title: High warfarin sensitivity in carriers of CYP2C9*35 is determined by the impaired interaction with P450 oxidoreductase.
abstract: Cytochrome P450 2C9 (CYP2C9) metabolizes many clinically important drugs including warfarin and diclofenac. We have recently reported a new allelic variant, CYP2C9*35, found in a warfarin hypersensitive patient with Arg125Leu and Arg144Cys mutations. Here, we have investigated the molecular basis for the functional consequences of these polymorphic changes. CYP2C9.1 and CYP2C9-Arg144Cys expressed in human embryonic kidney 293 cells effectively metabolized both S-warfarin and diclofenac in NADPH-dependent reactions, whereas CYP2C9-Arg125Leu or CYP2C9.35 were catalytically silent. However, when NADPH was replaced by a direct electron donor to CYPs, cumene hydroperoxide, hereby bypassing the CYP oxidoreductase (POR), all variant enzymes were active, indicating unproductive interactions between CYP2C9.35 and POR. In silico analysis revealed a decrease of the electrostatic potential of CYP2C9-Arg125Leu-POR interacting surface and the loss of stabilizing salt bridges between these proteins. In conclusion, our data strongly suggest that the Arg125Leu mutation in CYP2C9.35 prevents CYP2C9-POR interactions resulting in the absence of NADPH-dependent CYP2C9-catalyzed activity in vivo, thus influencing the warfarin sensitivity in the carriers of this allele.
authors: Section of Pharmacogenetics, Department of Physiology and Pharmacology, Karolinska Institutet, Stockholm, Sweden.
authors: M-Y
authors: 0000000289636165
authors: MY
authors: Lee
authors: Department of Biomedicine and Prevention, Genetics Unit, University of Rome 'Tor Vergata', Rome, Italy.
authors: P
authors: P
authors: Borgiani
authors: Section of Pharmacogenetics, Department of Physiology and Pharmacology, Karolinska Institutet, Stockholm, Sweden.
authors: I
authors: I
authors: Johansson
authors: Department of Biology, University of Rome 'Tor Vergata' and Interuniversity Consortium, National Institute Biostructures and Biosystems (INBB), Rome, Italy.
authors: F
authors: F
authors: Oteri
authors: Section of Pharmacogenetics, Department of Physiology and Pharmacology, Karolinska Institutet, Stockholm, Sweden.
authors: S
authors: S
authors: Mkrtchian
authors: Department of Biology, University of Rome 'Tor Vergata' and Interuniversity Consortium, National Institute Biostructures and Biosystems (INBB), Rome, Italy.
authors: M
authors: M
authors: Falconi
authors: Section of Pharmacogenetics, Department of Physiology and Pharmacology, Karolinska Institutet, Stockholm, Sweden.
authors: M
authors: M
authors: Ingelman-Sundberg
mesh_terms: D000925:Anticoagulants / Q000494:pharmacology*; D001555:Benzene Derivatives / Q000378:metabolism; D002478:Cells, Cultured; D065729:Cytochrome P-450 CYP2C9 / Q000235:genetics*; D004008:Diclofenac / Q000378:metabolism; D006579:Heterozygote; D006801:Humans; D006900:Hydroxylation; D009249:NADP / Q000378:metabolism; D009251:NADPH-Ferrihemoprotein Reductase / Q000378:metabolism*; D014859:Warfarin / Q000378:metabolism / Q000494:pharmacology*
chemical_list: D000925:Anticoagulants; D001555:Benzene Derivatives; D004008:Diclofenac; D009249:NADP; D014859:Warfarin; D065729:Cytochrome P-450 CYP2C9; D009251:NADPH-Ferrihemoprotein Reductase; C007164:cumene hydroperoxide