Arachidonic Acid Metabolism by Human Cardiovascular CYP2J2 Is Modulated by Doxorubicin

Article abstract of DOI:10.1021/acs.biochem.7b01025

Doxorubicin (DOX) is a chemotherapeutic that is used in the treatment of a wide variety of cancers. However, it causes cardiotoxicity partly because of the formation of reactive oxygen species. CYP2J2 is a human cytochrome P450 that is strongly expressed in cardiomyocytes. It converts arachidonic acid (AA) into four different regioisomers of epoxyeicosatrienoic acids (EETs). Using kinetic analyses, we show that AA metabolism by CYP2J2 is modulated by DOX. We show that cytochrome P450 reductase, the redox partner of CYP2J2, metabolizes DOX to 7-deoxydoxorubicin aglycone (7-de-aDOX). This metabolite then binds to CYP2J2 and inhibits and alters the preferred site of metabolism of AA, leading to a change in the ratio of the EET regioisomers. Furthermore, molecular dynamics simulations indicate that 7-de-aDOX and AA can concurrently bind to the CYP2J2 active site to produce these changes in the site of AA metabolism. To determine if these observations are unique to DOX/7-de-aDOX, we use noncardiotoxic DOX analogues, zorubicin (ZRN) and 5-iminodaunorubicin (5-IDN). ZRN and 5-IDN inhibit CYP2J2-mediated AA metabolism but do not change the ratio of EET regioisomers. Altogether, we demonstrate that DOX and 7-de-aDOX inhibit CYP2J2-mediated AA metabolism and 7-de-aDOX binds close to the active site to alter the ratio of cardioprotective EETs. These mechanistic studies of CYP2J2 can aid in the design of new alternative DOX derivatives.

Full text of DOI:10.1021/acs.biochem.7b01025

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Article
Arachidonic Acid Metabolism by Human
Cardiovascular CYP2J2 is Modulated by Doxorubicin
William R. Arnold, Javier L. Baylon, Emad Tajkhorshid, and Aditi Das
Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.7b01025 • Publication Date (Web): 04 Dec 2017
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Arachidonic Acid Metabolism by Human
Cardiovascular CYP2J2 is
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Modulated by Doxorubicin
William R. Arnold1, Javier L. Baylon1,2,3, Emad Tajkhorshid*,2,3, and Aditi Das*,†,1,2,3,4
†Department of Comparative Biosciences, 1Department of Biochemistry, 2Center for Biophysics
and Computational Biology, 3Beckman Institute for Advanced Science and Technology,
4Department of Bioengineering, Neuroscience program, University of Illinois Urbanaꢀ
Champaign, Urbana IL 61801
Corresponding Author
* To whom correspondence should be addressed:
Aditi Das, Ph.D., University of Illinois UrbanaꢀChampaign, 3836 VMBSB, 2001 South Lincoln
Avenue, Urbana IL 61802, Phone: 217ꢀ244ꢀ0630. aditidas@illinois.edu
Emad Tajkhorshid, Ph.D., University of Illinois UrbanaꢀChampaign, 3009 Beckman Institute,
405 N. Mathews Avenue, Urbana IL 61801, Phone: 217ꢀ244ꢀ6914. emad@life.illinois.edu
Funding Sources
American Heart Association Scientist Development grant [15SDG25760064] (AD), NIH R01
GM1155884 (AD) and in part by the National Institutes of Health R01ꢀGM101048, U54ꢀ
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GM087519,
and P41ꢀGM104601 (ET).
All
simulations
have
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using XSEDE resources (grant MCA06N060 to ET).
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KEYWORDS. Doxorubicin, arachidonic acid, cardiotoxicity, CYP2J2, epoxyeicosatrienoic acids
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ABBREVIATIONS
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5ꢀiminodaunorubicin, 5ꢀIDN; 7ꢀdeoxydoxorubicin aglycone, 7ꢀdeꢀaDOX; arachidonic acid,
AA; cytochrome b5, CYB5; cytochrome P450, CYP; cytochrome P450 reductase, CPR;
doxorubicin, DOX; ebastine, EBS; epoxyeicosatrienoic acid, EET; hydroxyebastine, EBSꢀOH;
hydroxyeicosatetraenoic acid, HETE; liquid chromatographyꢀtandem mass spectrometry,
LC/MSꢀMS; molecular dynamics, MD; polyunsaturated fatty acid, PUFA; reactive oxygen
species, ROS; Zorubicin, ZRN.
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ABSTRACT
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Doxorubicin (DOX) is a chemotherapeutic that is used in the treatment of a wide variety of
cancers. However, it causes cardiotoxicity partly due to the formation of reactive oxygen species
(ROS). CYP2J2 is a human cytochrome P450 that is highly expressed in cardiomyocytes. It
converts arachidonic acid (AA) into four different regioisomers of epoxyeicosatrienoic acids
(EETs). Using kinetic analyses we show that AA metabolism by CYP2J2 is modulated by DOX.
We show that cytochrome P450 reductase (CPR), the redox partner of CYP2J2, metabolizes
DOX to 7ꢀdeoxydoxorubicin aglycone (7ꢀdeꢀaDOX). This metabolite then binds to CYP2J2, and
inhibits and alters the preferred site of metabolism of AA leading to change in the ratio of the
EET regioisomers. Furthermore, molecular dynamics (MD) simulations indicate that 7ꢀdeꢀaDOX
and AA can concurrently bind to the CYP2J2 active site to produce these changes in the site of
AA metabolism. To see if these observations are unique to DOX/7ꢀdeꢀaDOX, we use nonꢀ
cardiotoxic DOX analogues, zorubicin (ZRN) and 5ꢀiminodaunorubicin (IDN). ZRN and 5ꢀIDN
inhibit CYP2J2ꢀmediated AA metabolism, but does not change the ratio of EET regioisomers.
Taken together, we demonstrate that DOX and 7ꢀdeꢀaDOX inhibit CYP2J2ꢀmediated AA
metabolism and 7ꢀdeꢀaDOX binds close to the active site to alter the ratio of cardioprotective
EETs. These mechanistic studies of CYP2J2 can aid in the design of new alternative DOX
derivatives.
