Chemico-Biological Interactions 226 (2015) 23–29
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Mechanism-based inactivation of cytochrome P450 2B6 by isoimperatorin q Jiaojiao Cao a, Liwei Zheng a, Lin Ji a, Dan Lu a, Ying Peng a,⇑, Jiang Zheng b,c,⇑ a
School of Pharmacy, Shenyang Pharmaceutical University, Shenyang, Liaoning 110016, PR China Key Laboratory of Structure-Based Drug Design & Discovery of Ministry of Education, Shenyang Pharmaceutical University, Shenyang, Liaoning 110016, PR China c Center for Developmental Therapeutics, Seattle Children’s Research Institute, Division of Gastroenterology and Hepatology, Department of Pediatrics, University of Washington School of Medicine, Seattle, WA 98101, United States b
a r t i c l e
i n f o
Article history: Received 15 August 2014 Received in revised form 1 November 2014 Accepted 2 December 2014 Available online 11 December 2014 Keywords: Isoimperatorin Cytochrome P450 2B6 Mechanism-based inactivator Reactive metabolite
a b s t r a c t Isoimperatorin (IIMP), a 6,7-furanocoumarin derivative, occurs in many common medicinal herbs. Human exposure to IIMP mainly results from intake of fruits, foods and medicinal herbs. We examined the irreversible inhibitory effect of IIMP on cytochrome P450 2B6. IIMP was found to cause time-dependent inhibition of CYP2B6. In addition, the loss of CYP2B6 activity occurred in a NAPDH- and concentration-dependent manner. About 60% of activity of CYP2B6 was suppressed after its incubation with IIMP at 25 lM for 9 min. Enzyme kinetic studies were performed, kinact for IIMP was found to be 0.071 min1, and KI was 17.1 lM, respectively. Glutathione and catalase/superoxide dismutase showed little protective effects on CYP2B6 against the inactivation by IIMP. S-Mephenytoin, a substrate of CYP2B6, mildly prevented the enzyme from the inactivation induced by IIMP. The estimated partition ratio of the inactivation was approximately 211. Additionally, a c-ketoenal intermediate was identified in microsomal incubations with IIMP. CYPs 2B6, 2D6, and 1A2 were the major enzymes responsible for the metabolic activation of IIMP. In conclusion, IIMP is a mechanism-based inactivator of CYP2B6. The formation of c-ketoenal intermediate may be responsible for the enzyme inactivation. Ó 2014 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Isoimperatorin (IIMP), a linear furanocoumarin, occurs in the Apiaceae families, which include a variety of medicinal herbs, such as Angelica Dahurica and Radix Glehniae [1,2]. In addition, many Abbreviations: CE, collision energy; DMSO, dimethyl sulfoxide; EPI, enhanced product ion; GSH, glutathione; IDA, information-dependent acquisition; IIMP, isoimperatorin; LC, liquid chromatography; LC–MS/MS, liquid chromatography coupled to tandem mass spectrometry; MRM, multiple-reaction monitoring; MS, mass spectrometry; MS/MS, tandem mass spectrometry; NADPH, b-nicotinamide adenine dinucleotide 20 -phosphate reduced tetrasodium salt; RLMs, rat liver microsomes; SOD, superoxide dismutase. q This work was supported in part by the National Natural Science Foundation of China [Grant 81373471 and 81430086]. ⇑ Corresponding authors at: Center for Developmental Therapeutics, Seattle Children’s Research Institute, Division of Gastroenterology and Hepatology, Department of Pediatrics, University of Washington School of Medicine, Seattle, WA 98101, United States. Key Laboratory of Structure-Based Drug Design & Discovery of Ministry of Education, Shenyang Pharmaceutical University, Shenyang, 110016, PR China. Tel.: +1 206 884 7651; fax: +1 206 987 7660 (J. Zheng). School of Pharmacy, Shenyang Pharmaceutical University, Shenyang, 110016, PR China. Tel.: +86 24 23986361 (Y. Peng). E-mail addresses: [email protected] (Y. Peng), jiang.zheng@ seattlechildrens.org (J. Zheng).
http://dx.doi.org/10.1016/j.cbi.2014.12.009 0009-2797/Ó 2014 Elsevier Ireland Ltd. All rights reserved.
fruits and foods contain IIMP, such as citrus fruits (grapefruit and lemon), umbelliferous vegetables, and culinary herbs (parsley) [3,4]. IIMP is also included in Yuan-hu-zhi-tong (in Chinese) tablets and Huo-xiang-zheng-qi (in Chinese) aqua widely used in China for the treatment of stomachache and headache. As a bioactive component, IIMP exhibited anti-inflammatory, analgesic, anti-spasmodic, and anti-cancer activities [5–8]. It also showed inhibitory effect on b-secretase that has been considered as a valuable target for the treatment of Alzheimer’s disease [9,10]. The multiple pharmacological activities of IIMP, along with its natural abundance, have attracted much attention. Many furanocoumarins have been documented as potent inhibitors of cytochrome P450s (CYPs). Epoxybergamottin and bergapten (5-methoxypsoralen) from grapefruit were proven to inhibit CYP3A4 activity [11,12]. Psoralen and isopsoralen showed inhibitory effects on CYP1A2 [13,14]. 8-Methoxypsoralen 5-methoxypsoralen, 5-hydroxypsoralen, 8-hydroxypsoralen, and psoralen were found to be mechanism-based inactivators of CYP2B1 [15]. Psoralen and 8-methoxypsoralen were reportedly oxidized on the furan ring to form the corresponding furanoepoxides that bind to CYP2B1. We speculated that IIMP might show similar irreversible inhibitory effect on CYP2B subfamily.
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CYP2B6 has been found in liver, brain, kidney, and heart in humans [16,17]. Although CYP2B6 comprises about 0.2% of the total CYPs in human liver microsomes, it is responsible for the metabolism of more than 3% of drugs widely used in clinic [18], such as bupropion, efavirenz, methadone, ifosfamide, and cyclophosphamide, which are preferentially metabolized or stereoselectively metabolized by CYP2B6 [19–22]. To confirm the role of specific CYPs in the clearance of various drugs and to prevent drug–drug interactions induced by the inhibition of CYPs, prediction and identification of compounds that act as mechanism-based inactivators of CYPs have become an important issue in drug discovery process. The objectives of the study were to examine the irreversible inhibitory effect of IIMP on cytochrome P450 2B6, to characterize the reactive metabolites responsible for the enzyme inactivation, and to identify the P450 enzymes responsible for the metabolic activation of IIMP.
of CYP2B6. Control incubations were performed in the absence of IIMP or S-mephenytoin in parallel. 2.4. Effects of GSH and catalase/superoxide dismutase on the enzyme inactivation The primary mixtures including CYP2B6 (0.1 lM), IIMP (10 lM)), and GSH (2 mM) were preincubated at 30 °C for 3 min. The reactions were initiated by the addition of NADPH (1.0 mM) and were incubated at 30 °C for 0, 3 and 9 min. Aliquots (40 lL) were taken to the secondary incubation mixture for determining the remaining enzyme activity. In control samples, GSH was replaced by an equal volume of phosphate buffer. In a separate study, CYP2B6 was incubated with IIMP and NADPH in the presence or absence of a mixture of catalase and superoxide dismutase. The concentrations of superoxide dismutase and catalase were both 800 unit/mL. After incubation for 0, 3, and 9 min, the residual CYP2B6 activities were examined as described below.
