Characterization of the molecular mechanism of 5-lipoxygenase inhibition by 2-aminothiazoles

Article abstract of DOI:10.1016/j.bcp.2016.09.021

5-Lipoxygenase (5-LO, EC1.13.11.34) has been implicated in the pathogenesis of inflammatory and immune diseases. Recently, aminothiazole comprising inhibitors have been discovered for this valuable target. Yet, the molecular mode of action of this class of substances is only poorly understood. Here, we present the detailed molecular mechanism of action of the compound class and the in vitro pharmacological profile of two lead compounds ST-1853 and ST-1906. Mechanistic studies with recombinant proteins as well as intact cell assays enabled us to define this class as a novel type of 5-LO inhibitors with unique characteristics. The parent compounds herein presented a certain reactivity concerning oxidation and thiol binding: Unsubstituted aminophenols bound covalently to C159 and C418 of human 5-LO. Yet, dimethyl substitution of the aminophenol prevented this reactivity and slowed down phase II metabolism. Both ST-1853 and ST-1906 confirmed their lead likeness by retaining their high potency in physiologically relevant 5-LO activity assays, high metabolic stability, high specificity and non-cytotoxicity.

Full text of DOI:10.1016/j.bcp.2016.09.021

Biochemical Pharmacology 123 (2017) 52–62
Contents lists available at ScienceDirect
Biochemical Pharmacology
journal homepage: www.elsevier.com/locate/biochempharm
Characterization of the molecular mechanism of 5-lipoxygenase
inhibition by 2-aminothiazoles
Simon B.M. Kretschmer a, Stefano Woltersdorf a, Dominik Vogt a, Felix F. Lillich a, Michael Rühl a,
Michael Karas a, Isabelle V. Maucher a, Jessica Roos a, Ann-Kathrin Häfner a, Astrid Kaiser a,
Mario Wurglics a, Manfred Schubert-Zsilavecz a, Carlo Angioni b, Gerd Geisslinger b, Holger Stark c,
Dieter Steinhilber a, Bettina Hofmann a,
⇑
a Institute of Pharmaceutical Chemistry, Goethe-University Frankfurt, Max-von-Laue-Strasse 9, D-60438 Frankfurt, Germany
b Institute of Clinical Pharmacology, Pharmazentrum Frankfurt, Goethe-University Frankfurt, Theodor Stern Kai 7, D-60590 Frankfurt/Main, Germany
c Institute of Pharmaceutical and Medicinal Chemistry, Heinrich Heine University Düsseldorf, Universitätsstrasse 1, D-40225 Düsseldorf, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 29 July 2016
Accepted 21 September 2016
Available online 23 September 2016
5-Lipoxygenase (5-LO, EC1.13.11.34) has been implicated in the pathogenesis of inflammatory and
immune diseases. Recently, aminothiazole comprising inhibitors have been discovered for this valuable
target. Yet, the molecular mode of action of this class of substances is only poorly understood. Here, we
present the detailed molecular mechanism of action of the compound class and the in vitro pharmaco-
logical profile of two lead compounds ST-1853 and ST-1906. Mechanistic studies with recombinant pro-
teins as well as intact cell assays enabled us to define this class as a novel type of 5-LO inhibitors with
unique characteristics. The parent compounds herein presented a certain reactivity concerning oxidation
and thiol binding: Unsubstituted aminophenols bound covalently to C159 and C418 of human 5-LO. Yet,
dimethyl substitution of the aminophenol prevented this reactivity and slowed down phase II metabo-
lism. Both ST-1853 and ST-1906 confirmed their lead likeness by retaining their high potency in physi-
ologically relevant 5-LO activity assays, high metabolic stability, high specificity and non-cytotoxicity.
Ó 2016 Elsevier Inc. All rights reserved.
Keywords:
5-Lipoxygenase
Aminothiazole
Leukotrienes
Inflammation
ST-1853
ST-1906
1. Introduction
Unfortunately the use of Zileuton is limited, partly due to hepato-
toxic adverse effects [14]. Therefore many novel compounds were
Inflammation is a protective response of the microcirculation to
harmful stimuli. Yet, excessive inflammation is potentially harmful
and a characteristic of many chronic diseases [1]. 5-Lipoxygenase
(5-LO, EC1.13.11.34) contributes to the inflammatory reaction by
catalyzing the conversion of arachidonic acid (AA) to potent lipid
mediators, namely leukotriene (LT)B4 and LTC4 [2]. These media-
tors drive chemotaxis and leukocyte activation, or in case of LTC4
lead to increased vasopermeability and bronchoconstriction [3].
Possible 5-LO linked indications therefore range from asthma [4]
and allergic rhinitis [5], to atherosclerosis [6], Alzheimer’s disease
[7], leukemia [8,9] and certain types of cancer [10]. In order to
widen the therapeutic armamentarium in these diseases many
efforts have been made to develop anti-leukotriene drugs,
which climaxed in the approval of the direct 5-LO inhibitor
Zileuton [11,12] (FDA approval for the therapy of asthma [13]).
designed, resulting in various promising strategies. Given the cat-
alytic cycle of 5-LO, direct 5-LO inhibitors can act via chelating
the active site iron (iron ligand inhibitors; e.g. Zileuton) or by keep-
ing the catalytic iron in the inactive ferrous state by reduction
(redox-active inhibitors). Further on there are active site directed,
non-redox type inhibitors which compete with AA for binding to
5-LO and allosteric inhibitors, which e.g. act at the regulatory C2-
like domain (e.g. Hyperforin [15], EP6 [16]). Despite these rational
approaches, besides Zileuton, a great number of investigated com-
pounds subsequently failed in preclinical or clinical trials [17]. This
was partly due to insufficient in vitro screening. Apart from toxic-
ities, several compounds suffer from a lack of specificity or display
only a poor efficacy in physiologically relevant conditions. The
potencies of ZM 230487 or L-739,010 for example depend on the
phosphorylation status of 5-LO [18]. Thus there is still a pressing
need to develop novel well-characterized 5-LO inhibitors.
In a previous study we presented comprehensive structure-
activity relationships (SAR) of a class of potent aminothiazole-
comprising 5-LO inhibitors [19]. Based on the parent compound
⇑
Corresponding author at: Institute of Pharmaceutical Chemistry, Goethe
University Frankfurt, Max-von-Laue-Strasse 9, D-60438 Frankfurt, Germany.
E-mail address: hofmann@pharmchem.uni-frankfurt.de (B. Hofmann).
http://dx.doi.org/10.1016/j.bcp.2016.09.021
0006-2952/Ó 2016 Elsevier Inc. All rights reserved.