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INTRODUCTION
Anthracyclines are one of the major classes of chemotherapeutics that are used in the treatment
of a wide variety of cancers.1, 2 The prototypical anthracyclines are daunorubicin, which was
naturally discovered in Streptomyoces peucetius, and the 14ꢀOH derivative, doxorubicin
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(DOX).3, Their anticancer mechanisms involve DNA intercalation and topoisomerase II
inhibition.2, 5 DOX produces reactive oxygen species (ROS) leading to nonspecific cytotoxicity.
DOX is especially cardiotoxic and causes arrhythmia as a result of QT interval prolongation.6, 7
Though ROSꢀrelated injury, including mitochondrial damage and lipid peroxidation, has been
suggested to cause cardiotoxicity, there remain unknown mechanisms.8-10 For instance, zorubicin
(ZRN) and 5ꢀiminodaunorubicin (5ꢀIDN) are daunorubicin analogues that have reduced
cardiotoxicity; however, whereas the former produces ROS, the latter does not,1, 11 indicating
there are other potential mechanisms of DOX cardiotoxicity. It is also known that DOX
cardiotoxicity is dependent on the cumulative dose, indicating that in addition to DOX, the DOX
metabolites are highly likely to be involved in longꢀterm cardiotoxicity.
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Cytochromes P450 (CYPs) are involved in firstꢀpass drug metabolism.12 After metabolism by
liver, the drug metabolites are delivered to the heart. Therefore, it is important to understand the
interaction of drugs and their metabolites with cardiovascular CYPs such as CYP2J2. Similar to
13-17
other CYPs such CYP3A4,
our hypothesis is that CYP2J2 should be capable of binding
several substrates at once, which might explain the potential cardiotoxicity of certain drugs.
There is an absence of any direct metabolism study of DOX with CYPs. In one study it was
shown that CYP2B1 was able to reduce DOX to a semiquinone using EPR measurements.18
However, DOX is typically reduced by the CYP redox partner, cytochrome P450 reductase
(CPR),19 to a semiquinone, producing ROS and leading to reductive aglycosylation, producing
mostly 7ꢀdeoxydoxorubicin aglycone (7ꢀdeꢀaDOX) (Figure 1).18, 20, 21 Since CPR reduces DOX
directly and CYPs require CPR to do catalysis, delineating the role of CYPs in DOX metabolism
has been challenging. Herein, we carefully delineate the role of heart CYP2J2 in the metabolism
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of DOX, and the modulation of CYP2J2 activity by DOX and its primary metabolite 7ꢀdeꢀ
aDOX.
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CYP2J2 is highly expressed in human cardiomyocytes22 and is responsible for the endogenous
oxidation of arachidonic acid (AA) into four regioisomers of epoxyeicosatrienoic acids (EETs)22-
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(Figure 1) and two hydroxyeicosatetraenoic acids (HETEs).23,
Together, these
metabolites are responsible for mediating several cardiac physiologies, including antiꢀ
inflammation, vasodilation, and cellular proliferation.29-31 They also protect against postꢀ
ischemia reperfusion injury and reduce myocardium infarct size.31-33 Therefore, these CYP2J2ꢀ
derived metabolites are cardioprotective.34 However, the efficacy of these EET regioisomers are
not similar to each other.30 For instance, 5,6ꢀEET and 8,9ꢀEET have been shown to be both
vasoconstrictory and vasodilatory depending on the tissue,35, 36 and 8,9ꢀEET did not reduce
myocardial infarct size.29 Overexpression of CYP2J2 was found to protect against DOXꢀinduced
injury in humanꢀCYP2J2ꢀoverexpressed mice with an unknown mechanism.37 In this study, a
decrease in mitochondrial damage was observed when 11,12ꢀEET was administered to H9c2
cells. This effectively demonstrates the importance of different regioisomers of EETs (e.g.11,12ꢀ
EET) in mediating cardioprotection.
CYP2J2 has also been found to metabolize several cardiotoxic drugs, such as terfenadine,
astemizole, amiodarone, and dronedarone.38-41 Our hypothesis is if cardiotoxic drugs inhibit
CYP2J2ꢀmediated drug metabolism, they should also inhibit CYP2J2ꢀmediated AA metabolism.
The inhibition of endogenous AA metabolism by CYP2J2 can have deleterious cardiovascular
effects as EETs play an important role in cardiovascular function.
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Herein, we use fluorescence polarization (FP) measurements, molecular dynamics (MD)
simulations, and substrate kinetics to delineate the mechanism of the DOX/CYP2J2/AA
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interactions.
We demonstrate that DOX inhibits CYP2J2ꢀmediated AA metabolism.
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Additionally, it alters the site of AA metabolism indirectly through its metabolite 7ꢀdeꢀaDOX. 7ꢀ
deꢀaDOX modulates the binding of AA in the active site of CYP2J2 leading to significant
change in the regioselectivity of AA epoxidation. We further show that DOX analogues, 5ꢀIDN
and ZRN, inhibit CYP2J2 with different potencies, but neither of them alter the regioselectivity
of AA metabolism. FP binding measurements and kinetics studies indicate that 7ꢀdeꢀaDOX can
bind at a separate site from AA. These studies are supported by MD simulations, which
demonstrate that 7ꢀdeꢀaDOX binds into a binding site we had previously determined to be crucial
for PUFA binding. 25 This positions AA into a conformation to support our observed changes in
the site of metabolism.
EXPERIMENTAL PROCEDURES
Materials expression and purification of CYP2J2, CPR, and CYPB5; EBS kinetics and
quantification; EET LC-MS/MS quantification; and IC50 calculations. See supplemental
information, methods section.
Use of reactive oxygen species (ROS) scavengers in in vitro kinetic experiments. To account
for ROSꢀmediated effects, kinetic experiments were performed with and without ROS
scavengers (10 U/mL of catalase and 10 U/mL of superoxide dismutase final, each an order of
magnitude larger than required). Scavenger stocks were made in 0.1 M KPi buffer, pH 7.4, and
handled and stored according to manufacturer’s recommendations. There were no significant
differences in the data with and without the scavengers, and all the data presented is in the
presence of scavengers.
