Functional analysis of phenylalanine residues in the active site of cytochrome P450 2C9

The two published crystal structures of cytochrome P450 2C9, complexed with ( S)-warfarin or flurbiprofen, implicate a cluster of three active site phenylalanine residues (F100, F114, F476) in ligand binding. However, these three residues appear to interact differently with these two ligands based on the static crystal structures. To elucidate the importance of CYP2C9's active site phenylalanines on substrate binding, orientation, and catalytic turnover, a series of leucine and tryptophan mutants were constructed and their interactions with ( S)-warfarin and ( S)-flurbiprofen examined. The F100-->L mutation had minor effects on substrate binding and metabolism of each substrate. In contrast, the F114L and F476L mutants exhibited substantially reduced ( S)-warfarin metabolism and altered hydroxy metabolite profiles but only modestly decreased nonsteroidal antiinflammatory drug (NSAID) turnover while maintaining product regioselectivity. The F114-->W and F476-->W mutations also had opposing effects on ( S)-warfarin versus NSAID turnover. Notably, the F476W mutant increased the efficiency of ( S)-warfarin metabolism 5-fold, yet decreased the efficiency of ( S)-flurbiprofen turnover 20-fold. (1)H NMR T 1 relaxation studies suggested a slightly closer positioning of ( S)-warfarin to the heme in the F476W mutant relative to the wild-type enzyme, and stoichiometry studies indicated enhanced coupling of reducing equivalents to product formation for ( S)-warfarin, again in contrast to effects observed with ( S)-flurbiprofen. These data demonstrate that F114 and F476, but not F100, influence ( S)-warfarin's catalytic orientation. Differential interactions of F476 mutants with the two substrates suggest that their catalytically productive binding modes are not superimposable.     

Structural and functional insights into polymorphic enzymes of cytochrome P450 2C8

The cytochrome P450 (CYP) superfamily plays a key role in the oxidative metabolism of a wide range of drugs and exogenous chemicals. CYP2C8 is the principal enzyme responsible for the metabolism of the anti-cancer drug paclitaxel in the human liver. Nearly all previous works about polymorphic variants of CYP2C8 were focused on unpurified proteins, either cells or human liver microsomes; therefore their structure–function relationships were unclear. In this study, two polymorphic enzymes of CYP2C8 (CYP2C8.4 (I264M) and CYP2C8 P404A) were expressed in E. coli and purified. Metabolic activities of paclitaxel by the two purified polymorphic enzymes were observed. The activity of CYP2C8.4 was 25% and CYP2C8 P404A was 30% of that of WT CYP2C8, respectively. Their structure–function relationships were systematically investigated for the first time. Paclitaxel binding ability of CYP2C8.4 increased about two times while CYP2C8 P404A decreased about two times than that of WT CYP2C8. The two polymorphic mutant sites of I264 and P404, located far from active site and substrate binding sites, significantly affect heme and/or substrate binding. This study indicated that two important nonsubstrate recognition site (SRS) residues of CYP2C8 are closely related to heme binding and/or substrate binding. This discovery could be valuable for explaining clinically individual differences in the metabolism of drugs and provides instructed information for individualized medication.     

Differential contribution of active site residues in substrate recognition sites 1 and 5 to cytochrome P450 2C8 substrate selectivity and regioselectivity

