Experimental Procedures

Materials.

Chemicals

The Saccharomyces cerevisiae strain INVSc1-HR bearing the human reductase gene was a gift from the LINK Project (a program of the University of Dundee/Biotechnology and Biology Research Council/Department of Trade and Industry/Pharmaceutical Industry). The galactose-inducible expression vector pYeD60 was a gift from Dr. Magnus Ingelman-Sundberg (Karolinska Institute, Stockholm, Sweden). cDNAs for CYP1A1, CYP1A2, CYP2C19, CYP2E1, and CYP2D6 were also gifts from Dr Magnus Ingelman-Sundberg. cDNAs for CYP2C9, CYP3A4, and CYP3A5 were received from the LINK Project and the cDNA of CYP2C8 was cloned from a human liver cDNA library. Isoform-specific primers were synthesized in our laboratory. Polymerase chain reaction and sequencing chemicals were of the highest grade commercially available.

Reagents used in the culturing of yeast and preparation of microsomes were: yeast nitrogen base, yeast extract, and peptone (Difco, Detroit, MI); histidine, leucine, tryptophan, glucose, and galactose (Sigma Chemical Co., St. Louis, MO); PEG 4000 (Kebo, Stockholm, Sweden); d-sorbitol (Sigma); yeast lytic enzyme (ICN Biochemicals Inc., Costa Mesa, CA); and Pefabloc and dithiothreitol (DTT; Boehringer Mannheim, Mannheim, Germany).

Substrates, metabolites, and inhibitors of the various CYPs were purchased from different companies: 7-ethoxyresorufin, resorufin, diclofenac, α-naphthoflavone, and quinidine (Sigma); 4-OH-diclofenac, paclitaxel, and 6α-hydroxypaclitaxel (GeneTest, Woburn, MA); bufuralol, 1′-OH-bufuralol and sulfaphenazole,S-mephenytoin, and 4-OH-mephenytoin (Ultrafine, Manchester, UK); testosterone and 6β-OH-testosterone (Steraloids Inc., Newport, RI); chlorzoxazone, 6-OH-chlorzoxazone, and quercetin (Aldrich Chemical Co., Milwaukee, WI); ketoconazole (Janssen Biotech, Flanders, NJ); and ticlopidine (ICN Biochemicals Inc.). NADPH and cytochrome c were obtained from Sigma.

Equipment.

Primers were synthesized on a Beckman Oligo 100 M DNA synthesizer and polymerase chain reaction done on a PTC-200 Engine (MS Research, Inc., Watertown, NY) and automated sequencing on an ABI Prism 377 DNA Sequencer (Perkin-Elmer Cetus Instruments, Eden Prairie, MN). Fermentations were done in a 20-liter Biostat C Fermentor (B. Braun Biotech International GmbH, Melzungen, Germany). Centrifugations were done on an Avanti J-20 centrifuge (Beckman Instruments, Berkeley, CA). A Rannie high pressure laboratory homogenizer 8.30H (ADV Homogeniser Group, Copenhagen, Denmark) and Ultraturrax homogenizer (Janke and Kunkel IKA Labortechnik, Germany) were used in the preparation of microsomes.

Measurement of CYP content and NADPH-P-450 reductase activity was done on a UV-24001PC UV-visible spectrophotometer (Shimadzu, Tokyo, Japan). Fluorometric assays were done on a Fluoroskan II fluorometer (Labsystems, Helsinki, Finland). Chromatographic assays were done with Zorbax C₁₈, 4.6 mm × 15 cm columns and C₁₈, 4.6 mm × 1.25 cm guard columns on a Hewlett Packard HPLC (Waldbronn, Germany) coupled with either a Diode Array detector (Hewlett-Packard) or fluorescence detector (JASCO, Tokyo, Japan).

Methods.

Construction of expression plasmids

The INVSc1-Hr yeast strain modified by the integration of the human NADPH-P-450 reductase gene into its genome (LINK project) was used for the expression of human CYPs. Figure 1shows the structure of the expression plasmid pYeDP60 (Urban et al., 1990) into which cDNAs of individual CYPs were cloned. The CYP cDNAs’ restriction sites at the 5-prime end were modified to be BamHI and the 3-prime modified to be EcoRI or KpnI sites. The cDNAs were cloned into BamHI-EcoRI orBamHI-KpnI sites of the pYePD60 vector (Fig. 1). The yeast bearing the human reductase was transformed with the pYeDP60-CYP constructs to give a panel of yeast strains for the production of the following CYPs: 1A1, 1A2, 2C8, 2C9, 2C19, 2D6, 2E1, 3A4, and 3A5.

