Catalytic Specificity of CYP2D Isoforms in Rat and Human

Hiroi, Toyoko; Chow, T. L.; Imaoka, Shingi; Funae, Yoshihiko

doi:10.1124/dmd.30.9.970

Cited by 73 publications

(45 citation statements)

References 27 publications

(25 reference statements)

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“…To date, there is little information about the catalytic specificity of CYP2D isoforms, especially CYP2D3 (Chow et al, 1999;Hiroi et al, 2002). In this study, we demonstrated that CYP2D3 was involving in diazepam p-hydroxylation in rats.…”

Section: Role Of Cyp2d3 In Polymorphism Of Diazepam P-hydroxylationsupporting

confidence: 48%

“…In addition, antibodies generated against the purified P450 enzymes are often not specific. Despite the structural similarities among CYP2D isoforms, significant differences in the ability to metabolize drugs have been observed among these CYP2D isoforms (Wan et al, 1997;Chow et al, 1999;Hiroi et al, 2002). In this study, we identified the enzyme involved in diazepam p-hydroxylation to be CYP2D3 using yeast recombinant CYP2D isoforms.…”

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confidence: 96%

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IMPORTANCE OF CYP2D3 IN POLYMORPHISM OF DIAZEPAMP-HYDROXYLATION IN RATS

Sakai

Saito²,

Kim³

et al. 2005

Drug Metab Dispos

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ABSTRACT:Diazepam was metabolized to three primary metabolites, 3-hydroxy-diazepam, N-desmethyl-diazepam, and p-hydroxy-diazepam. Our previous studies reported metabolic position-specific inter-or intrastrain differences in diazepam metabolism among Sprague-Dawley, Brown Norway, Dark Agouti, and Wistar rats. Especially, there were marked (ϳ300 fold) inter-or intrastrain differences in diazepam p-hydroxylation activity at low concentration of substrate. In this study, we investigated the enzyme that catalyzes diazepam p-hydroxylation. The activity toward diazepam phydroxylation was inhibited by anti-cytochrome P450 2D (CYP2D) antibody, suggesting that this activity was catalyzed by CYP2D isoforms. Comparing the expression levels of the CYP2D subfamily in liver microsomes from various strains of rats using anti-CYP2D2 antibody, we found that there was a band of protein that was consistent with the phenotype of diazepam p-hydroxylation. Nterminal amino acid sequences of the specific protein exactly corresponded to those of CYP2D3, indicating that CYP2D3 might be involved in diazepam p-hydroxylation. Moreover, using rat CYP2D isoforms expressed in yeast, we tested CYP2Ds to catalyze diazepam p-hydroxylation. CYP2D1 and CYP2D2 practically did not participate in diazepam metabolism. On the other hand, diazepam p-hydroxylation was catalyzed by CYP2D3. CYP2D4 had high activity toward diazepam N-desmethylation, but not p-hydroxylation. In conclusion, the polymorphic expression of CYP2D3 caused the inter-or intrastrain differences in diazepam p-hydroxylation among rat strains or individuals.

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Section: Role Of Cyp2d3 In Polymorphism Of Diazepam P-hydroxylationsupporting

confidence: 48%

mentioning

confidence: 96%

IMPORTANCE OF CYP2D3 IN POLYMORPHISM OF DIAZEPAMP-HYDROXYLATION IN RATS

Sakai

Saito²,

Kim³

et al. 2005

Drug Metab Dispos

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“…Phe-483 is also notably absent in these orthologues. 2D4 is essentially inactive toward debrisoquine, although metabolism by 2D2 can proceed smoothly (71). Venhorst et al (21) have attributed this difference in 2D2/4/6 metabolism to a large negative electrostatic potential in the cavities of 2D2 and 2D6, which is quite different in 2D4.…”

Section: Resultsmentioning

confidence: 99%

Crystal Structure of Human Cytochrome P450 2D6

Rowland¹,

Blaney²,

Smyth

et al. 2006

Journal of Biological Chemistry

414

381

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Cytochrome P450 2D6 is a heme-containing enzyme that is responsible for the metabolism of at least 20% of known drugs. Substrates of 2D6 typically contain a basic nitrogen and a planar aromatic ring. The crystal structure of human 2D6 has been solved and refined to 3.0 Å resolution. The structure shows the characteristic P450 fold as seen in other members of the family, with the lengths and orientations of the individual secondary structural elements being very similar to those seen in 2C9. There are, however, several important differences, the most notable involving the F helix, the F-G loop, the B helix, ␤ sheet 4, and part of ␤ sheet 1, all of which are situated on the distal face of the protein. The 2D6 structure has a well defined active site cavity above the heme group, containing many important residues that have been implicated in substrate recognition and binding, including Asp-301, Glu-216, Phe-483, and Phe-120. The crystal structure helps to explain how Asp-301, Glu-216, and Phe-483 can act as substrate binding residues and suggests that the role of Phe-120 is to control the orientation of the aromatic ring found in most substrates with respect to the heme. The structure has been compared with published homology models and has been used to explain much of the reported site-directed mutagenesis data and help understand the metabolism of several compounds. The cytochromes P4504 constitute a superfamily of heme-containing enzymes that catalyze the metabolism of a wide variety of endogenous and xenobiotic compounds. This is accomplished through the activation of molecular oxygen by the heme group, a process that involves the delivery of two electrons to the P450 system followed by cleavage of the dioxygen bond, yielding water and an activated iron-oxygen species (Compound 1), which reacts with substrates through a variety of mechanisms (1). In eukaryotic species, the electron source is a single flavoprotein, the FAD/FMN-containing cytochrome P450 reductase, which binds to the largely basic proximal face of the cytochrome through a number of salt bridges. Of the known human isoforms, cytochrome P450 2D6 is responsible for the metabolism of at least 20% of known drugs (2), with only 3A4 being responsible for a higher (50%) percentage.The cDNA encoding human P450 2D6 has been characterized (3) and subsequently localized to chromosome 22 in the q13.1 region (4). A relatively large number of genetic polymorphisms have been described for 2D6, some of which can either result in rapid or very poor metabolism. One well characterized allelic variant is responsible for a condition known as debrisoquine/sparteine type polymorphism (5, 6). This arises as a result of various genetic mutations and affects a significant percentage of the Caucasian population (7). It results in the defective metabolism of a number of important drug molecules, including debrisoquine, from which the condition got its name. The inability of patients to turn over compounds such as debrisoquine eventually leads to toxic levels of the drug in t...

