Mechanism-Based Inactivation of Cytochrome P450 2B1 by 7

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Mechanism-Based Inactivation of Cytochrome P450 2B1 by 7-Ethynylcoumarin: Verification of Apo-P450 Adduction by Electrospray Ion Trap Mass Spectrometry Kelly A. Regal,† Michael L. Schrag,‡ Ute M. Kent,† Larry C. Wienkers,‡ and Paul F. Hollenberg*,† Department of Pharmacology, The University of Michigan, Ann Arbor, Michigan 48109, and Drug Metabolism Research, Pharmacia & Upjohn, Inc., Kalamazoo, Michigan 49007 Received November 24, 1999

7-Ethynylcoumarin was synthesized as a potential mechanism-based inhibitor, and it was found to be an effective inactivator of 7-ethoxy-4-(trifluoromethyl)coumarin (7EFC) Odeethylation catalyzed by purified, reconstituted P450 2B1. In contrast, 7-ethynylcoumarin demonstrated minimal inactivation of P450 2A6-mediated 7-hydroxycoumarin formation. The inactivation of P450 2B1 demonstrated pseudo-first-order kinetics and was NADPH- and inhibitor-dependent. The maximal rate constant for the inactivation of 2B1 was 0.39 min-1 at 30 °C, and thus, the time required to inactivate 50% of the P450 2B1 that was present (t1/2) was 1.8 min. The estimated concentration which led to half-maximal inactivation (KI) was 25 µM. No protection from inactivation was seen in the presence of nucleophiles (glutathione and sodium cyanide), an iron chelator (deferroxamine), or superoxide dismutase and catalase. Addition of the substrate (7EFC) protected P450 2B1 from inactivation, in a concentrationdependent manner. The partition ratio for P450 2B1 was 25; i.e., the number of metabolic events was 25-fold higher than the number of inactivating events. Incubations of 7-ethynylcoumarin with P450 2B1 for 10 min resulted in an 80% loss in enzymatic activity, while 90% of the ability to form a reduced-CO complex remained. This activity loss was not recovered following dialysis, indicative of irreversible inactivation. Covalent attachment of the entire inhibitor and oxygen to apo-P450 2B1, in a 1:1 ratio, was shown via electrospray ion trap mass spectrometry. This method also verified the absence of modification to the heme or the cytochrome P450 reductase. Taken together, the characterization of the inhibition seen with P450 2B1 and 7-ethynylcoumarin was consistent with all of the criteria required to distinguish a mechanism-based inactivator. In addition, electrospray ion trap mass spectrometry has the potential to be applied to protein adducts above and beyond those associated with the mechanism-based inactivation of cytochrome P450s.

Introduction (P450s)1

Cytochrome P450s are a large family of microsomal heme-containing monooxygenases involved in the detoxification of a wide variety of xenobiotics as well as the metabolism of endobiotics. Despite the elucidation of the first mammalian crystal structure (1, 2), techniques which facilitate the identification of critical amino acids within the active site will continue providing a means to verify homology models for the remaining mammalian P450s. The techniques used to pinpoint critical residues include site-directed mutagenesis (35), sequencing of naturally occurring allelic variants (69), homology models based on the bacterial P450 crystal structures (10, 11), combinations of these techniques [site-directed mutagenesis and homology modeling (12* To whom correspondence should be addressed. E-mail: phollen@ umich.edu. Phone: (734) 764-8166. Fax: (734) 763-5387. † The University of Michigan. ‡ Pharmacia & Upjohn, Inc. 1 Abbreviations: BSA, bovine serum albumin; DLPC, dilauroyl-L-Rphosphatidylcholine; 7EC, 7-ethynylcoumarin; 7EFC, 7-ethoxy-4-(trifluoromethyl)coumarin; ESI, electrospray; GSH, glutathione; HFC, 7-hydroxy-4-(trifluoromethyl)coumarin; Mr, relative molecular mass; P450, cytochrome P450; PB, phenobarbital; SOD, superoxide dismutase.

14), and allelic variants and site-directed mutagenesis (15)], chemical modification of P450s (16), and mechanism-based inhibition (17-20). Within this latter category, mechanism-based inactivators which target the apo-P450 are especially important. Several criteria are necessary to prove that an inhibitor behaves in a mechanism-based fashion. These include the following: (1) time- and concentration-dependent inactivation, (2) NADPH and inhibitor dependence, (3) substrate protection, (4) lack of an effect of superoxide dismutase and catalase, (5) lack of an effect of exogenous nucleophiles, (6) irreversibility of the inactivation, and (7) a 1:1 stoichiometry in the adduction of the reactive species to the apoprotein, the heme, or both. The proof of stoichiometric adduction to the apo-P450 has relied almost solely on the retention of radiolabel with the P450 following inactivation with a radiolabeled inhibitor (18, 21-23). Electrospray LC-MS (ESI-LC-MS) has recently been shown to be applicable to the analysis of apo-P450 adducts (24). Analysis of the apoprotein adducts in this fashion facilitates the determination of the site of adduction (heme vs apo-P450 vs reductase) as well as the extent of adduction and stoichiometry, without the need for a radiolabeled inhibitor. In addition, the

