Molecular modelling of CYP2B6 based on homology with the CYP2C5 crystal structure: analysis of enzyme-substrate interactions

The results of homology modelling of CYP2B6 based on the CYP2C5 crystal structure is described in terms of substrates and inhibitors binding within the putative active site. In general these results are in agreement with currently available evidence from substrate metabolism, mode of inhibitor action and site-directed mutagenesis experiments within the CYP2B subfamily of enzymes. Consequently, the model based on the CYP2C5 template represents an advance on those models produced from bacterial P450s, such as CYP101 and CYP102. Quantitative Structure-Activity Relationships (QSARs) for substrates binding to CYP2B6 indicate a key role for hydrogen bonding, and lipophilic character, as determined by the log P parameter (where P is the octanol/water partition coefficient), is also of importance for explaining the variation in experimental binding affinity for CYP2B6 substrates. It is possible to estimate the binding energies for typical CYP2B6 substrates based on their properties and interactions with the enzyme, which show good concordance with experimental data in the form of apparent Km values.     

Molecular modelling of CYP1 family enzymes CYP1A1, CYP1A2, CYP1A6 and CYP1B1 based on sequence homology with CYP102

Molecular modelling of a number of CYP1 family enzymes from rat, plaice and human is described based on amino acid sequence homology with the haemoprotein domain of CYP102, a unique bacterial P450 of known structure. The interaction of various substrates and inhibitors within the putative active sites of rat CYP1A1, human CYP1A2, a fish CYP1 enzyme CYP1A6 (from plaice) and human CYP1B1, is shown to be consistent with P450-mediated oxidation in each example or, in the case of inhibitors, mechanism of inhibition. It is reported that relatively small changes between the enzymes' active site regions assist in the rationalization of CYP1 enzyme preferences for particular substrate types, and a template of superimposed CYP1A2 substrates is shown to fit the putative active site of the human CYP1A2 enzyme.     

Homology modelling of human CYP1A2 based on the CYP2C5 crystallographic template structure

1. The results of homology modelling of human cytochrome P4501A2 (CYP1A2) based on the CYP2C5 crystal structure are reported. It exhibits improved sequence homology relative to that of CYP102. 2. It was demonstrated that many selective substrates for CYP1A2 could fit within the putative active site of the enzyme, and in orientations which agree with documented evidence for CYP1A2-mediated metabolism. 3. Furthermore, a number of amino acid residues lining the haem pocket have been shown, via site-directed mutagenesis, to have an influence on substrate metabolism, and these experimental findings from the literature are consistent with the modelled interactions for selective substrates. 4. The binding affinities of several CYP1A2 substrates have also been calculated from the CYP1A2 active site interactions and they agree closely with experimental values.     

Homology modelling of human CYP1A2 based on the CYP2C5 crystallographic template structure

1. The results of homology modelling of human cytochrome P4501A2 (CYP1A2) based on the CYP2C5 crystal structure are reported. It exhibits improved sequence homology relative to that of CYP102. 2. It was demonstrated that many selective substrates for CYP1A2 could fit within the putative active site of the enzyme, and in orientations which agree with documented evidence for CYP1A2-mediated metabolism. 3. Furthermore, a number of amino acid residues lining the haem pocket have been shown, via site-directed mutagenesis, to have an influence on substrate metabolism, and these experimental findings from the literature are consistent with the modelled interactions for selective substrates. 4. The binding affinities of several CYP1A2 substrates have also been calculated from the CYP1A2 active site interactions and they agree closely with experimental values.     

Homology modelling of human CYP2E1 based on the CYP2C5 crystal structure: investigation of enzyme-substrate and enzyme-inhibitor interactions.

The construction of a homology model of human cytochrome P450 2E1 (CYP2E1) is reported, based on the CYP2C5 crystallographic template. A relatively high degree of primary sequence homology (identity=59%), as expected for proteins of the same CYP family, ensured a straightforward generation of the 3-dimensional model due to relatively few deletions and insertions of amino acid residues with respect to the CYP2C5 crystal structure. Probing the CYP2E1 model with typical substrates of the enzyme showed a good agreement with experimental information in the form of positions of metabolism for substrates, and with site-directed mutagenesis data on certain residues. Furthermore, quantitative relationships between substrate binding affinity and various structural parameters associated with the substrate molecules facilitated the formulation of a procedure for estimating relative binding energy and, consequently, K(m) or K(D) values towards the CYP2E1 enzyme. This method has been based on a consideration of the active site interactions between substrates and key amino acid residues lining the haem pocket, together with compound lipophilicity data from partition coefficients.     

