Design and characterization of the first selective and potent mechanism-based inhibitor of cytochrome p450 4z1

Article abstract of DOI:10.1021/acs.jmedchem.0c00101

Mammary-tissue-restricted cytochrome P450 4Z1 (CYP4Z1) has garnered interest for its potential role in breast cancer progression. CYP4Z1-dependent metabolism of arachidonic acid preferentially generates 14,15-epoxyeicosatrienoic acid (14,15-EET), a metabolite known to influence cellular proliferation, migration, and angiogenesis. In this study, we developed time-dependent inhibitors of CYP4Z1 designed as fatty acid mimetics linked to the bioactivatable pharmacophore, 1-aminobenzotriazole (ABT). The most potent analogue, 8-[(1H-benzotriazol-1-yl)amino]octanoic acid (7), showed a 60-fold lower shifted-half-maximal inhibitory concentration (IC₅₀) for CYP4Z1 compared to ABT, efficient mechanism-based inactivation of the enzyme evidenced by a K_I = 2.2 μM and a k_inact = 0.15 min^-1, and a partition ratio of 14. Furthermore, 7 exhibited low off-target inhibition of other CYP isozymes. Finally, low micromolar concentrations of 7 inhibited 14,15-EET production in T47D breast cancer cells transfected with CYP4Z1. This first-generation, selective mechanism-based inhibitor (MBI) will be a useful molecular tool to probe the biochemical role of CYP4Z1 and its association with breast cancer.

Full text of DOI:10.1021/acs.jmedchem.0c00101

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Article
Design and Characterization of the First Selective and
Potent Mechanism-Based Inhibitor of Cytochrome P450 4Z1
John P. Kowalski, Matthew G. McDonald, Robert D. Pelletier,
Helmut Hanenberg, Constanze Wiek, and Allan E Rettie
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.0c00101 • Publication Date (Web): 17 Apr 2020
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Design and Characterization of the First Selective and Potent
Mechanism-Based Inhibitor of Cytochrome P450 4Z1
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John P. Kowalski†, Matthew G. McDonald†, Robert D. Pelletier†, Helmut Hanenberg‡, Constanze
Wiek§, Allan E. Rettie†,*
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†Department of Medicinal Chemistry, School of Pharmacy, University of Washington, Seattle,
WA 98105, USA
‡Department of Pediatrics III, University Children’s Hospital Essen, University of Duisburg-
Essen, 45122 Essen, Germany
§Department of Otorhinolaryngology and Head/Neck Surgery, Heinrich-Heine University, 40225
Düsseldorf, Germany
*Corresponding author, email: rettie@uw.edu
Abstract
Mammary tissue-restricted cytochrome P450 4Z1 (CYP4Z1) has garnered interest for its
potential role in breast cancer progression. CYP4Z1-dependent metabolism of arachidonic acid
preferentially generates 14,15-epoxyeicosatrienoic acid (14,15-EET), a metabolite known to
influence cellular proliferation, migration, and angiogenesis. In the present study, we developed
time-dependent inhibitors of CYP4Z1 designed as fatty acid mimetics linked to the
bioactivatable pharmacophore, 1-aminobenzotriazole (ABT). The most potent analog, 8-[(1H-
benzotriazol-1-yl)amino]octanoic acid (7), showed a 60-fold lower shifted IC50 for CYP4Z1
compared to ABT, efficient mechanism-based inactivation of the enzyme evidenced by a KI =
2.2 μM, kinact = 0.15 min-1 and a partition ratio of 14. Furthermore, 7 exhibited low off-target
inhibition of other CYP isozymes. Finally, low micromolar concentrations of 7 inhibited 14,15-
EET production in T47D breast cancer cells transfected with CYP4Z1. This first-generation,
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selective MBI will be a useful molecular tool to probe the biochemical role of CYP4Z1 and its
association with breast cancer.
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Introduction
Cytochrome P450 (CYP) enzymes are a critical family of hemoproteins that are involved
in the metabolism of both xenobiotic and endogenous molecules.1 In 2004, CYP4Z1 was
cloned from a breast carcinoma line and subsequently shown by several groups to be
up-regulated in breast carcinoma.2,58,59 Other expression profiling studies also
demonstrate consistently that CYP4Z1 transcript level is associated with an elevated
tumor grade and increased tumor aggressiveness.56,57,60 Some mechanistic insight has
been provided by the finding that overexpression of CYP4Z1 promotes angiogenesis
and tumor growth both in vitro and in vivo3.
Many CYP4 enzymes are capable of metabolizing polyunsaturated fatty acids (PUFAs)
to biological compounds that participate in a complex web of signaling.4 Key metabolites of
arachidonic acid (AA, Fig. 1), namely 20-hydroxyeicosatetraenoic acid (20-HETE) and 14,15-
epoxyeicosatrienoic acid (14,15-EET), are thought to play roles in human cancer progression,
by influencing angiogenesis, tumor growth and metastasis.5-7 We recently established that
14,15-EET is a dominant metabolite of CYP4Z1-dependent AA metabolism and have
speculated that a role for CYP4Z1 in breast cancer may involve this signaling ligand.8
Breast cancer is one of the leading causes of death in women, with more than 2 million
new cases diagnosed worldwide in 2018.9 Novel therapeutic modalities are needed urgently,
especially for recurrent and metastatic tumors.10 Endogenous mammary-specific expression of
CYP4Z1 offers a new avenue to explore for breast cancer treatment, and there has already
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been success with CYP inhibitors developed for anti-cancer therapy. For example, aromatase
(CYP19) inhibitors, such as exemestane, letrozole and anastrozole, are useful in breast cancer
treatment because they effectively limit estrogen production.11
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Therefore, the goal of this study was to develop a potent and selective mechanism-
based inhibitor (MBI) of CYP4Z1 to enable biochemical characterization of this enzyme and help
unravel the role that bioactive lipid metabolites play in breast cancer progression. Herein, we
show that analogs of 1-aminobenzotriazole (ABT) are potent time-dependent inhibitors of
CYP4Z1. Two analogs (6, 7) showed pronounced selectivity as reversible inhibitors of CYP4Z1,
compared to other CYP enzymes, and even higher selectivity when they were evaluated for
their time-dependent effects on off-target CYPs. Further, we confirmed mechanism-based
inhibition for our most potent and selective analog, 8-[(1H-benzotriazol-1-yl)amino]octanoic acid
(7), determined the kinetics of inactivation, and assessed the potency of 7 towards inhibition of
CYP4Z1-mediated 14,15-EET production in a breast cancer cell line.