Selected active site residues in substrate recognition sites (SRS) 1 and 5 of cytochrome P450 2C8 (CYP2C8) were mutated to the corresponding amino acids present in CYP2C9 to investigate the contribution of these positions to the unique substrate selectivity and regioselectivity of CYP2C8. The effects of mutations, singly and in combination, were assessed from changes in the kinetics of paclitaxel 6alpha-hydroxylation, a CYP2C8-specific pathway, and the tolylmethyl and ring hydroxylations of torsemide, a mixed CYP2C9/CYP2C8 substrate. Within SRS1, the single mutation S114F abolished paclitaxel 6alpha-hydroxylation, while the I113V substitution resulted in modest parallel reductions in K(m) and V(max). Mutations in SRS5 (viz., V362L, G365S, and V366L) reduced paclitaxel intrinsic clearance (V(max)/K(m)) by 88-100%. Torsemide is preferentially metabolized by CYP2C9, and it was anticipated that the mutations in CYP2C8 might increase activity. However, methyl and ring hydroxylation intrinsic clearances were either unchanged or decreased by the mutations, although hydroxylation regioselectivity was often altered relative to wild-type CYP2C8. The mutations significantly increased (28-968%) K(m) values for both torsemide methyl and ring hydroxylation but had variable effects on V(max). The effects of the combined mutations in SRS1, SRS5, and SRS1 plus SRS5 were generally consistent with the changes produced by the separate mutations. Mutation of CYP2C8 at position 359 (S359I), a site of genetic polymorphism in CYP2C9, resulted in relatively minor changes in paclitaxel- and torsemide-hydroxylase activities. The results are consistent with multiple substrate binding orientations within the CYP2C8 active site and a differential contribution of active site residues to paclitaxel and torsemide binding and turnover.     

Homology modeling and substrate binding study of human CYP2C9 enzyme

The CYP2C subfamily of human liver P450 isozymes is of major importance in drug metabolism. The most abundant 2C isozyme, CYP2C9, regioselectively hydroxylates a wide variety of substrates. A major obstacle to understanding this specificity in human CYP2C9 is the absence of a 3D structure. A 3D model of CYP2C9 was built, assessed, and used to characterize explicit enzyme-substrate complexes using methods previously developed in our laboratory. The 3D model was assessed by determining its stability to unconstrained molecular dynamics and by comparison of specific properties with those of known protein structures. The CYP2C9 model was then used to characterize explicit enzyme complexes with three structurally and chemically diverse substrates: (S)-naproxen, phenytoin, and progesterone. Each substrate was found to bind to the enzyme with a favorable interaction energy and to remain in the binding site during unconstrained molecular dynamics. Moreover, the mode of binding of each substrate led to calculated preferred hydroxylation sites consistent with experiment. Binding-site residues identified for the models included Arg 105 and Arg97 as key cationic residues, as well as Asn 202, Asp 293, Pro 101, Leu 102, Gly 296, and Phe 476. Site-specific mutations are proposed for further integrated computational and experimental study.     

Substrate selectivity of human cytochrome P450 2C9: importance of residues 476, 365, and 114 in recognition of diclofenac and sulfaphenazole and in mechanism-based inactivation by tienilic acid

A series of six site-directed mutants of CYP 2C9 were constructed with the aim to better define the amino acid residues that play a critical role in substrate selectivity of CYP 2C9, particularly in three distinctive properties of this enzyme: (i) its selective mechanism-based inactivation by tienilic acid (TA), (ii) its high affinity and hydroxylation regioselectivity toward diclofenac, and (iii) its high affinity for the competitive inhibitor sulfaphenazole (SPA). The S365A mutant exhibited kinetic characteristics for the 5-hydroxylation of TA very similar to those of CYP 2C9; however, this mutant did not undergo any detectable mechanism-based inactivation by TA, which indicates that the OH group of Ser 365 could be the nucleophile forming a covalent bond with an electrophilic metabolite of TA in TA-dependent inactivation of CYP 2C9. The F114I mutant was inactive toward the hydroxylation of diclofenac; moreover, detailed analyses of its interaction with a series of SPA derivatives by difference visible spectroscopy showed that the high affinity of SPA to CYP 2C9 (K(s)=0.4 microM) was completely lost when the phenyl substituent of Phe 114 was replaced with the alkyl group of Ile (K(s)=190+/-20 microM), or when the phenyl substituent of SPA was replaced with a cyclohexyl group (K(s)=120+/-30 microM). However, this cyclohexyl derivative of SPA interacted well with the F114I mutant (K(s)=1.6+/-0.5 microM). At the opposite end, the F94L and F110I mutants showed properties very similar to those of CYP 2C9 toward TA and diclofenac. Finally, the F476I mutant exhibited at least three main differences compared to CYP 2C9: (i) big changes in the k(cat) and K(m) values for TA and diclofenac hydroxylation, (ii) a 37-fold increase of the K(i) value found for the inhibition of CYP 2C9 by SPA, and (iii) a great change in the regioselectivity of diclofenac hydroxylation, the 5-hydroxylation of this substrate by CYP 2C9 F476I exhibiting a k(cat) of 28min(-1). These data indicate that Phe 114 plays an important role in recognition of aromatic substrates of CYP 2C9, presumably via Pi-stacking interactions. They also provide the first experimental evidence showing that Phe 476 plays a crucial role in substrate recognition and hydroxylation by CYP 2C9.     

