Differential Time-Dependent Inactivation of P450 3A4 and P450 3A5

The role of C239 as the active-site residue responsible for forming the covalent linkage with raloxifene during P450 3A4 time-dependent inactivation (...
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Chem. Res. Toxicol. 2007, 20, 1778–1786

Differential Time-Dependent Inactivation of P450 3A4 and P450 3A5 by Raloxifene: A Key Role for C239 in Quenching Reactive Intermediates Josh T. Pearson,†,§ Jan L. Wahlstrom,†,§ Leslie J. Dickmann,† Santosh Kumar,‡ James R. Halpert,‡ Larry C. Wienkers,† Robert S. Foti,† and Dan A. Rock*,† Department of Pharmacokinetics and Drug Metabolism, Amgen Inc., 1201 Amgen Court West, Seattle, Washington 98119-3105, and Department of Pharmacology and Toxicology, UniVersity of Texas Medical Branch, 301 UniVersity BouleVard, GalVeston, Texas 77555-1031 ReceiVed June 7, 2007

The role of C239 as the active-site residue responsible for forming the covalent linkage with raloxifene during P450 3A4 time-dependent inactivation (TDI) was recently identified. The corresponding residue in CYP3A5 is S239, and when the potential for TDI in P450 3A5 was investigated, only reversible inhibition was observed against midazolam and testosterone, with median inhibitory concentration (IC50) values of 2.4 and 2.9 µM, respectively. In a similar fashion, when C239 was replaced with alanine in P450 3A4, TDI was successfully engineered out, and the reversible inhibition was characterized by IC50 values of 3.7 and 3.5 µM against midazolam and testosterone, respectively. Metabolism studies confirmed that the reactive diquinone methide intermediate required for P450 3A4 inactivation formed in all of the P450 3A enzymes investigated. Furthermore, the absence of TDI in P450 3A5 led to an increase in the formation of GSH-related adducts of raloxifene compared with that for P450 3A4. Consequently, the absence of the nucleophilic cysteine leads to differential TDI and generation of reactive metabolites in the P450 3A enzyme, providing the foundation for pharmacogenetics that contributes to individual differences in susceptibility to adverse drug reactions. Introduction The pharmacogenetics associated with cytochrome P450 enzymes represents an important determinant in relating interindividual variability of drug exposure to clinical pharmacological and toxicological end points. Initial insights into the role of genetic variability of P450 enzymes were first linked with the P450 2D6 polymorphism (1, 2). In this instance, patients receiving debrisoquine for the treatment of high blood pressure who also possessed the poor metabolizer phenotype of P450 2D6 were susceptible to an unsafe drop in blood pressure due to the reduced clearance of debrisoquine (3, 4). Additional population studies revealed that the inactive P450 2D6 enzyme was based on a homozygous null variant found in 5–10% of Caucasians and 1–2% of Southeast Asians (5). More recently, enzyme activities from single amino acid variants were linked with over-anticoagulation due in part to P450 2C9 polymorphism in patients taking (S)-warfarin (6, 7). Despite a strong understanding of the importance of pharmacogenetics and associated toxicity based on varying exposure to parent or active metabolites, there is a gap in linking potential mechanisms of toxicity related to the effects of pharmacogenetics on the generation of reactive intermediates. P450-mediated bioactivation generally leads to formation of reactive metabolites that inactivate the enzyme via a covalent linkage with residue(s) of the P450 (apoprotein) or through modification of the heme * To whom correspondence should be addressed: 1201 Amgen Court West, Seattle, WA 98119. Tel: (206) 265-7139. Fax: (206) 265-1149. E-mail: [email protected]. † Amgen Inc. ‡ University of Texas Medical Branch. § These authors contributed equally to this work.

by either direct covalent linkage or coordination of the heme iron to form a metabolic intermediate complex (MIC).1 Alternatively, bioactivated intermediates may migrate from the endoplasmic reticulum to react with other cellular constituents, including DNA. Both events have the propensity to increase the risk of adverse drug reactions (8). In the liver, P450 3A4 and P450 3A5 account for approximately 50% of the total P450 content (9). Moreover, P450 3A4 and P450 3A5 are the predominant P450 contributors to metabolism in the adult human liver, accounting for 40–60% of the oxidative metabolism of marketed drugs. While the importance of the role of P450 3A4 is well characterized for both liver and intestinal metabolism, the role of P450 3A5 in drug metabolism remains controversial. Initial reports suggested that P450 3A5 content was minimal, representing up to 20% of adult human livers (10). More recent data suggest that P450 3A5 may account for more than 50% of the P450 3A content in 25–30% of human livers (11). Although P450 3A4 and P450 3A5 share ∼85% sequence homology, enzyme activity and regioselectivity differences have been observed for a variety of substrates, suggesting that differing active-site architecture may lead to distinct inhibition profiles for the two P450 3A enzymes. Several studies have reported preferential inactivation of P450 3A4 over P450 3A5. Notably, mifepristone exhibited timedependent inactivation (TDI) of P450 3A4 but not P450 3A5 (12). Furthermore, the potency of MIC formation is increased in P450 3A4 compared with P450 3A5 for diltiazem, erythromycin, nicardipine, and verapamil (13, 14). Reaction rates and 1 Abbreviations: MIC, metabolic intermediate complex; DLPC, L-Rdilauroylphosphatidylcholine; DLPS, L-R-dilauroyl-sn-glycero-3-phosphoserine; DOPC, L-R-dioleoyl-sn-glycero-3-phosphocholine; TDI, timedependent inactivation; TFA, trifluoroacetic acid.

10.1021/tx700207u CCC: $37.00  2007 American Chemical Society Published on Web 11/15/2007

Differential Time-Dependent InactiVation of P450 3A

specific differences in regioselectivity between the two P450 3A enzymes may offer a partial explanation of the aforementioned examples. Therefore, exposure to reactive metabolites from P450 3A depends not only on the enzyme’s capacity to metabolize the molecular site necessary to produce bioactivation but also on the appropriate enzyme architecture to dictate whether the electrophilic species acts locally (at the P450) or escapes the active site to react with other cellular constituents. Recent studies from this laboratory established a role for C239 in the mechanism-based inactivation of P450 3A4 by the secondgeneration selective estrogen receptor modulator raloxifene (15). From sequence alignments, S239 in P450 3A5 corresponds to C239 in P450 3A4. In light of the potential for differences in metabolic profiles due to differences in P450 3A active-site architecture, it was postulated that this variability could also extend to structure-based mechanisms of TDI. In the current study, we hypothesized that P450 3A5 may exhibit reduced or minimal TDI inactivation at comparable rates of raloxifene metabolism, leading to an enhanced potential for systemic exposure to bioactivated intermediates. In view of this, the P450 3A genotype could play an important role in raloxifene oxidation and reactive metabolite generation.