2. Materials and methods
2.5. Partition coefficient
2.1. Chemicals and materials
To estimate the partition coefficient, IIMP was added to the primary reaction mixtures (final concentrations: 0, 1.5, 2.5, 5, 7.5, 10, 25, 50, and 150 lM) containing CYP2B6 (0.1 lM). The reactions were initiated by the addition of NADPH (1.0 mM) and incubated at 30 °C for 9 min to assure that the inactivation had proceeded to completion. Negative control incubations lacked NADPH. Aliquots (40 lL) were withdrawn from the primary reaction mixtures and transferred to the secondary incubation. The samples were analyzed as follows.
Glutathione (GSH), hexyl glutathione, bupropion, and NADPH were purchased from Sigma–Aldrich (St. Louis, MO). Isoimperatorin (IIMP) with purity of 98% was acquired from Shanghai Yuanye Biological Technology Co., Ltd. (Shanghai, China). Recombinant human CYP2B6 was purchased from BD Gentest (Woburn, MA). Rat liver microsomes (RLMs) were prepared by our lab, according to our early work [23]. All organic solvents were from Fisher Scientific (Springfield, NJ). Distilled water was purchased from Wahaha Co. Ltd (Hangzhou, China). All solvents and reagents were either analytical or HPLC grade. 2.2. Time-, concentration-, and NADPH-dependent inactivation of CYP2B6 by IIMP The composition of the primary incubation mixtures was CYP2B6 (0.1 lM), MgCl2 (3.2 mM), and IIMP at a series of concentrations (0, 2.5, 5.0, 7.5, 10, and 25 lM) in potassium phosphate buffer (pH 7.4). The total volume was 0.2 mL. To determine the requirement of metabolism for the enzyme inactivation, IIMP (25 lM) and CYP2B6 were incubated in the absence of NADPH as negative control. The primary mixture was preincubated at 30 °C for 3 min. The reactions were initiated by addition of NADPH (1.0 mM) and were incubated at 30 °C. At various time points, aliquots (40 lL) of the primary mixtures were transferred into the secondary incubation mixtures containing bupropion (100 lM) and NADPH (0.45 mM) in 0.1 M potassium phosphate buffer (pH 7.4). The secondary incubation mixtures were further incubated at 30 °C for 30 min, followed by the addition of 0.12 mL ice-cold acetonitrile containing propranolol as internal standard. After vortexed for 3 min, the mixture was centrifuged at 16,000 rpm for 10 min. The supernatant was subjected to LC–MS/MS analysis as described below. 2.3. Substrate protection Substrate protection from IIMP-induced inactivation of CYP2B6 was determined by including IIMP and S-mephenytoin (1:4) in the primary reaction mixture. The primary mixture was preincubated at 30 °C for 3 min. The reactions were initiated by the addition of NADPH (1.0 mM), and aliquots (40 lL) of the primary mixtures were transferred at 0, 3, and 9 min to the secondary incubation mixtures for the determination of bupropion hydroxylase activity
2.6. Irreversibility of enzyme inhibition The primary reaction mixture was incubated with 25 lM IIMP (inactivated sample) or without of IIMP (control sample) at 30 °C. Aliquots of the control and inactivated samples were withdrawn at 9 min and dialyzed using Slide-A-lyzer membranes (molecular mass cut off, 3,500 Da, Pierce, Rockford, IL) against 0.1 M potassium phosphate buffer (pH 7.4, 3 2 h) at 4 °C. The dialyzed samples were brought to room temperature and were added to the secondary incubation mixture for the determination of the residual enzyme activities as below. 2.7. CYP2B6 Assay CYP2B6 activity was monitored by measuring the formation of hydroxybupropion analyzed by LC–MS/MS, using an Accuore C18 column (2.1 50 mm, 2.6 lM, ThermoFisher, Pittsburgh, PA). The LC–MS/MS system consisted of AB Sciex Instruments 4000 Q-Trap mass spectrometry (Applied Biosystems, Foster City, CA) interfaced online with an ekspert ultra LC 100 system (Applied Biosystems, Foster City, CA). The mobile phases consisted of 0.1% formic acid in acetonitrile (mobile phase A) and 0.1% formic acid in water (mobile phase B). The flow rate was 0.5 mL/min, and the column temperature was maintained at 30 °C. The gradient elution was set as follow: 0–1.0 min, 10% A; 1.0–1.5 min, 10–30% A; 1.5– 4.0 min, 30% A; 4.0–5.5 min; 30–10% A; 5.5–8.0 min, 10% A. Injection volume was 5.0 lL. Quantification was performed by multiple reaction monitoring (MRM), and ion pairs of m/z 256.2 ? 238.3 for hydroxybupropion and m/z 260.7 ? 116.3 for propranolol (internal standard) were acquired in positive mode. 2.8. Reactive intermediate trapping by GSH IIMP (120 lM), rat liver microsomes (1.0 mg protein/mL), and GSH (1.0 mM) were incubated in the presence or absence of
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NADPH (1.0 mM) at 37 °C for 60 min. In a separate study, recombinant human CYPs including 1A2, 2A6, 2B6, 2C9, 2C19, 2D6, 2E1, 3A4, or 3A5 (0.1 lM for each) replaced the rat liver microsomes, and the other condition was equivalent to that for the microsomal incubations. The resulting samples were injected into LC–MS/MS for analysis. The separation of GSH conjugates was achieved on an Accuore C18 column (4.6 150 mm, 5 lM, ThermoFisher, Pittsburgh, PA). The solvent system consisted of mobile phase A (acetonitrile with 0.1% formic acid) and mobile phase B (water with 0.1% formic acid), and the flow rate was 0.8 mL/min. The column temperature was maintained at 30 °C. The gradient elution was set as follow: 0–2.0 min, 10% A; 2.0–8.0 min, 10–90% A; 8.0–10 min, 90% A; 10–13 min; 90–10% A; 13–15 min, 10% A. GSH conjugates were monitored by MRM scanning in positive mode (m/z 594.0 ? 465.0 for IIMP–GSH conjugates; and m/z 392.2 ? 246.3 for hexyl glutathione as internal standard). The informationdependent acquisition (IDA) standard was followed by triggering the enhanced product ion (EPI) scans. IDA was used to initiate acquisition of EPI spectra for ions exceeding 5000 cps with exclusion of former target ions after three occurrences for 10 s. The EPI scan was run in positive mode at a scan range for product ions from m/z 50 to 650. The EPI scanning parameters were listed as follows: scan mode = profile; step size = 0.08 Da; and scan rate = 1000 Da/s, 5 ms pause between mass ranges. 2.9. Synthesis of IIMP–GSH conjugates IIMP (2.5 mg) was dissolved in acetone (200 lL) and mixed with saturated sodium bicarbonate solution (40 lL) and Oxone (6.5 mg). The mixture was stirred for 30 min at room temperature, followed by addition of GSH (35 mg). The mixture was further stirred for 1 h at room temperature and was centrifuged. The supernatant was divided into two equal portions. One was directly analyzed by LC–MS/MS. The other was mixed with sodium borohydride (10 mg, NaBH4). The resulting mixture was gently vortexed for 5 min, followed by LC–MS/MS analysis. 3. Results 3.1. Time-, concentration- and NADPH-dependent CYP2B6 inactivation
Fig. 1. (A) Time- and concentration-dependent inactivation of CYP2B6 by IIMP. Recombinant human CYP 2B6 was incubated with IIMP at concentrations of 0 (), 2.5 (j), 5.0 (N), 7. 5( ), 10 (), and 25 lM (h) in the presence of NADPH at 30 °C for 0, 3, 6, and 9 min. (B) Wilson plot. The observed inactivation rate constant Kobs was calculated from the slope of the regression lines shown in (A). (C) NADPHdependent inactivation of P450 2B6 by IIMP. CYP2B6 was incubated with vehicle (N) and IIMP (25 lM) in the presence (j) or absence () of NADPH. Data represent the mean ± SD (n = 3).
