Wild-type Cytochrome P450 2D6 Research Articles

Spontaneous Ligand Access Events to Membrane-Bound Cytochrome P450 2D6 Sampled at Atomic Resolution

The membrane-anchored enzyme Cytochrome P450 2D6 (CYP2D6) is involved in the metabolism of around 25% of marketed drugs and its metabolic performance shows a high interindividual variation. While it was suggested that ligands access the buried active site of the enzyme from the membrane, no proof from unbiased simulations has been provided to support this hypothesis. Laboratory experiments fail to capture the access process which is suspected to influence binding kinetics. Here, we applied unbiased molecular dynamics (MD) simulations to investigate the access of ligands to wild-type CYP2D6, as well as the allelic variant CYP2D6*53. In multiple simulations, substrates accessed the active site of the enzyme from the protein-membrane interface to ultimately adopt a conformation that would allow a metabolic reaction. We propose the necessary steps for ligand access and the results suggest that the increased metabolic activity of CYP2D6*53 might be caused by a facilitated ligand uptake.

Functional characterization of 50 CYP2D6 allelic variants by assessing primaquine 5-hydroxylation

Cytochrome P450 2D6 (CYP2D6) is responsible for the metabolic activation of primaquine, an antimalarial drug. CYP2D6 is genetically polymorphic, and these polymorphisms are associated with interindividual variations observed in the therapeutic efficacy of primaquine. To further understand this association, we performed in vitro enzymatic analyses of the wild-type CYP2D6.1 and 49 CYP2D6 allelic variants, which were expressed in 293FT cells, using primaquine as a substrate. The concentrations of CYP2D6 variant holoenzymes were measured by using carbon monoxide (CO)-reduced difference spectroscopy, and the wild type and 27 variants showed a peak at 450 nm. The kinetic parameters Km, Vmax, and intrinsic clearance (Vmax/Km) of primaquine 5-hydroxylation were characterized. The kinetic parameters of the wild type and 16 variants were measured, but the values for the remaining 33 variants could not be determined because of low metabolite concentrations. Among the variants, six (i.e., CYP2D6.17, .18, .35, .39, .53, and .70) showed significantly reduced intrinsic clearance compared with that of CYP2D6.1. Three-dimensional structural modeling analysis was performed to elucidate the mechanism of changes in the kinetics of CYP2D6 variants. Our findings provide insights into the allele-specific activity of CYP2D6 for primaquine, which could be clinically useful for malaria treatment and eradication efforts.

Molecular Dynamics Simulations Reveal Structural Differences among Allelic Variants of Membrane-Anchored Cytochrome P450 2D6.

Cytochrome P450 2D6 (CYP2D6) is an enzyme that is involved in the metabolism of roughly 25% of all marketed drugs and therefore belongs to the most important enzymes in drug metabolism. CYP2D6 features a high degree of genetic polymorphism that can significantly affect the metabolic activity of an individual. In extreme cases, structural changes at the level of single amino acids can either increase its enzymatic activity abolishing the drug therapeutic effect or completely disable the enzyme and elevate drug plasma level potentially leading to adverse effects. In this study, starting from the crystal structure, we built a full-length membrane-anchored all-atom model of the wild-type CYP2D6 as well as five of its variants differing in the enzymatic activity. We validated our models with available experimental data and compared their structural properties with molecular dynamics simulations. The main focus of this study was to identify differences that could mechanistically explain the altered activity of the variants and improve our understanding of their functioning. We observed differences in the opening frequencies and minimal diameters of tunnels that connect the buried active site to the surrounding solvent environment. The variants CYP2D6*4 and CYP2D6*10 associated with missing or decreased activity showed less frequent opening of the tunnels compared to the wild-type. Both CYP2D6*10 and CYP2D6*17 showed a deprivation of an important ligand tunnel suggesting a feasible reason for their altered substrate specificity. Next, the altered fold at the N-terminal anchor region and the decreased active site volume caused by the amino acid mutations of the CYP2D6*4 variant offer an explanation for the absence of its metabolic activity. The mutations in CYP2D6*53 contributed to a significant enlargement of an important ligand tunnel and an extension of the active site cavity. This could explain the altered metabolic profile as well as the enhanced metabolic rates of this particular variant supporting its designation as a possible cause for the ultrarapid metabolizer phenotype. We believe these novel structural insights could advance the fields of personalized medicine and enzyme engineering. Furthermore, they could aid in guiding laboratory as well as computational experiments in the future.