10.1021/tx990195s CCC: $19.00 © 2000 American Chemical Society Published on Web 03/21/2000

Inactivation of P450 2B1 by 7-Ethynylcoumarin

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Scheme 1. Synthesis of 7-Ethynylcoumarin

magnitude of the mass shift relative to the control can yield structural information about the adducting species. Finally, this method provides a means of ensuring that the adducted species is not modified or lost in the process of cleaving the adducted P450 into peptides. The massto-charge ratios (m/z) within the apo-P450 and reductase protein envelopes were less than 2000 which made such analyses amenable to ion trap MS analysis. Some of the naturally occurring coumarins seem to have a predilection for mechanism-based inactivation of P450s, e.g., bergamottin and P450 3A4 (25), coriandrin and P450 1A1 (26), various psoralen derivatives, especially 8-methoxypsoralen, and P450 2A6 (27-29). Interestingly, some coumarin-related, mechanism-based inactivation has been noticed with the phenobarbitalinducible (2B) family of P450s (27, 30), but the main emphasis has been with the P450 2A enzyme(s). The 7-position of coumarin is the main site of P450 oxidation (31, 32). Both 7-ethoxycoumarin and 7-ethoxy-4-(trifluoromethyl)coumarin (7EFC) are known substrates for the P450 2B enzymes (33), and the major metabolites have resulted from dealkylation at the 7-position (34, 35). Thus, in an attempt to rationally design a mechanismbased inhibitor, an ethynyl group was placed at the 7-position of coumarin. The 7-ethynylcoumarin (7EC)dependent inhibition of the 7EFC O-deethylation activity of purified, reconstituted rat P450 2B1 was characterized. This inhibition was found to be consistent with all of the criteria that are necessary for establishing mechanismbased inactivation. Utilization of ESI ion trap MS has provided evidence for apo-P450 2B1 adduction, a mass shift consistent with the postulated ketene mechanism, and the stoichiometry of adduction as well as verification of the irreversibility of this inactivation.

Materials and Methods Materials. 7-Hydroxycoumarin, coumarin, trifluoromethanesulfonyl anhydride, triphenylphosphine, palladium(II) acetate, trimethylsilylacetylene, and tetrabutylammonium fluoride were obtained from Aldrich (Milwaukee, WI). Dilauroyl-L-R-phosphatidylcholine (DLPC), NADPH, superoxide dismutase (SOD), GSH, deferroxamine, NaCN, catalase, myoglobin (horse heart), and bovine serum albumin (BSA) were purchased from Sigma Chemical Co. (St. Louis, MO). 7-Ethoxy-4-(trifluoromethyl)coumarin (7EFC) was obtained from Molecular Probes Inc. (Eugene, OR), and 7-hydroxy-4-(trifluoromethyl)coumarin (HFC) was from Enzyme System Products (Livermore, CA). Slide-ALyzer cassettes were from Pierce (Rockford, IL). All other chemicals and solvents were reagent-grade. Synthesis of 7-Ethynylcoumarin (III). 7EC was prepared according to the reaction shown in Scheme 1. The identification of all of the intermediates was established by 1H NMR (300 MHz; Varian, Palo Alto, CA). 7-Hydroxycoumarin (1 g, 6.2 mmol) was initially combined with anhydrous pyridine (20 mL). The reaction mixture was placed on ice and allowed to cool prior to the addition of trifluoromethanesulfonic anhydride (1 mL, 5.9 mmol). The mixture was stirred on ice for 4 h, after which diethyl ether (100 mL) was added. The first precipitate was separated by filtration and discarded. Following a second precipitation after the addition of 1 M HCl (5 mL), the product

