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Department of Pharmacology and Toxicology, University of Utah, Salt Lake City, Utah
(Received July 26, 2007; accepted October 22, 2007)
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Abstract |
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). Elucidation of the molecular events that mediate mechanism-based inactivation can provide vital structural and mechanistic information about the active sites of the P450 enzymes.
3-Methylindole (3MI) is a selective pneumotoxicant that requires cytochrome P450-mediated bioactivation to elicit its toxicity. P450-mediated metabolism of 3MI occurs through two primary pathways; oxygenation and dehydrogenation (Yost, 1996). Cytochrome P450-mediated oxygenation of 3MI produces the stable metabolites 3-methyloxindole and 3-hydroxy-3-methyloxindole via a common 2,3-epoxide precursor. P450-mediated dehydrogenation of 3MI leads to the formation of the highly reactive intermediate 3-methyleneindolenine (Skordos et al., 1998b). Two intermediates formed from the 2,3-oxygenation of 3MI, the epoxide and 3-hydroxy-3-methylindolenine (Scheme 1), are highly reactive and can possibly form adducts with proteins and DNA. However, extensive studies have shown that dehydrogenation of 3-methyindole, leading to the formation of the highly reactive 3-methyleneindolenine, appears to be the major route of toxicity (Yost, 2001). Glutathione adducts of this reactive methylene imine intermediate have been isolated from cattle and from microsomal incubations with 3MI (Nocerini et al., 1985a,b; Yost et al., 1986). In addition, covalent adducts of 3-methyleneindolenine to both protein (Thornton-Manning et al., 1991, 1996) and DNA (Regal et al., 2001) have also been identified. Extensive studies of 3MI bioactivation by major cytochrome P450 enzymes have been performed (Lanza and Yost, 2001). Interestingly, it was seen that P450 enzymes CYP2F1 and CYP2F3 (goat form), selectively catalyzed the 3-methyl dehydrogenation of 3MI. P450 2F gene families are selectively expressed in lung tissues and are responsible for the bioactivation of several pneumotoxic and carcinogenic chemicals such as benzene, styrene, naphthalene, and 1,1-dichloroethylene. (Nakajima et al., 1994; Lanza et al., 1999; Powley and Carlson, 2001; Sheets et al., 2004; Simmonds et al., 2004).
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; Fan et al., 2000; Forkert et al., 2001). In addition to the generation of toxic reactive intermediates, P450-mediated dehydrogenation pathways can also generate reactive intermediates that act as suicide or mechanism-based inactivators (Racha et al., 1998). Preliminary experiments on the rate of 3MI dehydrogenation by CYP2F1 have shown that formation of the dehydrogenated product, 3-methyleneindolenine, was not linear with time. In addition, higher concentrations of 3MI strongly inhibited the formation of the dehydrogenated product (Lanza et al., 1999). The concentration-dependent inhibition of CYP2F1 was probably a substrate inhibition mechanism, but the nonlinearity in the rate of product formation was most likely caused by a mechanism-based inhibition process. In this process, the highly reactive 3-methyleneindolenine electrophile would form covalent bonds with residues within the CYP2F1 active site and irreversibly inactivate the enzyme. The highly reactive nature of the dehydrogenated metabolite, 3-methyleneindolenine, and the fact that both protein and DNA adducts of this reactive intermediate have been identified, lend further credence to this hypothesis.
Like CYP2F1, CYP2F3 is also selectively expressed in pulmonary tissues and, as mentioned before, previous studies from our laboratory indicated that CYP2F3 also selectively dehydrogenates 3MI. In addition, CYP2F3 is 84% identical to CYP2F1 and shares similar biochemical and metabolic characteristics. Therefore, it is a reasonable hypothesis that 3MI is a mechanism-based inactivator of CYP2F3 as well. The goal of the present study was to determine whether 3MI acts as a mechanism-based inactivator of CYP2F1 and CYP2F3. These results may provide insights into the molecular events that are involved in the relatively rare but nonetheless fascinating dehydrogenation reaction pathway, because dehydrogenation may be linked to the inactivation process.
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Materials and Methods |
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-aminolevulinic acid, ammonium acetate, and 3MI were obtained from Sigma-Aldrich (St. Louis, MO). Isopropyl thiogalactopyranoside and agarose for electrophoresis were obtained from Fisher (Pittsburgh, PA). Ampicillin was obtained from Boehringer Mannheim (Indianapolis, IN). All other chemicals were purchased from Sigma-Aldrich.
