Tandem benzophenone amino pyridines, potent and selective inhibitors of human leukotriene C4 synthase

Article abstract of DOI:10.1124/jpet.115.227157

Cysteinyl leukotrienes (cys-LTs) are lipid mediators of inflammation. The enzyme catalyzing synthesis of cys-LTs, leukotriene C₄ synthase (LTC4S), is considered an important drug target. Here we report the synthesis and characterization of thre

Full text of DOI:10.1124/jpet.115.227157

Supplemental material to this article can be found at:
http://jpet.aspetjournals.org/content/suppl/2015/08/17/jpet.115.227157.DC1.html
1521-0103/355/1/108–116$25.00
THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
http://dx.doi.org/10.1124/jpet.115.227157
J Pharmacol Exp Ther 355:108–116, October 2015
Copyright ª 2015 by The American Society for Pharmacology and Experimental Therapeutics
Tandem Benzophenone Amino Pyridines, Potent and Selective
Inhibitors of Human Leukotriene C4 Synthases
Thea K. Kleinschmidt, Martin Haraldsson, Devaraj Basavarajappa, Erik Lundeberg,
Madhuranayaki Thulasingam, Maria Ekoff, Alexander Fauland, Christoph Lehmann,
Astrid S. Kahnt, Lennart Lindbom, and Jesper Z. Haeggström
Department of Medical Biochemistry and Biophysics (T.K.K., D.B., M.T., A.F., J.Z.H.) and Chemical Biology Consortium Sweden,
Science for Life Laboratory Stockholm, Department of Medical Biochemistry and Biophysics (M.H.), and Microvascular
Physiology Research Group, Department of Physiology and Pharmacology (E.L., L.L.), Karolinska Institutet, Stockholm Sweden;
Clinical Immunology and Allergy Unit, Department of Medicine, Karolinska University Hospital, Stockholm, Sweden (M.E.); and
Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Project Group Translational Medicine and Pharmacology,
Frankfurt/Main, Germany (C.L., A.S.K.)
Received July 7, 2015; accepted August 12, 2015
ABSTRACT
Cysteinyl leukotrienes (cys-LTs) are lipid mediators of inflamma- Arg104 and Arg31. The TK compounds potently inhibited cys-LT
tion. The enzyme catalyzing synthesis of cys-LTs, leukotriene C4 biosynthesis in immune cells. In coincubations of platelets and
synthase (LTC4S), is considered an important drug target. Here we polymorphonuclear leukocytes, inhibition of LTC4S led to shunting
report the synthesis and characterization of three tandem ben- of LTA4 toward anti-inflammatory lipoxin A4, which was signifi-
zophenone amino pyridines as inhibitors of LTC4S in vitro and in cantly enhanced by simultaneous inhibition of LTA4H. Finally, we
vivo. The inhibitors were characterized in vitro using recombinant found that TK05 (6 mg×kg21×body weight) reduces LTE4 levels in
human LTC4S, MonoMac 6 cells, and a panel of peripheral human peritoneal lavage fluid by 88% and significantly decreases vascular
immune cells. In vivo, the compounds were tested in the Zymosan permeability in vivo. Our findings indicate that the TK compounds
A-induced peritonitis mouse model. The molecules, denoted TK04, are valuable experimental tools in eicosanoid research in vitro and
TK04a, and TK05, were potent and selective inhibitors of LTC4S in vivo. Their chemical structures may serve as leads for further
with IC50 values of 116, 124, and 95 nM, respectively. Molecular inhibitor design. Novel drugs depleting cys-LT production could be
docking revealed binding in a hydrophobic crevice between two beneficial for treatment of inflammatory diseases associated with
enzyme monomers and interaction with two catalytic residues, overexpression of LTC4S.
highly unstable epoxide can either be hydrolyzed by LTA4
hydrolase into LTB4 or conjugated with glutathione to form
LTC4. The target protein of our study, LTC4 synthase (LTC4S),
Introduction
Cysteinyl leukotrienes (cys-LTs), namely leukotriene (LT) C4,
D4, and E4, are lipid mediators derived from arachidonic acid
catalyzes this coupling reaction, e.g., in mast cells, eosinophils,
(AA), which is liberated from the nuclear membrane of leuko-
and monocytes. In patients suffering from asthma, the bron-
cytes by cytosolic phospholipase A2 (cPLA2) upon cell stimula-
chial mucosa is rich in these immune cells and here synthesis
tion (Haeggstrom and Funk, 2011). 5-Lipoxygenase (5-LO)
translocates to the nuclear envelope and with the help of
5-LO-activating protein (FLAP), it converts AA into LTA4. This
and release of cys-LTs causes smooth muscle contraction and
mucus secretion, contributing severely to asthmatic symptoms.
LTC4S is an 18-kDa enzyme embedded in the outer
membrane of the nucleus and the endoplasmic reticulum of
myeloid cells. LTC4S, together with FLAP, microsomal pros-
taglandin E synthase-1 (mPGES-1), and the microsomal
glutathione transferases 1, 2, and 3 (MGST1-3) constitute the
superfamily of membrane-associated proteins in eicosanoid
and glutathione metabolism (Hebert and Jegerschöld, 2007).
We acknowledge the financial support of the Else Kröner-Fresenius-
Graduiertenkolleg funded by the Else Kröner-Fresenius Stiftung, the LOEWE
initiative Anwendungsorientierte Arzneimittelforschung, and Fraunhofer
IME-TMP, as well as the Lars Hierta Memorial foundation. This study was
supported by the Swedish Research Council [10350, 20854 Linneus Grant
CERIC], the Cardiovascular Program, and Thematic Center for Inflammation.
J.Z.H. is supported by a Distinguished Professor Award from Karolinska
Institutet.