Quantitative structure-activity relationships (QSARs) for substrates of human cytochromes P450 CYP2 family enzymes.

The results of quantitative structure-activity relationship (QSAR) studies on substrates of human CYP2 family enzymes are reported, together with those of a small number of CYP2A6, CYP2C19 and CYP2D6 inhibitors. In general, there are good correlations (R = 0.90-0.99) between binding affinity (based on Km or KD values) and various parameters relating to active site interactions such as hydrogen bonding and pi-pi stacking. There is also evidence for the role of compound lipophilicity (as determined by either log P or log D7.4 values) in overall substrate binding affinity, and this could reflect the desolvation energy involved in substrate interaction within the enzyme active site. It is possible to estimate the substrate binding energy for a given P450 from a combination of energy terms relating to hydrogen bonding, pi-pi stacking, desolvation and loss in rotatable bond energy, which agree closely (R = 0.98) with experimental data based on either Km or KD values. Consequently, it is likely that active site interactions represent the major contributory factors to the overall binding affinities for human CYP2 family substrates and, therefore, their estimation is of potential importance for the development of new chemical entities (NCEs) as this can facilitate an assessment of likely metabolic clearance.     

Investigation of enzyme selectivity in the human CYP2C subfamily: homology modelling of CYP2C8, CYP2C9 and CYP2C19 from the CYP2C5 crystallographic template

Homology modelling of human CYP2C subfamily enzymes, CYP2C8, CYP2C9 and CYP2C19, based on the rabbit CYP2C5 crystal structure template is reported. The relatively high sequence homologies (75-80%) between the rabbit CYP2C5 and human CYP2C subfamily enzymes tend to indicate that the resulting structures should prove adequate models of these major catalysts of human drug metabolism. Selective substrates of all three human CYP2C enzymes are found to fit closely within the putative active sites in a manner which is consistent with site-directed mutagenesis experiments and known positions of substrate metabolism. The specific interactions between substrates and enzymes can be used to rationalize the variation in substrate binding affinity and generate QSAR models for both inhibition and metabolism via CYP2C family enzymes, yielding a generally good agreement with experimental binding data obtained from Km values, with correlation coefficients (R values) of between 0.97 and 0.99 depending on the QSAR equation produced.     

Polymorphisms of CYP2A6 and its practical consequences

CYP2A6 is an hepatic enzyme predominantly with some expression in specialized extrahepatic cell types. The CYP2A6 enzyme has a somewhat restricted active site, accepting only a few xenobiotics as substrates. Interest in CYP2A6 has risen considerably after nicotine and some tobacco specific nitrosamines were established as high-affinity substrates for this enzyme. Recently, the organization and structures of the CYP2A gene cluster and several polymorphic alleles of the CYP2A6 gene have been characterized. Two alleles with a point mutation and at least three different types of gene deletion, all leading to deficient gene function, have been found. The frequencies of these alleles vary considerably among different ethnic populations, the deletion alleles being most common in Orientals (up to 20%). The frequency of point mutations are low in all populations studied thus far (< 3%). Several case-control studies have addressed the relationship between CYP2A6 status and smoking habits as well as the role of CYP2A6 polymorphism in lung cancer risk. Studies in Japanese suggest that CYP2A6 poor metabolizer genotypes result in altered nicotine kinetics and may lower cigarette smoking elicited lung cancer risk, whereas similar studies in Caucasian populations have not revealed any clear associations between variant CYP2A6 genotypes and smoking behaviour or lung cancer predisposition.     

