ACS Publications. Most Trusted. Most Cited. Most Read
My Activity

Effector-Mediated Alteration of Substrate Orientation in Cytochrome P450 2C9

View Author Information
Department of Basic Pharmaceutical Sciences, West Virginia University School of Pharmacy, Morgantown, West Virginia 26506, and Department of Natural Sciences and Mathematics, West Liberty State College, West Liberty, West Virginia 26074
Cite this: Biochemistry 2004, 43, 22, 7207–7214
Publication Date (Web):May 8, 2004
Copyright © 2004 American Chemical Society

    Article Views





    Other access options


    Cytochrome P450 2C9 (CYP2C9)-mediated flurbiprofen 4‘-hydroxylation is activated by the presence of dapsone resulting in reduction of the Km for flurbiprofen hydroxylation and an increase in Vm. Previous spectral binding studies have demonstrated that the binding of flurbiprofen with CYP2C9 is increased (decrease in KS) by the presence of dapsone. We hypothesized that the two compounds are simultaneously in the active site with the presence of dapsone causing flurbiprofen to be oriented more closely to the heme. T1 relaxation rates determined by NMR were used to estimate the distances of protons on these compounds from the paramagnetic heme-iron center. Samples contained 0.014 μM CYP2C9 and 145 μM flurbiprofen in the presence and absence of 100 μM dapsone. Estimated distances of various flurbiprofen protons from the heme ranged from 4.2 to 4.5 Å in the absence of dapsone and from 3.2 to 3.8 Å in the presence of dapsone. The 4‘ proton of flurbiprofen, the site of metabolism, showed one of the greatest differences in distance from the heme in the presence of dapsone, 3.50 Å, as compared to the absence of dapsone, 4.41 Å. Dapsone protons were less affected, being 4.40 Å from the heme in the absence of flurbiprofen and 4.00−4.01 Å from the heme in the presence of flurbiprofen. Molecular modeling studies were also performed to corroborate the relative orientations of flurbiprofen and dapsone in the active site of CYP2C9. Shift of the 4‘ proton of flurbiprofen closer to the heme iron of CYP2C9 in the presence of dapsone may play a role in activation.

    Read this article

    To access this article, please review the available access options below.

    Get instant access

    Purchase Access

    Read this article for 48 hours. Check out below using your ACS ID or as a guest.


    Access through Your Institution

    You may have access to this article through your institution.

    Your institution does not have access to this content. You can change your affiliated institution below.

     This work was supported in part by grants from the Public Health Service (Grant GM-63215 to T.S.T), National Center for Research Resources (Grant 1P20RR16477 to J.S.A.) and NSF (Grant 1002165R to P.M.G.).

     West Virginia University School of Pharmacy.

     Current Address:  Department of Experimental and Clinical Pharmacology, College of Pharmacy, University of Minnesota, Minneapolis, MN 55455.


     West Liberty State College.


     Corresponding Author. Mailing address:  Department of Experimental and Clinical Pharmacology, College of Pharmacy, University of Minnesota, 7-168 Weaver-Densford Hall, 308 Harvard St. SE, Minneapolis, MN 55455. Tel:  612-625-7665. Fax:  612-625-3927. E-mail:  [email protected].

    Cited By

    This article is cited by 37 publications.

