Skip to main content
Advertisement

Main menu

  • Home
  • Articles
    • Current Issue
    • Fast Forward
    • Latest Articles
    • Special Sections
    • Archive
  • Information
    • Instructions to Authors
    • Submit a Manuscript
    • FAQs
    • For Subscribers
    • Terms & Conditions of Use
    • Permissions
  • Editorial Board
  • Alerts
    • Alerts
    • RSS Feeds
  • Virtual Issues
  • Feedback
  • Submit
  • Other Publications
    • Drug Metabolism and Disposition
    • Journal of Pharmacology and Experimental Therapeutics
    • Molecular Pharmacology
    • Pharmacological Reviews
    • Pharmacology Research & Perspectives
    • ASPET

User menu

  • My alerts
  • Log in
  • My Cart

Search

  • Advanced search
Drug Metabolism & Disposition
  • Other Publications
    • Drug Metabolism and Disposition
    • Journal of Pharmacology and Experimental Therapeutics
    • Molecular Pharmacology
    • Pharmacological Reviews
    • Pharmacology Research & Perspectives
    • ASPET
  • My alerts
  • Log in
  • My Cart
Drug Metabolism & Disposition

Advanced Search

  • Home
  • Articles
    • Current Issue
    • Fast Forward
    • Latest Articles
    • Special Sections
    • Archive
  • Information
    • Instructions to Authors
    • Submit a Manuscript
    • FAQs
    • For Subscribers
    • Terms & Conditions of Use
    • Permissions
  • Editorial Board
  • Alerts
    • Alerts
    • RSS Feeds
  • Virtual Issues
  • Feedback
  • Submit
  • Visit dmd on Facebook
  • Follow dmd on Twitter
  • Follow ASPET on LinkedIn
Research ArticleArticle

Mechanistic Studies of Cytochrome P450 3A4 Time-Dependent Inhibition Using Two Cysteine-Targeting Electrophiles

John T. Barr, Zhican Wang, Xiaoshan Min, Henry J. Wienkers, Brooke M. Rock, Dan A. Rock and Larry C. Wienkers
Drug Metabolism and Disposition June 2020, 48 (6) 508-514; DOI: https://doi.org/10.1124/dmd.119.089813
John T. Barr
Amgen Research, Pharmacokinetics and Drug Metabolism, Amgen Inc., South San Francisco, California
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Zhican Wang
Amgen Research, Pharmacokinetics and Drug Metabolism, Amgen Inc., South San Francisco, California
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Xiaoshan Min
Amgen Research, Pharmacokinetics and Drug Metabolism, Amgen Inc., South San Francisco, California
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Henry J. Wienkers
Amgen Research, Pharmacokinetics and Drug Metabolism, Amgen Inc., South San Francisco, California
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Brooke M. Rock
Amgen Research, Pharmacokinetics and Drug Metabolism, Amgen Inc., South San Francisco, California
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Dan A. Rock
Amgen Research, Pharmacokinetics and Drug Metabolism, Amgen Inc., South San Francisco, California
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Larry C. Wienkers
Amgen Research, Pharmacokinetics and Drug Metabolism, Amgen Inc., South San Francisco, California
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
  • Article
  • Figures & Data
  • Info & Metrics
  • eLetters
  • PDF + SI
  • PDF
Loading

Article Figures & Data

Figures

  • Tables
  • Additional Files
  • Fig. 1.
    • Download figure
    • Open in new tab
    • Download powerpoint
    Fig. 1.

    Structures of PM and PIA. PM (left) and PIA (right) differ by the electrophilic group attached to a pyrene core. Arrows indicate the flow of electrons from an attacking cysteine thiolate anion nucleophile.

  • Fig. 2.
    • Download figure
    • Open in new tab
    • Download powerpoint
    Fig. 2.

    Inactivation of CYP3A4 by PM and PIA. A solution containing CYP3A4 Supersomes in phosphate buffer was preincubated with PM or PIA (50 µM) for various times. After preincubation, mixture solutions were 20-fold diluted into a secondary incubation containing 10 µM midazolam. 1-hydroxymidazolam formation was monitored, and the activity was calculated as a percentage of the product formation observed at the 0-minute preincubation time point. The mean of transformed data for relative reaction rate of triplicate determinations ± S.E. is shown. Circles and squares denote treatment by PM and PIA, respectively.

