Introduction

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The antiprogestin mifepristone (RU 486), in combination with misoprostol, is the first clinically approved method of chemical abortion available for women in the US (DeHart and Morehead, 2001). The antiprogesterone activity of mifepristone has also shown promising potential against certain types of breast cancer, prostate cancer, meningioma, and uterine leiomyoma, as well as in the treatment of endometriosis (see review by Cadepond et al., 1997 and references therein). Mifepristone has also been reported to reverse P-glycoprotein-mediated drug resistance in vitro (Gruol et al., 1994; Lecureur et al., 1994), which could enhance its effectiveness as an anticancer agent alone or in combination therapy. Furthermore, the antiglucocorticoid activity of mifepristone also has several potential uses including the treatment of Cushing's syndrome, ectopic corticotropin secretion, adrenal carcinoma, and depression of human immunodeficiency virus replication (Cadepond et al., 1997).

The principal enzyme involved in the oxidation of mifepristone in human liver microsomes is cytochrome P450 3A4 (Jang et al., 1996), which is also inactivated by the drug (He et al., 1999). CYP3A4 and CYP3A5 are generally considered the two most important CYP3A enzymes in human liver. CYP3A5 is polymorphically expressed in 10 to 97% of adult livers and constitutes 6 to 100% of the entire hepatic CYP3A content (Wrighton et al., 1989, 1990; Hustert et al., 2001; Kuehl et al., 2001). These two CYP3A enzymes metabolize many of the same compounds and exhibit high (84%) amino acid sequence identity (Aoyama et al., 1989). However, compounds such as cyclosporin A (Aoyama et al., 1989), midazolam (Gorski et al., 1994), quinidine (Wrighton et al., 1990; Nielsen et al., 1999), and aflatoxin B₁ (Wang et al., 1998) are known to show significant differences in their oxidation by CYP3A4 and CYP3A5. In contrast, to date not a single selective inhibitor/inactivator of CYP3A4 or CYP3A5 has been identified that could be used unequivocally to differentiate between these two major drug-metabolizing enzymes. Azole inhibitors such as ketoconazole and fluconazole, although showing slightly higher potency toward CYP3A4, also inhibit CYP3A5 (Gibbs et al., 1999). Triacetyloleandomycin, which forms a complex between the ferrous form of the heme iron and a nitroso intermediate, and the acetylenic steroid gestodene, which probably inactivates the enzyme via a ketene, also fail to cause selective inhibition of these two CYP3A enzymes (Wrighton et al., 1990; Chang et al., 1994). In the present study, we compare and contrast P450 inactivation and oxidation of mifepristone by recombinant CYP3A4 and CYP3A5. The study finds that mifepristone oxidation causes selective CYP3A4 inactivation. The structural basis of this differential susceptibility has been further explored using site-directed mutants.

	Experimental Procedures

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Materials. Mifepristone, progesterone, 6-hydroxyprogesterone, NADPH, CHAPS,¹ and DOPC were purchased from Sigma-Aldrich (St. Louis, MO). Radiolabeled progesterone was obtained from PerkinElmer Life Sciences (Boston, MA). HEPES was obtained from Calbiochem Corp. (San Diego, CA). Oligonucleotide primers for PCR were either obtained from the University of Texas Medical Branch Molecular Biology Core Laboratory (Galveston, TX) or from Sigma-Genosys (The Woodlands, TX). The Expand PCR kit and Rapid Ligation kits were obtained from Roche Diagnostics (Indianapolis, IN). The QuikChange site-directed mutagenesis kit and GeneClean kit were from Stratagene (La Jolla, CA) and Qbiogene Inc. (Carlsbad, CA), respectively. Talon and Ni²⁺-metal affinity resin were from BD Biosciences Clontech (Palo Alto, CA) and Qiagen (Valencia, CA), respectively. Thin layer chromatography plates [silica gel, 250 µm, Si 250F (C19)] were purchased from J. T. Baker, Inc. (Phillipsburg, NJ). Recombinant NADPH-cytochrome P450 reductase and cytochrome b₅ from rat liver were prepared as described earlier (Harlow and Halpert, 1997). All other chemicals were of the highest grade available and were obtained from standard commercial sources.

