Effects of Eleven Isothiocyanates on P450 2A6- and 2A13-Catalyzed

Aug 3, 2007 - Two of these 11 ITCs, phenylpropyl isothiocyanate (PPITC) and phenylhexyl isothiocyanate (PHITC), were potent inhibitors of P450 2A13...
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Chem. Res. Toxicol. 2007, 20, 1252–1259

Effects of Eleven Isothiocyanates on P450 2A6- and 2A13-Catalyzed Coumarin 7-Hydroxylation Linda B. von Weymarn,† Jamie A. Chun, Gabriel A. Knudsen, and Paul F. Hollenberg* Department of Pharmacology, UniVersity of Michigan, Ann Arbor, Michigan 48109-0632 ReceiVed March 12, 2007

Many isothiocyanates (ITCs), both naturally occurring and synthetic, are potent and selective inhibitors of carcinogenesis in animal models and are now viewed as a class of promising chemopreventive agents. We have investigated the ability of 11 ITCs to inhibit and/or inactivate P450 2A6- and 2A13-mediated coumarin 7-hydroxylation. Two of these 11 ITCs, phenylpropyl isothiocyanate (PPITC) and phenylhexyl isothiocyanate (PHITC), were potent inhibitors of P450 2A13. The KI values for the inhibition of P450 2A13-mediated coumarin 7-hydroxylation by PPITC and PHITC were approximately 0.14 and 1.1 µM, respectively. P450 2A6 was also inhibited by these two ITCs; however, the KI values indicated they were approximately 10–20-fold less potent for P450 2A6 than for P450 2A13. Most of the ITCs tested, including PPITC and PHITC, showed some degree of inactivation of both P450s; however, only one compound, tert-butyl isothiocyanate (tBITC), showed significant inactivation of P450 2A13 at a concentration of 10 µM. None of the ITCs caused significant inactivation of P450 2A6 at this concentration. tBITC inactivated P450 2A13 with an apparent KI of 4.3 µM and a kinact of 0.94 min-1. Inactivation of P450 2A6 by tBITC was observed only at high concentrations and long incubation times. The observed differences in inhibition and/or inactivation of P450 2A6 and 2A13 by a few of the isothiocyanates suggest that these compounds may be useful for structure–function studies. Introduction Many isothiocyanates (ITCs), both naturally occurring and synthetic, are potent and selective inhibitors of carcinogenesis in animal models and are now viewed as a class of promising chemopreventive agents (reviewed in ref 1). The naturally occurring ITCs are found in high levels in cruciferous vegetables where they occur as thioglucoside conjugates which upon chewing or maceration are hydrolyzed by the enzyme myrosinase to release the isothiocyanate (2, 3). The chemopreventive properties of ITCs are thought to be due in part to the direct inhibition of the P450 enzymes involved in the metabolic activation of many chemical carcinogens. The ITCs can inhibit P450 activity by three different mechanisms: (1) by competitive inhibition, (2) by reacting directly with one or more nucleophilic amino acids crucial for P450 catalysis, and (3) through mechanism-based inactivation of the enzyme (4–9). Different ITCs have been shown to be mechanism-based inactivators of several rat (1A1, 1A2, 2B1, and 2E1), rabbit (2E1), and human (2B6 and 2D6) P450s (5, 6, 8, 9). Most ITCs inactivate the P450s through the formation of covalent adducts to the protein. Therefore, in addition to being potential chemopreventive agents, these compounds may also be very useful for determining structural features of the active site involved in catalytic turnover and substrate specificity. Several members of the P450 2A subfamily of enzymes efficiently catalyze the metabolic activation of a number of carcinogenic nitrosamines in Vitro (10–14). These P450s have been shown to play significant roles in the in ViVo metabolic * To whom correspondence should be addressed: Department of Pharmacology, 2301 Medical Science Research Building III, 1150 W. Medical Center Dr., Ann Arbor, MI 48109-0632. Telephone: (734) 7648166. Fax: (734) 763-5387. E-mail: [email protected]. † Current address: Cancer Center, University of Minnesota, Minneapolis, MN 55455.

