- Email: [email protected]
Psoralen, a mechanism-based inactivator of CYP2B6
Accepted Manuscript Psoralen, a mechanism-based inactivator of CYP2B6 Lin Ji, Dan Lu, Jiaojiao Cao, Liwei Zheng, Ying Peng, Jiang Zheng PII:
S0009-2797(15)30048-X
DOI:
10.1016/j.cbi.2015.08.020
Reference:
CBI 7450
To appear in:
Chemico-Biological Interactions
Received Date: 26 May 2015 Revised Date:
15 July 2015
Accepted Date: 28 August 2015
Please cite this article as: L. Ji, D. Lu, J. Cao, L. Zheng, Y. Peng, J. Zheng, Psoralen, a mechanismbased inactivator of CYP2B6, Chemico-Biological Interactions (2015), doi: 10.1016/j.cbi.2015.08.020. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Psoralen, a mechanism-based inactivator of CYP2B6
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Lin Ji1, Dan Lu1, Jiaojiao Cao1, Liwei Zheng1, Ying Peng1,*, and Jiang Zheng2, 3,*
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of Ministry of Education, Shenyang Pharmaceutical University, Shenyang, Liaoning,
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110016, P. R. China
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Division of Gastroenterology and Hepatology, Department of Pediatrics, University of
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Washington School of Medicine, Seattle, WA 98101
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Center for Developmental Therapeutics, Seattle Children’s Research Institute,
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College of Pharmacy, 2Key Laboratory of Structure-Based Drug Design & Discovery
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ACCEPTED MANUSCRIPT Corresponding Authors:
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Jiang Zheng, PhD
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Center for Developmental Therapeutics, Seattle Children's Research Institute, Division of Gastroenterology and Hepatology, Department of Pediatrics, University of Washington, Seattle, WA 98101 Key Laboratory of Structure-Based Drug Design & Discovery of Ministry of Education, Shenyang Pharmaceutical University, Shenyang, Liaoning, 110016, P. R. China Email: [email protected]
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Tel: 206-884-7651; Fax: 206-987-7660.
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Ying Peng, PhD
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School of Pharmacy, Shenyang Pharmaceutical University, Shenyang, Liaoning, 110016, P. R. China
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Email: [email protected]
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Tel: +86-24-23986361; Fax: +86-24-23986510.
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ACCEPTED MANUSCRIPT Abbreviations: PRN, psoralen; DMSO, dimethyl sulfoxide; GSH, glutathione;
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NADPH, β-nicotinamide adenine dinucleotide 2′-phosphate reduced tetrasodium salt;
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RLMs, rat liver microsomes; SOD, superoxide dismutase; LC, liquid chromatography;
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MS, mass spectrometry; LC-MS/MS, liquid chromatography coupled to tandem mass
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spectrometry; MRM, multiple-reaction monitoring; CE, collision energy; EPI,
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enhanced product ion; IDA, information-dependent acquisition
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Abstract Furanocoumarin compound psoralen (PRN) is a major active ingredient found in
3
herbaceous plants.
PRN has been used for the treatment of various dermal diseases
4
in China.
5
(CYP2B6)
6
NADPH-dependent inactivation of CYP2B6 with the values of KI and kinact being
7
110.2 µM and 0.200 min-1, respectively.
8
prevented the enzyme from the inactivation induced by PRN.
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nucleophile glutathione (GSH) and catalase/superoxide dismutase showed limited
that
PRN
induced
a
time-,
concentration-,
and
Ticlopidine, a CYP2B6 substrate,
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found
Exogenous
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and
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We evaluated the inhibitory effect of PRN on cytochrome P450 2B6
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protection of CYP2B6 from the inactivation.
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inactivation was approximately 400.
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epoxide or/and γ-ketoenal intermediate was formed in microsomal incubations with
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PRN.
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CYP2B6.
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GSH trapping experiments indicates that an
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In summary, PRN was characterized as a mechanism-based inactivator of
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The estimated partition ratio of the
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1. Introduction Psoralen (PRN) is a coumarin derivative fused with a furan and is the core of furanocoumarins widely found in nature.
4
of legumes Psoralea corylifolia L, as well as in the common fig, celery, parsley and in
5
all citrus fruits.
6
double teeth angelica root and coastal glehnia root [1].
7
herbal formulas such as Wenweishu tables, Sishen pills, and Yaotong pills, and it is
8
also used in ultraviolet light therapy for psoriasis, eczema, and vitiligo [2].
9
reported pharmacological activities of PRN included anti-inflammatory and
10
antipyretic, antibacterial, antiviral, hepato-protective, female hormone like effects
11
[3-7].
12
in four cancer cell lines, including KB, KBv200, K562, and K562/ADM [8]. PRN
13
has attracted much attention, due to its potential to be a pharmaceutical agent and
14
wide occurrence in nature.
PRN is also found in many traditional Chinese medicines, such as
The
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PRN has been widely used in
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In addition, PRN was reported to exhibit a dose-dependent anticancer activity
A number of furanocoumarin compounds have been reported to exhibit inhibition
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PRN mainly occurs in the fruits and seeds
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of cytochrome P450s (CYPs).
17
inhibitory effects on CYP1A2 [1, 9-10].
18
tin, major components of grapefruit, are reportedly mechanism-based inactivators of
19
CYP3A4 [11-12].
20
mechanism-based inactivators of CYP2B6 [13-15].
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exhibited mechanism-based inactivation of CYP2A6 [16].
22
its derivatives, including 8-methoxypsoralen, 5-methoxypsoralen, 5-hydroxypsoralen,
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PRN and its isomer isopsoralen were found to show Bergamottin and 6’,7’-dihydroxybergamot-
Bergamottin, imperatorin, and isoimperatorin were claimed as
5
PRN and 8-methoxypsoralen Additionally, PRN and
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8-hydroxypsoralen,
bergapten,
and
isopimpinellin,
2
mechanism-based inactivators of CYP2B1 [17-18].
were
found
to
be
CYP2B is an important member of the P450 family, and the content of CYP2B6
4
ranges from 2 to 10% of the whole P450 [19]. With the contribution of 3-6 % of
5
total hepatic P450 content [20], CYP2B6 is also found in extrahepatic tissue, such as
6
brain, kidney, and heart.
7
including biotransformation of endogenous and exogenous substances [21].
8
CYP2B6 is responsible for the metabolism of more than 4% of clinically used drugs,
9
for example, bupropion, efavirenz, methadone, ifosfamide, and cyclophosphamide
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The enzyme participates in a variety of metabolic reactions
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[22-26].
With the rapidly growing global interest in the use of natural products as medical
12
remedies and dietary supplements, much attention has been paid to drug-drug
13
interactions associated with natural product-mediated inhibition of cytochromes P450
14
enzymes.
15
PRN with CYP2B6, to characterize the reactive metabolites of PRN, and to identify
16
the P450 enzymes responsible for metabolic activation of PRN.
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The objectives of the present studies were to investigate the interaction of
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2. Materials and Methods
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2.1 Chemicals and Materials Psoralen (98% purity), superoxide dismutase (SOD), and catalase were obtained
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from Shanghai Yuanye Biological Technology Co., Ltd (Shanghai, China).
5
Glutathione (GSH), hexyl glutathione, bupropion, Oxone, and NADPH were acquired
6
from Sigma-Aldrich (St. Louis, MO).
7
purchased from BD Gentest (Woburn, MA).
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Scientific (Springfield, NJ).
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(Hangzhou, China).
Recombinant human P450 enzymes were
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All organic solvents were from Fisher
Distilled water was purchased from Wahaha Co. Ltd
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All solvents and reagents were either analytical or HPLC grade.
2.2 Time-, Concentration-, and NADPH-Dependent Inactivation of CYP2B6 by PRN The composition of the primary incubation mixtures contained CYP2B6 (0.1 µM),
12
MgCl2 (3.2 mM), and PRN at concentrations of 0, 40, 80, 120, 160, or 200 µM in
13
potassium phosphate buffer (pH 7.4) with a total volume of 0.2 mL.
14
components for the primary incubations were mixed at 4 °C and vortexed quickly.
15
The primary incubations were performed at 30 °C and preincubated for 3 min.
The
16
reactions were initiated by addition of NADPH (final concentration: 1.0 mM).
At
17
time points of 0, 3, 6, and 9 min, aliquots (40 µL) of the primary incubation mixtures
18
were transferred to the secondary incubation mixtures containing bupropion (100 µM)
19
and NADPH (0.45 mM) in 0.1 M potassium phosphate buffer (pH 7.4).
The reason
20
for
was
21
our preliminary study showed that a significant spontaneous CYP2B6 activity loss
22
took
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choosing
place
30
at
37
°C
°C
for
in
the
9
enzyme
min 7
in
incubations
the
absence
of
The
that
PRN,
ACCEPTED MANUSCRIPT but no such enzyme activity loss occurred at 30 °C in the same period of time.
The
2
secondary incubation mixtures were further incubated at 30 °C for 30 min, followed
3
by addition of ice-cold acetonitrile (120 µL) containing propranolol as internal
4
standard.
After vortexing for 3 min, the mixtures were centrifuged at 16,000 rpm for
5
10 min.
The supernatants were subjected to LC-MS/MS analysis. To ensure whether
6
the enzyme inactivation is NADPH-dependent, PRN (80 µM) and CYP2B6 were
7
incubated in the absence of NADPH as negative control in the primary incubation.
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2.3 Substrate Protection
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PRN (80 µM) and ticlopidine (at mole ratio of 1:2.5) were included in the
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primary reaction mixtures to study the substrate protection from PRN-induced
11
inactivation of CYP2B6.
12
min.