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Arachidonic Acid (AA) metabolism kinetics. The kinetics of AA metabolism by CYP2J2
with and without DOX were determined as follows. CYP2J2 and CPR (0.6 µM each) were
incubated in a 20% POPS lipid reconstituted system (see Supplementary Methods) in 0.5 mL of
potassium phosphate buffer (pH 7.4). This mixture was then preincubated with 100 µM AA with
varying concentrations of DOX or 7ꢀdeꢀaDOX (0ꢀ50 µM from 1 mM and 10 mM stocks in
DMSO) for 10 min. Reactions were started with 6 mM NADPH and terminated after 5 min with
100 µL acetic acid. Metabolites were extracted and quantified as previously described25. AA
epoxidation by CYP2J2 was determined to be linear up to 8 min under these conditions. ꢀꢁꢂꢃ and
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ꢄ values were determined as stated in the Supplementary Methods. To investigate the effects of
ꢅ
ROSꢀmediated epoxidation of AA from the reduction of DOX, the same procedure was repeated
with CPR and without CYP2J2 and without ROS scavengers.
Fluorescence polarization binding of DOX and 7-de-aDOX. Steadyꢀstate fluorescence
measurements were conducted with a K2 multiꢀfrequency phase and modulation fluorimeter
(ISS, Urbana, IL). Spectra were recorded using Vinci 2 software (Urbana, IL). Measurements
were taken with a 1 nm slit width filter. Fluorescence emission spectra between 500 nm and 650
nm of DOX and 7ꢀdeꢀaDOX were determined using an excitation wavelength of 480 nm, with
both showing optimal absorbance between 590 and 595 nm. Experiments were conducted at
37oC using a circulating water bath and monomeric protein stocks were solubilized in 0.1%
cholate. The presence of CYPs or CYB5 did not alter the fluorescence of DOX or 7ꢀdeꢀaDOX.
Polarization measurements were taken using a 594ꢀnm cutꢀoff filter, and the initial G and
polarization (r0) values were determined. For all measurements, G ≈ 1.000 and r0 ≈ 0.900 and
did not significantly change between experiments. 0ꢀ40 ꢁM of CYP2J2 and CYB5 were titrated
into 1 ꢁM DOX or 7ꢀdeꢀaDOX in 0.1% cholate and 0.1 M potassium phosphate (KPi) buffer (pH
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7.4) at 37oC and incubated for 2 min before each measurement. For concentrations of protein
greater than 10 ꢁM, 1 ꢁM of DOX or 7ꢀdeꢀaDOX were titrated into diluted protein stocks. The
polarization values were plotted and fitted to either linear (CYB5) or oneꢀbinding site (Eq 1)
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(CYP2J2) equations
[ꢋ]
ꢌ
ꢈ ꢊ
ꢉ
ꢍ
ꢆ = ꢆ +
(1)
ꢇ
[ꢋ]
ꢌ
ꢎꢏꢊ
ꢍ
where ꢆꢃ = the intrinsic (initial) polarization value and ꢆ = the maximum polarization value.
ꢎ
Competitive FP binding. EBS and AA competitive binding experiments to probe where DOX
and 7ꢀdeꢀaDOX bind to CYP2J2 were performed as above with the following modifications.
CYP2J2 protein stocks were preincubated with 60 µM EBS or 100 ꢁM AA prior to the titration.
Initial fluorescence measurements using 1 µM compound were also determined in the presence
of 60 µM EBS and 100 ꢁM. The CYP2J2 and substrate mixture was then titrated into the
solution and the FP determined as above. Data fit to a competitive binding equation whenever
inhibition was present.
Doxorubicin (DOX) Metabolism Kinetics. In vitro DOX metabolism was determined by preꢀ
incubating CPR and CYP2J2 (0.6 µM each) with DOX (10 mM and 1 mM stocks in DMSO) and
a 20% POPS lipid reconstituted system for 10 min at 37oC in 0.1 M KPi buffer (pH 7.4).
Reactions were initiated upon the addition of 6 mM NADPH based upon our initial NADPH
kinetic rates. Reactions were terminated with 100 ꢁL acetic acid and were extracted thrice with
equal volumes of ethyl acetate to purify doxorubicin aglycone (aDOX) metabolites. Undigested
DOX remained in the aqueous layer with no noticeable accumulation of aDOX metabolites. The
organic extracts were then dried under a stream of N2 gas and resuspended in 100 ꢁL acetonitrile
for HPLC quantification. Samples were heated with warm water to facilitate solubilization.
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HPLC separation and quantification of DOX metabolites. A highꢀperformance liquid
chromatography (HPLC) system consisting of an Alliance 2695 analytical separation module
(Waters, Milford, MA) and a Waters 996 photodiode diode array detector (Waters) was used for
the separation and quantification of DOX metabolites. Metabolites were separated using a
Phenomenex Prodigy® 5ꢁm ODSꢀ2, 150 x 4.60 mm column (Phenomenex, PN 00Fꢀ3300ꢀE0,
Torrance, CA) and a linearꢀgradient reverseꢀphase method at a flow rate of 0.5 mL/min. The
mobile phase consisted of Mobile Phase A (filtered ultrapure water titrated with formic acid to
pH = 3.0) and Mobile Phase B (acetonitrile). The gradient consisted of 95% Mobile Phase A to
100% Mobile Phase B over a 35ꢀmin period and held at 100% Solvent B for 5 more min (40 min
total) to wash the column. DOX eluted at 15 min and aDOX metabolites (DOX metabolites
without the daunosamine sugar) eluted between 17 and 24 min, with a major peak at 21.7 min.
Incubations with CPR produced a major 21.7 min peak and a minor peak at 23 min. The major
peak was identified as 7ꢀdeoxyꢀdoxorubicin aglycone (7ꢀdeꢀaDOX) by comparing elution time
and mass spectroscopy fragmentation against an authentic standard. DOX metabolism was
quantified at λ = 484 nm using an authentic standard curve.
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High-resolution LC-MS/MS identification of Anthracycline metabolites. For high
resolution LC/MS, the samples were analyzed by using the QꢀExactive MS system
(Thermo. Bremen, Germany) in the Metabolomics Laboratory of Roy J. Carver Biotechnology
Center, University of Illinois at UrbanaꢀChampaign. Software Xcalibur 3.0.63 was used for data
acquisition and analysis. The Dionex Ultimate 3000 series HPLC system (Thermo, Germering,
Germany) used includes a degasser, an autosampler, and a binary pump. The LC separation was
performed using the same Phenomenex Prodigy® 5ꢁm ODSꢀ2 column as for HPLC. The
autosampler was set to 10°C. The injection volume was 10 ꢁL. Mass spectra were acquired
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under positive electrospray ionization (sheath gas flow rate, 49; aux gas flow rate: 12; sweep gas
flow rate, 2; spray voltage, 3.5 kV; capillary temp, 259 °C; Aux gas heater temp, 419 °C). Mass
scan range is 70 ꢀ 1,000. For full scan, the resolution was set to 70,000, and the AGC target was
1E6 with a maximum injection time of 50 ms. For MS/MS scan, the resolution was set to 17,500
and the AGC target was 5E4 with a maximum injection time of 50 ms. NCE was 25 and 30.