Construction of CYP3A4 Mutants. Construction of mutants L210F and I369V was described previously (He et al., 1997; Wang et al., 1998). Mutants P107S, I120L, N206S, V376T, and L479T in the pCW expression vector and V296A in the pBS vector were reported by Wang et al. (1998). The V296A mutant was subcloned from pBS into the pSE380 expression vector using unique PstI/StuI restriction sites. The mutant M371I was constructed by site-directed mutagenesis using the Expand High Fidelity PCR system (Roche Diagnostics). F108L, S478D, G480Q, and the multiple mutant R212K/D214G/F219L/T224I/V225L/I230T were generated using the QuikChange site-directed mutagenesis kit (Stratagene). To construct this multiple mutant, the R212K/D214G and F219L/T224I/V225L/I230T mutants were first generated, and the BamHI/PstI fragment containing the F219L//T224I/V225L/I230T mutation was subcloned into pSE3A4 R212K/D214G. The primers used for the construction of the various mutants are listed in Table 1. All constructs were sequenced to ensure that only the desired mutation was present (Protein Chemistry Core Laboratory, The University of Texas Medical Branch Galveston, TX).

Expression and Purification of CYP3A4, CYP3A4 Mutants, and CYP3A5. Wild-type CYP3A4 and most previously generated mutants were expressed as His-tagged proteins in Escherichia coli TOPP3 (or DH5) cells and purified using Talon metal affinity resin (BD Biosciences Clontech) as described earlier (Domanski et al., 1998, 2001b; Khan et al., 2002, and references therein). The CYP3A4  CYP3A5 mutants P107S, F108L, I120L, N206S, V296A, M371I, V376T, S478D, L479T, and G480Q, as well as the multiple mutant R212K/D214G/F219L/T224I/V225L/I230T were devoid of a His-tag and were assayed as preparations of CHAPS-solubilized membranes. Heterologous expression and purification of (His)₆-tagged CYP3A5 using Ni²⁺-metal affinity chromatography (Qiagen) was performed as described previously (Domanski et al., 2001a; Hustert et al., 2001). The P450 contents were determined by carbon monoxide difference spectra in the presence of 1% Triton X-100. The Triton was added to the protein sample before dilution with 100 mM potassium phosphate, pH 7.3, 20% glycerol, 0.5% sodium cholate, 0.4% Renex, and 1.0 mM EDTA.

Mifepristone Oxidation Assay. The reconstituted system contained 5 pmol of purified P450 enzyme, 10 pmol of rat liver cytochrome b₅, 20 pmol of recombinant NADPH-cytochrome P450 reductase, 0.04% CHAPS, and 0.1 mg/ml DOPC. The mixture was incubated for 10 min at room temperature. A fixed concentration of mifepristone dissolved in 2% methanol (final) was added to the reconstituted protein system in 50 mM HEPES buffer, pH 7.6, and 15 mM MgCl₂. The mixture was preincubated for 10 min at 37°C, and the reaction was initiated by adding NADPH (1 mM final). The total reaction volume of the assay was 100 µl. After 4 min of incubation at 37°C, the reactions were stopped by the addition of 200 µl acetonitrile. After centrifugation (3000 rpm, 5 min), 50 µl of the reaction mixture was analyzed by HPLC.