CYP2F3 Expression and Purification. Restriction enzymes NdeI, XbaI, and DpnI were obtained from Invitrogen (Gaithersburg, MD). T4 DNA ligase was obtained from Invitrogen. Escherichia coli:DH5
was obtained from Invitrogen. Bacterial media, Luria-Bertani (LB) and Terrific Broth (TB), were obtained from Sigma-Aldrich as dry premixtures. For DNA purification, a large-scale plasmid purification kit (Midi), Ni-NTA beads, and the QIAEX II gel extraction kit were obtained from QIAGEN (Santa Clarita, CA). The Slide-A-Lyzer dialysis cassette was obtained from Pierce Chemical (Rockford, IL). All reagents were of the highest quality possible.
Enzymes. Lymphoblastoid microsomes containing CYP2F1 coexpressed with NADPH-cytochrome P450 oxidoreductase were obtained from BD Gentest (Woburn, MA). In addition, NADPH-cytochrome P450 oxidoreductase was also separately purchased from BD Gentest. CYP2F3 was expressed and purified according to previous methodology.
Mechanism-Based Inactivation Studies with CYP2F1. Time- and concentration-dependent CYP2F1 inhibition by 3MI. Primary incubations containing 200 pmol of CYP2F1 were preincubated with 3MI (0, 15, 30, 50, 70, and 100 µM) in the presence of NADPH (1 mM) at 37°C for 0, 10, 15, and 30 min. The determination of partition ratio for CYP2F1 was modified to measure the conditions of maximal inactivation by increasing the primary incubation times to 40 min. Aliquots containing 50 pmol of CYP2F1 were transferred to secondary incubations containing EC (500 µM) and 100 mM potassium phosphate buffer (pH 7.4) in a total reaction volume of 1.5 ml. A 3-fold dilution factor was obtained from the primary to the secondary incubations. Secondary reactions were initiated with 1 mM NADPH and were incubated for 25 min at 37°C in a shaking water bath. Control incubations were done in the presence of NADPH, but without 3MI. 7-Hydroxycoumarin formation was determined with slight modifications of a previously established method (Greenlee and Poland, 1978). Briefly, incubations were stopped by the addition of 0.1 ml of 2.0 N HCl and 2.0 ml of CHCl3. The tubes were mixed and centrifuged for 10 min at 6000g. One milliliter of the lower chloroform phase (containing both substrate and product) was transferred to a clean tube, and 1.0 ml of 0.01 N NaOH-0.1 M NaCl was added. The contents of the tubes were vortexed and centrifuged again for 10 min at 6000g. The upper phase, containing the phenolic product, was transferred to a new tube. 7-Ethoxycoumarin O-deethylase activity of CYP2F1 was determined by measuring the production of 7-hydroxycoumarin with an SLM-Aminco model SPF-500C spectrofluorometer with the excitation wavelength set at 338 nm and the emission wavelength set at 458 nm. The amount of product formed was quantified based on a standard curve of 7-hydroxycoumarin.
Incubations with glutathione. GSH (2 mM) was included in the primary incubation mixture along with 50 µM 3MI, 100 pmol of CYP2F1, and 1 mM NADPH. Secondary incubations containing EC and NADPH were conducted for 20 min. The EC O-deethylase activity of CYP2F1 was determined as above.
Expression and purification of CYP2F3. The expression and purification steps were based on subtle modifications of previously established methods (Scott et al., 2001). To facilitate expression of CYP2F3, the hydrophobic membrane-embedded domain was removed (residues 3–21), and several residues with positive charges were substituted near the N terminus. In addition, a C-terminal [His]4 tag was incorporated to aid in the purification of CYP2F3 by affinity chromatography. The truncated proteins are highly expressed in E coli and can be released from the membrane using high salt conditions (Scott et al., 2001). Modification of N-terminal region of functional P450 significantly enhances the level of expression (Barnes et al., 1991) but does not affect the catalytic properties of the enzyme (Larson et al., 1991). In addition to incorporating the N-terminal mutations, 5' NdeI and 3' XbaI restriction endonuclease sites were incorporated into CYP2F3 cDNA. This was done to facilitate cloning into the expression vector, pCWOri+ (a generous gift from Dr. Fred Guengerich, Vanderbilt University, Nashville, TN). Amplification of the modified CYP2F3 cDNA was performed by polymerase chain reaction and verified by gel electrophoresis. A double restriction digest using NdeI and XbaI was performed and CYP2F3 cDNA was subcloned into the same unique sites in the expression vector, pCW.
