Mechanism-based inactivation of CYP2D6 by methylenedioxymethamphetamine.

The potency of methylenedioxymethamphetamine (MDMA) as a mechanism-based inhibitor of CYP2D6 has been defined using microsomes prepared from yeast expressing the enzyme and from three human livers. The inhibitory effect was increased by preincubation through formation of a metabolic intermediate complex. Inactivation parameters (kinact and KI), defined with respect to the O-demethylation of dextromethorphan, were 0.29 +/- 0.03 (S.E.) min(-1) and 12.9 +/- 3.6 (S.E.) microM for yeast-expressed CYP2D6, and 0.26 +/- 0.02 min(-1) and 14.4 +/- 2.5 microM, 0.15 +/- 0.01 min(-1) and 8.8 +/- 2.6 microM, and 0.12 +/- 0.05 min(-1) and 45.3 +/- 32.1 microM for the liver microsomal preparations. The rate of inactivation of CYP2D6 by MDMA decreased when quinidine, a competitive inhibitor of CYP2D6, was added to the primary incubation mixture. However, inactivation was unaffected by the addition of glutathione. The results indicate that MDMA is a potent mechanism-based inhibitor of CYP2D6, with implications for understanding its in vivo disposition and drug interaction potential.

Inter-individual variability in levels of human microsomal protein and hepatocellularity per gram of liver.

AIMS: To determine levels of microsomal protein (MPPGL) and hepatocellularity (HPGL) per gram of human liver and their interindividual variability. METHODS: Triplicate liver samples were used to determine values of MPPGL (n = 20) and HPGL (n = 7) after accounting for the fractional loss of microsomal protein or hepatocytes during processing. Repeated measurements from each liver sample allowed the estimation of true interindividual variability in MPPGL and HPGL using ANOVA. RESULTS: The value of MPPGL ranged from 26 to 54 mg g(-1) (mean(geo)= 33 mg g(-1)). The value of HPGL ranged from 65 to 185 x 10(6) cells g(-1) (mean(geo)= 10(7) x 10(6) cells g(-1)). CONCLUSIONS: There is significant interindividual variability in MPPGL, which has implications for the accurate extrapolation of in vitro data on drug metabolism to predict in vivo metabolic clearance.

A discordance between cytochrome P450 2D6 genotype and phenotype in patients undergoing methadone maintenance treatment.

AIMS: To assess CYP2D6 activity and genotype in a group of patients undergoing methadone maintenance treatment (MMT). METHODS: Blood samples from 34 MMT patients were genotyped by a polymerase chain reaction-based method, and results were compared with CYP2D6 phenotype (n = 28), as measured by the molar metabolic ratio (MR) of dextromethorphan (DEX)/dextrorphan (DOR) in plasma. RESULTS: Whereas 9% of patients (3/34) were poor metabolizers (PM) by genotype, 57% (16/28) were PM by phenotype (P < 0.005). Eight patients, who were genotypically extensive metabolizers (EM), were assigned as PM by their phenotype. The number of CYP2D6*4 alleles and sex were significant determinants of CYP2D6 activity in MMT patients, whereas other covariates (methadone dose, age, weight) did not contribute to variation in CYP2D6 activity. CONCLUSIONS: There was a discordance between genotype and in vivo CYP2D6 activity in MMT patients. This finding is consistent with inhibition of CYP2D6 activity by methadone and may have implications for the safety and efficacy of other CYP2D6 substrates taken by MMT patients.

In-vitro analysis of the contribution of CYP2D6.35 to ultra-rapid metabolism.