NADPH kinetics. The rate of NADPH consumption in the presence of anthracyclines (0ꢀ50
µM from 1 mM and 10 mM stocks in DMSO) was determined using UVꢀVis spectroscopy as
previously described 42. Experiments were performed using 0.6 ꢁM CPR with or without 0.6 ꢁM
CYP2J2 and a 20% POPS lipid reconstituted system. 200 ꢁM of NADPH was added to initiate
the reaction to keep the absorbance values below 1. Rates were determined based on the initial
linear rate of three experiments.
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Statistical Analysis. All data were produced from the means of 3 repeats. Statistical
significance was based on a twoꢀtailed student’s tꢀtest confidence level of P < 0.05. All data
curve fittings were determined using OriginPro 9.1 software (Origin Labs Inc., Northhampton,
MA).
Modeling and simulation of membrane-bound CYP2J2 with AA and 7-de-aDOX. Initial
structural models of membraneꢀbound CYP2J2 bound to AA and 7ꢀdeꢀaDOX were generated
with molecular docking performed with AutoDock Vina.43 We started by docking 7ꢀdeꢀaDOX to
our previously derived models of CYP2J2 bound to AA based upon the homology model of
Lafite, P. et al.25, 44 For this docking step, we employed configurations of AA in which sites
involved in the regioselectivity shift (i.e., carbons C5,C6 and C8,C9) were close to the heme
(with distance < 5 ˚A), resulting in about 20 initial structures used for docking. A grid box of
dimension 22 ˚A in x, y and z and centered in the active site of CYP2J2 was employed. The
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resulting docked poses of 7ꢀdeaDOX were then clustered based on the RMSD of its heavy atoms,
resulting in four clusters. Finally, for each cluster, the configurations of CYP2J2 with both AA
and 7ꢀdeꢀaDOX bound with the best docking score were selected as a starting point for MD
simulations. Each system was then minimized for 2,000 steps, and equilibrated for 1 ns with the
Cα of CYP2J2 and the heavy atoms of AA and 7ꢀdeꢀaDOX harmonically restrained (with force
constant k=1 kcal/mol/˚A2). Following this preparation step, the four systems were simulated for
a short time (20 ns) to assess the stability of 7ꢀdeꢀaDOX binding. After this step, only one system
showed stable binding of 7ꢀdeꢀaDOX to CYP2J2, and this system was further extended to 200 ns
of simulation without restraints. As a control, an additional simulation in which 7ꢀdeꢀaDOX was
removed from this initial model was also performed following the same preparation protocol,
with 200 ns of production simulation.
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As a control, we also performed docking of DOX both to apo membraneꢀbound CYP2J2 and to
the CYP2J2ꢀAA complex, following the similar protocol as for 7ꢀdeꢀaDOX. Snapshots of apo
membraneꢀbound CYP2J2 were obtained from our simulations previously reported.45 From DOX
docking to CYP2J2, two main configurations of DOX in the active site were identified through
clustering and employed starting points for MD simulations (see Supplementary Movies). For
DOX docking to the CYP2J2ꢀAA complex, only one main configuration of DOX was identified
(Figure 7). In this configuration, DOX was located ~17 Å from the heme (center of mass
distance) and not within the active site, and thus this configuration was not simulated.
Docking of EBS to the CYP2J2/7-de-aDOX complex. We generated a model of EBSꢀbound
CYP2J2 by employing the last snapshot of the CYP2J2ꢀ7ꢀdeꢀaDOX/AA simulation for
molecular docking. For this, we removed AA from the structure, while 7ꢀdeꢀaDOX was
preserved. A grid box with same dimensions as previously described and centered in the active
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site of CYP2J2 was employed for EBS docking. From this step, 10 different docked poses of
EBS with the CYP2J2/7ꢀdeꢀaDOX complex were generated, and sorted based on docking score.
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Simulation protocol. The simulations were performed using NAMD2.46 The CHARMM27
force field with cMAP47, 48 corrections was used for the protein and the CHARMM3649, 50 force
field was used for lipids. Parameters for AA and 7ꢀdeꢀaDOX were obtained by analogy from the
CHARMM General Force Field.51 The TIP3P model was used for water.52 Simulations were
performed with the NPT ensemble with a time step of 2 fs. A constant pressure of 1 atm was
maintained using the Nos´eꢀHoover Langevin piston method.53, 54 Temperature was maintained
at 310 K using Langevin dynamics with a damping coefficient γ of 0.5 psꢀ1applied to all atoms.
Nonbonded interactions were cut off at 12 ˚A, with smoothing applied at 10 ˚A. The particle
mesh Ewald (PME) method55 was used for longꢀrange electrostatic calculations with a grid
density of > 1 ˚Aꢀ3 .
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Figure 1. Schematic of DOX and AA metabolism by CYP2J2 and CPR. (A) CYP2J2ꢀCPR metabolizes
arachidonic acid (AA) into four regioisomers of epoxyeicosatrienoic acids (EETs) (A1). (B) CPR reduces
doxorubicin (DOX) to a semiquinone (DOXꢀsQ) (B1) that leads to the formation of ROS and the reductive
aglycosylation of DOX to form 7ꢀdeoxydoxorubicin aglycone (7ꢀdeꢀaDOX) (B2).
RESULTS
CYP2J2-mediated AA metabolism in the presence of DOX and 7-de-aDOX. We firstly
investigated the effect of DOX on CYP2J2ꢀmediated arachidonic acid (AA) metabolism. We
used AA at a concentration near the previously determined ꢄꢐ.25 The concentration ranges for
DOX covers plasma levels upon immediate exposure (~1ꢀ10 µM) to the toxic cumulative dose
(~50 µM).56, 57 We see that DOX inhibits the total AA metabolism with an ꢀꢁꢂꢃ of 5.48 ꢑ 0.04
µM and a ꢄ of 3.11 ꢑ 0.02 µM.