13
mixtures were transferred to the secondary incubation mixtures for the determination
14
of bupropion hydroxylase activities of CYP2B6.
15
PRN or ticlopidine were performed in parallel.
16
2.4 Effects of GSH and Catalase/SOD on the Inactivation of CYP2B6
Control incubations lacking of
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After incubation at 30 °C for 0, 3, and 9 min, aliquots (40 µL) of the primary
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The primary mixtures were preincubated at 30 °C for 3
The primary reaction mixtures containing CYP2B6 (0.1 µM), PRN (80 µM), and
18
GSH (2.0 mM) were preincubated at 30 °C for 3 min.
The reactions were initiated
19
by addition of NADPH (1.0 mM).
20
transferred to the secondary incubation mixtures to determine the residual enzyme
21
activities.
At the time of 9 min, aliquots (40 µL) were
In control samples, phosphate buffer with an equal volume was in place
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of GSH solution.
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NADPH in the presence or absence of a mixture of catalase and superoxide dismutase
3
(SOD).
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enzyme per milliliter.
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2.5 Partition Coefficient
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The concentrations of SOD and catalase used were 800 units of each
To determine the partition ratio, CYP2B6 (0.1 µM) was mixed with PRN at
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In a separate study, CYP2B6 was incubated with PRN and
concentrations of 0, 10, 20, 80, 160, 200, and 300 µM.
NADPH at a final
8
concentration of 1.0 mM was added to initiate the reactions.
The incubations were
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accomplished after 9 min at 30 °C in a water bath.
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At 0 and 9 min, aliquots (40 µL)
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were withdrawn and transferred to the secondary incubations for determination of
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CYP2B6 activities.
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2.6 Irreversibility of Inhibition
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Incubations that lacked NADPH served as negative controls.
Primary incubations containing PRN (80 µM) and CYP2B6 (0.1 µM) were
14
performed in the presence of NADPH at 30 °C, along with the control incubations
15
lacking of PRN.
16
were withdrawn and transferred into the secondary mixtures for 30 min incubations
17
(non-dialyzed samples), and then the primary incubations were dialyzed using
18
Slide-A-lyzer membranes (molecular mass cut off: 3,500 Da, Pierce, Rockford, IL)
19
against 0.1 M potassium phosphate buffer (pH 7.4, 2 × 3 h) at 4 °C.
20
samples were brought to room temperature and were withdrawn -(40 µL) into the
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secondary incubations.
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At 0 and 9 min, aliquots of the control and inactivated samples
The dialyzed
After incubation for 30 min, the enzyme activities of the
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resulting samples were assessed as described below.
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2.7 CYP2B6 Assay
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To assess the activity of CYP2B6，the production of hydroxybupropion from bupropion was monitored by LC-MS/MS.
The HPLC-MS/MS system consisted of
5
an ekspert ultraLC 100 system (AB SCIEX, Foster City, CA) and a 4000 hybrid triple
6
quadrupole linear ion trap tandem mass spectrometer (AB SCIEX, Foster City, CA)
7
equipped with an electrospray ionization (ESI) interface.
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separation was performed on a C18 column (2.1 × 50 mm, 2.6 µm, ThermoFisher,
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Pittsburgh, PA) at the temperature of 30 °C.
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The chromatographic
The gradient elution phase was
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composed of 0.1% formic acid in acetonitrile (A) and 0.1% formic acid in water (B)
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with the flow rate of 0.3 mL/min.
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directed to waste for the initial 1.0 min via a divert valve and then turned directly into
13
the mass spectrometer.
14
1.0-1.5 min, 10-30% A; 4.0-5.5 min, 30-10% A; and 5.5-8.0 min, 10% A.
15
injection volume was 5 µL.
16
the positive ion detection.
17
to monitor the transition of m/z 256.0 protonated precursor ion to m/z 238.0 product
18
ion for product hydroxybupropion and m/z 260.7 protonated precursor ion to m/z
19
116.3 product ion for internal standard propranolol.
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2.8 Reactive Intermediate Trapping by GSH
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The eluates from the analytical column were
The gradient elution was set as follows: 0-1.0 min, 10% A;
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The mass spectrometer was operated with ESI source in
The multiple reaction monitoring (MRM) mode was used
PRN (80 µM), GSH (1.0 mM), and rat liver microsomes (1.0 mg protein/mL)
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ACCEPTED MANUSCRIPT prepared in our lab [27] or individual human recombinant P450 enzymes, including
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CYPs 1A2, 2A6, 2B6, 2C9, 2C19, 2D6, 2E1, 3A4, or 3A5 (0.1 µM for each) were
3
incubated in the presence or absence of NADPH (1.0 mM) at 37 °C for 60 min.
4
Equal volume of ice-cold acetonitrile containing hexyl glutathione as internal standard
5
was mixed with the final reactions, followed by vortexing and centrifuging.
6
supernatants were dried by blowing with a steam of nitrogen.
7
reconstituted with 50% acetonitrile (50 µL) and submitted to LC-MS/MS for analysis.
8
An Accuore C18 column (4.6 × 150 mm, 5 µm, ThermoFisher, Pittsburgh, PA) was
9
employed to separate the GSH conjugates with the solvent system consisted of 0.1%
10
formic acid in acetonitrile (A) and 0.1% formic acid in water (B), and the flow rate
11
was 0.8 mL/min.
12
min, 10-90% A; 8.0-10 min, 90% A; 10-13 min, 90-10% A; and 13-15 min, 10% A.
13
Under positive ESI condition, the PRN-GSH conjugates (m/z 510→381) and internal
14
standard hexyl glutathione (m/z 392→246) were monitored in MRM mode,
15
respectively.
16
trigger the enhanced product ion (EPI) scans by analyzing MRM signals.
17
was aimed at initiating acquisition of EPI spectra for ions exceeding 500 cps with
18
exclusion of former target ions after three occurrences for 10s.
19
EPI scan was set at a scan range for product ions from m/z 50 to 650.
20
scanning parameters are listed with the scan mode: profile; step size: 0.08 Da; and
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scan rate: 1000 Da/s, 5 ms pause between mass ranges.
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2.9 Chemical Synthesis of PRN-GSH Conjugates
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The
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The residue was
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The gradient elution was set as follow: 0-2.0 min, 10% A; 2.0-8.0
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The IDA
In positive mode, the The EPI
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Saturated sodium bicarbonate solution (40 µL) and Oxone (4.5 mg) were mixed
2
with PRN (1.72 mg) dissolved in acetone (200 µL).
3
room temperature, GSH (24 mg) was added with further stirring for 1 h at room
4
temperature, followed by centrifugation.
5
two portions.
6
mixed with sodium borohydride (7 mg, NaBH4).
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vortexed for 5 min and submitted to LC-MS/MS analysis.
One was subjected to LC-MS/MS analysis directly.
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The other was
The final mixture was gently
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The supernatant was equally allocated into
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After being stirred for 30 min at
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3. Results
2
3.1 Time-, Concentration-, and NADPH-Dependent Inactivation of CYP2B6 by PRN
3
The remaining activities of CYP2B6 were monitored by quantifying the amount of hydroxybupropion produced in the secondary incubations.
The residual
5
enzymatic activities of each concentration at 0 min were normalized to 100%.
6
1A and 1C were prepared by a semilogarithmic plot of percent remaining activity vs
7
incubation time.
8
activity in the absence of PRN (control group).
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and NADPH-dependent inhibition of CYP2B6 activity.
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Fig.
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As shown in Fig. 1A and 1C, CYP2B6 retained the catalytic
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PRN caused a time-, concentration-, In the incubations
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containing PRN (200 µM), about 60% of CYP2B6 activity was suppressed.
11
double-reciprocal plot (Wilson plot) of values for the observed rates of inactivation
12
(kobs) and PRN concentrations was employed to calculate the kinetic constants KI (a
13
concentration of PRN needed for half-maximal inactivation) and kinact (maximal rate
14
constant for inactivation).
15
0.200 min-1, respectively (Fig .1B).
16
3.2 Substrate Protection
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As a result, KI and kinact were found to be 110.2 µM and
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A
Ticlopidine, a CYP2B6 substrate, was used to study the PRN-dependent
18
inactivation of CYP2B6.
In the primary incubations, PRN and ticlopidine were
19
added at the molar ratio of 1:2.5.
20
inactivation of CYP2B6 induced by PRN with the residual CYP2B6 activities of
21
75.7±1.5% and 66.2±2.2% at 3 and 9 min, while the remaining activities of CYP2B6
22
were 70.8±2.7% and 40.6±1.3% in the absence of ticlopidine in the primary
The presence of ticlopidine attenuated the
13
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incubations.
2
CYP2B6 induced by PRN (Fig. 2).
3
3.3 Effects of GSH and Catalase/Superoxide Dismutase After incubation for 9 min with PRN (80 µM) and NADPH, the remaining
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This indicates the protection effect of ticlopidine on the inactivation of
CYP2B6 activity was 51.3±2.7%.
Inclusion of GSH (2 mM), an electrophile
6
trapping agent, showed limited protective effect on CYP2B6 from the inactivation
7
with residual CYP2B6 activity of 55.6±2.3%.
8
superoxide dismutase (SOD) acted as scavengers of reactive oxygen species produced
9
slight protection against the inactivation of CYP2B6 by PRN with the remaining
In addition, a mixture of catalase and
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enzyme activity of 57.8±3.4% at 9 min.
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3.4 Partition coefficient
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The partition ratio (P value) was estimated graphically using the previously published method [28] as shown in Figure. 3.
14
of molecules of PRN metabolized per molecule of CYP2B6 inactivated.
15
shows a plot of the percentage remaining activity vs the PRN/CYP2B6 molar ratio.