was collected by filtration, and dried under vacuum to give 1.66 g of I (91% yield). 7-(Trifluoromethanesulfonyl)coumarin, I (0.5 g, 1.7 mmol), was dissolved in pyridine (5 mL), followed by the addition of triethylamine (50 mL), triphenylphosphine (0.6 g, 2.25 mmol), and a catalytic amount of palladium(II) acetate. After the solvent mixture had been flushed with dry argon (5 min), trimethylsilylacetylene (0.5 mL, 3.5 mmol) was added and the reaction mixture was refluxed under argon for 5 h, followed by concentration under vacuum. Water (50 mL) was then added to the concentrate, and the resultant precipitate was collected by filtration and dried under vacuum, yielding 0.37 g of II (90% yield). Without further purification, 7-(trimethylsilylethynyl)coumarin, II (0.25 g, 1.0 mmol), was dissolved in methanol (50 mL), followed by the addition of tetrabutylammonium flouride (0.58 mL, 2.1 mmol). The reaction mixture was heated to 60 °C for 15 min. Water was added until precipitation occurred. The precipitate was collected, and the final product, III, was obtained in pure form following isocratic silica column chromatography in a 60:40 hexane/ethyl acetate mixture. The final yield was 0.165 g (97%). Normal-phase TLC analysis in the same solvent system resulted in a Rf of 0.5. 1H NMR (DMSO-d6): δ 3.20 (s, 1H, acetylene H), 6.60 (d, J ) 10 Hz, 1H, H3), 7.52 (dd, J ) 1.5, 8 Hz, 1H, H6), 7.78 (d, J ) 1.5 Hz, 1H, H8), 7.94 (d, J ) 8 Hz, 1H, H5), 8.12 (d, J ) 10 Hz, 1H, H4). UV (0.1%, 1:1 DMSO/CH3CN): 294 (294 = 13 960 M-1 cm-1), 324 nm (324 = 9465 M-1 cm-1). Electrospray MS: positive ion at m/z 171 (100) (MH+). Preparation of Microsomes. Microsomal membranes were prepared from the livers of fasted male Fischer rats (175-190 g, Harlan Sprague-Dawley, Indianapolis, IN), according to previously published methods (36). P450 2B1 had been induced by ip injection of 100 mg of phenobarbital (PB) in water/kg of body weight for 3 days. Human liver microsomes were prepared as previously reported (37). Purification of P450s, Reductase, and Cytochrome b5. P450 2B1 and cytochrome b5 were purified from microsomes isolated from livers of fasted male Long Evans rats (175-190 g, Harlan Sprague-Dawley) given 0.1% PB in their drinking water for 12 days according to the procedures of Saito and Strobel (36) and Waxman and Walsh (38), respectively. Reductase was purified after expression in Escherichia coli as previously described (39). Enzyme Inactivation and Activity Assays. The methods described here are similar to those of Kent et al. (22). Briefly, purified P450 2B1 and reductase were reconstituted with lipid for 45 min at 4 °C. The primary reaction mixture contained 0.5 µM P450, 0.5 µM reductase, 200 µg/mL DLPC, 7EC, and 50 mM potassium phosphate buffer (pH 7.4). After incubation at 30 °C for 3 min, the reactions were started by adding NADPH (final concentration of 0.8 mM). The 7EFC O-deethylation activity was measured spectrofluorometrically as previously described (35). At the indicated times, 10 µL samples of the primary reaction mixture were removed and mixed with 990 µL of the secondary reaction mixture containing 0.2 mM NADPH, 100 µM 7EFC, and 40 µg/mL BSA in 50 mM potassium phosphate buffer (pH 7.4). Attempts to modulate the inactivation of P450 2B1 with various trapping agents used one of the following: catalase (800 units/mL), SOD (800 units/mL), GSH (2 mM), NaCN (1 mM), or deferroxamine (100 µM). Each complete experiment was conducted from a single batch of reconstituted enzyme, to obtain consistent data from which a rate constant could be calculated. Enzyme activity in the secondary assay was stopped by adding ice-cold acetonitrile to a final concentration of 25% (v/v). The

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fluorescence of the samples was measured directly at room temperature on an SLM-Aminco model SPF-500C spectrofluorometer with excitation at 410 nm and emission at 510 nm. Enzyme activity was calculated from a standard curve generated with HFC. 7EC inactivation of P450 2A6 was assayed in a similar fashion, using human liver microsomes. Briefly, the primary assays (1 mL) contained 100 mM potassium phosphate buffer (pH 7.4), 1 mM EDTA, 1 µM P450, 1 mM NADPH, and 100 µM 7EC. The secondary assay (1 mL) contained 100 mM potassium phosphate buffer, 50 µM coumarin, and 1 mM NADPH. Prior to the initiation of the reaction by NADPH, the primary incubation mixtures were equilibrated at 37 °C for 5 min. After initiation and at appropriate time intervals, 50 µL transfers were made from the primary to the secondary incubation mixtures. The secondary incubations were terminated after 5 min by the addition of 50 µL of 30% TFA in methanol. The contents of each tube were then directly transferred to 1.5 mL HPLC autosampler vials, and 50 µL of each was injected onto a Hewlett-Packard Spherisorb ODS2 125 mm × 4 mm column (5 µm; Roseville, CA). A Hewlett-Packard series 1050 pump provided solvent delivery at a rate of 0.5 mL/min, and 7-hydroxycoumarin was detected postcolumn with a HewlettPackard 1046A fluorescence detector (excitation at 371 nm and emission at 454 nm). Elution of the metabolite was performed isocratically with a solvent consisting of 20% acetonitrile, 5% methanol, and 70% water. Analysis for Product and Metabolite Protection. 7EC was incubated with reconstituted P450 2B1 (with or without NADPH) under the conditions described above except that the incubation time was extended to 30 min. At this time, the individual incubations were transferred to 10K molecular weight cutoff membrane filters and centrifuged at 8000g. The resultant supernatant (100 µL) was added to fresh 7EC, NADPH, and reconstituted P450 2B1, and following incubation at 30 °C for 10 min, the remaining enzyme activity was determined using the 7EFC assay. Spectrophotometric Analysis. At the indicated times, the primary incubations were stopped by transfer to ice. ReducedCO versus CO spectra were recorded on a DW2 UV/vis spectrophotometer (SLM Aminco, Urbana, IL) equipped with an OLIS spectroscopy operating system (On-Line Instruments, Inc., Bogart, GA; 40). Absolute spectral values were determined by scanning from 375 to 500 nm relative to catalase, lipid, and potassium phosphate buffer (pH 7.4) in the reference. Substrate Protection. Substrate protection from 7ECdependent inactivation of the P450s was assayed by including 100 µM 7EC together with 7EFC at molar ratios of 1:0, 1:1, and 1:2 in the primary reaction mixture. At the indicated times, duplicate 10 µL aliquots were removed and assayed for remaining activity as described above. Partition Ratio. Samples were incubated in the presence of 0-800 µM 7EC for 10 min to ensure that the inactivation was complete. Duplicate aliquots were removed and assayed for 7EFC activity as described above. Irreversibility of Inactivation of P450 2B1. The P450 was inactivated with 300 µM 7EC in a total volume of 500 µL. Duplicate aliquots were transferred to the secondary assay at 0 min and at 10 min. Control and inactivated samples were dialyzed overnight against 50 mM potassium phosphate buffer (pH 7.4), 20% (w/v) glycerol, and 1 mM EDTA (2 × 1 L). All samples were reconstituted with lipid (50 µg) and again assayed for activity using the 7EFC assay. In addition, nondialyzed and dialyzed incubations were compared by ESI-LC-MS (LCQ) for the extent of apo-P450 adduction (see below). ESI-LC-MS Analysis of Apo-P450 Adducts (Stoichiometry and Specificity of Binding). Initially, the LCQ was optimized for whole protein analysis while 50 µg/mL BSA in 75% CH3CN and 0.1% TFA (rate of 3 µL/min) was infused into the mass spectrometer via an ESI source. Later, myoglobin was utilized for the optimization, due to inconsistency in the purity of the BSA. The sheath gas was set at 80 (arbitrary units), while