activation of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in human lung and liver, as well as in mouse lung. There are also indications that P450 2A enzymes play a major role in the metabolism of N′-nitrosonornicotine, another tobaccospecific nitrosamine, in both rats and humans (12, 15). There are three members of the human P450 2A gene family (16): P450 2A6, a hepatic coumarin 7-hydroxylase (17); P450 2A13, an extrahepatic P450 (14); and P450 2A7, which has been reported to be a nonfunctional enzyme (18). P450 2A6 constitutes approximately 1–10% of the total P450 content in human liver and is a specific and efficient coumarin 7-hydroxylase (19). With regard to smoking behavior, P450 2A6 is the primary enzyme responsible for the metabolism of nicotine to its inactive metabolite cotinine in ViVo (20–22). P450 2A13 is 94% identical in its sequence to P450 2A6, differing by only 32 amino acids (14, 23). P450 2A13 was originally cloned from human nasal mucosa. It is a functional enzyme that metabolizes several P450 2A6 substrates, including coumarin, NNK, and N,N-dimethylaniline; however, the metabolic efficiencies for the two enzymes differ significantly (14). Of potential importance for human lung cancer, NNK is metabolized to potentially carcinogenic intermediates much more efficiently by P450 2A13 than by P450 2A6. The Vmax/ Km for methylene hydroxylation of NNK by 2A6 is 0.008 compared to 0.36 for 2A13 (14). The metabolism of nicotine and its metabolite cotinine by these two enzymes also differs significantly. The Km for nicotine 5′-oxidation by P450 2A13 is 6-fold lower than that for P450 2A6, although the product distribution is very similar (24). With cotinine, both the efficiency of metabolism and the product distribution are different (25). P450 2A13 is again a more efficient catalyst of cotinine metabolism than P450 2A6. The primary product of cotinine metabolism by P450 2A13 is 5′-hydroxycotinine, whereas N-(hydroxymethyl)norcotinine is the primary product

10.1021/tx700078v CCC: $37.00  2007 American Chemical Society Published on Web 08/03/2007

Effects of Isothiocyanates on Coumarin 7-Hydroxylation

for P450 2A6. The metabolism of coumarin by P450s 2A6 and 2A13 follows a similar pattern. In this case, P450 2A6 exclusively catalyzes the hydroxylation at the 7 position of coumarin, whereas P450 2A13 catalyzes both 7-hydroxylation and 3,4-epoxidation (26). In addition, we have previously demonstrated that P450 2A13 is a testosterone 15R-hydroxylase, whereas P450 2A6 does not catalyze this reaction (27). Although these two enzymes are highly homologous and have overlapping substrate specificity, there seems to be a significant difference in the metabolic efficiency and preferential site of metabolism. In this study, we utilize expressed and purified human P450s 2A6 and 2A13 to screen 11 ITCs, several of them structurally related, for their ability to inhibit P450 2A6- and 2A13-mediated coumarin 7-hydroxylation as well as their ability to act as inactivators of these two enzymes. The results of this study both evaluate these different isothiocyanates for their potential as chemopreventive agents for smokers and give some insights into the structural characteristics that influence the efficiency and specificity of inhibition and/or inactivation of the human P450 2A enzymes.

Materials and Methods Materials. Dilauroyl-L-R-phosphatidylcholine (DLPC), NADPH, bovine serum albumin (BSA), coumarin, 7-hydroxycoumarin (7OHC), and catalase were purchased from Sigma Chemical Co. (St. Louis, MO). All isothiocyanates were purchased from Trans World Chemicals, Inc. (Rockville, MD), except for PPITC and PHITC, which were a kind gift from G. Stoner of the The Ohio State University (Columbus, OH). All other reagents were of analytical grade and purchased from Sigma Chemical Co. Enzyme Expression and Purification. The cDNA for P450 2A13 was a kind gift from X. Ding (Wadsworth Center, New York State Department of Health, Albany, NY), and the expression vector containing His-tagged P450 2A6 was a kind gift from F. Guengerich (Vanderbilt University, Nashville, TN). Expression of P450 2A6 in DH5R cells was accomplished using essentially the same protocol used previously for the expression of P450 2A6 and P450 3A4 (28, 29). P450 2A13 was expressed in C41(DE3) cells and purified according to previously published protocols (30). The NADPHP450 oxidoreductase (reductase) was expressed in Escherichia coli Topp 3 cells and purified according to a previously published protocol (31, 32). Inhibition of P450 2A6- and 2A13-Catalyzed 7-Hydroxylation of Coumarin. P450 2A6 or P450 2A13 was reconstituted with reductase and lipid (DLPC) for 45 min at 4 °C (27). After the reconstitution, catalase and Tris buffer were added to the reconstituted enzymes to give an incubation mixture containing 1 pmol of P450/µL, 2 pmol of reductase/µL, 0.1 µg of lipid/µL, 26 units of catalase/µL, and 50 mM Tris buffer (pH 7.4). The molar ratio of P450 to reductase was 1:2 unless otherwise noted. Aliquots of the reconstituted enzyme solution [containing 5 pmol of P450 (5 µL)] were added to reaction mixtures containing coumarin (20 µM), ITC (0, 0.2, or 2 µM), and NADPH (0.2 mM) in 50 mM Tris buffer (pH 7.4). The final reaction volume was 300 µL. After a 10 min incubation at 30 °C, the reaction was terminated by the addition of 20 µL of 15% trichloroacetic acid (TCA). The 7-OHC formed was analyzed using reverse phase HPLC with fluorescence detection (excitation wavelength, 350 nm; and emission wavelength, 453 nm) as previously described (33). 7-OHC was eluted isocratically with a 65% H2O/34% methanol/1% acetic acid mixture using a Varian Microsorb-MV C18, 5 µm, 100 Å column (250 mm × 4.6 mm). The flow rate was 0.8 mL/min. Quantitation was achieved by comparison to a standard curve of picomoles of 7-OHC versus peak area. For the kinetics experiments, aliquots of the reconstituted enzyme solution were added to reaction mixtures containing coumarin (0–20 µM), PPITC (0, 1, 3, or 9 µM for P450 2A6 and 0, 0.1, 0.5, or 1 µM for P450 2A13) or PHITC (0, 6, 12, or 24 µM for P450 2A6 or 0, 0.5, 1, and 3 µM for P450 2A13), and NADPH