16
The turnover number (P+1) was about 401, and the extrapolated partition ratio of
17
PRN was approximately 400.
18
further inactivate below about 30.8±2.8%.
19
3.5 Irreversibility of Inhibition
20 21
The value of the P means the number
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The figure
The ratio of PRN to CYP2B6 above 800 did not
By determining the remaining activities of the enzyme before and after dialysis, we investigated the irreversibility of CYP2B6 inhibition. 14
After the incubation of
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CYP2B6 with PRN (80 µM) at 30 °C for 9 min, the remaining CYP2B6 activity was
2
8.0% of control 0 min.
3
recovered after dialysis.
4
3.6 Reactive Metabolite Trapping
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The CYP2B6 activity was only 7.5% of control 0 min
To trap the reactive metabolites, GSH was incorporated in the PRN incubation
6
systems including rat liver microsomes or individual recombinant P450 enzymes in
7
the presence or absence of NADPH, followed by LC-MS/MS analysis.
8
GSH conjugate as a peak with protonated molecular ion [M+H]+ at m/z 510 at
9
retention time of 5.5 min was observed (Fig. 4A), while no such peak was found in
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The major
10
the absence of NADPH as the control sample (data not shown).
11
scanning mode with the ion transition m/z 510/381 was employed on identifying the
12
MS/MS spectrum of the conjugate.
13
m/z 510, the product ions at m/z 435 and 381 observed indicate the neutral losses of
14
glycinyl portion (-75 Da) and γ-glutamyl portion (-129 Da) which represent the
15
cleavage of the characteristic fragment ions of the GSH moiety.
16
product ion at m/z 203 was detected, resulting from the cleavage of S-C (PRN site)
17
bond of the PRN-GSH conjugate.
18
higher than that of the protonated molecular ion (m/z 187, Fig. S1), indicating an
19
insertion of an oxygen in the PRN moiety of the GSH conjugate.
20
The MRM-EPI
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As shown in Fig. 4B, based on the parent ion
In addition, a
The mass of the observed product ion was 16 Da
In order to verify the characterization, PRN was chemically oxidized by Oxone in
21
acetone before reacting with GSH.
As expected, the product formed in the
22
Oxone-based reaction showed identical chromatographic and mass spectrum 15
ACCEPTED MANUSCRIPT 1
behaviors (Fig. 4C and 4D) as that of the PRN-derived GSH conjugate generated in
2
the microsomal systems (Fig. 4A and 4B).
3
other portion of the reaction mixtures for chemical reduction.
4
depicted a new peak at retention time of 5.4 min (Fig. 5A) with the m/z of [M+H]+
5
512 (Fig. 5B), which was 2 Da higher than the conjugate detected in the mixture
6
without the treatment with sodium borohydride.
7
3.7 P450 Enzymes Responsible for PRN Bioactivation
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LC-MS/MS analysis
PRN was incubated with individual recombinant human P450 enzymes, including
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Sodium borohydride was added to the
CYPs 1A2, 2A6, 2B6, 2C9, 2C19, 2D6, 2E1, 3A4, or 3A5 (0.1 µM for each).
GSH
was included in the incubations as the trapping agent.
11
analyzed and quantified by the LC-MS/MS method.
12
2B6, 2C19, and 2D6 were the major enzymes involved in the formation of the
13
reactive metabolite.
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The PRN-GSH conjugate was
As shown in Fig. 6, CYPs 1A2,
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4. Discussion
2
The present study demonstrated that PRN inhibited CYP2B6 in time- and
3
concentration-dependent manners with the characteristic (in enzyme kinetics) of
4
mechanism-based inactivation.
5
absence of NADPH in the primary incubations, indicating the critical role of
6
metabolism in PRN-induced CYP2B6 inactivation.
7
instead of the parent compound exerted the inhibitory action.
8
PRN-induced enzyme inhibition is reversible was probed by determining the enzyme
9
activities of PRN-treated CYP2B6 before and after dialysis.
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No such enzyme inactivation was observed in the
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This implies that metabolite(s) Whether the
Limited enzyme
activities were recovered after the dialysis, possibly due to the protein covalent
11
modification by reactive metabolite(s) of PRN. This provided the further evidence
12
for mechanism-based inactivation of CYP2B6 by PRN.
13
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GSH is a nucleophilic agent, and it is often used to react with reactive electrophilic species produced.
The supplement of GSH in the enzyme incubations
15
revealed little protection of CYP2B6 against PRN-induced inactivation.
16
indicates that CYP2B6 was covalently modified by electrophilic metabolites of PRN
17
before escaping from the active site of the host enzyme and the modification of
18
protein induced the inactivation of the enzyme.
19
capable of quenching superoxide anion, hydrogen peroxide, and other reactive oxygen
20
species which are potential agents to inactivate enzymes [29].
21
that SOD/catalase showed minor protective effect on CYP2B6 from the enzyme
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17
This
SOD and catalase are enzymes
The results showed
ACCEPTED MANUSCRIPT 1
inactivation, indicating that reactive oxygen species did not make significant
2
contribution to the enzyme inactivation. The presence of ticlopidine in the enzyme incubation system was found to
3
attenuate the PRN-induced CYP2B6 inactivation (Fig. 2).
5
ticlopidine and PRN competed to bind to the active site of CYP2B6, resulting in
6
decreased generation of reactive metabolites of PRN responsible for covalent
7
modification of the enzyme.
8
that bioactivation of PRN occurred in the active site of CYP2B6.
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The observed protective effect of ticopidine indicates
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We speculated that
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The partition ratio reflects the efficiency of enzyme inactivator.
The smaller the
10
number is, the more efficient the inactivator is [30].
11
400.
12
enzymes ranged from 3 (very highly efficient inactivators) to >1000 (inefficient) [30].
13
Compared
14
4’,5’-dihydro-8-methoxypsoralen (partition ratio: 840) [16], PRN (partition ratio: 400)
15
may be classified as a moderately efficient inactivator.
furafylline
(partition
ratio:
3.8–5.6)
[31]
and
EP
with
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Reported P values in literatures for mechanism-based inactivators of P450
Metabolic formation of reactive metabolites is an essential step for
AC C
16
The P value of PRN was about
17
mechanism-based
enzyme
inactivation.
18
intermediates 2 and 3 as depicted in Scheme 1, were proposed to respond the
19
inactivation of CYP2B6.
Intermediate 2 is a furanoepoxide derivative, resulting
20
from epoxidation of PRN.
Intermediate 3 is a γ-ketoenal formed by direct oxidation
21
of PRN or/and sigmatropic rearrangement of furanoepoxide 2.
22
structures of the reactive metabolites, we trapped the electrophilic species with GSH 18
Two
reactive
intermediates,
i.e.
To characterize the
ACCEPTED MANUSCRIPT 1
in the microsomal incubations.
A PRN-derived GSH conjugate was detected by
2
LC-MS/MS.
3
molecular weight of GSH conjugates 4-6 (Scheme 1).
4
metabolite showed the indicative characteristic fragments of GSH, such as neutral
5
losses of 75 (glycinyl) and 129 Da (γ-glutamyl).
6
responsible for molecular formula of C9H6O3+ was observed (Fig. 4B and 4D),
7
implicating the opening of the furan ring (Scheme 1).
8
formation of GSH conjugate 4, since the conjugate is a thioacetal and relatively stable
9
against hydrolysis.
The detected protonated molecular ion [M+H]+ (m/z 510) matched the
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The MS/MS spectrum of the
A fragment ion at m/z 162
M AN U
SC
This allows us to exclude the
Only GSH conjugate 6 has the ring opening structure, and
conjugate 5 (a hemiacetal) and conjugate 6 could be converted to each other.
11
verify the structure of conjugate 6, we chemically reduced the conjugate with sodium
12
borohydride, followed by LC-MS/MS analysis.
13
512 (Fig. 5B) was detected by LC-MS/MS.
14
matched the molecular weight of conjugate 7 (Scheme 1) whose molecular ion was
15
2.0 Da higher than that of the one detected before the reductive reaction.
16
comparison with the MS/MS spectra of conjugates 6 and 7, the two conjugates shared
17
a same fragment ion of m/z 162 in response to molecular formula C9H6O3+ (Fig. 4B
18
and 5B).
19
reductive reaction.
20
4B) of conjugate 6 was responsible for the fragment which contained the aldehyde
21
group.
22
m/z 205 was instead observed by LC-MS/MS (Fig. 5B).
To
A new product with [M+H]+ at m/z
The molecular ion of the product
In
AC C
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10
This implies no reduction occurring within the coumarin ring during the In addition, the ion of m/z 203 observed in the MS spectrum (Fig.
After the reductive reaction, no such ion was detected, and a fragment ion of
19
This possibly resulted from
ACCEPTED MANUSCRIPT 1
the reduction of the aldehyde group to the corresponding alcohol.
2
further excludes the formation of conjugate 4, since the thioacetal group of the
3
conjugate should be stable enough to resist the reductive reaction.
4
magnetic resonance spectrum is needed to fully characterize the conjugate.
5
Unfortunately, the low yield of the reaction made us unable to obtain enough amount
6
of the product for NMR characterization.
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A nuclear
Multiple P450 enzymes, particularly CYPs 1A2, 2B6, 2C19, and 2D6, were found
SC
7
The observation
to catalyze the formation of the reactive intermediate.
PRN was reportedly the
9
mechanism-based inactivator of CYP2A6 and CYP2B1 [16-17].
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8
The identification
of the enzymes responsible for the reactive intermediate formation, along with the
11
reactive intermediate characterization, facilitates the understanding of the mechanisms
12
of the observed PRN-induced inactivation of CYP2B6 as well as that of the two
13
enzymes reported.