Results and Discussion
CYP4Z1 Assay for Rapid Inhibitor Screening. A mammalian enzyme source was
preferred and, historically, HepG2 cells have been suitable for heterologous expression of CYPs
due to their very low basal CYP levels and catalytically sufficient amounts of the co-enzymes
cytochrome P450 reductase and cytochrome b5.12,13 Therefore, a CYP4Z1-expressing HepG2
cell line was generated via lentiviral transduction (Figure S1A) and membranes prepared from
these cells to use as the primary enzyme source for screening. This expression system
provided an adequate amount of CYP4Z1, with no detectable enzyme in a control vector cell
line, as analyzed by western blot (Figure S1B). Quantification via carbon monoxide-binding
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spectra14 showed that HepG2 membrane preparations contained 1 M of active CYP4Z1
enzyme (Figure S1D). To test for CYP4Z1 inhibition, we established a simple, sensitive
functional assay using the recently identified CYP4Z1 substrate luciferin-benzyl ether15 (Luc-BE,
Figure 1). Utilization of this probe substrate enabled higher throughput evaluation of inhibitor
potency versus the use of more technically cumbersome fatty acid(s) such as laurate and
arachidonate.8 Luc-BE turnover to its luminescent metabolite, luciferin, is easily analyzed with
Promega’s P450 Glo kit.16 The reaction is NADPH-dependent, turnover is >500-fold higher than
background (Figure 2A) and displays apparent Michaelis-Menten kinetics (Figure 2B). For
determination of half maximal inhibitory concentration (IC50) values for CYP4Z1 inhibition, the
probe substrate Luc-BE was used at a concentration equal to the determined Km (29 M). To
evaluate both the reversible, and time-dependent nature of potential inhibitors, we used a non-
dilution (also termed addition) method IC50 shift assay.17 – 19 Typically, if a compound elicits an
IC50 shift of ≥1.5 it is considered to be a time-dependent inhibitor of the enzyme being
evaluated.20
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Design and Synthesis of ABT-analogs for CYP4Z1 Inhibition. We undertook a targeted
design strategy for a CYP4Z1 mechanism-based inhibitor in order to capitalize on potential
selectivity benefits. The rationale for increased specificity is based on the following features: i)
reversible binding of the inhibitor to the enzyme must be of appropriate affinity, ii) the inhibitor
must also serve as a substrate for bioactivation by the target, and iii) the reactive metabolite that
is generated requires an active site target to inactivate the enzyme.1,21
The known inhibitors of CYP4Z1 are promiscuous towards the CYP4 family and other
hepatic CYP enzymes.8,15 Our preliminary work demonstrated that ABT, a mechanism-based
pan-CYP inhibitor,21,22 displayed significant time-dependent inhibition of CYP4Z1. Thus, ABT
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appeared to offer a promising bioactivatable pharmacophore, and our data agreed with prior
research, which indicated that ABT effectively inactivates AA-oxidizing CYPs.23 Additionally,
there was already considerable precedent for exploration of ABT-analogs, as prior studies have
characterized N-benzyl-aminobenzotriazole, N--methylbenzyl-aminobenzotriazole, and N--
ethylbenzyl-aminobenzotriazole as isozyme-selective MBIs of rabbit, guinea pig, and rat
CYPs.24 – 26,53
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CYP4B1 is the only CYP4 member that has been crystallized,27 therefore, structural
inferences that can be made about CYP4Z1 are limited. However, catalytic work with CYP4Z1
provided a useful basis for simple inhibitor design. Unlike most other CYP4 enzymes, CYP4Z1
catalyzes oxidations internally rather than at the ω-terminus.8,15,54 In addition to AA metabolism,
the internal preference for oxidation is evident for the substrates lauric acid (LA) and Luc-BE
(Figure 1). Therefore, we made simple chemical modifications to the ABT scaffold to generate
CYP4Z1 inhibitors by adding carbon tails to ABT to incorporate structural elements of the
known CYP4Z1 substrate, LA. Ideally, we wanted to take advantage of the relatively high mid-
chain oxidation rate of LA to 8-hydroxy-LA (Vmax = ~40 min-1) in the hope that this would
translate to CYP4Z1-mediated bioactivation of ABT-analogs. The position of the exocyclic
nitrogen that was expected to undergo P450-mediated activation in these new N-alkylated ABT
congeners approximates the internal site of CYP4Z1-dependent metabolism of LA. Moreover,
as illustrated by the docking of LA to the recently published CYP4Z1 homology model,8
hydrophilic residues Asn381 and Ser383 are positioned to potentially engage with the free acid
moiety of fatty acid substrates. This hypothesis was tested by evaluating ABT-analog inhibitors
which contained a terminal carboxylic acid group alongside inhibitors where that moiety had
been removed.
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Neutral chain analogs were prepared from readily available aliphatic aldehyde building
blocks using methodology based on that previously used in the synthesis of N-benzyl-
aminobenzotriazole.24 Acid-containing analogs were synthesized using terminal acid, primary
alcohol starting materials and employing PCC-mediated oxidation to generate the aldehyde
moiety. Condensation of the aldehyde with ABT formed an imine which was then reduced to the
target compounds (Scheme 1).
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Evaluation of Time-Dependent Inhibition (TDI) of CYP4Z1 by ABT-analogs. Representative
dose-response curves are provided in Figure 3, which shows the potency comparison between
ABT and inhibitor 7 (all dose-response curves have been provided in the Supporting
Information, Figure S2). IC50 values for all ABT-analogs are shown in Table 1. ABT itself only
weakly inhibited CYP4Z1 without a prior pre-incubation with NADPH. However, the addition of
aliphatic tails (compounds 1 – 5) dramatically increased affinity for CYP4Z1 over that of ABT,
exemplified by a 50-fold decrease in the reversible IC50 value from 154 to 2.9 M for inhibitor 2.
While ABT itself showed a potent time-dependent effect for inhibition of CYP4Z1 (i.e. a 30-
minute pre-incubation of NADPH with CYP4Z1 produced a shifted-IC50 of 12 M), inhibitors 2-9
exhibited much lower shifted-IC50 values that indicated up to 120-fold higher potency compared
to ABT.
Analysis of the terminal acid-containing analogs showed similar gains in affinity over
ABT itself for CYP4Z1 inhibition. The most potent congeners, 7 and 8 (with eight and ten
carbon tails respectively), exhibited shifted-IC50 values of 200 nM, i.e. a 60-fold increase in
potency over ABT. Interestingly, the addition of the acid moiety did not have a dramatic effect
on CYP4Z1 inhibition potency (neither affinity nor inactivation) compared to the neutral-chain
aliphatic variants. However, the benefits of adding a terminal acid group became apparent as
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counter-screening proceeded as described below. Finally, despite changes in apparent
potencies for reversible and time-dependent inhibition across the entire analog series, the shift
ratios (IC50 / Shifted-IC50) varied only from 13 – 30 (omission of 3 narrowed this range to 23 –
30), which seems consistent with all inhibitors inactivating CYP4Z1 via a common mechanism.
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Counter-Screening Against CYP4 Family Enzymes. Due to the complex nature of the role
that fatty acid metabolites play in signaling in both healthy and disease states,4,5 non-specific
CYP4Z1 inhibitors such as ABT15,23 and HET00163,8,28,29 do not provide adequately
discriminatory molecular tools with which to assess the physiological role of CYP4Z1.
Consequently, we next evaluated the selectivity of the new ABT-analogs against recombinantly-
expressed, and commercially available, CYP4 enzymes; CYP4A11, -4F2, -4F3a, -4F3b, and -
4F12. As it is not commercially available, but was still of interest to us, CYP4F8 was expressed
in HepG2 cells and membranes were prepared identically to that for CYP4Z1. Pro-luciferin
probe substrates at concentrations  Km (see Experimental Section) were used for all CYP4
isozymes to determine inhibition, including CYP4F8 (as elucidated from in-house testing, Figure
S8), an isozyme in which limited substrate information has been published. A TDI assay at a
single, moderate, concentration of 30 M was utilized in order to assess whether any analogs
significantly inhibited the off-target enzymes. A 30-minute enzyme/NADPH pre-incubation step
was used to assess time-dependent effects (akin to a full IC50-shift assay), which were modest
for all enzymes and analogs tested (Figure 4). Initial screening against off-target CYP4
isozymes was performed with inhibitors 2 – 9 (inhibitor 1 was excluded due to its low potency).
The alkyl-modified analogs 2 – 5 inhibited CYP4F2, CYP4F3a and b and CYP4F12
substantially, whereas the acid modified analogs containing 10 and 12 carbon tails, 8 and 9,
respectively, substantially inhibited CYP4A11, CYP4F8 and 4F12. Importantly, however, two
acid analogs 6 and 7 exhibited negligible or very low activity towards the CYP4 enzymes tested
and were therefore selected for further study.
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Evaluation of TDI for Inhibitors 6 and 7 Against Off-target CYP isozymes. Full IC50-shift
assays (Figures S3, S4) were performed with 6 and 7 to thoroughly assess off-target inhibition
towards the other recombinantly-expressed CYP4 enzymes (Table 2). As determined from the
specificity ratios (off-target CYP IC50 / CYP4Z1 IC50), both 6 and 7 were comparatively poor
reversible inhibitors of these isozymes compared to CYP4Z1. Relatively small shift ratios (~1-3)
were obtained for both inhibitors against the six CYP4 isozymes tested, indicating low
propensity of 6 and 7 for enzyme-mediated bioactivation. Of note, any shift values <1 (indicative
of inhibitors with lowered potency after a plus NADPH pre-incubation) likely result from
significant metabolism of the inhibitor by the CYP to non-bioreactive species. The contrasting
reactivity of 6 and 7 between these off-target isozymes and CYP4Z1 is readily apparent from
the resultant TDI specificity ratios (off-target CYP shifted-IC50 / CYP4Z1 shifted-IC50) that ranged
from 137 to 2235. Therefore, CYP4Z1 selectivity becomes greatly enhanced as the result of TDI
for these two inhibitors.