ꢅ
DOX is converted to 7ꢀdeꢀaDOX by cytochrome P450 reductase. In order to do the studies, we
synthesized 7ꢀdeꢀaDOX from DOX through reduction (Supplementary Methods and Figure S1).
We next determined the effect of 7ꢀdeꢀaDOX on AA metabolism. 7ꢀdeꢀaDOX also inhibits AA
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metabolism, with an ꢀꢁꢂꢃ of 10.2 ꢑ 0.1 µM and a ꢄ of 5.78 ꢑ 0.03 µM. However, the
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maximum inhibition by 7ꢀdeꢀaDOX appears to be almost half as effective compared to DOX,
with a ꢒꢃ of 42.9 ꢑ 5.6 pmol (min)ꢀ1 (nmolCYP2J2)ꢀ1 for 7ꢀdeꢀaDOX and a ꢒꢃ of 19.8 ꢑ 3.8 pmol
(min)ꢀ1 (nmolCYP2J2)ꢀ1 for DOX (Figure 2B). This implies that 51% of the enzyme activity is
retained in the presence of saturating amounts of 7ꢀdeꢀaDOX, compared to 23% in the presence
of DOX. In theory, competitive inhibition of an enzyme would result in a ꢒꢃ = 0 due to the
inhibitor completely outcompeting the substrate. Since the ꢒꢃ of the 7ꢀdeꢀaDOX data does not
approach 0, this demonstrates that the inhibition is not competitive and suggests a possible
second binding site for binding 7ꢀdeꢀaDOX.
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Figure 2. AA Inhibition by DOX, 7ꢀdeꢀaDOX, and the nonꢀtoxic analogues ZRN and 5ꢀIDN. (A) Structures of the
anthracyclines doxorubicin (DOX), 7ꢀdeoxydoxorubicin aglycone (7ꢀdeꢀaDOX), 5ꢀiminodaunorubicin (5ꢀIDN),
and zorubicin (ZRN). (B) Rate of epoxidation of 100 ꢁM AA to EETs by CYP2J2ꢀCPr in the presence of
increasing concentrations of cardiotoxic DOX, 7ꢀdeꢀaDOX, or a 50:50 mixture of DOX:7ꢀdeꢀaDOX. The
theoretical fit based on the linear combination of DOX and 7ꢀdeꢀaDOX is shown as a grey, dashed line to
demonstrate the overlap of the theoretical fit to the experimental (see Results for details). (C) Rate of epoxidation
in the presence of nonꢀcardiotoxic analogues, zorubicin (ZRN) and 5ꢀiminodaunorubicin (5ꢀIDN) with the DOX
data from panel (B) shown in grey for comparison. 100 ꢁM of AA was used in all experiments. Concentrations of
anthracyclines represent the total amount of anthracyclines present. All inhibition data fit to eq S1. Error represents
the SEM of 3 experiments.
To confirm these observations, we proceeded to measure the AA metabolism with a 50:50
mixture of DOX and 7ꢀdeꢀaDOX, maintaining the total anthracycline concentration (Figure 2B).
If the incomplete inhibition of 7ꢀdeꢀaDOX is due to 7ꢀdeꢀaDOX and AA concurrently binding,
then the data would be a linear combination of the individual DOX and 7ꢀdeꢀaDOX data. The
theoretical values for this mixture based on the DOX and 7ꢀdeꢀaDOX data are as follows: ꢀꢁꢂꢃ
=
6.68 ꢑ 0.05 µM (ꢄ = 3.79 ꢑ 0.03 µM) and ꢒꢃ = 32.1 ꢑ 4.2. Our experimental values were: ꢀꢁꢂꢃ
ꢅ
= 10.1 ꢑ 0.03 µM (ꢄ = 5.73 ꢑ 0.02 µM) and ꢒꢃ = 24.8 ꢑ 5.5. These values agree well to the
ꢅ
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theoretical values and support that DOX competitively inhibits AA metabolism, and that 7ꢀdeꢀ
aDOX allows for the concurrent metabolism of AA and an incomplete inhibition.
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Figure 3. Regioselectivity of the epoxidation of AA by CYP2J2 in the presence of anthracyclines. (A)
EET regioisomers. (B) Regioselectivity of EETs in the presence of DOX, 7ꢀdeꢀaDOX, and the 50:50
mixture of DOX:7ꢀdeꢀaDOX. Each EET is shown as a percentage of the total EETs. The colors of the
bars represent the colors associated with EETs in panel (A). (C) Regioselectivity of epoxidation in the
presence of ZRN and 5ꢀIDN. (D) Foldꢀchange of the regioselectivity for 5,6ꢀEET and 14,15ꢀEET of the
data in Panels (B) and (C). Fold change = (Percent of regioisomer in the presence of 50 ꢁM
anthracycline) / (Percent of regioisomer in the presence of 0 ꢁM anthracycline). Data presented in (AꢀD)
is obtained from the same dataset as presented in Figure 2. (E) Rate of epoxidation of AA by CYP2J2
and/or CPR in presence and absence of ROS scavengers. Rates are normalized to the amount of CPR
(0.3 nmol) or CYP2J2 (0.3 nmol). (F) Regioselectivity of epoxidation by CYP2J2 vs. nonspecific
epoxidation. In all these experiments 50 ꢁM DOX and 100 ꢁM of AA was used. Concentrations of
anthracyclines represent the total amount of anthracyclines present. Error represents the SEM of 3
experiments. P values: * < 0.05; ** < 0.01; *** < 0.001; **** < 0.0001.
DOX and 7-de-aDOX changes the regioselectivity of the AA epoxidation. AA metabolism by
Formation of different EET regioisomers by CYP2J2-mediated AA metabolism in the presence
of DOX or 7-de-aDOX. CYP2J2 leads to the formation of four different EET regioisomers. We
further observed that DOX had a concentrationꢀdependent change on the regioselectivity of AA
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epoxidation (Figure 3B and Figure 3D). With increasing concentrations of DOX, the ratio of 5,6ꢀ
EET significantly increases (1.41ꢀfold with 50 µM DOX) (Figure 3D) and 14,15ꢀEET
significantly decreases (0.78ꢀfold with 50 µM DOX), with lessꢀpronounced albeit significant
changes in 8,9ꢀEET and 11,12ꢀEET (Figure 3B and D).