Experimental Procedures Materials. Raloxifene, midazolam, testosterone, reduced glutathione, CHAPS, potassium HEPES, MgCl2, guanidine hydrochloride, CaCl2, ZnSO4, and NADPH were purchased from SigmaAldrich (St. Louis, MO). Supersomes containing P450 3A4 or P450 3A5 were purchased from BD Gentest (Woburn, MA). A Zorbax RX-C8 column (2.1 × 250 mm) was obtained from Agilent (Palo Alto, CA). The QuikChange XL site-directed mutagenesis kit was obtained from Stratagene (La Jolla, CA). For P450 3A5, oligonucleotide primers for the polymerase chain reaction (PCR) were obtained from Sigma-Genosys (The Woodlands, TX). Instrumentation. For metabolite identification studies, an LTQ mass spectrometer (Thermo Scientific, San Jose, CA) was connected on-line with an Agilent 1100 series HPLC system equipped with a degasser, pumps, an autoinjector, a column oven, and a diode-array detector. For the analysis of midazolam and testosterone TDI studies as well as raloxifene substrate depletion studies, a 4000Q-Trap system (Applied Biosystems, San Jose, CA) was connected to a Shimadzu HPLC system equipped with a degasser. Mutagenesis of P450 3A4 and P450 3A5. A human P450 3A4 NF14 construct containing a C-terminal polyhistidine tag cloned into the pCWori vector was a gift from Professor William Atkins (University of Washington). The C239A mutation was introduced into the P450 3A4 clone with the sense 5′-ctc atc cca att ctt gaa gta tta aat atc gct gtg ttt cca aga gaa gtt ac-3′ and antisense 5′-gta act tct ctt gga aac aca gcg ata ttt aat act tca aga att ggg atg ag-3′ oligonucleotides according to the manufacturer’s suggested protocol. The mutation was verified by whole-protein mass spectrometry using purified protein. The S239C mutation was introduced into the P450 3A5 clone with the sense 5′-gca tta aat gtc tgt ctg ttt cca-3′ and antisense 5′-tgg aaa cag aca gac att taa tgc-3′ oligonucleotides according to the manufacturer’s suggested protocol. To confirm the formation of the desired mutation and verify the absence of any unintended mutations, the construct was sequenced at the University of Texas Medical Branch Protein Chemistry Laboratory (Galveston, TX). Protein Expression and Purification. P450 3A4 enzymes were expressed in the Escherichia coli DH5R strain. Complete expression conditions were the same as those described by Gillam et al. (16). Briefly, E. coli was freshly transformed with either the P450 3A4 wild-type or the C239A mutant plasmid, plated under ampicillin (50 µg/mL) selection, and grown overnight at 37 °C. Cells were shaken at 180 rpm for 48 h at 27 °C in a 2.8 L Fernbach flask. Pelleted cells were resuspended in buffer containing 50 mM potassium phosphate (pH 7.4), 500 mM NaCl, 20% glycerol, 50

Chem. Res. Toxicol., Vol. 20, No. 12, 2007 1779 µM testosterone (a stabilizing ligand), 20 mM β-mercaptoethanol, 1% Emulgen 911, and Sigma protease inhibitor cocktail (0.5 mL/L of initial culture volume). Cells were lysed using a French press operated at 10000 psi and then spun at 150000g. The supernatant was loaded directly onto NTA-Ni resin equilibrated with 50 mM potassium phosphate (pH 7.4), 500 mM NaCl, 20% glycerol, 50 µM testosterone, and 0.2% Emulgen 911. The column was washed with 200 mL of wash buffer containing 50 mM potassium phosphate (pH 7.4), 20% glycerol, 20 mM imidazole, 0.2% cholate, and 50 µM testosterone. P450 3A4 was eluted from the column with buffer containing 50 mM potassium phosphate (pH 7.4), 20% glycerol, 500 mM imidazole, and 0.2% cholate. The eluted protein was dialyzed against 100 mM potassium phosphate (pH 7.4) in 20% glycerol and stored at -80 °C. P450 3A5 and the S239C mutant were expressed as His-tagged proteins in E. coli TOPP3 and purified using a Ni-NTA affinity column as described previously (17). Protein concentrations were determined using the Bradford protein assay kit (BioRad, Hercules, CA). The specific contents of P450 3A5 and S239C were 17.4 and 11.6 nmol of P450/mg of protein, respectively. Rat cytochrome b5 and P450 reductase were expressed and purified as described previously (18, 19). In Silico Modeling. Maestro (Schro¨dinger, Portland, OR) was used to generate a homology model of P450 3A5 based on the crystal structure of P450 3A4 (PDB entry 1TQN) (20). Sequence alignment was performed using BIOEDIT version 7.0.5.2 (Ibis Therapeutics, Carlsbad, CA). The optimal alignment was obtained by allowing the ends of both sequences to slide. The Induced Fit Docking module of Maestro was applied to raloxifene with each of P450 3A4 and P450 3A5 in order to provide a relative idea of raloxifene binding orientations in each enzyme. Incubations with Purified P450. Enzyme reconstitution has been described previously (15, 16). Briefly, P450 (3A4, 3A5, 3A4C239A, or 3A5S239C) (100 pmol) was combined with NADPH-P450 reductase (200 pmol), cytochrome b5 (100 pmol), 0.1 mg/mL Chaps, 20 µg/mL liposomes [1:1:1 (w/w/w) DLPC/DOPC/DLPS], 3 mM reduced glutathione, 50 mM potassium HEPES (pH 7.4), and 30 mM MgCl2 in a total volume of 0.5 mL. Raloxifene was added to a final concentration of 50 µM. Each reconstituted P450 system was incubated for 10 min at 37 °C prior to the addition of 1 mM NADPH. Each reaction was allowed to proceed for 20 min unless otherwise noted. Time-Dependent Inactivation of P450 3A Enzymes. Primary reactions (200 µL) were carried out by incubating a P450 3A enzyme or the corresponding mutant (10 pmol) with different concentrations (0–20 µM) of raloxifene, and for each reaction, 5 µL aliquots were removed at 0, 2, 4, 6, 8, and 10 min and placed into secondary incubations containing 1 mM NADPH and 25 µM midazolam. The secondary reactions were quenched with 100 µL of 1 µM tolbutamide in acetonitrile after 5 min. The final volume of each secondary reaction was 100 µL, providing a 1:20 dilution of the primary incubation. Product formation from midazolam was determined to be linear over the 5 min incubation period. The reported measurements represent the average of duplicate incubations. Quantitation of 1′-hydroxymidazolam formation by mass spectrometry is described below. Measurement of Raloxifene IC50 Values. Median inhibitory concentration (IC50) values of raloxifene were determined against both testosterone and midazolam. Prior to the IC50 measurements, Km and kcat values for both testosterone and midazolam were determined for each enzyme studied (data not shown), and the results were consistent with published data (16, 21). Varying concentrations of raloxifene (0–100 µM) were preincubated with enzyme (10 nM) and substrate (at the predetermined Km value) for 5 min before initiation of the reaction with NADPH. After 5 min for midazolam and 20 min for testosterone, the reactions were quenched with an equal volume (100 µL) of 1 µM tolbutamide in acetonitrile. Concentrations of 1′-hydroxymidazolam and 6βhydroxytestosterone were measured using the multiple reaction monitoring (MRM) scan function of the 4000Q-Trap. 1′-Hydroxymidazolam was monitored with Q1 and Q3 set at m/z 342.1 and 203.1, respectively, with a declustering potential of 66 and a