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Overall, inhibitor 7 possessed a higher degree of selectivity for CYP4Z1 than 6.
Additionally, although 7 showed relatively similar affinity across the off-target CYP4 isozymes,
only CYP4F8 and CYP4F12 were well above the significance threshold for TDI (i.e. shift ratio 
1.5). This indicates that these two enzymes are more likely to bind 7 in a position which leads
directly to bioactivation. We suggest that this is the result of structural similarities with CYP4Z1
that are not present in the other CYP4 enzymes tested, namely, a lack of the active site
glutamate residue necessary to form a covalent attachment with the heme.8 The recent
publication on the crystal structure of octane-bound CYP4B1, an -hydroxylase with a
covalently attached heme, showed that this structural element is an important determinant of the
enzyme’s (and most likely other similar CYP4 isozymes) regio-specificity for oxidation at the
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terminal position of alkyl-chain containing substrates.27 Indeed, both CYP4F8 and CYP4F12
enzymes, neither of which possess a covalently attached heme, primarily produce an internal -
3-hydroxylated AA metabolite, 18-HETE,30,31 and thus seem to more closely mimic CYP4Z1
than the 20-HETE producing CYP4 family members. However, although the shift ratios pass the
threshold of significance for these two isozymes, they are ~10-fold lower than the shift ratio
observed for 7 and CYP4Z1. This results in CYP4F8 and CYP4F12 being inhibited 285-fold and
230-fold less, respectively, than CYP4Z1 after a pre-incubation with cofactor. We speculate that
this observation reflects the fact that 18-HETE is still three to four C-C bond lengths away from
the major site of CYP4Z1-mediated AA metabolism, and thus bioactivation of 7 is limited for
CYP4F8 and CYP4F12 because the exocyclic nitrogen for this analog is not positioned
optimally for these two isozymes.
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Full IC50-shift assays were also performed with 6 and 7 to evaluate off-target inhibition
towards hepatic CYP isozymes (Figures S5, S6), chosen based on the seven FDA
recommended CYPs for drug-drug interaction (DDI) testing.32 Human liver microsomes (HLM)
served as the enzyme source and a cocktail method was employed to evaluate inhibition of
CYP1A2, -2B6, -2C8, -2C9, -2C19, -2D6, and -3A4/5 concurrently (Table 3). Of note, in-house
testing for inhibition, using isozyme-specific inhibitor probes, of metabolism by the seven hepatic
CYPs in the cocktail assays yielded data in agreement with that reported in the literature33
(Figure S9). Similar to the results obtained with the CYP4 isozymes, inhibitors 6 and 7 did not
potently inhibit off-target hepatic CYPs, either reversibly or irreversibly, with specificity and TDI
specificity ratios spanning 23 – 140 and 134 – 1650, respectively. For both inhibitors, the largest
shift ratios were observed for CYP1A2, at 12.2 and 11.2 for 6 and 7, respectively. The large
degree of bioactivation observed may be explained by CYP1A2, like CYP4Z1, having a
propensity itself for internal oxidation of PUFAs. The 14,15-EET synthase activity for CYP1A2
has been reported to be ~0.54 pmol/pmol/min (at 50 M AA),34,35 and is in a similar range to that
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observed for CYP4Z1 of 1.9 pmol/pmol/min (at 75 M AA).8 Furthermore, all seven of the
hepatic CYP isozymes that we analyzed have been shown previously to metabolize AA to
14,15-EET, albeit with a large range of efficiencies and regio-specificities.35 Although for most
AA-metabolizing CYPs their role in breast cancer progression is largely unknown, CYP3A4
expression has been specifically linked to decreased survival and is required for growth in
several breast cancer cell lines.36 Therefore, the low off-target inhibition of the generally liver-
localized CYPs studied herein was a desired trait for both broad selectivity purposes and will
also allow for discriminatory probing of the role of CYP4Z1-dependent epoxygenase activity in a
breast cancer setting. Overall, both inhibitors 6 and 7 demonstrated their lack of activity towards
off-target enzymes primarily through low rates of bioactivation that resulted in modest time-
dependent inhibition and high TDI specificity ratios. Due to its combined properties of high
potency and excellent selectivity for CYP4Z1, inhibitor 7 was chosen for in-depth
characterization as an MBI.
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Characterization of Mechanism-based Inactivation of CYP4Z1 by 7. Mechanism-based
inactivation is typically viewed as a characteristic to be designed away from in drug
development, as the enzymes affected by this process are frequently off-target CYPs. When
this occurs, DDIs are common liabilities as these off-target enzymes are rendered catalytically
incompetent and must then be newly biosynthesized. However, numerous irreversible inhibitors
(including MBIs) have emerged recently as therapeutics and have the benefits of increased
biochemical efficiency, a longer duration of action, efficient inactivation of the target, and the
potential to avoid some resistance mechanisms.37 Examples of CYP MBI drugs include some
aromatase (CYP19A) inhibitors that have shown efficacy in the clinic and are currently used for
breast cancer treatment; an example being the steroidal mimetic exemestane.38 Evaluating the
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characteristics of an MBI (not simply establishing TDI) enables insight into inhibitor structure-
activity relationships, mechanisms of action, guides future analog work, and provides a better
understanding of the biochemistry of the target CYP. Therefore, the action of inhibitor 7 towards
CYP4Z1 was considered in light of the Silverman criteria for an MBI.39
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As seen in Figure 5A, 7 displayed concentration- and time-dependent loss of enzyme
activity, as first indicated by the single time point assays described above. When the values for
kobs were plotted against concentrations of 7, a KI = 2.2 μM and a kinact = 0.15 min-1 (Figure 5B)
were determined; the observed maximal rate of inactivation indicating saturation kinetics.
Comparison with exemestane, where a kinact for aromatase inhibition of 0.05 min-1 has been
determined, emphasizes how rapidly 7 inactivates CYP4Z1, although it must be noted that
exemestane inactivation of aromatase is highly efficient due to its tight-binding affinity (KI = 26
nM) for the enzyme.40 In order to show irreversibility of mechanism-based inactivation, we
assessed the fraction of activity remaining, from CYP4Z1 treated with 7, before and after
overnight dialysis to remove excess inhibitor (Figure 5C). No increased activity was observed
after dialysis, indicating irreversible enzyme destruction. Multiple experiments confirmed the
necessity of a catalytic step for the time-dependent inhibition of CYP4Z1 by 7. This is directly
shown in the dialysis experiment where lack of a pre-incubation step with the NADPH cofactor
prior to the substrate turnover reaction resulted in no inactivation of the enzyme. The addition of
hard or soft nucleophiles, at commonly used concentrations for reactive intermediate trapping
experiments41 – glutathione (GSH), N-acetyl lysine (NAL), methoxyamine (MeONH2), N-acetyl
cysteine (NAC), semicarbazide (Semicarb.), potassium cyanide (KCN) – did not abrogate
inactivation of CYP4Z1 by 7 (Figure 5D). Additionally, the presence of the reactive oxygen
species scavenging enzymes, catalase (CAT) and superoxide dismutase (SOD), did not rescue
CYP4Z1 activity, eliminating the possibility of non-specific, and non-enzymatic, oxidation
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reactions causing inactivation of CYP4Z1. These data indicate that inactivation of CYP4Z1
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occurs prior to release of bioactivated 7 from the active site.
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Next, the partition ratio for inactivation of CYP4Z1 by 7 was determined (Figure 5E). As
a measure of efficiency, the partition ratio is the number of latent inactivator molecules
converted and released as a product relative to each catalytic cycle resulting in enzyme
inactivation.39,42 Partition ratios for MBIs of CYPs can span a large range, with values reported
from 1 to >5000.43 Therefore, the partition ratio of 14 for 7 lies towards the efficient end of this
spectrum.
