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Research ArticleArticle

Identification of Selective Mechanism-Based Inactivators of Cytochromes P-450 2B4 and 2B5, and Determination of the Molecular Basis for Differential Susceptibility

Sharon M. Strobel, Grazyna D. Szklarz, You qun He, Maryam Foroozesh, William L. Alworth, Elizabeth S. Roberts, Paul F. Hollenberg and James R. Halpert
Journal of Pharmacology and Experimental Therapeutics July 1999, 290 (1) 445-451;
Sharon M. Strobel
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Grazyna D. Szklarz
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You qun He
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Maryam Foroozesh
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William L. Alworth
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Elizabeth S. Roberts
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Paul F. Hollenberg
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James R. Halpert
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Abstract

Rabbit cytochromes P-450 (P-450) 2B4 and 2B5 differ by only 12 amino acid residues yet they exhibit unique steroid hydroxylation profiles. Previous studies have led to the identification of active site residues that are determinants of these specificities. In this study, mechanism-based inactivators were identified that discriminate between the closely related 2B4 and 2B5 enzymes. A previously characterized inhibitor, 2-ethynylnaphthalene (2EN), was found to be selective for 2B4 inactivation. As inhibitor metabolism and the partition ratio affect susceptibility, molecular dynamics simulations were performed to assess the stability of the productive binding orientation of 2EN within 2B4 and 2B5 three-dimensional models. Although 2EN was stable within the 2B4 model, it exhibited substantial movement away from the heme moiety in the 2B5 model. However, heterologously expressed 2B5 was found to catalyze the oxidation of 2EN to the stable product 2-naphthylacetic acid. Thus, the increased mobility of 2EN may result in reduced susceptibility of 2B5 by increasing the probability that the reactive ketene intermediate hydrolyzes with water instead of reacting with active site residues. Another compound, 1-adamantyl propargyl ether (1APE), selectively inactivated 2B5. The structural basis for 2EN and 1APE susceptibility was assessed using active site mutants. Interconversion of 2EN susceptibility was observed for 2B4 or 2B5 mutants containing a single alteration at residue 363. Single substitutions in 2B4 also conferred susceptibility to 1APE; however, multiple alterations were required to reduce the susceptibility of 2B5. These alterations may influence inhibitor susceptibility by affecting the stability of the productive binding orientation.

Footnotes

  • Send reprint requests to: Dr. James R. Halpert, Department of Pharmacology and Toxicology, University of Texas Medical Branch at Galveston, 301 University Blvd., Galveston, TX 77555-1031. E-mail:jhalpert{at}utmb.edu

  • ↵1 This work was supported by National Institutes of Health Grants ES03619 (J.R.H.), CA38192 (W.L.A), CA16954 (P.F.H.), and Center Grant ES06694 (University of Arizona).

  • ↵2 Current address: MDS Panlabs, 11804 North Creek Pkwy. S., Bothell, WA 98011.

  • Abbreviations:
    P-450
    cytochrome P-450
    DMSO
    dimethyl sulfoxide
    EPh
    ethynylphenanthrene
    1APE
    1-adamantyl propargyl ether
    5Ph1P
    5-phenyl-1-pentyne
    2EN
    2-ethynylnaphthalene
    2NA
    2-naphthylacetic acid
    BBT
    N-benzyl-1-aminobenzotriazole
    • Received December 10, 1998.
    • Accepted March 12, 1999.
  • The American Society for Pharmacology and Experimental Therapeutics
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Journal of Pharmacology and Experimental Therapeutics: 290 (1)
Journal of Pharmacology and Experimental Therapeutics
Vol. 290, Issue 1
1 Jul 1999
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Research ArticleArticle

Identification of Selective Mechanism-Based Inactivators of Cytochromes P-450 2B4 and 2B5, and Determination of the Molecular Basis for Differential Susceptibility

Sharon M. Strobel, Grazyna D. Szklarz, You qun He, Maryam Foroozesh, William L. Alworth, Elizabeth S. Roberts, Paul F. Hollenberg and James R. Halpert
Journal of Pharmacology and Experimental Therapeutics July 1, 1999, 290 (1) 445-451;

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Research ArticleArticle

Identification of Selective Mechanism-Based Inactivators of Cytochromes P-450 2B4 and 2B5, and Determination of the Molecular Basis for Differential Susceptibility

Sharon M. Strobel, Grazyna D. Szklarz, You qun He, Maryam Foroozesh, William L. Alworth, Elizabeth S. Roberts, Paul F. Hollenberg and James R. Halpert
Journal of Pharmacology and Experimental Therapeutics July 1, 1999, 290 (1) 445-451;
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