  • Fig. 3.
    • Download figure
    • Open in new tab
    • Download powerpoint
    Fig. 3.

    Alkylation of CYP3A4 apo-protein by PM and PIA. Purified recombinant CYP3A4 (0.5 µM) was reacted with of PIA or PM (5 µM) in separate tubes for 10 minutes. The samples were analyzed by high-resolution mass spectrometry, and resultant deconvoluted spectra are shown in the chromatogram for PM (top panel) and PIA (bottom panel).

  • Fig. 4.
    • Download figure
    • Open in new tab
    • Download powerpoint
    Fig. 4.

    Effect of small molecule treatment on CYP3A4 CO binding capacity. (A) Incubations containing CYP3A4 Supersomes were treated with PM (50 µM). After incubation of CYP3A4 with PM for 0, 0.5, 2, and 5 minutes at 37°C, CO binding was measured using UV-visible difference spectra as described in Materials and Methods. (B) Incubation CYP3A4 Supersomes with PIA (50 µM). After incubation of CYP3A4 with PM for 0, 5, 10, and 20 minutes, CO binding was measured using UV-visible difference spectra as described in Materials and Methods. (C) Incubation CYP3A4 Supersomes with either ABT or raloxifene in the presence of NADPH. Reaction was initiated by the addition of NADPH and incubated for 30 minutes at 37°C.

  • Fig. 5.
    • Download figure
    • Open in new tab
    • Download powerpoint
    Fig. 5.

    Quantitative assessment of intact heme loss after CYP3A4 treatment with PM and PIA. CYP3A4 Supersomes (4 pmol) were incubated with vehicle (0.5% DMSO), PM (50 µM), PIA (50 µM), ABT (200 µM), and raloxifene (100 µM) as described in the Materials and Methods section. After an incubation of 0, 1, 2, 5, 10, and 20 minutes, aliquots were quenched and extracted for LC-UV-MS/MS analysis. The percentage of intact heme remaining after treatment is shown. Points and error bars represent the mean of triplicate experiments ± S.E.

  • Fig. 6.
    • Download figure
    • Open in new tab
    • Download powerpoint
    Fig. 6.

    Free heme release from the CYP3A4 after PM and PIA treatment. CYP3A4 Supersomes (40 pmol/ml, without b5) were incubated with vehicle (0.5% DMSO), PM (50 µM), and PIA (50 µM) in separate incubations. The incubations were quenched, and the total and free heme were differentially extracted as described in Materials and Methods. Left panel shows the time course of total intact heme, whereas the right panel shows the percentage of heme released into solution over time. Points and error bars represent the mean of triplicate experiments ± S.E.

  • Fig. 7.
    • Download figure
    • Open in new tab
    • Download powerpoint
    Fig. 7.

    Effect of PIA and PM pretreatment on recombinant 3A5 activity and carbon monoxide binding spectra. (A) A solution containing CYP3A5 Supersomes in phosphate buffer was preincubated with PM or PIA (50 µM) for various incubation times. After preincubation, mixture solutions were 20-fold diluted into a secondary incubation containing 10 µM midazolam. Formation of 1-hydroxymidazolam was monitored, and the activity was calculated as a percentage of the product formation observed relative to vehicle-treated control at each time point. Transformed data for relative reaction rate of triplicate determinations ± S.E. is shown. Circles and squares denote treatment by PM and PIA, respectively. (B) Incubations containing CYP3A5 Supersomes were treated with PM (50 µM, red line), PIA (50 µM, blue line), or DMSO (green line) for 5 minutes at 37°C. CO binding was measured using UV-visible spectrometer difference spectra scanning from 400 to 500 nm as described in Materials and Methods.

Tables

  • Figures
  • Additional Files
    • View popup
    TABLE 1

    Effect of PIA and PM pretreatment on recombinant CYP3A4 onset Tagg

    Values represent means of duplicate determinations.