From 10 to 30% of CYP2D6 ultra-rapid metabolizers of Caucasian origin harbor alleles with duplicated or amplified functional CYP2D6 genes. Recently, the CYP2D6*35 allele has been reported to be more frequent in ultra-rapid metabolizing subjects than in extensive metabolizers, suggesting a possible role of this variant in CYP2D6 duplication-negative ultra-rapid metabolizing subjects. In this study, we examined the functional consequences of the Val11Met, Arg296Cys and Ser486Thr amino acid substitutions associated with the CYP2D6*35 on the expression and catalytic activity of the variant enzyme, heterologously expressed in yeast. Our results indicate that the functional activity and level of expression of recombinant CYP2D6.35 are comparable with those of the wild-type enzyme, thus precluding the hypothesis that the high level of enzyme activity in CYP2D6 duplication-negative ultra-rapid metabolizing subjects is a consequence of the expression of a more catalytically effective CYP2D6.35 enzyme.

Influence of phenylalanine-481 substitutions on the catalytic activity of cytochrome P450 2D6.

Homology models of the active site of cytochrome P450 2D6 (CYP2D6) have identified phenylalanine 481 (Phe(481)) as a putative ligand-binding residue, its aromatic side chain being potentially capable of participating in pi-pi interactions with the benzene ring of ligands. We have tested this hypothesis by replacing Phe(481) with tyrosine (Phe(481)-->Tyr), a conservative substitution, and with leucine (Phe(481)-->Leu) or glycine (Phe(481)-->Gly), two non-aromatic residues, and have compared the properties of the wild-type and mutant enzymes in microsomes prepared from yeast cells expressing the appropriate cDNA-derived protein. The Phe(481)-->Tyr substitution did not alter the kinetics [K(m) (microM) and V(max) (pmol/min per pmol) respectively] of oxidation of S-metoprolol (27; 4.60), debrisoquine (46; 2.46) or dextromethorphan (2; 8.43) relative to the respective wild-type values [S-metoprolol (26; 3.48), debrisoquine (51; 3.20) and dextromethorphan (2; 8.16)]. The binding capacities [K(s) (microM)] of a range of CYP2D6 ligands to the Phe(481)-->Tyr enzyme (S-metoprolol, 22.8; debrisoquine, 12.5; dextromethorphan, 2.3; quinidine, 0.13) were also similar to those for the wild-type enzyme (S-metoprolol, 10.9; debrisoquine, 8.9; dextromethorphan, 3.1; quinidine, 0.10). In contrast, the Phe(481)-->Leu and Phe(481)-->Gly substitutions increased significantly (3-16-fold) the K(m) values of oxidation of the three substrates [S-metoprolol (120-124 microM), debrisoquine (152-184 microM) and dextromethorphan (20-31 microM)]. Similarly, the K(s) values of the ligands to Phe(481)-->Leu and Phe(481)-->Gly mutants were also increased 3 to 10-fold (S-metoprolol, 33.2-41.9 microM; debrisoquine, 85-90 microM; dextromethorphan, 15.7-18.8 microM; quinidine 0.35-0.53 microM). However, contrary to a recent proposal that Phe(481) has the dominant role in the binding of substrates that undergo CYP2D6-mediated N-dealkylation routes of metabolism, the Phe(481)-->Gly substitution did not substantially decrease the capacity of the enzyme to N-deisopropylate metoprolol (wild-type, 1.12 pmol/min per pmol of P450; Phe(481)-->Gly, 0.71), whereas an Asp(301)-->Gly substitution decreased the N-dealkylation reaction by 95% of the wild-type rate. Overall, our results are consistent with the proposal that Phe(481) is a ligand-binding residue in the active site of CYP2D6 and that the residue interacts with ligands via a pi-pi interaction between its phenyl ring and the aromatic moiety of the ligand. However, the relative importance of Phe(481) in binding is ligand-dependent; furthermore, its importance is secondary to that of Asp(301). Finally, contrary to predictions of a recent homology model, Phe(481) does not seem to have a primary role in CYP2D6-mediated N-dealkylation.

Regioselective hydroxylation of debrisoquine by cytochrome P4502D6: implications for active site modelling.