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Figure 1

Structure of the pYeDP60 vector with cloning sites.

Enzyme expression and microsomal preparation.

Selective medium (yeast nitrogen base, glucose, 20 μg/ml His, 20 μg/ml Leu) was inoculated with yeast cells, INVSc1-HR, transformed with plasmids carrying specific CYPs, and incubated at 30°C for 24 h. A rich fermentation medium (200 g yeast extract/10 liters, 200 g peptone/10 liters, 2% glucose) at pH 6.7 was kept at 30°C in a 20-liter fermentor. The fermentation was started by inoculation with a preculture grown in selective medium at 1 liter innoculum/10 liter rich medium and kept at pH 6.7. After 12 h of fermentation, 600 ml of 50% glucose and 300 ml of ethanol were added. After another 18 h of fermentation, induction of CYP expression was initiated by addition of 1 liter of 20% galactose. After an induction period of 8 h, the cells were harvested by centrifugation (10,000g, 10 min).

The cells were suspended in 2 volumes of 50 mM Tris, 0.1 mM EDTA, 0.1 mM DTT, 2 M sorbitol (pH 7.4). Yeast lytic enzyme (2 mg/g; w/w) was dissolved in the suspension and homogenized with Ultraturrax (low speed, 20 s). The suspension was incubated at 30°C with gentle shaking for 1 h. After centrifugation at 3000g for 10 min, the pellet was resuspended in 2 volumes of 50 mM Tris, 1.0 mM EDTA, 0.1 mM DTT, 0.6 M sorbitol, 10% glycerol (pH 7.4). Pefabloc (4 mM) was added and the suspension passed through a Rannie 8.30 high pressure homogenizer at 1000 bar three times. The homogenate was centrifuged at 3000g for 5 min and at 10,000g for 10 min and the pellet discarded. PEG 4000 and (50%) 5 M NaCl were added to the supernatant to give final concentrations of 10% and 0.1 M respectively. After 1 h on ice, the precipitated microsomes were collected by centrifugation at 12,000g for 15 min. The resulting microsomal pellet was homogenized with Ultraturrax (low speed, 20 s) in 50 mM potassium phosphate buffer (pH 7.4) with 1.0 mM EDTA and 20% glycerol.

The protein concentration was measured according to the Bio-Rad Protein Assay (Bio-Rad Laboratories, Inc., Richmond, CA). The CYP content was determined according to Omura and Sato (1964) and the NADPH-P-450 reductase activity was measured according to Pearce et al. (1996; Table 2).

Enzyme Kinetics.

General incubation conditions

All incubations were done in a total volume of 200 μl. Final concentrations of 1 mM NADPH and 0.1 M potassium phosphate buffer (pH 7.4) were used. The substrates were dissolved in ethanol, water, or methanol and 2 to 5 μl was added to the incubation mixture. The stock enzyme concentrations were diluted to 0.5 to 1 pmol CYP/μl with 0.1 M potassium phosphate buffer (pH 7.4), and the appropriate volume was added to get the required enzyme concentration. Each reaction was optimized for CYP concentration and incubation time linearity (Table4). The stop solutions used were: 1) 50 μl of a 94% acetonitrile, 6% glacial acetic acid solution for CYP2C9; 2) 100 μl of 6% perchloric acid for CYP2C19; 3) 10 μl of 60% perchlorate for CYP2D6; and 4) 100 μl methanol for all other CYPs. The protein was then sedimented by centrifugation at 10,000g for 5 min. Analysis of the metabolites formed was done as described in Table1. Quantitation of metabolite formed was done from external standard curves made using authentic metabolites.

Evaluation of enzyme-substrate specificity and Michaelis-Menten kinetics.