The high-resolution crystal structure of LTC4S, solved by
This article has supplemental material available at jpet.aspetjournals.org. Martinez Molina et al. (2007), revealed a homotrimeric structure.
dx.doi.org/10.1124/jpet.115.227157.
s
ABBREVIATIONS: AA, arachidonic acid; CBMC, cord blood-derived mast cell; cys-LT, cysteinyl leukotrienes; DMSO, dimethylsulfoxide; EtOH,
ethanol; EBD, Evans blue dye; FLAP, 5-lipoxygenase-activating protein; GSH, glutathione; 5-HETE, (5S, 6E, 8Z, 11Z, 14Z)-5-hydroxyeicosa-6, 8, 11,
14-eicosatetraenoic acid; LC-MS/MS, liquid chromatography tandem mass spectrometry; 5-LO, 5-lipoxygenase; LT, leukotriene; LTA4, leukotriene
A4; LTC4, leukotriene C4; LTC4S, leukotriene C4 synthase; LXA4, lipoxin A4; MeOH, methanol; MGST, microsomal glutathione transferase; MM6,
MonoMac 6; mPGES-1, microsomal prostaglandin E synthase-1; PBMC, peripheral blood mononuclear cell; PMNL, polymorphonuclear leukocytes;
SPE, solid phase extraction.
108

Potent and Selective Inhibitors of Human LTC4S
109
Enzyme Inhibition Assay. The assay was performed, and sam-
ples were analyzed as described before (Niegowski et al., 2014a).
MonoMac 6 Cell Culture and Assay. MM6 cells, originally
developed by Ziegler-Heitbrock et al. (1988), were cultured and
differentiated as described before (Esser et al., 2011). Differentiated
MM6 cells (about 2 Â 106 cells) suspended in 1 ml PGC buffer (PBS
containing 1 mg×ml21 glucose and 1 mM CaCl2) were treated with TK04,
TK04a, and TK05 (0.2–8 mM) or vehicle [,1% dimethylsulfoxide
(DMSO)] for 30 minutes at 37°C. LT production was initiated with
calcium ionophore A23187 (5 mM) with or without AA (40 mM) for
10 minutes before stopping the reaction with 1 ml ice-cold MeOH.
Mast Cell Preparation and Assay. Cord blood-derived mast
cells (CBMCs) were derived and then maintained as previously de-
scribed by Xiang et al. (2006) and Gela et al. (2015). Before activation,
cells were incubated with IL-4 (10 ng×ml21) and IL-3 (5 ng×ml21) for
4 days. For activation, cells were incubated with 1 mg×ml21 IgE
(Calbiochem, Merck Millipore, Darmstadt, Germany) overnight and
activated using 2 mg×ml21 a-IgE (Sigma, St Louis, MO). For inhibition
studies, the TK04, TK04a, and TK05 were added 30 minutes before
activation (final concentrations 0.2–8 mM). After 30 minutes of activa-
Each monomer consists of five a-helices, four of which penetrate
the membrane. It has been proposed that LTC4S binds its
substrate LTA4 in a hydrophobic crevice on the surface of two
adjacent monomers, indicated by a bound detergent molecule of
dodecyl maltoside in the crystal structure. The second substrate
glutathione (GSH) binds in a deeper pocket below LTA4 in a
unique “horseshoe”-shaped conformation.
There are two established receptors for LTC4, D4, and E4,
i.e., CysLT1 (Lynch et al., 1999) and CysLT2 (Heise et al.,
2000), the former of which signals classic cys-LT bioactions
and is targeted by the lukast class of antileukotrienes,
established drugs against asthma. Unfortunately, about 40%
of the patients do not respond to this medication (Malmstrom
et al., 1999), which may be explained by a more complex
receptor system, involving a receptor selective for LTE4, as
suggested in several studies (Kanaoka et al., 2013).
5-LO antagonists are another pharmacological approach in
asthma therapy, with Zileuton Abbott Laboratories, Abbott
Park, IL as the Food and Drug Administration-approved
representative (Rubin et al., 1991). Inhibition of the enzyme
highest upstream in LT biosynthesis decreases LTA4 levels
along with LTB4 and cys-LTs. Despite the resulting beneficial
effects of reducing all LTs, 5-LO inhibition additionally leads
to reduction of anti-inflammatory and proresolving lipoxins
that are also derived from LTA4, which may lead to delayed
resolution of inflammation (Rao et al., 2007).
tion, cells (80,000 cells per well, equivalent to 0.4 Â 106 cells×ml21
)
were spun down, and supernatants were collected.
Liquid Chromatography-Mass Spectrometry Analysis of
CBMC Supernatant. A stable isotope dilution liquid chromatogra-
phy tandem mass spectrometry (LC-MS/MS) method was used for the
quantification of lipid mediators in CBMC supernatant, previously
reported by Balgoma et al. (2013). The method was modified to
quantify the following lipid mediators: 5-HETE, LTC4, and LTD4.
The detailed LC-MS platform is described in the Supplemental Data.
Briefly, CBMC supernatant was mixed with a cocktail of deuterated
internal standards and extracted onto Waters Oasis HLB solid phase
extraction (SPE) cartridges (Waters Corporation, Milford, MA) before
LC-MS/MS analysis. Samples were injected onto an Waters Acquity
Selective pharmacological intervention further downstream
of 5-LO, at the committed step in cys-LT biosynthesis, may
spare lipoxin formation as well as provide higher efficacy than
CysLT1 receptor antagonists. LTC4S is thus considered to be
a promising approach in the treatment of asthma and in- Ultra-Performance Liquid Chromatography (UPLC) equipped with
a BEH C18 column and analyzed on a Waters Xevo TQS-MS in
negative and positive electrospray ionization mode. The calibration
levels and method parameters of the analyzed compounds are pro-
vided in the Supplemental Data and Supplemental Table S1.