Chem. Res. Toxicol., Vol. 20, No. 9, 2007 1253 (0.2 mM) in 50 mM Tris buffer (pH 7.4). Km, Vmax, and KI values were determined using the Ez-Fit 7 kinetics program from Perrella Scientific (Amherst, NH) (33). This program uses nonlinear regression to calculate kinetic constants. Inactivation of P450 2A6 and P450 2A13. P450 2A6 or 2A13 was reconstituted with reductase and lipid for 45 min at 4 °C. The primary reaction mixture contained 1 pmol of P450/µL, 2 pmol of reductase/µL, 0.1 µg of lipid/µL, 26 units of catalase/µL, ITC (0, 10, or 100 µM), and 1 mM NADPH in 50 mM Tris buffer (pH 7.4). The primary reaction mixture was incubated for 5 min at 30 °C prior to the addition of NADPH. Aliquots were removed from the primary reactions (5 µL ) 5 pmol of P450) at 0 and 10 min following NADPH addition and were diluted into a secondary reaction mixture containing coumarin (20 µM) and NADPH (0.2 mM) in 50 mM Tris buffer (pH 7.4). The coumarin 7-hydroxylation activity remaining was determined as described above. For the tBITC kinetics experiments, the primary reaction mixture contained 0.5 pmol of P450/µL,1 pmol of reductase/µL, 0.1 µg of lipid/µL, 26 units of catalase/µL, tBITC (0, 2.5, 5, 7.5, or 10 µM), and 1 mM NADPH in 50 mM Tris buffer (pH 7.4), and 5 µL aliquots (2.5 pmol of P450) were transferred into the secondary reaction mixtures at different time points (0, 0.5, 1, 1.5, 2, and 3 min) following addition of NADPH. Inactivation of P450 2A6 by tBITC was performed as described for the tBITC kinetics experiment except 0, 50, 75, or 100 µM tBITC was used and aliquots were transferred at 16 and 30 min. Due to the large amount of competitive inhibition by PPITC and PHITC, an aliquot of each sample (control and 10 and 100 µM ITC) was put through a G50 Sephadex spin column to remove the ITCs, and the flow-through from the spin column was then assayed for the amount of coumarin 7-hydroxylation activity remaining. Computer Modeling and Docking. X-ray crystal structure coordinates for P450 2A13 (PDB entry 2P85) and P450 2A6 (PDB entry 1Z10) were used to model P450 2A13 and P450 2A6 in MAESTRO (version 3.5, Schrodinger, LLC, New York, NY). Isothiocyanate ligands (PPITC and tBITC) were generated and minimized in MAESTRO prior to docking. Docking studies were conducted using GLIDE within MAESTRO to generate the top 20 possible binding orientations (out of 10 000) for the ligands within the active sites of P450 2A13 and P450 2A6. Substrates were allowed to dock flexibly within the active sites.

Results The effects of 11 different ITCs on the catalytic activities of P450s 2A6 and 2A13 that had been expressed and purified from E. coli were determined using coumarin as the probe substrate. The structures of the 11 ITCs that were screened for their ability to inhibit P450 2A6- and 2A13-mediated coumarin 7-hydroxylation are shown in Figure 1. One of the ITCs, IPrITC, is present in cruciferous vegetables; the others are synthetic (1). The 11 ITCs fall into three general categories. Two ITCs contain a phenyl ring with alkyl chains of different lengths; four ITCs contain two benzene rings connected by different linkers, and the remaining ITCs are straight and branched alkyl chains of different lengths. Of the 11 ITCs that were screened, only PPITC resulted in significant (∼30%) inhibition of the P450 2A6-mediated coumarin hydroxylation at a concentration of 0.2 µM (Table 1). At an ITC concentration of 2 µM, six ITCs (PPITC, NITC, BiPylITC, PrITC, IBuITC, and CITC) inhibited the P450 2A6 activity by more than 30% (Table 1). PPITC and PHITC, the two ITCs that contained a single phenyl group, both caused significant inhibition (>40%) of the P450 2A13-catalyzed coumarin 7-hydroxylation at the low (0.2 µM) concentration of ITC. Five ITCs (PPITC, PHITC, NITC, BiPylITC, and tBITC) inhibited P450 2A13 by g40% at a concentration of 2 µM (Table 1). PPITC, NITC, and BiPylITC were the only ITCs

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Figure 1. Structures of the 11 isothiocyanates.