Through measuring the amount of hydroxybupropion produced, the remaining CYP2B6 activity was monitored. As shown in Fig. 1A and C, the inactivation of CYP2B6 by IIMP was timeand concentration-dependent and required NADPH. The residual enzymatic activities at 0 min were normalized to 100% to facilitate vivid comparison of the rates of inactivation at each concentrations. The inactivation of CYP2B6 increased progressively with increasing time and concentrations of IIMP applied. About 60% of activity of CYP2B6 was suppressed after its incubation with 25 lM IIMP for 9 min at 30 °C. Nevertheless, no loss of enzyme activity was observed in the absence of IIMP or NADPH. A double-reciprocal plot (Wilson plot) of the observed rates of inactivation (Kobs) and IIMP concentrations was used to estimate the kinetic constants KI and kinact (Fig. 1B). KI equaled to 17.1 lM, and kinact was 0.071 min1. 3.2. Substrate protection
Fig. 2. Substrate protection against inactivation of CYP2B6 by IIMP. CYP2B6 was incubated with vehicle () and IIMP (10 lM) in the presence (N) or absence (j) of Smephenytoin (40 lM). Data represent the mean ± SD (n = 3).
To study substrate protection from the IIMP-induced enzyme inactivation, CYP2B6 (0.1 lM) was incubated with IIMP in the presence of S-mephenytoin (a CYP2B6 substrate) at a molar ratio of 1:4 (IIMP: S-mephenytoin) in the primary reaction mixture. Compared with samples containing IIMP without S-mephenytoin, residual
CYP2B6 activities in samples incubated with IIMP and S-Mephenytoin were 84.9 ± 1.4% and 55.8 ± 1.6% at 3 and 9 min, respectively, which were higher than the 71.4 ± 4.5% and 53.7 ± 5.3%. This indicates that the presence of S-mephenytoin slightly slowed down the CYP2B6 inactivation induced by IIMP (Fig. 2).
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superoxide dismutase (SOD), scavengers of reactive oxygen species, was included in the primary incubation mixtures. 3.4. Partition coefficient The partition ratio (P) is a measure of efficiency for inactivation, which was evaluated according to previously published method [24]. The percentage of activity remaining was plotted as a function of the IIMP/CYP2B6 molar ratio (Fig. 3). The turnover number (P + 1) was approximately 211, and the partition ratio was found to be about 210 (Fig. 3). Fig. 3. Partition ratio determination for 2B6 inactivation by IIMP. CYP2B6 was incubated with IIMP at various concentrations. The extrapolated P was determined from the X-axis intercept of regression line of the lower ratios. Data represent the mean ± SD (n = 3).
3.3. Effects of GSH and catalase/superoxide dismutase on the enzyme inactivation To evaluate the protective effect of GSH against the enzymatic inactivation induced by IIMP, the primary incubation was performed in the presence or absence of GSH (2 mM). After incubation for 9 min, the remaining CYP2B6 activities were found to be 54.1 ± 2.9% (with GSH) and 49.5 ± 4.3% (without GSH), respectively. This agent produced minor protective effect on the enzyme from inactivation. Additionally, there was only slight protection against the inactivation of CYP2B6 by IIMP, when a mixture of catalase and
3.5. Irreversibility of enzyme inhibition Irreversibility of CYP2B6 inhibition was examined by determining whether dialysis can recover the enzyme activity after exposure to IIMP. A loss of CYP2B6 activity (11.7 ± 1.7% of control) was observed after the enzyme was incubated with IIMP (25 lM) at 30 °C for 9 min. The enzyme remained inhibited (8.5 ± 0.5% of control) after dialysis, indicating that dialysis did not recover the enzyme activity. 3.6. Reactive metabolite trapping by GSH IIMP was incubated with rat liver microsomes or recombinant human P450 enzymes supplemented with glutathione (GSH) to trap the reactive metabolites. The mixtures in the presence or
Fig. 4. (A) Extracted ion (m/z 594 ? 465) chromatograms obtained from LC-Q-Trap mass spectrometry analysis of rat liver microsomal incubations containing IIMP and GSH. (B) MS/MS spectrum of IIMP–GSH conjugate generated in microsomal incubation. (C) Extracted ion (m/z 594 ? 465) chromatogram obtained from LC-Q-Trap MS analysis of chemical oxidation of IIMP trapped by GSH. (D) MS/MS spectrum of synthetic IIMP–GSH conjugate.
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reduction of the conjugate and then analyzed by LC–MS/MS. A new peak was observed at retention time of 7.19 min with [M+H]+ at m/z 596.2 (Fig. 5A), 2 Da higher than that of the conjugate observed before the reduction by sodium borohydride. 3.7. P450 Enzymes responsible for IIMP bioactivation To determine which P450 enzymes preferentially catalyze the oxidation of IIMP, recombinant human CYPs 1A2, 2A6, 2B6, 2C9, 2C19, 2D6, 2E1, 3A4, or 3A5 (0.1 lM for each) was incubated with IIMP in the presence of GSH as a trapping agent, follow by LC–MS/ MS analysis. As showed in the Fig. 6, the IIMP–GSH conjugate was assessed in the incubation with CYPs 2B6, 2D6, or 1A2, and limited amount of the GSH conjugate was found in the incubations with the other P450 enzymes. This indicates that CYPs 2B6, 2D6, and 1A2 were the major enzymes responsible for the metabolic activation of IIMP. 4. Discussion
Fig. 5. (A) Extracted ion (m/z 596 ? 467) chromatogram obtained from LC-Q-Trap MS analysis of the chemical oxidation reaction followed by reaction with NaBH4. (B) MS/MS spectrum of the reduced product.
