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

Abstract

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.

Fig. 1.

Determination of K_i for inhibition of 7-MFC O-deethylation by CYP2B6dH and K262R in the presence of inhibitors (A–H). 7-MFC concentrations included in the assay were 2.5, 5, 10, and 50 μM, and the concentrations of the inhibitors used are provided in the plot. Global fitting of all the data from each experiment was used to obtain K_i. The fitting was done using SpectraLab as described under Materials and Methods.

Fig. 2.

Representative type I difference spectra of ticlopidine, clopidogrel, sertraline, and itraconazole binding (A–D inset). The data were fit to the tight-binding equation, as described under Materials and Methods, to derive the K_s values as listed in Table 1.

Fig. 3.

Determination of K_i for inhibition of 7-MFC O-deethylation by Q172H/K262R in the presence of clopidogrel (A) or sertraline (B). 7-MFC concentrations included in the assay were 25, 50, 100, and 150 μM, and the concentrations of the inhibitors used are provided in the plot. Global fitting of all the data from each experiment was used to obtain K_i. Experiments were done in duplicate. The individual K_i values were clopidogrel (0.59, 0.63 μM) and sertraline (2.20, 2.16 μM).

Fig. 4.

Schematic representation of a three-dimensional homology model of CYP2B6dH. The heme is shown in red, whereas the wild-type Gln-172 and Lys-262 are in green and the variant His-172 and Arg-262 are in yellow.