1. Debrisoquine, a prototypic probe substrate for human cytochrome P4502D6 (CYP2D6), is hydroxylated at the alicyclic C4-position by this enzyme. Phenolic metabolites of debrisoquine (5-, 6-, 7- and 8-hydroxydebrisoquine) have also been reported as in vivo metabolites, but the role of CYP2D6 in their formation is unclear. 2. As part of studies to develop a predictive model of the active site of CYP2D6 using pharmacophore and homology modelling techniques, it became important to determine the precise regioselective hydroxylation of debrisoquine by CYP2D6. 3. Data from studies with human liver microsomes and yeast microsomes containing cDNA-derived CYP2D6 demonstrated unequivocally that debrisoquine was hydroxylated by CYP2D6 at each aromatic site in the molecule, as well as at the alicyclic 4-position. The four phenolic metabolites amounted to > 60% of the total identified products and the pattern of regioselective hydroxylation (4-HD > 7-HD > 6-HD > 8-HD > 5-HD) was similar in both in vitro systems. 4. A pharmacophore model for CYP2D6 indicated that while the hydroxylation of debrisoquine at alternative positions could arise from the substrate adopting multiple binding orientations, the energy constraints for the aromatic hydroxylations were unfavourable. An alternative proposal involving essentially a single binding orientation and a mechanism of hydroxylation based on benzylic radical spin delocalization could satisfactorily rationalize all the hydroxylations of debrisoquine. 5. This latter proposal demonstrates the need to consider the mechanism of oxidation as well as the spatial orientation of the substrate in the development of a predictive model of the active site of CYP2D6.

Evidence that serine 304 is not a key ligand-binding residue in the active site of cytochrome P450 2D6.

Homology models of cytochrome P450 2D6 (CYP2D6) have identified serine 304 as an active-site residue and implicated a putative role for this residue in substrate enantioselectivity and the differential inhibition of enzyme activity by the diastereoisomers quinine and quinidine. The role of serine 304 in selectivity is thought to be achieved through a preferential hydrogen-bond interaction between the hydroxyl group of the residue and one of the stereoisomers of each ligand. We have tested this hypothesis by substituting serine 304 with alanine, a non-hydrogen-bonding residue, and compared the properties of the wild-type and mutant enzymes in microsomes prepared from yeast cells expressing the appropriate cDNA-derived enzyme. The Ser(304)Ala substitution did not alter the enantioselective oxidation of metoprolol; the O-demethylation reaction remained R-(+)-enantioselective (wild-type, R/S, 1.7; mutant, R/S, 1.6), whereas alpha-hydroxylation remained S-(-)-enantioselective (wild-type and mutant, R/S, 0.7). Similarly, the selective oxidation of the R-(+) and S-(-) enantiomers of propranolol to the major 4-hydroxy metabolite was identical with both wild-type and mutant forms of the enzyme (R/S 0.9), although the formation of minor metabolites (5-hydroxy and deisopropylpropranolol) did show some slight alteration in enantioselectivity. The differential inhibition of enzyme activity by quinine and quinidine was also identical with both forms of CYP2D6, the IC(50) values for each enzyme being approx. 10 microM and 0.1 microM for quinine and quinidine, respectively. The kinetics of formation of alpha-hydroxymetoprolol and 4-hydroxydebrisoquine by wild-type and the Ser(304)Ala mutant was also very similar. However, modest changes in the regioselective oxidation of metoprolol and debrisoquine were observed with the Ser(304)Ala mutant. The regio- and enantioselective oxidation of an analogue of metoprolol, in which the hydroxyl group attached to the chiral carbon was replaced by a methyl moiety, was again identical with both wild-type and Ser(304)Ala mutant. However, the observed selectivity was the reverse of that observed with metoprolol. Collectively, these data indicate that Ser(304) is unlikely to be a key ligand-binding residue, although the residue may indeed be located in the active-site cavity. The reversal of selectivity with the methyl analogue of metoprolol indicates that the hydroxyl group attached to the chiral centre of ligands, such as metoprolol, is important in defining the enzyme's selective properties, and that a hydrogen-bonding residue, other than Ser(304), may be involved in this interaction. Current homology models of the active site of CYP2D6 that predict a hydrogen-bond interaction between Ser(304) and specific ligands will need to be re-evaluated, and other candidate residues capable of such an interaction nominated and tested by site-directed mutagenesis studies.