LC-MS Analysis. LC-MS analyses were performed on two different instruments. The initial LC-MS was performed at the School of Pharmacy, University of Arizona, Tucson, AZ. In this case, samples were prepared from incubations containing 100 pmol CYP3A4. The supernatant fractions were collected after centrifugation (3000 rpm, 10 min) and dried under a stream of nitrogen gas at room temperature. The dried sample was resuspended in the initial mobile phase before loading for LC-MS analysis. The LC-MS system consisted of a Finnigan-MAT LCQ instrument equipped with a Spectrasystem TSP P4000 HPLC system and an AS3000 auto sampler (Thermo Finnigan MAT, San Jose, CA). Automated acquisition of tandem mass spectometry spectra was carried out by data-dependent scanning with Finnigan Excalibur software. The total run time was set to 35 min. Metabolites were analyzed using the same Ultrasphere ODS C₁₈ column (5 µm × 250 mm × 4.6 mm; Beckman Coulter, Inc.) used for HPLC analysis. The initial mobile phase composition of water/acetonitrile/methanol in ratios of 55:25:20 (v/v/v) was held for 5 min, then changed to 25:40:35 over the course of 15 min, and held for 10 min. A flow rate of 0.7 ml/min was achieved with a splitter tee upstream of a Microm Magic flow splitter box (Michrom BioResources Inc., Auburn, CA). The mass spectra were recorded under positive ion electrospray conditions, and the tandem mass spectometry spectra were obtained by collisional activation of MH⁺ parent ion species. The retention times of mifepristone and its three metabolites on the LC-MS instrument were slightly different from when using HPLC, because of different instrumentation and a small change in flow rate, but their elution order was the same on both instruments and as described in the literature (Heikinheimo et. al., 1990).

Inactivation Assays. The inactivation assays were similar to those described previously (Khan et al., 2002). The preincubation mixture contained 35 pmol of purified P450, 70 pmol of rat liver cytochrome b₅, 140 pmol of recombinant NADPH-cytochrome P450 reductase, 0.04% CHAPS, and 0.1 mg/ml DOPC. After incubation for 10 min at room temperature, mifepristone (dissolved in methanol, 1% final) was added to the reconstituted protein system in 50 mM HEPES buffer, pH 7.6, and 15 mM MgCl₂. The protein-substrate mixture was further incubated for 2 min at 37°C, and the reaction was initiated by adding NADPH (1 mM final concentration). The total volume of the mixture was 560 µl. An aliquot (80 µl) of this mixture was taken at various time intervals (1-6 min), and added to a secondary reaction mixture (20 µl of volume) for determination of the residual progesterone hydroxylase activity ([progesterone] = 25 µM). The reaction was stopped after 6 min by the addition of 50 µl tetrahydrofuran and extracted with 1.0 ml of chloroform. The extracted solution (0.9 ml) was dried under nitrogen at 37°C, resuspended in 50 µl of chloroform, and spotted on the preadsorbent loading zone of a thin layer chromatography plate (Baker silica gel, 250 ml, Si 250F; J. T. Baker). The plate was developed twice in benzene:ethyl acetate:acetone (10:1:1, v/v/v). Metabolites of progesterone were visualized by autoradiography and identified by comparison with unlabeled standards. The radioactive areas from the plate were scraped into scintillation vials, and the metabolites were quantified by liquid scintillation counting.

	Results

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Mifepristone Oxidation and Inactivation of CYP3A4 and CYP3A5. The time-course of mifepristone oxidation by CYP3A4 showed nonlinear behavior (data not shown), consistent with the inactivation of the enzyme (He et al., 1999). At 20 µM mifepristone, the k_i as measured by the decrease in the rate of progesterone 6-hydroxylation was 0.17 min¹ (Fig. 1A, Table 2). The values for K_I and k_inactivation have been reported as 4.7 µM and 0.09 min¹, respectively (He, et al., 1999). In contrast to CYP3A4, CYP3A5 exhibited no time-dependent inhibition at mifepristone concentrations as high as 150 µM (Fig. 1B), although time-independent (presumably competitive) inhibition was clearly evident. Mifepristone at 20 µM also appears to be a more potent inhibitor of CYP3A4 than CYP3A5.

