Identification of a New Site of Metabolism for Phenprocoumon by Modeling it’s CYP2C9 Hydroxylation Pattern

Molecular modeling was conducted to study the observed sites of metabolism for cytochrome P450 enzyme CYP2C9 and find a hitherto unknown hydroxylation site for phenprocoumon. Four hydroxylation sites of phenprocoumon metabolism by CYP2C9 were taken from our previous site of metabolism (SoM) identification by mass-spectrometric and selected-reaction-monitoring chromatographic analyses. Phenprocoumon (PPC) and warfarin (WFN) were successfully docked into CYP2C9 and CYP3A4. Docking results for the four monohydroxylation metabolites (6-OH-PPC, 7-OH-PPC, C2'-OH-PPC and C4'-OH-PPC) showed final docking poses in good keeping with the experimental data. The catalytic cycle was revised and ternary coordination complexes between heme B, reactive oxygen and substrate with a tetrahedral geometry (atoms Fe-O-CH) were identified under the molecular mechanics force field (Yeti). Additional quantum mechanics calculations (Gaussian09) also focused on the ternary coordination complex (iron IV - activated oxygen - sp3 hybrid carbon atom). Transition state and equilibrium structures for OH-Ph-S complexes of iron porphyrin moiety were computed with density functional theory (DFT) at B3LYP level of theory. To establish the most preferred mode of interaction between the complexes and the protein structure, the DFT calculations have been used to accomplish the molecular docking studies. As a direct result the hitherto unknown structure of a PPC metabolite could be identified as the 10-hydroxy-metabolite of PPC. To simulate the hydroxylation of the ortho position (2') at the phenyl ring, however, a customary model (rotamer library) was generated. After rotation of three binding-relevant side chains (Val113, Leu365 and Thr301) the sterically congested 2' position became accessible to enzymatic hydroxylation.