Primary Cells. Peripheral blood mononuclear cells (PBMCs),
polymorphonuclear leukocytes (PMNL), eosinophils, and platelets
were isolated from buffy coats prepared from nonallergic healthy
volunteers at the Department of Clinical Immunology and Trans-
fusion Medicine of Karolinska University Hospital Stockholm.
PBMC Preparation and Assay. Human monocytes were pre-
pared from the PBMC fraction after Ficoll-Paque PREMIUM density
centrifugation (GE Healthcare, Little Chalfont, UK). Monocytes were
isolated by attachment to culture dishes for 2 hours at 37°C followed
by washing off nonadherent cells with PBS. Monocytes (4 Â 106 cells)
in 1 ml PGC buffer (PBS containing 1 mg ml21 glucose and 1 mM
CaCl2) were treated with TK04, TK04a, and TK05 in DMSO (1 mM) or
vehicle (,0.5% DMSO) for 30 minutes at 37°C. The cells were
stimulated with A23187 (5 mM) and AA (20 mM) for 10 minutes at
37°C before stopping the reaction with 1 ml ice cold MeOH.
Primary Eosinophil Assay. Cells were isolated using Ficoll-
Paque PREMIUM centrifugation (GE Healthcare) followed by an eosin-
ophil isolation kit (Milteny Biotech, Bergisch Gladbach, Germany),
according to the manufacturer’s description. Cells (3 Â 106) in PGC
buffer were incubated with TK04, TK04a, and TK05 in DMSO (1 mM)
or vehicle (,0.5% DMSO) for 30 minutes at 37°C. LT production was
stimulated with A23187 (5 mM) for 10 minutes at 37°C before stopping
the reaction with 1 ml ice-cold MeOH.
flammation (Haeggström et al., 2010, Devi and Doble, 2012).
To date, only a small number of LTC4S inhibitors have thus
far been reported and none has evolved to a drug candidate.
In this study we present the synthesis and in vitro and in
vivo characterization of three potent LTC4S inhibitors (Nilsson
et al., 2011a), here referred to as TK04, TK04a, and TK05, that
could serve as experimental tools as well as lead structures for
novel inhibitors.
Materials and Methods
Materials. LTA4 methyl ester (BIOMOL) in tetrahydrofuran was
saponified with 1 M LiOH (6%, v/v) for 48 hours at 4°C. All other
chemicals were obtained from common commercial sources.
Synthesis of Inhibitors TK04, TK04a, and TK05. See Supple-
mental Data.
Molecular Docking. Molecular docking was performed using
AutoDock 4.2 Molecular Graphics Laboratory, La Jolla, CA, and the
input files were prepared using AutoDockTools (Morris et al., 2009).
Apo LTC4S structure (PDB ID: 2UUI) as a macromolecular target was
retrieved from Research Collaboratory for Structural Bioinformatics
(RCSB)-PDB. The PDB coordinates for TK04, TK04a, and TK05 were
obtained using eLBOW Lawrence Berkeley Laboratory, Berkeley, CA
(Moriarty et al., 2009). The input files were prepared by adding polar
hydrogen atoms and Gasteiger charges using AutoDockTools. The grid
box size was kept as 60, 58, 72 Å for X, Y, Z with 0.392-Å grid spacing
that encompasses the active site of LTC4S. Lamarckian genetic
Isolation of Platelets and Coincubations with PMNL. Plate-
algorithm was used with default settings, and the docking simulation lets and neutrophils were isolated from the same buffy coat. Platelet-
yielded 20 docked conformations for each inhibitor. The resulting rich plasma was separated from the blood by centrifugation at 600 g
conformations were screened based on free energy binding (DG) and for 20 minutes. The supernatant was collected and supplemented with
conservation of key interactions with LTC4S. Structure visualizations 1/10 volume of ACD buffer (85 mM sodium citrate dehydrate, 666 mM
and distance measurements were done using PyMOL (Schrodinger,
NY, https://www.pymol.org/).
citric acid, 111 mM glucose). For the assay, platelets were dissolved in
PGC buffer supplemented with 1 g×l21 human albumin.

110
Kleinschmidt et al.
Neutrophils were obtained by dextran sedimentation of the remain- using the equation Y 5 bottom 1 (top 2 bottom)/(1110X-LogIC50), and
ing blood followed by Ficoll-Paque PREMIUM density centrifugation Ki was determined using the Michaelis-Menten equation edited for
(GE Healthcare) of the supernatant at 2100 g for 20 minutes (no brake competitive inhibition.
applied) followed by hypotonic lysis of erythrocytes. Neutrophils were
pelleted (300 g, 10 minutes) and resuspended in PGC buffer.
Platelets (1 Â 109 cells) in 1 ml PGC buffer were preincubated with
Results
10 mM TK04, TK04a, TK05, or vehicle (,0.5% DMSO) for 30 minutes
Synthesis of inhibitors TK04, TK04a, and TK05. In
at 37°C followed by addition of 10 mM LTA4 for 5 minutes at 37°C
this study, we present a detailed chemical synthesis of the
compounds TK04, TK04a, and TK05 in 8, 8, and 10 steps,
respectively. We based our synthesis on a procedure published
by Nilsson et al. (2011a). Our protocol involves the use of
aldehydes instead of carboxylic acid chlorides in the first step
of the synthesis. Moreover, we chose a convergent approach,
as opposed to the original linear strategy, to increase the
efficiency of the reactions including three crucial steps. A
Grignard reaction of an aromatic iodide with an aryl aldehyde
on the one hand and a Buchwald coupling of a monoalkylated
arylamine and an aromatic bromide on the other led to two bis-
aromatic building blocks. Those were coupled in the second
Grignard reaction. The cyclopropyl moiety demanded two
extra steps in the synthesis of TK05 before the Buchwald
coupling: a condensation of cyclopropanecarbaldehyde and
4-chloroaniline followed by the reduction of the resulting
imine to the corresponding secondary amine. The entire syn-
thesis is shown in Supplemental Fig. S1, and the chemical
structures of the three inhibitors are depicted in Fig. 1A.