Regal et al. the auxiliary gas was set at 15 (arbitrary units). The spray voltage was 5 kV, and the capillary temperature was 200 °C. Initial LC separation was carried out on a 150 mm × 2.1 mm Vydac C4 column (5 µm, Vydac Advances, Hesperia, CA). However, this column rapidly lost the ability to resolve the P450 peak from the lipid, which complicated the deconvolution. As a result, a longer C4 column (250 mm × 2.1 mm, 5 µm, Vydac Advances) was used. Fifty microliters of the primary incubations described above was injected onto the long C4 column which had been previously equilibrated in 10% B (B being 0.1% TFA in CH3CN and A being 0.1% TFA in H2O) at a rate of 0.5 mL/ min. After the column had been washed for 15 min, a linear gradient was applied to the column over the course of 15 min, bringing the concentration of B up to 90%. The LC eluent was directed into the LCQ during the gradient without splitting. After spectra collection for the control (without 7EC or NADPH) and complete P450 incubations, the protein envelopes were deconvoluted with Bioexplore 1.0 (Thermoquest) to give the mass associated with each protein envelope. Retention times were as follows: residual cholate, 20.9 min; heme, 21.5 min; clipped reductase 1 (70 354 Da), 22.9 min; clipped reductase 2 (74 540 Da), 23.3 min; active reductase (77 719 ( 2 Da), 24.2 min; P450 2B1 (55 899 ( 1 Da), 28.1 min; and DLPC, 28.4 min.

Results P450 Inactivation by 7EC. 7EC (0-100 µM) was incubated with both human microsomal P450 2A6 and purified, reconstituted P450 2B1 in the presence of NADPH. Preliminary experiments demonstrated that 7EC only marginally inactivated P450 2A6 in a timedependent manner, i.e., a loss of 15-20% activity after a 15 min preincubation with 100 µM 7EC (37 °C). In contrast, the attenuation of P450 2B1 activity was rapid, demonstrating a loss of >90% activity over a 10 min preincubation with 100 µM 7EC at 37 °C. Semilog plots of the percent of remaining activity versus time quickly became nonlinear with increasing time. A representative plot is shown in Figure 1A. Therefore, experiments were conducted at 30 °C so that aliquots could be taken during the initial linear phase of the inactivation and transferred to the secondary incubations containing 7EFC and NADPH. The duration of this latter assay was 10 min, and the rate of turnover for the uninhibited reaction was 9.2 pmol of metabolite (pmol of P450 2B1)-1 min-1, which was comparable to the number reported by Hanna et al. (39). Thus, this assay was chosen for its sensitivity and quick turnaround time. No significant loss of activity was observed in the absence of 7EC over the time course of the experiments. Notably, a 30% loss in activity was observed in the absence of 7EC when the purified P450 2B1 had been multiply thawed (at least three times; data not shown). Thus, the majority of the work presented here was done with enzyme that had not been thawed more than twice. 7EC was found to cause a decrease in the extent of oxidative dealkylation of 7EFC in a timeand concentration-dependent manner (Figure 1B). This inhibition was NADPH- and 7EC-dependent, indicating that catalytic processing was mandatory (Table 1). Under conditions leading to maximal inactivation, minimal changes in the inactivation occurred in the presence of nucleophiles (GSH and NaCN), an iron chelator (deferroxamine), or superoxide dismutase and catalase. Maximal inactivation did not lead to a substantial loss in the ability of P450 2B1 to form a reduced-CO complex (Table 1). Since there was a 100-fold dilution in enzyme concentration in the transfer of an aliquot from the primary to the secondary reaction mixture, the competitive effects of 7EC within the secondary reaction were minimal. A



