Design, synthesis and cellular characterization of a dual inhibitor of 5-lipoxygenase and soluble epoxide hydrolase

Article abstract of DOI:10.3390/molecules22010045

The arachidonic acid cascade is a key player in inflammation, and numerous well-established drugs interfere with this pathway. Previous studies have suggested that simultaneous inhibition of 5-lipoxygenase (5-LO) and soluble epoxide hydrolase (sEH) results in synergistic anti-inflammatory effects. In this study, a novel prototype of a dual 5-LO/sEH inhibitor KM55 was rationally designed and synthesized. KM55 was evaluated in enzyme activity assays with recombinant enzymes. Furthermore, activity of KM55 in human whole blood and endothelial cells was investigated. KM55 potently inhibited both enzymes in vitro and attenuated the formation of leukotrienes in human whole blood. KM55 was also tested in a cell function-based assay. The compound significantly inhibited the LPS-induced adhesion of leukocytes to endothelial cells by blocking leukocyte activation.

Full text of DOI:10.3390/molecules22010045

molecules
Communication
Design, Synthesis and Cellular Characterization of a
Dual Inhibitor of 5-Lipoxygenase and Soluble
Epoxide Hydrolase
Karin Meirer 1, Daniel Glatzel 2, Simon Kretschmer 1, Sandra K. Wittmann 1, Markus Hartmann 1,
René Blöcher 1, Carlo Angioni 3, Gerd Geisslinger 3, Dieter Steinhilber 1, Bettina Hofmann 1,
Robert Fürst 2,* and Ewgenij Proschak 1,
*
1
Institute of Pharmaceutical Chemistry, Goethe-University of Frankfurt, Max-von-Laue Str. 9,
D-60438 Frankfurt am Main, Germany; karin.meirer@boehringer-ingelheim.com (K.M.);
simkrets@pharmchem.uni-frankfurt.de (S.K.); wittmann@pharmchem.uni-frankfurt.de (S.K.W.);
hartmann@pharmchem.uni-frankfurt.de (M.H.); bloecher@ucdavis.edu (R.B.);
steinhilber@em.uni-frankfurt.de (D.S.); hofmann@pharmchem.uni-frankfurt.de (B.H.)
Institute of Pharmaceutical Biology, Goethe-University of Frankfurt, Max-von-Laue Str. 9,
D-60438 Frankfurt am Main, Germany; Glatzel@em.uni-frankfurt.de
Institute of Clinical Pharmacology, Goethe-University of Frankfurt, Theodor-Stern-Kai 7,
D-60590 Frankfurt am Main, Germany; angioni@em.uni-frankfurt.de (C.A.);
geisslinger@em.uni-frankfurt.de (G.G.)
2
3
*
Correspondence: fuerst@em.uni-frankfurt.de (R.F.); proschak@pharmchem.uni-frankfurt.de (E.P.);
Tel.: +49-69-798-29655 (R.F.); +49-69-798-29301 (E.P.)
Academic Editor: Cornelis J. Van der Schyf
Received: 7 November 2016; Accepted: 23 December 2016; Published: 29 December 2016
Abstract: The arachidonic acid cascade is a key player in inflammation, and numerous well-established
drugs interfere with this pathway. Previous studies have suggested that simultaneous inhibition of
5-lipoxygenase (5-LO) and soluble epoxide hydrolase (sEH) results in synergistic anti-inflammatory
effects. In this study, a novel prototype of a dual 5-LO/sEH inhibitor KM55 was rationally designed
and synthesized. KM55 was evaluated in enzyme activity assays with recombinant enzymes.
Furthermore, activity of KM55 in human whole blood and endothelial cells was investigated. KM55
potently inhibited both enzymes in vitro and attenuated the formation of leukotrienes in human whole
blood. KM55 was also tested in a cell function-based assay. The compound significantly inhibited
the LPS-induced adhesion of leukocytes to endothelial cells by blocking leukocyte activation.
