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Research ArticleArticle

Re-engineering of CYP2C9 to Probe Acid-Base Substrate Selectivity

Guoying Tai, Leslie J. Dickmann, Nicholas Matovic, James J. DeVoss, Elizabeth M. J. Gillam and Allan E. Rettie
Drug Metabolism and Disposition October 2008, 36 (10) 1992-1997; DOI: https://doi.org/10.1124/dmd.108.022186
Guoying Tai
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Leslie J. Dickmann
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Nicholas Matovic
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James J. DeVoss
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Elizabeth M. J. Gillam
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Allan E. Rettie
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Abstract

A common feature of many CYP2C9 ligands is their weak acidity. As revealed by crystallography, the structural basis for this behavior involves a charge-pairing interaction between an anionic moiety on the substrate and an active site R108 residue. In the present study we attempted to re-engineer CYP2C9 to better accept basic ligands by charge reversal at this key residue. We expressed and purified the R108E and R108E/D293N mutants and compared their ability with that of native CYP2C9 to interact with (S)-warfarin, diclofenac, pyrene, propranolol, and ibuprofen amine. As expected, the R108E mutant maintained all the native enzyme's pyrene 1-hydroxylation activity, but catalytic activity toward diclofenac and (S)-warfarin was abrogated. In contrast, the double mutant displayed much less selectivity in its behavior toward these control ligands. Neither of the mutants displayed significant enhancement of propranolol metabolism, and all three preparations exhibited a type II (inhibitor) rather than type I (substrate) spectrum with ibuprofen amine, although binding became progressively weaker with the single and double mutants. Collectively, these data underscore the importance of the amino acid at position 108 in the acid substrate selectivity of CYP2C9, highlight the accommodating nature of the CYP2C9 active site, and provide a cautionary note regarding facile re-engineering of these complex cytochrome P450 active sites.

Footnotes

  • Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.

  • doi:10.1124/dmd.108.022186.

  • ABBREVIATIONS: P450, cytochrome P450; NSAID, nonsteroidal anti-inflammatory drug; HPLC, high-performance liquid chromatography; LC, liquid chromatography; MS, mass spectrometry; TFA, trifluoroacetic acid; MAO, monoamine oxidase.

  • ↵1 Current affiliation: GlaxoSmithKline, King of Prussia, PA.

  • ↵2 Current affiliation: Department of Pharmacokinetics and Drug Metabolism, Amgen, Seattle, WA.

  • The American Society for Pharmacology and Experimental Therapeutics
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Drug Metabolism and Disposition: 36 (10)
Drug Metabolism and Disposition
Vol. 36, Issue 10
1 Oct 2008
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Research ArticleArticle

Re-engineering of CYP2C9 to Probe Acid-Base Substrate Selectivity

Guoying Tai, Leslie J. Dickmann, Nicholas Matovic, James J. DeVoss, Elizabeth M. J. Gillam and Allan E. Rettie
Drug Metabolism and Disposition October 1, 2008, 36 (10) 1992-1997; DOI: https://doi.org/10.1124/dmd.108.022186

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Research ArticleArticle

Re-engineering of CYP2C9 to Probe Acid-Base Substrate Selectivity

Guoying Tai, Leslie J. Dickmann, Nicholas Matovic, James J. DeVoss, Elizabeth M. J. Gillam and Allan E. Rettie
Drug Metabolism and Disposition October 1, 2008, 36 (10) 1992-1997; DOI: https://doi.org/10.1124/dmd.108.022186
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