Table 1. Inhibition of P450 2A6- and 2A13-Mediated Coumarin 7-Hydroxylation by Various Isothiocyanatesa % activityb P450 2A6 PPITC PHITC NITC BHyITC DPEITC BiPylITC PrITC IPrITC IBITC tBITC CITC

P450 2A13

0.2 µM

2 µM

0.2 µM

2 µM

72 ( 10 101 ( 9 92 ( 8 84 ( 9 94 ( 10 95 ( 10 103 ( 11 108 ( 13 87 ( 8 93 ( 8 93 ( 13

37 ( 8 77 ( 5 63 ( 7 75 ( 6 82 ( 8 61 ( 9 59 ( 8 92 ( 11 68 ( 8 73 ( 8 50 ( 6

49 ( 6 57 ( 9 102 ( 11 115 ( 5 109 ( 6 101 ( 2 110 ( 19 95 ( 11 114 ( 7 108 ( 5 110 ( 16

9(5 12 ( 7 15 ( 3 98 ( 13 82 ( 8 62 ( 7 111 ( 5 88 ( 7 106 ( 12 57 ( 17 115 ( 11

a Samples were prepared as described in Materials and Methods. Reconstituted P450 2A6 or 2A13 (5 pmol) was incubated with 20 µM coumarin in the presence of 0, 0.2, or 2 µM isothiocyanate. The rate of coumarin 7-hydroxylation was determined in duplicate in three separate experiments. b The percent activity was calculated on the basis of control samples with no isothiocyanate present.

that significantly inhibited both P450s at 2 µM. PHITC and tBITC inhibited P450 2A13 by >30% but not P450 2A6, and PrICT, IBuITC, and CITC inhibited P450 2A6 but not P450 2A13. The inhibition of P450s 2A6 and 2A13 by the two most potent ITCs from the initial screen, PPITC and PHITC, was investigated further. The kinetic parameters for the inhibition of P450 2A6- and 2A13-catalyzed coumarin 7-hydroxylation were determined (Figure 2). The KI values for the inhibition of P450 2A6-mediated coumarin 7-hydroxylation by PPITC and PHITC were 2.6 and 19.9 µM, respectively (Figure 2A,B). The mode of inhibition for both compounds was noncompetitive. The KI

values for P450 2A13 were 0.1 µM with PPITC and 1.1 µM with PHITC (Figure 2C,D), and the inhibition was also noncompetitive. The abilities of the 11 ITCs to cause concentration-, NADPH-, and time-dependent losses in P450 2A6- and 2A13-mediated coumarin 7-hydroxylation were investigated. The results from the screen for NADPH- and concentration-dependent activity loss for nine of the 11 ITCs are listed in Table 2. The inactivation experiments were performed at 30 °C to minimize activity loss in the control samples due to the futile cycle. Significant inhibition by PPITC and PHITC of the coumarin hydroxylase activity in the secondary reaction mixture was observed for both P450 enzymes when using the standard technique for measuring mechanism-based inactivation to determine if these compounds are able to inactivate P450s 2A6 and 2A13 (data not shown), and therefore, these two compounds were not included in Table 2. The low KI values for the inhibition of P450 2A6- and 2A13-catalyzed coumarin 7-hydroxylation by PPITC and PHITC might be expected to mask any mechanism-based type of inactivation. Therefore, an alternate method had to be used. To determine whether PPITC and/or PHITC could inactivate P450s 2A6 and 2A13 in a timeand NADPH-dependent manner, we used a spin column to remove the ITCs, which have relatively low molecular weights, after incubation of the reconstituted P450 with the ITCs in the presence or absence of NADPH. Losses in activity were observed in both the exposed control samples and the inactive samples prior to the samples being passed through the spin columns (Table 3). However, as shown in Table 3, the loss in P450 2A6-catalyzed coumarin 7-hydroxylation activity was greater in the presence of NADPH (79% for PPITC, 64% for PHITC) than in its absence (46% for PPITC, 20% for PHITC). Similar results were observed with P450 2A13, although the loss in activity in the absence of NADPH before the spin column was significantly greater for P450 2A13 than for 2A6, consistent with the lower KI values for P450 2A13 with both ITCs. Following elution of the exposed control sample (+ITC, –NADPH) from the spin column, the ability of both P450 2A6 and 2A13 to catalyze coumarin 7-hydroxylation was fully restored, whereas for the isothiocyanate-exposed samples incubated in the presence of NADPH, only slight gains in coumarin 7-hydroxylation activity were observed (Table 3). The remaining loss in activity has to be due to inactivation of the enzymes since after the spin column essentially no free ITC is left in the sample to cause inhibition. These results demonstrate that PPITC and PHITC are both inhibitors and time- and NADPH-dependent inactivators of P450s 2A6 and 2A13. The other ITCs did not cause inhibition of either P450 2A6 or P450 2A13 in the secondary reaction (data not shown); therefore, the standard approach for assessing inactivation was used. Of the remaining nine ITCs only one, BiPylITC, inactivated P450 2A6 by g30% when incubated for 10 min at a concentration of 10 µM (Table 2). Increasing the concentration of the ITCs to 100 µM resulted in >30% inactivation of P450 2A6 by three additional ITCs (NITC, IBuITC, and CITC). As shown in Table 2, most of the ITCs gave rise to a greater activity loss with P450 2A13 than with P450 2A6. For example, 10 µM tBITC inactivated P450 2A13 by 72% compared to 18% for P450 2A6, and DPEITC (10 µM) resulted in approximately 30% inactivation of P450 2A13 and no inactivation of P450 2A6 following a 10 min incubation. At a concentration of 100 µM, all but two ITCs, PrITC and CITC, inactivated P450 2A13 by at least 50% following 10 min incubations. Only two ITCs,