Expression of CYP2F3. Expression of CYP2F3 was performed according to a previously established method for cytochrome P450 expression in bacterial systems (Gillam, 1998). Bacterial clones containing the enzyme were streaked on LB agar plates containing 100 µg/ml ampicillin and incubated at 37°C for 12 to 16 h. Individual colonies were picked and used to inoculate 5 ml of LB media containing 100 µg/ml ampicillin. The starter cultures were then used to inoculate 500 ml of TB media containing 100 µg/ml ampicillin, bactopeptone (1 g/0.5 liter), 1 mM thiamine, and the trace elements FeCl3, ZnCl2,Na2MoO4, CaCl2, CuCl2, and H3BO3. The culture was incubated at 37°C for 2 h at which time 0.5 mM -aminolevulinic acid was added. The culture was incubated until the absorbance at 600 nm (A600) was approximately 0.8 to 1.0. At this time, the culture was induced with 1 mM isopropyl thiogalactopyranoside and both the temperature and rotation rate were reduced to 30°C and 110 rpm, respectively. The incubation continued for 16 to 20 h postinduction, at which time the bacterial cells were harvested by centrifugation at 4000g for 20 min, the supernatant was decanted, and the cells were stored at -70°C until further use.
Preparation of bacterial membrane fractions. Bacterial membranes were prepared by subtle modifications of previously established methods (Guengerich et al., 1996; Scott et al., 2001). The bacterial cell pellets were resuspended in 50 ml (10% total culture volume) of 20 mM K2HPO4 (pH 7.4) containing 20% glycerol. Lysozyme (0.2 mg/ml) was added to the suspension and incubated for 30 min on ice. The suspension was centrifuged at 10,000g. The pellet containing the spheroblasts was then resuspended in 25 ml (5% total culture volume) of 10 mM K2HPO4 (pH 7.4) containing 10% glycerol and 0.5% CHAPS. A protease inhibitor cocktail was added in a 1000-fold dilution (25 µl for a 25-ml resuspension). The suspension was then sonicated with four 25-s bursts at 50 to 60% power from a 1.2-cm diameter probe. The suspension was transferred to a clean ultracentrifuge tube, and the membrane fraction was collected by ultracentrifugation at 105,000g for 1 h. The supernatant, which contained the protein, was stored at 4°C until further use. The reduced CO-binding P450 spectrum was checked at each step of the procedure as described below. The pellet left after ultracentrifugation containing the inclusion bodies and other undisrupted cells was resuspended in 5 ml of 20 mM K2HPO4 (pH 7.4) containing 20% glycerol and 0.5% CHAPS and the same procedure repeated to extract remaining P450 protein.
Assay of cytochrome P450 expression. An aliquot of the membrane fraction or whole cell lysate was diluted to 1 ml in 10 mM K2HPO4 buffer. The reduced CO-binding spectrum was measured on a Gilford Response UV/visible spectrophotometer using a previously established method (Omura and Sato, 1964a). Briefly, the sample was reduced by the addition of a few milligrams of solid Na2S2O4 and placed in a spectrophotometer. The reduced reference spectrum between 400 and 500 nm was obtained. The sample was then bubbled with carbon monoxide gas for approximately 30 s. The CO-bound spectrum was then obtained, and the difference between the bound and reduced reference spectra was determined. Cytochrome P450 content was then calculated by subtracting the absorbance at 490 nm from that of 450 nm and using the millimolar extinction coefficient (91 cm-1 mM-1) for P450 enzymes (Omura and Sato, 1964b).