Species differences in coumarin metabolism: a molecular modelling evaluation of CYP2A interactions

1. An account of the differences in coumarin metabolism between several mammalian species, including man, is reported. 2. The metabolism of coumarin via 7-hydroxylation in the human (CYP2A6) and mouse (CYP2A5) enzymes is explained in terms of molecular modelling of the active site interactions, whereas the rat orthologue (CYP2A1) exhibits 3,4-epoxidation of coumarin, which is also consistent with the modelled interaction between enzyme and substrate. 3. In addition, quantitative structure-activity relationships (QSARs) for coumarin 7-hydroxylation in wild-type and mutant CYP2A5 show the importance of amino acid residue properties for substrate binding, whereas QSARs for CYP2A6 substrates indicate the importance of hydrogen bonding and lipophilicity for favourable interactions with the enzyme.     

Substrates of human cytochromes P450 from families CYP1 and CYP2: analysis of enzyme selectivity and metabolism

A compilation of information relating to substrate metabolism via human cytochromes P450 (CYP) from the CYP1 and CYP2 families is reported. The data presented include details of preferred sites of metabolism and Km values (usually for the expressed enzymes) for each reaction for selected substrates of CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6 and CYP2E1. Although other P450 databases are available, they do not provide such information as is collated here, and which can prove useful for comparing P450 substrate characteristics. This information can be employed in analysing the structural requirements for human P450 enzyme selectivity and for establishing various rules regarding preferred site of metabolism for selective P450 substrates. For example, in most cases it would appear that there is a set number of intervening 'heavy' atoms (atoms other than hydrogen) between sites of metabolism and key hydrogen bond acceptors (or donors) for human P450 substrates, with the number of intervening atoms being dependent upon the type of P450 involved.     

Species differences in coumarin metabolism: a molecular modelling evaluation of CYP2A interactions

1. An account of the differences in coumarin metabolism between several mammalian species, including man, is reported. 2. The metabolism of coumarin via 7-hydroxylation in the human (CYP2A6) and mouse (CYP2A5) enzymes is explained in terms of molecular modelling of the active site interactions, whereas the rat orthologue (CYP2A1) exhibits 3,4-epoxidation of coumarin, which is also consistent with the modelled interaction between enzyme and substrate. 3. In addition, quantitative structure-activity relationships (QSARs) for coumarin 7-hydroxylation in wild-type and mutant CYP2A5 show the importance of amino acid residue properties for substrate binding, whereas QSARs for CYP2A6 substrates indicate the importance of hydrogen bonding and lipophilicity for favourable interactions with the enzyme.     

Cytochrome P450 fluorometric substrates: identification of isoform-selective probes for rat CYP2D2 and human CYP3A4.

We have tested a panel of 29 cDNA-expressed rat and human enzymes with 9 fluorometric substrates to determine the P450 isoform selectivity in the catalysis of the substrates to fluorescent products. The substrates examined were dibenzyl fluorescein, 7-benzyloxyquinoline (BQ), 3-cyano-7-ethoxycoumarin, 3-cyano-7-methoxycoumarin, 7-methoxy-4-trifluoromethylcoumarin, 3-[2-(N,N-diethyl-N-methylamino)ethyl]-7-methoxy-4-methylcoumarin (AMMC), 3-[2-(N,N-diethyl-N-methylamino)ethyl]-7-methoxy-4-trifluoromethylcoumarin, 7-benzyloxyresorufin, and 7-benzyloxy-4-trifluoromethylcoumarin (BFC). For most substrates, multiple cDNA-expressed cytochrome P450 isoforms were found to catalyze the formation of the fluorescent product. However, among the combinations tested, rat CYP2D2 displayed high selectivity for AMMC demethylation (a substrate selective for CYP2D6 in human liver microsomes). AMMC demethylation activity was 15-fold lower in microsomes isolated from female Dark Agouti rats, a model known to have a low abundance of CYP2D2, and apparent K(M) values were similar for cDNA-expressed CYP2D2 and male Sprague-Dawley liver microsomes. BFC dealkylation and BQ dealkylation were selective but not exclusive for human CYP3A4. A small role for CYP1A2 could be demonstrated. The CYP3A4 selectivity in hepatic microsomes was supported by studies using chemical and antibody inhibitors and a correlation analysis within a panel of liver microsomes from individual donors. BQ demonstrated a higher degree of selectivity for and higher rates of metabolism by CYP3A than BFC. However, per unit enzyme the fluorescent signal is lower for BQ than BFC. AMMC, BQ, and BFC should find uses as enzyme-selective probe substrates.     

