title: Comparison of in vitro metabolism of ticlopidine by human cytochrome P450 2B6 and rabbit cytochrome P450 2B4.
abstract: 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.
authors.affiliation: Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego, La Jolla, California, USA.
authors.forename: Jyothi C
authors.initials: JC
authors.lastname: Talakad
authors.forename: Manish B
authors.initials: MB
authors.lastname: Shah
authors.forename: Gregory S
authors.initials: GS
authors.lastname: Walker
authors.forename: Cathie
authors.initials: C
authors.lastname: Xiang
authors.forename: James R
authors.initials: JR
authors.lastname: Halpert
authors.forename: Deepak
authors.initials: D
authors.lastname: Dalvie
mesh_terms: D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D055162:Biocatalysis; D016642:Bupropion / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D003374:Coumarins / Q000378:metabolism; D065702:Cytochrome P-450 CYP2B6; D065607:Cytochrome P-450 Enzyme Inhibitors; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D000072467:Cytochrome P450 Family 2; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D015394:Molecular Structure; D010084:Oxidation-Reduction; D010975:Platelet Aggregation Inhibitors / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D011817:Rabbits; D011994:Recombinant Proteins / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization; D053719:Tandem Mass Spectrometry; D013988:Ticlopidine / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology
chemical_list: D003374:Coumarins; D065607:Cytochrome P-450 Enzyme Inhibitors; D004791:Enzyme Inhibitors; D010975:Platelet Aggregation Inhibitors; D011994:Recombinant Proteins; D016642:Bupropion; C056033:7-ethoxy-4-trifluoromethylcoumarin; D003577:Cytochrome P-450 Enzyme System; D001189:Aryl Hydrocarbon Hydroxylases; C585599:CYP2B6 protein, human; D065702:Cytochrome P-450 CYP2B6; D000072467:Cytochrome P450 Family 2; C465241:cytochrome P-450 CYP2B4 (rabbit); D013988:Ticlopidine