Comparison of In Vitro Metabolism of Ticlopidine by Human Cytochrome P450 2B6 and Rabbit Cytochrome P450 2B4S⃞

A recent X-ray crystal structure of a rabbit cytochrome P450 2B4 (CYP2B4)-ticlopidine complex indicated that the compound could be modeled with either the thiophene or chlorophenyl group oriented toward the heme prosthetic group. Subsequent NMR relaxation and molecular docking studies suggested that orientation with the chlorophenyl ring closer to the heme was the preferred one. To evaluate the predictive value of these findings, the oxidation of ticlopidine by reconstituted CYP2B4 was studied and compared with CYP2B6, in which the thiophene portion of the molecule likely orients toward the heme. In vitro incubation of ticlopidine with both enzymes yielded the same set of metabolites: 7-hydroxyticlopidine (M1), 2-oxoticlopidine (M2), 5-(2-chlorobenzyl)thieno[3,2-c]pyridin-5-ium metabolite (M3), 5-(2-chlorobenzyl)thieno[3,2-c]pyridin-5-ium metabolite (M4), ticlopidine N-oxide (M5), and ticlopidine S-oxide dimer, a dimerization product of ticlopidine S-oxide (M6). The rates of metabolite formation deviated markedly from linearity with time, consistent with the known inactivation of CYP2B6 by ticlopidine. Fitting to a first-order equation yielded similar rate constants (kobs) for both enzymes. However, the amplitude (Rmax) of M1 and M6 formation was 4 to 5 times higher for CYP2B6 than CYP2B4, indicating a greater residence time of ticlopidine with its thiophene ring closer to heme in CYP2B6. In contrast, CYP2B4 formed M4 and M5 in more abundance than CYP2B6, indicating an alternate orientation. Overall, the results suggest that the preferential orientation of ticlopidine in the active site of CYP2B4 predicted by X-ray crystallography and NMR studies is unproductive and that ticlopidine likely reorients within CYP2B4 to a more productive mode

Crystal Structure of a Cytochrome P450 2B6 Genetic Variant in Complex with the Inhibitor 4-(4-Chlorophenyl)imidazole at 2.0-Å Resolution

The structure of the K262R genetic variant of human cytochrome P450 2B6 in complex with the inhibitor 4-(4-chlorophenyl)imidazole (4-CPI) has been determined using X-ray crystallography to 2.0-Å resolution. Production of diffraction quality crystals was enabled through a combination of protein engineering, chaperone coexpression, modifications to the purification protocol, and the use of unique facial amphiphiles during crystallization. The 2B6-4-CPI complex is virtually identical to the rabbit 2B4 structure bound to the same inhibitor with respect to the arrangement of secondary structural elements and the placement of active site residues. The structure supports prior P450 2B6 homology models based on other mammalian cytochromes P450 and is consistent with the limited site-directed mutagenesis studies on 2B6 and extensive studies on P450 2B4 and 2B1. Although the K262R genetic variant shows unaltered binding of 4-CPI, altered binding affinity, kinetics, and/or product profiles have been previously shown with several other ligands. On the basis of new P450 2B6 crystal structure and previous 2B4 structures, substitutions at residue 262 affect a hydrogen-bonding network connecting the G and H helices, where subtle differences could be transduced to the active site. Docking experiments indicate that the closed protein conformation allows smaller ligands such as ticlopidine to bind to the 2B6 active site in the expected orientation. However, it is unknown whether 2B6 undergoes structural reorganization to accommodate bulkier molecules, as previously inferred from multiple P450 2B4 crystal structures