View larger version (13K): [in this window] [in a new window]   Fig. 1.   A, time-dependent inactivation of wild-type CYP3A4-dependent progesterone 6-hydroxylase activity at 0 () and 20 () µM mifepristone; details are described under Experimental Procedures; the lines shown through experimental points were generated by linear regression analysis of the natural logarithm of the residual activity as a function of time; the rate constant of inactivation (ki) is derived from the negative slope of the line, whereas the extent of reversible inhibition is reflected by a decrease in the extrapolated activity at zero preincubation time compared with the methanol control; B, time- and concentration-dependent percentage decrease in progesterone 6- hydroxylase activity of wild-type CYP3A5 at 0 (), 20 (), 100 (), and 150 () µM mifepristone.

View this table: [in this window] [in a new window]   TABLE 2 Inactivation by mifepristone (20 µM) of wild-type and mutant CYP3A enzymes CYP3A4  CYP3A5 mutants are shown in bold.

View larger version (27K): [in this window] [in a new window]   Fig. 2.   Mass spectra of (A) mifepristone and the three metabolites: (B) didemethylated mifepristone, (C) monodemethylated mifepristone, and (D) hydroxylated mifepristone, and their fragmentation patterns based on these peaks.

View larger version (17K): [in this window] [in a new window]   Fig. 3.   Steady-state kinetics of monodemethylated mifepristone formation by wild-type CYP3A4 () and CYP3A5 (). The lines through the experimental points show the fit to the Michaelis-Menten equation. Due to significant amount of substrate consumption at lower concentrations, the incubations were done using 5 pmol of the enzymes (see Experimental Procedures for details). Because of the low enzyme concentration used, the rates of didemethylated and hydroxylated mifepristone formation were too low to determine kinetic parameters for these metabolites.

Inactivation of CYP3A4 Site-Directed Mutants. CYP3A4 and CYP3A5 differ by 78 of 503 amino acids, 12 of which fall within the 5 putative SRS domains (Aoyama et al., 1989, Wang et al., 1998; Domanski et al., 2001a) shown to be responsible for the substrate specificity of 3A4 with a wide range of compounds (Domanski and Halpert, 2001). Interestingly, none of the CYP3A4  CYP3A5 mutants examined at these sites exhibited a significant decrease in susceptibility to inactivation by mifepristone (Table 2, bold). However, several of these residues have previously been reported as crucial for conversion of CYP3A4-catalyzed aflatoxin B₁ oxidation to that of CYP3A5 (Wang et al., 1998; Xue et al., 2001). A multiple mutant R212K/D214G/F219L/T224I/V225L/I230T) that spans the proposed P450 substrate access channel (Poulos et al., 1986; Hasemann et al., 1995, Nakayama et al., 2001; Pikuleva et al., 2001) was also made to examine whether the substitution of these residues has a significant effect on CYP3A4 inactivation. However, this multiple mutant also showed mifepristone inactivation similar to that of the wild type (Table 2).

View larger version (32K): [in this window] [in a new window]   Fig. 4.   A comparison of the metabolic profiles of wild-type CYP3A4, CYP3A4 S119A, CYP3A4 A370F, and wild-type CYP3A5. These reactions were performed using 25 pmol of enzyme and 100 µM mifepristone (see Experimental Procedures for details).

	Discussion

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CYP3A4 and 3A5 are the most important CYP3A enzymes found in adult human liver. Although 3A4 is generally considered the major CYP3A enzyme, a recent report suggests that the relative amount of 3A5 may be significantly higher in certain individuals than originally thought (Kuehl et al., 2001). Since CYP3A enzymes are involved in the oxidative metabolism of over half of the drugs available in the market, the variation in CYP3A content could be one of the most important contributors to interindividual and interracial differences in drug metabolism and disposition. However, the substrate specificities of CYP3A enzymes are well conserved within and across species, which has made it difficult to differentiate between these two human adult 3A forms. The absence of a suitable genetic marker has further complicated the process of assessing the contribution of these two enzymes in individuals (Hustert et al., 2001). The identification of mifepristone as a selective inactivator of CYP3A4 should provide an excellent probe for differentiating between the two major human CYP3A enzymes.