Purification of CYP2F3. Purification of the histidine-tagged CYP2F3 was done using Ni-NTA beads purchased from QIAGEN (Valencia, CA). The purification protocol was based on the manufacturer's instructions. To the supernatant from the bacterial membrane preparation, 0.25 M NaCl, 10 mM β-mercaptoethanol, and 15 mM imidazole were added. The supernatant was then loaded onto a 5-ml polypropylene column (QIAGEN) packed with 2 ml of Ni-NTA beads and flushed with this solution. The P450 content of the eluant was checked to ensure optimal binding. If optimal binding was not achieved, the eluant was reapplied to the column. The column was then washed three times with 10 mM potassium phosphate buffer containing 0.5% CHAPS, 0.5 M NaCl, 10% glycerol, and increasing concentrations of imidazole (10, 20, and 40 mM). High-salt concentrations and detergents reduce nonspecific binding to the Ni-NTA beads by a hydrophobic or ionic interaction, and they do not affect the binding of histidine-tagged proteins to the Ni-NTA beads. β-Mercaptoethanol was added to prevent the copurification of proteins that might have formed disulfide bonds with the P450 proteins. Imidazole prevents the binding of dispersed histidine residues in the nontagged background proteins and was added to further reduce any nonspecific interaction. The eluant from all three washes was analyzed for P450 content to ensure that the P450 was not lost during the washing steps. The protein in the column was then eluted with the same buffer, but the imidazole content in the buffer was increased to 300 mM. The eluted protein was then concentrated using Centricon tubes (molecular weight cutoff 30,000) and dialyzed overnight in a Slide-A-Lyzer dialysis cassette (0.5–3 ml; molecular weight 3500) in 2.5 liters of 10 mM K2HPO4 containing 10% glycerol. The P450 spectra and protein contents were checked at each step, and the specific activity of the protein was calculated. The purified protein was stored at -70°C for further use.
Mechanism-Based Inactivation Studies with CYP2F3. Time- and concentration-dependent CYP2F3 inhibition by 3MI. Primary incubations containing 400 pmol CYP2F3 and cytochrome P450 reductase microsomes in a 1:3 ratio, were preincubated at room temperature for 10 min. Potassium phosphate buffer (100 mM, pH 7.4) and varying concentrations of 3MI (50, 100, 150, and 200 µM) were added to the incubation mix. Primary incubations were initiated with 1 mM NADPH and allowed to incubate at 37°C. Aliquots containing 100 pmol of CYP2F3 were removed at 0, 10, 20, and 30 min and added to a secondary incubation mix containing CHZX (150 µM) in 100 mM potassium phosphate buffer (pH 7.4) in a total reaction volume of 0.5 ml. The activity of CYP2F3 was measured with CHZX rather than EC because CHZX is an excellent substrate for CYP2F3, and in this case CYP2F3 was a more efficient catalyst (i.e., higher V/K) of CHZX than EC. A 12-fold dilution factor was achieved from the primary to the secondary incubations. Control incubations contained NADPH, but no 3MI. Secondary incubations were initiated with 1 mM NADPH and allowed to incubate at 37°C for 20 min in a shaking water bath. Reactions were stopped by the addition of 20 µl of concentrated phosphoric acid and the formation of 6-hydroxychlorzoxazone was determined by high-performance liquid chromatography analysis using a previously described method (Lucas et al., 1993) with minor modifications. Briefly, analytes from the secondary incubation mixtures were extracted into 2 ml of ethyl acetate. The organic phase was dried down under a N2 stream, and the dry residues were brought up in 100 µl of the mobile phase. Samples (50 µl) were analyzed with a reversed-phase C18 column (2.5 µm, 4.5 x 250 mm, Ultrasphere ODS column; Beckman Coulter, Fullerton, CA). The isocratic mobile phase consisted of water-acetonitrile-glacial acetic acid [75:25:0.5% (v/v)] at a flow rate of 0.4 ml/min. The column effluent was monitored at 287 nm. 6-Hydroxychlorzoxazone eluted from the column at 4.4 min. Benzoxazolinone was used as the internal standard, which eluted at 5.7 min. Levels of CHZX hydroxylase activity were determined by extrapolating absorbance to a standard calibration curve of peak area ratios of known amounts of 6-hydroxychlorzoxazone, divided by the peak area of benzoxazolinone. The enzyme assay was carried out under conditions of linearity with respect to time and protein concentrations.
Incubations with glutathione. GSH (2 mM) was included in the primary incubation mixture along with 100 µM 3MI, 400 pmol of CYP2F3, and 2 mM NADPH. In addition, catalase and superoxide dismutase were also included in the primary incubation mixture in separate experiments. At 0-, 10-, 20-, and 30-min time points, aliquots containing 100 pmol of CYP2F3 were removed and added to a secondary incubation mixture containing CHZX (150 µM) and 1 mM NADPH. Secondary incubations were conducted for 20 min. The CHZX hydroxylase activity of CYP2F3 was determined as above.