Essential requirements for substrate binding affinity and selectivity toward human CYP2 family enzymes

A detailed analysis of substrate selectivity within the cytochrome P450 2 (CYP2) family is reported. From a consideration of specific interactions between drug substrates for human CYP2 family enzymes and the putative active sites of CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, and CYP2E1, it is likely that the number and disposition of hydrogen bond donor/acceptors and aromatic rings within the various P450 substrate molecules determines their enzyme selectivity and binding affinity, together with directing their preferred routes of metabolism by the CYP2 enzymes concerned. Although many aliphatic residues are present in most P450 active sites, it would appear that their main contribution centers around hydrophobic interactions and desolvation processes accompanying substrate binding. Molecular modeling studies based on the recent CYP2C5 crystal structure appear to show close agreement with site-directed mutagenesis experiments and with information on substrate metabolism and selectivity within the CYP2 family.     

Identification of human CYP2C19 residues that confer S-mephenytoin 4'-hydroxylation activity to CYP2C9

CYP2C19 is selective for the 4'-hydroxylation of S-mephenytoin while the highly similar CYP2C9 has little activity toward this substrate. To identify critical amino acids determining the specificity of human CYP2C19 for S-mephenytoin 4'-hydroxylation, we constructed chimeras by replacing portions of CYP2C9 containing various proposed substrate recognition sites (SRSs) with those of CYP2C19 and mutating individual residues by site-directed mutagenesis. Only a chimera containing regions encompassing SRSs 1--4 was active (30% of wild-type CYP2C19), indicating that multiple regions are necessary to confer specificity for S-mephenytoin. Mutagenesis studies identified six residues in three topological components of the proteins required to convert CYP2C9 to an S-mephenytoin 4'-hydroxylase (6% of the activity of wild-type CYP2C19). Of these, only the I99H difference located in SRS 1 between helices B and C reflects a change in a side chain that is predicted to be in the substrate-binding cavity formed above the heme prosthetic group. Two additional substitutions, S220P and P221T residing between helices F and G but not in close proximity to the substrate binding site together with five differences in the N-terminal portion of helix I conferred S-mephenytoin 4'-hydroxylation activity with a K(M) similar to that of CYP2C19 but a 3-fold lower K(cat). Three residues in helix I, S286N, V292A, and F295L, were essential for S-mephenytoin 4'-hydroxylation activity. On the basis of the structure of the closely related enzyme CYP2C5, these residues are unlikely to directly contact the substrate during catalysis but are positioned to influence the packing of substrate binding site residues and likely substrate access channels in the enzyme.     

Analysis of human cytochrome P450 2C8 substrate specificity using a substrate pharmacophore and site-directed mutants

