PT  - JOURNAL ARTICLE
AU  - Brooke M VandenBrink
AU  - John A Davis
AU  - Josh T Pearson
AU  - Robert S Foti
AU  - Larry C. Wienkers
AU  - Dan A Rock
TI  - Cytochrome P450 Architecture and Cysteine Nucleophile Placement Impacts Raloxifene Mediated Mechanism-Based Inactivation.
AID  - 10.1124/mol.112.080739
DP  - 2012 Aug 02
TA  - Molecular Pharmacology
PG  - mol.112.080739
4099  - http://molpharm.aspetjournals.org/content/early/2012/08/02/mol.112.080739.short
4100  - http://molpharm.aspetjournals.org/content/early/2012/08/02/mol.112.080739.full
AB  - The propensity for cytochrome P450 (CYP) enzymes to bioactivate xenobiotics is governed by the inherent chemistry of the xenobiotic itself and the active site architecture of the P450 enzyme(s). Accessible nucleophiles in the active site or egress channels of the P450 enzyme have the potential of sequestering reactive metabolites through covalent modification, thereby limiting their exposure to other proteins. Raloxifene, a drug known to undergo CYP3A-mediated reactive metabolite formation and time-dependent inhibition in vitro, was utilized to explore the potential for bioactivation and enzyme inactivation of additional CYP enzymes (CYP1A2, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1 and CYP3A5). Every CYP tested except CYP2E1 was capable of raloxifene bioactivation, based on glutathione adduct formation. However, raloxifene mediated time-dependent inhibition only occurred in CYP2C8 and CYP3A4. Comparable inactivation kinetics were achieved with a KI and kinact values of 0.26 μM and 0.10 min-1, 0.81 μM and 0.20 min-1 for CYP2C8 and CYP3A4, respectively. Proteolytic digests of CYP2C8 and CYP3A4 Supersomes™ revealed adducts to Cys225 and Cys239 for CYP2C8 and CYP3A4, respectively. For each CYP enzyme, proposed substrate/metabolite access channels were mapped and active site cysteines were identified, which revealed only CYP2C8 and CYP3A4 possess accessible cysteine residues near the active site cavities, a result consistent with the observed kinetics. The combined data suggest the extent of bioactivation across CYP enzymes does not correlate to CYP inactivation. In addition, multiple factors contribute to the ability of reactive metabolites to form apo-adducts with a CYP enzyme