Partition ratio. The partition ratio was estimated using the titration method (Silverman, 1995). Primary reaction mixtures for CYP2F1 and CYP2F3 were prepared as described above, with the exception that increasing concentrations of the inactivator, 3MI, were added to the primary mixture, until a saturation of the inactivation was obtained. Aliquots were removed from the primary incubation mixture and added to a secondary incubation containing either EC or CHZX as a substrate for CYP2F1 and CYP2F3, respectively. To obtain a saturation line for CYP2F1 that approached a horizontal line, the primary incubations were conducted for 40 min. The rest of the assay procedure was identical to that described for the inactivation studies. The partition ratio was obtained by plotting the percent activity remaining versus [3MI]/[P450]. The partition coefficient was then extrapolated from the intercept between the linear regression line obtained at low concentrations of 3MI and the straight line obtained at saturating 3MI concentrations.
Spectrophotometric analysis. CYP2F3 was preincubated with reductase at room temperature for 10 min. 3-Methylindole (150 µM) and 100 mM potassium phosphate buffer were added to the samples. Reactions were started by the addition of 1 mM NADPH. At 0- and 20-min time points, an aliquot containing 50 pmol of the enzyme was added to 900 µl of ice-cold buffer containing 10 mM potassium phosphate (pH 7.4) and 10% glycerol. Control samples contained 3MI, but lacked NADPH. The reduced CO-binding spectrum was measured on a Gilford Response UV/visible spectrophotometer as described previously. Negative controls lacking both 3MI and NADPH were also performed.
Substrate protection. Substrate protection from 3MI-mediated inactivation of 400 pmol of CYP2F3 was determined by including either EC or CHZX in the primary incubation. 7-EC concentrations of 300 and 600 µMor750 µM and 1 mM CHZX were added along with 150 µM 3MI to the primary incubations. Reactions were initiated with 1 mM NADPH and at 0-, 10-, 20-, and 30-min time points, aliquots containing 100 pmol of CYP2F3 were transferred to a secondary incubation mixture, containing 500 µM CHZX. A 12-fold dilution was achieved from the primary to the secondary incubations. The CHZX hydroxylase activity of CYP2F3 was assayed as before. Control incubations lacking the inactivator and the alternate substrate were also used.
Irreversibility of inactivation. CYP2F3 and cytochrome P450 reductase in a 1:3 ratio were preincubated at room temperature for 10 min. Primary incubations containing 250 µM 3MI were performed as described earlier. At 0- and 30-min time points, aliquots containing 100 pmol of CYP2F3 were removed from the primary incubation mixture and applied to Slide-A-Lyzer membranes (molecular weight cutoff, 3500; Pierce Chemical). Samples were dialyzed for 7 h at 4°C against 2 liters of a buffer containing 0.1M potassium phosphate (pH 7.4) and 10% glycerol. The dialyzed samples were collected and added to a secondary incubation mixture containing 500 µM CHZX and the CHZX hydroxylase activity of CYP2F3 was determined as described previously. Control incubations lacked 3MI. The samples were compared with the controls for decreases in activity. In a separate experiment, fresh reductase was added to the dialyzed samples before determination of the CHZX hydroxylase activity of CYP2F3.
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Results |
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; Silverman, 1995) (inset, Fig. 1). The inactivator concentration at half-maximal inactivation rate, KI, was obtained from the x-intercept and determined to be 160 µM. The inactivation rate constant, kinact, was obtained from the reciprocal of the y-intercept, which was equal to kinact/2.303 = 0.09 min-1.
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; Kent et al., 2001). The partition ratio was estimated using the titration method (Silverman, 1995). The percent enzymatic activity remaining, after a 30-min preincubation (longest time point tested), was plotted against the ratio of the concentrations of 3MI to P450. Figures 3 and 4 show the plots of the percentage activity remaining versus the ratio of [3MI]/[P450] for CYP2F3 and CYP2F1, respectively. The x-intercept between the linear regression line obtained at low concentrations of 3MI and the straight line obtained at saturating 3MI concentrations was extrapolated graphically to obtain the turnover number (P + 1), which is equal to the partition ratio + 1. The partition ratio was then calculated from the turnover number to be 280 for CYP2F1 and CYP220 for CYP2F3.