Keywords: soluble epoxide hydrolase; 5-lipoxygenase; inflammation; designed multitarget ligands;
leukocyte-endothelial cell interaction
1. Introduction
The arachidonic acid (AA) cascade is one of the main regulatory pathways involved in the
inflammatory response and associated diseases. The initial step of the AA cascade is the release of AA
from membrane phospholipids by phospholipases [1]. Free AA is subsequently metabolized in three
distinct branches of the AA cascade. The cyclooxygenase (COX) branch leads to the formation
of various prostaglandins and thromboxane [
enzymes of the lipoxygenase (LO) branch [ ]. Oxidation of AA by cytochrome P450 (CYP) enzymes
results in the biosynthesis of epoxyeicosatrienoic acids (EETs), which are subsequently degraded to
dihydroxyeicosatrienoic acids (DHETs) by the enzyme soluble epoxide hydrolase (sEH) [ ]. Almost
2]. Leukotrienes and lipoxins are formed by the
3
4
all key enzymes of the AA cascade are of interest in pharmaceutical research, however, only COX
inhibitors, one 5-LO inhibitor, and antagonists of cysteinyl leukotriene receptors and thromboxane
receptors are in clinical use.
Molecules 2017, 22, 45; doi:10.3390/molecules22010045
www.mdpi.com/journal/molecules

Molecules 2017, 22, 45
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Modulators of the AA cascade not only affect their primary targets; the accumulation of the
substrates and regulatory crosstalk between the products leads to pronounced shunting effects. One of
the best investigated effects is the accumulation of leukotrienes as a consequence of COX inhibition,
leading to analgesic-induced asthma [
the inhibition of leukotriene A4 hydrolase leads to increased formation of lipoxin A4, a pro-resolvatory
lipid mediator [ ]. An interesting crosstalk involves the lipoxygenase and CYP branch of the AA
cascade. The inhibition of sEH and subsequent accumulation of AA-derived epoxides leads on the one
hand to the formation of lipoxins [ ] and on the other hand to increased levels of pro-inflammatory
LOX products [ ]. Most interestingly, the simultaneous inhibition of sEH and 5-lipoxygenase activating
5]. The shunting phenomena can have beneficial effects; e.g.,
6,7
8
9
protein (FLAP) led to a synergistic anti-inflammatory response in vivo [10].
The described effects support the paradigm change in drug discovery from selective ligands
towards designed multi-target ligands (DMLs), which might exhibit improved safety and efficacy [11].
The possibilities and limitations of DMLs in the context of the AA cascade have been excessively
reviewed [12–14]. Several approaches have been made to design DMLs that inhibit 5-LO and sEH
simultaneously. Moser et al. identified a benzimidazole derivative as a dual sEH/5-LO inhibitor using
virtual screening [15]. A hybrid imidazo-[1,2a]-pyridine-based inhibitor was designed by Meirer et al.
and exhibited high potency in vitro [16], but failed to affect cellular systems due to unfavorable plasma
stability (data not shown). A fragment-based approach was followed by Achenbach et al. who
identified and subsequently optimized a 2-aminothiazole derivative as a dual sEH/5-LO inhibitor [17].
In this study, we present the rational design of a novel hybrid dual sEH/5-LO as a chemical tool for
the investigation of cellular effects of the simultaneous inhibition of sEH and 5-LO.