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Inhibition of CYP4Z1 AA metabolism in T47D Cells by 7. We next evaluated the inhibition of
CYP4Z1-dependent metabolism of AA in breast cancer derived T47D cells.44 Although a low
level of CYP4Z1 mRNA is detectable in breast cancer cell lines, which may be induced ~15-fold
from dexamethasone treatment,55 immortalization appears to eliminate expression of
endogenous CYP4Z1.3 Therefore, T47D cells were modified to constitutively express CYP4Z1
(T47D-CYP4Z1) using a recombinant lentivirus. Western blot analysis showed ample CYP4Z1
expression in the CYP4Z1-T47D cells and no observable endogenous CYP4Z1 expression in
the Vector-T47D cells (Figure S1C). Concentrations of free AA in human plasma span a wide
range, from 2.7 to 50 M.45,46 Therefore, a 50 M dose of AA was used to reflect this upper
boundary. The previously characterized CYP4Z1 metabolites of AA, 19-HETE and 14,15-EET,
along with the metabolite commonly generated by other CYP4 isozymes, 20-HETE, were
monitored.8 Also included in the AA metabolite profile was the degradation product 14,15-
dihydroxy eicosatrienoic acid (14,15-DiHET), as this could be generated by 14,15-EET
hydrolysis both non-enzymatically, in the tissue culture environment, and enzymatically through
the action of epoxide hydrolases potentially present in T47D cells.35,47
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The T47D-CYP4Z1 cells were exposed to 7, incorporating a 24-hour inhibitor pre-
incubation step prior to introduction of substrate, to assess TDI. At 3 M, potent inhibition of AA
metabolism was observed (Figure 6). Comparing peak area ratios (PAR), of metabolites divided
by their respective deuterated internal standards, showed that production of 14,15-EET, 19-
HETE, and 14,15-DiHET was inhibited 83, 86, and 80%, respectively. When inhibitor and
substrate were administered together to T47D-CYP4Z1 cells, without a pre-incubation step, 3
M of 7 produced lower levels of inhibition against production of 14,15-EET, 19-HETE, and
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14,15-DiHET, (Table 6), as expected. When dosed at 1 M, a comparable time-dependent
effect was observed with a ~2-fold decrease in potency. Similar results were also attained when
investigating AA metabolism in CYP4Z1-HepG2 membranes, where a time-dependent, ~90%
loss in CYP4Z1 activity (Figure S10) was observed with inhibitor 7 utilized at 3 M.
Finally, we confirmed the preference for CYP4Z1-mediated 14,15-EET production over
that of 20-HETE (only a trace amount was detected, slightly above lower limit of detection) in a
mammalian whole cell setting. Although other internal oxidation products from CYP4Z1 AA
metabolism were observed in both the HepG2 membranes and T47D cells (decreasing
proportionally with inhibitor and tentatively identified as 15-, 16-, 17-, and 18-HETE), these
apparently minor metabolites were not characterized further here.
These data demonstrate the efficacy of inhibitor 7 in a whole cell environment, and are
comparable to results we obtained for the inhibition of CYP4Z1-mediated Luc-BE O-
debenzylation in HepG2 cells (Figure S11A). Therefore, although not tested directly, potentially
lower inhibitor permeability and stability in whole cells appear not to be major issues.
Additionally, these results are not confounded by possible cytotoxicity of inhibitor 7, as a 3 M
treatment did not cause noticeable changes in viability 24 hours later for any of the cell lines
assayed (Figure S11B). Although we hypothesize 7 to have anti-proliferative effects against
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breast cancer cells expressing CYP4Z1, the specific conditions required to capture this effect
(e.g. temporal, growth factor, and PUFA substrate variables) were not assessed here as it was
beyond the scope of this study. To our knowledge, no studies utilizing primary cells have been
performed to interrogate CYP4Z1. However, due to its effectiveness in a breast-cancer derived
cell line for inhibiting the generation of the pro-angiogenic and pro-proliferative metabolite 14,15-
EET, we posit that inhibitor 7 will be an effective molecular tool to probe the druggability of
CYP4Z1 as a target for breast cancer therapy in varied in vitro settings.
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Conclusion
In summary, we leveraged our knowledge of the product selectivity of CYP4Z1 to
generate ABT-analogs that are efficient time-dependent inhibitors of the enzyme with
substantially increased potency over ABT. The design strategy of incorporating a terminal acid
to produce fatty acid mimetics resulted in high selectivity for CYP4Z1 for two analogs, 6 and 7.
Further characterization, monitoring isozyme-specific inhibition in a large panel of off-target
CYPs, identified 7 as the inhibitor with the best combination of isozyme selectivity and potency.
7 was further characterized as a relatively efficient mechanism-based inhibitor (kinact/Ki = 0.07
min-1M-1), with demonstrated time-dependent inhibition of CYP4Z1 in transfected breast
cancer-derived whole cells.
Future studies will investigate the mechanism of CYP4Z1 inactivation by 7 through
trapping of reactive metabolite(s) and analysis of heme and apoprotein adducts. These first-
generation inhibitors are potentially useful lead compounds for the development of more potent
CYP4Z1 inhibitors with appropriate pharmacokinetic features to support in vivo studies and
potential clinical applicability.
Experimental Section
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General Information for Synthesis. All solvents and reagents were purchased from
commercial sources (Alfa Aesar, Acros Organics, AstaTech, Fisher, Oakwood Chemical,
Sigma-Aldrich, and TCI) and used as received. All reactions were monitored by thin-layer
chromatography, visualized by either UV (254 nm) or potassium permanganate stain. For
isolation of purified compounds, normal phase chromatography was carried out on a Teledyne
Isco Combiflash system (Lincoln, NE). 1H and 13C NMR spectra were recorded at 25 °C in
deuterated chloroform (CDCl3) on either a 499.73 MHz Agilent DD2 (Santa Clara, CA) or 499.96
MHz Bruker Avance series (Billerica, MA) spectrometer, respectively. Chemical shifts are
reported below relative to the solvent peak in CDCl3 at 7.26 ppm (1H NMR), or tetramethylsilane
at 0.00 ppm (13C NMR). Coupling constants (J) are noted in hertz (Hz), and peak multiplicities
as either a singlet (s), doublet (d), triplet (t), pentet (p), or multiplet (m). All 1H and 13C NMR
spectra have been provided in the Supporting Information. Accurate mass and purity analyses
of the synthesized compounds were determined via UPLC−MS on a Waters Acquity UPLC
(Milford, MA) coupled in tandem with a Waters Acquity tunable UV detector and a Thermo LTQ-
Orbitrap mass spectrometer (San Jose, CA). Purity as assessed by UPLC−UV (254 and 280
nm) and 1H NMR spectroscopy were  95% for all compounds.
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Alcohol Oxidation (General Procedure I). A modified version of an existing method
that has been published was utilized48 and was as follows: A dried and N2-purged round-bottom
flask containing 1-2 g celite was charged with pyridinium chlorochromate (PCC, 3-5 mmol, 1.5
eq), solvated in ~50 mL anhydrous dichloromethane. The appropriate primary alcohol (2-3
mmol), solvated in a minimal amount of anhydrous dichloromethane, was subsequently added
to the reaction flask, and the mixture was stirred at room temperature under N2 for 90 minutes.
Next, anhydrous diethyl ether was added to the slurry and the mixture was filtered through a
pad of celite, rinsed with several portions of diethyl ether, and solvent was removed from the
combined organic fractions under vacuum. The residue was then washed thrice with portions of
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diethyl ether to harvest the soluble oxo-product and the combined diethyl ether fractions were
once again filtered through celite and solvent was removed under vacuum to afford the
corresponding aldehyde.
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Imine Formation (General Procedure II). Modified versions of existing methods that
have been published were utilized for both general procedures II and III24 and were as follows: A
dried and N2-purged round-bottom flask was charged with ABT (0.75 mmol), the appropriate
aldehyde (4.5 mmol, 6.0 eq), and ~20 mL glacial acetic acid. For inhibitors 6 – 9, the oxo-
product derived from general procedure I (0.5-0.8 mmol) was reacted with 1.5 eq of ABT. The
reaction mixture was stirred at room temperature overnight and the acetic acid was
subsequently removed under vacuum. The crude product was purified via flash chromatography
using a dichloromethane/ethyl acetate gradient to afford the corresponding imine intermediate
(for analogs containing terminal carboxylic acid groups, 2% acetic acid was added to the mobile
phase for improved chromatographic separation).