1780 Chem. Res. Toxicol., Vol. 20, No. 12, 2007 collision energy of 39. Concentrations of 6β-hydroxytestosterone were measured with Q1 and Q3 set at m/z 305.0 and 269.0, respectively, with a declustering potential of 55 and a collision energy of 25. Tolbutamide was monitored with Q1 and Q3 set at m/z 271.2 and 91.1, respectively, with a declustering potential of 66 and a collision energy of 39. The following instrument settings were the same for all three compounds: dwell time, 500 ms; curtain gas, 10; ion-spray voltage, 4500 V; source temperature, 400 °C; and ion-spray gas 1 and 2, 40. Incubations were run in triplicate, and IC50 values were determined using Prism 5.0 (GraphPad Software, San Diego, CA, www.graphpad.com). Substrate Depletion of Raloxifene. Enzyme (10 pmol) was preincubated for 5 min at 37 °C prior to the addition of 10 mM NADPH to a final concentration of 1 mM (1 mL total volume). Aliquots (50 µL) were removed from the incubation at 0, 3, 6, 9, 12, and 15 min and placed into 100 µL of cold acetonitrile spiked with 1 µM tolbutamide as an internal standard. All incubations were run in triplicate. Concentrations of unreacted raloxifene were measured using the 4000Q-Trap in MRM mode with Q1 and Q3 set at m/z 474.28 and 111.9, respectively, with a declustering potential of 66 and a collision energy of 47. Tolbutamide was monitored with Q1 and Q3 set at m/z 271.2 and 91.1, respectively. The following instrument settings were the same for both compounds: dwell time, 500 ms; curtain gas, 10; ion-spray voltage, 4500 V; source temperature, 400 °C; and ion-spray gas 1 and 2, 40. Measurement of Raloxifene Metabolites Produced by P450 3A Enzymes. Following a 30 min incubation of P450 3A4, P450 3A5, or P450 3A4C239A (10 pmol) with 50 µM raloxifene, 5 mM GSH, and 1 mM NADPH as described previously (15), the reaction was quenched with 20 µL of trifluoroacetic acid (TFA). Control reactions without NADPH were conducted simultaneously. The samples were centrifuged at 13000 rpm for 30 min at 4 °C. Supernatant (100 µL) was injected onto an Agilent Zorbax RX-C8 column (2.1 × 250 mm) at a flow rate of 1 mL/min, with 25% of the flow diverted into the mass spectrometer. Initial HPLC conditions were 90% solvent A (0.05% TFA in H2O) and 10% solvent B (0.05% TFA in acetonitrile). The following elution gradient was used: B increased from 10 to 50% over 30 min, then increased from 50 to 95% over 2 min, and then was held at 95% for an additional 3 min. In the absence of metabolite standards, the metabolite levels were determined on the basis of a comparison of their UV–vis responses with the signature absorbance of raloxifene at 285–290 nm. Even though the molar absorptivities of the metabolites may differ subtly from that of raloxifene as a result of oxidation and glutathione conjugation, this method provides at least a semiquantitative measure of metabolite levels. Metabolite formation was reported as the percentage of the total raloxifene from each of the incubations. Reactions were performed in triplicate in order to obtain standard deviations. The diglutathione metabolite (GSH1, m/z 550) eluted at 19.7 min, and the hydroxyglutathione adduct (GSH2, m/z 779) eluted at 24.4 min. The three monoglutathione metabolites (GSH3-GSH5, m/z 779) eluted from 25.1 to 25.5 min. Hydroxyraloxifenes (m/z 490) eluted between 29 and 31 min, and raloxifene (m/z 474) eluted at 33.7 min.

Results Structural Comparison of P450 3A4 and P450 3A5. Both P450 3A4 and P450 3A5 contain 503 residues; of these, 84% are identical and 92% are similar, yielding a strong alignment score of 2244 for these enzymes. Furthermore, from the sequence alignment, C239 in P450 3A4 corresponds to S239 in P450 3A5. Residue 239 is located in substrate recognition site 3 (SRS-3) in both P450 3A enzymes (22). To better elucidate the potential role of C239 in sequestering reactive metabolites, a homology model of P450 3A5 was constructed. Overall, the three-dimensional structure of P450 3A5 accurately overlaid the structural folds in P450 3A4. The distance between the heme iron and the sulfur of C239 in P450 3A4 was measured

Pearson et al.