Expression, purification, and characterization of mouse CYP2d22.

Metabolism of the prototype human CYP2D6 substrates debrisoquine and bufuralol proceeds at a much slower rate in mice; therefore, the mouse has been proposed as an animal model for the human CYP2D6 genetic deficiency. To interpret the molecular mechanism of this deficiency, a cDNA belonging to the CYP2D gene subfamily (Cyp2d22) has been cloned and sequenced from a mouse mammary tumor-derived cell line. In the current study, Cyp2d22 enzyme was overexpressed and purified from insect cells using a baculovirus-mediated system. The activity of this purified enzyme was directly compared with purified human CYP2D6 toward codeine, dextromethorphan, and methadone as substrates. Purified Cyp2d22 was found to catalyze the O-demethylation of dextromethorphan with significantly higher K(m) values (250 microM) than that (4.2 microM) exhibited by purified human CYP2D6. The K(m) for dextromethorphan N-demethylation by Cyp2d22 was found to be 418 microM, much lower than that observed with human CYP2D6 and near the K(m) for dextromethorphan N-demethylation catalyzed by CYP3A4. CYP2D6 catalyzed codeine O-demethylation, whereas Cyp2d22 and CYP3A4 mediated codeine N-demethylation. Furthermore, methadone, a known CYP3A4 substrate and CYP2D6 inhibitor, was N-demethylated by Cyp2d22 with a K(m) of 517 microM and V(max) of 4.9 pmol/pmol/min. Quinidine and ketoconazole, potent inhibitors to CYP2D6 and CYP3A4, respectively, did not show strong inhibition toward Cyp2d22-mediated dextromethorphan O- or N-demethylation. These results suggest that mouse Cyp2d22 has its own substrate specificity beyond CYP2D6-like-deficient activity.     

Homology modelling of CYP3A4 from the CYP2C5 crystallographic template: analysis of typical CYP3A4 substrate interactions

1. The results of homology modelling of cytochrome P4503A4 (CYP3A4), which is a human enzyme of major importance for the Phase 1 metabolism of drug substrates, from the CYP2C5 crystal structure is reported. 2. The overall homology between the two protein sequences was generally good (46%) with 24% of amino acid residues being identical and a 22% similarity between matched pairs in the CYP3A4 and CYP2C5 aligned sequences, thus indicating that CYP2C5 represents a viable template for modelling CYP3A4 by homology. 3. The CYP3A4 model appears to show consistency with the reported findings from the extensive site-directed mutagenesis studies already published. 4. Typical CYP3A4 substrates, such as midazolam, testosterone, nifedipine and verapamil, are shown to fit the putative active site of the enzyme structure in a manner consistent with their known positions of metabolism.     

Gene-gene interactions between CYP2B6 and CYP2A6 in nicotine metabolism

OBJECTIVES: CYP2A6 is the major enzyme involved in nicotine metabolism, yet large interindividual variations in the rate of nicotine metabolism exist within groups of individuals having the same CYP2A6 genotype. We investigated the influence of genetic variation in another potential nicotine-metabolizing enzyme, CYP2B6, and its interaction with CYP2A6, on the metabolism of nicotine. METHODS: Two hundred and twelve healthy Caucasian adult twin volunteers underwent an intravenous infusion of stable isotope-labeled nicotine and its major metabolite, cotinine, for characterization of pharmacokinetic and metabolism phenotypes. Five CYP2B6 genetic polymorphisms causing amino acid substitutions (R22C, Q172 H, S259R, K262R, and R487C) were analyzed. RESULTS: We observed that the CYP2B6*6 haplotype (defined as having both Q172 H and K262R variants) was associated with faster nicotine and cotinine clearance, and that such associations were more prominent among individuals having decreased-activity CYP2A6 genotypes. Statistically significant interactions between CYP2B6 and CYP2A6 genotypes were observed (P<0.003 for nicotine clearance and P<0.002 for cotinine clearance). CONCLUSIONS: Our results indicate that CYP2B6 genetic variation is associated with the metabolism of nicotine and cotinine among individuals with decreased CYP2A6 activity. Further investigation of the roles of CYP2B6 and the interaction between CYP2B6 and CYP2A6 genotypes in mediating nicotine dependence and tobacco-related diseases is merited.