Imine Reduction (General Procedure III). A dried round-bottom flask was charged with
the appropriate imine intermediate (0.3-0.7 mmol), NaBH4 (~20 eq), and ~40 mL of a 2:1
solution of methanol/dichloromethane. The reaction mixture was stirred at room temperature for
1 hour and then the solvents were removed under vacuum. The remaining mixture was solvated
in a 20:1 solution of dichloromethane/methanol and then washed thrice with H2O; for the
carboxylic acid-containing analogs, the H2O was adjusted with 1N HCl to a pH of ~2 prior to
washing. The organic fraction was dried over Na2SO4, filtered, and the crude product was
purified via flash chromatography using a hexane/ethyl acetate gradient to afford the
corresponding inhibitors 1 – 9.
N‐butyl‐1H‐1,2,3‐benzotriazol‐1‐amine (1). Synthesized from butanal and ABT according
to general procedures II and III, with respective yields of 89 and 80%. 1H NMR (500 MHz,
CDCl3)  8.02 (d, J = 8.4 Hz, 1H), 7.61 (d, J = 8.3, 1H), 7.50 (t, J = 7.9 1H), 7.37 (t, J = 7.4, 1H),
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3.42 (t, J = 7.1, 2H), 1.52 (p, J = 7.0, 2H), 1.46 (sx, J = 7.5, 2H), 0.93 (t, J = 7.2, 3H); 13C NMR
(125 MHz, CDCl3)  144.4, 132.3, 127.7, 124.1, 119.9, 109.8, 52.91, 29.91, 19.96, 13.80; HRMS
(ESI+) m/z [M+H] calculated (C10H15N4) 191.1291, observed 191.1292,  ppm 0.5.
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N‐hexyl‐1H‐1,2,3‐benzotriazol‐1‐amine (2). Synthesized from hexanal and ABT
according to general procedures II and III, with respective yields of 80 and 84%. 1H NMR (500
MHz, CDCl3)  8.02 (d, J = 8.4 Hz, 1H), 7.61 (d, J = 8.3, 1H), 7.50 (t, J = 7.5 1H), 7.37 (t, J =
7.2, 1H), 3.41 (t, J = 7.2, 2H), 1.53 (p, J = 7.3, 2H), 1.42 (p, J = 8.2, 2H), 1.29 (m, 4H), 0.88 (t, J
= 6.9, 3H); 13C NMR (125 MHz, CDCl3)  144.4, 132.3, 127.6, 124.1, 119.9, 109.8, 53.22, 31.53,
27.84, 26.45, 22.52, 13.97; HRMS (ESI+) m/z [M+H] calculated (C12H19N4) 219.1604, observed
219.1606,  ppm 0.9.
N‐octyl‐1H‐1,2,3‐benzotriazol‐1‐amine (3). Synthesized from octanal and ABT according
to general procedures II and III, with respective yields of 77 and 70%. 1H NMR (500 MHz,
CDCl3)  8.00 (d, J = 8.4 Hz, 1H), 7.60 (d, J = 8.3, 1H), 7.48 (t, J = 7.3 1H), 7.35 (t, J = 7.2, 1H),
3.40 (t, J = 7.2, 2H), 1.52 (p, J = 7.3, 2H), 1.40 (p, J = 7.6, 2H), 1.26 (m, 8H), 0.87 (t, J = 7.1,
3H); 13C NMR (125 MHz, CDCl3)  144.4, 132.3, 127.7, 124.1, 119.9, 109.8, 53.27, 31.77,
29.31, 29.17, 27.88, 26.79, 22.62, 14.06; HRMS (ESI+) m/z [M+H] calculated (C14H23N4)
247.1917, observed 247.1922,  ppm 2.0.
N‐decyl‐1H‐1,2,3‐benzotriazol‐1‐amine (4). Synthesized from decanal and ABT
according to general procedures II and III, with respective yields of 67 and 70%. 1H NMR (500
MHz, CDCl3)  8.01 (d, J = 8.0 Hz, 1H), 7.60 (d, J = 8.3, 1H), 7.49 (t, J = 7.2 1H), 7.36 (t, J =
8.1, 1H), 3.40 (t, J = 6.8, 2H), 1.52 (p, J = 7.1, 2H), 1.41 (p, J = 6.9, 2H), 1.25 (m, 12H), 0.87 (t,
J = 7.2, 3H); 13C NMR (125 MHz, CDCl3)  144.4, 132.3, 127.6, 124.1, 119.9, 109.8, 53.24,
31.88, 29.52, 29.35, 29.28, 27.89, 26.79, 22.67, 14.10; HRMS (ESI+) m/z [M+H] calculated
(C16H27N4) 275.2230, observed 275.2234,  ppm 1.5.
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N‐dodecyl‐1H‐1,2,3‐benzotriazol‐1‐amine (5). Synthesized from dodecanal and ABT
according to general procedures II and III, with respective yields of 65 and 74%. 1H NMR (500
MHz, CDCl3)  8.02 (d, J = 8.4 Hz, 1H), 7.61 (d, J = 8.3, 1H), 7.50 (t, J = 7.7 1H), 7.37 (t, J =
7.3, 1H), 3.41 (t, J = 7.2, 2H), 1.53 (p, J = 7.4, 2H), 1.41 (p, J = 7.6, 2H), 1.25 (m, 16H), 0.88 (t,
J = 6.9, 3H); 13C NMR (125 MHz, CDCl3)  144.4, 132.3, 127.7, 124.1, 119.9, 109.8, 53.28,
31.92, 29.63, 29.56, 29.52, 29.35, 27.89, 26.79, 22.69, 14.11; HRMS (ESI+) m/z [M+H]
calculated (C18H31N4) 303.2543, observed 303.2546,  ppm 1.0.
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6‐[(1H‐1,2,3‐benzotriazol‐1‐yl)amino]hexanoic acid (6). Synthesized from 6-
hydroxyhexanoic acid and ABT according to general procedures I, II and III, with respective
yields of 23, 50 and 76%. 1H NMR (500 MHz, CDCl3)  8.01 (d, J = 8.4 Hz, 1H), 7.60 (d, J = 8.3,
1H), 7.49 (t, J = 7.8 1H), 7.36 (t, J = 7.9, 1H), 3.42 (t, J = 6.8, 2H), 2.36 (t, J = 7.3, 2H), 1.66 (p,
J = 7.4, 2H), 1.52 (m, 4H); 13C NMR (125 MHz, CDCl3)  178.7, 144.3, 132.3, 127.9, 124.3,
119.9, 109.8, 52.87, 33.74, 27.56, 26.19, 24.34; HRMS (ESI+) m/z [M+H] calculated
(C12H17N4O2) 249.1346, observed 249.1357,  ppm 4.4.
8‐[(1H‐1,2,3‐benzotriazol‐1‐yl)amino]octanoic acid (7). Synthesized from 8-
hydroxyoctanoic acid and ABT according to general procedures I, II and III, with respective
yields of 25, 60 and 80%. 1H NMR (500 MHz, CDCl3)  8.02 (d, J = 8.4 Hz, 1H), 7.60 (d, J = 8.3,
1H), 7.49 (t, J = 7.8 1H), 7.36 (t, J = 7.5, 1H), 3.40 (t, J = 7.1, 2H), 2.34 (t, J = 7.5, 2H), 1.63 (p,
J = 7.3, 2H), 1.53 (p, J = 7.4, 2H), 1.43 (p, J = 6.7, 2H), 1.33 (m, 4H); 13C NMR (125 MHz,
CDCl3)  179.3, 144.3, 132.3, 127.8, 124.3, 119.8, 109.8, 53.13, 33.99, 28.91, 28.88, 27.75,
26.53, 24.56; HRMS (ESI+) m/z [M+H] calculated (C14H21N4O2) 277.1659, observed 277.1670, 
ppm 4.0.
10‐[(1H‐1,2,3‐benzotriazol‐1‐yl)amino]decanoic acid (8). Synthesized from 10-
hydroxydecanoic acid and ABT according to general procedures I, II and III, with respective
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yields of 21, 43 and 56%. 1H NMR (500 MHz, CDCl3)  8.01 (d, J = 8.4 Hz, 1H), 7.60 (d, J = 8.3,
1H), 7.49 (t, J = 7.8 1H), 7.36 (t, J = 7.5, 1H), 3.40 (t, J = 7.1, 2H), 2.34 (t, J = 7.5, 2H), 1.62 (p,
J = 7.4, 2H), 1.52 (p, J = 7.5, 2H), 1.40 (p, J = 6.7, 2H), 1.28 (m, 8H); 13C NMR (125 MHz,
CDCl3)  179.2, 144.3, 132.2, 127.7, 124.2, 119.9, 109.8, 53.23, 33.96, 29.21, 29.08, 28.97,
27.82, 26.69, 24.65; HRMS (ESI+) m/z [M+H] calculated (C16H25N4O2) 305.1972, observed
305.1984,  ppm 3.9.