    1. David L. Tierney . Jahn–Teller Dynamics in a Series of High-Symmetry Co(II) Chelates Determine Paramagnetic Relaxation Enhancements. The Journal of Physical Chemistry A 2012, 116 (45) , 10959-10972.
    2. Arthur G. Roberts, Sara E. A. Sjögren, Nadezda Fomina, Kathy T. Vu, Adah Almutairi, and James R. Halpert . NMR-Derived Models of Amidopyrine and Its Metabolites in Complexes with Rabbit Cytochrome P450 2B4 Reveal a Structural Mechanism of Sequential N-Dealkylation. Biochemistry 2011, 50 (12) , 2123-2134.
    3. Sean C. Gay, Arthur G. Roberts, Keiko Maekawa, Jyothi C. Talakad, Wen-Xu Hong, Qinghai Zhang, C. David Stout, and James R. Halpert . Structures of Cytochrome P450 2B4 Complexed with the Antiplatelet Drugs Ticlopidine and Clopidogrel,. Biochemistry 2010, 49 (40) , 8709-8720.
    4. Carrie M. Mosher, Matthew A. Hummel, Timothy S. Tracy and Allan E. Rettie . Functional Analysis of Phenylalanine Residues in the Active Site of Cytochrome P450 2C9. Biochemistry 2008, 47 (45) , 11725-11734.
    5. Michael D. Cameron, Bo Wen, Arthur G. Roberts, William M. Atkins, A. Patricia Campbell and Sidney D. Nelson. Cooperative Binding of Acetaminophen and Caffeine within the P450 3A4 Active Site. Chemical Research in Toxicology 2007, 20 (10) , 1434-1441.
    6. Pramod C. Nair, Kushari Burns, Nuy Chau, Ross A. McKinnon, John O. Miners. The molecular basis of dapsone activation of CYP2C9-catalyzed nonsteroidal anti-inflammatory drug oxidation. Journal of Biological Chemistry 2023, 299 (12) , 105368.
    7. Juan Cai, Rongwei Shi. Structural dynamics of the cooperative binding of small inhibitors in human cytochrome P450 2C9. Journal of Molecular Graphics and Modelling 2022, 113 , 108151.
    8. Libo Zhang, Dumei Ma, Yingwu Yin, Qian Wang. Using Small Molecules to Enhance P450 OleT Enzyme Activity in Situ. Chemistry – A European Journal 2021, 27 (35) , 8940-8945.
    9. Tomohiko Ichikawa, Hirofumi Tsujino, Takahiro Miki, Masaya Kobayashi, Chiaki Matsubara, Sara Miyata, Taku Yamashita, Kohei Takeshita, Yasushige Yonezawa, Tadayuki Uno. Allosteric activation of cytochrome P450 3A4 by efavirenz facilitates midazolam binding. Xenobiotica 2018, 48 (12) , 1227-1236.
    10. Qingbiao Huang, Grazyna Szklarz. Increased Phenacetin Oxidation upon the L382V Substitution in Cytochrome P450 1A2 is Associated with Altered Substrate Binding Orientation. International Journal of Molecular Sciences 2018, 19 (6) , 1580.
    11. Peter Hlavica. Challenges in assignment of allosteric effects in cytochrome P450-catalyzed substrate oxidations to structural dynamics in the hemoprotein architecture. Journal of Inorganic Biochemistry 2017, 167 , 100-115.
    12. Ying Peng, Hui Wu, Xueyuan Zhang, Fengyi Zhang, Huanhuan Qi, Yunxi Zhong, Yu Wang, Hua Sang, Guangji Wang, Jianguo Sun. A comprehensive assay for nine major cytochrome P450 enzymes activities with 16 probe reactions on human liver microsomes by a single LC/MS/MS run to support reliable in vitro inhibitory drug–drug interaction evaluation. Xenobiotica 2015, 45 (11) , 961-977.
    13. Christopher D Bostick, Darcy R Flora, Peter M Gannett, Timothy S Tracy, David Lederman. Nanoscale electron transport measurements of immobilized cytochrome P450 proteins. Nanotechnology 2015, 26 (15) , 155102.
    14. Xue Li, Jinping Hu, Baolian Wang, Li Sheng, Zhihao Liu, Shuang Yang, Yan Li. Inhibitory effects of herbal constituents on P-glycoprotein in vitro and in vivo: Herb–drug interactions mediated via P-gp. Toxicology and Applied Pharmacology 2014, 275 (2) , 163-175.
    15. Qingbiao Huang, Rahul S. Deshmukh, Spencer S. Ericksen, Youbin Tu, Grazyna D. Szklarz. Preferred Binding Orientations of Phenacetin in CYP1A1 and CYP1A2 Are Associated with Isoform-Selective Metabolism. Drug Metabolism and Disposition 2012, 40 (12) , 2324-2331.
    16. Márton Vass, Ákos Tarcsay, György M. Keserű. Multiple ligand docking by Glide: implications for virtual second-site screening. Journal of Computer-Aided Molecular Design 2012, 26 (7) , 821-834.
    17. Anja Keubler, Johanna Weiss, Walter E. Haefeli, Gerd Mikus, Jürgen Burhenne. Drug Interaction of Efavirenz and Midazolam: Efavirenz Activates the CYP3A-Mediated Midazolam 1′-Hydroxylation In Vitro. Drug Metabolism and Disposition 2012, 40 (6) , 1178-1182.
    18. Sean C Gay, Arthur G Roberts, James R Halpert. Structural features of cytochromes P450 and ligands that affect drug metabolism as revealed by x-ray crystallography and NMR. Future Medicinal Chemistry 2010, 2 (9) , 1451-1468.
    19. Charles W. Locuson, Timothy S. Tracy. Metabolism Kinetics. 2010, 1-18.
