Comparison of in vitro metabolism of ticlopidine by human cytochrome P450 2B6 and rabbit cytochrome P450 2B4.

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 (k(obs)) for both enzymes. However, the amplitude (R(max)) 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.

Structures of cytochrome P450 2B4 complexed with the antiplatelet drugs ticlopidine and clopidogrel .

Prior X-ray crystal structures of rabbit cytochrome P450 2B4 (2B4) in complexes with various imidazoles have demonstrated markedly different enzyme conformations depending on the size of the inhibitor occupying the active site. In this study, structures of 2B4 were determined with the antiplatelet drugs clopidogrel and ticlopidine, which were expected to have greater freedom of movement in the binding pocket. Ticlopidine could be modeled into the electron density maps in two distinct orientations, both of which are consistent with metabolic data gathered with other mammalian P450 enzymes. Results of ligand docking and heme-induced NMR relaxation of drug protons showed that ticlopidine was preferentially oriented with the chlorophenyl group closest to the heme. Because of its stereocenter, clopidogrel was easier to fit in the electron density and exhibited a single orientation, which points the chlorophenyl ring toward the heme. The C(α) traces of both complexes aligned very well with each other and revealed a compact, closed structure that resembles the conformation observed in two previously determined 2B4 structures with the small molecule inhibitors 4-(4-chlorophenyl)imidazole and 1-(4-chlorophenyl)imidazole. The 2B4 active site is able to accommodate small ligands by moving only a small number of side chains, suggesting that ligand reorientation is energetically favored over protein conformational changes for binding of these similarly sized molecules. Adjusting both protein conformation and ligand orientation in the active site gives 2B4 the flexibility to bind to the widest range of molecules, while also being energetically favorable.

Crystal structure of a cytochrome P450 2B6 genetic variant in complex with the inhibitor 4-(4-chlorophenyl)imidazole at 2.0-A 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-A 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.

Rational engineering of cytochromes P450 2B6 and 2B11 for enhanced stability: Insights into structural importance of residue 334.

Rational mutagenesis was used to improve the thermal stability of human cytochrome P450 2B6 and canine P450 2B11. Comparison of the amino acid sequences revealed seven sites that are conserved between the stable 2B1 and 2B4 but different from those found in the less stable 2B6 and 2B11. P334S was the only mutant that showed increased heterologous expression levels and thermal stability in both 2B6 and 2B11. The mechanism of this effect was explored with pressure-perturbation spectroscopy. Compressibility of the heme pocket in variants of all four CYP2B enzymes containing proline at position 334 are characterized by lower compressibility than their more stable serine 334 counterpart. Therefore, the stabilizing effect of P334S is associated with increased conformational flexibility in the region of the heme pocket. Improved stability of P334S 2B6 and 2B11 may facilitate the studies of these enzymes by X-ray crystallography and biophysical techniques.

Decreased susceptibility of the cytochrome P450 2B6 variant K262R to inhibition by several clinically important drugs.

Cytochrome P450 (P450) 2B6 metabolizes a number of clinically relevant drugs and is one of the most highly polymorphic human P450 enzymes, with the Lys(262)-->Arg substitution being especially common in several genetic variants. Therefore, K262R (2B6*4) was created in the CYP2B6dH background (N-terminal-modified and C-terminal His-tagged) and expressed in Escherichia coli. The recombinant CYP2B6dH and K262R were purified and studied to investigate the effect of the Lys(262)-->Arg substitution with six of the most potent drug inhibitors of CYP2B6, namely, clopidogrel, clotrimazole, itraconazole, raloxifene, sertraline, and ticlopidine. K262R showed a >3-fold increase in the K(i) values with clopidogrel, itraconazole, and raloxifene and approximately 6-fold increase in K(i) with sertraline compared with CYP2B6dH. Likewise, K262R showed 2-, 4-, and >20-fold higher K(s) values than CYP2B6dH with clopidogrel, sertraline, and itraconazole, respectively. In contrast, when tested with several known type II inhibitors of CYP2B enzymes, K262R showed a 10-fold lower IC(50) with 4-(phenyl)pyridine and approximately 2-fold lower IC(50) with 4-(4-nitrobenzyl)pyridine or 1-(4-phenyl)benzylimidazole than CYP2B6dH. Subsequent analysis predicted possible in vivo drug-drug interactions between the CYP2B6 substrate efavirenz and drug inhibitors clopidogrel, clotrimazole, itraconazole, sertraline, and ticlopidine. Furthermore, Q172H/K262R (2B6*6), which is the most common genetic variant of CYP2B6 harboring K262R, was created in CYP2B6dH, expressed, purified, and characterized for inhibition. Q172H/K262R showed a >6-fold increase in K(i) with sertraline and clopidogrel compared with CYP2B6dH. The results suggest that individuals, especially homozygotes, with the 2B6*4 or 2B6*6 allele might be less susceptible to drug interactions resulting from P450 inhibition.