absence of NADPH were all conducted and then analyzed by LC– MS/MS. A peak with a protonated molecular ion [M+H]+ at m/z 594 (retention time = 6.94 min) as the major GSH conjugate was observed (Fig. 4A), but no such peak was found in the incubations without NADPH (the control samples). The product ion spectrum of the conjugate obtained by MRM–EPI scanning (ion transition m/z 594/465) showed fragment ions m/z 519, 465, 245, and 177 (Fig. 4B). The product ions at m/z 519 and 465 were derived from the loss of glycine portion (-75 Da) and c-glutamyl portion (129 Da) from m/z 594, respectively. For further structural characterization, the IIMP-derived GSH conjugate was chemically synthesized by oxidation with Oxone in acetone, followed by reaction with GSH. As expected, the resulting product showed the same retention time and the same fragmentation behavior (Fig. 4C and D) as that of the conjugate generated in the microsomal reactions (Fig. 4A and B). Due to poor reaction yield, we were unable to have enough amount of the product for NMR characterization. In a separate study, the other portion of the reaction solution was mixed with sodium borohydride for
Our kinetic study clearly demonstrated that IIMP produced a time-dependent inhibition of CYP2B6. The enzyme inactivation by IIMP was also found to be concentration-dependent, and it attained saturation at the concentration of 25 lM. IIMP-induced CYP2B6 inactivation was not observed unless NADPH was supplemented in the first incubation mixtures. This indicates that IIMP itself is not an inactivator of CYP2B6 and that the enzyme inactivation by IIMP needs to be initiated through biotransformation with assistance of P450 cofactor NADPH. The irreversibility of enzyme inhibition induced by IIMP was examined by dialysis of the enzyme which was exposed to IIMP, and no recovery of enzyme activity was observed after dialysis. The results clearly demonstrate that IIMP is a mechanism-based inactivator of CYP2B6. Catalase and SOD are known to be the enzymes responsible for quenching reactive oxygen species, such as superoxide anion and hydrogen peroxide [25]. Only minor protection of enzyme inactivation by IIMP from catalase/SOD was observed, indicating that reactive oxygen species may not play a major role in CYP2B6 inactivation induced by IIMP. In addition, GSH as a nucleophilic agent showed weak protection of CYP2B6 against IIMP. This indicates in part that CYP2B6 is covalently modified by reactive metabolites of IIMP before escaping from the active site. The mildly protective effect of S-mephenytoin on CYP2B6 inactivation observed in the substrate competition experiment implies that Smephenytoin competed with IIMP binding to the active site of CYP2B6 and reduced the rate of generation of the reactive metabolites of IIMP. The critical finding of substrate protection further indicates that bioactivation of IIMP occurred in the active site of CYP2B6. The partition ratio, reflecting the efficiency of the inactivator, is defined as the number of molecules of product molecules per mole-
Fig. 6. Human P450 enzymes involved in the formation of IIMP-conjugate. IIMP was incubated with individual human recombinant P450 enzymes in the presence of NADPH and GSH. Data represent the mean ± SD (n = 3).
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Scheme 1. Proposed pathways for the formation of reactive intermediate(s) and GSH adducts during the metabolism of IIMP.
cule of enzyme inactivated. Reported P values in literatures for mechanism-based inactivators of P450 enzymes vary from 3 (very highly efficient inactivators) to >1000 (inefficient) [26]. For example, 4-ipomeanol, a furan-containing compound, that was a moderately efficient inactivator of CYP3A4 with an estimated P of 257 [27]. Therefore, the efficiency of CYP2B6 inactivation by IIMP (P value: 210) is similar to that of CYP3A4 inactivation by 4-ipomeanol. Generation of reactive metabolites is an essential step for mechanism-based enzyme inactivation. Here we propose two reactive metabolites responsible the inactivation of CYP2B6 by IIMP, and they are furanoepoxide intermediate 2 (Scheme 1) and c-ketoenal intermediate 3, and the ketoenal is also suggested to be generated by rearrangement of the furanoepoxide. To identify the structures of the reactive metabolites, we trapped the electrophilic intermediates using GSH. An IIMP–GSH conjugate with its [M+H]+ ion at m/z 594 was detected by LC–MS/MS (Fig. 4A) after incubation of IIMP in rat liver microsomes in the presence of NADPH and GSH. The observed molecular ion of m/z 594 matched the molecular weight of GSH conjugates 4–6 (Scheme 1). The MS/MS spectrum showed the indicative characteristic fragments of the GSH moiety and some other fragments valuable for metabolite identification (Fig. 4D). Ion m/z 162 responsible for molecular formula of C9H6O+3 was detected, suggesting that the 5-member ring fused with the coumarin ring was opened (Scheme 1). This could also allow us to exclude the formation of GSH conjugate 4, since the ring opening of conjugate 4 would produce a thioester. Only the structure of GSH conjugate 6 is consistent with the observed fragment of C9H6O+3, and conjugate 5 could be spontaneously converted to conjugate 6. To verify conjugate 6, we chemically reduced the conjugate with NaBH4. The reduction reaction afforded a new product with [M+H]+ at m/z 596.2 (Fig. 5A), which is higher 2.0 Da than that of the one detected before the reduction reaction. Additionally, the fragment at m/z 162 was retained in the MS/MS spectrum of the reduced GSH conjugate, indicating no reduction took place on the coumarin ring. Furthermore, the MS/MS spectrum of the original IIMP–GSH conjugate showed a fragment at m/z 177 that results from the elemental composition of C11H9O+5 of the conjugate by loss of the side chain, GSH, and CO2. In other words, fragment of m/z 177 contained the aldehyde group (Fig. 4D). No such fragment was found in the MS/MS spectrum of the reduced GSH–IIMP conjugate (Fig. 5B). Instead, a fragment of m/z 179 resulting from the reduced IIMP–GSH conjugate was detected, which was 2 Da higher than that (m/z 177) observed in the MS/MS spectrum of the original IIMP–GSH conjugate (Fig. 5B). The observation indicates that the reduction took place
on the aldehyde not on the lactone group. A separate experiment demonstrated that IIMP was resistant to the reduction by NaBH4 (data not shown). This provided the additional evidence that the NaBH4–mediated reduction of conjugate 6 gave conjugate 7. The observed conjugate 7 indicates that conjugate 6 was the primary GSH conjugate resulting from metabolic activation of IIMP. However, we were unable to tell whether conjugate 6 came from intermediate 2 or 3, since conjugate 5 might be spontaneously converted to conjugate 6, and vice versa. In other words, whether intermediate 2 or 3 was the primary metabolic intermediate remains unknown. We speculate that IIMP was oxidized to epoxides/c-ketoenal (on the furan rings), which chemically attacked the host enzyme to form covalent binding and the enzyme modification initiated the process of the enzyme inactivation. The bioactivation studies with individual recombinant enzymes demonstrated that multiple P450 enzymes catalyzed the metabolism of IIMP to the reactive intermediate (Fig. 6). CYPs 1A2, 2B6 and 2D6 were found to be the major enzyme responsible for the bioactivation of IIMP. Whether IIMP is a mechanism-based inactivator of CYPs 1A2 and 2D6 is under investigation. 5. Conclusions We have demonstrated that IIMP is enzymatically activated by CYP2B6 and is a characterized mechanism-based inactivator of CYP2B6. A GSH conjugate derived from c-ketoenal intermediate was identified in microsomal incubations with IIMP trapped with GSH. The electrophilic intermediate is possibly responsible for the enzyme inactivation. Conflict of Interest None. Transparency Document The Transparency document associated with this article can be found in the online version. References [1] N.I. Baek, E.M. Ahn, H.Y. Kim, Y.D. Perk, Furanocoumarins from the root of Angelica dahurica, Arch. Pharm. Res. 23 (2000) 467–470. [2] A. Satoh, Y. Narita, N. Endo, H. Nishimura, Potent allelochemical falcalindiol from Glehnia littoralis F. Schm, Biosci. Biotechnol. Biochem. 60 (1996) 152–153.