    TreatmentOnset Tagg (°C)
    DMSO48.8
    Pyrene48.6
    Pyrene iodoacetamide48.3
    Pyrene maleimide36.0

Additional Files

  • Figures
  • Tables
  • Data Supplement

    • Supplemental Figure -

      Supplementary Figure 1 - Representative aggregation curves for four treatments of recombinant CYP3A4 (as measured by scattering intensity at 266 nm) over a thermal ramp.

PreviousNext
Back to top

In this issue

Drug Metabolism and Disposition: 48 (6)
Drug Metabolism and Disposition
Vol. 48, Issue 6
1 Jun 2020
  • Table of Contents
  • Table of Contents (PDF)
  • About the Cover
  • Index by author
  • Editorial Board (PDF)
  • Front Matter (PDF)
Download PDF
Article Alerts
Sign In to Email Alerts with your Email Address
Email Article

Thank you for sharing this Drug Metabolism & Disposition article.

NOTE: We request your email address only to inform the recipient that it was you who recommended this article, and that it is not junk mail. We do not retain these email addresses.

Enter multiple addresses on separate lines or separate them with commas.
Mechanistic Studies of Cytochrome P450 3A4 Time-Dependent Inhibition Using Two Cysteine-Targeting Electrophiles
(Your Name) has forwarded a page to you from Drug Metabolism & Disposition
(Your Name) thought you would be interested in this article in Drug Metabolism & Disposition.
CAPTCHA
This question is for testing whether or not you are a human visitor and to prevent automated spam submissions.
Citation Tools
Research ArticleArticle

CYP3A4 Time-Dependent Inhibition by Cysteine-Targeting Electrophiles

John T. Barr, Zhican Wang, Xiaoshan Min, Henry J. Wienkers, Brooke M. Rock, Dan A. Rock and Larry C. Wienkers
Drug Metabolism and Disposition June 1, 2020, 48 (6) 508-514; DOI: https://doi.org/10.1124/dmd.119.089813

Citation Manager Formats

  • BibTeX
  • Bookends
  • EasyBib
  • EndNote (tagged)
  • EndNote 8 (xml)
  • Medlars
  • Mendeley
  • Papers
  • RefWorks Tagged
  • Ref Manager
  • RIS
  • Zotero

Share
Research ArticleArticle

CYP3A4 Time-Dependent Inhibition by Cysteine-Targeting Electrophiles

John T. Barr, Zhican Wang, Xiaoshan Min, Henry J. Wienkers, Brooke M. Rock, Dan A. Rock and Larry C. Wienkers
Drug Metabolism and Disposition June 1, 2020, 48 (6) 508-514; DOI: https://doi.org/10.1124/dmd.119.089813
del.icio.us logo Digg logo Reddit logo Twitter logo Facebook logo Google logo Mendeley logo
  • Tweet Widget
  • Facebook Like
  • Google Plus One

Jump to section

  • Article
    • Abstract
    • Introduction
    • Materials and Methods
    • Results
    • Discussion
    • Acknowledgments
    • Authorship Contributions
    • Footnotes
    • Abbreviations
    • References
  • Figures & Data
  • Info & Metrics
  • eLetters
  • PDF + SI
  • PDF

Related Articles

Cited By...

More in this TOC Section

  • Human ADME properties of abrocitinib
  • Impact of physiological microenvironments on HepaRG cells
  • New Dog, Cat, and Pig P450 2J Enzymes
Show more Articles

Similar Articles

Advertisement
  • Home
  • Alerts
Facebook   Twitter   LinkedIn   RSS

Navigate

  • Current Issue
  • Fast Forward by date
  • Fast Forward by section
  • Latest Articles
  • Archive
  • Search for Articles
  • Feedback
  • ASPET

More Information

  • About DMD
  • Editorial Board
  • Instructions to Authors
  • Submit a Manuscript
  • Customized Alerts
  • RSS Feeds
  • Subscriptions
  • Permissions
  • Terms & Conditions of Use

ASPET's Other Journals

  • Journal of Pharmacology and Experimental Therapeutics
  • Molecular Pharmacology
  • Pharmacological Reviews
  • Pharmacology Research & Perspectives
ISSN 1521-009X (Online)

Copyright © 2022 by the American Society for Pharmacology and Experimental Therapeutics