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12‐[(1H‐1,2,3‐benzotriazol‐1‐yl)amino]dodecanoic acid (9). Synthesized from 12-
hydroxydodecanoic acid and ABT according to general procedures I, II and III, with respective
yields of 22, 55 and 71%. 1H NMR (500 MHz, CDCl3)  8.02 (d, J = 8.4 Hz, 1H), 7.61 (d, J = 8.3,
1H), 7.49 (t, J = 8.0 1H), 7.36 (t, J = 7.8, 1H), 3.40 (t, J = 7.2, 2H), 2.35 (t, J = 7.5, 2H), 1.63 (p,
J = 7.4, 2H), 1.53 (p, J = 7.5, 2H), 1.41 (p, J = 7.6, 2H), 1.26 (m, 12h); 13C NMR (125 MHz,
CDCl3)  179.5, 144.3, 132.2, 127.7, 124.2, 119.9, 109.8, 53.27, 34.06, 29.39, 29.32, 29.28,
29.16, 29.02, 27.83, 26.73, 24.70; HRMS (ESI+) m/z [M+H]+ calculated (C18H29N4O2) 333.2285,
observed 333.2297,  ppm 3.6.
CYP4Z1-Mediated Luciferin-Benzyl Ether (Luc-BE) O-Debenzylation. To compare
Luc-BE (Promega, Madison, Wisconsin) O-debenzylation activity between the CYP4Z1-HepG2
(81 pmol CYP4Z1/mg membrane protein) and Vector-HepG2 membrane stocks (Supporting
Information: Method for Membrane Preparation), metabolic reactions were set up in triplicate
on ice and contained 0.6 mg/mL HepG2 membrane protein in 100 mM KPi, pH 7.4, in a final
volume of 100 L. Luc-BE was added to achieve a final concentration of 50 M and samples
were equilibrated at 37 C for 5 min. NADPH was added for a final concentration of 1mM, or KPi
was added for the minus NADPH controls. Metabolic reactions were allowed to proceed for 20
min at 37 C. To assess the kinetics of CYP4Z1-mediated Luc-BE O-debenzylation, metabolic
reactions were set up in triplicate on ice and contained 3 pmol of CYP4Z1, in 100 mM KPi, pH
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7.4, in a final volume of 100 L. Luc-BE was added for final concentrations of 1, 5, 10, 25, 50,
75, 100, and 150 M and the reactions were equilibrated at 37 C for 5 min. NADPH was added
for a final concentration of 1 mM, and metabolism was allowed to proceed for 10 minutes at 37
C.
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Luminescence Assay (General Procedure IV). The CYP4Z1-mediated Luc-BE O-
debenzylation reactions outlined above, those discussed below to assess inhibition of CYP4Z1
activity, and the other CYP-mediated pro-luciferin metabolism reactions, were quenched (with
P450 glo) and assayed as follows: Reactions were transferred from their incubation tubes to
white walled 96-well plates containing an equal volume of P450-glo luciferase detection reagent
(Promega, Madison, Wisconsin). Beetle luciferin metabolite standards (Promega), prepared in
identical matrices, were added at the same time as the metabolic reactions to the plates. After
gently mixing and a 20-minute incubation at room temperature, the plates were read on a Biotek
Synergy HTX Microplate Reader (Winooski, VT). The luminescence generated (relative light
units, RLU) from the calibration luciferin standards was used to generate a standard curve to
quantitate the metabolite concentrations in the reactions. These data were subsequently used to
determine rates of CYP4Z1 activity and for comparisons between inhibitor and vehicle treated
reactions.
CYP4 Isozyme IC50-Shift Experiments. IC50-shift experiments to test inhibition of
recombinantly expressed CYP4 isozymes were each performed separately, with duplicate
technical replicates, and the experiments were repeated two to three times on separate days.
Each metabolic reaction in the experiments contained one of the following: 4 pmol of CYP4Z1
(HepG2 membranes), 1.5 pmol CYP4F8 (HepG2 membranes), 4 pmol of CYP4A11, CYP4F2,
CYP4F3a, CYP4F3b, CYP4F12 (Corning SupersomesTM, New York, NY). Total protein
concentration for all reactions was ~0.20 – 0.25 mg/mL, except for CYP4F8, which was 0.35
mg/mL due to the lower specific content of the enzyme in the HepG2 membrane preparation.
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Reactions were set up on ice containing the specific CYP4 isozyme in 100 mM KPi, pH 7.4, in a
final volume of 200 L. All inhibitors were solvated in DMSO to make 200 stocks and added to
the reactions to achieve final concentrations spanning 0.001 – 100 M (1 – 9 for CYP4Z1), 0.03
9
– 1000 M (ABT for CYP4Z1), 1 – 3000 M (6 for off-target CYP4 isozymes), or 1 – 1000 M (7
for off-target CYP4 isozymes). An equal volume of DMSO was added to all vehicle control
samples and DMSO concentrations did not exceed 0.5% v/v for all reactions. In order to assess
time-dependent inhibition for the inhibitors, half of the metabolic reactions (for each isozyme)
included a pre-incubation step plus the cofactor NADPH and the other half a pre-incubation step
minus NADPH. The temporal and reagent addition scheme used was as follows: After a 5-min
equilibration at 37 C, NADPH was added from a 50 stock solution for a final concentration of 1
mM to the plus NADPH pre-incubation samples, and an equal volume of buffer to the minus
NADPH pre-incubation samples. After a subsequent 30-minute pre-incubation at 37 C, pro-
luciferin probe substrates for each isozyme (detailed below) were added to both sets of pre-
incubation reactions from 50 stocks (all at final concentrations  Km values). Next, NADPH was
added to the minus NADPH pre-incubation reactions and an equal volume of buffer to the plus
NADPH pre-incubation reactions. The probe substrate reactions were allowed to proceed for 10
minutes at 37 C and reactions were assayed according to general procedure IV. The pro-
luciferins (all from Promega) that were used as CYP4 isozyme probe substrates
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(concentrations) and the biotransformations that were monitored were as follows: CYP4Z1 Luc-
BE (29 M), CYP4F8 Luc-BE (12 M), and CYP4F12 Luc-BE (23 M) O-debenzylation;
CYP4A11 Luc-4A (80 M) O-demethylation; CYP4F2, CYP4F3a, and CYP4F3b, Luc-4F2/3 (2
M, due to substrate inhibition kinetics that was observed for all three enzymes, Figure S7) O-
(4-methylthio)-debenzylation. As no metabolite standard for Luc-4A or Luc-4F2/3 was available,
enzymatic activity was assessed in a semi-quantitative fashion for isozymes metabolizing these
probe substrates. The counter-screening TDI experiment that is presented in Figure 4 used the
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same methods as detailed above except with only a single concentration of 30 M for inhibitors
2 – 9 and HET0016, as a general CYP4 inhibitor positive control, at 0.5 M.
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Hepatic CYP Isozyme IC50-Shift Experiments. IC50-shift experiments to test Inhibition
of hepatic CYP isoforms by 6 and 7 were performed with duplicate technical replicates and
experiments were repeated three times on separate days. The assays utilized pooled human
liver microsomes (HLM) made up from eight random, individual donor livers, that had been
prepared for a previous study52 and followed a cocktail approach similar to previously published
methods.19,33 The metabolic reactions contained HLM at 0.25 mg/mL total protein, inhibitor 6 or
7 at 1 – 3000 or 1 – 1000 M, respectively, and used the same setup, timing, and reagent
addition scheme as that followed for the CYP4 isozyme IC50-shift experiments. However, after
the pre-incubation period, a cocktail of seven selective probe substrates (see below) combined
was added from a 50 stock. The CYP isoforms evaluated, substrates (concentrations), and
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biotransformations were as follows: CYP1A2 phenacetin (100 M) O-deethylation; CYP2B6
bupropion (12 M) 1’-hydroxylation; CYP2C8 amodiaquine (1 M) N-deethylation; CYP2C9
tolbutamide (100 M) methyl-hydroxylation; CYP2C19 (S)-mephenytoin (50 M) 4’-
hydroxylation; CYP2D6 dextromethorphan (2.5 M) O-demethylation; and CYP3A4/5
midazolam (2.5 M) 1’-hydroxylation. All probe substrates were used at concentrations  Km.