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Incubations with Glutathione. In a true mechanism-based inactivation process, addition of exogenous nucleophiles should not protect the enzyme against inactivation, because the reactive intermediate should inactivate the enzyme by covalent bond formation within the enzyme active site and not diffuse out of the active site before binding to critical residues of the enzyme. In cases where the reactive species slowly diffuses out of the enzyme active site, it is believed that irreversible inactivation has occurred before release of the reactive species (Silverman, 1995). To determine the protective effects of glutathione on 3MI-mediated inactivation of CYP2F1 and CYP2F3, 2 mM glutathione was included in the primary incubation. Inclusion of glutathione did not ameliorate inactivation of either enzyme. Recovery of CHZX hydroxylase activity of CYP2F3 was not observed (Table 1).
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Spectrophotometric Analysis. To determine whether enzyme inactivation occurred by destruction of the heme group, the ability of reduced CYP2F3 to bind CO was determined after preincubation with 150 µM 3MI for 20 min. In the presence of NADPH, CYP2F3 incubated with 3MI lost approximately half of its activity compared with a control that lacked both the inactivator and NADPH (Table 2). However, the amount of P450, as determined by its CO-binding spectrum, remained about the same (82% of control) as that in a sample that was incubated for the normal substrate incubation time (20 min) with CHZX (87% of control). This minimal loss in CO binding of CYP2F3 suggests that the heme moiety was not significantly altered during the inactivation process.
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Substrate Protection. When the normal substrate (CHZX) or another substrate, 7-ethoxycoumarin, is added to the preincubation, it competes with the inactivator (3MI) for binding at the active site. This decreases the rate of CYP2F3 inactivation by preventing enzyme inactivator complex formation (Silverman, 1995). Therefore, to study whether the inactivation occurred in the active site of the enzyme, CYP2F3 was incubated with alternate substrates, 7-ethoxycoumarin or CHZX, in the primary incubations, along with 150 µM 3MI. The remaining CHZX hydroxylase activity of CYP2F3 was measured as described previously and normalized to control activity at time 0 (100% activity). In samples containing 3MI that lacked the alternate substrate, there was a 67 ± 4% loss of activity (Fig. 5). The inclusion of a 4-fold molar excess of EC (600 µM) significantly reduced (23 ± 6%) the level of CYP2F3 inhibition. Substrate protection was also observed in the incubations containing CHZX as an alternate substrate. In the presence of a 7-fold molar excess of CHZX (1 mM) a 30 ± 4% reduction in the level of CYP2F3 inactivation by 3MI was observed.
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Discussion |
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,b; Yost et al., 1986; Huijzer et al., 1987). However, we have recently shown (Yan et al., 2007) that several P450 enzymes catalyzed the formation of at least two novel electrophilic quinoneimines (one putative isomer is illustrated in Scheme 1) that were trapped with GSH and identified by mass spectrometry. Previous results from our laboratory have also demonstrated that the 2,3-epoxide and 3-hydroxy-3-methyleneindolenine are potential reactive intermediates (Skordos et al., 1998a,b). None of these intermediates appear to be produced by the lung-selective CYP2F1 or CYP2F3 enzymes, but it is feasible that they might participate in the adverse effects of 3MI.
If dehydrogenation of the benzylic position of 3-substituted indoles is a facile process, other indole-containing molecules should be bioactivated at this position, and could potentially inactivate the P450 enzyme(s) that catalyze dehydrogenation. Several recent intriguing examples (Scheme 1) have shown that this conjecture is often true. A dopamine D4-selective antagonist, L-745,870, contains a 3-substituted-7-azaindole moiety that is highly similar to 3MI. This drug was metabolized in rats, rhesus monkeys, and humans to a novel mercapturate that was probably formed from processing the GSH adduct of the exocyclic methylene position of the dehydrogenated imine methide (Zhang et al., 2000). However, it is not known whether L-745,870 inactivates P450 enzymes.
Another example is the dehydrogenation of zafirlukast, a leukotriene receptor antagonist (Kassahun et al., 2005) that contains a 3-alkylindole moiety. The dehydrogenation of the 3-alkylindole moiety of zafirlukast formed the same type of imine methide electrophilic intermediate as 3MI and L-745,870. In addition, this drug was a reasonably potent mechanism-based inactivator of CYP3A4, presumably through alkylation of active site residues by the imine methide intermediate. Surprisingly, CYP3A4 was the only susceptible P450 enzyme; CYP2D6, CYP2C9, CYP2C19, and CYP1A2 were not inactivated.