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Chemico-Biological Interactions journal homepage: www.elsevier.com/locate/chembioint
Mechanism-based inactivation of cytochrome P450 2B6 by isoimperatorin q Jiaojiao Cao a, Liwei Zheng a, Lin Ji a, Dan Lu a, Ying Peng a,⇑, Jiang Zheng b,c,⇑ a
School of Pharmacy, Shenyang Pharmaceutical University, Shenyang, Liaoning 110016, PR China Key Laboratory of Structure-Based Drug Design & Discovery of Ministry of Education, Shenyang Pharmaceutical University, Shenyang, Liaoning 110016, PR China c Center for Developmental Therapeutics, Seattle Children’s Research Institute, Division of Gastroenterology and Hepatology, Department of Pediatrics, University of Washington School of Medicine, Seattle, WA 98101, United States b
a r t i c l e
i n f o
Article history: Received 15 August 2014 Received in revised form 1 November 2014 Accepted 2 December 2014 Available online 11 December 2014 Keywords: Isoimperatorin Cytochrome P450 2B6 Mechanism-based inactivator Reactive metabolite
a b s t r a c t Isoimperatorin (IIMP), a 6,7-furanocoumarin derivative, occurs in many common medicinal herbs. Human exposure to IIMP mainly results from intake of fruits, foods and medicinal herbs. We examined the irreversible inhibitory effect of IIMP on cytochrome P450 2B6. IIMP was found to cause time-dependent inhibition of CYP2B6. In addition, the loss of CYP2B6 activity occurred in a NAPDH- and concentration-dependent manner. About 60% of activity of CYP2B6 was suppressed after its incubation with IIMP at 25 lM for 9 min. Enzyme kinetic studies were performed, kinact for IIMP was found to be 0.071 min1, and KI was 17.1 lM, respectively. Glutathione and catalase/superoxide dismutase showed little protective effects on CYP2B6 against the inactivation by IIMP. S-Mephenytoin, a substrate of CYP2B6, mildly prevented the enzyme from the inactivation induced by IIMP. The estimated partition ratio of the inactivation was approximately 211. Additionally, a c-ketoenal intermediate was identified in microsomal incubations with IIMP. CYPs 2B6, 2D6, and 1A2 were the major enzymes responsible for the metabolic activation of IIMP. In conclusion, IIMP is a mechanism-based inactivator of CYP2B6. The formation of c-ketoenal intermediate may be responsible for the enzyme inactivation. Ó 2014 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Isoimperatorin (IIMP), a linear furanocoumarin, occurs in the Apiaceae families, which include a variety of medicinal herbs, such as Angelica Dahurica and Radix Glehniae [1,2]. In addition, many Abbreviations: CE, collision energy; DMSO, dimethyl sulfoxide; EPI, enhanced product ion; GSH, glutathione; IDA, information-dependent acquisition; IIMP, isoimperatorin; LC, liquid chromatography; LC–MS/MS, liquid chromatography coupled to tandem mass spectrometry; MRM, multiple-reaction monitoring; MS, mass spectrometry; MS/MS, tandem mass spectrometry; NADPH, b-nicotinamide adenine dinucleotide 20 -phosphate reduced tetrasodium salt; RLMs, rat liver microsomes; SOD, superoxide dismutase. q This work was supported in part by the National Natural Science Foundation of China [Grant 81373471 and 81430086]. ⇑ Corresponding authors at: Center for Developmental Therapeutics, Seattle Children’s Research Institute, Division of Gastroenterology and Hepatology, Department of Pediatrics, University of Washington School of Medicine, Seattle, WA 98101, United States. Key Laboratory of Structure-Based Drug Design & Discovery of Ministry of Education, Shenyang Pharmaceutical University, Shenyang, 110016, PR China. Tel.: +1 206 884 7651; fax: +1 206 987 7660 (J. Zheng). School of Pharmacy, Shenyang Pharmaceutical University, Shenyang, 110016, PR China. Tel.: +86 24 23986361 (Y. Peng). E-mail addresses: [email protected] (Y. Peng), jiang.zheng@ seattlechildrens.org (J. Zheng).
http://dx.doi.org/10.1016/j.cbi.2014.12.009 0009-2797/Ó 2014 Elsevier Ireland Ltd. All rights reserved.
fruits and foods contain IIMP, such as citrus fruits (grapefruit and lemon), umbelliferous vegetables, and culinary herbs (parsley) [3,4]. IIMP is also included in Yuan-hu-zhi-tong (in Chinese) tablets and Huo-xiang-zheng-qi (in Chinese) aqua widely used in China for the treatment of stomachache and headache. As a bioactive component, IIMP exhibited anti-inflammatory, analgesic, anti-spasmodic, and anti-cancer activities [5–8]. It also showed inhibitory effect on b-secretase that has been considered as a valuable target for the treatment of Alzheimer’s disease [9,10]. The multiple pharmacological activities of IIMP, along with its natural abundance, have attracted much attention. Many furanocoumarins have been documented as potent inhibitors of cytochrome P450s (CYPs). Epoxybergamottin and bergapten (5-methoxypsoralen) from grapefruit were proven to inhibit CYP3A4 activity [11,12]. Psoralen and isopsoralen showed inhibitory effects on CYP1A2 [13,14]. 8-Methoxypsoralen 5-methoxypsoralen, 5-hydroxypsoralen, 8-hydroxypsoralen, and psoralen were found to be mechanism-based inactivators of CYP2B1 [15]. Psoralen and 8-methoxypsoralen were reportedly oxidized on the furan ring to form the corresponding furanoepoxides that bind to CYP2B1. We speculated that IIMP might show similar irreversible inhibitory effect on CYP2B subfamily.
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CYP2B6 has been found in liver, brain, kidney, and heart in humans [16,17]. Although CYP2B6 comprises about 0.2% of the total CYPs in human liver microsomes, it is responsible for the metabolism of more than 3% of drugs widely used in clinic [18], such as bupropion, efavirenz, methadone, ifosfamide, and cyclophosphamide, which are preferentially metabolized or stereoselectively metabolized by CYP2B6 [19–22]. To confirm the role of specific CYPs in the clearance of various drugs and to prevent drug–drug interactions induced by the inhibition of CYPs, prediction and identification of compounds that act as mechanism-based inactivators of CYPs have become an important issue in drug discovery process. The objectives of the study were to examine the irreversible inhibitory effect of IIMP on cytochrome P450 2B6, to characterize the reactive metabolites responsible for the enzyme inactivation, and to identify the P450 enzymes responsible for the metabolic activation of IIMP.
of CYP2B6. Control incubations were performed in the absence of IIMP or S-mephenytoin in parallel. 2.4. Effects of GSH and catalase/superoxide dismutase on the enzyme inactivation The primary mixtures including CYP2B6 (0.1 lM), IIMP (10 lM)), and GSH (2 mM) were preincubated at 30 °C for 3 min. The reactions were initiated by the addition of NADPH (1.0 mM) and were incubated at 30 °C for 0, 3 and 9 min. Aliquots (40 lL) were taken to the secondary incubation mixture for determining the remaining enzyme activity. In control samples, GSH was replaced by an equal volume of phosphate buffer. In a separate study, CYP2B6 was incubated with IIMP and NADPH in the presence or absence of a mixture of catalase and superoxide dismutase. The concentrations of superoxide dismutase and catalase were both 800 unit/mL. After incubation for 0, 3, and 9 min, the residual CYP2B6 activities were examined as described below.