Effects of Isothiocyanates on Coumarin 7-Hydroxylation

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Figure 2. Inhibition of P450 2A6- and 2A13-catalyzed 7-hydroxylation of coumarin by PPITC and PHITC. The inhibition of P450 2A6- and 2A13-catalyzed coumarin 7-hydroxylation was assessed as described in Materials and Methods. (A) Inhibition of P450 2A6 activity by 0 (9), 1.0 (2), 3.0 (1), and 9.0 µM PPITC ([). (B) Inhibition of P450 2A6 by 0 (9), 6 (2), 12.0 (1), and 24.0 µM PHITC ([). (C) Inhibition of P405 2A13 by 0 (9), 0.1 (2), 0.5 (1), and 1.0 µM PPITC ([). (D) Inhibition of P450 2A13 by 0 (9), 0.5 (2), 1.0 (1), and 3.0 µM PHITC ([). The curves were generated using nonlinear regression analysis. The data represent the mean and standard deviations from three experiments carried out in duplicate.

Table 2. Inactivation of P450s 2A6 and 2A13 by Different Isothiocyanatesa % activity remainingb P450 2A6 NITC BHyITC DPEITC BiPylITC PrITC IPrITC IBITC tBITC CITC

P450 2A13

10 µM

100 µM

10 µM

100 µM

95 ( 7 114 ( 13 113 ( 10 70 ( 8 82 ( 5 101 ( 1 89 ( 11 82 ( 9 93 ( 7

53 ( 4 89 ( 6 78 ( 7 42 ( 3 72 ( 10 76 ( 9 69 ( 5 80 ( 14 56 ( 7

103 ( 12 88 ( 6 70 ( 9 81 ( 9 90 ( 8 94 ( 13 82 ( 3 28 ( 3 101 ( 11

52 ( 11 40 ( 3 28 ( 9 38 ( 2 94 ( 2 41 ( 4 51 ( 3 18 ( 5 92 ( 10

a Samples were prepared as described in Materials and Methods. Control samples and those containing isothiocyanates (10 or 100 µM) were incubated for 10 min at 30 °C in the presence or absence of NADPH prior to assaying for the remaining coumarin 7-hydroxylation activity. All the values are in the presence of NADPH. No activity loss was observed with any of the ITCs in the absence of NADPH. Each value is the average from three experiments that were carried out in duplicate. b The percent activity remaining was calculated on the basis of the 0 min time point for each concentration of isothiocyanate.

PrITC and CITC, were better inactivators of P450 2A6 than of P450 2A13 (Table 2). The ability of tBITC to inactivate P450s 2A6 and 2A13 differs significantly. tBITC inactivates P450 2A13 by 72% at a concentration of 10 µM but does not show any significant inactivation of P450 2A6 even at a concentration of 100 µM.

To further characterize the differences in inactivation of these two enzymes by tBITC, we determined the kinetics of inactivation. tBITC appears to be a very potent inactivator of P450 2A13 but not P450 2A6. We had to resort to 30 s time points at relatively low tBITC concentrations to be able to gain kinetic information with P450 2A13. These time points were still not sufficiently short to obtain linear activity loss past the first 1.5 min of inactivation. Therefore, the values reported here are estimates. The kinetic parameters were estimated from the slopes of the lines when the logarithm of the percent activity remaining (coumarin) was plotted against time (Figure 3, inset). Because of the nonlinearity, the kinetic parameters were estimated using only the first four time points. The rate of inactivation at a saturating tBITC concentration, kinact, was 0.94 min-1, and the t1/2 was 0.73 min. The KI was estimated to be 4.3 µM. The kinetics for the inactivation of P450 2A6 by tBITC was not determined since no measurable inactivation was observed at concentrations as high as 50 µM at times as long as 30 min (data not shown). Some time- and concentration-dependent inactivation was observed at tBITC concentrations of g75 µM at times longer than 16 min (data not shown). The crystal structures of both P450s 2A6 and 2A13 have recently been published (34, 35), and the coordinates for the structures are available online in the RCSB Protein Data Bank. We docked PPITC, the most potent inhibitor of the ITCs that we tested, into the active sites of both enzymes using homology modeling software (Figure 4). The orientation of PPITC in the active sites of P450s 2A6 and 2A13 differed significantly. In