2. Results
2.1. Design and Synthesis of KM55, a Dual sEH/5-LO Inhibitor
A critical step in the design of a hybrid DML is the careful choice of pharmacophores for the
individual targets and the linker to interconnect them [11]. The only approved inhibitor of 5-LO,
zileuton (1, Scheme 1), exhibits an iron-chelating moiety, which interacts with the non-heme iron
ion in the catalytic center and an aromatic ring [18]. Substituted ureas are well-established epoxide
mimetics interacting with the catalytic center of sEH [19]. One of the substituents, a 4-trifluoromethoxy
phenyl moiety, was shown to contribute to the high potency and excellent pharmacokinetic profile of
a range of sEH inhibitors [20], represented by 1-trifluoromethoxyphenyl-3-(1-acetylpiperidin-4-yl)-
urea (TPAU, 2, Scheme 1). Considering the linker between the two pharmacophores, studies by
Meirer et al. [16] and Hwang et al. [21] have shown that a propyl spacer is most suitable. These
considerations lead to the design of 1-(3-{5-(hydroxyureido)methyl-2-methoxyphenoxy}propyl)-3-[4-
(trifluoromethoxy)phenyl]urea 5 (KM55) as a potential dual sEH/5-LO inhibitor.
The synthesis of KM55 was accomplished in five linear steps (Scheme 2). First, the urea
was formed from 3-chloropropyl isocyanate and 4-(trifluoromethoxy)aniline in dichloromethane.
A Williamson ether synthesis procedure was used to couple the linker to 3-hydroxy-4-methoxy-
benzaldehyde. The following steps for the introduction of the hydroxyurea moiety were performed
according to the procedure described by Malamas et al. [22]. Therefore, the hydroxyimine was formed
and subsequently reduced with sodium cyanoborohydride. After the introduction of the terminal
hydroxyurea, a purification by reversed-phase HPLC was required to obtain compound
sufficient purity for biological testing.
5 (KM55) in

Molecules 2017, 22, 45
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2 (TPAU)
1 (zileuton)
aromatic
ring
iron-chelating
moiety
hydrophobic
moiety
urea hydrophobic
moiety
3
4
urea hydrophobic
moiety
linker
urea hydrophobic
linker
moiety
5 (KM55)
aromatic
ring
urea hydrophobic
iron-chelating
moiety
linker
moiety
Scheme 1. Design strategy for a dual sEH/5-LOX inhibitor.
6
7
8
9
5 (KM55)
Scheme 2. Synthesis of KM55. Reagents and Conditions: (a) DCM, 0 ◦C to r.t.; overnight (b) Cs2CO3,
◦
DMF, 70 C; overnight (c) NH2OH *HCl, NaAc, EtOH/MeOH (2:1), H2O, r.t., 24 h (d) NaCNBH3,
methyl orange, HCl, MeOH, THF, argon; r.t. (e) Me3SiNCO, dioxane, r.t., 2 h.

Molecules 2017, 22, 45
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2.2. In Vitro Evalulation of KM55 on Recombinant Enzymes and in Human Whole Blood
In order to evaluate the potency of KM55
proteins were performed. KM55 potently inhibited sEH in a fluorescence-based activity assay
with an IC50 of 29 nM (logIC50 7.5; std. error(logIC50) = 0.03) which is comparable to the
reference inhibitor N-cyclohexyl-N0-(4-iodophenyl)urea (CIU) [23] (IC50 = 140 nM; logIC50
6.8;
std. error(logIC50) = 0.02; Supplementary Materials Figure S1) Recombinant 5-LO activity was
measured using the natural substrate arachidonic acid. KM55 inhibited 5-LO with an IC50 of 1.3
(logIC50 5.9; std. error(logIC50) = 0.3) (Figure 1) which is in the same range as 5-LO reference
, in vitro enzyme activity assays using recombinant
=
−
=
−
µM
=
−
inhibitor zileuton (IC50 = 650 nM; logIC50 = −6.5; std. error(logIC50) = 0.13; Figure S2).
150
5-LO
sEH
100
50
0
-10
-8
-6
-4
c [µM]
Figure 1. Inhibitory effect of KM55 on recombinant enzymes.
The effect of KM55 on lipoxygenase product formation was further evaluated in a human whole
blood assay. KM55 inhibited the formation of leukotriene B4 (LTB4) and 5-hydroxyeicosatrienoic acid
(5-HETE). The formation of 12-HETE and 15-HETE remained unaffected (Figure 2).