The structural determinants of substrate specificity of human liver cytochrome P450 2C8 (CYP2C8) were investigated using site-directed mutants chosen on the basis of a preliminary substrate pharmacophore and a three-dimensional (3D) model. Analysis of the structural features common to CYP2C8 substrates exhibiting a micromolar K(m) led to a substrate pharmacophore in which the site of oxidation by CYP2C8 is 12.9, 8.6, 4.4, and 3.9 A from features that could establish ionic or hydrogen bonds, and hydrophobic interactions with protein amino acid residues. Comparison of this pharmacophore with a 3D model of CYP2C8 constructed using the X-ray structure of CYP2C5 suggested potential CYP2C8 amino acid residues that could be involved in substrate recognition. Twenty CYP2C8 site-directed mutants were constructed and expressed in yeast to compare their catalytic activities using five CYP2C8 substrates that exhibit different structures and sizes [paclitaxel, fluvastatin, retinoic acid, a sulfaphenazole derivative (DMZ), and diclofenac]. Mutation of arginine 241 had marked effects on the hydroxylation of anionic substrates of CYP2C8 such as retinoic acid and fluvastatin. Serine 100 appears to be involved in hydrogen bonding interactions with a polar site of the CYP2C8 substrate pharmacophore, as shown by the 3-4-fold increase in the K(m) of paclitaxel and DMZ hydroxylation after the S100A mutation. Residues 114, 201, and 205 are predicted to be in close contact with substrates, and their mutations lead either to favorable hydrophobic interactions or to steric clashes with substrates. For instance, the S114F mutant was unable to catalyze the 6alpha-hydroxylation of paclitaxel. The S114F and F205A mutants were the best catalysts for retinoic acid and paclitaxel (or fluvastatin) hydroxylation, respectively, with k(cat)/K(m) values 5 and 2.1 (or 2.4) times higher, respectively, than those found for CYP2C8. Preliminary experiments of docking of the substrate into the experimentally determined X-ray structure of substrate-free CYP2C8, which became available quite recently [Schoch, G. A., et al. (2004) J. Biol. Chem. 279, 9497], were consistent with key roles for S100, S114, and F205 residues in substrate binding. The results suggest that the effects of mutation of arginine 241 on anionic substrate hydroxylation could be indirect and result from alterations of the packing of helix G with helix B'.     

Topological role of cytochrome P450 2D6 active site residues

Recent reports have identified Phe120, Asp301, Thr309, and Glu216 as important residues in cytochrome P450 2D6 (CYP2D6) substrate binding and catalysis. Complementary homology models have located these amino acids within the binding pocket of CYP2D6 and in the present study we have used aryldiazenes to test these models and gain further insight in the role these amino acids have in maintaining the integrity of the active site cavity. When Phe120 was replaced to alanine, there was a significant increase in probe migration to pyrrole nitrogens C and D, in agreement with homology models which have located the phenyl side-chain of Phe120 above these two pyrrole rings. No changes in topology were observed with the D301Q mutant, supporting claims that in this mutant the electrostatic interactions with the B/C-loop are largely maintained and the loop retains its native orientation. The T309V mutation resulted in significant topological alteration suggesting that, in addition to its potential role in dioxygen activation, Thr309 plays an important structural role within the active site crevice. Replacement of Ile106 with Glu, engineered to cause electrostatic repulsion with Glu216, had a profound topological effect in the higher region within the active site cavity and impaired the catalytic activity towards CYP2D6 probe substrates.     

Combination of docking,molecular dynamics and quantum mechanical calculations for metabolism prediction of 3,4-methylenedioxybenzoyl-2-thienylhydrazone

In modern drug discovery process, ADME/Tox properties should be determined as early as possible in the test cascade to allow a timely assessment of their property profiles. To help medicinal chemists in designing new compounds with improved pharmacokinetics, the knowledge of the soft spot position or the site of metabolism (SOM) is needed. In silico methods based on docking, molecular dynamics and quantum chemical calculations can bring us closer to understand drug metabolism and predict drug–drug interactions. We report herein on a combined methodology to explore the site of metabolism prediction of a new cardioactive drug prototype, LASSBio-294 (1), using MetaPrint2D to predict the most likely metabolites, combined with structure-based tools using docking, molecular dynamics and quantum mechanical calculations to predict the binding of the substrate to CYP2C9 enzyme, to estimate the binding free energy and to study the energy profiles for the oxidation of (1). Additionally, the computational study was correlated with a metabolic fingerprint profiling using LC-MS analysis. The results obtained using the computational methods gave valuable information about the probable metabolites of (1) (qualitatively) and also about the important interactions of this lead compound with the amino acid residues of the active site of CYP2C9. Moreover, using a combination of different levels of theory sheds light on the understanding of (1) metabolism by CYP2C9 and its mechanisms. The metabolic fingerprint profiling of (1) has shown that the metabolites founded in highest concentration in different species were metabolites M1, M2 and M3, whereas M8 was found to be a minor metabolite. Therefore, our computational study allowed a qualitative prediction for the metabolism of (1). The approach presented here has afforded new opportunities to improve metabolite identification strategies, mediated by not only CYP2C9 but also other CYP450 family enzymes.     