Figure 1. Homology model of P450 3A5 based on the crystal structure of P450 3A4 (PDB entry 1TQN) (36). (A) Residue 239 is displayed in yellow in both P450 3A4 (cyan) and P450 3A5 (green). The sulfur atom of C239 (P450 3A4) is 19.5 Å from the heme iron, and the hydroxyl oxygen atom of S239 (P450 3A5) is 22.1 Å from the heme iron. (B) Induced-fit docking of raloxifene in P450 3A4 illustrates a single binding mode prone to oxidation at the 6 position of the benzothiophene, leading to formation of a diquinone methide followed by nucleophilic attack at the 3′ or 5′ position at the other end of raloxifene.

to be 21 Å, while the corresponding hydroxyl group of S239 in P450 3A5 was 19 Å from the heme iron. In the structural representations, both side chains point toward the distal face of the heme and closely overlap one another when the enzymes are superimposed (Figure 1A). Docking studies with raloxifene produced an enzyme–substrate conformation in which the benzothiophene alcohol of raloxifene was oriented toward the heme iron at a distance of 3.9 Å, which subsequently positioned the phenol 6.1 Å from the sulfur of C239 (Figure 1B). A similar binding motif was observed in P450 3A5 (data not shown). Raloxifene Inhibition of P450 3A4 and P450 3A5. Raloxifene inhibited P450 3A4 in Supersomes in a time-dependent manner, with KI and kinact values of 0.78 µM and 0.18 min-1, respectively (Figure 2A). These results are similar to those previously reported by Chen et al. (24). In P450 3A5, raloxifene

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Figure 2. Inactivation kinetics of 1′-hydroxymidazolam formation in Supersomes was investigated using varying concentrations of raloxifene (0-10 µM) with (A) P450 3A4 and (B) P450 3A5. (C) Substrate depletion of raloxifene with P450 3A4 and P450 3A5. Incubations (500 µL) of 50 pmol of enzyme in combination with 1 µM raloxifene were used to determine raloxifene consumption. Aliquots (50 µL) were extracted at each time point and quenched with internal standard for sample analysis. Experiments were performed in triplicate.

Table 1. Results from Studies of Raloxifene Inhibition Kinetics in P450 3A4 and P450 3A5a enzyme

IC50 (µM)b

IC50 (µM)c

KI (µM)

kinact (min-1)

P450 3A4 P450 3A5

0.8 ( 0.2 2.4 ( 0.1

0.4 ( 0.1 2.9 ( 0.1

0.8 ND

0.17 ND

a IC50 values were obtained in triplicate for raloxifene in Supersomes containing P450 3A4 or P450 3A5 against two probes (midazolam and testosterone) to ensure the active site was thoroughly probed. Inactivation kinetics parameters (KI and kinact) for P450 3A4 were determined using midazolam as the probe substrate; values were obtained using nonlinear regression, with each data point being the average of duplicate incubations. Parameters for P450 3A5 were not determined (ND). b Value for 1′-hydroxymidazolam formation. c Value for 6β-hydroxytestosterone formation.

showed no time-dependent inactivation. Instead, only reversible inhibition in the secondary incubations was observed (Figure 2B). Therefore, the potential for reversible inhibition was characterized by measuring the metabolism of midazolam and testosterone by P450 3A5 in the presence of varying raloxifene concentrations. Raloxifene reversibly inhibited P450 3A5, with IC50 values of 2.4 and 2.9 µM against midazolam and testosterone, respectively (Table 1). To ensure that raloxifene was a substrate for P450 3A5, a prerequisite for TDI, raloxifene

Figure 3. UV–vis (285–290 nm) metabolite profiles from incubations (30 min) of Supersomes containing 50 µM raloxifene with (A) P450 3A4 and (B) P450 3A5. (C) Percentages of glutathionyl metabolites and hydroxyraloxifene formed in raloxifene incubations with P450 3A4 and P450 3A5.

metabolism was measured in Supersomes containing P450 3A4 or P450 3A5. Both isoforms were capable of metabolizing raloxifene, but with different efficiencies (Figure 2C). P450 3A5 turnover was 4 times greater than that of P450 3A4, with only 4 ( 2% of raloxifene remaining from P450 3A5 incubations. Raloxifene Metabolites Produced by P450 3A4 and P450 3A5. Comparison of the overall metabolite profiles revealed that P450 3A4 and P450 3A5 produced similar metabolites (Figure 3A,B). However, the regioselectivity of the glutathionyl metabolites and hydroxyraloxifene varied between the two enzymes (Figure 3C). Incubations with 50 µM raloxifene and P450 3A5 yielded 29 ( 5% of the total incubate as the glutathionyl metabolites, an approximately 10-fold increase in formation of GSH-related adducts compared with the 2.7 ( 0.1% yield in incubations with P450 3A4. Furthermore, focusing on the amount of GSH-related metabolites formed from the diquinone methide intermediate may be relevant, since this species is the precursor to P450 3A4 inactivation. This analysis revealed that 1.8 ( 0.1% of the GSH-related adducts formed by P450 3A4 resulted from the diquinone methide, whereas P450 3A5 produced 6.6 ( 2.5% of its GSH-related material from the diquinone methide. The hydroxyraloxifene formed represented 2.0 ( 0.1 and 15.1 ( 5.7% of the total metabolites produced by P450 3A4 and P450 3A5, respectively. Incubations without NADPH produced no signal for any of the measured metabolites. Raloxifene Inhibition of Reconstituted P450 3A4 and Reconstituted P450 3A4C239A. To engineer out TDI from P450 3A4, C239 was replaced with a nonnucleophilic residue.

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Pearson et al. Table 2. Results from Studies of Raloxifene Inhibition Kinetics with Reconstituted P450 3A4 and P450 3A4C239A Mutanta enzyme P450 3A4 P450 3A4C239A

IC50 (µM)b

IC50 (µM)c

4.4 ( 0.1 3.7 ( 0.1

2.0 ( 0.1 3.5 ( 0.3

KI (µM)

kinact (min-1)

3.1 ND

0.07 ND

a IC50 values were obtained in triplicate for raloxifene with reconstituted P450 3A4 or P450 3A4C239A against two probes (midazolam and testosterone) to ensure the active site was thoroughly probed. Inactivation kinetics parameters (KI and kinact) for reconstituted P450 3A4 were determined using midazolam as the probe substrate; values were obtained using nonlinear regression, with each data point being the average of duplicate incubations. Parameters for P450 3A4C239A were not determined (ND). b Value for 1′-hydroxymidazolam formation. c Value for 6β-hydroxytestosterone formation.

Figure 4. Inactivation kinetics of 1′-hydroxymidazolam formation were investigated using varying concentrations of raloxifene (0-10 µM) with (A) reconstituted P450 3A4 and (B) reconstituted P450 3A4C239A. (C) Substrate depletion of raloxifene with reconstituted P450 3A4 and reconstituted P450 3A4C239A. Incubations (500 µL) of 50 pmol of enzyme in combination with 1 µM raloxifene were used to determine raloxifene consumption. Aliquots (50 µL) were extracted at each time point and quenched with internal standard for sample analysis. Experiments were performed in triplicate.