After probe substrate turnover had proceeded for 10 minutes at 37 C, reactions were
subsequently terminated with an equal volume of ice-cold acetonitrile containing 200 nM of each
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deuterated internal standard (listed below) and centrifuged at 15,000
g for 10 min. A
supernatant aliquot (5 μL) was analyzed by UPLC−MS/MS on a Waters Acquity UPLC
connected to a Waters Xevo TQ-S instrument in ESI+ mode, with the following settings:
capillary 3.1 kV, source offset 60.0 V, source temperature 150 °C, desolvation temperature 350
°C, cone gas flow 150 L/h, desolvation gas flow 800 L/hour, and collision gas flow 0.15 mL/min.
Cone voltage and collision energies varied between analytes and are noted below. Analytes
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were separated chromatographically using a Waters HSS T3 column (2.1 × 100 mm, 1.8 μm),
starting with 95% mobile phase A (0.1% formic acid in H2O) and 5% mobile phase B (0.1%
formic acid in acetonitrile) at a flow rate of 0.3 mL/min. After holding for 1 min, B was increased
linearly from 5 to 60% between 1 and 7 min, immediately elevated to 95%, and then held at
95% between 7 and 8.5 min; total run time was 11 min following a 2.5 min equilibration period.
The metabolites were detected by multiple reaction monitoring (MRM) with the following
transitions, cone voltages (CV), and collision energies (CE): acetaminophen m/z 152.2 > 110,
CV = 40 V, CE = 20 eV; hydroxybupropion m/z 256.1 > 238.1, CV = 32 V, CE = 10 eV; N-
desethylamodiaquine m/z 328.1 > 283, CV = 40 V, CE = 20 eV; hydroxytolbutamide m/z 287.1 >
107, CV = 40 V, CE = 20 eV; 4-hydroxymephenytoin m/z 235.1 > 150.1, CV = 30 V, CE = 20
eV; dextrorphan m/z 258.1 > 157, CV = 40 V, CE = 30 eV; and 1’-hydroxymidazolam m/z 342.1
> 203, CV = 40 V, CE = 30 eV. The corresponding isotope-labeled internal standards and MRM
transitions were as follows: acetaminophen-d3 m/z 155.2 > 111.06, hydroxybupropion-d6 m/z
262.2 > 244.2, N-desethylamodiaquine-d5 m/z 333.1 > 283.1, hydroxytolbutamide-d9 m/z 296.1
> 107, 4-hydroxymephenytoin-d3 m/z 238.1 > 150.1, dextrorphan-d3 m/z 261.2 > 157, and 1’-
hydroxymidazolam-d4 m/z 346.1 > 203.05. Metabolite standards, using identical matrix and
work-up conditions, were used to create standard curves that were utilized in quantitation of the
metabolites and subsequent comparison of enzymatic activity between inhibitor and vehicle
treated metabolic reactions.
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Kinetics of CYP4Z1 Inactivation From 7. Inhibition reactions were set up on ice in
triplicate and contained 5 pmol CYP4Z1 in 100 mM KPi, pH 7.4, in a final volume of 100 μL.
Inhibitor 7 was added to achieve final concentrations of 0.3, 1, 3, 6, 10, and 20 μM, and DMSO
alone was utilized as a vehicle control. After a 5 min equilibration at 37 C, the reactions were
initiated by the addition of NADPH (1 mM final concentration). Aliquots of the inhibition reaction
(10 μL) were taken at times 0, 3, 5, 10, 15, and 20 min, diluted 10-fold into a 100 μL probe
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substrate reaction containing Luc-BE, at 10 the determined Km for CYP4Z1 (290 μM), in 100
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mM KPi buffer with 1 mM NADPH. The probe substrate reactions progressed for 10 min at 37
°C and were quenched with an equal volume of ice-cold acetonitrile containing 200 nM Luc-4A
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as an internal standard and centrifuged at 15,000
g for 10 min. Note: due to the timing
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constraints of assaying for the kinetics of enzyme inactivation, LC-MS/MS was utilized to detect
the luciferin metabolite from CYP4Z1 Luc-BE O-debenzylation. A supernatant aliquot (3 μL) was
analyzed by UPLC−MS/MS on a Waters Acquity UPLC connected to a Waters Xevo TQ-S
instrument in ESI+ mode, with the following settings: capillary 2.8 kV, cone 40.0 V, collision 20.0
V, source offset 60.0 V, source temperature 150 °C, desolvation temperature 350 °C, cone gas
flow 150 L/h, desolvation gas flow 800 L/hour, and collision gas flow 0.15 mL/min. Analytes
were separated chromatographically using a Waters BEH C18 column (2.1 × 50 mm, 1.7 μm),
starting with 95% mobile phase A (0.1% formic acid in H2O) and 5% mobile phase B (0.1%
formic acid in acetonitrile) at a flow rate of 0.3 mL/min. After holding for 1 min, B was increased
linearly from 5 to 95% between 1 and 4 min and held at 95% between 4 and 6 min; total run
time was 8.5 min following a 2.5 min equilibration period. The metabolite luciferin was detected
by MRM at m/z 281.0 > 234.92 and the internal standard Luc-4A was detected by MRM at m/z
289.1 > 201.96. The metabolite luciferin, using identical matrix and work-up conditions, was
used to create a standard curve that was utilized in quantitation of the metabolite concentration
in the reactions and subsequent comparison of enzymatic activity between inhibitor and vehicle
treated metabolic reactions. The natural logarithm of the remaining CYP4Z1 activity after
treatment with varying concentrations of 7 was plotted versus pre-incubation time. The resultant
differential slopes generated kobs rates, which were then plotted against the concentrations of 7
to allow for the determination of the inactivation parameters KI and kinact
.
Examination of the Effects of Dialysis on CYP4Z1 Inactivation by 7. Inhibition
reactions were set up on ice in triplicate and contained 3 pmol CYP4Z1 in 100 mM KPi, pH 7.4,
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in a final volume of 100 μL. Inhibitor 7 was added for a final concentration of 3 M, and DMSO
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was added to vehicle reactions. After a 5 min equilibration at 37 C, the reactions were initiated
by the addition of NADPH (1 mM final concentration), or KPi buffer only for the minus NADPH
samples. The reactions were allowed to proceed for 15 min and then, for the pre-dialysis
samples, aliquots (10 μL) were removed and diluted 10-fold into a 100 μL probe substrate
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reaction containing Luc-BE, at 10 the determined Km for CYP4Z1 (290 μM), in 100 mM KPi
buffer with 1 mM NADPH. The probe substrate reactions progressed for 10 min at 37 °C and
were assayed according to general procedure IV. For the post-dialysis samples (following the
15 min inhibition reaction), the entirety of the reaction was immediately transferred to 10,000
molecular weight cut-off mini dialysis cups (Thermo Scientific Slide-A-Lyzer) suspended in 1 L
of 100 mM KPi, pH 7.4, and incubated at 4 C with gentle stirring overnight. The next morning,
aliquots were removed, diluted into probe substrate reactions, and assayed as described for the
pre-dialysis samples.
Examination of the Effects of Trapping Agents and Reactive Oxygen-Scavenging
Enzymes on CYP4Z1 Inactivation by 7. Inhibition reactions were set up on ice in triplicate and
contained 3 pmol CYP4Z1 in 100 mM KPi, pH 7.4, in a final volume of 100 μL. Inhibitor 7 was
added for a final concentration of 3 M, and DMSO was added to vehicle reactions. Reduced
glutathione, N-acetyl-lysine, methoxyamine, N-acetyl-cysteine, and semicarbazide were added
individually to reactions for final concentrations of 5 mM, and potassium cyanide was added to a
reaction for a final concentration of 1 mM. Catalase (CAT) and super oxide dismutase (SOD),
from Sigma (St. Louis, MO), were both added to an inhibition reaction together at 80 units each.
