with that of human liver microsomes. CYP3A4, combined with WT CYPOR, demonstrated kinetic parameters (k(cat) and K-m) equal to those for pooled human liver microsomes. CYPOR variants Y181D, Y459H, V492E, L565P, and R616X all demonstrated maximal loss of CYP3A4 catalytic efficiency, whereas R457H and G539R retained similar to 10 and 30% activities, respectively. Conversely, variants P228L, M263V, A287P, and G413S each showed WT-like capacity (k(cat)/K-m), with the A287P variant being formerly reported to exhibit substantially lower catalytic efficiency. In addition, Q153R exhibited 60% of WT CYPOR capacity to support the DBF O-debenzylation reaction, contradicting increased catalytic efficiency (k(cat)/K-m) relative to that for the WT, reported previously. Our data indicate the importance of use of simulated, validated in vitro systems, employing full-length proteins with appropriate stoichiometric incorporation of protein partners, when pharmacogenetic predictions are to be made for P450-mediated biotransformation.
Original languageEnglish
Pages (from-to)754-760
Number of pages7
JournalDrug Metabolism And Disposition
Volume40
Issue number4
DOIs
Publication statusPublished - Apr 2012

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Antley-Bixler Syndrome Phenotype
Cytochrome P-450 CYP3A
NADPH-Ferrihemoprotein Reductase
Human Activities
Cytochrome P-450 Enzyme System
Oxidoreductases
Liver Microsomes
Sex Differentiation
Pharmacogenetics
Biotransformation
In Vitro Techniques
Pharmaceutical Preparations
Proteins

Cite this

@article{59ac387c88c049828ad69256d4cfa983,
title = "Altered Human CYP3A4 Activity Caused by Antley-Bixler Syndrome-Related Variants of NADPH-Cytochrome P450 Oxidoreductase Measured in a Robust In Vitro System",
abstract = "NADPH-cytochrome P450 oxidoreductase (CYPOR) variants have been described in patients with perturbed steroidogenesis and sexual differentiation, related to Antley-Bixler syndrome (ABS). It is important to determine the effect of these variants on CYP3A4, the major drug-metabolizing cytochrome P450 (P450) in humans. In this study, 12 CYPOR_ABS variants were separately coexpressed with CYP3A4 in a robust in vitro system to evaluate the effects of these variants on CYP3A4 activity in a milieu that recapitulates the stoichiometry of the mammalian systems. Full-length CYPOR variants were coexpressed with CYP3A4, resulting in relative expression levels comparable to those found in hepatic tissue. Dibenzylfluorescein (DBF), a CYP3A-specific reporter substrate (Biopharm Drug Dispos 24:375-384, 2003), was used to compare the variants and wild-type (WT) CYPOR activities with that of human liver microsomes. CYP3A4, combined with WT CYPOR, demonstrated kinetic parameters (k(cat) and K-m) equal to those for pooled human liver microsomes. CYPOR variants Y181D, Y459H, V492E, L565P, and R616X all demonstrated maximal loss of CYP3A4 catalytic efficiency, whereas R457H and G539R retained similar to 10 and 30{\%} activities, respectively. Conversely, variants P228L, M263V, A287P, and G413S each showed WT-like capacity (k(cat)/K-m), with the A287P variant being formerly reported to exhibit substantially lower catalytic efficiency. In addition, Q153R exhibited 60{\%} of WT CYPOR capacity to support the DBF O-debenzylation reaction, contradicting increased catalytic efficiency (k(cat)/K-m) relative to that for the WT, reported previously. Our data indicate the importance of use of simulated, validated in vitro systems, employing full-length proteins with appropriate stoichiometric incorporation of protein partners, when pharmacogenetic predictions are to be made for P450-mediated biotransformation.",
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year = "2012",
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Altered Human CYP3A4 Activity Caused by Antley-Bixler Syndrome-Related Variants of NADPH-Cytochrome P450 Oxidoreductase Measured in a Robust In Vitro System. / Moutinho, Daniela; Marohnic, Christopher C; Panda, Satya Prakash; Rueff, J. ; Masters, Bettie Sue S.S.S.; Kranendonk, Michel.

In: Drug Metabolism And Disposition, Vol. 40, No. 4, 04.2012, p. 754-760.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Altered Human CYP3A4 Activity Caused by Antley-Bixler Syndrome-Related Variants of NADPH-Cytochrome P450 Oxidoreductase Measured in a Robust In Vitro System

AU - Moutinho, Daniela

AU - Marohnic, Christopher C

AU - Panda, Satya Prakash

AU - Rueff, J.