Molecular simulation study of CYP2B6 polymorphism with and without psoralen

CYP2B6 is a polymorphic enzyme with a large number of variants which may lead to functional changes in enzyme activity and substrate selectivity. In this study, CYP2B6 and its three variants with and without psoralen, a mechanism-based inactivator, were investigated using molecular simulation method. The obtained docking orientation of psoralen was in agreement with previously identified site of metabolism. Stability analysis showed that the three variants displayed more flexibility than CYP2B6.1, and CYP2B6.34 was the most flexible one without psoralen binding. However, in the presence of psoralen, CYP2B6.34 became more rigidity. Tunnel analysis indicates that the bottleneck change of tunnels may be correlated to the increased or decreased activity of variants. Binding free energy analysis shows that van der Waals interaction dominates the binding of psoralen. CYP2B6.34 has the highest affinity to psoralen with lowest binding free energy. Ile114, Phe115 and heme contribute largely to the binding of psoralen with CYP2B6.6, while Phe206 and Leu363 play important roles for CYP2B6.1 and CYP2B6.4. These computational observations suggest that the increased activity of CYP2B6.4 and reduced activity of CYP2B6.6 may be due to changes in regional structures.     

Stereoselective hexobarbital 3'-hydroxylation by CYP2C19 expressed in yeast cells and the roles of amino acid residues at positions 300 and 476

We examined the enzymatic function of recombinant CYP2C19 in enantiomeric hexobarbital (HB) 3'-hydroxylation, and searched the roles of amino acid residues, such as Phe-100, Phe-114, Asp-293, Glu-300, and Phe-476 of CYP2C19 in the stereoselective HB 3'-hydroxylation, using a yeast cell expression system and site-directed mutagenesis method. CYP2C19 wild-type exerted substrate enantioselectivity of (R)-HB>(S)-HB and metabolite diastereoselectivity of 3'(R)<3'(S) in 3'-hydroxylation of HB enantiomers. The substitution of Asp-293 by alanine failed to yield an observable peak at 450 nm in its reduced carbon monoxide-difference spectrum. CYP2C19-E300A and CYP2C19-E300V with alanine and valine, respectively, in place of Glu-300 exerted total HB 3'-hydroxylation activities of 45 and 108%, respectively, that of the wild-type. Interestingly, these two mutants showed substrate enantioselectivity of (R)-HB<(S)-HB, which is opposite to that of the wild-type, while metabolite diasteroselectivity remained unchanged. The replacement of Phe-476 by alanine increased total HB 3'-hydroxylation activity to approximately 3-fold that of the wild-type. Particularly, 3'(S)-OH-(S)-HB-forming activity elevated to 7-fold that of the wild-type, resulting in the reversal of the substrate enantioselectivity. In contrast, the substitution of phenylalanine at positions 100 and 114 by alanine did not produce a remarkable change in the total activity or the substrate enantioselectivity. These results indicate that Glu-300 and Phe-476 are important in stereoselective oxidation of HB enantiomers by CYP2C19.     

Targeting of Splice Variants of Human Cytochrome P450 2C8 (CYP2C8) to Mitochondria and Their Role in Arachidonic Acid Metabolism and Respiratory Dysfunction