14
P450 enzymes participating in the generation of the reactive intermediate is under
15
investigation.
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10
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Whether PRN is a mechanism-based inactivator of the other
In summary, our study demonstrated that PRN is a mechanism-based inactivator
17
of CYP2B6 and metabolism of PRN was required for the inactivation of CYP2B.
18
An epoxide or/and γ-ketoenal intermediate trapped by GSH was identified as the
19
reactive intermediate(s) in microsomal incubations with PRN.
20
was chemically synthesized by Oxone-mediated oxidation of PRN.
21
of the intermediate(s) may be responsible for the inactivation of CYP2B6.
AC C
16
22 20
The intermediate(s) The generation
ACCEPTED MANUSCRIPT References
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clinical relevance, Drug. Metab. Drug. Interact. 27 (2012) 185-197.
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[21] S. Miksys, C. Lerman, P.G. Shields, D.C. Mash, R.F. Tyndale, Smoking
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alcoholism and genetic polymorphisms alter CYP2B6 levels in human brain,
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Neuropharmacol. 45(2003) 122-132.
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[22] C. Sridar, C. Kenaan, P.F. Hollenberg, Inhibition of bupropion metabolism by
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selegiline: mechanism-based inactivation of human CYP2B6 and characterization of
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glutathione and peptide adducts, Drug Metab. Dispos. 40 (2012) 2256-226.
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[23] S.R. Faucette, R.L. Hawke, E.L. Lecluyse, S.S. Shord, B. Yan, R.M. Laethem,
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C.M. Lindley, Validation of bupropion hydroxylation as a selective marker of human
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cytochrome P450 catalytic activity, Drug Metab. Dispos. 28 (2000) 1222-1230.
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[24] J.G. Gerber, R.J. Rhodes, J. Gal, Stereoselective metabolism of methadone
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N-demethylation by cytochrome P450 2B6 and 2C19, Chirality. 16 (2004) 36-44.
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[25] Z. Huang, P. Roy, and D.J. Waxman, Role of human microsomal CYP3A4 and
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CYP2B6 in catalyzing N-dechlorethylation of cyclophosphamide and ifosfamide,
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Biochem. Pharmaco. l59 (2000) 961-972.
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2
cytochrome P450 2B6 (CYP2B6) is the main catalyst of efavirenz primary and
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secondary metabolism: implication for HIV/AIDS therapy and utility of efavirenz as a
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substrate marker of CYP2B6 catalytic activity, J. Pharmcol. Exp. Ther. 306 (2003)
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287-300.
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[27] G. Lin, J. Tang, X.Q. Liu, Y. Jiang, J. Zheng, Deacetylclivorine: a
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gender-selective metabolite formed in female SD rat liver microsome, Drug Metab.
8
Disp. 35 (2007) 607-613.
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[28] R.B. Silverman, Mechanism-based enzyme inactivation, in: D.L. Purich (ed),
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contempoary enzyme kinetics and mechanism, Acafemic Press, San Diego, 1996, pp.
11
291-335.
12
[29] C.D. Klaassen, M.O. Amdur, and J. Doull, Biotransformation of xenobiotics, in:
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McGraw-Hill (Eds.), Casarett and Doull’s Toxicology, the Basic Science of Poisons,
14
New York, 1986, pp. 113-186.
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[30] M. K. Ute, M. I. Jushchyshyn, P. F. Hollenberg, Mechanism-based inactivators as
16
probes of cytochrome P450 structure and function, Curr. Drug Metab. 2 (2001) 215
17
-243.
18
[31] K.L. Kunze, and W.F. Trager, Isoform-selective mechanism-based inhibition of
19
human cytochrome P450 1A2 by furafylline, Chem. Res. Toxicol. 6 (1993) 649-656.
21
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ACCEPTED MANUSCRIPT 1
Footnotes. This work was supported in part by the National Natural Science
2
Foundation of China [Grants 81430086 and 81373471].
3
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4 5 6
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ACCEPTED MANUSCRIPT 1
Scheme Legends
2
Scheme 1. Proposed pathways for the formation of reactive intermediate(s) and GSH
3
adducts during metabolism of PRN.
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4
Figure Legends
6
Fig. 1. (A) Time- and concentration-dependent inactivation of CYP2B6 by PRN.
7
Recombinant human CYP2B6 was incubated with PRN at concentrations of 0 (●), 40
8
(■), 80 (▲), 120 (□), 160 (△), and 200 (○) µM in the presence of NADPH at 30 °C
9
for 0, 3, 6, and 9 min.
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5
Aliquots of incubation mixtures were transferred to the
10
secondary incubation mixtures for the determination of residual enzymatic activities.
11
The residual enzymatic activities at 0 min were normalized to 100% at each
12
concentration.
13
Double reciprocal plot of the rates of inactivation as a function of PRN concentrations.
14
The observed inactivation rate constant kobs was calculated from the slope of the
15
regression lines shown in Figure. 1A.
16
CYP2B6 by PRN.
17
absence (▲) or presence (■) of NADPH.
18
Fig. 2. Substrate protection against inactivation of CYP2B6 by PRN.
19
incubated with vehicle (●) and PRN (80 µM) in the absence (■) or presence of
20
ticlopidine (200 µM) (▲).
21
Fig. 3. Partition ratio determination for CYP2B6 inactivation by PRN.
(B)
TE D
Each data represent the average of four separate experiments.
EP
(C) NADPH-dependent inactivation of
AC C
CYP2B6 was incubated with vehicle (●) and PRN (80 µM) in the Data represent mean±SD (n=3).
CYP2B6 was
Data represent mean±SD (n=3).
1
CYP2B6 was
ACCEPTED MANUSCRIPT 1
incubated with PRN at various concentrations.
The extrapolated P+1 was
2
determined from the point of intersection to the abscissa.
3
(n=3).
4
Fig. 4. (A) Extracted ion (m/z 510→381) chromatogram obtained from LC-Q-Trap
5
MS analysis of microsomal incubations containing PRN. (B) MS/MS spectrum of
6
PRN-GSH conjugate generated in microsomal incubation.
7
510→381) chromatogram obtained from LC-Q-Trap MS analysis of chemical
8
oxidation of PRN trapped by GSH.
9
conjugate.
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Data represent mean±SD
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(C) Extracted ion (m/z
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(D) MS/MS spectrum of synthetic PRN-GSH
Fig. 5. (A) Extracted ion (m/z 512→383) chromatogram obtained from LC-Q-Trap
11
MS analysis of the chemical oxidation reaction followed by reaction with NaBH4.
12
(B) MS/MS spectrum of the product in the reductive reaction.
13
Fig. 6. Recombinant human P450 enzymes involved in the formation of
14
PRN-conjugate.
15
presence of NADPH and GSH.
17
EP
PRN was incubated with individual human P450 enzymes in the
AC C
16
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10
Data represent mean±SD (n=3).
Supplementary material
Fig. S1. MS/MS spectrum of PRN.
18 19 20
2
ACCEPTED MANUSCRIPT Scheme 1
O
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O
O
1
P450
O
H
O
O
O
GSH
O
O
GSH
O
O
OH
SG 4
EP AC C
O
O
O
SG
5
TE D
OH
OH H
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O
O
3
2
O
O
SC
O
O
SG
6
OH H
NaBH4
O
O
OH SG 7
ACCEPTED MANUSCRIPT
AC C
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Figure. 1
ACCEPTED MANUSCRIPT
2.1
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1.9
1.7
1.5 3
Time (min) 6
AC C
EP
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0
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Log% Remaining Activity
Figure. 2
9
ACCEPTED MANUSCRIPT
AC C
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Figure. 3
ACCEPTED MANUSCRIPT Figure. 4 OH
1.0e4 2.0
4.0
C
6.0 5.54
8.0
10.0 12.0 14.0
D 9.7e4
-H2O
345.0
510.2
8.0e4
2.0e4
6.0e4
1.6e4 1.2e4
4.0e4
8000.0
2.0e4
2.0
4.0
6.0 8.0 10.0 12.0 14.0 Time (min)
AC C
EP
TE
D
4000.0 0.0
363.0
0.0 50 100 150 200 250 300 350 400 450 500 550
M AN U
4000.0 0.0
-H2O
381.2 317.0363.0 435.0 203.1259.9 417.2 162.0 233.0 345.0
2.0e4
8000.0
O
381.2
3.0e4
1.2e4
2.7e4 2.4e4
COOH
4.0e4
1.6e4
NH
NH
5.0e4
2.0e4
NH2
-H O 435.0 2 417.2 COOH 510.1
RI PT
5.8e4
162.0
S
O
SC
2.7e4 2.4e4
B
5.50
O
O
203.1
A
O
0.0
363.0 317.1 381.2 435.0 203.1259.8 162.0 345.0 417.2 492.1
50 100 150 200 250 300 350 400 450 500 550 m/z, Da
ACCEPTED MANUSCRIPT
AC C
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Figure. 5
ACCEPTED MANUSCRIPT Figure. 6
80
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60 40 20 0 2A6
2B6
2C9
2C19
2D6
2E1
AC C
EP
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M AN U
1A2
SC
%(Formation of GSH-conjugate)
100
3A4
3A5
ACCEPTED MANUSCRIPT Supplementary material Figure.S1 115.0
130.8
1.10e8
RI PT
1.00e8 9.00e7 8.00e7
6.00e7
5.00e7
76.9
4.00e7
SC
Intensity, cps
103.0
7.00e7
89.0
2.00e7
159.1
88.2 86.4 70
80
124.1 127.2
90
100
110
120 130 m/z, Da
AC C
EP
TE
D
60
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95.0
1.00e7 0.00 50
187.3
143.0
3.00e7
140
150
160
170
180
190
200
ACCEPTED MANUSCRIPT Psoralen (PRN) is a mechanism-based inactivator of CYP2B6. A γ-ketoenal intermediate was identified in rat liver microsomes after exposed to PRN. The γ-ketoenal intermediate may be responsible for the enzyme inactivation.