2. Materials and methods
2.5. Partition coefficient
2.1. Chemicals and materials
To estimate the partition coefficient, IIMP was added to the primary reaction mixtures (final concentrations: 0, 1.5, 2.5, 5, 7.5, 10, 25, 50, and 150 lM) containing CYP2B6 (0.1 lM). The reactions were initiated by the addition of NADPH (1.0 mM) and incubated at 30 °C for 9 min to assure that the inactivation had proceeded to completion. Negative control incubations lacked NADPH. Aliquots (40 lL) were withdrawn from the primary reaction mixtures and transferred to the secondary incubation. The samples were analyzed as follows.
Glutathione (GSH), hexyl glutathione, bupropion, and NADPH were purchased from Sigma–Aldrich (St. Louis, MO). Isoimperatorin (IIMP) with purity of 98% was acquired from Shanghai Yuanye Biological Technology Co., Ltd. (Shanghai, China). Recombinant human CYP2B6 was purchased from BD Gentest (Woburn, MA). Rat liver microsomes (RLMs) were prepared by our lab, according to our early work [23]. All organic solvents were from Fisher Scientific (Springfield, NJ). Distilled water was purchased from Wahaha Co. Ltd (Hangzhou, China). All solvents and reagents were either analytical or HPLC grade. 2.2. Time-, concentration-, and NADPH-dependent inactivation of CYP2B6 by IIMP The composition of the primary incubation mixtures was CYP2B6 (0.1 lM), MgCl2 (3.2 mM), and IIMP at a series of concentrations (0, 2.5, 5.0, 7.5, 10, and 25 lM) in potassium phosphate buffer (pH 7.4). The total volume was 0.2 mL. To determine the requirement of metabolism for the enzyme inactivation, IIMP (25 lM) and CYP2B6 were incubated in the absence of NADPH as negative control. The primary mixture was preincubated at 30 °C for 3 min. The reactions were initiated by addition of NADPH (1.0 mM) and were incubated at 30 °C. At various time points, aliquots (40 lL) of the primary mixtures were transferred into the secondary incubation mixtures containing bupropion (100 lM) and NADPH (0.45 mM) in 0.1 M potassium phosphate buffer (pH 7.4). The secondary incubation mixtures were further incubated at 30 °C for 30 min, followed by the addition of 0.12 mL ice-cold acetonitrile containing propranolol as internal standard. After vortexed for 3 min, the mixture was centrifuged at 16,000 rpm for 10 min. The supernatant was subjected to LC–MS/MS analysis as described below. 2.3. Substrate protection Substrate protection from IIMP-induced inactivation of CYP2B6 was determined by including IIMP and S-mephenytoin (1:4) in the primary reaction mixture. The primary mixture was preincubated at 30 °C for 3 min. The reactions were initiated by the addition of NADPH (1.0 mM), and aliquots (40 lL) of the primary mixtures were transferred at 0, 3, and 9 min to the secondary incubation mixtures for the determination of bupropion hydroxylase activity
2.6. Irreversibility of enzyme inhibition The primary reaction mixture was incubated with 25 lM IIMP (inactivated sample) or without of IIMP (control sample) at 30 °C. Aliquots of the control and inactivated samples were withdrawn at 9 min and dialyzed using Slide-A-lyzer membranes (molecular mass cut off, 3,500 Da, Pierce, Rockford, IL) against 0.1 M potassium phosphate buffer (pH 7.4, 3 2 h) at 4 °C. The dialyzed samples were brought to room temperature and were added to the secondary incubation mixture for the determination of the residual enzyme activities as below. 2.7. CYP2B6 Assay CYP2B6 activity was monitored by measuring the formation of hydroxybupropion analyzed by LC–MS/MS, using an Accuore C18 column (2.1 50 mm, 2.6 lM, ThermoFisher, Pittsburgh, PA). The LC–MS/MS system consisted of AB Sciex Instruments 4000 Q-Trap mass spectrometry (Applied Biosystems, Foster City, CA) interfaced online with an ekspert ultra LC 100 system (Applied Biosystems, Foster City, CA). The mobile phases consisted of 0.1% formic acid in acetonitrile (mobile phase A) and 0.1% formic acid in water (mobile phase B). The flow rate was 0.5 mL/min, and the column temperature was maintained at 30 °C. The gradient elution was set as follow: 0–1.0 min, 10% A; 1.0–1.5 min, 10–30% A; 1.5– 4.0 min, 30% A; 4.0–5.5 min; 30–10% A; 5.5–8.0 min, 10% A. Injection volume was 5.0 lL. Quantification was performed by multiple reaction monitoring (MRM), and ion pairs of m/z 256.2 ? 238.3 for hydroxybupropion and m/z 260.7 ? 116.3 for propranolol (internal standard) were acquired in positive mode. 2.8. Reactive intermediate trapping by GSH IIMP (120 lM), rat liver microsomes (1.0 mg protein/mL), and GSH (1.0 mM) were incubated in the presence or absence of
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NADPH (1.0 mM) at 37 °C for 60 min. In a separate study, recombinant human CYPs including 1A2, 2A6, 2B6, 2C9, 2C19, 2D6, 2E1, 3A4, or 3A5 (0.1 lM for each) replaced the rat liver microsomes, and the other condition was equivalent to that for the microsomal incubations. The resulting samples were injected into LC–MS/MS for analysis. The separation of GSH conjugates was achieved on an Accuore C18 column (4.6 150 mm, 5 lM, ThermoFisher, Pittsburgh, PA). The solvent system consisted of mobile phase A (acetonitrile with 0.1% formic acid) and mobile phase B (water with 0.1% formic acid), and the flow rate was 0.8 mL/min. The column temperature was maintained at 30 °C. The gradient elution was set as follow: 0–2.0 min, 10% A; 2.0–8.0 min, 10–90% A; 8.0–10 min, 90% A; 10–13 min; 90–10% A; 13–15 min, 10% A. GSH conjugates were monitored by MRM scanning in positive mode (m/z 594.0 ? 465.0 for IIMP–GSH conjugates; and m/z 392.2 ? 246.3 for hexyl glutathione as internal standard). The informationdependent acquisition (IDA) standard was followed by triggering the enhanced product ion (EPI) scans. IDA was used to initiate acquisition of EPI spectra for ions exceeding 5000 cps with exclusion of former target ions after three occurrences for 10 s. The EPI scan was run in positive mode at a scan range for product ions from m/z 50 to 650. The EPI scanning parameters were listed as follows: scan mode = profile; step size = 0.08 Da; and scan rate = 1000 Da/s, 5 ms pause between mass ranges. 2.9. Synthesis of IIMP–GSH conjugates IIMP (2.5 mg) was dissolved in acetone (200 lL) and mixed with saturated sodium bicarbonate solution (40 lL) and Oxone (6.5 mg). The mixture was stirred for 30 min at room temperature, followed by addition of GSH (35 mg). The mixture was further stirred for 1 h at room temperature and was centrifuged. The supernatant was divided into two equal portions. One was directly analyzed by LC–MS/MS. The other was mixed with sodium borohydride (10 mg, NaBH4). The resulting mixture was gently vortexed for 5 min, followed by LC–MS/MS analysis. 3. Results 3.1. Time-, concentration- and NADPH-dependent CYP2B6 inactivation
Fig. 1. (A) Time- and concentration-dependent inactivation of CYP2B6 by IIMP. Recombinant human CYP 2B6 was incubated with IIMP at concentrations of 0 (), 2.5 (j), 5.0 (N), 7. 5( ), 10 (), and 25 lM (h) in the presence of NADPH at 30 °C for 0, 3, 6, and 9 min. (B) Wilson plot. The observed inactivation rate constant Kobs was calculated from the slope of the regression lines shown in (A). (C) NADPHdependent inactivation of P450 2B6 by IIMP. CYP2B6 was incubated with vehicle (N) and IIMP (25 lM) in the presence (j) or absence () of NADPH. Data represent the mean ± SD (n = 3).