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As a control, incubating AA with CPR and 50 µM DOX, without ROS scavengers produces a
negligible epoxidation of AA (Figure 3E) with a different distribution of EETs compared to that
produced by CYP2J2 (Figure 3F). Furthermore, since all these experiments with CYP2J2 were
conducted in the presence of ROS scavengers, this EET regioselective change is CYP2J2ꢀ
mediated and not ROSꢀmediated.
The experiments with 7ꢀdeꢀaDOX also resulted in a similar regioselectivity change of the AA
epoxidation (1.41ꢀfold for 5,6ꢀEET and 0.86ꢀfold for 14,15ꢀEET with 50 µM 7ꢀdeꢀaDOX)
(Figure 3B and D). Similar changes were observed in the 50:50 mixture (Figure 3B). Since DOX
displays competitive inhibition and 7ꢀdeꢀaDOX displays incomplete inhibition, 7ꢀdeꢀaDOX
appears to be the modulator of the EET regioselectivity. The regioselective change with DOX is
likely due to its rapid conversion to 7ꢀdeꢀaDOX. Coincidentally, 11,12ꢀEET and 14,15ꢀEET have
been shown to be the most effective at reducing ischemic injury, whereas 5,6ꢀEET and 8,9ꢀEET
have not.29
CYP2J2-mediated AA metabolism in the presence of non-cardiotoxic DOX analogues, ZRN
and 5-IDN. DOX and 7ꢀdeꢀaDOX inhibited AA metabolism and altered the site of metabolism.
In order to determine if these observations are unique to DOX and 7ꢀdeꢀaDOX, we repeated the
study using two DOX analogues. The rationale for choosing the analogues is that both ZRN and
5ꢀIDN are chemotherapeutic DOX analogues with lowered cardiotoxicity compared to DOX.
ZRN redox cycles and produces ROS as effectively as DOX, while 5ꢀIDN has a lowered rate of
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redox cycling.1, 11, 58 The structural differences are shown in Figure 2A. ZRN contains a large
benzohydrazide modification, and the presence of an imino group in place of a keto makes 5ꢀ
IDN inhibit redox cycling. ZRN inhibits AA metabolism as effectively as DOX, with inhibition
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parameters of: ꢀꢁꢂꢃ = 6.46 ꢑ 0.03 µM (ꢄ = 3.67 ꢑ 0.02 µM) and ꢒꢃ = 18.7 ꢑ 3.5 (Figure 2C).
ꢅ
Table 1. Calculated fitting parameters of the fluorescence polarization data of DOX and 7ꢀdeꢀ
aDOX binding (Figure 4). Ki refers to the inhibition of DOX or 7ꢀdeꢀaDOX by either AA or
EBS as indicated. Error represents the SEM of 3 experiments.
Summary of Binding Parameters
r0
r1
Ki (µM)
ꢓꢔ (µM)
DOX
DOX 0.083 ꢑ 0.007
0.219 ꢑ 0.010
5.32 ꢑ 0.89
ꢀꢀꢀ
DOX + AA
ꢀꢀꢀ
ꢀꢀꢀ
ꢀꢀꢀ
ꢀꢀꢀ
0.094 ꢑ 0.005
0.101 ꢑ 0.003
176 ꢑ 64
13.8 ꢑ 1.6
DOX + EBS
7-de-aDOX
7-de-aDOX
ꢀꢀꢀ
210 ꢑ 26
∞
0.055 ꢑ 0.006
0.084 ꢑ 0.002
0.351 ꢑ 0.022
13.1 ꢑ 1.1
7-de-aDOX + AA
ꢀꢀꢀ
ꢀꢀꢀ
7-de-aDOX + EBS 0.159 ꢑ 0.006 0.383 ꢑ 0.021 (app) 13.4 ꢑ 1.4 (app)
5ꢀIDN displayed a lower inhibition affinity compared to ZRN or DOX, with an ꢀꢁꢂꢃ = 27.4 ꢑ
0.03 µM (ꢄ = 15.6 ꢑ 0.02 µM) (Figure 2C).
ꢅ
However, there is not a significant change in the epoxide regioselectivity for either 5ꢀIDN or
ZRN compared to DOX or 7ꢀdeꢀaDOX (Figure 3C and D). The minor changes in the
regioselectivity (Figure 3D) are likely due to the conversion of 5ꢀIDN and ZRN to their
respective aglycones, which do not modulate AA metabolism as effectively as 7ꢀdeꢀaDOX
(Figures S2ꢀS3). For ZRN, the bulky benzohydrazide would significantly change the binding of
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Figure 4. Binding and metabolism studies of DOX and 7ꢀdeꢀaDOX. (AꢀB) Fluorescence polarization
measurements of DOX (A) and 7ꢀdeꢀaDOX (B) binding to CYP2J2. Both DOX and 7ꢀdeꢀaDOX
demonstrate oneꢀsite binding (eq 1). Binding was repeated in the presence of 60 ꢁM ebastine (EBS) or 100
ꢁM AA. DOX data in the presence of EBS or AA fit to a competitive binding model and 7ꢀdeꢀaDOX is
fitted to eq 1 (no inhibition). For all experiments, [DOX] and [7ꢀdeꢀaDOX] = 1 ꢁM. (C) EBS metabolism
in the presence of DOX, 7ꢀdeꢀaDOX, and a 50:50 mixture of DOX:7ꢀdeꢀaDOX. DOX data fit to eq S1 and
7ꢀdeꢀaDOX data is linear. The theoretical fit based on the linear combination of DOX and 7ꢀdeꢀaDOX is
shown as a grey, dashed line to demonstrate the closeness of the theoretical fit to the experimental fit.
Concentrations of anthracyclines represent the total amount of anthracyclines present. Error represents ±
SEM of 3 experiments.
the ZRNꢀaglycone. In summary, ZRN and IDN differ in their inhibition potential towards
CYP2J2ꢀmediated AA metabolism and do not produce a significant concentrationꢀdependent
regioselectivity change (Figure 3 C and D).
Fluorescence polarization (FP) binding of DOX and 7-de-aDOX to CYP2J2. To further
investigate the mechanistic details of the AA metabolism modulation by DOX and 7ꢀdeꢀaDOX,
we measured their binding to CYP2J2 using FP measurements. Heme Soret titration could not be
used to measure the binding, as DOX and its derivatives have a high absorbance between 400ꢀ
500 nm. Cytochrome b5 (CYB5) was used as a nonspecific binding control, as it does not
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interact with DOX. None of these hemoproteins altered the fluorescent properties of DOX
(Figure S4). CPR, however, has an intrinsic fluorescence in the same region as DOX
fluorescence and could not be used in this assay (Figure S4D).