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CYPs 1A2, 2B6, 2C19, and 2D6 are the major enzymes responsible for the metabolic activation of PRN.
S0009-2797(15)30048-X
DOI:
10.1016/j.cbi.2015.08.020
Reference:
CBI 7450
To appear in:
Chemico-Biological Interactions
Received Date: 26 May 2015 Revised Date:
15 July 2015
Accepted Date: 28 August 2015
Please cite this article as: L. Ji, D. Lu, J. Cao, L. Zheng, Y. Peng, J. Zheng, Psoralen, a mechanismbased inactivator of CYP2B6, Chemico-Biological Interactions (2015), doi: 10.1016/j.cbi.2015.08.020. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Psoralen, a mechanism-based inactivator of CYP2B6
1 2 3 4
Lin Ji1, Dan Lu1, Jiaojiao Cao1, Liwei Zheng1, Ying Peng1,*, and Jiang Zheng2, 3,*
5
1
6
of Ministry of Education, Shenyang Pharmaceutical University, Shenyang, Liaoning,
7
110016, P. R. China
8
3
9
Division of Gastroenterology and Hepatology, Department of Pediatrics, University of
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Washington School of Medicine, Seattle, WA 98101
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17 18 19 20
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Center for Developmental Therapeutics, Seattle Children’s Research Institute,
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College of Pharmacy, 2Key Laboratory of Structure-Based Drug Design & Discovery
21 22 23 24 1
ACCEPTED MANUSCRIPT Corresponding Authors:
3
Jiang Zheng, PhD
4 5 6 7 8 9
Center for Developmental Therapeutics, Seattle Children's Research Institute, Division of Gastroenterology and Hepatology, Department of Pediatrics, University of Washington, Seattle, WA 98101 Key Laboratory of Structure-Based Drug Design & Discovery of Ministry of Education, Shenyang Pharmaceutical University, Shenyang, Liaoning, 110016, P. R. China Email: [email protected]
11
Tel: 206-884-7651; Fax: 206-987-7660.
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1 2
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Ying Peng, PhD
14 15
School of Pharmacy, Shenyang Pharmaceutical University, Shenyang, Liaoning, 110016, P. R. China
16
Email: [email protected]
17
Tel: +86-24-23986361; Fax: +86-24-23986510.
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ACCEPTED MANUSCRIPT Abbreviations: PRN, psoralen; DMSO, dimethyl sulfoxide; GSH, glutathione;
2
NADPH, β-nicotinamide adenine dinucleotide 2′-phosphate reduced tetrasodium salt;
3
RLMs, rat liver microsomes; SOD, superoxide dismutase; LC, liquid chromatography;
4
MS, mass spectrometry; LC-MS/MS, liquid chromatography coupled to tandem mass
5
spectrometry; MRM, multiple-reaction monitoring; CE, collision energy; EPI,
6
enhanced product ion; IDA, information-dependent acquisition
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ACCEPTED MANUSCRIPT 1 2
Abstract Furanocoumarin compound psoralen (PRN) is a major active ingredient found in
3
herbaceous plants.
PRN has been used for the treatment of various dermal diseases
4
in China.
5
(CYP2B6)
6
NADPH-dependent inactivation of CYP2B6 with the values of KI and kinact being
7
110.2 µM and 0.200 min-1, respectively.
8
prevented the enzyme from the inactivation induced by PRN.
9
nucleophile glutathione (GSH) and catalase/superoxide dismutase showed limited
that
PRN
induced
a
time-,
concentration-,
and
Ticlopidine, a CYP2B6 substrate,
SC
found
Exogenous
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and
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We evaluated the inhibitory effect of PRN on cytochrome P450 2B6
10
protection of CYP2B6 from the inactivation.
11
inactivation was approximately 400.
12
epoxide or/and γ-ketoenal intermediate was formed in microsomal incubations with
13
PRN.
14
CYP2B6.
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GSH trapping experiments indicates that an
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In summary, PRN was characterized as a mechanism-based inactivator of
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15
The estimated partition ratio of the
4
ACCEPTED MANUSCRIPT 1 2
1. Introduction Psoralen (PRN) is a coumarin derivative fused with a furan and is the core of furanocoumarins widely found in nature.
4
of legumes Psoralea corylifolia L, as well as in the common fig, celery, parsley and in
5
all citrus fruits.
6
double teeth angelica root and coastal glehnia root [1].
7
herbal formulas such as Wenweishu tables, Sishen pills, and Yaotong pills, and it is
8
also used in ultraviolet light therapy for psoriasis, eczema, and vitiligo [2].
9
reported pharmacological activities of PRN included anti-inflammatory and
10
antipyretic, antibacterial, antiviral, hepato-protective, female hormone like effects
11
[3-7].
12
in four cancer cell lines, including KB, KBv200, K562, and K562/ADM [8]. PRN
13
has attracted much attention, due to its potential to be a pharmaceutical agent and
14
wide occurrence in nature.
PRN is also found in many traditional Chinese medicines, such as
The
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PRN has been widely used in
TE D
In addition, PRN was reported to exhibit a dose-dependent anticancer activity
A number of furanocoumarin compounds have been reported to exhibit inhibition
EP
15
PRN mainly occurs in the fruits and seeds
RI PT
3
of cytochrome P450s (CYPs).
17
inhibitory effects on CYP1A2 [1, 9-10].
18
tin, major components of grapefruit, are reportedly mechanism-based inactivators of
19
CYP3A4 [11-12].
20
mechanism-based inactivators of CYP2B6 [13-15].
21
exhibited mechanism-based inactivation of CYP2A6 [16].
22
its derivatives, including 8-methoxypsoralen, 5-methoxypsoralen, 5-hydroxypsoralen,
AC C
16
PRN and its isomer isopsoralen were found to show Bergamottin and 6’,7’-dihydroxybergamot-
Bergamottin, imperatorin, and isoimperatorin were claimed as
5
PRN and 8-methoxypsoralen Additionally, PRN and
ACCEPTED MANUSCRIPT 1
8-hydroxypsoralen,
bergapten,
and
isopimpinellin,
2
mechanism-based inactivators of CYP2B1 [17-18].
were
found
to
be
CYP2B is an important member of the P450 family, and the content of CYP2B6
4
ranges from 2 to 10% of the whole P450 [19]. With the contribution of 3-6 % of
5
total hepatic P450 content [20], CYP2B6 is also found in extrahepatic tissue, such as
6
brain, kidney, and heart.
7
including biotransformation of endogenous and exogenous substances [21].
8
CYP2B6 is responsible for the metabolism of more than 4% of clinically used drugs,
9
for example, bupropion, efavirenz, methadone, ifosfamide, and cyclophosphamide
SC
The enzyme participates in a variety of metabolic reactions
M AN U
10
RI PT
3
[22-26].
With the rapidly growing global interest in the use of natural products as medical
12
remedies and dietary supplements, much attention has been paid to drug-drug
13
interactions associated with natural product-mediated inhibition of cytochromes P450
14
enzymes.
15
PRN with CYP2B6, to characterize the reactive metabolites of PRN, and to identify
16
the P450 enzymes responsible for metabolic activation of PRN.
EP
The objectives of the present studies were to investigate the interaction of
AC C
17
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11
6
ACCEPTED MANUSCRIPT 1
2. Materials and Methods
2
2.1 Chemicals and Materials Psoralen (98% purity), superoxide dismutase (SOD), and catalase were obtained
4
from Shanghai Yuanye Biological Technology Co., Ltd (Shanghai, China).
5
Glutathione (GSH), hexyl glutathione, bupropion, Oxone, and NADPH were acquired
6
from Sigma-Aldrich (St. Louis, MO).
7
purchased from BD Gentest (Woburn, MA).
8
Scientific (Springfield, NJ).
9
(Hangzhou, China).
Recombinant human P450 enzymes were
SC
All organic solvents were from Fisher
Distilled water was purchased from Wahaha Co. Ltd
M AN U
10
RI PT
3
All solvents and reagents were either analytical or HPLC grade.
2.2 Time-, Concentration-, and NADPH-Dependent Inactivation of CYP2B6 by PRN The composition of the primary incubation mixtures contained CYP2B6 (0.1 µM),
12
MgCl2 (3.2 mM), and PRN at concentrations of 0, 40, 80, 120, 160, or 200 µM in
13
potassium phosphate buffer (pH 7.4) with a total volume of 0.2 mL.
14
components for the primary incubations were mixed at 4 °C and vortexed quickly.
15
The primary incubations were performed at 30 °C and preincubated for 3 min.
The
16
reactions were initiated by addition of NADPH (final concentration: 1.0 mM).
At
17
time points of 0, 3, 6, and 9 min, aliquots (40 µL) of the primary incubation mixtures
18
were transferred to the secondary incubation mixtures containing bupropion (100 µM)
19
and NADPH (0.45 mM) in 0.1 M potassium phosphate buffer (pH 7.4).
The reason
20
for
was
21
our preliminary study showed that a significant spontaneous CYP2B6 activity loss
22
took
AC C
EP
TE D
11
choosing
place
30
at
37
°C
°C
for
in
the
9
enzyme
min 7
in
incubations
the
absence
of
The
that
PRN,
ACCEPTED MANUSCRIPT but no such enzyme activity loss occurred at 30 °C in the same period of time.
The
2
secondary incubation mixtures were further incubated at 30 °C for 30 min, followed
3
by addition of ice-cold acetonitrile (120 µL) containing propranolol as internal
4
standard.
