Amino Acid Residues Critical for Differential Inhibition of CYP2B4, CYP2B5, and CYP2B1 by Phenylimidazoles
- Margit Spatzenegger1,
- Qinmi Wang1,
- You Qun He1,
- Michael R. Wester2,
- Eric F. Johnson2 and
- James R. Halpert1
- 1Department of Pharmacology and Toxicology, University of Texas Medical Branch, Galveston, Texas (M.S., Q.W., Y.Q.H., J.R.H.);2Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, California (M.R.W., E.F.J.)
Abstract
The molecular basis for reversible inhibition of rabbit CYP2B4 and CYP2B5 and rat CYP2B1 by phenylimidazoles was assessed with active-site mutants and new three-dimensional models based on the crystal structure of CYP2C5. 4-Phenylimidazole was 17- to 32-fold more potent toward CYP2B4 and CYP2B1 than CYP2B5. The 3D models, along with site-directed mutagenesis data, revealed the importance of residue 114 for sensitivity to inhibition of all three CYP2B enzymes. Besides Ile 114, Val 367 was also found to be critical for inhibition of CYP2B4 and CYP2B1. The most interesting new insights were obtained from analysis of the CYP2B5 model and the CYP2B5 active-site mutants. Simultaneous substitution of residues 114, 294, 363, and 367 with the corresponding residues of CYP2B4 decreased the IC50 value for inhibition by 4-phenylimidazole 12-fold. Docking 4-phenylimidazole into the models of CYP2B5 mutants demonstrated that the inhibitor-binding site is strongly influenced by residue-residue interactions, especially between residues 114 and 294. A chlorine substitution at position 4 of the phenyl moiety of 4- and 1-phenylimidazole resulted in IC50values 95- and 130-fold lower for CYP2B4 than for CYP2B5, respectively, suggesting that these compounds are selective inhibitors of CYP2B4. Overall, the study revealed that differences in the determinants of inhibition between CYP2B4 and CYP2B5 are caused not only by single residue inhibitor contacts but also by residue-residue interactions. This new generation of CYP2B models may provide valuable information for the design of selective inhibitors of human CYP2B6 and for the development of drugs that avoid drug interactions due to P450 inhibition.
Footnotes
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Send reprint requests to: Dr. Margit Spatzenegger, Department of Pharmacology and Toxicology, University of Texas Medical Branch at Galveston, 301 University Blvd., Galveston, TX 77555-1031. E-mail: maspatze{at}utmb.edu
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↵1 A very similar effect was observed in CYP2B2 when Leu 58 and Ile 114 were replaced simultaneously with Phe residues. The IC50 value of 4-phenylimidazole increased from 0.8 μM for the wild-type enzyme to 44 μM for CYP2B2 L58F-I114F.
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↵2 The distances reported underResults are those between the nearest atoms of the residue and 4-phenylimidazole.
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This work was supported by AstraZeneca and National Institutes of Health Grants ES03619 (J.R.H.), GM31001 (E.F.J.), and Center Grant ES06676. An abstract of this work appeared in FASEB J14:158.
- Abbreviations:
- P450
- cytochrome P450
- SRS
- substrate recognition site
- CHAPS
- 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid
- BROD
- benzyloxyresorufin O-debenzylase
- PROD
- pentoxyresorufinO-dealkylase
- WT
- wild-type
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- Received August 18, 2000.
- Accepted November 2, 2000.
- The American Society for Pharmacology and Experimental Therapeutics