Figure 1. (A) Linearity of the inactivation of P450 2B1 by 7EC. (B) Time- and concentration-dependent inactivation of the 7EFC O-deethylation activity by 7EC. Incubation conditions were as described in Materials and Methods. Each point represents the average of duplicate incubations. The concentrations of 7EC were (0) 0, (]) 7.6, (O) 15.6, (4) 18.8, (!) 37.6, (crossed ]) 56.4, and (x) 75.2 µM. (C) Double-reciprocal plot of the rates of inactivation of 7EFC O-deethylation activity as a function of inhibitor concentration. Plots are representative of the results obtained in three separate experiments. Table 1. Effect of NADPH and Trapping Agents on P450 2B1 Inhibition by 7-Ethynylcoumarina

inactivation assay components

% control activity (10 min)

% P450 remaining (10 min)

NADPH 7-ethynylcoumarin 7-ethynylcoumarin, NADPH 7-ethynylcoumarin, NADPH, SOD, catalase 7-ethynylcoumarin, NADPH, glutathione 7-ethynylcoumarin, NADPH, deferroxamine 7-ethynylcoumarin, NADPH, NaCN

95 92 16 23 22 15 19

95 100 94 89 88 90 90

a Primary and secondary assays were performed in duplicate at 30 °C as described in Materials and Methods. Concentrations of trapping agents were as follows: SOD, 800 units/mL; catalase, 800 units/mL; GSH, 2 mM; deferroxamine, 100 µM; and NaCN, 1 mM.

Figure 2. Substrate protection against P450 2B1 inactivation by 7EC. Incubation conditions were as described in Materials and Methods. The primary reaction mixtures contained the following molar ratios of 7EFC to 7EC: (0) 0:0, (]) 0:1, (O) 1:1, and (4) 2:1.

double-reciprocal plot of the resultant inactivation rates and 7EC concentrations gave a straight line (Figure 1C), indicative of saturable inhibition. The concentration which produced half-maximal inactivation (KI) was 25 ( 2 µM, and the maximal rate constant for inactivation (kinact) at 30 °C was 0.39 ( 0.01 min-1. Thus, the half-life of inactivation (t1/2) was 1.8 min. Substrate Protection. Simultaneous incorporation of increasing concentrations of 7EFC with 100 µM 7EC in the primary incubations reduced the efficiency of the 7EC-induced inactivation of P450 2B1 in a concentrationdependent manner (Figure 2). In the absence of 7EFC, only 55% of the control activity remained after 2 min at 30 °C. However, 86% of the control activity was still present when the molar concentration of 7EFC was twice

Figure 3. Loss of P450 2B1 activity as a function of the ratio of 7EC to P450. The enzyme was incubated with increasing concentrations of 7EC as described in Materials and Methods. The extrapolated partition ratio was determined from the intercept or the linear regression line from the lower ratios with the x-axis.

that of 7EC, indicating that 7EFC competes with 7EC for oxidation by P450 2B1. It also suggests that the inactivation of P450 2B1 proceeded via attachment of the reactive intermediate of 7EC to the active site. Partition Ratio Determination. The number of molecules of 7EC metabolized per molecule of 2B1 inactivated, i.e., the partition ratio (41), was estimated from the percent of remaining activity following incubation with various concentrations of 7EC. The reactions were allowed to progress until the inactivation was complete (10 min). The turnover number (partition ratio + 1) was estimated by plotting the percent of remaining activity as a function of the molar ratio of 7EC to P450 2B1, as previously demonstrated by Kent et al. (21, 22). The turnover number was 26, and therefore, the partition ratio was 25 (Figure 3). Irreversibility of Inactivation. Control samples or samples inactivated with 7EC were dialyzed overnight. Following reconstitution with fresh lipid, they were again sampled for the remaining activity. No recovery in activity was observed for the inactivated sample (Table 2). Fresh reductase after dialysis was not required since the MS data demonstrated that the reductase was modification-free. Dialyzed and nondialyzed samples of inactivated P450 2B1 were analyzed by ESI-LC/MS. The relative proportion of unmodified and adducted apo-2B1 was independent of whether the sample had been dialyzed (data not shown). Stoichiometry and Specificity of Adduction (LCMS). Reverse-phase HPLC separation of the proteins and

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Table 2. Irreversibility of the Inactivation of P450 2B1 by 7-Ethynylcoumarina before dialysis % remaining activity

% P450

0 min 10 min 10 min 100 µM 7EC without NADPH 100 µM 7EC with NADPH

after dialysisb % remaining activity % P450 10 min

10 min

100

99

92

93

NDc

99

18

88

26

86

a Assay conditions were as described in Materials and Methods. The activities are representative of the mean of duplicate samples from two separate experiments. b The numbers were normalized to adjust for the volume change seen after dialysis. c Not determined.