Given the difficulty of reconstituting P450 3A enzymes, purified P450 3A4 was reconstituted alongside the P450 3A4C239A mutant to ensure that the reconstitution conditions were appropriate to achieve TDI as observed with the microsomal P450 3A4 preparation. The reconstituted P450 3A4 system showed TDI and produced KI and kinact values of 3.1 µM and 0.07 min-1, respectively (Figure 4A). Importantly, the P450 3A4C239A mutant showed no TDI, although it was still inhibited reversibly, as evidenced by IC50 values of 3.7 and 3.5 µM against midazolam and testosterone, respectively (Table 2). Turnover was also assessed to determine whether raloxifene served as a substrate as well as an inhibitor in the incubations with reconstituted P450 3A4 and P450 3A4C239A mutant. The results showed turnover, with 25 ( 1 and 62 ( 3% of raloxifene metabolized after 15 min by the reconstituted P450 3A4 and P450 3A4C239A mutant, respectively. Raloxifene Metabolites Produced by Reconstituted P450 3A4 and Reconstituted P450 3A4C239A. Comparison of the overall metabolite profiles revealed that reconstituted P450 3A4 and reconstituted P450 3A4C239A produced similar

Figure 5. UV–vis (285–290 nm) metabolite profiles from 50 µM raloxifene incubations (30 min) with (A) reconstituted P450 3A4 and (B) reconstituted P450 3A4C239A. (C) Percentages of glutathionyl metabolites and hydroxyraloxifene formed in raloxifene incubations with reconstituted P450 3A4 and reconstituted P450 3A4C239A.

metabolites (Figure 5A,B). Incubations with 50 µM raloxifene and reconstituted P450 3A4C239A mutant yielded 3.5 ( 0.4% of the total incubate as the glutathionyl metabolites, a similar but slightly greater amount of GSH-related adduct formation compared to the 1.7 ( 0.1% yield in incubations with reconstituted P450 3A4. Determination of the amount of GSHrelated metabolites formed from the diquinone methide revealed that reconstituted P450 3A4C239A also slightly favored the formation of the diquinone methide, with 3.0 ( 0.1% of diquinone-related adducts measured for the mutant compared with 1.0 ( 0.1% for the reconstituted P450 3A4. Overall, the

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hydroxyraloxifene metabolites were favored in the reconstituted P450 3A4 compared to the P450 3A4C239A mutant, which predominantly formed the glutathionyl metabolites (Figure 5C). Incubations without NADPH produced no signal for any of the measured metabolites. P450 3A5 and Reconstituted P450 3A5S239C. In an effort to confer susceptibility to TDI onto P450 3A5, the S239C mutant was created. Reconstituted P450 3A5 wild type showed an IC50 value for inhibition of testosterone hydroxylation comparable to that for the enzyme in Supersomes. However, the IC50 value for the reconstituted mutant was 10-fold higher, and the efficiency of midazolam and testosterone oxidation was greatly impaired, precluding accurate assessment of TDI. Raloxifene Metabolite Characterization in P450 3A Enzymes. In total, five glutathione adducts were observed and arbitrarily designated as GSH1-GSH5. GSH1 represented a diglutathione adduct and GSH2 a hydroxyglutathione adduct, whereas GSH3-GSH5 were monoglutathione adducts arising from the diquinone methide. GSH1 had an [M + 2H]2+ ion at m/z 550 that produced base fragments at m/z 486 and 421 (consistent with losses of one and two pyroglutamates, respectively) when subjected to collision-induced dissociation (CID) (Figure 6A); this metabolite was consistent with the previously observed diglutathione adduct produced from rat liver microsomes. The proposed metabolite resulted from multiple oxidations of raloxifene to yield 7-hydroxy-4,5diglutathionylraloxifene (23). The diglutathionylraloxifene adduct has not previously been observed with human P450s. GSH2 had an [M + H]+ ion at m/z 795 (Figure 6B) and was consistent with o-quinone generation followed by glutathione adduction similar to that described for GSH1. The major diagnostic fragment (m/z 666) upon CID of m/z 795 was also consistent with loss of pyroglutamate. GSH3, GSH4, and GSH5, the monoglutathione adducts of unmodified raloxifene, all had an [M + H]+ ion at m/z 779 (Figure 6C) produced by oxidation of raloxifene to the diquinone methide with subsequent quenching by the addition of glutathione. CID of m/z 779 produced base fragments at m/z 704 and 650 characteristic of glycine and pyroglutamate losses, respectively. GSH4 and GSH5 coeluted under the HPLC conditions used here and had to be separated as described previously (24). For the purposes of the present work, they were not routinely separated, since they both resulted from the diquinone methide intermediate. Up to three oxidative metabolites were observed to various degrees in all P450 3A incubations and were characterized by an [M + H]+ ion at m/z 490.

Discussion The current investigation was aimed at understanding whether P450 pharmacogenetics has the potential to impact TDI and reactive metabolite generation. Kinetic reports have shown that P450 3A4 and P450 3A5 are prone to different metabolic behavior. Recently, C239 was identified as the key amino acid responsible for facilitating TDI of raloxifene in P450 3A4 (15). These findings provide a unique opportunity to investigate the potential role of C239 versus that of exposure to reactive metabolites in enzyme inactivation. Previous efforts have subdivided the P450 3A4 active site into unique regions known as substrate recognition sites (22, 25, 26). Alteration of a single amino acid in four of the six P450 3A4 SRS regions conferred P450 3A5-like regioselectivity to the metabolism of aflatoxin B1, further indicating similarity between the two enzymes (22). Therefore, a homology model of P450 3A5 based on P450 3A4

Figure 6. CID spectra of GSH-related raloxifene adducts: (A) GSH1, the diglutathione adduct; (B) GSH2, the hydroxyglutathione adduct; (C) GSH3, GSH4, and GSH5, the monoglutathione adducts.

was generated. The three-dimensional superposition demonstrated overlap of the major active-site architectural features of P450 3A4 and P450 3A5 (Figure 1A). A key feature revealed by this analysis was the presence of a serine in P450 3A5 that corresponds to C239 in P450 3A4 (highlighted in yellow in Figure 1A). Both side chains are oriented toward the distal face of the heme at approximately the same distance and are therefore likely to have similar exposures to reactive intermediates generated within the enzyme active sites. This is further illustrated by the docking study of raloxifene and P450 3A4, which showed raloxifene poised for dehydrogenation and indicated that subsequent trapping of the raloxifene diquinone

1784 Chem. Res. Toxicol., Vol. 20, No. 12, 2007

methide is feasible on the basis of a single binding conformation (Figure 1B). Furthermore, solvent accessibility of C239 in P450 3A4 was determined on the basis of selective alkylation of the protein at C239 with the addition of iodoacetamide (15). The differing nucleophilicities of the cysteine thiol group and the serine hydroxyl group may contribute to the differences in their abilities to trap reactive metabolites (22). Precedence has been established for preferential reactivities of electrophilic reactive intermediates with nucleophiles that retain a similar degree of “hardness” or “softness” (27–29). Iminium ions formed from the metabolism of cyclic amines, such as nicotine, react preferentially with nucleophiles such as cyanide (30). Reactive intermediates such as aldehydes are often trapped with amines (31). Glutathione has a weaker propensity to trap hard electrophiles but reacts well with soft electrophiles such as arene oxides, quinones, and thiophene sulfoxides (29). Correspondingly, it may be expected that protein residues such as cysteine will trap soft electrophiles such as the diquinone methide intermediate of raloxifene, while lysine and serine residues are more likely to trap harder electrophiles. In order to test this hypothesis, TDI experiments using raloxifene were carried out using P450 3A4, P450 3A5, and P450 3A4C239A. Studies of raloxifene inactivation kinetics to compare TDI in P450 3A4 and P450 3A5 were conducted in order to examine the potential for differential inhibition of P450 3A4 and P450 3A5. Raloxifene exhibited TDI of midazolam 1′-hydroxylation in P450 3A4 but did not show TDI in P450 3A5 at concentrations up to 10 µM, which is well above the measured IC50 values against midazolam and testosterone (Table 1). The metabolic capacities of P450 3A4 and P450 3A5 toward raloxifene were also investigated by measuring raloxifene depletion, and the results showed that raloxifene was a substrate for both P450 3A4 and P450 3A5. The activity from P450 3A4 decreased rapidly, consistent with enzyme inactivation and the moderate capacity for P450 3A4 to metabolize raloxifene (Figure 2C). Under these circumstances, it may have been expected that minimal amounts of bioactivated raloxifene would be generated, since the activity of P450 3A4 was simultaneously being reduced within the first few minutes of the incubation as a result of irreversible inhibition of P450 3A4. The rapid enzyme inactivation resulted from efficient trapping of a large percentage of the reactive intermediate and was consistent with a low partition ratio of 1.8 (32). The exponential decay of raloxifene resulting from P450 3A5mediated metabolism (Figure 2C) was consistent with the results shown in Figure 2B, which indicate that raloxifene did not display TDI in P450 3A5. Consequently, P450 3A5 was capable of continually producing reactive species from raloxifene throughout the course of the experiment. Extending these findings to a potential in vivo scenario suggests that the cysteine of P450 3A4 could minimize exposure to P450 3A4-generated reactive metabolites by inactivating the enzyme, the source of the reactive metabolites, via a pathway akin to a biological feedback inhibition mechanism (Scheme 1). Alternatively, in the case of P450 3A5 where enzyme inactivation does not occur, the reactive intermediates could be continually produced. This hypothesis may be extended to explain the correlation between aflatoxin-albumin adducts and P450 3A5 enzyme levels (33). Moreover, this notion is also consistent with the lack of TDI observed for aflatoxin in P450 3A5 (data not shown). Metabolite profiles (indicating regioselectivity) for P450 3A4 and P450 3A5 enzymes were also examined in order to ensure that the lack of TDI in P450 3A5 did not result simply from the inability of the enzyme to generate glutathione adducts, in

Pearson et al. Scheme 1

particular those arising from the diquinone methide, which has previously been determined as the precursor to inactivation of P450 3A4 (15). Incubations with P450 3A4 confirmed diquinone methide formation as indicated by the presence of the monoglutathionyl adducts, which were consistent with previously obtained results based on mass spectrometry (23, 24). In addition, the monohydroxylated metabolites and trace amounts of a diglutathione adduct were also present. The diglutathione metabolite has previously been reported in rat liver microsomes and is postulated to be formed via sequential enzyme oxidation reactions (23). P450 3A5 favored the formation of oxidative glutathione adducts as well as significantly greater amounts of all the other metabolites, including the monoglutathione and diglutathione adducts, observed from P450 3A4 incubations (Figure 3B and 3C). The resultant CID fragmentations were consistent with previous mass spectral data generated with the mono- and diglutathione raloxifene metabolites from the rat as well as the monoglutathione adducts from human liver microsomes (23, 24). Most importantly, P450 3A5 generated the diquinone methide intermediate required for P450 3A4 inactivation, yet no inactivation of P450 3A5 occurred; this is consistent with the hypothesis that P450 3A5 is capable of yielding increased amounts of reactive metabolites relative to P450 3A4 when the resulting bioactivated compound (in this case raloxifene) preferentially reacts with a soft nucleophile such as cysteine (Figure 3C). The two mutants P450 3A4C239A and P450 3A5S239C were engineered on the basis of the unique difference between P450 3A4 and P450 3A5 at residue 239 in SRS-3. The P450 3A5S239C mutation significantly impacted the kinetics profile of midazolam and testosterone turnover, to the extent that further studies could not be carried out because of solubility limitations. For the P450 3A4C239A mutant, alanine instead of serine was chosen to replace the cysteine in P450 3A4, in order to eliminate the potential for any electrophilic quenching of raloxifene bioactivation at residue 239. This precaution was taken because different micro environments are known to activate the nucleophilic behavior of serine residues (34). Furthermore, serine has previously been implicated in covalent binding of other P450 enzyme systems (35). Raloxifene failed to produce TDI of the P450 3A4C239A mutant with respect to midazolam 1′-hydroxylation, whereas reversible inhibition of P450 3A4C239A was similar to that observed for reconstituted P450 3A4, suggesting that raloxifene still binds to the mutant enzyme (Table 2). While TDI was not observed for the P450 3A4C239A mutant in these experiments, it was critical to determine that raloxifene is a substrate for the enzyme prior to drawing any conclusions regarding the significance of the cysteine/serine difference as the determining factor for differential TDI of P450 3A4 compared with P450 3A5. The turnover of raloxifene for the P450 3A4C239A mutant was compared to that for reconstituted P450 3A4 in order to ensure that raloxifene behaved as a substrate in both systems.

Differential Time-Dependent InactiVation of P450 3A

Both enzymes showed activity toward raloxifene (Figure 4C). A difference in activity was observed by the end of the incubation, which showed that the P450 3A4C239A mutant was more efficient than the reconstituted P450 3A4, consistent with the lack of TDI observed (Figure 4B). This study confirmed that raloxifene was a substrate for both P450 3A4C239A and the reconstituted P450 3A4. Examination of the metabolite profiles further revealed that the putative precursor to P450 3A4 inactivation forms in both reconstituted systems and thus should facilitate TDI (Figure 5A,B). Furthermore, the P450 3A4C239A mutant produced a 2-fold increase in the amount of GSH adducts formed, with the majority of this GSH-related material originating from the diquinone methide precursor. These results confirm that the P450 3A4C239A is capable of forming the necessary metabolites for inactivation, while the lack of inactivation indicates that the C239A mutation protects the enzyme from inactivation. Several mechanisms can be postulated for the differential TDI by raloxifene of P450 3A4 compared with P450 3A5: (1) different rates of substrate turnover, (2) alteration in regioselectivity due to differences in active site architecture, or (3) the presence of varying nucleophilic active-site residues. To date, literature reports attempting to explain the differences between P450 3A4 and P450 3A5 have focused on differential regioselectivity produced by the enzymes. For instance, the antibacterial agent erythromycin forms a metabolite-inhibitor complex in P450 3A4 but is unable to inhibit P450 3A5 in this manner. Reports suggest that a sequential oxidation pathway leading to the reactive intermediate is available in P450 3A4 but does not occur in P450 3A5, possibly as a result of different substrate orientations or mechanisms of oxidation (13). In addition, mifepristone also selectively inactivates P450 3A4. Interestingly, examining the metabolic profile of mifepristone revealed that both C-hydroxylated and N-dealkylated metabolites were formed by P450 3A4, while only N-dealkylated products were formed by P450 3A5. Although the exact structural basis for the observed difference was not elucidated, the authors proposed that differential substrate binding did not allow the putative alkynyl reactive metabolite precursor to access the reactive oxygen species, thereby preventing generation of the reactive intermediate (12). Moreover, others have reported that verapamil, diltiazem, and nicardipine selectively form MICs in P450 3A4 compared with P450 3A5, although the mechanism of this inactivation selectivity is not well understood (13). We believe that the current study is the first report describing select P450 genotype-mediated inactivation based on alkylation of active-site residues. This proposal is based on concordance of results generated from kinetic analysis, computational modeling, and site-directed mutagenesis. These results firmly establish a unique role for C239 in P450 3A4 (in contrast with S239 in P450 3A5) as a nucleophilic “hook” for capturing enzymegenerated reactive intermediates before they are able to leave the active site. Furthermore, the current study also provides a possible mechanistic rationale for observed differences between P450 3A4 and P450 3A5 pharmacogenetic drug-related toxicities observed across human populations. Acknowledgment. This research was supported in part by NIH Grant GM54995 (to J.R.H.).

References (1) Mahgoub, A., Idle, J. R., Dring, L. G., Lancaster, R., and Smith, R. L. (1977) Polymorphic hydroxylation of Debrisoquine in man. Lancet 2, 584–586.

Chem. Res. Toxicol., Vol. 20, No. 12, 2007 1785 (2) Dayer, P., Gasser, R., Gut, J., Kronbach, T., Robertz, G. M., Eichelbaum, M., and Meyer, U. A. (1984) Characterization of a common genetic defect of cytochrome P-450 function (debrisoquinesparteine type polymorphism)sincreased Michaelis constant (Km) and loss of stereoselectivity of bufuralol 1′-hydroxylation in poor metabolizers. Biochem. Biophys. Res. Commun. 125, 374–380. (3) Tucker, G. T., Silas, J. H., Iyun, A. O., Lennard, M. S., and Smith, A. J. (1977) Polymorphic hydroxylation of debrisoquine. Lancet 2, 718. (4) Idle, J. R., Mahgoub, A., Lancaster, R., and Smith, R. L. (1978) Hypotensive response to debrisoquine and hydroxylation phenotype. Life Sci. 22, 979–983. (5) Bradford, L. D. (2002) CYP2D6 allele frequency in European Caucasians, Asians, Africans and their descendants. Pharmacogenomics 3, 229–243. (6) Heimark, L. D., Wienkers, L., Kunze, K., Gibaldi, M., Eddy, A. C., Trager, W. F., O’Reilly, R. A., and Goulart, D. A. (1992) The mechanism of the interaction between amiodarone and warfarin in humans. Clin. Pharmacol. Ther. 51, 398–407. (7) Steward, D. J., Haining, R. L., Henne, K. R., Davis, G., Rushmore, T. H., Trager, W. F., and Rettie, A. E. (1997) Genetic association between sensitivity to warfarin and expression of CYP2C9*3. Pharmacogenetics 7, 361–367. (8) Becking, G. C. (1995) Use of mechanistic information in risk assessment for toxic chemicals. Toxicol. Lett. 77, 15–24. (9) Shimada, T., Yamazaki, H., Mimura, M., Inui, Y., and Guengerich, F. P. (1994) Interindividual variations in human liver cytochrome P-450 enzymes involved in the oxidation of drugs, carcinogens and toxic chemicals: studies with liver microsomes of 30 Japanese and 30 Caucasians. J. Pharmacol. Exp. Ther. 270, 414–423. (10) Wrighton, S. A., Brian, W. R., Sari, M. A., Iwasaki, M., Guengerich, F. P., Raucy, J. L., Molowa, D. T., and Vandenbranden, M. (1990) Studies on the expression and metabolic capabilities of human liver cytochrome P450IIIA5 (HLp3). Mol. Pharmacol. 38, 207–213. (11) Lin, Y. S., Dowling, A. L., Quigley, S. D., Farin, F. M., Zhang, J., Lamba, J., Schuetz, E. G., and Thummel, K. E. (2002) Co-regulation of CYP3A4 and CYP3A5 and contribution to hepatic and intestinal midazolam metabolism. Mol. Pharmacol. 62, 162–172. (12) Khan, K. K., He, Y. Q., Correia, M. A., and Halpert, J. R. (2002) Differential oxidation of mifepristone by cytochromes P450 3A4 and 3A5: selective inactivation of P450 3A4. Drug Metab. Dispos. 30, 985–990. (13) McConn, D. J., 2nd, Lin, Y. S., Allen, K., Kunze, K. L., and Thummel, K. E. (2004) Differences in the inhibition of cytochromes P450 3A4 and 3A5 by metabolite-inhibitor complex-forming drugs. Drug Metab. Dispos. 32, 1083–1091. (14) Wang, Y. H., Jones, D. R., and Hall, S. D. (2005) Differential mechanism-based inhibition of CYP3A4 and CYP3A5 by verapamil. Drug Metab. Dispos. 33, 664–671. (15) Baer, B. R., Wienkers, L. C., and Rock, D. A. (2007) Time-dependent inactivation of P450 3A4 by raloxifene: identification of Cys239 as the site of apoprotein alkylation. Chem. Res. Toxicol. 20, 954–264. (16) Gillam, E. M., Baba, T., Kim, B. R., Ohmori, S., and Guengerich, F. P. (1993) Expression of modified human cytochrome P450 3A4 in Escherichia coli and purification and reconstitution of the enzyme. Arch. Biochem. Biophys. 305, 123–131. (17) Domanski, T. L., He, Y. A., Khan, K. K., Roussel, F., Wang, Q., and Halpert, J. R. (2001) Phenylalanine and tryptophan scanning mutagenesis of CYP3A4 substrate recognition site residues and effect on substrate oxidation and cooperativity. Biochemistry 40, 10150–10160. (18) Holmans, P. L., Shet, M. S., Martin-Wixtrom, C. A., Fisher, C. W., and Estabrook, R. W. (1994) The high-level expression in Escherichia coli of the membrane-bound form of human and rat cytochrome b5 and studies on their mechanism of function. Arch. Biochem. Biophys. 312, 554–565. (19) Rock, D., Rock, D., and Jones, J. P. (2001) Inexpensive purification of P450 reductase and other proteins using 2′,5′-adenosine diphosphate agarose affinity columns. Protein Expression Purif. 22, 82–83. (20) Yano, J. K., Wester, M. R., Schoch, G. A., Griffin, K. J., Stout, C. D., and Johnson, E. F. (2004) The structure of human microsomal cytochrome P450 3A4 determined by X-ray crystallography to 2.05-Å resolution. J. Biol. Chem. 279, 38091–38094. (21) Shaw, P. M., Hosea, N. A., Thompson, D. V., Lenius, J. M., and Guengerich, F. P. (1997) Reconstitution premixes for assays using purified recombinant human cytochrome P450, NADPH-cytochrome P450 reductase, and cytochrome b5. Arch. Biochem. Biophys. 348, 107–115. (22) Wang, H., Dick, R., Yin, H., Licad-Coles, E., Kroetz, D. L., Szklarz, G.,Harlow,G.,Halpert,J.R.,andCorreia,M.A.(1998)Structure-function relationships of human liver cytochromes P450 3A: aflatoxin B1 metabolism as a probe. Biochemistry 37, 12536–12545. (23) Yu, L., Liu, H., Li, W., Zhang, F., Luckie, C., van Breemen, R. B., Thatcher, G. R., and Bolton, J. L. (2004) Oxidation of raloxifene to

1786 Chem. Res. Toxicol., Vol. 20, No. 12, 2007

(24)

(25)

(26)

(27) (28)

(29)

(30)

quinoids: potential toxic pathways via a diquinone methide and o-quinones. Chem. Res. Toxicol. 17, 879–888. Chen, Q., Ngui, J. S., Doss, G. A., Wang, R. W., Cai, X., DiNinno, F. P., Blizzard, T. A., Hammond, M. L., Stearns, R. A., Evans, D. C., Baillie, T. A., and Tang, W. (2002) Cytochrome P450 3A4-mediated bioactivation of raloxifene: irreversible enzyme inhibition and thiol adduct formation. Chem. Res. Toxicol. 15, 907–914. Hasemann, C. A., Kurumbail, R. G., Boddupalli, S. S., Peterson, J. A., and Deisenhofer, J. (1995) Structure and function of cytochromes P450: a comparative analysis of three crystal structures. Structure 3, 41–62. Xue, L., Wang, H. F., Wang, Q., Szklarz, G. D., Domanski, T. L., Halpert, J. R., and Correia, M. A. (2001) Influence of P450 3A4 SRS-2 residues on cooperativity and/or regioselectivity of aflatoxin B1 oxidation. Chem. Res. Toxicol. 14, 483–491. Coles, B. (1984) Effects of modifying structure on electrophilic reactions with biological nucleophiles. Drug Metab. ReV. 15, 1307– 1334. Mutlib, A. E., Goosen, T. C., Bauman, J. N., Williams, J. A., Kulkarni, S., and Kostrubsky, S. (2006) Kinetics of acetaminophen glucuronidation by UDP-glucuronosyltransferases 1A1, 1A6, 1A9, and 2B15. Potential implications in acetaminophen-induced hepatotoxicity. Chem. Res. Toxicol. 19, 701–709. Stark, K. L., Harris, C., and Juchau, M. R. (1989) Influence of electrophilic character and glutathione depletion on chemical dysmorphogenesis in cultured rat embryos. Biochem. Pharmacol. 38, 2685– 2692. Argoti, D., Liang, L., Conteh, A., Chen, L., Bershas, D., Yu, C. P., Vouros, P., and Yang, E. (2005) Cyanide trapping of iminium ion reactive intermediates followed by detection and structure identification

Pearson et al.

(31)

(32)

(33)

(34) (35)

(36)

using liquid chromatography-tandem mass spectrometry (LC-MS/ MS). Chem. Res. Toxicol. 18, 1537–1544. Schnetz-Boutaud, N., Daniels, J. S., Hashim, M. F., Scholl, P., Burrus, T., and Marnett, L. J. (2000) Pyrimido[1,2-R]purin-10(3H)-one: a reactive electrophile in the genome. Chem. Res. Toxicol. 13, 967– 970. Zhou, S., Chan, E., Lim, L. Y., Boelsterli, U. A., Li, S. C., Wang, J., Zhang, Q., Huang, M., and Xu, A. (2004) Therapeutic drugs that behave as mechanism-based inhibitors of cytochrome P450 3A4. Curr. Drug Metab. 5, 415–442. Wojnowski, L., Turner, P. C., Pedersen, B., Hustert, E., Brockmoller, J., Mendy, M., Whittle, H. C., Kirk, G., and Wild, C. P. (2004) Increased levels of aflatoxin-albumin adducts are associated with CYP3A5 polymorphisms in The Gambia, West Africa. Pharmacogenetics 14, 691–700. Anderson, B. M., Cordes, E. H., and Jencks, W. P. (1961) Reactivity and catalysis in reactions of the serine hydroxyl group and of O-acyl serines. J. Biol. Chem. 236, 455–463. Melet, A., Assrir, N., Jean, P., Pilar Lopez-Garcia, M., Marques-Soares, C., Jaouen, M., Dansette, P. M., Sari, M. A., and Mansuy, D. (2003) 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. Arch. Biochem. Biophys. 409, 80–91. Yano, J. K., Denton, T. T., Cerny, M. A., Zhang, X., Johnson, E. F., and Cashman, J. R. (2006) Synthetic inhibitors of cytochrome P-450 2A6: inhibitory activity, difference spectra, mechanism of inhibition, and protein cocrystallization. J. Med. Chem. 49, 6987–7001.

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