AU - Masters, Bettie Sue S.S.S.

AU - Kranendonk, Michel

PY - 2012/4

Y1 - 2012/4

N2 - NADPH-cytochrome P450 oxidoreductase (CYPOR) variants have been described in patients with perturbed steroidogenesis and sexual differentiation, related to Antley-Bixler syndrome (ABS). It is important to determine the effect of these variants on CYP3A4, the major drug-metabolizing cytochrome P450 (P450) in humans. In this study, 12 CYPOR_ABS variants were separately coexpressed with CYP3A4 in a robust in vitro system to evaluate the effects of these variants on CYP3A4 activity in a milieu that recapitulates the stoichiometry of the mammalian systems. Full-length CYPOR variants were coexpressed with CYP3A4, resulting in relative expression levels comparable to those found in hepatic tissue. Dibenzylfluorescein (DBF), a CYP3A-specific reporter substrate (Biopharm Drug Dispos 24:375-384, 2003), was used to compare the variants and wild-type (WT) CYPOR activities with that of human liver microsomes. CYP3A4, combined with WT CYPOR, demonstrated kinetic parameters (k(cat) and K-m) equal to those for pooled human liver microsomes. CYPOR variants Y181D, Y459H, V492E, L565P, and R616X all demonstrated maximal loss of CYP3A4 catalytic efficiency, whereas R457H and G539R retained similar to 10 and 30% activities, respectively. Conversely, variants P228L, M263V, A287P, and G413S each showed WT-like capacity (k(cat)/K-m), with the A287P variant being formerly reported to exhibit substantially lower catalytic efficiency. In addition, Q153R exhibited 60% of WT CYPOR capacity to support the DBF O-debenzylation reaction, contradicting increased catalytic efficiency (k(cat)/K-m) relative to that for the WT, reported previously. Our data indicate the importance of use of simulated, validated in vitro systems, employing full-length proteins with appropriate stoichiometric incorporation of protein partners, when pharmacogenetic predictions are to be made for P450-mediated biotransformation.

AB - NADPH-cytochrome P450 oxidoreductase (CYPOR) variants have been described in patients with perturbed steroidogenesis and sexual differentiation, related to Antley-Bixler syndrome (ABS). It is important to determine the effect of these variants on CYP3A4, the major drug-metabolizing cytochrome P450 (P450) in humans. In this study, 12 CYPOR_ABS variants were separately coexpressed with CYP3A4 in a robust in vitro system to evaluate the effects of these variants on CYP3A4 activity in a milieu that recapitulates the stoichiometry of the mammalian systems. Full-length CYPOR variants were coexpressed with CYP3A4, resulting in relative expression levels comparable to those found in hepatic tissue. Dibenzylfluorescein (DBF), a CYP3A-specific reporter substrate (Biopharm Drug Dispos 24:375-384, 2003), was used to compare the variants and wild-type (WT) CYPOR activities with that of human liver microsomes. CYP3A4, combined with WT CYPOR, demonstrated kinetic parameters (k(cat) and K-m) equal to those for pooled human liver microsomes. CYPOR variants Y181D, Y459H, V492E, L565P, and R616X all demonstrated maximal loss of CYP3A4 catalytic efficiency, whereas R457H and G539R retained similar to 10 and 30% activities, respectively. Conversely, variants P228L, M263V, A287P, and G413S each showed WT-like capacity (k(cat)/K-m), with the A287P variant being formerly reported to exhibit substantially lower catalytic efficiency. In addition, Q153R exhibited 60% of WT CYPOR capacity to support the DBF O-debenzylation reaction, contradicting increased catalytic efficiency (k(cat)/K-m) relative to that for the WT, reported previously. Our data indicate the importance of use of simulated, validated in vitro systems, employing full-length proteins with appropriate stoichiometric incorporation of protein partners, when pharmacogenetic predictions are to be made for P450-mediated biotransformation.

KW - ESCHERICHIA-COLI

KW - DRUG-METABOLISM

KW - MODULATION

KW - P-450 REDUCTASE

KW - MUTATIONS

KW - STEROIDOGENESIS

KW - GENETIC-VARIANTS

KW - HUMAN LIVER-MICROSOMES

KW - PHARMACOGENOMICS

KW - TESTER STRAIN

U2 - 10.1124/dmd.111.042820

DO - 10.1124/dmd.111.042820

M3 - Article

VL - 40

SP - 754

EP - 760

JO - Drug Metabolism And Disposition

JF - Drug Metabolism And Disposition

SN - 0090-9556

IS - 4

ER -