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The binding parameters are shown in Table 1. CYB5 showed polarization values, r, that are
linear as a function of protein concentration indicating nonspecific binding (Figure S5). CYP2J2
demonstrated oneꢀsite binding with a ꢄꢕ of 5.32 ꢑ 0.89 ꢁM (Figure 4A), agreeing well with the
ꢀꢁꢂꢃ/ꢄ of DOX for the AA inhibition. AA, however, weakly inhibits DOX binding.
ꢅ
To determine if DOX is binding to the active site of CYP2J2, we performed these studies with
CYP2J2 and DOX in the presence of 60 ꢁM ebastine (EBS) to saturate the active site (Figure
4A). EBS is a substrate of CYP2J2 with a similar ꢄꢕ as DOX (8.16 ꢁM) and demonstrates oneꢀ
site binding to the active site.25, 59 EBS competitively inhibited DOX binding to CYP2J2, with a
ꢄ of 13.8 ꢑ 1.6 ꢁM that agrees well with the ꢄꢕ and ꢄꢐ values for EBS.25 These data conclude
ꢅ
that DOX and EBS are competing for binding to the active site of CYP2J2 and that AA weakly
inhibits DOX binding (Figure 4A).
CYP2J2 had a larger amplitude of change on the 7ꢀdeꢀaDOX polarization compared to DOX
and demonstrated oneꢀsite binding with a ꢄꢕ of 13.1 ꢑ 1.1 ꢁM (Figure 4B). AA showed little
inhibition of 7ꢀdeꢀaDOX binding (ꢄ > 200 ꢁM). Despite EBS having a tighter ꢄꢕ than 7ꢀdeꢀ
ꢅ
aDOX (8.16 ꢁM25 vs. 13.1 ꢁM), EBS did not inhibit 7ꢀdeꢀaDOX binding to CYP2J2 (Figure
4B). The inability of EBS to inhibit 7ꢀdeꢀaDOX binding suggests that these two molecules do not
compete for binding to the same site. Since EBS binds the active site, this proposes a peripheral
binding site for 7ꢀdeꢀaDOX. The presence of peripheral binding sites is common in CYPs.13-17
FP also measures the “rigidity” of binding, where an FP value of r = 0.4 represents the
theoretical maximum value and the most restriction to rotational mobility. Therefore, the r
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maximum (r1) value of the oneꢀsite model can be used as a semiꢀquantitative measurement of the
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rotational flexibility of the molecule in the active site of the protein . The binding of DOX to
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CYP2J2 produced an r1 value of 0.219 ꢑ 0.010 and thus DOX maintains some rotational
freedom while bound to the active site. Contrariwise, CYP2J2 binding of 7ꢀdeꢀaDOX showed an
r1 value of 0.351 ꢑ 0.022. This indicates that 7ꢀdeꢀaDOX has more rotational restriction when
bound to CYP2J2.
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Effects of DOX on CYP2J2-mediated EBS metabolism. In order to further probe the
inhibitory characteristics of DOX and 7ꢀdeꢀaDOX, we investigated their effects on EBS
metabolism by CYP2J2. The binding and kinetics of EBS metabolism are closer to the
parameters of DOX and therefore provides clearer insights into the interactions. We determined
the ꢄꢐ of EBS metabolism by CYP2J2 to be 19.4 µM.25 Therefore, we looked at the effect that
DOX has on the CYP2J2ꢀmediated metabolism with 20 µM of EBS. DOX inhibited EBS
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metabolism with an ꢀꢁꢂꢃ of 17.9 ꢑ 0.0 µM and a ꢄ of 8.97 ꢑ 0.01 µM (Figure 4C). Incubations
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with 7ꢀdeꢀaDOX, however, did not result in an inhibition of EBS metabolism (Figure 4C). This
is similar to the observations from the FP binding experiments, in which 7ꢀdeꢀaDOX binding to
CYP2J2 was not inhibited by EBS. Therefore, EBS and 7ꢀdeꢀaDOX likely bind to different sites
in CYP2J2. To confirm, we measured the EBS metabolism in the presence of a 50:50 mixture of
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DOX and 7ꢀdeꢀaDOX. The theoretical combination of the DOX (ꢒꢃ = 5.87 ꢑ 2.84) and 7ꢀdeꢀ
aDOX (ꢖ = 22.5 ꢑ 1.2) data results in a ꢒꢃ = 13.2. The 50:50 DOX:7ꢀdeꢀaDOX data match the
theoretical model as a linear combination of the 7ꢀdeꢀaDOX and DOX data quite well, resulting
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in an ꢀꢁꢂꢃ of 9.07 ꢑ 0.08 µM (ꢄ = 4.53 ꢑ 0.04 µM) and a ꢒꢃ of 14.2 ꢑ 2.1. This confirms our
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observations that DOX competes with EBS for binding to the active site of CYP2J2, whereas 7ꢀ
deꢀaDOX binds in a site that allows for the binding of a second ligand such as EBS or AA.
Furthermore, this demonstrates that the effects of 7ꢀdeꢀaDOX are not universal for all CYP2J2
substrates, since 7ꢀdeꢀaDOX alters the metabolism of AA but not the metabolism of EBS.
Metabolism of DOX, ZRN, and 5-IDN by CPR. We next investigated if CYP2J2 is capable of
metabolizing DOX. This is difficult to delineate as CPR, the obligate redox partner of CYPs, can
Figure 5. Dox metabolism kinetics. (A) Rate of NADPH oxidation by CPR (red, squares) and by
CPR with CYP2J2 (dark red, circles) to reduce DOX. CPR displayed linear kinetics towards the
reduction of DOX (equation shown on plot) and the presence of CYP2J2 displayed oneꢀbindingꢀ
site kinetics. (B) Rate of NADPH oxidation by CPR (orange, squares) and by CPR with CYP2J2
(gold, circles) to reduce 7ꢀdeꢀaDOX. (C) DOX metabolism to 7ꢀdeꢀaDOX by CPR and in the presence
of CPR + CYP2J2. Concentrations of anthracyclines represent the total amount of anthracyclines present.