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1256 Chem. Res. Toxicol., Vol. 20, No. 9, 2007 Table 3. Effect of PPITC and PHITC on P450 2A6- and 2A13-Catalyzed Coumarin 7-Hydroxylationa % activity remainingb PPITC PHITC

before spin column

P450 2A6

P450 2A13

–PPITC, +NADPH +PPITC, –NADPH + PPITC, +NADPH –PHITC, +NADPH +PHITC, –NADPH +PHITC, +NADPH

85 ( 4 54 ( 9 21 ( 7c 100 ( 5 80 ( 5 36 ( 2c

82 ( 10 24 ( 2c 11 ( 1c 92 ( 7 29 ( 9c 26 ( 4c

% activity remainingb after spin column PPITC PHITC

–PPITC, +NADPH +PPITC, –NADPH +PPITC, +NADPH –PHITC, +NADPH +PHITC, –NADPH +PHITC, +NADPH

P450 2A6 100 ( 5 83 ( 13 62 ( 11c 100 ( 15 93 ( 6 57 ( 3c

P450 2A13 102 ( 7 92 ( 7 24 ( 7c 96 ( 4 86 ( 4 40 ( 2c

a Samples were prepared as described in Materials and Methods. Control samples and those containing PPITC or PHITC (100 µM) were incubated for 10 min at 30 °C in the presence or absence of NADPH. The coumarin 7-hydroxylase activity was measured before and after the samples were passed through a spin column to remove small molecules such as PPITC or PHITC and NADPH. All samples were run in duplicate (before spin column) or triplicate (after spin column) on two separate occasions. b The percent activity remaining was calculated on the basis of control samples at 0 min time points (-PPITC/PHITC, +NADPH). c p < 0.005 when compared to -ITC, +NADPH controls.

Figure 3. Inactivation of P450 2A13 by tBITC. Experimental conditions are described in Materials and Methods. The tBITC following concentrations were used: 0 (b), 2.5 (O), 5 (1), 7.5 (3), and 10 µM (9). The percent activity remaining refers to the amount of coumarin 7-hydroxylase activity remaining compared to control sample (0 µM tBITC and 0 min). The data represent the mean and standard deviation of three independent experiments. The inset represents the doublereciprocal plot generated from the slopes of the lines at the various concentrations using only the first four time points.

four of the top five most energetically favorable orientations of PPITC in the P450 2A13 active site, the methylene carbon closest to the isothiocyanate moiety was pointing toward the heme iron with both the phenyl ring and the isothiocyanate moiety pointing away from the heme (Figure 4B). In contrast, the preferred position of PPITC in the 2A6 active site in three of the five top orientations had the para position of the phenyl ring closest to the heme iron with the carbon linker curving away from the heme (Figure 4A). The two remaining orientations of PPITC in the P450 2A6 active site corresponded to the preferred position with P450 2A13.

We also docked tBITC into the active sites of both P450 2A6 and 2A13 to determine if there were any major differences in how tBITC prefers to orient in the active site that might explain the difference in inactivity that we observed with the two enzymes. The predominant orientation when comparing the top 20 orientations for tBITC in the active sites of P450 2A6 and 2A13 is shown in Figure 5. However, the small tBITC molecule could fit in the active sites in many different orientations (data not shown). With both enzymes, the isothiocyanate group was pointed toward the heme iron more often than the tert-butyl group. When the isothiocyanate moiety was facing the heme iron, the tert-butyl group preferentially pointed toward the “roof” of the active site in P450 2A6 (Figure 5A), whereas the tertbutyl group was more parallel to the heme pointing away from residue 297 in P450 2A13 (Figure 5B). P450 2A6 did adopt the orientation that is observed predominantly with P450 2A13 in three of the 20 best fit orientations, although none were among the best 10 orientations. The orientation that was predominant in P450 2A6 (Figure 5A) was not observed among the top 20 orientations for P450 2A13.