Molecular Docking. The docked conformation for each
ligand was chosen based on the lowest predicted free energy of
binding. All three inhibitors have taken up an extended
conformation as a favored mode of binding at the active site
before stopping the reaction with 1 ml cold MeOH.
For the coincubations, 1 Â 109 platelets were allowed to equilibrate
with 10 Â 106 neutrophils in 1 ml PGC buffer for 5 minutes at 37°C.
Next, they were treated with either TK05 in DMSO (final concen-
trations 0.5–3 mM), with or without additional 0.2 mM SC 57461A in
DMSO, or vehicle (,0.5% DMSO) for 30 minutes at 37°C followed by
A23187 stimulation (5 mM) for 30 minutes. The reaction was stopped
with 1 ml ice cold MeOH.
Analysis of LTs, 5-HETE, and Lipoxins. LTs, 5-HETE, and
lipoxins were obtained from the aliquots (2 ml) of the stopped reaction
mixtures by SPE before reversed-phase HPLC as described before
(Basavarajappa et al., 2014). Prostaglandin B2 (100 pmol) and 17-OH-
C22:4 (100 pmol) were added as internal standards, kind gifts from
Mats Hamberg (Karolinska Institutet).
mPGES-1 Activity Assay. The effect of the inhibitors on mPGES-1
was determined using a protocol described by Hammarberg et al.
(2009). Reaction mixtures were pretreated with vehicle (0.1% DMSO)
or TK04, TK04a, and TK05 (10 mM) for 30 minutes.
MGST2 Activity Assay. The affinity of the inhibitors toward
MGST2 was determined essentially as described by Ahmad et al.
(2013). Purified recombinant enzyme (0.5 mM) supplemented with
5 mM GSH was incubated with vehicle (2% DMSO) or TK04, TK04a,
or TK05 (10 mM) for 30 minutes. The reaction was started by addition
of 0.5 mM CDNB. Initial reaction rates were analyzed spectrophoto-
metrically at 340 nm over 1–2 minutes.
5-LO Activity Assay. Purified human recombinant stable 5-LO of LTC4S. The inhibitors prefer the same location in the
(5.6 nM) in 100 ml of the reaction buffer (50 mM Tris HCl, 1 mM MgCl2,
0.25 mg 13-HpODE, 25 mg PC, pH 7.5) was preincubated with vehicle
(DMSO ,1%) or TK04, TK04a, or TK05 (10 mM) for 30 minutes. AA
(40 mM) was added, and the initial reaction rate was monitored
spectrophotometrically at 234 nm for 2 minutes.
hydrophobic cleft of the active site at the dimer interface
(Fig. 1B), the same region where a bound dodecyl maltoside
molecule was observed in the crystal structure of LTC4S
(PDB ID: 2UUH), possibly mimicking the LTA4 substrate.
The binding site of the compounds in this docking study
supports the competitive mode of inhibition for all three
inhibitors observed in the present study (see below). Addi-
tionally, we observed that these inhibitors are accommodated
within the active site in alignment with Trp-116, which has
been suggested to act as a molecular ruler for the positioning of
LTA4 substrate but also as a lid involved in product release
from the active site (Martinez Molina et al., 2007, Niegowski
et al., 2014b). The orientation of all three inhibitors is consis-
tent by facing its chlorine end toward Trp-116 and the methyl
end pointing opposite to the hydrophobic pocket.
At this docked conformation, all three inhibitors exhibit
three key ionic interactions with two arginine residues of
LTC4S. The carbonyl oxygen between rings b and c of the
inhibitor establishes the interaction with Arg-104 and Arg-31
from the adjacent monomer interacts with the oxygen of the
carboxyl group on ring b. The predicted docked conformations
were further strengthened by a third interaction created by
the carbonyl oxygen between rings a and b with Arg-31 (Fig. 1,
C–E). Because of the presence of a methoxy group on ring a,
TK04a and TK05 were further stabilized by interaction with
Ser-36 (Fig. 1, D and E).
Animals. All animal protocols were carried out as approved by the
regional ethical committee for animal experimentation. The study
included male wild-type C57BL/6 mice (Harlan, The Netherlands),
weighing 20–25 g (n 5 18, age 7–8 weeks). Animals were kept in
a temperature-controlled room (21 6 2°C), exposed to light-dark cycles
of 12 hours each, and were allowed ad libitum access to water and food.
Zymosan A Induced Peritonitis Mouse Model. Mice were
divided into five groups (n 5 3–4) to receive intraperitoneal injections
of either 1) control (0.25% ethanol in PBS, 500 ml); 2) Zymosan
(1 mg×ml21 in 0.25% ethanol in PBS, 500 ml); 3) Zymosan 1 low dose
TK05 (1 mg×ml21 1 10 mM, respectively, in 0.25% EtOH in PBS,
500 ml); 4) Zymosan 1 high dose TK05 (1 mg×ml21 1 500 mM,
respectively, in 0.25% EtOH in PBS, 500 ml), or 5) high-dose TK05
(500 mM in 0.25% EtOH in PBS, 500 ml). The low and high dose of
TK05 corresponds to exposure of 0.12 and 6 mg×kg21 body weight,
respectively. Evans blue dye (EBD, 1% in PBS, 50 mg×kg21) was injected
into the tail vein followed by an intraperitoneal injection of Zymosan
and/or compound as described above. After 60 minutes, mice were
euthanized and peritoneal exudates were collected using 4 ml PBS.