The Michaelis-Menten kinetics of each CYP with respect to its marker substrate (Table 4) were done using modifications of analytical methods reported in the literature (Table 1): 7-ethoxyresorufinO-dealkylation (Burke and Mayer, 1985), paclitaxel 6α-hydroxylation (Rahman et al., 1994), diclofenac 4-hydroxylation (Leemann et al., 1993), bufuralol 1′-hydroxylation (Kronbach et al., 1987), S-mephenytoin 4-hydroxylation, chlorzoxazone 6-hydroxylation, and testosterone 6β-hydroxylation (Pearce et al., 1996). The catalytic activities of all the CYPs were assessed on each reaction (Table 3).

Evaluation of inhibitor-enzyme selectivity.

After a cursory review of the literature on inhibitor concentrations that would show inhibitor-CYP selectivity, an average of 10 μM was considered suitable for a one-concentration screening study. The inhibitors evaluated, α-naphthoflavone, quercetin dihydrate, quinidine, sulfaphenazole, and ketoconazole, were dissolved in methanol. Two microliters of the 1 mM stock solutions were added to the 200-μl incubation mixtures to give a final inhibitor concentration of 10 μM and an added methanol content of 1%. The effect of the methanol was assessed for each set of inhibition studies as a baseline activity level. The inhibitory effect of each compound was assessed on the catalytic activity of each CYP toward its maker reaction at conditions similar to those used in the enzyme-substrate specificity evaluation.

Data Analysis.

K_m andV_max were determined by nonlinear least-squares fitting using Graphfit Version 3.0 (Erithacus Software Limited, Middlesex, UK). Sigmaplot 4.0 (SPSS, Inc., Chicago, IL) was used for graphical representations.

Results and Discussion

We have successfully cloned and expressed human CYPs in S. cerevisae. The CYPs have been characterized with respect to their Michaelis-Menten kinetics, substrate specificity, and inhibitor-enzyme selectivity properties and have been found to be comparable to those of human liver microsomes and other recombinant CYPs. This means that we can use our enzymes in metabolism research and obtain valid in vitro data for extrapolation to in vivo metabolism.

The choice of yeast (S. cerevisiae) for the heterologous expression of human CYPs was based on a number of factors: 1) simplicity of setting up, 2) high yield production of stable enzymes without having to modify the cDNA sequence for most of the CYPs, and 3) the presence of an endoplasmic reticulum in yeast. The alternative systems where E. coli, which, although easy to grow and extract enzyme, requires modification of the CYP protein amino terminus for efficient enzyme production and also lacks an endoplasmic reticulum (Iwata et al., 1998). The lymphoblastoid and Hep G₂ cell systems with the apparent advantage of being mammalian are, however, difficult to optimize for maximal CYP expression (Gonzalez and Korzekwa, 1995). The baculovirus-insect cell system produces large amounts of enzyme per milligram of protein but is technically more difficult to set up and maintain because CYP expression is transient (Friedberg and Wolf, 1996). Many university laboratories, pharmaceutical companies, and biotech companies selling or using recombinant CYPs are using one or a combination of the above mentioned heterologous systems with success after appropriate modifications unique to each system. To avoid having to add exogenous NADPH-P-450-reductase to reaction mixtures, the systems have been engineered to coexpress the NADPH-P-450 reductase and cytochrome b₅ with the human CYP of interest (Gonzalez and Korzekwa, 1995).

In our system, the CYP yield ranged from 22 to 132 pmols/mg microsomal protein (Table 2). No CYP was detectable in the microsomes from yeast not transformed with recombinant CYP, implying that the yeast has very little constitutive CYP. The NADPH-P-450 reductase activity averaged 75 nmols reduced cytochromec/min/mg (Table 2). In comparison, human liver microsomes have an average total CYP content of 300 pmols/mg microsomal protein and average reductase activity of 150 nmols reduced cytochromec/min/mg (Pearce et al., 1996). By comparing the CYP content-reductase activity ratio in yeast and human liver microsomes, it can be concluded that the reductase will probably not be a limiting factor in supporting individual CYP reactions in our system. Expression system-related factors and incubation conditions can, however, have an effect on the coupling efficiency of the reductase and CYPs.