150
LTB4
5-HETE
12-HETE
100
15-HETE
50
0
Figure 2. Inhibitory effect of zileuton and KM55 on lipoxygenase product formation in human whole
blood. Data are expressed as mean ± S.E.M. (n = 3).
2.3. Effects of KM55 on Leukocyte-Endothelial Cell Interaction
In order to evaluate the effect of KM55 in a cell function-based assay, we analyzed the action of
the compound on the adhesion of leukocytes to endothelial cells, a key process in acute and chronic
inflammation. Two sets of experiments were performed, in which either human leukocytes (monocytic

Molecules 2017, 22, 45
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THP-1 cells) or human primary endothelial cells (HUVECs) were treated with KM55. Figure 3a
shows that KM55, which was only applied to leukocytes, strongly inhibited the lipopolysaccharide
(LPS)-induced adhesion of the leukocytes to endothelial cells. The 5-LO inhibitor zileuton and the
sEH inhibitor CIU were applied for comparison. Used at the same concentration as KM55, they only
slightly inhibited cell adhesion. In the second setting, in which only endothelial cells were treated with
KM55, the leukocyte-endothelial cell interaction was not reduced (Figure 3b).
a)
4
3
2
1
0
10
8
b)
6
*
4
2
0
-
-
-
-
-
-
-
-
+
-
-
-
CIU
zileuton
KM55
LPS
+
-
-
KM55 (µM)
TNF
0
-
0
+
30 10
3
+
1
+
+
+
+
+
+
+
+
Figure 3. KM55 blocks leukocyte-endothelial cell interaction by impairing leukocyte activation,
whereas endothelial cells are not affected. ( ) The adhesion of monocyte-like THP-1 cells onto HUVECs
is significantly reduced by 30 KM55 (* p < 0.05 vs. LPS alone). THP-1 cells where pretreated
with CUI (10 M), zileuton (10 M) or KM55 (30 M, each) for 30 min and were then activated with
g/mL LPS for 24 h; ( ) HUVECs were pretreated with 30 KM55 for 30 min before they were
activated with 10 ng/mL TNF- for 24 h. THP-1 cells were left untreated. Adhesion assays were
a
µM
µ
µ
µ
1
µ
b
µM
α
performed as described in the section Materials and Methods. Data are expressed as mean
(n = 3).
±
S.E.M.
3. Discussion
KM55 is a designed multi-target ligand which inhibits the enzymatic activity of 5-LO and sEH.
In the case of KM55 the combination of the essential pharmacophore features of both enzymes was
successful. One of the reasons might be the similarity of the endogenous substrates of 5-LO–arachidonic
acid and sEH–EETs, which facilitates the dual activity. The potency of KM55 is not balanced.
The reason for this might be the fact that ureas are transition state mimetics of sEH [24,25], which are
commonly known as very potent inhibitors and is comparable to the reference compound AUDA and
CIU in our assay, while the inhibitory potency of KM55 is comparable to that of zileuton [16]. However,
in the whole blood setting, KM55 inhibits about 50% of the 5-LO product formation compared to
zileuton, which might result from higher plasma protein binding.
In this study, we could show that the dual 5-LO/sEH inhibitor KM55 potently inhibits the
adhesion of leukocytes onto endothelial cells by impairing leukocyte function. The adhesive
properties of KM55-treated monocyte-like THP-1 cells were completely reduced down to control
levels. Equimolar concentrations of CIU or zileuton showed a clear, but statistically not significant
tendency to also reduce leukocyte adhesion. Thus, the dual inhibitor KM55 exhibited a stronger action
compared to both inhibitors in this cell function-based assay. In contrast, endothelial cells are not
affected by KM55. This is in line with several studies indicating that the 5-LO pathway plays a minor
role or is even not existing in endothelial cells [26,27].
Overall, KM55 can be regarded as a novel prototype of a dual 5-LO/sEH inhibitor and can be
used for cellular investigation and as a starting point for optimization.