After vortexing for 3 min, the mixtures were centrifuged at 16,000 rpm for
5
10 min.
The supernatants were subjected to LC-MS/MS analysis. To ensure whether
6
the enzyme inactivation is NADPH-dependent, PRN (80 µM) and CYP2B6 were
7
incubated in the absence of NADPH as negative control in the primary incubation.
8
2.3 Substrate Protection
M AN U
SC
RI PT
1
9
PRN (80 µM) and ticlopidine (at mole ratio of 1:2.5) were included in the
10
primary reaction mixtures to study the substrate protection from PRN-induced
11
inactivation of CYP2B6.
12
min.
13
mixtures were transferred to the secondary incubation mixtures for the determination
14
of bupropion hydroxylase activities of CYP2B6.
15
PRN or ticlopidine were performed in parallel.
16
2.4 Effects of GSH and Catalase/SOD on the Inactivation of CYP2B6
Control incubations lacking of
EP
TE D
After incubation at 30 °C for 0, 3, and 9 min, aliquots (40 µL) of the primary
AC C
17
The primary mixtures were preincubated at 30 °C for 3
The primary reaction mixtures containing CYP2B6 (0.1 µM), PRN (80 µM), and
18
GSH (2.0 mM) were preincubated at 30 °C for 3 min.
The reactions were initiated
19
by addition of NADPH (1.0 mM).
20
transferred to the secondary incubation mixtures to determine the residual enzyme
21
activities.
At the time of 9 min, aliquots (40 µL) were
In control samples, phosphate buffer with an equal volume was in place
8
ACCEPTED MANUSCRIPT 1
of GSH solution.
2
NADPH in the presence or absence of a mixture of catalase and superoxide dismutase
3
(SOD).
4
enzyme per milliliter.
5
2.5 Partition Coefficient
RI PT
The concentrations of SOD and catalase used were 800 units of each
To determine the partition ratio, CYP2B6 (0.1 µM) was mixed with PRN at
SC
6
In a separate study, CYP2B6 was incubated with PRN and
concentrations of 0, 10, 20, 80, 160, 200, and 300 µM.
NADPH at a final
8
concentration of 1.0 mM was added to initiate the reactions.
The incubations were
9
accomplished after 9 min at 30 °C in a water bath.
M AN U
7
At 0 and 9 min, aliquots (40 µL)
10
were withdrawn and transferred to the secondary incubations for determination of
11
CYP2B6 activities.
12
2.6 Irreversibility of Inhibition
TE D
Incubations that lacked NADPH served as negative controls.
Primary incubations containing PRN (80 µM) and CYP2B6 (0.1 µM) were
14
performed in the presence of NADPH at 30 °C, along with the control incubations
15
lacking of PRN.
16
were withdrawn and transferred into the secondary mixtures for 30 min incubations
17
(non-dialyzed samples), and then the primary incubations were dialyzed using
18
Slide-A-lyzer membranes (molecular mass cut off: 3,500 Da, Pierce, Rockford, IL)
19
against 0.1 M potassium phosphate buffer (pH 7.4, 2 × 3 h) at 4 °C.
20
samples were brought to room temperature and were withdrawn -(40 µL) into the
21
secondary incubations.
EP
13
AC C
At 0 and 9 min, aliquots of the control and inactivated samples
The dialyzed
After incubation for 30 min, the enzyme activities of the
9
ACCEPTED MANUSCRIPT 1
resulting samples were assessed as described below.
2
2.7 CYP2B6 Assay
3
To assess the activity of CYP2B6，the production of hydroxybupropion from bupropion was monitored by LC-MS/MS.
The HPLC-MS/MS system consisted of
5
an ekspert ultraLC 100 system (AB SCIEX, Foster City, CA) and a 4000 hybrid triple
6
quadrupole linear ion trap tandem mass spectrometer (AB SCIEX, Foster City, CA)
7
equipped with an electrospray ionization (ESI) interface.
8
separation was performed on a C18 column (2.1 × 50 mm, 2.6 µm, ThermoFisher,
9
Pittsburgh, PA) at the temperature of 30 °C.
SC
RI PT
4
M AN U
The chromatographic
The gradient elution phase was
10
composed of 0.1% formic acid in acetonitrile (A) and 0.1% formic acid in water (B)
11
with the flow rate of 0.3 mL/min.
12
directed to waste for the initial 1.0 min via a divert valve and then turned directly into
13
the mass spectrometer.
14
1.0-1.5 min, 10-30% A; 4.0-5.5 min, 30-10% A; and 5.5-8.0 min, 10% A.
15
injection volume was 5 µL.
16
the positive ion detection.
17
to monitor the transition of m/z 256.0 protonated precursor ion to m/z 238.0 product
18
ion for product hydroxybupropion and m/z 260.7 protonated precursor ion to m/z
19
116.3 product ion for internal standard propranolol.
20
2.8 Reactive Intermediate Trapping by GSH
21
TE D
The eluates from the analytical column were
The gradient elution was set as follows: 0-1.0 min, 10% A;
AC C
EP
The
The mass spectrometer was operated with ESI source in
The multiple reaction monitoring (MRM) mode was used
PRN (80 µM), GSH (1.0 mM), and rat liver microsomes (1.0 mg protein/mL)
10
ACCEPTED MANUSCRIPT prepared in our lab [27] or individual human recombinant P450 enzymes, including
2
CYPs 1A2, 2A6, 2B6, 2C9, 2C19, 2D6, 2E1, 3A4, or 3A5 (0.1 µM for each) were
3
incubated in the presence or absence of NADPH (1.0 mM) at 37 °C for 60 min.
4
Equal volume of ice-cold acetonitrile containing hexyl glutathione as internal standard
5
was mixed with the final reactions, followed by vortexing and centrifuging.
6
supernatants were dried by blowing with a steam of nitrogen.
7
reconstituted with 50% acetonitrile (50 µL) and submitted to LC-MS/MS for analysis.
8
An Accuore C18 column (4.6 × 150 mm, 5 µm, ThermoFisher, Pittsburgh, PA) was
9
employed to separate the GSH conjugates with the solvent system consisted of 0.1%
10
formic acid in acetonitrile (A) and 0.1% formic acid in water (B), and the flow rate
11
was 0.8 mL/min.
12
min, 10-90% A; 8.0-10 min, 90% A; 10-13 min, 90-10% A; and 13-15 min, 10% A.
13
Under positive ESI condition, the PRN-GSH conjugates (m/z 510→381) and internal
14
standard hexyl glutathione (m/z 392→246) were monitored in MRM mode,
15
respectively.
16
trigger the enhanced product ion (EPI) scans by analyzing MRM signals.
17
was aimed at initiating acquisition of EPI spectra for ions exceeding 500 cps with
18
exclusion of former target ions after three occurrences for 10s.
19
EPI scan was set at a scan range for product ions from m/z 50 to 650.
20
scanning parameters are listed with the scan mode: profile; step size: 0.08 Da; and
21
scan rate: 1000 Da/s, 5 ms pause between mass ranges.
22
2.9 Chemical Synthesis of PRN-GSH Conjugates
RI PT
1
The
M AN U
SC
The residue was
TE D
The gradient elution was set as follow: 0-2.0 min, 10% A; 2.0-8.0
AC C
EP
The information-dependent acquisition (IDA) method was employed to
11
The IDA
In positive mode, the The EPI
ACCEPTED MANUSCRIPT 1
Saturated sodium bicarbonate solution (40 µL) and Oxone (4.5 mg) were mixed
2
with PRN (1.72 mg) dissolved in acetone (200 µL).
3
room temperature, GSH (24 mg) was added with further stirring for 1 h at room
4
temperature, followed by centrifugation.
5
two portions.
6
mixed with sodium borohydride (7 mg, NaBH4).
7
vortexed for 5 min and submitted to LC-MS/MS analysis.
One was subjected to LC-MS/MS analysis directly.
SC
10 11
17 18
EP
16
AC C
15
TE D
12
12
The other was
The final mixture was gently
M AN U
9
14
RI PT
The supernatant was equally allocated into
8
13
After being stirred for 30 min at
ACCEPTED MANUSCRIPT 1
3. Results
2
3.1 Time-, Concentration-, and NADPH-Dependent Inactivation of CYP2B6 by PRN
3
The remaining activities of CYP2B6 were monitored by quantifying the amount of hydroxybupropion produced in the secondary incubations.
The residual
5
enzymatic activities of each concentration at 0 min were normalized to 100%.
6
1A and 1C were prepared by a semilogarithmic plot of percent remaining activity vs
7
incubation time.
8
activity in the absence of PRN (control group).
9
and NADPH-dependent inhibition of CYP2B6 activity.
RI PT
4
Fig.
SC
As shown in Fig. 1A and 1C, CYP2B6 retained the catalytic
M AN U
PRN caused a time-, concentration-, In the incubations
10
containing PRN (200 µM), about 60% of CYP2B6 activity was suppressed.
11
double-reciprocal plot (Wilson plot) of values for the observed rates of inactivation
12
(kobs) and PRN concentrations was employed to calculate the kinetic constants KI (a
13
concentration of PRN needed for half-maximal inactivation) and kinact (maximal rate
14
constant for inactivation).
15
0.200 min-1, respectively (Fig .1B).
16
3.2 Substrate Protection
TE D
As a result, KI and kinact were found to be 110.2 µM and
EP
AC C
17
A
Ticlopidine, a CYP2B6 substrate, was used to study the PRN-dependent
18
inactivation of CYP2B6.
In the primary incubations, PRN and ticlopidine were
19
added at the molar ratio of 1:2.5.