Detection of EBD in Lavage Fluid. The collected lavage fluid
was spun at 3000 rpm for 10 minutes. Lavage fluid (200 ml) was
subjected to absorbance spectrophotometry (Multiskan FC Microplate
Photometer, Thermo Fisher Scientific, Waltham, MA) at 620 nm.
Analysis of LTs in Mouse Peritoneal Lavage Fluid. LTE4, the
only cys-LT detected in lavage fluid, was analyzed by SPE followed by
HPLC as described above. LTB4 levels were analyzed using an LTB4
EIA Kit (Cayman, Ann Arbor, MI).
Inhibition of Human Recombinant LTC4S. We found
that TK04, TK04a, and TK05 are nanomolar inhibitors of the
isolated human enzyme in agreement with Nilsson et al.
(2011a). At constant substrate concentrations, we determined
Statistical Analysis. GraphPad Prism 6 (La Jolla, CA) was used
to calculate the kinetic parameters IC50 and Ki. IC50 was determined the IC50 values to 116 6 18 nM (TK04), 124 6 10 nM (TK04a),

Potent and Selective Inhibitors of Human LTC4S
111
Fig. 1. Chemical structure of the TK
inhibitors and molecular docking. (A)
Chemical structure of TK04, TK04a, and
TK05. (B) Superposition of docked inhib-
itors TK04 (yellow), TK04a (cyan), and
TK05 (green) located at the hydrophobic
cleft of the LTC4S active site. (C) In-
teraction of docked inhibitor TK04 (yel-
low), TK04a (cyan), and TK05 (green) with
Arg-104 (wheat) and Arg-31 (violet).
and 95 6 25 nM (TK05). When varying the amount of the intermediate during LTA4 production and thus an indicator of
substrate LTA4, we observed a competitive mode of inhibition, effects on the 5-LO/FLAP complex. We also determined effects
consistent with the docking data. Ki values were calculated to on other LTA4-derived products, namely LTB4, as well as the
13 6 7 nM (TK04), 53 6 15 nM (TK04a), and 6 6 5 nM (TK05). nonenzymatic degradation products all-trans-LTB4 and 12-epi
Interestingly, LTC4S inhibition was time dependent, with all-trans-LTB4. When treated with the TK compounds, we
a maximum effect after 30 minutes on both the isolated and observed increased levels of LTB4 and its all-trans isomers
the cellular enzyme (Supplemental Fig. S2).
peaking at 1 mM inhibitor concentration. In contrast, 5-HETE
Specificity of TK04, TK04a, and TK05 for LTC4S. To was reduced to 40% by 8 mM TK04a and to 40% and 20% by
assess their specificity, we treated the membrane-associated 3 and 8 mM TK05, respectively (Fig. 3B).
proteins in eicosanoid and glutathione metabolism enzymes
When challenged with additional 40 mM AA, the IC50 values
mPGES-1 and MGST2 as well as the upstream biosynthetic of TK04, TK04a, and TK05 slightly increased to 1289 6 391 nM,
enzyme 5-LO with the TK inhibitors. We found that TK04, 2217 6 659 nM, and 446 6 150 nM, respectively.
TK04a, and TK05 had no significant inhibitory effect on
mPGES-1, MGST2, or 5-LO at a concentration of 10 mM (Fig. 2).
Inhibition of LTC4S in Differentiated MM6 Cells. To
study the cellular activity of the inhibitors, we chose differen-
tiated MM6 cells, a human monocytic cell line with high
expression of 5-LO and LTC4S and low expression of LTA4
hydrolase. We found that all three compounds reduced LTC4
production in a dose-dependent manner with IC50 values of
666 6 79 nM (TK04) and 1820 6 208 nM (TK04a), and 318 6
60 nM (TK05) as shown in Fig. 3A. Consistent with the
isolated enzyme assay, TK05 and TK04a were found to be the
strongest and weakest inhibitor, respectively. To further elu-
cidate the compound’s effect on the LT pathway in MM6 cells,
we additionally measured formation of 5-HETE, the reduced
hydroxyl derivative of the hydroperoxide 5-HpETE, an
Fig. 2. Specificity of TK inhibitors for LTC4S. The activity of MGST2,
mPGES-1 and stable 5-LO was not affected by 10 mM TK04, TK04a, or
TK05. Data are presented as percentage of control (ctrl; 100%), mean 6
S.E.M. (n = 3).

112
Kleinschmidt et al.
Fig. 3. Inhibition of LTC4 formation in the cell line MM6 and peripheral immune cells. (A) Differentiated MM6 cells (1 Â 106 cells×ml21) were
preincubated with TK inhibitors (0.2–8 mM) or vehicle (,1% DMSO) for 30 minutes at 37°C. LT formation was initiated with A23187 (5 mM) for
10 minutes, and LTC4 was analyzed with HPLC. Data are presented as percentage of ctrl (100%), mean 6 S.E.M. (n = 3), and each observation represents
one out of two or three independent experiments. LTC4 concentrations of ctrl samples were 30.4 6 6.0 pmol×106 cells. (B) The effect of TK inhibitors on
5-HETE formation in MM6 cells. (C) CBMCs were incubated with 10 ng×ml21 IL-4 and 5 ng×ml21 IL-3 for 4 days followed by IgE treatment (1 mg)
overnight. Cells were incubated with TK04, TK04a, and TK05 (0.2–8 mM) for 30 minutes and then activated using 2 mg×ml21 a-IgE for 30 minutes. Data
are presented as percentage of ctrl (100%), mean 6 S.E.M. (n = 4), ctrl samples had a LTC4 concentration of 1.46 6 0.22 ng×ml cell supernatant. (D) Effect
of TK04, TK04a, and TK05 on 5-HETE formation in mast cells. (E) Effect of the TK inhibitors (1 mM) on LTC4S in MM6, eosinophils, PBMCs, platelets,
and CBMCs. In contrast to the other cell types, CBMCs were stimulated with IgE and LTC4 formation analyzed with LC-MS. Data are presented as
percentage of ctrl (100%), mean 6 S.E.M. (n = 3) and represents one out of two independent experiments.
