title: Drug metabolism by CYP2C8.3 is determined by substrate dependent interactions with cytochrome P450 reductase and cytochrome b5.
abstract: Genetic polymorphisms in CYP2C8 can influence the metabolism of important therapeutic agents and cause interindividual variation in drug response and toxicity. The significance of the variant CYP2C8*3 has been controversial with reports of higher in vivo but lower in vitro activity compared to CYP2C8*1. In this study, the contribution of the redox partners cytochrome P450 reductase (CPR) and cytochrome b5 to the substrate dependent activity of CYP2C8.3 (R139K, K399R) was investigated in human liver microsomes (HLMs) and Escherichia coli expressed recombinant CYP2C8 proteins using amodiaquine, paclitaxel, rosiglitazone and cerivastatin as probe substrates. For recombinant CYP2C8.3, clearance values were two- to five-fold higher compared to CYP2C8.1. CYP2C8.3's higher k(cat) seems to be dominated by a higher, but substrate specific affinity, towards cytochrome b5 and CPR (K(D) and K(m,red)) which resulted in increased reaction coupling. A stronger binding affinity of ligands to CYP2C8.3, based on a two site binding model, in conjunction with a five fold increase in amplitude of heme spin change during binding of ligands and redox partners could potentially contribute to a higher k(cat). In HLMs, carriers of the CYP2C8*1/*3 genotype were as active as CYP2C8*1/*1 towards the CYP2C8 specific reaction amodiaquine N-deethylation. Large excess of cytochrome b5 compared to CYP2C8 in recombinant systems and HLMs inhibited metabolic clearance, diminishing the difference in k(cat) between the two enzymes, and may provide an explanation for the discrepancy to in vivo data. In silico studies illustrate the genetic differences between wild type and variant on the molecular level.
authors: Department of Medicinal Chemistry, University of Washington, Seattle, WA 98195-7610, USA.
authors: Rüdiger
authors: R
authors: Kaspera
authors: Suresh B
authors: SB
authors: Naraharisetti
authors: Eric A
authors: EA
authors: Evangelista
authors: Kristin D
authors: KD
authors: Marciante
authors: Bruce M
authors: BM
authors: Psaty
authors: Rheem A
authors: RA
authors: Totah
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases / Q000235:genetics / Q000378:metabolism*; D002384:Catalysis; D065727:Cytochrome P-450 CYP2C8; D015786:Cytochromes b5 / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology; D006801:Humans; D066298:In Vitro Techniques; D008658:Inactivation, Metabolic; D007700:Kinetics; D008862:Microsomes, Liver / Q000187:drug effects / Q000201:enzymology; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009251:NADPH-Ferrihemoprotein Reductase / Q000235:genetics / Q000378:metabolism*; D010084:Oxidation-Reduction; D004364:Pharmaceutical Preparations / Q000378:metabolism*; D011110:Polymorphism, Genetic; D011485:Protein Binding; D051381:Rats; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D013379:Substrate Specificity
chemical_list: D004364:Pharmaceutical Preparations; D011994:Recombinant Proteins; D015786:Cytochromes b5; D001189:Aryl Hydrocarbon Hydroxylases; C450259:CYP2C8 protein, human; D065727:Cytochrome P-450 CYP2C8; D009251:NADPH-Ferrihemoprotein Reductase