Through measuring the amount of hydroxybupropion produced, the remaining CYP2B6 activity was monitored. As shown in Fig. 1A and C, the inactivation of CYP2B6 by IIMP was timeand concentration-dependent and required NADPH. The residual enzymatic activities at 0 min were normalized to 100% to facilitate vivid comparison of the rates of inactivation at each concentrations. The inactivation of CYP2B6 increased progressively with increasing time and concentrations of IIMP applied. About 60% of activity of CYP2B6 was suppressed after its incubation with 25 lM IIMP for 9 min at 30 °C. Nevertheless, no loss of enzyme activity was observed in the absence of IIMP or NADPH. A double-reciprocal plot (Wilson plot) of the observed rates of inactivation (Kobs) and IIMP concentrations was used to estimate the kinetic constants KI and kinact (Fig. 1B). KI equaled to 17.1 lM, and kinact was 0.071 min1. 3.2. Substrate protection
Fig. 2. Substrate protection against inactivation of CYP2B6 by IIMP. CYP2B6 was incubated with vehicle () and IIMP (10 lM) in the presence (N) or absence (j) of Smephenytoin (40 lM). Data represent the mean ± SD (n = 3).
To study substrate protection from the IIMP-induced enzyme inactivation, CYP2B6 (0.1 lM) was incubated with IIMP in the presence of S-mephenytoin (a CYP2B6 substrate) at a molar ratio of 1:4 (IIMP: S-mephenytoin) in the primary reaction mixture. Compared with samples containing IIMP without S-mephenytoin, residual
CYP2B6 activities in samples incubated with IIMP and S-Mephenytoin were 84.9 ± 1.4% and 55.8 ± 1.6% at 3 and 9 min, respectively, which were higher than the 71.4 ± 4.5% and 53.7 ± 5.3%. This indicates that the presence of S-mephenytoin slightly slowed down the CYP2B6 inactivation induced by IIMP (Fig. 2).
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superoxide dismutase (SOD), scavengers of reactive oxygen species, was included in the primary incubation mixtures. 3.4. Partition coefficient The partition ratio (P) is a measure of efficiency for inactivation, which was evaluated according to previously published method [24]. The percentage of activity remaining was plotted as a function of the IIMP/CYP2B6 molar ratio (Fig. 3). The turnover number (P + 1) was approximately 211, and the partition ratio was found to be about 210 (Fig. 3). Fig. 3. Partition ratio determination for 2B6 inactivation by IIMP. CYP2B6 was incubated with IIMP at various concentrations. The extrapolated P was determined from the X-axis intercept of regression line of the lower ratios. Data represent the mean ± SD (n = 3).
3.3. Effects of GSH and catalase/superoxide dismutase on the enzyme inactivation To evaluate the protective effect of GSH against the enzymatic inactivation induced by IIMP, the primary incubation was performed in the presence or absence of GSH (2 mM). After incubation for 9 min, the remaining CYP2B6 activities were found to be 54.1 ± 2.9% (with GSH) and 49.5 ± 4.3% (without GSH), respectively. This agent produced minor protective effect on the enzyme from inactivation. Additionally, there was only slight protection against the inactivation of CYP2B6 by IIMP, when a mixture of catalase and
3.5. Irreversibility of enzyme inhibition Irreversibility of CYP2B6 inhibition was examined by determining whether dialysis can recover the enzyme activity after exposure to IIMP. A loss of CYP2B6 activity (11.7 ± 1.7% of control) was observed after the enzyme was incubated with IIMP (25 lM) at 30 °C for 9 min. The enzyme remained inhibited (8.5 ± 0.5% of control) after dialysis, indicating that dialysis did not recover the enzyme activity. 3.6. Reactive metabolite trapping by GSH IIMP was incubated with rat liver microsomes or recombinant human P450 enzymes supplemented with glutathione (GSH) to trap the reactive metabolites. The mixtures in the presence or
Fig. 4. (A) Extracted ion (m/z 594 ? 465) chromatograms obtained from LC-Q-Trap mass spectrometry analysis of rat liver microsomal incubations containing IIMP and GSH. (B) MS/MS spectrum of IIMP–GSH conjugate generated in microsomal incubation. (C) Extracted ion (m/z 594 ? 465) chromatogram obtained from LC-Q-Trap MS analysis of chemical oxidation of IIMP trapped by GSH. (D) MS/MS spectrum of synthetic IIMP–GSH conjugate.
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reduction of the conjugate and then analyzed by LC–MS/MS. A new peak was observed at retention time of 7.19 min with [M+H]+ at m/z 596.2 (Fig. 5A), 2 Da higher than that of the conjugate observed before the reduction by sodium borohydride. 3.7. P450 Enzymes responsible for IIMP bioactivation To determine which P450 enzymes preferentially catalyze the oxidation of IIMP, recombinant human CYPs 1A2, 2A6, 2B6, 2C9, 2C19, 2D6, 2E1, 3A4, or 3A5 (0.1 lM for each) was incubated with IIMP in the presence of GSH as a trapping agent, follow by LC–MS/ MS analysis. As showed in the Fig. 6, the IIMP–GSH conjugate was assessed in the incubation with CYPs 2B6, 2D6, or 1A2, and limited amount of the GSH conjugate was found in the incubations with the other P450 enzymes. This indicates that CYPs 2B6, 2D6, and 1A2 were the major enzymes responsible for the metabolic activation of IIMP. 4. Discussion
Fig. 5. (A) Extracted ion (m/z 596 ? 467) chromatogram obtained from LC-Q-Trap MS analysis of the chemical oxidation reaction followed by reaction with NaBH4. (B) MS/MS spectrum of the reduced product.