Inhibition of LTC4S in Human Cord Blood Derived another type of inflammatory cells that express LTC4S. One
Mast Cells. Cord blood-derived mast cells (CBMCs) were micromole of TK04, TK04a, and TK05 reduced LTC4 forma-
treated with the same concentrations of the inhibitors as the tion by 65, 49, and 80%, respectively, compared with control
MM6 cells. Surprisingly, we found an increase in the IC50 cells.
values for all compounds with 2194 nM (TK04), 5644 nM
The effects of the TK inhibitors (1 mM) on LTC4S in all
(TK04a), and 5098 nM (TK05) (Fig. 3C). LTD4 was inhibited tested cell types are compared in Fig. 3E.
in a similar fashion, whereas 5-HETE levels were not affected
by the inhibitors (Fig. 3D).
Inhibition of LTC4 in Isolated Human Platelets and
Coincubations with Neutrophils. Human platelets ex-
Inhibition of LTC4S in Human Monocytes. To verify press not only LTC4S but also 12-lipoxygenase, allowing them
our MM6 cell data with primary cells, we treated human to produce both inflammatory LTC4 and anti-inflammatory
monocytes with our inhibitors. We observed 45, 60, and 69% lipoxins. On the HPLC-UV system, LXA4, LXB4, as well as
inhibition of LTC4 production when treated with 1 mM TK04, their all-trans isomers, could be detected (Serhan and
TK04a, and TK05, whereas 5-HETE, LTB4, and its two all-trans Sheppard, 1990). First, we incubated untreated platelets
isomers, nonenzymatic hydrolysis products of LTA4, were not and platelets treated with 1 mM of TK04, TK04a, and TK05
affected (data not shown).
with 10 mM exogenous LTA4 and found reduced LTC4
Inhibition of LTC4S in Human Eosinophils. We con- production by .95% for all three inhibitors along with
ducted the same experiments with isolated human eosinophils, a significant increase in the overall lipoxin production and

Potent and Selective Inhibitors of Human LTC4S
113
LXA4 alone by TK04 and TK05 (Supplemental Fig. S3). In
a second experimental setting, we coincubated platelets
with neutrophils producing LTA4, allowing transcellular
LT metabolism. Upon LTC4S inhibition with TK05 (Fig.
4A), we again observed significantly increased levels of
LXA4 to 1.6-fold of control levels for inhibitor concentra-
tions of 0.5 mM, whereas overall lipoxin formation was
increased, but not statistically significant. Formation of
LTB4 was increased in a similar fashion to about 1.4-fold
upon LTC4S inhibition, whereas 5-HETE levels did
not differ from controls (Fig. 4B). Moreover, we observed that
this effect was further boosted with the addition of an
LTA4 hydrolase inhibitor (SC 57461A). Incubations of plate-
lets together with neutrophils with both 0.2 mM SC 57461A
and 1 mM TK05 resulted in more than threefold increased
lipoxin and LXA4 levels compared with control cells (Fig. 4,
C and D).
Inhibition of LTC4S In Vivo. Zymosan A-induced peri-
toneal inflammation in mice is driven by cys-LTs (Kanaoka
et al., 2001). Using this model, we found that 0.12 mg×kg21 of
TK05 significantly reduces LTE4 levels, whereas 6 mg×kg21
body weight of the compound resulted in almost complete
inhibition compared with mice treated with only Zymosan A.
LTE4 could neither be detected in the control mice nor in
animals treated with only 6 mg×kg21 (Fig. 5A). Cys-LT
production leads to increased permeability of small blood
vessels at the sites of inflammation. The leakage of intrave-
nously injected EBD serves as a marker for the extravasation
of plasma proteins. The lower dose of TK05 slightly reduced
plasma exudation evoked by Zymosan, whereas the effect of
the high dose was significant (Fig. 5B). LTB4 levels were not
Discussion
Cys-LTs are involved in many inflammatory conditions,
especially asthma and allergic rhinitis, and inhibitors of
5-LO and CysLT1 receptor antagonists are established medi-
cations for these diseases. However, there is a need for more
effective and safe treatments. Drugs that inhibit the com-
mitted step in cys-LT biosynthesis, i.e., the enzyme LTC4S,
may have fewer side effects than 5-LO inhibitors because
they do not interfere with prostaglandin or lipoxin metabo-
lism. Additionally, responsiveness may increase in compar-
ison with receptor antagonist, because all cys-LT signaling
will be interrupted.
As a membrane protein, LTC4S is a difficult target for
inhibitor design. Potential inhibitors require both hydrophilic
and lipophilic features to enter and bind well to the catalytic
site. The proposed hydrophobic binding site for LTA4 provides
few amino acid residues for strong hydrogen or electrostatic
bonds. Inhibition has to occur mainly via weaker van der
Waals and hydrophobic forces. This has made drug develop-
ment challenging (Devi and Doble, 2012).
In this study we synthesized three potent inhibitors of
LTC4S, denoted TK04, TK04a, and TK05, and characterized
them both in vitro against purified enzyme and immune cells
as well as in vivo in a mouse model of peritonitis.