Figure 4. ESI-LC-MS analysis of inactivated P450 2B1. (A) Reconstructed reverse-phase HPLC chromatogram of the primary incubation(s). Incubations were performed as described in Materials and Methods, and 50 µL was injected, i.e., 25-50 pmol of P450 2B1 on-column. The peaks were identified as cholate (I), heme plus an unidentified protein (II), clipped reductase 1 (III), clipped reductase 2 (IV), active reductase (V), P450 2B1 (VI), and DLPC (VII). (B) Deconvoluted spectrum of adducted apo-2B1 (Mr ) 56 084 ( 3 Da; n ) 6) as well as nonadducted apo-2B1 (Mr ) 55 899 ( 1 Da; n ) 6). The inset shows the deconvoluted spectrum of the apo-P450 2B1 envelope from a control incubation (7EC without NADPH).

the lipid was performed in conjunction with ESI-LC-MS analysis of the eluting proteins. The accompanying LC method was capable of separating the heme, two clipped reductases, active reductase, P450 2B1, and DLPC (Figure 4A). The resultant spectra after deconvolution of the protein envelopes demonstrated that the inactivated P450 2B1 had increased in size by 185 mass units (Figure 4B), in the absence of heme or reductase modification (data not shown). This mass difference was equivalent to one molecule of 7EC plus oxygen, within experimental error (≈0.005%). Therefore, P450 2B1 was inactivated via adduction to the apoprotein, and the stoichiometry of this labeling was 1:1. This latter conclusion as well as the competitive substrate studies and the lack of an effect of the exogenous nucleophiles (GSH and NaCN; Table 1) indicated that the nucleophilic adduction by 7EC occurred in an active site specific manner.

Discussion 7EC was rationally synthesized as a potential mechanism-based inhibitor on the basis of the following information. First, it has been noted that there is a

preference for oxidation of coumarin, 7-ethoxycoumarin, and 7EFC at the 7-position (32, 34, 35). P450 2B1 has been shown to efficiently O-deethylate 7-ethoxycoumarin and 7EFC (18, 39), while P450 2A6 preferentially carries out the 7-hydroxylation of coumarin (42, 43). Second, the ethynyl group is known to be associated with mechanismbased inactivation of numerous P450s (44), and it has previously been incorporated into various molecules in an attempt to introduce selective mechanism-based inactivation (45-49). Therefore, an ethynyl group was placed at the 7-position of coumarin. While no significant loss in P450 2A6-mediated coumarin 7-hydroxylation activity was observed (data not shown), there was a substantial loss in the extent of P450 2B1-mediated O-deethylation of 7EFC. Characterization of the P450 2B1 inhibition was consistent with all of the criteria needed to distinguish a mechanism-based inactivator. Nonlinear inactivation kinetics were observed at 37 °C, and in accordance with previous studies, the temperature of the incubations was lowered to 30 °C in an attempt to regain linearity. Despite the characterization of the key kinetic parameters (KI, kinact, and t1/2) at the less than optimal 30 °C, biphasic kinetics were still observed (Figure 1A). This phenomenon has been previously reported and seems to be relatively common for the P450 2B isoforms (21, 23, 50, 51). There are several potential reasons for such nonlinear plots. The first relates to cases with a high partition ratio, where the turnover of the inhibitor is rapid, leading to inhibitor depletion and deviation from the kinetic models which are meant to describe the loss of activity (41, 52). However, this was not the case since a minimum of 90% of both high (100 µM) and low (10 µM) inhibitor concentrations was still present after a 10 min incubation at 30 °C (data not shown). A second possible cause for the non-pseudo-firstorder kinetics was the loss of inactivated enzyme due to instability and breakdown. However, the ability to repeatedly freeze the incubations and identify the apoP450 2B1 adduct by ESI-LC-MS would not support this possibility. It had previously been suggested that the involvement of multiple isoforms could be responsible (50), although there was no evidence for multiple apoP450s in the deconvoluted ESI-LC-MS spectra obtained here. The biphasic plot seen in Figure 3 has previously been suggested to be indicative of either product inhibition or product protection (41). To address this issue, 30 min incubations with 7EC and P450 2B1 (with or without NADPH) were performed, followed by centrifugation across a 10K molecular weight cutoff membrane filter, to obtain the protein-free supernatant with the metabolite(s). This supernatant was again incubated with the inhibitor and P450. The extent of P450 2B1 inactivation was independent of the presence or absence of NADPH in the supernatants (data not shown); i.e., product inhibition or protection was not responsible for the biphasic kinetics. The possibilities remain that there could have been multiple inhibition mechanisms (50) or that the change in the rate simply reflected the inactivation of a significant amount of protein (41). Without a clear explanation for the curved plots, the problem was circumvented by calculating the key kinetic parameters (KI, kinact, and t1/2) from the initial linear rates. Previously, Hopkins et al. (49) screened a series of polycyclic aromatic acetylenes for the inactivation of P450 2B1 and Roberts et al. (23) screened a similar series of



Scheme 2. Potential Mechanisms for the Oxidation of 7-Ethynylcoumarin Leading to either Metabolite Formation or Apo-P450 2B1 Adduction

compounds for the inactivation of P450s 2B1, 2B4, and 2B11. While the first study concluded that only relatively small, compact arylacetylenes were capable of mechanism-based inactivation of P450 2B1, both studies established that the size and shape of the aromatic ring system as well as the placement of the ethynyl group were important determinants for mechanism-based inactivation. Under experimental conditions identical to those utilized within this study, the maximal P450 2B1 inactivation rate constants (kinact) for these arylalkynes, in the presence of reconstituted P450 2B1, ranged from 0.02 to 0.88 min-1. 7EC, although structurally different, compared favorably with these inhibitors because its kinact (0.39 min-1) fell well within the reported range. In fact, only two of the 11 inhibitors screened by Roberts et al. (23) had higher rate constants of inactivation. Calculation of the turnover number associated with the inactivation of P450 2B1 by 7EC was possible via incubation with increasing inhibitor concentrations and extrapolation of the line through the lower 7EC:P450 ratios (Figure 3; 41). In contrast to the classical turnover number of enzyme kinetics (kcat), this turnover number represents the number of inactivator molecules required for complete inactivation and is directly related to the partition ratio (turnover number ) partition ratio + 1). The partition ratio represents the number of molecules leading to released metabolite per each inactivation event (kcat/kinact) or the efficiency of inactivation (53). The resultant partition ratio was 25 (Figure 3) and falls well within the range of partition ratios seen for the inactivation of purified, reconstituted P450 2B1 by various arylacetylene compounds (16, 18, 54). Given an estimated partition ratio (kcat/kinact; 23) and the kinact (0.39 min-1), the overall oxidation rate or kcat would be expected to be 9.8 min-1. However, since the relationship between these constants is occasionally more complicated, continuing work will determine the oxidation rate via quantification of the metabolite(s) of 7EC. The phenylacetylenes inactivated P450 2B1 via heme alkylation (54), while 2-ethynylnaphthalene and 9-ethynylphenanthrene inactivated the same isoform via apoprotein adduction (18, 19). It is believed that the oxidative sequence leading to metabolites and/or apoprotein adduction diverges from that leading to heme alkylation

prior to, or concurrent with, transfer of oxygen to the π-bond (55), on the basis of the isotope effects observed for both processes. The assumption has been made that the respective ketene species was generated in all of the cases described above, due to the identification of the corresponding carboxylic acid metabolite. Thus, in the presence of more than one nucleophile (heme vs apoprotein vs H2O), binding affinities as well as the orientation of the inhibitor within the P450 active site seem to play a role in the identification of the particular nucleophile that will be poised to react immediately (55). Oxidation on the internal carbon of phenylacetylene led to inactivation via heme alkylation before protein acylation became significant (56, 57). Meanwhile, 2-ethynylnaphthalene and 7EC seem to bind to P450 2B1 in a manner which suppresses delivery of the ferryl oxygen to the internal carbon and results in apoprotein adduction as well as the formation of the carboxylic acid metabolite(s)2 (18). In the studies presented here, heme adduction was ruled out because the level of CO binding was not substantially reduced and ESI-LC-MS analysis did not demonstrate modification to the heme. Hence, formation of acid metabolite or protein adduct is likely to result from pathway a or b in Scheme 2. The exclusion of either pathway may not be possible since the multiple intermediates may form the basis of the multiple inactivation mechanisms that produce the biphasic kinetics often associated with the P450 2B isoforms. The expected mass shift relative to the mass of the apoP450 was 185, assuming the formation of the adduct shown in Scheme 2. Repeated injections onto the LCQ were carried out to determine the intra- and interday variability in the mass assignment for both the native and the adducted apo-P450 2B1. The mass of the native apo-P450 was 55 899 Da ((1; Figure 4). The mass of the adducted apo-P450 was 56 084 Da ((3), and thus, the mass difference was 185. With approximately 0.005% variability in the mass assignment, these results were conclusive evidence that the entire 7EC molecule plus oxygen was adducted to the apo-P450 2B1, in a 1:1 stoichiometry, as a result of an attack by an active site 2

Unpublished data (7EC).

268


nucleophile on the external carbon. At this time, it is not possible to rule out the potential involvement of an epoxide intermediate, which could lead to either an alkylated or an acylated apoprotein adduct (pathway a in Scheme 2). Work to identify the adducted peptide and the nature of the covalent linkage is in progress. Despite the loss of 80-90% of the 7EFC O-deethylation activity of P450 2B1, there was a significant native apoP450 2B1 peak present in all of the ESI-LC-MS spectra that were examined, in addition to the adducted apoP450. In some instances, the nonadducted apo-2B1 was greater than 50% of the total P450, assuming that there was no difference in the ionization of the adducted and native P450. Therefore, in some instances, there appeared to be a differential of 10-30% between the level of adduction and loss of catalytic activity, with activity loss being the greater of the two. Typically, stoichiometry has been calculated as the ratio of the protein-bound radiolabel to the estimated P450 content. Using these methods, we believe that the inactivation of P450 2B1 by 7EC would have resulted in less than stoichiometric labeling of the apo-P450. Previous explanations for substoichiometric labeling included the presence of additional P450s and the P450 not being catalytically active despite its ability to form the reduced-CO complex (29) as well as acid lability of the adduct (58) or loss of the radiolabel due to splitting of the molecule into two radicals (21). The latter explanation did not apply to the mechanism of inactivation by 7EC; i.e., the entire 7EC molecule plus oxygen was adducted to the apo-P450 (∆Mr ) 185; Figure 4 and Scheme 2). ESI-LC-MS gave no evidence for the presence of more than one P450 isoform. The P450 2B1-7EC adduct would be expected to be relatively acid-stable on the basis of work with the adducts of 2-ethynylnaphthalene and 9-ethynylphenanthrene (18, 19). In addition, ESI-LC-MS analysis following dialysis of the inactivated P450 demonstrated that there was no change in the extent of adduction, relative to that of a nondialyzed sample (data not shown). Two possibilities remain. There may have been uncoupling of the P450 catalytic cycle, which led to a loss of activity via the generation of reactive oxygen species, in addition to the loss associated with the mechanism-based inactivation. Alternatively, there may have been a significant amount of apo-P450 2B1 copurified with the holoenzyme. Although the discrepancy between activity loss and adduction is currently not accounted for, it is apparent that the use of ESI-LC-MS is superior to the radiolabel methodology in its ability to directly report stoichiometry as well as to provide additional mass data for the reductase, and the heme. In addition, as evidenced here, ion trap MS is a convenient and precise method for generating whole protein mass spectra and seems to be as applicable as the recently published quadrupole MS method (24). The predicted mass for P450 2B1 was 55 934 Da (Swiss data bank access number P00176), while the mass obtained here was 55 899 Da. The experimental mass for the active reductase (77 719 ( 2 Da) was within 0.006% of its theoretical mass, while the error in the mass determination for P450 2B1 was 0.005%, in line with previous ESI-LC-MS protein data (24). There is precedent for allelic variants within the P450 2B1 subfamily, and this polymorphism can vary between different strains 3

Long Evans rats.

Regal et al.

and colonies of rats (59, 60). The mass of 55 934 Da was based on two mRNAs isolated from PB-induced SpragueDawley rats (PB-1 and PB-2; 61, 62). The two resultant cDNAs were 97% identical, and it was concluded that the resultant proteins would differ by six amino acids (∆Mr ) 16). Importantly, the predicted identities of the last seven amino acids at the C-terminus of the 55 934 Da protein do not match the last seven residues determined for a P450 2B1 isolated from PB-induced LE3 rats (63). Despite the difference between the expected and experimental masses, P450 2B1 isolated from the latter rats demonstrated normal activity with marker substrates (39). In addition, multiple rounds of peptide mapping with inactivated LE3 P450 2B1 led to adducted fragments which were predicted from the Swiss data bank sequence (16, 18, 19, 47). Thus, ESI-LC-MS analysis also holds the potential of identifying or verifying the existence of allelic variants. In summary, 7EC was conclusively shown to be a mechanism-based inactivator of P450 2B1. Despite the fact that mechanism-based inactivation of this particular isoform has been prevalent in the literature, this is the first report of whole protein mass data which are consistent with the postulated ketene (or oxirene) species leading to protein adduction. Analysis on an ion trap mass spectrometer resulted in the determination of the mass and the probable identity of the adducted species as well as the stoichiometry of adduction, with a maximum of 50 pmol of P450 2B1 injected on-column. It also provided evidence for the lack of heme or reductase modification. Thus, ESI ion trap MS can easily be applied to the detection of apoprotein adducts as well as the identification of naturally occurring allelic variants. Future work will be directed toward characterization of the metabolite(s) and the potential GSH conjugate(s), enzymatic cleavage of the modified P450 2B1 to identify the adducted amino acid, and elucidation of the type of bond involved in the peptide adduction as well as the potential inactivation of closely associated isoforms.

Acknowledgment. We thank Dr. William Trager (University of Washington, Seattle, WA) for overseeing the synthesis of the 7-ethynylcoumarin. Special thanks to Chitra Sridar (The University of Michigan) for the purification of the reductase and Hsia-lien Lin (The University of Michigan) for the preparation of the PB microsomes and the purification of the P450 2B1 and cytochrome b5. This research was supported by NIH Grant CA 16954 (P.F.H.) and by U.S. Public Health Service Grant T32-ES07062.

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Mechanism-Based Inactivation of Cytochrome P450 2B1 by 7

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