absence of NADPH were all conducted and then analyzed by LC– MS/MS. A peak with a protonated molecular ion [M+H]+ at m/z 594 (retention time = 6.94 min) as the major GSH conjugate was observed (Fig. 4A), but no such peak was found in the incubations without NADPH (the control samples). The product ion spectrum of the conjugate obtained by MRM–EPI scanning (ion transition m/z 594/465) showed fragment ions m/z 519, 465, 245, and 177 (Fig. 4B). The product ions at m/z 519 and 465 were derived from the loss of glycine portion (-75 Da) and c-glutamyl portion (129 Da) from m/z 594, respectively. For further structural characterization, the IIMP-derived GSH conjugate was chemically synthesized by oxidation with Oxone in acetone, followed by reaction with GSH. As expected, the resulting product showed the same retention time and the same fragmentation behavior (Fig. 4C and D) as that of the conjugate generated in the microsomal reactions (Fig. 4A and B). Due to poor reaction yield, we were unable to have enough amount of the product for NMR characterization. In a separate study, the other portion of the reaction solution was mixed with sodium borohydride for
Our kinetic study clearly demonstrated that IIMP produced a time-dependent inhibition of CYP2B6. The enzyme inactivation by IIMP was also found to be concentration-dependent, and it attained saturation at the concentration of 25 lM. IIMP-induced CYP2B6 inactivation was not observed unless NADPH was supplemented in the first incubation mixtures. This indicates that IIMP itself is not an inactivator of CYP2B6 and that the enzyme inactivation by IIMP needs to be initiated through biotransformation with assistance of P450 cofactor NADPH. The irreversibility of enzyme inhibition induced by IIMP was examined by dialysis of the enzyme which was exposed to IIMP, and no recovery of enzyme activity was observed after dialysis. The results clearly demonstrate that IIMP is a mechanism-based inactivator of CYP2B6. Catalase and SOD are known to be the enzymes responsible for quenching reactive oxygen species, such as superoxide anion and hydrogen peroxide [25]. Only minor protection of enzyme inactivation by IIMP from catalase/SOD was observed, indicating that reactive oxygen species may not play a major role in CYP2B6 inactivation induced by IIMP. In addition, GSH as a nucleophilic agent showed weak protection of CYP2B6 against IIMP. This indicates in part that CYP2B6 is covalently modified by reactive metabolites of IIMP before escaping from the active site. The mildly protective effect of S-mephenytoin on CYP2B6 inactivation observed in the substrate competition experiment implies that Smephenytoin competed with IIMP binding to the active site of CYP2B6 and reduced the rate of generation of the reactive metabolites of IIMP. The critical finding of substrate protection further indicates that bioactivation of IIMP occurred in the active site of CYP2B6. The partition ratio, reflecting the efficiency of the inactivator, is defined as the number of molecules of product molecules per mole-
Fig. 6. Human P450 enzymes involved in the formation of IIMP-conjugate. IIMP was incubated with individual human recombinant P450 enzymes in the presence of NADPH and GSH. Data represent the mean ± SD (n = 3).
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Scheme 1. Proposed pathways for the formation of reactive intermediate(s) and GSH adducts during the metabolism of IIMP.
cule of enzyme inactivated. Reported P values in literatures for mechanism-based inactivators of P450 enzymes vary from 3 (very highly efficient inactivators) to >1000 (inefficient) [26]. For example, 4-ipomeanol, a furan-containing compound, that was a moderately efficient inactivator of CYP3A4 with an estimated P of 257 [27]. Therefore, the efficiency of CYP2B6 inactivation by IIMP (P value: 210) is similar to that of CYP3A4 inactivation by 4-ipomeanol. Generation of reactive metabolites is an essential step for mechanism-based enzyme inactivation. Here we propose two reactive metabolites responsible the inactivation of CYP2B6 by IIMP, and they are furanoepoxide intermediate 2 (Scheme 1) and c-ketoenal intermediate 3, and the ketoenal is also suggested to be generated by rearrangement of the furanoepoxide. To identify the structures of the reactive metabolites, we trapped the electrophilic intermediates using GSH. An IIMP–GSH conjugate with its [M+H]+ ion at m/z 594 was detected by LC–MS/MS (Fig. 4A) after incubation of IIMP in rat liver microsomes in the presence of NADPH and GSH. The observed molecular ion of m/z 594 matched the molecular weight of GSH conjugates 4–6 (Scheme 1). The MS/MS spectrum showed the indicative characteristic fragments of the GSH moiety and some other fragments valuable for metabolite identification (Fig. 4D). Ion m/z 162 responsible for molecular formula of C9H6O+3 was detected, suggesting that the 5-member ring fused with the coumarin ring was opened (Scheme 1). This could also allow us to exclude the formation of GSH conjugate 4, since the ring opening of conjugate 4 would produce a thioester. Only the structure of GSH conjugate 6 is consistent with the observed fragment of C9H6O+3, and conjugate 5 could be spontaneously converted to conjugate 6. To verify conjugate 6, we chemically reduced the conjugate with NaBH4. The reduction reaction afforded a new product with [M+H]+ at m/z 596.2 (Fig. 5A), which is higher 2.0 Da than that of the one detected before the reduction reaction. Additionally, the fragment at m/z 162 was retained in the MS/MS spectrum of the reduced GSH conjugate, indicating no reduction took place on the coumarin ring. Furthermore, the MS/MS spectrum of the original IIMP–GSH conjugate showed a fragment at m/z 177 that results from the elemental composition of C11H9O+5 of the conjugate by loss of the side chain, GSH, and CO2. In other words, fragment of m/z 177 contained the aldehyde group (Fig. 4D). No such fragment was found in the MS/MS spectrum of the reduced GSH–IIMP conjugate (Fig. 5B). Instead, a fragment of m/z 179 resulting from the reduced IIMP–GSH conjugate was detected, which was 2 Da higher than that (m/z 177) observed in the MS/MS spectrum of the original IIMP–GSH conjugate (Fig. 5B). The observation indicates that the reduction took place
on the aldehyde not on the lactone group. A separate experiment demonstrated that IIMP was resistant to the reduction by NaBH4 (data not shown). This provided the additional evidence that the NaBH4–mediated reduction of conjugate 6 gave conjugate 7. The observed conjugate 7 indicates that conjugate 6 was the primary GSH conjugate resulting from metabolic activation of IIMP. However, we were unable to tell whether conjugate 6 came from intermediate 2 or 3, since conjugate 5 might be spontaneously converted to conjugate 6, and vice versa. In other words, whether intermediate 2 or 3 was the primary metabolic intermediate remains unknown. We speculate that IIMP was oxidized to epoxides/c-ketoenal (on the furan rings), which chemically attacked the host enzyme to form covalent binding and the enzyme modification initiated the process of the enzyme inactivation. The bioactivation studies with individual recombinant enzymes demonstrated that multiple P450 enzymes catalyzed the metabolism of IIMP to the reactive intermediate (Fig. 6). CYPs 1A2, 2B6 and 2D6 were found to be the major enzyme responsible for the bioactivation of IIMP. Whether IIMP is a mechanism-based inactivator of CYPs 1A2 and 2D6 is under investigation. 5. Conclusions We have demonstrated that IIMP is enzymatically activated by CYP2B6 and is a characterized mechanism-based inactivator of CYP2B6. A GSH conjugate derived from c-ketoenal intermediate was identified in microsomal incubations with IIMP trapped with GSH. The electrophilic intermediate is possibly responsible for the enzyme inactivation. Conflict of Interest None. Transparency Document The Transparency document associated with this article can be found in the online version. References [1] N.I. Baek, E.M. Ahn, H.Y. Kim, Y.D. Perk, Furanocoumarins from the root of Angelica dahurica, Arch. Pharm. Res. 23 (2000) 467–470. [2] A. Satoh, Y. Narita, N. Endo, H. Nishimura, Potent allelochemical falcalindiol from Glehnia littoralis F. Schm, Biosci. Biotechnol. Biochem. 60 (1996) 152–153.
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