Previous attempts to develop LTC4S inhibitors have led to
several inhibitors with IC50 values in the micromolar range,
e.g., LL-699.333 and MK886 from Merck (Kenilworth, NJ),
which, however, are unselective (Gupta et al., 1997; Sala
et al., 1997). The crystal structure of LTC4S, published in
2007, allowed new efforts in the field. Apart from the series
significantly altered by either doses of TK05 compared with of bis-aromatic compounds that include our TK inhibitors,
the Zymosan group (Fig. 5C).
libraries of methylenebisphenyl, bis-aryl, and indole molecules
Fig. 4. Effect on LTC4 and lipoxin formation in co-
incubations of platelets and neutrophils. 1 Â 109 platelets
and 10 Â 106 PMNL were mixed for equilibration for
5 minutes at 37°C. TK05 (0.5–3 mM) or vehicle (,0.5%
DMSO) was added for 30 minutes with or without addition
of the LTA4H inhibitor SC 57461A (0.2 mM) at 37°C
followed by A23187 (5 mM) stimulation for 30 minutes
at 37°C. Effect of TK05 on the biosynthesis of LTC4 (A),
5-HETE (B), and overall lipoxin levels (C) and LXA4 (D)
specifically. Data are presented as percentage of ctrl (100%),
mean 6 S.E.M. (n = 3, **P , 0.01, ***P , 0.001) and
represents one out of two independent experiments.

114
Kleinschmidt et al.
Fig. 5. Effect of TK05 on Zymosan
A-induced peritonitis in mice. Eighteen male
wild-type mice received a tail vein injection
of 1% EBD, immediately followed by a
peritoneal injection of PBS + 0.5% EtOH
(ctrl), Zymosan, Zymosan and 0.12 mg×kg21
TK05, Zymosan and 6 mg×kg21 TK05, or
6 mg×kg21 TK05. The Peritoneal lavage
fluid was analyzed for LTE4 formation (A),
leakage of EBD (B), and LTB4 formation
(C). Data are presented as mean 6 S.E.M.
(n = 3–4, *P , 0.05, **P , 0.01, ***P ,
0.001, ns = not significant).
have been developed and patented (Pelcman and Nilsson, 2008, by secreting cytotoxic agents and bronchoconstrictive LTs
Nilsson, 2010, Nilsson et al., 2011b). Some of those exhibit low (Nakagome and Nagata, 2011) and it has been shown that
nM IC50 values, but to our knowledge, they have not been further eosinophils are the predominant source of cys-LTs in patients
studied. More recently, Ago et al. (2013) published an in silico suffering from aspirin-intolerant asthma (Cowburn et al.,
screening focused on the GSH binding site of LTC4S, which 1998). Our data show that 1 mM of our compound can reduce
resulted in a series of 5-(5-methylene-4-oxo-4,5-dihydrothiazol- eosinophil-derived LTC4 by up to 80%.
2-ylamino) isophthalic acid compounds. The strongest candi-
date (IC50 1.9 mM) was active in vitro and in cell assays but was LT production. TK04, TK04a, and TK05 were comparably
shown to be a dual inhibitor of LTC4S and 5-LO. effective as in the monocytic cell line MM6. Monocytes migrate
Monocytes are another type of immune cells capable of cys-
TK04, TK04a, and TK05 are hydrophobic compounds with from the blood stream to the site of infection or tissue damage,
a backbone of four aromatic rings connected by two benzophe- where they eliminate noxious molecules and cells with phago-
none moieties and an alkylated nitrogen connecting rings c cytosis or differentiate into macrophages or dendritic cells.
and d. Addition of a cyclopropyl ring to that nitrogen in TK05 Their recruitment is associated with inflammatory conditions,
increases its activity. Our results show that our TK com- e.g., atherosclerosis (Shi and Pamer, 2011).
pounds potently inhibit LTC4S in cell free and whole cell
As a result, targeting LTC4 production of resident mast cells
assays. We did not observe any cell toxicity during incubations and recruited eosinophils may be a successful approach in the
of MM6 cells with 10 mM compound for 24 hours (data not clinical management of many acute and chronic inflammatory
shown). Furthermore, TK04, TK04a, and TK05 are specific and allergic diseases. Our inhibitors were less potent against
toward LTC4S without interacting with mPGES-1, MGST-2, mast cells compared with monocytes, eosinophils, platelets, and
or 5-LO. However, the effect of TK04a and TK05 on 5-HETE MM6 cells. A possible explanation for this may be different
production may suggest a weak interference with FLAP at degrees of LTC4S phosphorylation among cell types, which in
higher concentrations.
The results of the molecular docking study suggest that currently under investigation in our laboratory.
TK04, TK04a, and TK05 inhibit the action of LTC4S compet- Platelets play a fundamental role in primary hemostasis,
itively, by occupying the proposed binding site for LTA4 and but more recent work has also implicated these corpuscles in
interacting with the key catalytic arginine residues. inflammation (Gros et al., 2014). Overproduction of cys-LTs by
turn may influence binding of the inhibitors, a subject that is
Several cys-LT producing cells are notorious for their role in platelet-adherent leukocytes has been shown to amplify tissue
inflammatory and allergic disease, recently reviewed by Liu inflammation in aspirin-exacerbated respiratory disease
and Yokomizo (2015). Allergic reactions are largely driven by (Laidlaw et al., 2012) as well as contribute to pathologic
IgE-mediated degranulation of mast cells. In nasal allergy, vascular events (Cerletti et al., 2010). We found that LTC4
cys-LTs are released during both the early- and late-phase production in isolated platelets incubated with exogenous
response to allergens (Haberal and Corey, 2003) and result in LTA4 is almost completely blocked by TK04, TK04a, and TK05
reinforced symptoms, e.g., sneezing, rhinorrhea, and nasal in vitro. In parallel, we observed a significant increase in
obstruction (Miadonna et al., 1987). Mast cells are also a key lipoxin formation. In vivo, platelet-dependent LTC4 and LXA4
player in the pathogenesis of abdominal aortic aneurysm biosynthesis requires a neighboring leukocyte capable of
(Swedenborg et al., 2011). Elevated levels of LTC4S expres- donating LTA4 through transcellular routes. Therefore we
sion along with production of cys-LTs were discovered in tested our LTC4S inhibitors in mixed platelet neutrophil
human abdominal aortic aneurysm thrombus and wall tissue suspensions. In this setting, we found that TK05 decreased
(Di Gennaro et al., 2010). In our hands, TK04, TK04a, and LTC4 synthesis, whereas LXA4 formation was significantly
TK05 inhibited cys-LT formation with IC50 values in the low increased at a TK05 concentration of 0.5 mM. Furthermore, we
micromolar range in human mast cells activated by cross- show that this effect became more pronounced upon simulta-
linking of the IgE receptor, indicating that these compounds neous inhibition of both LTC4S and LTA4H, presumably
are able to target cys-LT biosynthesis during mast cell reflecting increased export of LTA4 from the neutrophils
dependent inflammation.