The mechanism-based inactivation of CYP3A4 by mifepristone has been ascribed to protein rather than heme modification (He et al., 1999). By analogy with compounds bearing a terminal acetylenic or propylenic group (Ortiz de Montellano and Kunze, 1980; Foroozesh et al., 1997; Roberts et al., 1997), it has been proposed that mifepristone is oxidized and converted via a 1,2-methyl shift to a ketene, which subsequently reacts with one or more nucleophiles present in the active site of CYP3A4. A series of earlier studies involving the isolation and characterization of the radiolabeled peptide containing the covalently attached inactivator and the use of site-directed mutagenesis implicated a conserved threonine residue in the I-helix of P450 2B1, 2B4, and 2E1 in the inactivation of these enzymes by various compounds (Roberts et al., 1993, 1994, 1996; Moreno et al., 2001). However, substitution of the corresponding residue (Thr-309) in CYP3A4 by alanine showed no significant effect on enzyme inactivation by mifepristone. Previous mutagenesis studies from our laboratory also have indicated that conservation of this threonine residue is not essential for substrate metabolism by CYP3A4 (Domanski et al., 1998, 2001b; Khan et al., 2002). The unaltered susceptibility to mifepristone inactivation of various CYP3A4  CYP3A5 mutants was rather surprising, since several of these substitutions (N206S and L210F) are crucial for conversion of CYP3A4 to CYP3A5 regioselectivity with aflatoxin B₁ and testosterone (Wang et al., 1998; Xue et al., 2001). A recent study by Lightning et al. (2000) has implicated residue Glu-307 in the mechanism-based inactivation of CYP3A4 by L-754,394 based on molecular modeling. However, CYP3A4 and CYP3A5 contain the same amino acid at this position, so it is less likely that this residue may be involved in mifepristone selectivity. Thus, the identity of the residue(s) that leads to the differential susceptibility of CYP3A4 and CYP3A5 to mifepristone remains elusive. It is possible that CYP3A4 inactivation by mifepristone involves more than one amino acid or a residue not explored in this study.

The other major difference between mifepristone oxidation by CYP3A4 and CYP3A5 is the absence of C-hydroxylated product formation in the case CYP3A5 (Fig. 4). Since formation of this product and of the ketene intermediate proposed to lead to CYP3A4 inactivation involves the same propylenic group, the results suggest that mifepristone is not able to orient itself in the CYP3A5 active site in such a way that the propylenic group is accessible for oxidation. In contrast, the inactivation of CYP3A4 by mifepristone is likely to depend upon the factors that influence the partitioning between the oxidation of the terminal methyl group and formation of the putative ketene intermediate.² In the case of CYP3A4 A370F, which gets inactivated but does not produce the C-hydroxylated product, substitution of the active site residue appears to favor only formation of the putative ketene intermediate and not the terminal alcohol.

An earlier study indicated that altered pharmacokinetics and metabolism of mifepristone have no role in its ineffectiveness in the termination of pregnancy in 10-15% of women (Heikinheimo et al., 1990). Therefore, the differences in the metabolism of mifepristone by CYP3A4 and CYP3A5 may not alter its efficacy as an abortifacient. However, the use of mifepristone for the treatment of other diseases may need to be further evaluated in light of our findings. In addition, selective inactivation of CYP3A4 but not CYP3A5 by mifepristone could significantly affect the bioavailability and clearance of various coadministered drugs that are metabolized by these two CYP3A enzymes. Drugs such as midazolam, quinidine, and cyclosporin A, which are differentially metabolized by CYP3A4 and CYP3A5, may be of greatest concern. Similarly, the relative risk upon exposure to the heptocarcinogen aflatoxin B₁ may be significantly enhanced by this selective enzyme inactivation, because oxidation by CYP3A5 largely produces the genotoxic exo-8, 9-epoxide, as opposed to the innocuous 3-hydroxylated product predominantly formed by CYP3A4. A recent report suggesting that CYP3A5 is more frequently expressed in livers of African Americans than those of Caucasians (Kuehl et al., 2001) further highlights the importance of proper evaluation of the selective contribution of these two enzymes in individuals to avoid adverse drug reactions.