In this study, we found that the full-length CYP2C8 (WT CYP2C8) and N-terminal truncated splice variant 3 (∼44-kDa mass) are localized in mitochondria in addition to the endoplasmic reticulum. Analysis of human livers showed that the mitochondrial levels of these two forms varied markedly. Molecular modeling based on the x-ray crystal structure coordinates of CYP2D6 and CYP2C8 showed that despite lacking the N-terminal 102 residues variant 3 possessed nearly complete substrate binding and heme binding pockets. Stable expression of cDNAs in HepG2 cells showed that the WT protein is mostly targeted to the endoplasmic reticulum and at low levels to mitochondria, whereas variant 3 is primarily targeted to mitochondria and at low levels to the endoplasmic reticulum. Enzyme reconstitution experiments showed that both microsomal and mitochondrial WT CYP2C8 efficiently catalyzed paclitaxel 6-hydroxylation. However, mitochondrial variant 3 was unable to catalyze this reaction possibly because of its inability to stabilize the large 854-Da substrate. Conversely, mitochondrial variant 3 catalyzed the metabolism of arachidonic acid into 8,9-, 11,12-, and 14,15-epoxyeicosatrienoic acids and 20-hydroxyeicosatetraenoic acid when reconstituted with adrenodoxin and adrenodoxin reductase. HepG2 cells stably expressing variant 3 generated higher levels of reactive oxygen species and showed a higher level of mitochondrial respiratory dysfunction. This study suggests that mitochondrially targeted variant 3 CYP2C8 may contribute to oxidative stress in various tissues.     

Comparative homology modeling of human cytochrome P4501A1 (CYP1A1) and confirmation of residues involved in 7-ethoxyresorufin O-deethylation by site-directed mutagenesis and enzyme kinetic analysis

CYP1A1 homology models based on the CYP2C5 and a composite of CYP2C5, CYP2C8, and CYP2C9 X-ray crystal structures were compared to a model generated using the recently published coordinates of CYP1A2. The model using the CYP1A2 coordinates, CYP1A1-(1A2), gave near ideal stereochemical quality and was favored energetically. Docking studies identified the active-site residues potentially involved in binding of the prototypic CYP1A1 substrate 7-ethoxyresorufin. CYP1A1 mutants S122A, F123A, F224A, A317Y, T321G, and I386G were generated to explore the roles of these residues in 7-ethoxyresorufin binding and turnover, and generally confirmed the importance of aromatic interactions over hydrogen bonding in orientating 7-ethoxyresrufin in a catalytically favorable orientation. Although 7-ethoxyresorufin O-deethylation by CYP1A1 and several mutants exhibited substrate inhibition, it is unlikely that inhibition arises from the simultaneous binding of two substrates within the active-site given the geometry of the active site-cavity.     

Engineering the substrate specificity of cytochrome P450 CYP102A2 by directed evolution: production of an efficient enzyme for bioconversion of fine chemicals

The P450 cytochromes constitute a large family of hemoproteins that catalyze the monooxygenation of a diversity of hydrophobic substrates. CYP102A2 is a catalytically self-sufficient cytoplasmic enzyme from Bacillus subtilis, containing both a monooxygenase domain and a reductase domain on a single polypeptide chain. CYP102A2 was subjected to error-prone PCR to generate mutants with enhanced activity with fatty acids and other aromatic substrates. The library of CYP102A2 mutants was expressed in BL21(DE3) Escherichia coli cells and screened for their ability to oxidize different substrates by means of an activity assay. After a single round of error-prone PCR, the variant Pro15Ser exhibiting modified substrate specificity was generated. This variant showed approximately 6- to 9-fold increased activity with SDS, lauric acid and 1,4-naphthoquinone, and enhanced activity for other substrates such as ethacrynic acid and epsilon-amino-n-caproic acid. Molecular modeling of the CYP102A2 monooxygenase domain suggested that Pro15 is located in a short helical segment and is involved in extensive interactions between the N-terminal domain and the beta2 sheet, which contribute to the formation of the substrate binding site. Thus, Pro15 appears to affect substrate binding and catalysis indirectly. These results clearly demonstrate the importance of remote residues, not readily predicted by rational design, for the determination of substrate specificity. In addition, we report here that the Pro15Ser variant of CYP102A2 can be efficiently immobilized on epoxy-activated Sepharose at pH 8.5 and 4 degrees C. The immobilized variant of CYP102A2 retains most of its activity (81%) and shows improved stability at 37 degrees C. The approach offers the possibility of designing a P450 bioreactor that can be operated over a long period of time with high efficiency and which can be used in fine chemical synthesis.     