Another recent example showed that the prostaglandin D2 receptor 1 antagonist, MK-0524, was dehydrogenated predominantly by CYP3A4 in human liver to form an electrophilic imine methide, just like the other 3-substituted indoles presented here. However, reactive intermediates did not inactivate CYP3A4, despite the observation that CYP3A4 catalyzed the dehydrogenation of this 3-substituted indole. Finally, an intriguing new inhibitor of tumor necrosis factor- (SPD-304) has recently been identified (He et al., 2005). It also contains a 3-alkylindole group, and we have determined that it also is dehydrogenated by CYP3A4 to its electrophilic product that inactivates the enzyme in a mechanism-based manner (Sun and Yost, 2008). Thus, these multiple examples provide convincing evidence that the 3-alkylindole functional moiety is a potential "hot spot" to avoid when new drug entities are developed for therapeutic purposes.
The mechanisms that govern substrate binding and catalysis leading to the unusually specific dehydrogenation of 3MI by CYP2F1 and CYP2F3 are not well understood. Previous studies with CYP2F1 have shown that formation of the dehydrogenated product, 3-methyleneindolenine, is not linear with time. In addition, concentrations of 3MI >200 µM strongly inhibited formation of the dehydrogenated product (Lanza and Yost, 2001), suggesting unusual kinetics in 3MI turnover by the CYP2F1/CYP2F3 enzymes. The purpose of this study was, therefore, to determine whether indeed 3MI irreversibly inactivates CYP2F1/CYP2F3 in a time- and concentration-dependent manner. Kinetic studies confirmed that 3MI caused time- and concentration-dependent inactivation, and catalytic turnover of 3MI was a prerequisite for the inactivation. In a 30-min preincubation with 3MI, CYP2F3 lost approximately 80% of the CHZX deethylase activity, and inactivation reached saturation at a concentration of approximately 300 µM. The partition ratio P is an indicator of the efficiency of a compound as a mechanism-based inactivator. Mechanism-based inactivators having P values ranging from 1 to values >1000 (inefficient) have been reported (Kent et al., 2001). The partition ratio for inactivation of CYP2F3 by 3MI was estimated to be 220. The partition ratio along with the kinetic constants (KI = 160 µM and kinact = 0.09 min-1) suggested that 3MI is a slow but relatively efficient inactivator of CYP2F3. The addition of exogenous nucleophiles such as GSH did not alter the amount of inactivation, suggesting that the reactive intermediate formed irreversibly inactivates the enzyme by covalently binding to active site residues and does not diffuse out before inactivation events take place.
The protection of 3MI-mediated inactivation of CYP2F3 by the addition of an alternate substrate in the preincubation was investigated. Both CHZX and EC protected CYP2F3 from 3MI inactivation, putatively by competition with 3MI for binding at the active site. Irreversibility of the inactivation is another important criterion for determining whether a compound is a true mechanism-based inactivator. Overnight dialysis of a sample of 2F3 that was 70% inactivated did not bring about significant recovery of CHZX hydroxylase activity (less than 15%). Because removal of the excess inactivator did not bring about recovery of activity, the mechanism of inactivation appears to occur by the covalent attachment of the reactive species to the enzyme active site. Furthermore, addition of fresh reductase did not cause recovery of activity, suggesting that the reductase was the not the site of inactivation (data not shown). These data support the conclusion that 3MI is indeed a mechanism-based inactivator of CYP2F3.
As expected, the inactivation kinetics of CYP2F1 with 3MI were similar to those of CYP2F3. In the presence of NADPH, CYP2F1 lost approximately 68% of its 7-ethoxycoumarin O-deethylase activity. The kinetic constants, KI and kinact for the inactivation of CYP2F1 by 3MI were 49 µM and 0.04/min, respectively, and the partition ratio for 3MI-mediated inactivation of CYP2F1 was approximately 280. The exogenous nucleophile glutathione did not protect CYP2F1 from inactivation. Thus, the human CYP2F1 enzyme had very similar 3MI-mediated inactivation properties when compared with goat lung CYP2F3.
We have not successfully expressed CYP2F1 in E. coli, so this enzyme was purchased from BD Gentest. The enzyme was expressed in small quantities in microsomal fractions from lymphoblast cells. Therefore, only limited quantities of CYP2F1 were available, and we did not have enough enzyme to perform additional studies to measure inactivation reversibility or alternate substrate protection.