Polymorphic debrisoquine 4-hydroxylase activity in the rat is due to differences in CYP2D2 expression.

The female Dark Agouti rat is widely used as an animal model for the CYP2D6 poor metabolizer phenotype, males of other strains such as Sprague Dawley or Wistar serving as models for the extensive metabolizer phenotype. To determine the relative level of expression of CYP2D enzymes in the liver of female and male Dark Agouti, Sprague Dawley and Wistar rats, anti-peptide antibodies were raised in rabbits against short synthetic peptides representing the C-termini of the rat P450 enzymes CYP2D1, CYP2D2, CYP2D3, CYP2D4 and CYP2D5. In immunoblotting studies, it was found that the hepatic expression of CYP2D1 was greater in Dark Agouti rats than Sprague Dawley or Wistar rats. In contrast, hepatic CYP2D2 was 30-40-fold less abundant in female Dark Agouti than female Sprague Dawley or Wistar rats and six- to eightfold less abundant in male Dark Agouti than male Sprague Dawley or Wistar rats. No hepatic CYP2D3 could be detected in either sex of any of the three strains. Hepatic CYP2D4 expression was generally greater in male than female rats, and higher in Dark Agouti compared with Sprague Dawley or Wistar strains. CYP2D5 was expressed in the livers of female and male Dark Agouti rats but not in female Sprague Dawley or Wistar rats. This form was variably expressed in livers of male Sprague Dawley and Wistar rats. Hepatic debrisoquine 4-hydroxylase activity was markedly reduced in female and male Dark Agouti rats as compared to Sprague Dawley or Wistar rats and correlated (r = 0.88; P < 0.001) with the hepatic CYP2D2 content. Recombinant CYP2D2 was 18-fold more active at catalysing the 4-hydroxylation of debrisoquine than CYP2D1. Furthermore, quinine markedly inhibited CYP2D2-mediated debrisoquine and metoprolol oxidation, while quinidine, its diastereoisomer, inhibited the reactions to a lesser extent. In conclusion, these results show that impaired debrisoquine 4-hydroxylase activity in the female Dark Agouti rat is due to low levels of CYP2D2.

First principles investigation of singly reduced cytochrome P450.

1. The application of novel ab initio quantum mechanical methods to the states in the catalytic cycle of cytochrome P450 following the first reduction step is described. 2. A good correlation was found between the calculated energy of reduction and the experimentally determined redox potential for a range of substrate- and substrate analogue-bound systems. 3. On reduction of the haem system, the ground state of Fe remains Fe3+. On binding of a CO molecule, Fe adopts a low-spin Fe2+ state, in agreement with experiment. However, on binding of an O2 molecule, calculations indicate that the system adopts a ferric superoxide ground state, in which the Fe is in a low-spin Fe3+ state.

Study of the association between cytochromes P450 2D6 and 2E1 genotypes and the risk of drug and chemical induced idiosyncratic aplastic anaemia.