Both inhibitor 7 and DMSO vehicle reactions were set up for each additive listed above. After a
5 min equilibration at 37 C, the reactions were initiated by the addition of NADPH (1 mM final
concentration) or KPi for the minus NADPH samples. The reactions were allowed to proceed for
15 min at 37 C, and then aliquots of the inhibition reaction (10 μL) were diluted 10-fold into a
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(290 μM), in 100 mM KPi buffer with 1 mM NADPH. The probe substrate reactions progressed
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for 10 min at 37 °C and were assayed according to general procedure IV.
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Determination of the Partition Ratio. The number of molecules of 7 metabolized per
molecule of CYP4Z1 inactivated, the partition ratio, was estimated from a dilution method.
Inhibition reactions were set up on ice in triplicate and contained 5 pmol CYP4Z1 in 100 mM
KPi, pH 7.4, in a final volume of 100 μL. Inhibitor 7 was added at 11 different final
concentrations spanning 0 to 50 M. After a 5 min equilibration at 37 C, the inactivation
reactions were initiated by the addition of NADPH (1 mM final concentration). The reactions
were allowed to proceed for 45 minutes at 37 C to allow for maximal inactivation, and then
aliquots of the inhibition reaction (10 μL) were diluted 10-fold into a 100 μL probe substrate
×
reaction containing Luc-BE, at 10 the determined Km for CYP4Z1 (290 μM), in 100 mM KPi
buffer with 1 mM NADPH. The probe substrate reactions progressed for 10 min at 37 °C and
were assayed according to general procedure IV. The percentage of remaining CYP4Z1 activity
was plotted versus the molar ratio of [7]/[CYP4Z1]. Extrapolation of the initial linear portion to
the x-intercept generated the turnover number; the partition number was then calculated as the
turnover number minus 1.
Inhibition of CYP4Z1-Mediated AA Metabolism in T47D Cells by 7. T47D cells were
modified to express CYP4Z1 (CYP4Z1-T47D), or a control vector (Vector-T47D), in a similar
fashion to the HepG2 cell lines. A schematic outline of the lentiviral vector system has been
provided in the Supporting Information (Figure S1A). Western blot analysis was performed as
previously described.8 The stably transduced cell lines were grown in tissue culture media
consisting of RPMI-1640, 1%Glutamine, 10% FBS, and 0.01 mg/ml Insulin and selected with
puromycin treatment (2 μg/mL) over a 4-day period to ensure homogenous transduction
profiles. Both CYP4Z1- and Vector-T47D cell lines were seeded into 6-well tissue culture-
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treated plates at 2.5105 cells/mL in a volume of 2 mL/well. The CYP4Z1-T47D cells were then
dosed with inhibitor 7 to achieve final concentrations of 1 and 3 M from 1000 DMSO stocks,
or DMSO vehicle was added (plus 24-hour pre-incubation samples); Vector-T47D cells were
dosed with DMSO. The cells were then placed in a tissue culture incubator at 37 C. After 24
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hours, AA was added to all wells for a final concentration of 50 M from a 400 concentrated
stock solvated in degassed ethanol. Inhibitor 7 was then added to the CYP4Z1-T47D cells,
which had not been dosed the day prior (minus 24-hour pre-incubation samples), at 1 and 3 M,
or DMSO vehicle was added; Vector-T47D cells were dosed with DMSO. Cells were placed
back into the tissue culture incubator for 2 hours to allow for CYP4Z1-mediated AA oxidation.
Following this incubation period, media was collected from each well and 0.5 mL H2O was
added to the wells which were then incubated at room temperature for 1 minute. Subsequently,
1 mL of a 1:1 mixture of acetonitrile and methanol was added to each well and cells were
scraped to remove them from the tissue culture dish. The aqueous and organic mixture was
pooled with the respective media collections and a further 1 mL of the acetonitrile/methanol
mixture was added to rinse the wells of any remaining cells and/or extracted analytes. The
combined aqueous and organic fractions were then subjected to a previously described workup8
and a supernatant aliquot (10 L) was analyzed by UPLC−MS/MS on a Waters Acquity UPLC
connected to a Waters Xevo TQ-S instrument in ESI- mode using settings, chromatography
gradient, and column as previously described by McDonald et al.8 The oxidative AA metabolites
14,15-DiHET, 19-HETE, 20-HETE, and 14,15-EET were detected by MRM at m/z 337.10 >
207.00, 319.1 > 231, 319 > 245, and 319.1 > 219, respectively. The deuterated internal
standards 14,15-DiHET-d11, 20-HETE-d6, and 14,15-EET-d11 were detected by MRM at m/z
348.30 > 207.20, 325.10 > 281.10, and 330.20 > 218.80, respectively. Relative inhibition of AA
metabolite formation was determined by comparing the peak area ratios of the analytes divided
by their respective deuterated internal standards from cells treated with 7 compared to vehicle,
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at the various timepoints and conditions noted above. As no deuterated internal standard for 19-
HETE is available, 20-HETE-d6 was utilized as the internal standard for this AA metabolite. The
entire experiment was repeated under similar conditions and yielded comparable results (data
not shown).
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Data Analysis. GraphPad Prism 7.00 (San Diego, CA) was used for graphing all results,
to estimate all kinetic parameters (Km, Vmax, KI, and kinact), and to determine the half maximal
inhibitory concentration (IC50) values. IC50 values from each individual experiment were
averaged and standard deviations calculated. Mass spectral analysis was performed with
Waters MassLynx V4.1 and Thermo Xcalibur V2.0. 1H and 13C NMR spectral analysis was
performed with Bruker TopSpin V4.0.7.
Associated Content
Supporting Information
CYP4Z1 expression systems; Method for Membrane Preparation; IC50 and shifted-IC50
determination for inhibition of CYP4Z1 by 1 – 9; IC50 and shifted-IC50 determination for inhibition
of CYP4 isoforms by 6; IC50 and shifted-IC50 determination for inhibition of CYP4 isoforms by 7;
IC50 and shifted-IC50 determination for inhibition of hepatic CYP isoforms by 6; IC50 and shifted-
IC50 determination for inhibition of hepatic CYP isoforms by 7; Luc-4F2/3 metabolism kinetics for
CYP4F2, CYP4F3a, and CYP4F3b; Analysis of CYP4F8 metabolism of pro-luciferins; Internal
validation of HLM cocktail inhibition assay; Inhibition of CYP4Z1-mediated AA Metabolism by 7
in HepG2 Membranes; Inhibition of CYP4Z1 in HepG2 Cells and HepG2, T47D Cell Viability; 1H
and 13C NMR Spectra; Molecular Formula Strings Spreadsheet
Acknowledgements
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We thank Dale Whittington and J. Scott Edgar for assistance in the University of Washington,
Department of Medicinal Chemistry, Mass Spectrometry Center. We also thank Adrienne
Roehrich for assistance in the University of Washington, Department of Chemistry, NMR
Facility. Lastly, we thank Kent Kunze for helpful discussions regarding mechanism-based
inactivation of CYP450s.
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Abbreviations
CYP4Z1, cytochrome P450 4Z1; KPi, potassium phosphate buffer; ABT, 1-aminobenzotriazole;
Luc-BE, luciferin-benzyl ether; NADPH, Reduced β-nicotinamide-adenine dinucleotide
phosphate; MBI, mechanism-based inhibitor; TDI, time-dependent inhibition; IC50, half maximal
inhibitory concentration; AA, arachidonic acid; LA, lauric acid; 14,15-EET, 14,15-
epoxyeicosatrienoic acid; 20-HETE, 20-hydroxyeicosatetraenoic acid; PUFA, polyunsaturated
fatty acid; CAT, catalase; SOD, super oxide dismutase; GSH, glutathione; NAL, N-acetyl lysine;
MeONH2, methoxyamine; NAC, N-acetyl cysteine; KCN, potassium cyanide
References
(1) Ortiz de Montellano, P.R. Cytochrome P450: Structure, Mechanism, and Biochemistry. New
York, NY: Kluwer Academic/Plenum Publishers; 2005.
(2) Rieger, M. A.; Ebner, R.; Bell, D. R.; Kiessling, A.; Rohayem, J.; Schmitz, M.; Temme, A.;
Rieber, E. P.; Weigle, B. Identification of a novel mammary-restricted cytochrome P450,
CYP4Z1, with overexpression in breast carcinoma. Cancer Res 2004, 64, 2357-2364.
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