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“…Of the four rat CYP2D isoforms, substrate specificity (Wan et al, 1997;Hiroi et al, 2002;Grobe et al, 2012), region-selective metabolism (Suzuki et al, 1992;Masubuchi et al, 1993Masubuchi et al, , 1994Narimatsu et al, 1994), and a study on ligand-binding specificities by homology modeling (Venhorst et al, 2003) suggested that CYP2D2 functionally conserves human CYP2D6, and the markedly lower expression of CYP2D2 in female Dark Agouti rats than in Wistar and SD rats allowed female Dark Agouti rats to be used as a model of the CYP2D6 poor metabolizer phenotype in humans (Yamamoto et al, 1998;Schulz-Utermoehl et al, 1999). Homology modeling of rat and human CYP2D isoforms indicated that the negative electrostatic potential on the active site surface conferred common characteristics on both CYP2D2 and CYP2D6, which was distinct from the neutral electrostatic potentials calculated for the other rat CYP2D isoforms (Venhorst et al, 2003).…”

Section: Discussionmentioning

confidence: 99%

Interindividual Variations in Metabolism and Pharmacokinetics of 3-(6-Methylpyridine-3-yl-sulfanyl)-6-(4H-[1,2,4]triazole-3-yl-sulfanyl)-N-(1,3-thiazole-2-yl)-2-pyridine carboxamide, a Glucokinase Activator, in Rats Caused by the Genetic Polymorphism of CYP2D1

Hasegawa¹,

Eiki²,

Chiba³

2014

Drug Metab Dispos

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3-(6-Methylpyridine-3-yl-sulfanyl)-6-(4H-[1,2,4]triazole-3-yl-sulfanyl)-N-(1,3-thiazole-2-yl)-2-pyridine carboxamide (Cpd-D) is a novel glucokinase activator that is being developed for the treatment of type 2 diabetes. Large interindividual variations were observed in the pharmacokinetics of Cpd-D in male Sprague-Dawley (SD) rats, which were subsequently divided into two phenotypes; >6-fold longer terminal-phase half-life and ∼10-fold larger AUC 0-' values were observed in slow metabolizers (SM) than in fast metabolizers (FM) after the oral administration of Cpd-D. The thiohydantoic acid analog (M2) was the predominant metabolite detected in the urine, bile, and plasma after the oral administration of [ 14 C]Cpd-D to the FM phenotypes of bile-duct cannulated SD rats. The liver microsomes prepared from FM phenotyped rats extensively formed M2 with the highest affinity (K m = 0.09 mM) and largest V max /K m value in primary metabolism, whereas those from SM phenotypes had little capacity to form M2. Of the rat cytochrome P450 isoforms tested, the formation of M2 was only catalyzed by recombinant CYP2D1. Sequence substitutions (418A/421C and 418G/421T) were detected in the CYP2D1 gene and were designated F and S alleles, respectively. The genotypephenotype correlation analysis indicated that two S alleles were homozygous (S/S) in the SM phenotypes, whereas the FM phenotypes were either homozygous for the F-alleles (F/F) or heterozygous (F/S). These results indicated that the CYP2D1 polymorphism caused by nucleotide substitutions (418A/421C versus 418G/421T) was responsible for interindividual variations leading to the polymorphism in the major metabolism and pharmacokinetics of Cpd-D in male SD rats.

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Catalytic Specificity of CYP2D Isoforms in Rat and Human

Cited by 73 publications

References 27 publications

IMPORTANCE OF CYP2D3 IN POLYMORPHISM OF DIAZEPAMP-HYDROXYLATION IN RATS

IMPORTANCE OF CYP2D3 IN POLYMORPHISM OF DIAZEPAMP-HYDROXYLATION IN RATS

Crystal Structure of Human Cytochrome P450 2D6

Interindividual Variations in Metabolism and Pharmacokinetics of 3-(6-Methylpyridine-3-yl-sulfanyl)-6-(4H-[1,2,4]triazole-3-yl-sulfanyl)-N-(1,3-thiazole-2-yl)-2-pyridine carboxamide, a Glucokinase Activator, in Rats Caused by the Genetic Polymorphism of CYP2D1

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