A genetic susceptibility to drug or chemical toxicity may provide a basis for an increased risk of idiosyncratic aplastic anaemia (AA). The cytochrome P450 enzymes are responsible for the metabolism of many drugs, some of which have been linked to AA. Mutations in the cytochrome P450 CYP2D6 gene result in absent or impaired enzyme activity in about 7% of Caucasians, whereas a specific mutation in the 5'-regulatory region of the CYP2E1 gene causes overexpression of the gene. We evaluated the frequency of allelic variants of CYP2D6 and CYP2E1 using allele-specific PCR amplification and restriction enzyme analysis of blood mononuclear cell DNA among 54 Caucasian AA patients. CYP2D6 and CYP2E1 were chosen because of the link between AA and the antipsychotic drug remoxipride (CYP2D6 substrate) and benzene (CYP2E1 substrate), respectively. Results were compared with 53 controls matched for age, sex and ethnicity. The percentage of AA patients homozygous for the CYP2D6*3, CYP2D6*4 alleles (poor metabolizer phenotype) and the CYP2E1 mutant allele (overexpression) was 0%, 4% and 0%, respectively, and the percentage of heterozygotes was 2%, 28% and 15%, respectively. For normal controls the corresponding results for homozygous mutants were 0%, 4% and 0% and for heterozygotes 4%, 25% and 6%, respectively. We concluded that there were no major differences in the frequencies of the genetic polymorphisms between this series of AA patients and controls, but due to the low number of cases with the poor metabolizer phenotype and those with a history of drug exposure, the power of the study was too low to disprove an interaction.

An ab initio approach to the understanding of cytochrome P450-ligand interactions.

1. We describe the application of novel ab initio quantum mechanical methods to the study of ligand interactions with cytochrome P450cam (CYP101). 2. We find that our techniques accurately describe the transition from a low-spin state to a high-spin state of the haem Fe3+ on binding of a substrate. Furthermore, our methods correctly predict that a large fraction of low-spin character is retained on binding of an inhibitor. 3. We demonstrate the use of 'computational experiments' to elucidate key features of the mechanism of interaction. This leads us to identify a new mechanism for the suppression of the low- to high-spin transition on binding of an inhibitor, namely the shortening of the bond between the Fe atom and the coordinated S atom of the cysteine axial ligand.

Oxidation of methamphetamine and methylenedioxymethamphetamine by CYP2D6.

Methamphetamine (MeAmp) abuse has recently experienced a resurgence and approaches to the treatment of its addiction similar to those used with cocaine have been considered. As the treatment regimes are likely to use drugs whose metabolism is related to that of MeAmp, studies were initiated to establish the enzymology of the fate of MeAmp. This report describes investigations of the role of CYP2D6, the human isoform of the enzyme that catalyzes debrisoquine hydroxylation, in the 4-hydroxylation and N-demethylation of MeAmp. The results of studies with human liver microsomes including those from a genetically poor metabolizer with respect to CYP2D6, showing correlation between MeAmp and metoprolol hydroxylation and MDMA demethylenation, were consistent with a major involvement of CYP2D6 in the aromatic 4-hydroxylation of MeAmp. This was confirmed by studies with recombinant CYP2D6 expressed in yeast, which was also shown to effect the N-demethylation of MeAmp. The rate of the 4-hydroxylation reaction was substantially slower than the demethylenation of MDMA. In contrast to MeAmp, MDMA was not N-demethylated by CYP2D6. Since CYP2D6 participates in the major steps of MeAmp metabolism, pharmacokinetic interactions are likely with other drug substrates proposed for the treatment of MeAmp addiction. Furthermore, the genetic polymorphism associated with the enzyme could manifest itself in abnormal responses to MeAmp.

Cytochromes P450 mediating the N-demethylation of amitriptyline.