Molecules 2017, 22, 45
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4. Materials and Methods
4.1. General Information
All reagents and solvents were purchased from the suppliers Sigma-Aldrich (Taufkirchen,
Germany), Apollo Scientific (Stockport, UK), Acros Organics (Geel, Belgium) or Alfa Aesar (Karlsruhe,
Germany) and were used without further purification. Flash chromatography was performed on
packed silica columns (particle size 50 µM) from Varian Medical Systems GmbH (Darmstadt, Germany).
NMR spectra were measured on AV 250 nuclear magnetic resonance spectrometer from Bruker
(Fällanden, Switzerland). Chemical shifts are reported in parts per million (ppm) using TMS as
1
internal standard; H (250/300 MHz), 13C (63/75 MHz). Mass spectra were measured using ESI
with a VG Platform II spectrometer by Fisons (Loughborough, UK) or a Mariner Biospectrometry
Workstation by Perspective Biosystems (Waltham, MA, USA). HRMS spectra were measured by a
MALDI LTQ Orbitrap XL spectrometer from Thermo Scientific (Waltham, MA, USA). HPLC was
performed on a LC2020 system (Shimadzu, Duisburg, Germany), using the following conditions:
acetonitrile/water + 0.1% V/V formic acid gradient run (10%–90% AcN in 10 min, then hold 6 min
90% AcN, 90%–10% in 1 min, hold 10% for 3 min) over 20 min using a Luna 10 µm C18 (2) 100 Å,
250
×
4.6 mm (Phenomenex, Aschaffenburg, Germany) at room temperature, UV detection was set at
254 nm and 280 nm. All compounds were characterized by 1H-NMR and ESI MS, final compounds
additionally by 13C-NMR, HRMS and HPLC to determine purity >95%.
4.2. Synthesis
1-(3-Chloropropyl)-3-[4-(trifluoromethoxy)phenyl]urea (6). 3-Chloropropylisocyanate (1.50 g, 12.55 mmol)
was added to a solution of 4-(trifluoromethoxy)aniline (2.22 g, 12.55 mmol) in DCM (50 mL) at 0 ◦C.
The mixture was allowed to warm to room temperature and was stirred overnight. A yellow solution
was obtained. The solvent was removed under reduced pressure and the white solid was triturated
1
with hexane to form the pure product
6
(3.14 g, 84% yield). H-NMR (250 MHz, DMSO-d6):
δ
8.67
(s, 1H), 7.51–7.43 (m, 2H), 7.23–7.18 (m, 2H), 6.32 (t, J = 5.77 Hz, 1H), 3.66 (t, J = 6.50 Hz, 2H), 3.21 (q,
J = 7.50 Hz, 2H), 1.88 (quint, J = 6.61 Hz, 2H). MS (ESI, 70 eV) m/z (%): 297.0 (100) (M + H).
1-[3-(5-Formyl-2-methoxyphenoxy)propyl]-3-[4-(trifluoromethoxy)phenyl]urea (
7). 3-Hydroxy-4-methoxy-
benzaldehyde (1.54 g, 10.11 mmol) and cesium carbonate (3.29 g, 10.11 mmol) in DMF (40 mL) were
heated to 70 ◦C for 30 min.
6 (3.00 g, 10.11 mmol), dissolved in DMF (40 mL), was added and the
mixture was stirred overnight at 70 ◦C. The solvent was removed under reduced pressure and the
residue was diluted with EtOAc (20 mL). The organic phase was extracted with water (20 mL), NaOH
solution (1 M, 20 mL twice) and again with water (20 mL). After drying the organic layer over MgSO4,
the solvent was removed under reduced pressure. The product
(1.75 g, 42% yield) from hexane/EtOAc. H-NMR (250 MHz, DMSO-d6):
7
was recrystallized as a white solid
9.83 (s, 1H), 8.64 (s, 1H),
1
δ
7.57 (dd, J1 = 1.81, J2 = 8.25 Hz, 1H), 7.50–7.45 (m, 2H), 7.42–7.39 (m, 1H), 7.30–7.17 (m, 3H), 6.33 (t,
J = 5.66 Hz, 1H), 4.08 (t, J = 6.30 Hz, 2H), 3.88 (s, 3H), 3.29–3.19 (m, 2H), 2.00–1.88 (m, 2H). MS (ESI,
70 eV) m/z (%): 411.4 (100) (M − H).