when conversion into LTB4 is blocked in agreement with
Eosinophil infiltration is a well-known characteristic of Lehmann et al. (submitted manuscript). These data suggest
allergic asthma (Beasley et al., 1989), and the cell number that specific LTC4S inhibition may contribute to resolution of
correlates with the severity of the disease (Bousquet et al., airway inflammation in two ways: by reducing the formation
1990). They are believed to contribute to asthmatic symptoms of proinflammatory cys-LTs and by boosting generation of

Potent and Selective Inhibitors of Human LTC4S
115
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anti-inflammatory lipoxins (Levy et al., 2002). Moreover, a
dual LTC4S and LTA4H inhibitor will most likely have an
even stronger effect due to additional inhibition of proinflam-
matory LTB4 formation together with enhanced lipoxin
production.
For in vivo testing we used Zymosan A-induced peritonitis
in the mouse, an inflammation model in which cys-LTs
contribute to extravasation of plasma, as demonstrated with
LTC4S-deficient mice (Kanaoka et al., 2001). In our hands,
a dose of 6 mg×kg21 LTC4S inhibitor TK05 administered
intraperitoneally reduced amounts of LTE4 in lavage fluid by
almost 90%. Additionally, we observed decreased plasma
protein extravasation as judged by measurements of EBD in
lavage fluid. Together with earlier data showing that purified
human and mouse LTC4S are both inhibited by the TK
compounds (Niegowski et al., 2014a), the results of the present
study supports the notion that mouse models will be valuable
tools to assess the efficacy of LTC4S inhibitors.
There is a distinct need for new and improved medications
against asthma and LTC4S is one promising target. Pharma-
cological intervention of cys-LT production by blocking LTC4S
would be an approach that circumvents multiple receptors and
intervention with the lipoxin metabolism and has not yet been
explored. Our findings demonstrate that the LTC4S inhibitors
TK04, TK04a, and TK05 are potent and selective inhibitors of
human LTC4S, which block LTC4 and cys-LT synthesis both in
vitro and in vivo. Thus we now have useful experimental tools
in leukotriene research as well as lead structures for further
LTC4S inhibitor design. Finally, our data suggest that a com-
bination of LTC4S and LTA4H inhibitors would perhaps be
an even better approach that will block both arms of the
leukotriene signaling pathways, whereas boosting synthesis
of anti-inflammatory and proresolving lipoxins.
Acknowledgments
The authors thank Birger Sjöberg and Annika Jenmalm Jensen at
Chemical Biology Consortium Sweden, Karolinska Institutet, for pro-
viding excellent facilities, supervision, and valuable discussions re-
garding the chemical synthesis. Furthermore, the authors thank Mats
Hamberg for conducting the mPGES-1 activity assay as well as Gunnar
Nilsson, Craig Wheelock, and Dieter Steinhilber for expert advice.
Authorship Contributions
Levy BD, De Sanctis GT, Devchand PR, Kim E, Ackerman K, Schmidt BA, Szczeklik
W, Drazen JM, and Serhan CN (2002) Multi-pronged inhibition of airway hyper-
responsiveness and inflammation by lipoxin A(4). Nat Med 8:1018–1023.
Liu M and Yokomizo T (2015) The role of leukotrienes in allergic diseases. Allergol
Int 64:17–26.
Lynch KR, O’Neill GP, Liu Q, Im D-S, Sawyer N, Metters KM, Coulombe N, Abramovitz
M, Figueroa DJ, and Zeng Z, et al. (1999) Characterization of the human cysteinyl
leukotriene CysLT1 receptor. Nature 399:789–793.
Malmstrom K, Rodriguez-Gomez G, Guerra J, Villaran C, Piñeiro A, Wei LX, Seidenberg
BC, and Reiss TF; Montelukast/Beclomethasone Study Group (1999) Oral
montelukast, inhaled beclomethasone, and placebo for chronic asthma. A ran-
domized, controlled trial. Ann Intern Med 130:487–495.
Martinez Molina D, Wetterholm A, Kohl A, McCarthy AA, Niegowski D, Ohlson E,
Hammarberg T, Eshaghi S, Haeggström JZ, and Nordlund P (2007) Structural
basis for synthesis of inflammatory mediators by human leukotriene C4 synthase.
Nature 448:613–616.
Participated in research design: Kleinschmidt, Haraldsson,
Basavarajappa, Kahnt, Lindbom, Haeggström.
Conducted experiments: Kleinschmidt, Lundeberg, Thulasingam,
Ekoff, Fauland, Lehmann.
Contributed new reagents or analytic tools: Kleinschmidt.
Performed data analysis: Kleinschmidt, Lundeberg, Thulasingam,
Fauland.
Wrote or contributed to the writing of the manuscript:
Kleinschmidt, Haraldsson, Lundeberg, Thulasingam, Ekoff, Fauland,
Haeggström.
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