AIMS: Using human liver microsomes and heterologously expressed human enzymes, we have investigated the involvement of CYPs 1A2, 2C9, 2C19, 2D6 and 3A4 in the N-demethylation of amitriptyline (AMI), with a view to defining likely influences on its clinical pharmacokinetics. METHODS: The kinetics of formation of nortriptyline (NT) from AMI were measured over the substrate concentration range 1-500 microM, using liver microsomes from four extensive metabolisers (EM) and one poor metaboliser (PM) with respect to CYP2D6 activity. RESULTS: The data were best described by a two-site model comprising a Michaelis-Menten function for a high affinity site and a Hill function for a low affinity site. The activity at the low affinity site was eliminated by triacetyloleandomycin and ketoconazole, selective inhibitors of CYP3A4, such that the kinetics were then described by a two-site model comprising two Michaelis-Menten functions. A further decrease in activity was associated with the addition of the CYP2C9 inhibitor sulphaphenazole such that the residual kinetics were best described by a single Michaelis-Menten function. The addition of quinidine, a selective inhibitor of CYP2D6, along with triacetyloleandomycin and sulphaphenazole produced an additional decrease in the rate of NT formation in all but the PM liver, but did not completely eliminate the reaction. The remaining activity was best described by a single Michaelis-Menten function. Inhibitors of CYP1A2 (furafylline) and CYP2C19 (mephenytoin) did not impair NT formation. Microsomes from yeast cells expressing CYP2D6 and from human lymphoblastoid cells expressing CYP3A4 or CYP2C9-Arg N-demethylated AMI, but those from cells expressing CYPs 1A2 and 2C19 did not. CONCLUSIONS: We conclude that CYPs 3A4, 2C9 and 2D6 together with an unidentified enzyme, but not CYPs 1A2 and 2C19, mediate the N-demethylation of AMI. Thus, the clinical pharmacokinetics of AMI would be expected to depend upon the net activities of all of these enzymes. However, the quantitative importance of each isoform is difficult to predict without knowledge of the exposure of the enzymes in vivo to AMI.

Variable contribution of cytochromes P450 2D6, 2C9 and 3A4 to the 4-hydroxylation of tamoxifen by human liver microsomes.

4-Hydroxylation is an important pathway of tamoxifen metabolism because the product of this reaction is intrinsically 100 times more potent as an oestrogen receptor antagonist than is the parent drug. Although tamoxifen 4-hydroxylation is catalysed by human cytochrome P450 (CYP), data conflict on the specific isoforms responsible. The aim of this study was to define unequivocally the role of individual CYPs in the 4-hydroxylation of tamoxifen by human liver microsomes. Microsomes from each of 10 human livers catalysed the reaction [range = 0.6-2.9 pmol/mg protein/min (1 microM substrate concentration) and 6-25 pmol/mg protein/min (18 microM)]. Three of the livers with the lowest tamoxifen 4-hydroxylation activity were from genetically poor metabolisers with respect to CYP2D6. Inhibition of activity by quinidine (1 microM), sulphaphenazole (20 microM) and ketoconazole (2 microM), selective inhibitors of CYPs 2D6, 2C9 and 3A4, respectively, was 0-80%, 0-80% and 12-57%. The proportion of activity inhibited by quinidine correlated positively with total microsomal tamoxifen 4-hydroxylation activity (rs = 0.89, P < 0.01), indicating a major involvement of CYP2D6 in this reaction. Recombinant human CYPs 2D6, 2C9 and 3A4 but not CYPs 1A1, 1A2, 2C19 and 2E1 displayed significant 4-hydroxylation activity. Similar inhibition and correlation experiments confirmed that tamoxifen N-demethylation is catalysed predominantly by CYP3A4. These findings indicate that the 4-hydroxylation of tamoxifen is catalysed almost exclusively by CYPs 2D6, 2C9 and 3A4 in human liver microsomes. However, the marked between-subject variation in the contribution of these isoforms underlines the need to study metabolic reactions in a sufficient number of livers that are characterised with respect to a range of cytochrome P450 activities.

Ab initio molecular modeling in the study of drug metabolism.