1-(3-{5-(Hydroxyimino)methyl-2-methoxyphenoxy}propyl)-3-[4-(trifluoromethoxy)phenyl]urea (8). 7 (0.50 g,
1.21 mmol) was dissolved in EtOH/MeOH (2:1, 50 mL). Hydroxylamine hydrochloride (0.26 g,
2.43 mmol), sodium acetate (0.40 g, 4.85 mmol) and H2O (10 mL) were added to the solution and the
mixture was stirred at room temperature for 24 h. Afterwards, the mixture was poured into H2O and
extracted with EtOAc (100 mL). The organic layer was dried over MgSO4. After evaporation of the
◦
solvent, the crude product was crystallized from acetone/Et2O (0 C) to give
8
as a white solid (0.50 g,
1
97% yield). The product was used for the next step without further purification. H-NMR (250 MHz,
DMSO-d6):
δ 10.93 (s, 1H), 8.65 (s, 1H), 8.03 (s, 1H), 7.51–7.44 (m, 2H), 7.24–7.19 (m, 3H), 7.09 (dd,
J1 = 8.30 Hz, J2 = 1.70 Hz, 1H), 6.98 (d, J = 8.30 Hz, 1H), 6.32 (t, J = 5.40 Hz, 1H), 4.01 (t, J = 6.25 Hz, 2H),
3.78 (s, 3H), 3.31–3.22 (m, 2H), 2.02–1.94 (m, 2H). MS (ESI, 70 eV) m/z (%): 428.5 (100) (M + H).

Molecules 2017, 22, 45
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1-(3-{5-(Hydroxyamino)methyl-2-methoxyphenoxy}propyl)-3-[4-(trifluoromethoxy)phenyl]urea (
9
). Sodium
cyanoborohydride (NaCNBH3, 0.24 g, 3.74 mmol) in dry THF (10 mL) was added into a solution of 8
(0.50 g, 1.17 mmol) in MeOH (50 mL) under argon atmosphere. Methyl orange was added in order
to control the pH of the reaction (Warning! HCN develops under acidic conditions!). After 5 min of
stirring, a solution of 4 N HCl in dioxane was added dropwise to maintain pH at 3–4. When a steady
red color was obtained, the mixture was poured into H2O, basified with 1 N NaOH and extracted
with EtOAc (2
under reduced pressure. The product
step without further purification. H-NMR (250 MHz, MeOD-d4):
×
70 mL). The organic layer was dried over MgSO4 and the solvent was evaporated
9
as a white solid (0.50 g, quant. yield) was used for the next
7.46–7.40 (m, 2H), 7.17–7.12 (m,
1
δ
2H), 7.03 (s, 1H), 6.92–6.90 (m, 2H), 4.11 (t, J = 6.04 Hz, 2H), 3.90 (s, 2H), 3.81 (s, 3H), 3.42 (t, J = 6.60 Hz,
2H), 2.07–1.96 (m, 2H). MS (ESI, 70 eV) m/z (%): 428.4 (100) (M − H).
1-(3-{5-(Hydroxyureido)methyl-2-methoxyphenoxy}propyl)-3-[4-(trifluoromethoxy)phenyl]urea (KM55
Trimethylsilylisocyanate (0.19 mL, 1.40 mmol) was added into a solution of (0.50 g, 1.16 mmol) in
dioxane (30 mL). After stirring for 2 h, the mixture was poured into H2O, acidified with 2N HCl and
extracted with EtOAc (2 30 mL). The organic layer was dried over MgSO4 and the solvent was
, 5).