Error represents ± SEM of 3 experiments.
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reduce DOX to produce a DOX semiquinone (DOXꢀsQ) via NADPH oxidation (Figure 1B).
DOXꢀsQ can then produce reactive oxygen species or reductively aglycosylate the daunosamine
sugar moiety to produce doxorubicin aglycone (aDOX) metabolites.18, 20, 21 Previously, the
steadyꢀstate kinetics of these reactions have not been measured. We determined the kinetics of
DOX metabolism by CPR. HPLC analysis (Figure S6) of the reaction products showed a major
7ꢀdeꢀaDOX peak (20.5 min) that was confirmed using authentic standards (Figure S7), and a
minor peak (22.4 min) (Figure S6). The minor peak had MS/MS fragments that were +14 m/z
values of the 7ꢀdeꢀaDOX fragments (Figure S8), which may be due to the oxidation of the 7ꢀOH
of doxorubicin aglycone to a ketone. The product was not analyzed further due to poor yields.
A similar metabolism was observed for the reduction of ZRN and 5ꢀIDN by CPR (Figures S2
and S3). ZRN elutes at 17.2 min, and masses corresponding to 7ꢀdeoxyzorbucin aglycone were
identified at 22.2 and 25.7 min (Figure S2). 5ꢀIDN elutes at 16.4 min and a mass corresponding
to the 7ꢀdeoxy aglycone analogue was identified at 22.7 min (Figure S3).
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Kinetics of DOX, 7-de-aDOX, ZRN, and 5-IDN metabolism by CPR. The rate of NADPH
oxidation in the presence of DOX or 7ꢀdeꢀaDOX was negligible in the absence of any enzyme.
The rate of NADPH oxidation was linear when CPR is reducing DOX or 7ꢀdeꢀaDOX, indicating
that the reduction proceeds without a discernable binding event (Figure 5AꢀB and Table S1). The
rate with 7ꢀdeꢀaDOX (Figure 5B) was approximately tenfold lower than with DOX (Figure 5A)
and implies that 7ꢀdeꢀaDOX is not readily reduced by CPR to form ROS. Comparatively,
NADPH oxidation in the presence of ZRN is similar to DOX, and with 5ꢀIDN is significantly
slower (Figure S9 and Table S1). This confirms the observation that ZRN redox cycles as
effectively as DOX and 5ꢀIDN has a much lower rate of redox cycling. The metabolism of DOX
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to 7ꢀdeꢀaDOX by CPR is also linear, confirming the metabolism of DOX by CPR proceeds
without a binding event (Figure 5C and Table S1).
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NADPH oxidation and Kinetics of DOX metabolism by CYP2J2-CPR. With the addition of
CYP2J2, the NADPH oxidation with DOX follows a hyperbolic curve, with an apparent ꢄꢐ of
10.4 ꢑ 1.6 µM (Figure 5A). This implies that CYP2J2 reduces DOX. The NADPH oxidation
does not increase when 7ꢀdeꢀaDOX binds CYP2J2 and implies that 7ꢀdeꢀaDOX is not reduced by
CYP2J2 (Figure 5B).
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The conversion of DOX to 7ꢀdeꢀaDOX remained linear and is inhibited in the presence of
CYP2J2 (Figure 5C and Table S1). Therefore, we conclude that CYP2J2 does not contribute
towards the reductive aglycosylation of DOX (Figure 5C). Overall, this shows that CYP2J2 does
not metabolize DOX or 7ꢀdeꢀaDOX to form new products (none detected by LCꢀMS/MS
analysis), but it reduces DOX (Figure 5A).
Molecular dynamic (MD) simulations to investigate the effect of 7-de-aDOX on AA
orientation in the active site of CYP2J2. To gain insight into how 7ꢀdeꢀaDOX may be
modulating the AA metabolism by CYP2J2, we employed a combined approach of molecular
docking and MD simulations, as described in Experimental Procedures. We have successfully
used these techniques to identify key residues that differentially moderate PUFA binding to the
CYP2J2 active site.25 To investigate how the presence of 7ꢀdeꢀaDOX modulates AA
regioselectivity by CYP2J2, we performed MD simulations starting from structures of
membraneꢀbound CYP2J2 in complex with AA and 7ꢀdeꢀaDOX obtained with molecular
docking (see Methods). The initial model included both 7ꢀdeꢀaDOX and AA molecules in the
active site of CYP2J2. The initial orientation of AA was selected so that sites involved in the
regioselectivity shift (i.e., carbons C5,C6 and C8,C9) were initially close to the heme (with
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distance < 5 ˚A) (Figure 6). As a control, a simulation where 7ꢀdeꢀaDOX was removed from the
initial model was also performed to assess the stability of AA in its initial orientation.
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A table of binding interaction energies is shown in Table S2. The simulations suggest that the
regioselectivity shift is in part due to how 7ꢀdeꢀaDOX sits in the active site of CYP2J2 (Figure
6A). A specific orientation of 7ꢀdeꢀaDOX located near the active site interacts with key residues
that have been shown to be important for AA binding to CYP2J2 (i.e., R321 and S493) 25. In this
configuration, 7ꢀdeꢀaDOX prevents AA from fully extending to place its main epoxidation
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Figure 6. Effect of 7ꢀdeꢀaDOX on AA orientation in CYP2J2 simulations. (A) Representative snapshots
(i.e., most often observed configuration) of the active site of membraneꢀbound CYP2J2 in complex with
AA/7ꢀdeꢀaDOX in with AA only. Membrane Lipids are shown in stick representation. CYP2J2 in cartoon
representation, with helix I (longest helix in CYP2J2) in purple cartoon. AA and 7ꢀdeꢀaDOX molecules
are shown as sticks. Residues involved in AA interactions as previously reported are also shown as sticks.
Carbons involved in epoxidation are highlighted as spheres, with colors corresponding to the distributions
shown in the next panel. (B) Distribution of carbonꢀtoꢀheme distances obtained from 200 ns MD
simulations for the main epoxidation sites of AA. Colors correspond to the spheres shown in (A). (C)
Representative snapshot obtained from molecular docking of EBS to the CYP2J2/7ꢀdeꢀaDOX structure
obtained from MD simulations. Docking shows that EBS can be accommodated in a productive orientation
(i.e., with its methyl groups close to the heme) with 7ꢀdeꢀaDOX bound to CYP2J2 without hindrance.
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