20
inactivation of CYP2B6 induced by PRN with the residual CYP2B6 activities of
21
75.7±1.5% and 66.2±2.2% at 3 and 9 min, while the remaining activities of CYP2B6
22
were 70.8±2.7% and 40.6±1.3% in the absence of ticlopidine in the primary
The presence of ticlopidine attenuated the
13
ACCEPTED MANUSCRIPT 1
incubations.
2
CYP2B6 induced by PRN (Fig. 2).
3
3.3 Effects of GSH and Catalase/Superoxide Dismutase After incubation for 9 min with PRN (80 µM) and NADPH, the remaining
RI PT
4
This indicates the protection effect of ticlopidine on the inactivation of
CYP2B6 activity was 51.3±2.7%.
Inclusion of GSH (2 mM), an electrophile
6
trapping agent, showed limited protective effect on CYP2B6 from the inactivation
7
with residual CYP2B6 activity of 55.6±2.3%.
8
superoxide dismutase (SOD) acted as scavengers of reactive oxygen species produced
9
slight protection against the inactivation of CYP2B6 by PRN with the remaining
In addition, a mixture of catalase and
M AN U
enzyme activity of 57.8±3.4% at 9 min.
11
3.4 Partition coefficient
TE D
10
12
SC
5
The partition ratio (P value) was estimated graphically using the previously published method [28] as shown in Figure. 3.
14
of molecules of PRN metabolized per molecule of CYP2B6 inactivated.
15
shows a plot of the percentage remaining activity vs the PRN/CYP2B6 molar ratio.
16
The turnover number (P+1) was about 401, and the extrapolated partition ratio of
17
PRN was approximately 400.
18
further inactivate below about 30.8±2.8%.
19
3.5 Irreversibility of Inhibition
20 21
The value of the P means the number
AC C
EP
13
The figure
The ratio of PRN to CYP2B6 above 800 did not
By determining the remaining activities of the enzyme before and after dialysis, we investigated the irreversibility of CYP2B6 inhibition. 14
After the incubation of
ACCEPTED MANUSCRIPT 1
CYP2B6 with PRN (80 µM) at 30 °C for 9 min, the remaining CYP2B6 activity was
2
8.0% of control 0 min.
3
recovered after dialysis.
4
3.6 Reactive Metabolite Trapping
RI PT
The CYP2B6 activity was only 7.5% of control 0 min
To trap the reactive metabolites, GSH was incorporated in the PRN incubation
6
systems including rat liver microsomes or individual recombinant P450 enzymes in
7
the presence or absence of NADPH, followed by LC-MS/MS analysis.
8
GSH conjugate as a peak with protonated molecular ion [M+H]+ at m/z 510 at
9
retention time of 5.5 min was observed (Fig. 4A), while no such peak was found in
M AN U
SC
5
The major
10
the absence of NADPH as the control sample (data not shown).
11
scanning mode with the ion transition m/z 510/381 was employed on identifying the
12
MS/MS spectrum of the conjugate.
13
m/z 510, the product ions at m/z 435 and 381 observed indicate the neutral losses of
14
glycinyl portion (-75 Da) and γ-glutamyl portion (-129 Da) which represent the
15
cleavage of the characteristic fragment ions of the GSH moiety.
16
product ion at m/z 203 was detected, resulting from the cleavage of S-C (PRN site)
17
bond of the PRN-GSH conjugate.
18
higher than that of the protonated molecular ion (m/z 187, Fig. S1), indicating an
19
insertion of an oxygen in the PRN moiety of the GSH conjugate.
20
The MRM-EPI
AC C
EP
TE D
As shown in Fig. 4B, based on the parent ion
In addition, a
The mass of the observed product ion was 16 Da
In order to verify the characterization, PRN was chemically oxidized by Oxone in
21
acetone before reacting with GSH.
As expected, the product formed in the
22
Oxone-based reaction showed identical chromatographic and mass spectrum 15
ACCEPTED MANUSCRIPT 1
behaviors (Fig. 4C and 4D) as that of the PRN-derived GSH conjugate generated in
2
the microsomal systems (Fig. 4A and 4B).
3
other portion of the reaction mixtures for chemical reduction.
4
depicted a new peak at retention time of 5.4 min (Fig. 5A) with the m/z of [M+H]+
5
512 (Fig. 5B), which was 2 Da higher than the conjugate detected in the mixture
6
without the treatment with sodium borohydride.
7
3.7 P450 Enzymes Responsible for PRN Bioactivation
9
SC
RI PT
LC-MS/MS analysis
PRN was incubated with individual recombinant human P450 enzymes, including
M AN U
8
Sodium borohydride was added to the
CYPs 1A2, 2A6, 2B6, 2C9, 2C19, 2D6, 2E1, 3A4, or 3A5 (0.1 µM for each).
GSH
was included in the incubations as the trapping agent.
11
analyzed and quantified by the LC-MS/MS method.
12
2B6, 2C19, and 2D6 were the major enzymes involved in the formation of the
13
reactive metabolite.
EP AC C
14
TE D
10
16
The PRN-GSH conjugate was
As shown in Fig. 6, CYPs 1A2,
ACCEPTED MANUSCRIPT 1
4. Discussion
2
The present study demonstrated that PRN inhibited CYP2B6 in time- and
3
concentration-dependent manners with the characteristic (in enzyme kinetics) of
4
mechanism-based inactivation.
5
absence of NADPH in the primary incubations, indicating the critical role of
6
metabolism in PRN-induced CYP2B6 inactivation.
7
instead of the parent compound exerted the inhibitory action.
8
PRN-induced enzyme inhibition is reversible was probed by determining the enzyme
9
activities of PRN-treated CYP2B6 before and after dialysis.
RI PT
No such enzyme inactivation was observed in the
M AN U
SC
This implies that metabolite(s) Whether the
Limited enzyme
activities were recovered after the dialysis, possibly due to the protein covalent
11
modification by reactive metabolite(s) of PRN. This provided the further evidence
12
for mechanism-based inactivation of CYP2B6 by PRN.
13
TE D
10
GSH is a nucleophilic agent, and it is often used to react with reactive electrophilic species produced.
The supplement of GSH in the enzyme incubations
15
revealed little protection of CYP2B6 against PRN-induced inactivation.
16
indicates that CYP2B6 was covalently modified by electrophilic metabolites of PRN
17
before escaping from the active site of the host enzyme and the modification of
18
protein induced the inactivation of the enzyme.
19
capable of quenching superoxide anion, hydrogen peroxide, and other reactive oxygen
20
species which are potential agents to inactivate enzymes [29].
21
that SOD/catalase showed minor protective effect on CYP2B6 from the enzyme
AC C
EP
14
17
This
SOD and catalase are enzymes
The results showed
ACCEPTED MANUSCRIPT 1
inactivation, indicating that reactive oxygen species did not make significant
2
contribution to the enzyme inactivation. The presence of ticlopidine in the enzyme incubation system was found to
3
attenuate the PRN-induced CYP2B6 inactivation (Fig. 2).
5
ticlopidine and PRN competed to bind to the active site of CYP2B6, resulting in
6
decreased generation of reactive metabolites of PRN responsible for covalent
7
modification of the enzyme.
8
that bioactivation of PRN occurred in the active site of CYP2B6.
SC
The observed protective effect of ticopidine indicates
M AN U
9
We speculated that
RI PT
4
The partition ratio reflects the efficiency of enzyme inactivator.
The smaller the
10
number is, the more efficient the inactivator is [30].
11
400.
12
enzymes ranged from 3 (very highly efficient inactivators) to >1000 (inefficient) [30].
13
Compared
14
4’,5’-dihydro-8-methoxypsoralen (partition ratio: 840) [16], PRN (partition ratio: 400)
15
may be classified as a moderately efficient inactivator.
furafylline
(partition
ratio:
3.8–5.6)
[31]
and
EP
with
TE D
Reported P values in literatures for mechanism-based inactivators of P450
Metabolic formation of reactive metabolites is an essential step for
AC C
16
The P value of PRN was about
17
mechanism-based
enzyme
inactivation.
18
intermediates 2 and 3 as depicted in Scheme 1, were proposed to respond the
19
inactivation of CYP2B6.
Intermediate 2 is a furanoepoxide derivative, resulting
20
from epoxidation of PRN.
Intermediate 3 is a γ-ketoenal formed by direct oxidation
21
of PRN or/and sigmatropic rearrangement of furanoepoxide 2.
22
structures of the reactive metabolites, we trapped the electrophilic species with GSH 18
Two
reactive
intermediates,
i.e.
To characterize the
ACCEPTED MANUSCRIPT 1
in the microsomal incubations.
A PRN-derived GSH conjugate was detected by
2
LC-MS/MS.
3
molecular weight of GSH conjugates 4-6 (Scheme 1).
4
metabolite showed the indicative characteristic fragments of GSH, such as neutral
5
losses of 75 (glycinyl) and 129 Da (γ-glutamyl).
6
responsible for molecular formula of C9H6O3+ was observed (Fig. 4B and 4D),
7
implicating the opening of the furan ring (Scheme 1).
8
formation of GSH conjugate 4, since the conjugate is a thioacetal and relatively stable
9
against hydrolysis.
The detected protonated molecular ion [M+H]+ (m/z 510) matched the
RI PT
The MS/MS spectrum of the
A fragment ion at m/z 162
M AN U
SC
This allows us to exclude the
Only GSH conjugate 6 has the ring opening structure, and
conjugate 5 (a hemiacetal) and conjugate 6 could be converted to each other.
11
verify the structure of conjugate 6, we chemically reduced the conjugate with sodium
12
borohydride, followed by LC-MS/MS analysis.
13
512 (Fig. 5B) was detected by LC-MS/MS.