9
×
evaporated. Purification of the crude product was accomplished using reversed phase HPLC, yielding
1
5
as a waxy solid (0.03 g, 5.5%). H-NMR (250 MHz, CD3OD-d4):
δ
7.46–7.40 (m, 2H), 7.16–7.12 (m,
2H), 6.99 (bs, 1H) 6.93–6.87 (m, 2H), 4.54 (s, 2H), 4.09 (t, J = 6.09 Hz, 2H), 3.81 (s, 3H), 3.41 (t, J = 6.62 Hz,
2H), 2.01 (quint, J = 6.35 Hz, 2H). MS (ESI, 70 eV) m/z (%): 473.9 (100) (M + H). 13C-NMR (75 MHz,
CD3OD-d4):
δ 164.1, 158.1, 150.4, 149.6, 145.1, 140.3, 131.6, 123.7, 122.7, 122.6, 121.0, 115.5, 113.1, 68.2,
56.6, 54.4, 38.2, 30.8. HRMS calculated m/z 495.14674, found m/z 495.14619. HPLC: tR: 9.4 min,
purity >95%.
4.3. Recombinant sEH Enzyme Activity Assay
The IC50 values of the compounds were determined by a fluorescence-based assay system of
96-well format. As substrate non-fluorescent 3-phenyl-cyano-(6-methoxy-2-naphthalenyl)methyl
ester-2-oxirane-acetic acid (PHOME, Cayman Chemicals, Ann Arbor, MI, USA) was used, which can be
hydrolyzed by the sEH to the fluorescent 6-methoxynaphtaldehyde [28]. The formation of the product
was measured (
λ
= 330 nm,
λ
ex
= 465 nm) by a Tecan Infinite® F200 Pro plate reader (Tecan Trading
em
AG, Männedorf, Switzerland). Therefore, recombinant human sEH (2
pH 7 with 0.1 mg
mL−1 BSA containing a final concentration of 0.01% Triton-X 100. 100
solution were incubated with different concentrations of compounds (DMSO with final concentration
of 1%) for 30 min. at room temperature. After that 10 L of substrate were added (final concentration
50 M). The hydrolysed substrate was measured for 30 min (one point every minute). A blank control
µg per well) in Bis-Tris buffer
·
µL of protein
µ
µ
(no protein and no compound) as well as a positive control (no compound) was executed.
4.4. Recombinant 5-LO Activity Assay
5-LO was purified as described by Kretschmer et al. [29]. In brief, r5-LO was expressed in
a 2
×
500 mL culture (E. coli BL21(DE3)) and purified in an ÄKTA Xpress system (GE Healthcare,
Uppsala, Sweden) using ATP affinity (5 mL ATP-agarose column) and anion exchange chromatography
(ResourceQ 1 mL IEX column, GE Healthcare). Three
µ
g of r5-LO were then pre-incubated 15 min
◦
at 4 C with test compound or vehicle control in 1 mL PBS pH 7.4 containing 1 mM EDTA. After
◦
30 s pre-warming at 37 C, the reaction was started by addition of 2 mM CaCl2 and 20
µM AA.
The reaction was stopped after 10 min by addition of 1 mL methanol. Prostaglandin B1 (200 ng,
internal standard), HCl (1 N, 30 L), and PBS (500 L) were added for solid phase extraction. HPLC
µ
µ
analysis was performed according to PMC42826 and 2555374. Analysis included leukotriene (LT) B4,
its all-trans isomers, and 5-hydro(pero)xy-6,8,11,14(E,Z,Z,Z)-eicosatetraenoic acid (5-H(P)ETE). Data
of three independent measurements was normalized on vehicle control and means
values were calculated. For this a nonlinear fit (log (inhibitor) vs. normalized response—variable
±
SEM or IC50

Molecules 2017, 22, 45
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slope) by GraphPad Prism software version 7.00 for Windows (GraphPad Software, La Jolla, CA, USA)
was used.