We discuss the use of ab initio quantum mechanical methods in drug metabolism studies. These methods require only the positions and atomic numbers of the atoms to be specified and offer greater transferability than conventional molecular modeling techniques. This fact, coupled with the accuracy of our approach, permits 'computational experiments' to be performed, allowing details of reaction mechanisms to be understood. We review the application of these methods to the cytochrome P450 superfamily of enzymes. There is much interest in understanding the mechanisms of these enzymes due to their participation in a wide range of metabolic processes including drug activation/deactivation. We find that our methods accurately reproduce the low- to high-spin transition of the haem Fe on binding of a substrate. Furthermore, we identify a new mechanism for the suppression of this spin transition, namely the shortening of the bond between the Fe atom and the coordinated S atom of the cysteine axial ligand. These results indicate that ab initio molecular modeling may be usefully applied in the study of drug metabolism and that further study of intermediate states in the P450 reaction cycle would be beneficial, particularly those which are not accessible using conventional experimental approaches.

Expression of human cytochrome P4501A1 (CYP1A1) in Saccharomyces cerevisiae inhibits cell division.

1. Saccharomyces cerevisiae cells, genetically engineered to express human cytochrome P4501A1 (CYP1A1), have a mean doubling time of 5.8 h, which is considerably slower than that of control yeast cells that have undergone the same transformation process but with a plasmid lacking CYP1A1 cDNA (3.3 h). 2. A smaller reduction in the rate of cell division is observed in yeast cells expressing the closely related human P450, CYP1A2. No reduction is seen with plaice CYP1A, despite similar levels of P450 expression and enzyme activity (ethoxyresorufin O-deethylation) and no inhibition of growth is observed with yeast cells expressing higher levels of human CYP2D6. 3. Repeated culture of cells from a single CYP1A1 transformant colony results in a gradual loss of P450 expression and of CYP1A1-associated enzymatic activity over a 5-6 week period. In contrast, expression of human CYP2D6 by a single transformant colony is stable for at least 6 months. 4. The loss of CYP1A1 activity from transformed cells is accompanied by a return to normal growth rate, similar to that of control cells. 5. Inhibition of CYP1A1 enzyme activity during culture, by either type I (alpha-naphthoflavone), type II (ellipticine) or mechanism-based (1-(1'propynyl)pyrene) CYP1A inhibitors, does not affect growth rate, suggesting that some other property of human CYP1A1 protein is responsible for the growth inhibition observed.

Variable contribution of CYP2D6 to the N-dealkylation of S-(-)-propranolol by human liver microsomes.

Recombinant cDNA expression systems for CYP2D6 have been shown to have significant catalytic activity with respect to the N-dealkylation of propranolol. However, the involvement of CYP2D6 in this reaction in human liver is inconclusive. We have re-evaluated the role of CYP2D6 in the dealkylation of S-(-)-propranolol using a bank of 10 human livers characterized for their specific CYP2D6 and CYP1A2 activities, the latter enzyme being known to be involved substantially in the formation of N-desisopropylpropranolol. Using quinidine (1 microM) or LKM-1 antibodies as selective inhibitors of CYP2D6, the contribution of this enzyme to net N-desisopropylation of S-(-)-propranolol (10 microM) by microsomes from the range of livers was found to vary from nil (poor metabolizer genotype) to 60%. N-desisopropylpropranolol formation inhibitable by quinidine was highly correlated with specific CYP2D6 activity, as measured by the alpha-hydroxylation of metoprolol (rs = 0.90; P < 0.001). The two livers with the highest proportion of CYP2D6-mediated N-dealkylation had relatively high ratios of specific CYP2D6 to CYP1A2 activity. These findings emphasize that data obtained using microsomes from single human livers or pooled microsomes from several livers may be misleading inasmuch as the relative contribution of different isoenzymes to the same metabolic reaction may show considerable between-subject variation.

Active-site topologies of human CYP2D6 and its aspartate-301 --> glutamate, asparagine, and glycine mutants.