14
matched the molecular weight of conjugate 7 (Scheme 1) whose molecular ion was
15
2.0 Da higher than that of the one detected before the reductive reaction.
16
comparison with the MS/MS spectra of conjugates 6 and 7, the two conjugates shared
17
a same fragment ion of m/z 162 in response to molecular formula C9H6O3+ (Fig. 4B
18
and 5B).
19
reductive reaction.
20
4B) of conjugate 6 was responsible for the fragment which contained the aldehyde
21
group.
22
m/z 205 was instead observed by LC-MS/MS (Fig. 5B).
To
A new product with [M+H]+ at m/z
The molecular ion of the product
In
AC C
EP
TE D
10
This implies no reduction occurring within the coumarin ring during the In addition, the ion of m/z 203 observed in the MS spectrum (Fig.
After the reductive reaction, no such ion was detected, and a fragment ion of
19
This possibly resulted from
ACCEPTED MANUSCRIPT 1
the reduction of the aldehyde group to the corresponding alcohol.
2
further excludes the formation of conjugate 4, since the thioacetal group of the
3
conjugate should be stable enough to resist the reductive reaction.
4
magnetic resonance spectrum is needed to fully characterize the conjugate.
5
Unfortunately, the low yield of the reaction made us unable to obtain enough amount
6
of the product for NMR characterization.
RI PT
A nuclear
Multiple P450 enzymes, particularly CYPs 1A2, 2B6, 2C19, and 2D6, were found
SC
7
The observation
to catalyze the formation of the reactive intermediate.
PRN was reportedly the
9
mechanism-based inactivator of CYP2A6 and CYP2B1 [16-17].
M AN U
8
The identification
of the enzymes responsible for the reactive intermediate formation, along with the
11
reactive intermediate characterization, facilitates the understanding of the mechanisms
12
of the observed PRN-induced inactivation of CYP2B6 as well as that of the two
13
enzymes reported.
14
P450 enzymes participating in the generation of the reactive intermediate is under
15
investigation.
TE D
10
EP
Whether PRN is a mechanism-based inactivator of the other
In summary, our study demonstrated that PRN is a mechanism-based inactivator
17
of CYP2B6 and metabolism of PRN was required for the inactivation of CYP2B.
18
An epoxide or/and γ-ketoenal intermediate trapped by GSH was identified as the
19
reactive intermediate(s) in microsomal incubations with PRN.
20
was chemically synthesized by Oxone-mediated oxidation of PRN.
21
of the intermediate(s) may be responsible for the inactivation of CYP2B6.
AC C
16
22 20
The intermediate(s) The generation
ACCEPTED MANUSCRIPT References
2
[1] X.M. Zhuang, Y.H. Zhong, W.B. Xiao, H. Li, C. Lu, Identification and
3
characterization of psoralen and isopsoralen as potent CYP1A2 reversible and
4
time-dependent inhibitors in human and rat preclinical studies, Drug Metab. Dispos.
5
41 (2013) 1914-1922.
6
[2] R.S. Stern, Psoralen and ultraviolet a light therapy for psoriasis, N. Enql. J. Med.
7
357 (2007) 682-690.
8
[3] B.C. Nadine, C.L. Delporte, R.E. Negrete, S. Erazo, A. Zuñiga, A. Pinto, and B.K.
9
Cassels,
isolated
from
SC
constituents
psoralea
glandulosa
L.
with
M AN U
Active
RI PT
1
antiinflammatory and antipyretic activities, J. Ethnopharmacol. 78 (2001) 27-31.
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2
cytochrome P450 2B6 (CYP2B6) is the main catalyst of efavirenz primary and
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ACCEPTED MANUSCRIPT 1
Footnotes. This work was supported in part by the National Natural Science
2
Foundation of China [Grants 81430086 and 81373471].
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Scheme Legends
2
Scheme 1. Proposed pathways for the formation of reactive intermediate(s) and GSH
3
adducts during metabolism of PRN.
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4
Figure Legends
6
Fig. 1. (A) Time- and concentration-dependent inactivation of CYP2B6 by PRN.
7
Recombinant human CYP2B6 was incubated with PRN at concentrations of 0 (●), 40
8
(■), 80 (▲), 120 (□), 160 (△), and 200 (○) µM in the presence of NADPH at 30 °C
9
for 0, 3, 6, and 9 min.
M AN U
SC
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Aliquots of incubation mixtures were transferred to the
10
secondary incubation mixtures for the determination of residual enzymatic activities.
11
The residual enzymatic activities at 0 min were normalized to 100% at each
12
concentration.
13
Double reciprocal plot of the rates of inactivation as a function of PRN concentrations.
14
The observed inactivation rate constant kobs was calculated from the slope of the
15
regression lines shown in Figure. 1A.
16
CYP2B6 by PRN.
17
absence (▲) or presence (■) of NADPH.
18
Fig. 2. Substrate protection against inactivation of CYP2B6 by PRN.
19
incubated with vehicle (●) and PRN (80 µM) in the absence (■) or presence of
20
ticlopidine (200 µM) (▲).
21
Fig. 3. Partition ratio determination for CYP2B6 inactivation by PRN.
(B)
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Each data represent the average of four separate experiments.
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(C) NADPH-dependent inactivation of
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CYP2B6 was incubated with vehicle (●) and PRN (80 µM) in the Data represent mean±SD (n=3).
CYP2B6 was
Data represent mean±SD (n=3).
1
CYP2B6 was
ACCEPTED MANUSCRIPT 1
incubated with PRN at various concentrations.
The extrapolated P+1 was
2
determined from the point of intersection to the abscissa.
3
(n=3).
4
Fig. 4. (A) Extracted ion (m/z 510→381) chromatogram obtained from LC-Q-Trap
5
MS analysis of microsomal incubations containing PRN. (B) MS/MS spectrum of
6
PRN-GSH conjugate generated in microsomal incubation.
7
510→381) chromatogram obtained from LC-Q-Trap MS analysis of chemical
8
oxidation of PRN trapped by GSH.
9
conjugate.
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Data represent mean±SD
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(C) Extracted ion (m/z
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(D) MS/MS spectrum of synthetic PRN-GSH
Fig. 5. (A) Extracted ion (m/z 512→383) chromatogram obtained from LC-Q-Trap
11
MS analysis of the chemical oxidation reaction followed by reaction with NaBH4.
12
(B) MS/MS spectrum of the product in the reductive reaction.
13
Fig. 6. Recombinant human P450 enzymes involved in the formation of
14
PRN-conjugate.
15
presence of NADPH and GSH.
17
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PRN was incubated with individual human P450 enzymes in the
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Data represent mean±SD (n=3).
Supplementary material
Fig. S1. MS/MS spectrum of PRN.
18 19 20
2
ACCEPTED MANUSCRIPT Scheme 1
O
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O
O
1
P450
O
H
O
O
O
GSH
O
O
GSH
O
O
OH
SG 4
EP AC C
O
O
O
SG
5
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OH
OH H
M AN U
O
O
3
2
O
O
SC
O
O
SG
6
OH H
NaBH4
O
O
OH SG 7
ACCEPTED MANUSCRIPT
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Figure. 1
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2.1
RI PT
1.9
1.7
1.5 3
Time (min) 6
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0
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Log% Remaining Activity
Figure. 2
9
ACCEPTED MANUSCRIPT
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Figure. 3
ACCEPTED MANUSCRIPT Figure. 4 OH
1.0e4 2.0
4.0
C
6.0 5.54
8.0
10.0 12.0 14.0
D 9.7e4
-H2O
345.0
510.2
8.0e4
2.0e4
6.0e4
1.6e4 1.2e4
4.0e4
8000.0
2.0e4
2.0
4.0
6.0 8.0 10.0 12.0 14.0 Time (min)
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4000.0 0.0
363.0
0.0 50 100 150 200 250 300 350 400 450 500 550
M AN U
4000.0 0.0
-H2O
381.2 317.0363.0 435.0 203.1259.9 417.2 162.0 233.0 345.0
2.0e4
8000.0
O
381.2
3.0e4
1.2e4
2.7e4 2.4e4
COOH
4.0e4
1.6e4
NH
NH
5.0e4
2.0e4
NH2
-H O 435.0 2 417.2 COOH 510.1
RI PT
5.8e4
162.0
S
O
SC
2.7e4 2.4e4
B
5.50
O
O
203.1
A
O
0.0
363.0 317.1 381.2 435.0 203.1259.8 162.0 345.0 417.2 492.1
50 100 150 200 250 300 350 400 450 500 550 m/z, Da
ACCEPTED MANUSCRIPT
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Figure. 5
ACCEPTED MANUSCRIPT Figure. 6
80
RI PT
60 40 20 0 2A6
2B6
2C9
2C19
2D6
2E1
AC C
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1A2
SC
%(Formation of GSH-conjugate)
100
3A4
3A5
ACCEPTED MANUSCRIPT Supplementary material Figure.S1 115.0
130.8
1.10e8
RI PT
1.00e8 9.00e7 8.00e7
6.00e7
5.00e7
76.9
4.00e7
SC
Intensity, cps
103.0
7.00e7
89.0
2.00e7
159.1
88.2 86.4 70
80
124.1 127.2
90
100
110
120 130 m/z, Da
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60
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95.0
1.00e7 0.00 50
187.3
143.0
3.00e7
140
150
160
170
180
190
200
ACCEPTED MANUSCRIPT Psoralen (PRN) is a mechanism-based inactivator of CYP2B6. A γ-ketoenal intermediate was identified in rat liver microsomes after exposed to PRN. The γ-ketoenal intermediate may be responsible for the enzyme inactivation.
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CYPs 1A2, 2B6, 2C19, and 2D6 are the major enzymes responsible for the metabolic activation of PRN.