Mechanism-based inactivators can sometimes irreversibly inactivate the enzyme by arylating or alkylating the prosthetic heme group and cause destruction of the heme group, leading to heme-derived products that covalently modify the P450 apoprotein (Osawa and Pohl, 1989). Some inactivators such as 17-ethynylestradiol bind to both the heme prosthetic group and to the apoprotein (Lin and Hollenberg, 2007). To determine the site of adduction, spectral analysis of 3MI-inactivated 2F3 was performed, but a decrease in CO binding, concurrent with the loss in enzymatic activity, was not observed, suggesting that CYP2F3 heme alkylation or destruction by 3MI was not the mechanism of inactivation. Conversely, covalent bond formation with the apoprotein was apparently responsible for 3MI-mediated irreversible inactivation of CYP2F3. We tried a number of times to identify the site of 3MI adduct formation on CYP2F3, using prototypical (Lin et al., 2005) mass spectral analysis of the apoprotein or liquid chromatography/mass spectrometry analysis of peptide fragments from trypsin or CNBr protein cleavage methods. We successfully characterized the intact protein and a reasonable number (70% coverage) of its CNBr-treated peptides but were not able to characterize 3MI adducts to the enzyme.
In conclusion, these studies have shown that 3MI inactivates both CYP2F3 and CYP2F1 in a time- and concentration-dependent manner and the irreversible inactivation probably occurs through modification of active site residues. It is interesting to speculate that when human lung cells are exposed to reasonably high amounts of 3MI by chronic cigarette smoking, the enzyme that is a highly specific catalyst of the DNA-binding intermediate, 3-methyleneindolenine, might be essentially inactive. This process could contribute to the incongruous observation that many heavy smokers never develop lung cancer, because CYP2F1 would not be able to form the ultimate carcinogenic intermediates from 3MI or other cigarette smoke carcinogens.
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Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.
ABBREVIATIONS: P450, cytochrome P450; 3MI, 3-methylindole; CHZX, chlorzoxazone; LB, Luria-Bertani; TB, Terrific Broth; EC, 7-ethoxycoumarin; GSH, glutathione; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; L-745,870, 3-{[4-(4-chlorophenyl)piperazin-1-yl]-methyl}-1H-pyrrolo-2,3-β-pyridine; MK-0524, (3R)-4-(4-chlorobenzyl)-7-fluoro-5-(methylsulfonyl)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl acetic acid; SPD-304, 6,7-dimethyl-3-{[methyl-(2-{methyl-[1-(3-trifluoromethyl-phenyl)-1H-indol-3-ylmethyl]-amino}-ethyl)-amino]-methyl}-chromen-4-one.
Address correspondence to: Dr. Garold S. Yost, Department of Pharmacology and Toxicology, 30 South 2000 East, Room 201, University of Utah, Salt Lake City, UT 84112-5820. E-mail: gyost{at}pharm.utah.edu
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-hydroxylase in Escherichia coli. Proc Natl Acad Sci U S A 88: 5597-5601.. Science 310: 1022-1025., β-unsaturated iminium species that results in the selective mechanism-based inactivation of the enzyme. Chem Res Toxicol 18: 1427-1437.[CrossRef][Medline]-ethynylestradiol is cytochrome b5-dependent: metabolic activation of the ethynyl moiety leads to the formation of glutathione conjugates, a heme adduct, and covalent binding to the apoprotein. J Pharmacol Exp Ther 321: 276-287.
4-VPA, a toxic metabolite of valproic acid. Science 235: 890-893. inhibitor: dehydrogenation and inactivation of CYP3A4. Chem Res Toxicol, in press.
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), 50 (
), 100 (
), 150 (x), and 250 (
) µM. Control incubations were done in the presence of NADPH, but without 3MI, and the activity was 8.5 nmol/min/nmol P450. The data points correspond to the mean of three separate experiments. The slopes of the lines represent the observed first-order rate constants (kobs) of the inactivation reaction at a given 3MI concentration. The inset shows the double-reciprocal Kitz-Wilson plot for CYP2F3 inactivation. The KI (obtained from the x-intercept) was found to be 160 µM, and the maximal rate of inactivation kinact (obtained from the y-intercept) was 0.09/min.
), and (
) 100 µM. In incubations with 70 and 100 µM 3MI, saturation of inactivation was observed beyond the 15-min point. Hence, data points beyond 15 min were excluded when the kobs was calculated. The inset shows the double-reciprocal Kitz-Wilson plot for CYP2F1 inactivation. The KI (obtained from the x-intercept) was found to be 49 µM and the maximal rate of inactivation kinact (obtained from the y-intercept), was 0.025/min
280.