Homology model of human retinoic acid metabolising enzyme cytochrome P450 26A1 (CYP26A1): active site architecture and ligand binding

Homology models of cytochrome P450 RA1 (CYP26A1) were constructed using three human P450 structures, CYP2C8, CYP2C9 and CYP3A4 as templates for the model building. Using MOE software the lowest energy CYP26A1 model was then assessed for stereochemical quality and side chain environment. Further active site optimisation of the CYP26A1 model built using the CYP3A4 template was performed by molecular dynamics to generate a final CYP26A1 model. The natural substrate, all-trans-retinoic acid (atRA), and inhibitor R 15866, were docked into the model allowing further validation of the active site architecture. Using the docking studies structurally and functionally important residues were identified with subsequent characterisation of secondary structure. Multiple hydrophobic interactions, including the side chains of TRP112, PHE299, PHE222, PHE84, PHE374 and PRO371, are important for binding of atRA and R115866. Additional hydrogen bonding interactions were noted as follows: atRA-- C==O of the atRA carboxylate group and ARG86; R115866--benzothiazole nitrogen and the backbone NH of SER115.     

An in vivo pilot study characterizing the new CYP2A6*7, *8, and *10 alleles.

We developed genotyping assays for CYP2A6*7 (Ile471Thr) and CYP2A6*8 (Arg485Leu). We found higher allelic frequencies in Japanese and Chinese versus Caucasians and identified an allele in which both substitutions occur together (CYP2A6*10). We created a homology model for predicting the impact of allelic variants on enzymatic activity and subsequently tested this in vivo in a pilot kinetic study. Consistent with our homology model predictions, we found (i) that CYP2A6*7 produces an enzyme that has decreased (not inactive) activity for metabolizing nicotine and coumarin; (ii) that CYP2A6*8 is unlikely to affect catalytic activity in vivo; and (iii) that having both substitutions together on an allele (CYP2A6*10) dramatically reduces function and may be fully inactive for some substrates. In conclusion, this study identifies, at relatively high frequency in Asians, an allele with decreased activity (may be substrate selective), a fully functional allele, and an allele containing both substitutions in which function is dramatically reduced.     

Structural forms of phenprocoumon and warfarin that are metabolized at the active site of CYP2C9

Possible reasons for the observed differences in metabolic behavior and drug interaction liability between the structurally similar oral anticoagulants warfarin and phenprocoumon were explored. Incubating (S)-phenprocoumon with human liver microsomes and cDNA-expressed CYP2C9 and determining its metabolism both in the absence and presence of the CYP2C9 inhibitor, sulfaphenazole, confirmed that phenprocoumon is a substrate for CYP2C9. Comparing the metabolic behavior of (S)- and (R)-warfarin, (S)- and (R)-phenprocoumon, and fixed structural mimics of the various tautomeric forms [(S)- and (R)-4-methoxyphenprocoumon, (S)- and (R)-2-methoxyphenprocoumon, (S)- and (R)-4-methoxywarfarin, (S)- and (R)-2-methoxywarfarin, and 9(S)- and 9(R)-cyclocoumarol] available to these two drugs with expressed CYP2C9 provides compelling evidence indicating that the ring closed form of (S)-warfarin and the ring opened anionic form of (S)-phenprocoumon are the major and specific structural forms of the two drugs that interact with the active site of CYP2C9. The conclusion that (S)-warfarin and (S)-phenprocoumon interact with CYP2C9 in very different structural states provides a clear basis for the significant differences observed in their metabolic profiles. Moreover, in accord with a previously established CoMFA model these results are consistent with the hypothesis that the active site of CYP2C9 possesses at least two major substrate binding sites, a pi-stacking site for aromatic rings and an ionic binding site for organic anions. An additional electrostatic binding site also appears to contribute to the orientation of coumarin analogs in the CYP2C9 active site by interacting with the C2-carbonyl group of the coumarin nucleus.