The CYPs showed expected substrate specificity (Table3) with respect to the established marker reactions (Ono et al., 1996). We have also shown for the first time that chlorzoxazone 6-hydroxylation is catalyzed by CYP1A1 (V_max = 1.88) with a high turnover besides the documented role of CYP2E1 and 1A2 (Ono et al., 1995). Chlorzoxazone 6-hydroxylation is used as an in vivo probe for hepatic CYP2E1 activity (Frye et al., 1997). The use of chlorzoxazone as an in vivo probe for CYP2E1 activity might not be affected because CYP1A1 is not constitutively expressed in the liver, but results in subjects pre-exposed to CYP1A1 and 1A2 inducers should be interpreted with caution. Reaction conditions for each CYP-marker reaction were optimized with respect to time and enzyme concentration for Michaelis-Menten kinetic studies. Table 4summarizes the apparent Michaelis-Menten constants (K_m) and the maximum turnover of the reactions (V_max) for the different CYPs with respect to specific marker reactions. TheK_m values obtained for the same reactions using human liver microsomes are also shown for comparison. The values we obtained are within the ranges of those reported in the literature. It can also be noted that recombinant CYP2E1 in our study and others (Ono et al., 1995; Kudo et al., 1997) has a higherK_m (177 μM) than that obtained using human liver microsomes (40 μM; Peter et al., 1990). Additional investigations on this observation are in progress.

Chemical inhibitors showed significant selectivity toward specific CYPs as reported in literature (Ono et al., 1996); α-naphthoflavone (CYP1A1 and 1A2), quercetin (CYP2C8), sulfaphenazole (CYP2C9), quinidine (CYP2D6), and ketoconazole (CYP3A4 and 3A5) (Fig.2). Methanol (Fig. 2) and acetonitrile (not shown) are good solvents for substrates and inhibitors because they have minimal inhibitory effects on CYP at final concentration of less than 2%.

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Figure 2

Selective inhibition of CYPs by several chemicals.

Ticlopidine has been previously shown to inhibit phenytoin metabolism (Donahue et al., 1997) and to inhibit the CYP2C19-catalyzed omeprazole metabolism more than the CYP3A4 activity in vivo (Tateishi et al., 1999). Using human liver microsomes, Donahue et al. (1997) showed that ticlopidine was a potent competitive inhibitor ofS-mephenytoin hydroxylation and a weak inhibitor of tolbutamide hydroxylation, and concluded that the drug inhibited CYP2C19 more than CYP2C9. Screening for the inhibitory effect of ticlopidine on all the CYPs in this study showed the drug to be a potent inhibitor of CYP2C19 (IC₅₀ = 4.5 μM) and CYP2D6 (IC₅₀ = 3.5 μM) activities (Fig.3). Coadministration of ticlopidine with drugs that are substrates of CYP2C19 (Donahue et al., 1997) or CYP2D6 can result in clinically important drug-drug interactions.

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Figure 3

Inhibition of CYP2C19 and CYP2D6 activity by ticlopidine.

The Michaelis-Menten parameters from different published studies show a tremendous variation in the apparent K_m andV_max values. Variation in inhibition constants (K_i and IC₅₀) can also be observed in published literature. The causes of these differences could be: 1) interindividual variation from using liver microsomes from donors with different genetic/disease/drug backgrounds, 2) differences causedby the specific expression system used owing to physiological factors unique to each system affecting the CYP quantity/affinity/activity profiles, and 3) interlaboratory variations due to differing experimental conditions used. These variations and their causes have important implications on the validity of metabolic in vitro-in vivo quantitative extrapolations. It has been shown that incubation conditions such as type of buffer (Tris or phosphate) and ionic strength, amounts of added NADPH-P-450 reductase, cytochromeb₅, protein content, glutathione, phospholipids, and detergents can have a profound effect on enzyme kinetics (Shet et al., 1995; Crespi, 1998; Mäenpää et al., 1998). Although there are great variations in the enzyme kinetic values, the ranking with respect to substrate specificity and inhibitor selectivity for the panel of drug-metabolizing CYPs is generally the same irrespective of source of enzyme or incubation conditions used. This means that the different laboratories can still reach the same qualitative conclusions with respect to metabolism studies but very different quantitative deductions.

In conclusion, our recombinant CYPs have passed a three level validation criterion test with respect to enzyme kinetic properties. This unlimited source of enzyme will satisfy the demands from our automated drug-drug interaction high-throughput screening procedures, metabolic pathway identification studies, and elucidation of structure/functional properties of each CYP for better prediction of the roles of CYPs in drug metabolism.