Discussion The ability of a number of ITCs to inhibit carcinogenesis in animal models is well-documented (reviewed in ref 1). The ability of a particular ITC to inhibit tumor formation in animals is dependent on the carcinogen used in the study. For example, benzyl isothiocyanate (BITC) is a potent inhibitor of lung carcinogenesis in mice exposed to benzo[a]pyrene but not in mice exposed to NNK (36, 37). Similarly, PHITC inhibits tumor formation in rat lung after exposure to NNK but increases the carcinogenicity of N-nitrosobenzylmethylamine (NBzMA) in the rat esophagus (38–42). The ability of ITCs to inhibit and/or inactivate P450-mediated metabolic activation of a variety of carcinogens is thought to be one of the most important mechanisms by which these compounds act as chemopreventive agents. The ability of an ITC to inhibit tumor formation is thought to depend on whether it is an inhibitor of the specific isozyme of P450 that is primarily responsible for the metabolic activation of the carcinogen in question in the target tissue. The human P450 enzymes 2A6 and 2A13 can metabolize a number of carcinogens in Vitro and have been proposed to be important in the bioactivation of tobacco-specific nitrosamines in ViVo. These two enzymes are highly homologous, differing by only 32 amino acids (16, 17). There is significant overlap in substrate specificity between these two enzymes, although the rates of metabolism and the product distribution often differ significantly. In this study, we have screened 11 ITCs with different structural features for their ability to inhibit and/or inactivate P450s 2A6 and 2A13 to determine if there is a particular structural characteristic that is important in determining the inhibition/inactivation potency of the ITCs. Two of the 11 ITCs screened in this study, PPITC and PHITC, are potent inhibitors of P450 2A13-mediated metabolism. Both of these ITCs contain a single phenyl ring with an alkyl linker between the phenyl ring and the isothiocyanate moiety. In the initial screen, these were the only two ITCs that showed significant inhibition at a concentration of 0.2 µM (Table 1). PPITC was a 10-fold better inhibitor of P450 2A13-mediated coumarin 7-hydroxylation activity than PHITC. The fact that these two ITCs were potent inhibitors of P450 2A13 was not surprising since we have previously reported that benzyl isothiocyanate (BITC) and phenethyl isothiocyanate (PEITC), which differ from PPITC and PHITC only in the number of carbons in the alkyl chain, are both very potent inhibitors of

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Figure 4. Docking of PPITC into the active sites of P450 2A6 (A) and P450 2A13 (B). PPITC was docked in the active sites of the two P450 enzymes as described in Materials and Methods. (A) Preferred position of PPITC in the active site of P450 2A6. (B) Preferred position of PPITC in the active site of P450 2A13. The heme moiety is shown in both panels as a reference point.

Figure 5. Docking of tBITC in the active sites of P450 2A6 (A) and P450 2A13 (B). tBITC was docked in the active sites of these two P450 enzymes as described in Materials and Methods. (A) Preferred position of tBITC in the active site of P450 2A6. (B) Preferred position of tBITC in the active site of P450 2A13. The heme moiety and Asn297 are shown in both panels as reference points.

P450 2A13 (25). With both enzymes, the ITCs with a phenyl ring and two or three carbon atoms in the linker, i.e., PEITC and PPITC, were the most potent inhibitors (Table 1 and ref 30). BITC with a one-carbon linker is significantly less potent (30), as is PHITC with a six-carbon linker. The crystal structures for both P450 2A6 and 2A13 have recently been published (34, 35). The crystal structures for the two enzymes were, as expected, very similar to each other (34). Both enzymes have relatively small and hydrophobic active sites with the active site of P450 2A13 being 15–20% larger than that of P450 2A6. The small size of the active sites could potentially explain why PHITC with its six-carbon linker is a less potent inhibitor than the phenyl ring containing ITCs with shorter linkers. The roof of the active sites, opposite the heme moiety, is lined with phenylalanines in both enzymes and is therefore highly hydrophobic (34). Interactions between the hydrophobic phenylalanines lining the active site and the phenyl groups of ITCs such as BITC, PEITC, PPITC, and PHITC could be favorable and could potentially orient these ITCs with the isothiocyanate facing the heme, an orientation that could result in desulfuration of the ITCs. P450-catalyzed desulfuration has previously been reported for BITC with P450 2B1 and for 2-naphthyl isothiocyanate in rat liver microsomes and could lead to the formation of a reactive intermediate (8, 43). PPITC, the most potent inhibitor tested in this screen, was modeled into the active site of both P450 2A6 and 2A13. The preferred orientation of PPITC in P450 2A6 was the opposite of that in P450 2A13 (Figure 4). According to our models the main site of metabolism on PPITC would be on the para position on the phenyl ring for P450 2A6 and on the propyl chain for

P450 2A13. PPITC was not readily oriented in a position where the isothiocyanate moiety was directly pointing toward the heme iron, suggesting that desulfuration might not be the primary pathway of PPITC metabolism by the P450 2A enzymes. Whether this difference in preferred orientation of PPITC in the two active sites could influence the potency of inhibition is hard to predict without complete studies of the metabolism of PPITC by these two enzymes. Two of the four ITCs that contain two phenyl rings, NITC and BiPylITC, were able to inhibit P450 2A6 and 2A13 activity by >35% (Table 1). Both of these ITCs lack a linker carbon between the two rings. The ITCs having one- and two-carbon linkers between the two phenyl rings did not significantly inhibit either P450 2A6 or P450 2A13. Neither of the ITCs containing two phenyl rings was as potent an an inhibitor as the ITCs containing only one phenyl ring. With the straight and branched alkyl chain ITCs, there seem to be some interesting differences between the two P450 enzymes. None of the alkyl chain ITCs were able to inhibit the enzymes at the low concentration, 0.2 µM. PrITC and IBITC were both able to significantly inhibit P450 2A6 at the higher concentration, 2 µM, but not P450 2A13. With regard to branching versus straight alkyl chains, there was not a clear trend for either enzyme. PrITC, which is a straight chain ITC, is an inhibitor of P450 2A6, but IPrITC, which is branched, is not. IBITC and tBITC, which are both branched but to different degrees, did not display any difference in their ability to inhibit P450 2A6 in our screen. With P450 2A13, neither PrITC nor IPrITC showed any inhibition, tBITC inhibited P450 2A13, and IBITC did not.