Cytochrome P450 2D6 (CYP2D6) catalyzes the oxidation of substrates with a positively charged nitrogen atom 5-7 angstroms from the site of the oxidation. The active-site topology of CYP2D6 is examined here with phenyl-, 2-naphthyl-, and p-biphenyldiazene, which react with P450 enzymes to form sigma-bonded aryl-iron (Fe-Ar) complexes. Ferricyanide-mediated migration of the aryl group from the iron to the porphyrin nitrogens produces the N-arylprotoporphyrin IX regioisomers (NB:NA:NC:ND, in which the aryl group is bound to the nitrogen of pyrrole rings B, A, C, and D, respectively) in the following ratios (zero means <5%): phenyl, 10:90:00:00; 2-naphthyl, 09:91:00:00; and p-biphenyl, 16:84:00:00. These results suggest that the CYP2D6 active site is open above pyrrole ring A and to a small extent above pyrrole ring B but is closed above pyrrole rings C and D. This geometry differs from those determined by the same method for P450s for which crystal structures are available. Replacement of Asp-301 by a Glu, which preserves the carboxylate side chain, causes no detectable change in the N-aryl porphyrin regioisomer patterns and only minor changes in the catalytic activity. Replacement of Asp-301 by an Asn or Gly, which eliminates the negatively charged side chain, suppresses migration of the aryl groups to pyrrole ring B without impairing migration to pyrrole ring A and virtually abolishes catalytic activity. These results provide a refined model of the active site of CYP2D6. They confirm, furthermore, that the loss of activity observed when Asp-301 is replaced by a neutral residue is due to loss of the charge-pairing interaction with the substrate positive charge and/or subtle structural effects in the vicinity of pyrrole ring B, but not to major structural reorganization of the active site.

Influence of amino acid residue 374 of cytochrome P-450 2D6 (CYP2D6) on the regio- and enantio-selective metabolism of metoprolol.

Cytochrome P-450 2D6 (CYP2D6) is an important human drug-metabolizing enzyme responsible for the oxidation of more than 30 widely used therapeutic agents. The enzymes encoded by the published genomic [Kimura, Umeno, Skoda, Meyer and Gonzalez (1989) Am. J. Hum. Genet. 45, 889-904] and cDNA [Gonzalez, Skoda, Kimura, Umeno, Zanger, Nebert, Gelboin, Hardwick and Meyer (1988) Nature 331, 442-446] sequences of CYP2D6, and presumed to represent wild-type sequences, differ at residue 374 and encode valine (CYP2D6-Val) and methionine (CYP2D6-Met) respectively. The influence of this amino acid difference on cytochrome P-450 expression, ligand binding, catalysis and stereoselective oxidation of metoprolol was investigated by the heterologous expression of the corresponding cDNAs in the yeast Saccharomyces cerevisiae. The level of expression of apo- and holo-protein was similar with each form of CYP2D6 cDNA, and the binding affinities of a series of ligands to CYP2D6-Val and CYP2D6-Met were identical. The enantioselective O-demethylation and alpha-hydroxylation of metoprolol were also similar with each form of CYP2D6, O-demethylation being R-(+)- enantioselective (CYP2D6-Val: R/S, 1.6; CYP2D6-Met: R/S, 1.4), whereas alpha-hydroxylation showed a preference for S-(-)-metoprolol (CYP2D6-Val: R/S, 0.7; CYP2D6-Met: R/S, 0.8). However, although the favoured regiomer overall was O-demethylmetoprolol (ODM), the regioselectivity for O-demethylation of each metoprolol enantiomer was significantly greater for CYP2D6-Val [R-(+)-: ODM/alpha-hydroxymetoprolol (alpha OH), 5.9; S-(-)-: ODM/alpha OH, 2.5) than that observed for CYP2D6-Met [R-(+)-: ODM/alpha OH, 2.2; S-(-)-: ODM/alpha OH, 1.4]. The stereoselective properties of CYP2D6-Val were consistent with those observed for CYP2D6 in human liver microsomes. The difference in the stereoselective properties of CYP2D6-Val and CYP2D6-Met were rationalized with respect to a homology model of the active site of CYP2D6 based on an alignment with the crystal structure of the bacterial cytochrome P-450BM-3' CYP102.