Effect of 22 CYP2D6 variants found in the Chinese population on tolterodine metabolism in vitro

Cytochrome P450 2D6 (CYP2D6) is an important member of the cytochrome P450 enzyme superfamily. We recently identified 22 novel variants in the Chinese population using PCR and bidirectional sequencing methods. The aim of this study is to characterize the enzymatic activity of these variants and their effects on the metabolism of the antimuscarinic drug tolterodine in vitro. A baculovirus-mediated expression system was used to express wild-type CYP2D6 and 24 variants (CYP2D6*2, CYP2D6*10, and 22 novel CYP2D6 variants) at high levels. The insect microsomes expressing CYP2D6 proteins were incubated with 0.1–50 μM tolterodine at 37 °C for 30 min and the metabolites were analyzed by high-performance liquid chromatography–tandem mass spectrometry system. Of the 24 CYP2D6 variants tested, 2 variants (CYP2D6*92 and CYP2D6*96) were found to be catalytically inactive, 4 variants (CYP2D6*94, F164L, F219S and D336N) exhibited markedly increased intrinsic clearance values (Vmax/Km) compared with the wild-type (from 66.34 to 99.79%), whereas 4 variants (CYP2D6*10, *93, *95 and E215K) exhibited significantly decreased values (from 49.02 to 98.50%). This is the first report of all these rare alleles for tolterodine metabolism and these findings suggest that more attention should be paid to subjects carrying these infrequent CYP2D6 alleles when administering tolterodine in the clinic.

Effect of 22 Novel Cytochrome P450 2D6 (CYP2D6) Variants Found in the Chinese Population on Hemangeol Metabolism In Vitro.

Hemangeol, approved for the treatment of proliferative infantile hemangiomas requiring systemic therapy, is metabolized by cytochrome P450 2D6 (CYP2D6), which is a highly polymorphic enzyme that metabolizes a large number of drugs. More than 100 CYP2D6 allelic variants have been reported so far, including 22 novel variants that discovered in our lab in the Chinese population. Our study aimed to probe the enzymatic activity of these variants toward hemangeol in vitro with recombinant microsomes that expressed in sf21 insect cells using a baculovirus-mediated expression system. The wild-type CYP2D6.1 and other variants (CYP2D6.2, CYP2D6.10 and 22 novel CYP2D6 variants) were incubated with 1-200μM hemangeol for 50min at 37°C. Then the products were extracted, and signal detection was performed by high-performance liquid chromatography with fluorescence detector. All of the variants exhibited changed apparent Michaelis-Menten constant (Km) or maximum velocity of the reaction (V max) values compared with that of wild-type protein. The intrinsic clearances (V max /Km) were significantly decreased by 0.37 to 42.74%. However, CYP2D6.92 and CYP2D6.96 showed no or minimal enzymatic activity as no concentration of 4'-hydroxypropranolol was detected. The comprehensive in vitro assessment of CYP2D6 variants provides significant insights into allele-specific activity towards hemangeol in vivo.

Effects of 22 Novel CYP2D6 Variants Found in the Chinese Population on the Bufuralol and Dextromethorphan Metabolisms In Vitro.

Cytochrome P450 2D6 (CYP2D6) is a highly polymorphic enzyme that metabolizes a large number of therapeutic drugs. To date, more than 100 CYP2D6 allelic variants have been reported. Among these variants, we recently identified 22 novel variants in the Chinese population. The aim of this study was to functionally characterize the enzymatic activity of these variants in vitro. A baculovirus-mediated expression system was used to express wild-type CYP2D6.1 and other variants (CYP2D6.2, CYP2D6.10 and 22 novel CYP2D6 variants) at high levels. Then, the insect microsomes containing expressed CYP2D6 proteins were incubated with bufuralol or dextromethorphan at 37°C for 20 or 25 min., respectively. After termination, the metabolites were extracted and used for the detection with high-performance liquid chromatography. Among the 24 CYP2D6 variants tested, two variants (CYP2D6.92 and CYP2D6.96) were found to be catalytically inactive. The remaining 22 variants exhibited significantly decreased intrinsic clearance values for bufuralol 1'-hydroxylation and 20 variants showed significantly lower intrinsic clearance values for dextromethorphan O-demethylation than those of the wild-type CYP2D6.1. Our in vitro results suggest that most of the variants exhibit significantly reduced catalytic activities compared with the wild-type, and these data provide valuable information for personalized medicine in Chinese and other Asian populations.

In vitro functional assessment of 22 newly identified CYP2D6 allelic variants in the Chinese population.

Cytochrome P450 2D6 (CYP2D6) is one of the most widely investigated CYPs related to genetic polymorphisms and is responsible for one-quarter of the currently used clinical drugs. We previously detected 22 novel, non-synonymous, mutated sites in the Chinese population, but nothing is known about the functional effects of these mutations in terms of specific CYP2D6 substrates. In this study, wild-type CYP2D6, two common allelic variants and 22 newly reported CYP2D6 isoforms were transiently expressed in 293FT cells, and the enzymatic activities of these variants were systematically assessed using dextromethorphan and bufuralol as the probing substrates. Consequently, 19 and 21 allelic variants were found to exhibit significantly decreased enzymatic activities for dextromethorphan and bufuralol, respectively. Of 22 novel CYP2D6 variants, six allelic isoforms (CYP2D6.89, CYP2D6.92, CYP2D6.93, CYP2D6.96, E215K and R440C) exhibited absent or extremely reduced metabolic activities compared with those observed for the wild-type enzyme. Our in vitro functional data can be useful for CYP2D6 phenotype prediction and provide valuable information for the study of clinical impact of these newly found CYP2D6 variants in China.

Functional Characterization of Wild-type and 49 CYP2D6 Allelic Variants for N-Desmethyltamoxifen 4-Hydroxylation Activity

Genetic variations in cytochrome P450 2D6 (CYP2D6) contribute to interindividual variability in the metabolism of clinically used drugs, e.g., tamoxifen. CYP2D6 is genetically polymorphic and is associated with large interindividual variations in therapeutic efficacy and drug toxicity. In this study, we performed an in vitro analysis of 50 allelic variants of CYP2D6 proteins. Wild-type CYP2D6.1 and 49 variants were transiently expressed in COS-7 cells, and the enzymatic activities of the CYP2D6 variants were characterized using N-desmethyltamoxifen as a substrate. The kinetic parameters K(m), V(max), and intrinsic clearance (V(max)/K(m)) of N-desmethyltamoxifen 4-hydroxylation were determined. Among the 50 CYP2D6 variants, the kinetic parameters for N-desmethyltamoxifen 4-hydroxylation were determined for 20 CYP2D6 variants. On the other hand, the kinetic parameters of 30 CYP2D6 variants could not be determined because the amount of metabolite produced was at or below the detection limit at the lower substrate concentrations. Among them, 8 variants, i.e., CYP2D6.2, .9, .26, .28, .32, .43, .45, and .70, showed decreased intrinsic clearance at <50% of CYP2D6.1. The comprehensive in vitro assessment of CYP2D6 variants provides novel insights into allele-specific activity towards tamoxifen and may be valuable when interpreting in vivo studies.

CYP2D6 Genotype Should Not Be Used to Determine Endocrine Therapy in Postmenopausal Breast Cancer Patients

The antiestrogen tamoxifen is an effective treatment for all stages of estrogen receptor (ER)-positive breast cancer.1 Tamoxifen blocks estrogen-dependent breast cancer growth by competing with estrogen for binding to its receptor. Tamoxifen itself has antiestrogenic properties, but it is metabolized by a number of cytochrome P450 enzymes into many different metabolites that have varying degrees of antiestrogenic activity. Studies have consistently shown that cytochrome P450 2D6 (CYP2D6), a highly polymorphic drug-metabolizing enzyme, is the enzyme primarily responsible for the production of one of the most potent metabolites, endoxifen (4-hydroxy-N-desmethyl-tamoxifen). Preclinical studies of estrogen-dependent breast cancer have shown that endoxifen is an approximately 100-fold more potent antiestrogen than tamoxifen or the major tamoxifen metabolite, N-desmethyl-tamoxifen. Patients who are homozygous for two null CYP2D6 alleles, and are therefore poor metabolizers (PMs), as well as patients who are taking medications that are CYP2D6 inhibitors, have lower serum endoxifen concentrations as compared with patients who have one or more wild-type CYP2D6 alleles (intermediate metabolizers (IMs) and extensive metabolizers (EMs), respectively) (reviewed by Hertz et al.2).

Double site saturation mutagenesis of the human cytochrome P450 2D6 results in regioselective steroid hydroxylation

The human cytochrome P450 2D6 (CYP2D6) is one of the major human drug metabolizing enzymes and acts preferably on substrates containing a basic nitrogen atom. Testosterone - just as other steroids - is an atypical substrate and only poorly metabolized by CYP2D6. The present study intended to investigate the influence of the two active site residues 216 and 483 on the capability of CYP2D6 to hydroxylate steroids such as for example testosterone. All 400 possible combinatorial mutations at these two positions have been generated and expressed individually in Pichia pastoris. Employing whole-cell biotransformations coupled with HPLC-MS analysis the testosterone hydroxylase activity and regioselectivity of every single CYP2D6 variant was determined. Covering the whole sequence space, CYP2D6 variants with improved activity and so far unknown regio-preference in testosterone hydroxylation were identified. Most intriguingly and in contrast to previous literature reports about mutein F483I, the mutation F483G led to preferred hydroxylation at the 2β-position, while the slow formation of 6β-hydroxytestosterone, the main product of wild-type CYP2D6, was further reduced. Two point mutations have already been sufficient to convert CYP2D6 into a steroid hydroxylase with the highest ever reported testosterone hydroxylation rate for this enzyme, which is of the same order of magnitude as for the conversion of the standard substrate bufuralol by wild-type CYP2D6. Furthermore, this study is also an example for efficient human CYP engineering in P. pastoris for biocatalytic applications and to study so far unknown pharmacokinetic effects of individual and combined mutations in these key enzymes of the human drug metabolism.

Human Liver Enzymes Responsible for Metabolic Elimination of Tyramine, a Vasopressor Agent from Daily Food

Dietary tyramine is associated with hypertensive crises because of its ability to induce the release of catecholamines. The roles of monoamine oxidase (MAO); flavin-containing monooxygenase (FMO); and cytochrome P450 2D6 (CYP2D6) were studied in terms of the enzymatic elimination of tyramine in vitro at a substrate concentration of 1.0 µM; which is relevant to in vivo serum concentrations. Tyramine elimination by human liver supernatant fractions was decreased by ˜70% in the absence of NADPH. Pargyline; an MAO inhibitor; decreased tyramine elimination rates by ˜30%. Among recombinant P450 and FMO enzymes; CYP2D6 had a high activity in terms of tyramine elimination. Tyramine elimination rates were inhibited by quinidine and significantly correlated with bufuralol 1'-hydroxylation activities (a CYP2D6 marker). Liver microsomes genotyped for CYP2D6*10/*10 and CYP2D6*4/*4 showed low and undetectable activities; respectively; compared with the wild-type CYP2D6*1/*1. The present results suggest that tyramine is eliminated mainly by polymorphic CYP2D6. Tyramine toxicity resulting from differences in individual metabolic elimination is thus genetically determined.

Effects of monoamine oxidase inhibitor and cytochrome P450 2D6 status on 5-methoxy- N, N-dimethyltryptamine metabolism and pharmacokinetics

5-Methoxy- N, N-dimethyltryptamine (5-MeO-DMT) is a natural psychoactive indolealkylamine drug that has been used for recreational purpose. Our previous study revealed that polymorphic cytochrome P450 2D6 (CYP2D6) catalyzed 5-MeO-DMT O-demethylation to produce active metabolite bufotenine, while 5-MeO-DMT is mainly inactivated through deamination pathway mediated by monoamine oxidase (MAO). This study, therefore, aimed to investigate the impact of CYP2D6 genotype/phenotype status and MAO inhibitor (MAOI) on 5-MeO-DMT metabolism and pharmacokinetics. Enzyme kinetic studies using recombinant CYP2D6 allelic isozymes showed that CYP2D6.2 and CYP2D6.10 exhibited 2.6- and 40-fold lower catalytic efficiency ( V max/ K m), respectively, in producing bufotenine from 5-MeO-DMT, compared with wild-type CYP2D6.1. When co-incubated with MAOI pargyline, 5-MeO-DMT O-demethylation in 10 human liver microsomes showed significantly strong correlation with bufuralol 1′-hydroxylase activities ( R 2 = 0.98; P < 0.0001) and CYP2D6 contents ( R 2 = 0.77; P = 0.0007), whereas no appreciable correlations with enzymatic activities of other P450 enzymes. Furthermore, concurrent MAOI harmaline sharply reduced 5-MeO-DMT depletion and increased bufotenine formation in human CYP2D6 extensive metabolizer hepatocytes. In vivo studies in wild-type and CYP2D6-humanized (Tg- CYP2D6) mouse models showed that Tg- CYP2D6 mice receiving the same dose of 5-MeO-DMT (20 mg/kg, i.p.) had 60% higher systemic exposure to metabolite bufotenine. In addition, pretreatment of harmaline (5 mg/kg, i.p.) led to 3.6- and 4.4-fold higher systemic exposure to 5-MeO-DMT (2 mg/kg, i.p.), and 9.9- and 6.1-fold higher systemic exposure to bufotenine in Tg- CYP2D6 and wild-type mice, respectively. These findings indicate that MAOI largely affects 5-MeO-DMT metabolism and pharmacokinetics, as well as bufotenine formation that is mediated by CYP2D6.

Response: Re: Pharmacogenomic Variation of CYP2D6 and the Choice of Optimal Adjuvant Endocrine Therapy for Postmenopausal Breast Cancer: A Modeling Analysis

Adjuvant endocrine treatment with aromatase inhibitors improves disease-free survival compared with tamoxifen in postmenopausal women with estrogen receptor-positive breast cancer. This difference could be due to differences in tamoxifen metabolism because levels of endoxifen, the active tamoxifen metabolite, vary with the number of mutant alleles, including the *4 allele, of the gene encoding cytochrome P450 2D6 (CYP2D6).We created a Markov model to determine whether tamoxifen or aromatase inhibitor monotherapy maximized 5-year disease-free survival for patients with the wild-type CYP2D6 genotype (wt/wt). Annual risks of recurrence with aromatase inhibitors and tamoxifen in breast cancer patients who were not selected by CYP2D6 genotype were derived from the Breast International Group 1-98 trial. Genotype frequencies and the hazard ratio for cancer recurrence on tamoxifen among patients with the *4/*4 genotype relative to the wt/wt or wt/*4 genotypes (HR(*4/*4) = 1.86) were based on data from an analysis of the North Central Cancer Treatment Group trial of adjuvant tamoxifen. We explored the impact of CYP2D6(*4) heterozygosity on disease-free survival for wt/wt patients by studying a range of effect (ie, recurrence on tamoxifen) estimates, from no effect of the single mutation (Eff(wt/*4) = 0, recurrence rate in wt/*4 patients same as that in wt/wt patients) to complete effect (Eff(wt/*4) = 1 recurrence rate in wt/*4 patients same as that in *4/*4 patients).With HR(*4/*4) = 1.86 and Eff(wt/*4) = 0.5, the 5-year disease-free survival of tamoxifen-treated patients with no mutations (wt/wt) was 83.9%, that is, essentially the same as that (84.0%) for genotypically unselected patients who were treated with aromatase inhibitors. With greater HR(*4/*4) estimates, disease-free survival with tamoxifen exceed that with aromatase inhibitors in wt/wt patients, even at lower assumed Eff(wt/*4) ratios.Modeling suggests that among patients who are wild type for CYP2D6, 5-year disease-free survival outcomes are similar to or perhaps even superior with tamoxifen than with aromatase inhibitors. Endocrine therapy tailored to CYP2D6 genotype could be considered for women who are newly diagnosed with breast cancer, particularly those who have with concerns about either the relative toxicity or the increased cost of aromatase inhibitors.

Functional Characterization of 17CYP2D6Allelic Variants (CYP2D6.2, 10, 14A–B, 18, 27, 36, 39, 47–51, 53–55, and 57)

Cytochrome P450 2D6 (CYP2D6) is an enzyme of potential importance for the metabolism of drugs used clinically, and it exhibits genetic polymorphism with interindividual differences in metabolic activity. To date, 21 CYP2D6 allelic variants have been identified in the Japanese population. The aim of this study was to investigate the functional characterization of CYP2D6 variants identified in Japanese subjects. Wild-type CYP2D6 and its variants, namely, CYP2D6.2, CYP2D6.10, CYP2D6.14A, CYP2D6.14B, CYP2D6.18, CYP2D6.27, CYP2D6.36, CYP2D6.39, CYP2D6.47, CYP2D6.48, CYP2D6.49, CYP2D6.50, CYP2D6.51, CYP2D6.53, CYP2D6.54, CYP2D6.55, and CYP2D6.57 were transiently expressed in COS-7 cells, and enzymatic activities of the CYP2D6 variant proteins were characterized using bufuralol and dextromethorphan. Functional characterization of 17 CYP2D6 variants revealed an absence of enzyme activity in four (CYP2D6.14A, CYP2D6.36, CYP2D6.47, and CYP2D6.57), low activity in eight (CYP2D6.10, CYP2D6.14B, CYP2D6.18, CYP2D6.49, CYP2D6.50, CYP2D6.51, CYP2D6.54, and CYP2D6.55), and high activity in one (CYP2D6.53) compared with the wild type. Analysis of CYP2D6 variant proteins can be useful for predicting CYP2D6 phenotypes and could be applied to personalized drug therapy.

Pharmacogenomic Variation of CYP2D6 and the Choice of Optimal Adjuvant Endocrine Therapy for Postmenopausal Breast Cancer: A Modeling Analysis

Adjuvant endocrine treatment with aromatase inhibitors improves disease-free survival compared with tamoxifen in postmenopausal women with estrogen receptor-positive breast cancer. This difference could be due to differences in tamoxifen metabolism because levels of endoxifen, the active tamoxifen metabolite, vary with the number of mutant alleles, including the *4 allele, of the gene encoding cytochrome P450 2D6 (CYP2D6). We created a Markov model to determine whether tamoxifen or aromatase inhibitor monotherapy maximized 5-year disease-free survival for patients with the wild-type CYP2D6 genotype (wt/wt). Annual risks of recurrence with aromatase inhibitors and tamoxifen in breast cancer patients who were not selected by CYP2D6 genotype were derived from the Breast International Group 1-98 trial. Genotype frequencies and the hazard ratio for cancer recurrence on tamoxifen among patients with the *4/*4 genotype relative to the wt/wt or wt/*4 genotypes (HR(*4/*4) = 1.86) were based on data from an analysis of the North Central Cancer Treatment Group trial of adjuvant tamoxifen. We explored the impact of CYP2D6(*4) heterozygosity on disease-free survival for wt/wt patients by studying a range of effect (ie, recurrence on tamoxifen) estimates, from no effect of the single mutation (Eff(wt/*4) = 0, recurrence rate in wt/*4 patients same as that in wt/wt patients) to complete effect (Eff(wt/*4) = 1 recurrence rate in wt/*4 patients same as that in *4/*4 patients). With HR(*4/*4) = 1.86 and Eff(wt/*4) = 0.5, the 5-year disease-free survival of tamoxifen-treated patients with no mutations (wt/wt) was 83.9%, that is, essentially the same as that (84.0%) for genotypically unselected patients who were treated with aromatase inhibitors. With greater HR(*4/*4) estimates, disease-free survival with tamoxifen exceed that with aromatase inhibitors in wt/wt patients, even at lower assumed Eff(wt/*4) ratios. Modeling suggests that among patients who are wild type for CYP2D6, 5-year disease-free survival outcomes are similar to or perhaps even superior with tamoxifen than with aromatase inhibitors. Endocrine therapy tailored to CYP2D6 genotype could be considered for women who are newly diagnosed with breast cancer, particularly those who have with concerns about either the relative toxicity or the increased cost of aromatase inhibitors.

Impact of CYP2D6*10 on recurrence‐free survival in breast cancer patients receiving adjuvant tamoxifen therapy

The clinical outcomes of breast cancer patients treated with tamoxifen may be influenced by the activity of cytochrome P450 2D6 (CYP2D6) enzyme because tamixifen is metabolized by CYP2D6 to its active forms of antiestrogenic metabolite, 4-hydroxytamoxifen and endoxifen. We investigated the predictive value of the CYP2D6*10 allele, which decreased CYP2D6 activity, for clinical outcomes of patients that received adjuvant tamoxifen monotherapy after surgical operation on breast cancer. Among 67 patients examined, those homozygous for the CYP2D6*10 alleles revealed a significantly higher incidence of recurrence within 10 years after the operation (P = 0.0057; odds ratio, 16.63; 95% confidence interval, 1.75-158.12), compared with those homozygous for the wild-type CYP2D6*1 alleles. The elevated risk of recurrence seemed to be dependent on the number of CYP2D6*10 alleles (P = 0.0031 for trend). Cox proportional hazard analysis demonstrated that the CYP2D6 genotype and tumor size were independent factors affecting recurrence-free survival. Patients with the CYP2D6*10/*10 genotype showed a significantly shorter recurrence-free survival period (P = 0.036; adjusted hazard ratio, 10.04; 95% confidence interval, 1.17-86.27) compared to patients with CYP2D6*1/*1 after adjustment of other prognosis factors. The present study suggests that the CYP2D6 genotype should be considered when selecting adjuvant hormonal therapy for breast cancer patients.

Metabolic Regio- and Stereoselectivity of Cytochrome P450 2D6 towards 3,4-Methylenedioxy-N-alkylamphetamines: in Silico Predictions and Experimental Validation

A series of 3,4-methylenedioxy-N-alkylamphetamines (MDAAs) were automatically docked and subjected to molecular dynamics (MD) simulations in a cytochrome P450 2D6 (CYP2D6) protein model. The predicted substrate binding orientations, sites of oxidation, and relative reactivities were compared to the experimental data of wild-type and Phe120Ala mutant CYP2D6. Automated docking results were not sufficient to accurately rationalize experimental binding orientations of 3,4-methylenedioxy-N-methylamphetamine (MDMA) in the two enzymes as measured with spin lattice relaxation NMR. Nevertheless, the docking results could be used as starting structures for MD simulations. Predicted binding orientations of MDMA and sites of oxidation of the MDAAs derived from MD simulations matched well with the experimental data. It appeared the experimental results were best described in MD simulations considering the nitrogen atoms of the MDAAs in neutral form. Differences in regioselectivity and stereoselectivity in the oxidative metabolism of the MDAAs by the Phe120Ala mutant CYP2D6 were correctly predicted, and the effects of the Phe120Ala mutation could be rationalized as well.

Influence of phenylalanine 120 on cytochrome P450 2D6 catalytic selectivity and regiospecificity: crucial role in 7-methoxy-4-(aminomethyl)-coumarin metabolism

The polymorphic human debrisoquine hydroxylase, cytochrome P450 2D6 (CYP2D6), is one of the most important phase I drug metabolising enzymes. It is responsible for metabolising a large number of compounds that mostly share similarity in having a basic N-atom and an aromatic moiety. In homology modelling studies, it has been suggested that in fixation of this aromatic moiety, there may be an important role for phenylalanine 120 (Phe 120). In this study, the role of Phe 120 in ligand binding and catalysis was experimentally examined by mutating it into an alanine. Strikingly, this substitution led to a completely abolished 7-methoxy-4-(aminomethyl)-coumarin (MAMC) O-demethylating activity of CYP2D6. On the other hand, bufuralol metabolism was hardly affected ( K m of 1-hydroxylation mutant: 1.2 μM, wild-type: 2.9 μM, 4-hydroxylation mutant: 1.5 μM, and wild-type: 3.2 μM) and neither was affected dextromethorphan O-demethylation ( K m mutant: 1.2 μM, wild-type: 2 μM, k cat mutant: 4.5 min −1, and wild-type: 3.3 min −1). However, the Phe 120Ala mutant also formed 3-hydroxymorphinan, the double demethylated form of dextromethorphan, which was not detected using wild-type CYP2D6. 3,4-Methylenedioxymethamphetamine (MDMA) was demethylenated by both mutant and wild-type CYP2D6 to 3,4-dihydroxymethamphetamine (3,4-OH-MA K m of mutant: 55 μM and wild-type: 2 μM). In addition, the mutant formed two additional metabolites; 3,4-methylenedioxyamphetamine (MDA) and N-hydroxy-3,4-methylenedioxymethamphetamine ( N-OH-MDMA). Inhibition experiments of dextromethorphan O-demethylation showed a decreased affinity of the Phe 120Ala mutant for quinidine (IC 50 mutant: 240 nM and wild-type, 40 nM), while IC 50s for quinine were equal (1 μM). These data indicate the importance of Phe 120 in the selectivity and regiospecificity in substrate binding and catalysis by CYP2D6.

Residues Glutamate 216 and Aspartate 301 Are Key Determinants of Substrate Specificity and Product Regioselectivity in Cytochrome P450 2D6

Cytochrome P450 2D6 (CYP2D6) metabolizes a wide range of therapeutic drugs. CYP2D6 substrates typically contain a basic nitrogen atom, and the active-site residue Asp-301 has been implicated in substrate recognition through electrostatic interactions. Our recent computational models point to a predominantly structural role for Asp-301 in loop positioning (Kirton, S. B., Kemp, C. A., Tomkinson, N. P., St.-Gallay, S., and Sutcliffe, M. J. (2002) Proteins 49, 216-231) and suggest a second acidic residue, Glu-216, as a key determinant in the binding of basic substrates. We have evaluated the role of Glu-216 in substrate recognition, along with Asp-301, by site-directed mutagenesis. Reversal of the Glu-216 charge to Lys or substitution with neutral residues (Gln, Phe, or Leu) greatly decreased the affinity (K(m) values increased 10-100-fold) for the classical basic nitrogen-containing substrates bufuralol and dextromethorphan. Altered binding was also manifested in significant differences in regiospecificity with respect to dextromethorphan, producing enzymes with no preference for N-demethylation versus O-demethylation (E216K and E216F). Neutralization of Asp-301 to Gln and Asn had similarly profound effects on substrate binding and regioselectivity. Intriguingly, removal of the negative charge from either 216 or 301 produced enzymes (E216A, E216K, and D301Q) with elevated levels (50-75-fold) of catalytic activity toward diclofenac, a carboxylate-containing CYP2C9 substrate that lacks a basic nitrogen atom. Activity was increased still further (>1000-fold) upon neutralization of both residues (E216Q/D301Q). The kinetic parameters for diclofenac (K(m) 108 microm, k(cat) 5 min(-1)) along with nifedipine (K(m) 28 microm, k(cat) 2 min(-1)) and tolbutamide (K(m) 315 microm, k(cat) 1 min(-1)), which are not normally substrates for CYP2D6, were within an order of magnitude of those observed with CYP3A4 or CYP2C9. Neutralizing both Glu-216 and Asp-301 thus effectively alters substrate recognition illustrating the central role of the negative charges provided by both residues in defining the specificity of CYP2D6 toward substrates containing a basic nitrogen.

Reduced (±)-3,4-methylenedioxymethamphetamine (“Ecstasy”) metabolism with cytochrome P450 2D6 inhibitors and pharmacogenetic variants in vitro

“Ecstasy” [(±)-3,4-methylenedioxymethamphetamine or MDMA] is a CNS stimulant, whose use is increasing despite evidence of long-term neurotoxicity. In vitro, the majority of MDMA is demethylenated to (±)-3,4-dihydroxymethamphetamine (DHMA) by the polymorphic cytochrome P450 2D6 (CYP2D6). We investigated the demethylenation of MDMA and dextromethorphan (DEX), as a comparison drug, in reconstituted microsomes expressing the variant CYP2D6 alleles ∗ 2, ∗ 10, and ∗ 17, all of which have been linked to decreased enzyme activity. With MDMA, intrinsic clearances ( V max/ K m ) in CYP2D6.2, CYP2D6.17, and CYP2D6.10 were reduced 15-, 13-, and 135-fold, respectively, compared with wild-type CYP2D6.1. With DEX, intrinsic clearances were reduced by 37-, 51-, and 164-fold, respectively. It was evident that CYP2D6.17 displayed substrate-specific changes in drug affinity ( K m ). Compounds potentially used with MDMA [fluoxetine, paroxetine, (−)-cocaine] demonstrated significant inhibition of MDMA metabolism in both human liver and CYP2D6.1-expressing microsomes. These data demonstrate that individuals possessing the CYP2D6 ∗ 2, ∗ 17, and, particularly, ∗ 10 alleles may show significantly reduced MDMA metabolism. These individuals, and those taking CYP2D6 inhibitors, may demonstrate altered acute and/or long-term MDMA-related toxicity.