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Several ITCs have been shown to be fairly potent mechanismbased inactivators of a number of P450 enzymes (4–9). However, the ability of different ITCs to act as mechanismbased inactivators of enzymes from the P450 2A subfamily has not been extensively studied. The only data published on the effects of ITCs on P450 2A13 activity demonstrate that BITC and PEITC can inactivate P450 2A13 in a time- and NADPHdependent manner through formation of adducts to the apoprotein (30). There are a few reports on the effects of ITCs on P450 2A6. Nakajima et al. (9) have reported that PEITC is a competitive inhibitor of P450 2A6; however, in a previous publication (30), we reported that PEITC can also inactivate P450 2A6 but that this inactivation is masked by a very potent inhibition and therefore would not be observed when the standard experimental protocols for the study of mechanismbased inactivators are employed. Due to the potent inhibition of P450s 2A6 and 2A13 by PPITC and PHITC, all of the samples [control (–ITC, +NADPH), exposed control (+ITC, –NADPH), and inactive (+ITC, +NADPH)] were put through a spin column to remove the lowmolecular weight ITCs prior to assaying for remaining activity, hence eliminating any decrease in activity observed due to competitive inhibition. These experiments indicate that in addition to being potent inhibitors of P450 2A6- and 2A13catalyzed metabolism, both PPITC and PHITC are able to inactivate the enzymes. However, because of this potent inhibition, we were not able to determine if PPITC and PHITC are true mechanism-based inactivators of P450s 2A6 and 2A13. The most potent inactivator of P450 2A13 was tBITC (Table 2). None of the ITCs significantly inactivated P450 2A6 at the lower concentration, 10 µM. tBITC is a potent inactivator of P450 2A13. The inactivation of P450 2A13 by tBITC was very rapid even at 30 °C. At low concentrations, the inactivation was saturated after incubation for 1.5 min, and at high concentrations, the enzyme was saturated. Therefore, the KI of 4.3 µM and a t1/2 of 0.73 min reported here are just estimates. tBITC significantly inactivates P450 2A13 at very low tBITC concentrations and extremely short incubation times; however, P450 2A6 is inactivated only at very high tBITC concentrations and at very long incubation times. Docking of tBITC in the active site did not give any clear insight into why there is such a large discrepancy between P450 2A6 and 2A13 in terms of inactivation potency. The small tBITC molecule could adopt many different orientations in both active sites (data not shown). The preferred orientation for tBITC in the two active sites was different (Figure 5). In both cases, the isothiocyanate moiety was facing the heme iron more often than the tert-butyl group. The preferential orientation of tBITC in the P450 2A13 active site (Figure 5B) was also observed with P450 2A6 in three of the 20 best fit orientations. However, this orientation was the least favorable of the ones observed for P450 2A6. The orientation for tBITC in P450 2A13 that is shown in Figure 5B is favorable for oxidative desulfuration which has been implicated as a possible pathway for generating reactive intermediates (8, 43). If this orientation is important in the generation of reactive intermediates involved in the inactivation of P450 2A13 and to a minor extent P450 2A6, it could explain at least some of the differences we observe in the rates of inactivation. P450 2A6 and 2A13 are 94% identical, differing by only 32 amino acid residues, and they have somewhat overlapping substrate specificities (14, 26, 46). Further investigation of the active site amino acid residues responsible for the differences in inactivation of P450 2A6 and 2A13 by tBITC could result

in a better understanding of some of the structural features that govern the substrate specificity and metabolic efficiency of these two P450s. In addition, further studies of the inactivation of P450s 2A6 and 2A13 by these ITCs to determine if they are indeed true mechanism-based inactivators would be very useful. With more and more polymorphisms being identified in the human P450 2A enzymes (16, 47–50), it becomes increasingly important to better understand those factors governing substrate specificity and catalytic activity. Inactivators such as tBITC could prove to be very useful tools for the further study of the structure–function relationships of the P450 2A enzymes. Acknowledgment. We thank Kari Schlicht for her help with the molecular modeling and substrate docking analysis and the Supercomputing Institute at the University of Minnesota for the use of their facilities.The research described in this article was supported in part by a postdoctoral fellowship to L.B.v.W. from the Philip Morris External Research Program. The work was also supported in part by Grant CA 16954 from the National Institutes of Health.

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