4.5. Human Whole Blood Assay
HWB and leukocyte concentrates were taken with informed consent. In vitro human whole blood
assays were performed as described by Maier et al. [30]. In brief, heparinized venous blood was first
pre-warmed for 30 min, then pre-incubated with the test compound or vehicle control for 30 min at
37 ◦C. Reaction was started by addition of 20
previously dissolved in 50
and plasma supernatants were taken after centrifugation at 9391
LC-MS/MS analysis for LTB4 and 5-HETE was performed according to [
µ
M calcium ionophore (IO) A23187 (final concentration,
L autologous plasma). After 15 min the reaction was stopped on ice
g. Liquid-liquid extraction and
]. Data of three independent
µ
×
7
measurements was normalized to vehicle control (DMSO) and means ± SEM were calculated.
4.6. Cell Culture
THP-1 cells (ACC-16) were obtained from the Leibniz Institute DMSZ—German Collection of
Microorganisms and Cell Cultures (Braunschweig, Germany) and were cultured in HyClone
™ RPMI
1640 media (GE Healthcare, Chicago, IL, USA) containing 10% fetal bovine serum (FBS, Biochrom,
Berlin, Germany), 100 U/mL penicillin (PAA, Pasching, Austria) and 100 µg/mL streptomycin (PAA)
at 37 ◦C in an atmosphere with 5% CO2. Primary human umbilical vein endothelial cells (HUVECs)
were obtained from Provitro (Berlin, Germany) and cultured in Endothelial Cell Growth Medium
(PELOBiotech, Planegg, Germany) containing 10% FBS, 100 U/mL penicillin, 100
µg/mL streptomycin
and 2.5 g/mL amphotericin B (PAN-Biotech, Aidenbach, Germany). HUVECs were cultivated
µ
◦
until passage 2 and used for experiments at passage 3. Both cell types were cultured at 37 C in
an atmosphere with 5% CO2.
4.7. Cell Adhesion Assay
HUVECs were seeded in 24-well plates and were grown to confluence. HUVECs and THP-1 were
treated as indicated in the respective figure legend. After 30 min pretreatment with the respective
compound, either HUVECs were treated with 10 ng/mL TNF-α (PeproTech, Hamburg, Germany) or
THP-1 cells were treated with 1
µg/mL LPS (Sigma-Aldrich, Taufkirchen, Germany) for 24 h. THP-1
cells were labelled with Cell-Tracker
™
(10 M; Life Technologies, Carlsbad, CA, USA). 200,000 THP-1
µ
cells were seeded onto HUVEC monolayers and were allowed to adhere for 20 min. After a washing
step, which removes non-adherent THP-1 cells, cells were lysed. Fluorescence of the lysates, which
correlates with the number of adhered THP-1 cells, was finally measured in a Tecan Infinite® F200
plate reader (Tecan, Männedorf, Switzerland) at 535 nm.
Supplementary Materials: Supplementary materials can be accessed at: http://www.mdpi.com/1420-3049/22/
1/45/s1.
Acknowledgments: This work was supported the Deutsche Forschungsgemeinschaft (DFG; Sachbeihilfe PR1405/
2-2; SFB 1039 Teilprojekte A02, A07 and Z01). R.B., thanks to the graduate college for Translational Research
Innovation-Pharma (TRIP) and M.H. thanks German Cancer Consortium (DKTK) for the Ph.D. fellowship.
Author Contributions: K.M. performed the synthesis, D.G. performed the experiments with endothelial cells,
S.K.W. performed the sEH assays, S.K. performed 5-LO and human whole blood assays, R.B. and M.H. performed
HPLC experiments, C.A. performed LC-MS/MS analysis, G.G., D.S., B.H., R.F. and. E.P. planned the experiments
and conducted the research, R.F. and E.P. wrote the manuscript.
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of the compound KM55 are available from the authors.
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2016 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC-BY) license (http://creativecommons.org/licenses/by/4.0/).