    20. Murali Subramanian, Timothy S. Tracy. Allosteric Enzyme- and Transporter-Based Interactions. 2010, 497-515.
    21. Ilia G. Denisov, Daniel J. Frank, Stephen G. Sligar. Cooperative properties of cytochromes P450. Pharmacology & Therapeutics 2009, 124 (2) , 151-167.
    22. Dayong Si, Ying Wang, Yi-Han Zhou, Yingjie Guo, Juan Wang, Hui Zhou, Ze-Sheng Li, J. Paul Fawcett. Mechanism of CYP2C9 Inhibition by Flavones and Flavonols. Drug Metabolism and Disposition 2009, 37 (3) , 629-634.
    23. Emre M. Isin, F. Peter Guengerich. Substrate binding to cytochromes P450. Analytical and Bioanalytical Chemistry 2008, 392 (6) , 1019-1030.
    24. Matthew A. Hummel, Peter M. Gannett, Jarrett Aguilar, Timothy S. Tracy. Substrate proton to heme distances in CYP2C9 allelic variants and alterations by the heterotropic activator, dapsone. Archives of Biochemistry and Biophysics 2008, 475 (2) , 175-183.
    25. Melissa A Kramer, Timothy S Tracy. Studying cytochrome P450 kinetics in drug metabolism. Expert Opinion on Drug Metabolism & Toxicology 2008, 4 (5) , 591-603.
    26. Harshica Fernando, Dmitri R. Davydov, Christopher C. Chin, James R. Halpert. Role of subunit interactions in P450 oligomers in the loss of homotropic cooperativity in the cytochrome P450 3A4 mutant L211F/D214E/F304W. Archives of Biochemistry and Biophysics 2007, 460 (1) , 129-140.
    27. Charles W. Locuson, Peter M. Gannett, Robyn Ayscue, Timothy S. Tracy. Use of Simple Docking Methods To Screen a Virtual Library for Heteroactivators of Cytochrome P450 2C9. Journal of Medicinal Chemistry 2007, 50 (6) , 1158-1165.
    28. T.M. Cho, R.L. Rose, E. Hodgson. The Effect of Chlorpyrifos-Oxon and other Xenobiotics on the Human Cytochrome P450-Dependent Metabolism of Naphthalene and DEET. Drug Metabolism and Drug Interactions 2007, 22 (4)
    29. Vikas Kumar, Jan L. Wahlstrom, Dan A. Rock, Chad J. Warren, Lee A. Gorman, Timothy S. Tracy. CYP2C9 Inhibition: Impact of Probe Selection and Pharmacogenetics on in Vitro Inhibition Profiles. Drug Metabolism and Disposition 2006, 34 (12) , 1966-1975.
    30. William M Atkins. Current views on the fundamental mechanisms of cytochrome P450 allosterism. Expert Opinion on Drug Metabolism & Toxicology 2006, 2 (4) , 573-579.
    31. Alexander Seifert, Stephan Tatzel, Rolf D. Schmid, Jürgen Pleiss. Multiple molecular dynamics simulations of human p450 monooxygenase CYP2C9: The molecular basis of substrate binding and regioselectivity toward warfarin. Proteins: Structure, Function, and Bioinformatics 2006, 64 (1) , 147-155.
    32. Charles W. Locuson, Peter M. Gannett, Timothy S. Tracy. Heteroactivator effects on the coupling and spin state equilibrium of CYP2C9. Archives of Biochemistry and Biophysics 2006, 449 (1-2) , 115-129.
    33. Timothy S Tracy. Atypical Cytochrome P450 Kinetics. Drugs in R & D 2006, 7 (6) , 349-363.
    34. Allan E. Rettie, Jeffrey P. Jones. CLINICAL AND TOXICOLOGICAL RELEVANCE OF CYP2C9: Drug-Drug Interactions and Pharmacogenetics. Annual Review of Pharmacology and Toxicology 2005, 45 (1) , 477-494.
    35. Chaohong Sun, Jeffrey R. Huth, Philip J. Hajduk. NMR in Pharmacokinetic and Pharmacodynamic Profiling. ChemBioChem 2005, 6 (9) , 1592-1600.
    36. Matthew A. Hummel, Charles W. Locuson, Peter M. Gannett, Dan A. Rock, Carrie M. Mosher, Allan E. Rettie, Timothy S. Tracy. CYP2C9 Genotype-Dependent Effects on in Vitro Drug-Drug Interactions: Switching of Benzbromarone Effect from Inhibition to Activation in the CYP2C9.3 Variant. Molecular Pharmacology 2005, 68 (3) , 644-651.
    37. Annalise Di Marco, Isabella Marcucci, Ashok Chaudhary, Marina Taliani, Ralph Laufer. DEVELOPMENT AND VALIDATION OF A HIGH-THROUGHPUT RADIOMETRIC CYP2C9 INHIBITION ASSAY USING TRITIATED DICLOFENAC. Drug Metabolism and Disposition 2005, 33 (3) , 359-364.

    Pair your accounts.

    Export articles to Mendeley

    Get article recommendations from ACS based on references in your Mendeley library.

    Pair your accounts.

    Export articles to Mendeley

    Get article recommendations from ACS based on references in your Mendeley library.

    You’ve supercharged your research process with ACS and Mendeley!

    STEP 1:
    Click to create an ACS ID

    Please note: If you switch to a different device, you may be asked to login again with only your ACS ID.

    Please note: If you switch to a different device, you may be asked to login again with only your ACS ID.

    Please note: If you switch to a different device, you may be asked to login again with only your ACS ID.

    Your Mendeley pairing has expired. Please reconnect