Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Protocol
  • Published:

High-throughput fluorescence assay of cytochrome P450 3A4

Abstract

Cytochrome P450 mono-oxygenases (P450s) are the principal enzymes involved in the oxidative metabolism of drugs and other xenobiotics. In this protocol, we describe a fluorescence-based, high-throughput assay for measuring the activity of P450 3A4, one of the key enzymes involved in drug metabolism. The assay involves the oxidative debenzylation of a substituted coumarin, yielding an increase in fluorescence on reaction. The entire procedure can be accomplished in 1 h or less.

This is a preview of subscription content, access via your institution

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Prices may be subject to local taxes which are calculated during checkout

Figure 1
Figure 2: Calculated IC50 of ketoconazole on P450 3A4.

Similar content being viewed by others

References

  1. Isin, E.M. & Guengerich, F.P. Complex reactions catalyzed by cytochrome P450 enzymes. Biochim. Biophys. Acta. 1770, 314–329 (2007).

    Article  CAS  PubMed  Google Scholar 

  2. Guengerich, F.P. Human cytochrome P450 enzymes. in Cytochrome P450: Structure, Mechanism, and Biochemistry, 3rd edn. (ed. Ortiz de Montellano, P.R.) 377–530 (Kluwer Academic/Plenum Press, NY, USA, 2005).

    Chapter  Google Scholar 

  3. Williams, J.A. et al. Drug-drug interactions for UDP-glucuronosyltransferase substrates: a pharmacokinetic explanation for typically observed low exposure (AUCi/AUC) ratios. Drug Metab. Dispos. 32, 1201–1208 (2004).

    Article  CAS  PubMed  Google Scholar 

  4. Wienkers, L.C. & Heath, T.G. Predicting in vivo drug interactions from in vitro drug discovery data. Nat. Rev. Drug Discov. 4, 825–833 (2005).

    Article  CAS  PubMed  Google Scholar 

  5. Fowler, S. & Zhang, H. In vitro evaluation of reversible and irreversible cytochrome P450 inhibition: current status on methodologies and their utility for predicting drug-drug interactions. AAPS J. 10, 410–424 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Turpeinen, M., Uusitalo, J., Jalonen, J. & Pelkonen, O. Multiple P450 substrates in a single run: rapid and comprehensive in vitro interaction assay. Eur. J. Pharm. Sci. 24, 123–132 (2005).

    Article  CAS  PubMed  Google Scholar 

  7. Lin, T., Pan, K., Mordenti, J. & Pan, L. In vitro assessment of cytochrome P450 inhibition: strategies for increasing LC/MS-based assay throughput using a one-point IC50 method and multiplexing high-performance liquid chromatography. J. Pharm. Sci. 96, 2485–2493 (2007).

    Article  CAS  PubMed  Google Scholar 

  8. Youdim, K.A., Lyons, R., Payne, L., Jones, B.C. & Saunders, K. An automated, high-throughput, 384 well cytochrome P450 cocktail IC50 assay using a rapid resolution LC-MS/MS end-point. J. Pharm. Biomed. Anal. 48, 92–99 (2008).

    Article  CAS  PubMed  Google Scholar 

  9. Yamamoto, T., Suzuki, A. & Kohno, Y. Application of microtiter plate assay to evaluate inhibitory effects of various compounds on nine cytochrome P450 isoforms and to estimate their inhibition patterns. Drug Metab. Pharmacokinet. 17, 437–448 (2002).

    Article  CAS  PubMed  Google Scholar 

  10. Schaeffner, I., Petters, J., Aurich, H., Frohberg, P. & Christ, B. A microtiter plate-based screening assay to assess diverse effects on cytochrome P450 enzyme activities in primary rat hepatocytes by various compounds. Assay Drug Dev. Technol. 3, 27–38 (2005).

    Article  CAS  PubMed  Google Scholar 

  11. Kim, D. & Guengerich, F.P. Enhancement of 7-methoxyresorufin O-demethylation of human cytochrome P450 1A2 by molecular breeding. Arch. Biochem. Biophys. 432, 102–108 (2004).

    Article  CAS  PubMed  Google Scholar 

  12. Kim, D., Wu, Z.-L. & Guengerich, F.P. Analysis of coumarin 7-hydroxylation activity of cytochrome P450 2A6 using random mutagenesis. J. Biol. Chem. 280, 40319–40327 (2005).

    Article  CAS  PubMed  Google Scholar 

  13. Crespi, C.L., Miller, V.P. & Penman, B.W. Microtiter plate assays for inhibition of human, drug-metabolizing cytochromes P450. Anal. Biochem. 248, 188–190 (1997).

    Article  CAS  PubMed  Google Scholar 

  14. Onderwater, R.C.A., Venhorst, J., Commandeur, J.N.M. & Vermeulen, N.P.E. Design, synthesis, and characterization of 7-methoxy-4-(aminomethyl) coumarin as a novel and selective cytochrome P450 2D6 substrate suitable for high-throughput screening. Chem. Res. Toxicol. 12, 555–559 (1999).

    Article  CAS  PubMed  Google Scholar 

  15. Nakamura, K., Hanna, I.H., Cai, H., Nishimura, Y., Williams, K.M. & Guengerich, F.P. Coumarin substrates for cytochrome P450 2D6 fluorescence assays. Anal. Biochem. 292, 280–286 (2001).

    Article  CAS  PubMed  Google Scholar 

  16. Cali, J.J. et al. Luminogenic cytochrome P450 assays. Expert Opin. Drug Metab. Toxicol. 2, 629–645 (2006).

    Article  CAS  PubMed  Google Scholar 

  17. Parikh, A., Gillam, E.M.J. & Guengerich, F.P. Drug metabolism by Escherichia coli expressing human cytochromes P450. Nat. Biotechnol. 15, 784–788 (1997).

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

Cytochrome P450 research in this laboratory is supported by United States Public Health Service grant R37 CA090426.

Author information

Authors and Affiliations

Authors

Contributions

Q.C. performed and optimized the assays. Q.C. wrote most of the paper, with the assistance of F.P.G. and C.D.S. C.D.S. checked the protocols and helped Q.C. with data processing.

Corresponding author

Correspondence to F Peter Guengerich.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Cheng, Q., Sohl, C. & Guengerich, F. High-throughput fluorescence assay of cytochrome P450 3A4. Nat Protoc 4, 1258–1261 (2009). https://doi.org/10.1038/nprot.2009.123

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nprot.2009.123

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing