Spectral and 3D model studies of the interaction of orphan human cytochrome P450 2U1 with substrates and ligands.

BACKGROUND: Cytochrome P450 2U1 (CYP2U1) has been identified from the human genome and is highly conserved in the living kingdom. It is considered as an "orphan" protein as few data are available on its physiological function(s) and spectral characteristics. Its only known substrates reported so far are unsaturated fatty acids such as arachidonic acid (AA), and, more recently, N-arachidonoylserotonin (AS) and some xenobiotics related to debrisoquine (Deb) and terfenadine. METHODS: We have expressed CYP2U1 in E. coli and performed UV-vis and EPR spectroscopy experiments with purified CYP2U1 alone and in the presence of substrates and imidazole and pyridine derivatives. Docking experiments using a 3D homology model of CYP2U1 were done to explain the observed spectroscopic data and the different regioselectivities of the oxidations of AA and AS. RESULTS: The UV-vis and EPR spectra of native recombinant human CYP2U1 revealed a predominant low-spin hexacoordinate FeIII state. Imidazole (Im) derivatives, such as miconazole, acted as FeIII ligands, contrary to ketoconazole, whereas the previously described substrates AS and Deb led to "reverse type I" difference UV-vis spectra. These data, as well as the different regioselectivities of AA and AS oxidations, were supported by docking experiments performed on our previously reported CYP2U1 3D model. MAJOR CONCLUSION AND GENERAL SIGNIFICANCE: Our study describes for the first time the mode of interaction of several FeIII-heme ligands and substrates with the active site of CYP2U1 on the basis of spectroscopic and molecular docking data. The good agreement between these data validates the used CYP2U1 3D model which should help the design of new substrates or inhibitors of this orphan CYP.

Functional characterization of CYP2D6 novel allelic variants identified in the Chinese Han population.

AIM: This study was aimed to functionally characterize four novel CYP2D6 alleles identified in Chinese Han population. MATERIALS & METHODS: CYP2D6 proteins of wild-type and the four novel variants along with CYP2D6.2 and CYP2D6.10 were heterologously expressed in yeast cells and the kinetic parameters were determined. RESULTS: Compared with CYP2D6.1 (frequency in Chinese 24.65%), CYP2D6.X (1.63%), CYP2D6.Y (1.50%), CYP2D6.Z (0.81%), CYP2D6.10 (52.53%) and CYP2D6.75 (0.13%) exhibited low activity at different degrees, whereas the kinetic parameters of CYP2D6.2 (11.06%) were much the same with CYP2D6.1. The novel allele CYP2D6.75 showed decreased enzyme activity. CONCLUSION: This is the first study to conduct functional analysis of CYP2D6 four novel alleles in Chinese Han population, which might be helpful for optimizing pharmacotherapy and the design of personalized medicine.

Expression in yeast, new substrates, and construction of a first 3D model of human orphan cytochrome P450 2U1: Interpretation of substrate hydroxylation regioselectivity from docking studies.

BACKGROUND: Cytochrome P450 2U1 (CYP2U1) has been identified from the human genome and is highly conserved in the living kingdom. In humans, it has been found to be predominantly expressed in the thymus and in the brain. CYP2U1 is considered as an "orphan" enzyme as few data are available on its physiological function(s) and active site topology. Its only substrates reported so far were unsaturated fatty acids such as arachidonic acid, and, much more recently, N-arachidonoylserotonin. METHODS: We expressed CYP2U1 in yeast Saccharomyces cerevisiae, built a 3D homology model of CYP2U1, screened a library of compounds known to be substrates of CYP2 family with metabolite detection by high performance liquid chromatography-mass spectrometry, and performed docking experiments to explain the observed regioselectivity of the reactions. RESULTS: We show that drug-related compounds, debrisoquine and terfenadine derivatives, subtrates of CYP2D6 and CYP2J2, are hydroxylated by recombinant CYP2U1 with regioselectivities different from those reported for CYP2D6 and 2J2. Docking experiments of those compounds and of arachidonic acid allow us to explain the regioselectivity of the hydroxylations on the basis of their interactions with key residues of CYP2U1 active site. MAJOR CONCLUSION: Our results show for the first time that human orphan CYP2U1 can oxidize several exogenous molecules including drugs, and describe a first CYP2U1 3D model. GENERAL SIGNIFICANCE: These results could have consequences for the metabolism of drugs particularly in the brain. The described 3D model should be useful to identify other substrates of CYP2U1 and help in understanding its physiologic roles.

Molecular insight into affinities of drugs and their metabolites to lipid bilayers.

The penetration properties of drug-like molecules on human cell membranes are crucial for understanding the metabolism of xenobiotics and overall drug distribution in the human body. Here, we analyze partitioning of substrates of cytochrome P450s (caffeine, chlorzoxazone, coumarin, ibuprofen, and debrisoquine) and their metabolites (paraxanthine, 6-hydroxychlorzoxazone, 7-hydroxycoumarin, 3-hydroxyibuprofen, and 4-hydroxydebrisoquine) on two model membranes: dioleoylphosphatidylcholine (DOPC) and palmitoyloleoylphophatidylglycerol (POPG). We calculated the free energy profiles of these molecules and the distribution coefficients on the model membranes. The drugs were usually located deeper in the membrane than the corresponding metabolites and also had a higher affinity to the membranes. Moreover, the behavior of the molecules on the membranes differed, as they seemed to have a higher affinity to the DOPC membrane than to POPG, implying they have different modes of action in human (mostly PC) and bacterial (mostly PG) cells. As the xenobiotics need to pass through lipid membranes on their way through the body and the effect of some drugs might depend on their accumulation on membranes, we believe that detailed information of penetration phenomenon is important for understanding the overall metabolism of xenobiotics.

Comparative metabolic capabilities and inhibitory profiles of CYP2D6.1, CYP2D6.10, and CYP2D6.17.

Polymorphisms in the cytochrome P450 2D6 (CYP2D6) gene are a major cause of pharmacokinetic variability in human. Although the poor metabolizer phenotype is known to be caused by two null alleles leading to absence of functional CYP2D6 protein, the large variability among individuals with functional alleles remains mostly unexplained. Thus, the goal of this study was to examine the intrinsic enzymatic differences that exist among the several active CYP2D6 allelic variants. The relative catalytic activities (enzyme kinetics) of three functionally active human CYP2D6 allelic variants, CYP2D6.1, CYP2D6.10, and CYP2D6.17, were systematically investigated for their ability to metabolize a structurally diverse set of clinically important CYP2D6-metabolized drugs [atomoxetine, bufuralol, codeine, debrisoquine, dextromethorphan, (S)-fluoxetine, nortriptyline, and tramadol] and the effects of various CYP2D6-inhibitors [cocaine, (S)-fluoxetine, (S)-norfluoxetine, imipramine, quinidine, and thioridazine] on these three variants. The most significant difference observed was a consistent but substrate-dependent decease in the catalytic efficiencies of cDNA-expressed CYP2D6.10 and CYP2D6.17 compared with CYP2D6.1, yielding 1.32 to 27.9 and 7.33 to 80.4% of the efficiency of CYP2D6.1, respectively. The most important finding from this study is that there are mixed effects on the functionally reduced allelic variants in enzyme-substrate affinity or enzyme-inhibitor affinity, which is lower, higher, or comparable to that for CYP2D6.1. Considering the rather high frequencies of CYP2D6*10 and CYP2D6*17 alleles for Asians and African Americans, respectively, these data provide further insight into ethnic differences in CYP2D6-mediated drug metabolism. However, as with all in vitro to in vivo extrapolations, caution should be applied to the clinical consequences.

The roles of amino acid residues at positions 43 and 45 in microsomal contents and enzymatic functions of rat CYP2D1 and CYP2D2.

The effects of the substitution of amino acid residues at positions 43 and 45 of rat CYP2D1 and CYP2D2 on their microsomal contents and enzymatic functions were examined. The substitution of Val-45 of CYP2D1 by glycine decreased the microsomal content, whereas the substitution of Gly-45 of CYP2D2 by valine increased. The substitution of Leu-43 of CYP2D2 by tryptophan also increased the microsomal protein content. In reduced CO-difference spectra, CYP2D2 showed a P420 peak as well as a P450 peak, whereas CYP2D1 gave only a P450 peak. The substitution of Leu-43 and Gly-45 of CYP2D2 by valine and tryptophan, respectively, markedly decreased the P420 peak in parallel with an increase in P450 content. These substitutions did not cause remarkable changes in drug oxidation capacities (bufuralol 1''-hydroxylation and debrisoquine 4-hydroxylation) of the recombinant enzymes in terms of nmol/min/nmol CYP. The results indicate that amino acid residues at positions 43 and 45 are important for anchoring of the rat CYP2D proteins and their stabilities in the endoplasmic reticulum membrane.

4-Hydroxylation of debrisoquine by human CYP1A1 and its inhibition by quinidine and quinine.

A panel of 15 recombinant cytochromes P450 expressed in human B-lymphoblastoid cells was used to study debrisoquine 4-hydroxylation. Both CYP2D6 and CYP1A1 carried out the reaction. The apparent K(m) (micromolar) and V(max) (picomoles per minute per picomole of P450) for CYP2D6 were 12.1 and 18.2 and for CYP1A1 were 23.1 and 15.2, respectively. CYP1A1 debrisoquine 4-hydroxylase was inhibited by the CYP1A1 inhibitor alpha-naphthoflavone and the CYP1A1 substrate 7-ethoxyresorufin. Additionally and surprisingly, this reaction was also inhibited by quinidine and quinine, with respective IC(50) values of 1.38 +/- 0.10 and 3.31 +/- 0.14 microM, compared with those for CYP2D6 debrisoquine 4-hydroxylase of 0.018 +/- 0.05 and 3.75 +/- 2.07 microM, respectively. Anti-CYP1A1 monoclonal antibody (mAb) 1-7-1 abolished CYP1A1 debrisoquine hydroxylase and anti-CYP2D6 mAb 50-1-3 eradicated CYP2D6 debrisoquine 4-hydroxylase. Three further CYP2D6-specific reactions were tested: dextromethorphan O-demethylation, bufuralol 1'-hydroxylation, and sparteine dehydrogenation. The CYP2D6 specificity, judged by the CYP2D6/CYP1A1 activity ratios was 18.5, 7.0, 6.0, and 1.6 for dextromethorphan, bufuralol, sparteine, and debrisoquine, respectively. Thus, debrisoquine is not a specific CYP2D6 substrate and quinidine is not a specific CYP2D6 inhibitor. These findings have significant implications for the conduct of in vitro drug metabolism inhibition studies and underscore the fallacy of "specific chemical inhibitors" of a supergene family of enzymes that have overlapping substrate specificities. The use of highly specific mAbs in such studies is mandated. It is unclear as yet whether these findings have implications for the relationship between CYP2D6 genotype and in vivo debrisoquine 4-hydroxylase activity.

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.

Separation and quantitation of debrisoquine and 4-hydroxydebrisoquine in human urine by capillary electrophoresis and high-performance liquid chromatography.

A comparative study on the use of reversed-phase high-performance liquid chromatography (RP-HPLC) and capillary electrophoresis (CE) for the determination of debrisoquine (D) and its metabolite, 4-hydroxydebrisoquine (4-HD), in human urine is presented. Four different urine pre-treatments are compared for purification of samples prior to their injection in HPLC and CE. The use of a solid-phase extraction with a C18 cartridge provides the best results for the urine sample treatment, with good recoveries, i.e., 94.5% for D and 93.4% for 4-HD, and high reproducibility, i.e., R.S.D. N = 10 values of 1.7% and 1.2%, respectively. Under our separation conditions it is shown that CE is twice as fast and provides slightly better analysis time reproducibility than HPLC for this type of sample. Both the sensitivity and peak area reproducibility are better when HPLC is used. The two techniques show good agreement when employed for determination of phenotypes for hydroxylation, which seems to corroborate the usefulness of CE for this type of study.

Characterization of stereoselectivity and genetic polymorphism of the debrisoquine hydroxylation in man via analysis of urinary debrisoquine and 4-hydroxydebrisoquine by capillary electrophoresis.

Using capillary zone electrophoresis with a phosphate buffer at pH 2.5 containing 50 mM heptakis-(2,3,6-tri-O-methyl)-beta-CD as chiral selector, the separation of the enantiomers of the main metabolite of debrisoquine (DEB), 4-hydroxydebrisoquine (4-OHDEB), is reported. For extraction of underivatized urinary DEB, S-4-OHDEB and R-4-OHDEB, a procedure using disposable cartridges containing a polystyrene-based polymer was developed. A few nL of the extracts were analyzed in a 60 cm fused-silica capillary of 50 microns ID and solute detection was effected at 195 nm. For all three compounds, a mean (n = 5) recovery of about 73% and a detection limit of about 150 ng/mL were noted. Data obtained with urines that were received for routine phenotyping with DEB and mephenytoin confirmed the almost exclusive formation of S-4-OHDEB. Under the described conditions, no R-4-OHDEB could be detected. With these data and those obtained employing no chiral selector in the buffer, differentiation between extensive metabolizer phenotypes (EM) and poor metabolizer phenotypes (PM) for DEB was unambiguously possible by the presence of a significant peak and no (or minor) peak for 4-OHDEB, respectively. Data obtained for ten EM subjects and five PM subjects were found to agree with those generated by the routine assay based on gas chromatography. The capillary electrophoretic assays described are simple, reproducible (relative standard deviation of peak area ratios < 3%), require no sample derivatization, consume no halogenated organic solvents, and operate with inexpensive separation columns as well as small amounts of chemicals.

Evidence for the polymorphic oxidation of debrisoquine and proguanil in a Khmer (Cambodian) population.

The frequency distributions of the urinary metabolic ratios of debrisoquine and proguanil were measured in a population of unrelated Khmers. Out of 98 Khmer subjects studied, two were identified as poor metabolisers of debrisoquine when a metabolic ratio of 12.6 was used as the cut off point. This represents a prevalence of debrisoquine poor metabolisers of 2.1% (95% confidence interval 0.25-7.3%) which is similar to other Asian populations. Based on the distribution of the ratio of proguanil to cycloguanil excreted in urine, and using an antimode value of 10, the prevalence of poor metabolisers of proguanil in a Khmer population was estimated to be 18.4% (95% confidence interval 10.9-28.1%). The frequency of poor metabolisers of proguanil in Khmers was higher than that described for Caucasian populations, but similar to most reported results in Asian populations.

Stereoselective disposition of mianserin is related to debrisoquin hydroxylation polymorphism.

The pharmacokinetics of mianserin and its main metabolite desmethylmianserin were studied in poor and extensive metabolizers of debrisoquin and of S-mephenytoin after a single oral dose of racemic mianserin. The debrisoquin metabolic ratio (MR) correlated significantly with area under the serum concentration-time curves (AUC) for (+/-)-mianserin and (+/-)-desmethylmianserin. Enantioselective high-performance liquid chromatographic analysis of mianserin showed that debrisoquin MR was related to AUC(0-12) for S(+)-mianserin (rs = 0.87; p = 0.001; n = 15) but not for R(-)-mianserin. The ratio between the AUC(0-12) for S(+)-mianserin and that for R(-)-mianserin was higher in poor metabolizers than in extensive metabolizers. Two extremely rapid extensive metabolizer subjects had the lowest mianserin S/R ratios. No differences in the pharmacokinetics of mianserin or desmethylmianserin were found between extensive metabolizers and poor metabolizers of S-mephenytoin. The study shows that the elimination of both mianserin and its main metabolite desmethylmianserin is dependent on CYP2D6 activity. Furthermore, the CYP2D6-dependent elimination of mianserin shows marked enantioselectivity for the more active S(+)-enantiomer of mianserin.

A predictive model for substrates of cytochrome P450-debrisoquine (2D6).

Molecular modeling techniques were used to derive a predictive model for substrates of cytochrome P450 2D6, an isozyme known to metabolize only compounds with one or more basic nitrogen atoms. Sixteen substrates, accounting for 23 metabolic reactions, with a distance of either 5 A ("5-A substrates", e.g., debrisoquine) or 7 A ("7-A substrates", e.g., dextromethorphan) between oxidation site and basic nitrogen atom were fitted into one model by postulating an interaction of the basic nitrogen atom with a negatively charged carboxylate group on the protein. This acidic residue anchors and neutralizes the positively charged basic nitrogen atom of the substrates. In case of "5-A substrates" this interaction probably occurs with the carboxylic oxygen atom nearest to the oxidation site, whereas in the case of "7-A substrates" this interaction takes place at the other oxygen atom. Furthermore, all substrates exhibit a coplanar conformation near the oxidation site and have negative molecular electrostatic potentials (MEPs) in a part of this planar domain approximately 3 A away from the oxidation site. No common features were found in the neighbourhood of the basic nitrogen atom of the substrates studied so that this region of the active site can accommodate a variety of N-substituents. Therefore, the substrate specificity of P450 2D6 most likely is determined by the distance between oxidation site and basic nitrogen atom, by steric constraints near the oxidation site, and by the degree of complementarity between the MEPs of substrate and protein in the planar region adjacent to the oxidation site.(ABSTRACT TRUNCATED AT 250 WORDS)