S.B.M. Kretschmer et al. / Biochemical Pharmacology 123 (2017) 52–62
53
ST-1083 we developed two lead compounds (ST-1853 and
ST-1906) out of this SAR with high 5-LO inhibitory potency in puri-
fied enzyme and intact cell assays (IC50 = 50 nM in intact human
polymorphonuclear leukocytes (PMNL)) (Fig. 1) [19]. Yet, compre-
hensive evaluation of the lead likeness of the compounds, as well
as the detailed mechanism of action of this novel 5-LO inhibitor
type is still due. Therefore, we elaborated various in vitro assays
to clarify these unmet questions and present a characterization
of the 5-LO inhibition by N-phenyl-4-aryl-1,3-thiazole-2-amines
with focus on the two lead compounds ST-1853 and ST-1906.
Scientific, Bremen, Germany). Compound purity above 95% was
ensured by the use of liquid chromatography-mass spectrometry
(Shimadzu prominence LCMS 2020 (Duisburg, Germany)) with a
Luna 5
l
m C18(2) 100 Å, column (250 ꢀ 4.6 mm, Phenomenex
Ltd, Aschaffenburg, Germany). Here, a linear gradient of acetoni-
trile/water/formic acid (50/50/0.1–90/10/0.1 V/V) was used for
10 min, kept constant for 5 min, and switched back to (50/50/0.1)
in a 3 min linear gradient and kept constant for another 2 min.
For UV-detection (254 nm and 280 nm)
a SPD-20A UV/VIS-
detector (Shimadzu) was used. All reported yields are unoptimized.
2.1.2. 4-(4-(4-Chlorophenyl)thiazol-2-ylimino)cyclohexa-2,5-dienone
(ST-1905)
2. Materials & methods
ST-1083 (500 mg, 1.6 mmol) was heated to 45 °C in 70 ml ethyl
acetate. Lead(IV) acetate (805 mg, 1.8 mmol) was added. The solu-
tion instantly changed the color to cherry-red. After stirring for
10 min calcium carbonate (1 g, 10.0 mmol) was added. The formed
sponge was filtrated. The filtrate was consequently concentrated
under vacuum and purified by column chromatography (eluent:
dichloromethane), yielding a brown/black solid (60 mg, 0.20 mmol,
12%). 1H-NMR (250 MHz, DMSO-d6): d = 8.51 (1H, s, thiazole-5H),
8.43 (dd, 1H, J = 3.0 Hz, 10.0 Hz, dienone-5H), 8.02 (d, 2H,
J = 8.5 Hz, Ph-2H,6H), 7.54 (d, 2H, J = 8.5 Hz, Ph-3H,5H), 7.35 (dd,
1H, J = 2.9 Hz, 10.4 Hz, dienone-3H), 6.76 (d, 2H, J = 10.6 Hz, die-
none-2H,6H). 13C-NMR (75 MHz, DMSO-d6): d = 187.06, 169.08,
157.65, 153.46, 141.82, 134.05, 133.22, 133.13, 132.19, 130.69,
128.87, 127.85, 120.113. ESI-MS: m/z = 300.9 [M+H+]+; HR-MS: cal-
culated: m/z = 300.01241; found: m/z = 300.01295.
2.1. Chemistry
2.1.1. Chemicals and analytical methods
Synthetic procedures and analytical data for compounds
ST-1083, ST-1904, ST-1853, and ST-1906 were already described
previously [19,20]. All chemicals and solvents were purchased
from Sigma-Aldrich (Taufkirchen, Germany), Acros Organics (Geel,
Belgium), Alfa Aesar (Karlsruhe, Germany), TCI Europe (Eschborn,
Germany) and Apollo Scientific (Bredbury, UK) and used without
further purification unless otherwise stated. Analytical thin layer
chromatography (TLC) was performed with precoated TLC sheets
(ALUGRAMÒ Xtra SIL G UV254, Macherey-Nagel, Düren, Germany)
visualized with ultraviolet light (254 nm). Column chromatogra-
phy was performed with technical grade solvent mixtures specified
in the corresponding experiment with Fluka silica gel 60 (230 –
400 mesh ASTM). 1H-NMR spectra were recorded on a Bruker
DPX 250 (250 MHz), AV300 (300 MHz) or AV400 (400 MHz) spec-
trometer (Bruker, Karlsruhe, Germany). 1H-NMR data are reported
in the following order: chemical shift (d) in ppm downfield relative
to tetramethylsilane: internal reference non-deuterated solvent;
multiplicity (br, broad; s, singlet; d, doublet; dd, double doublet;
t, triplet; m, multiplet; p, pseudo); number of protons; approxi-
mate coupling constant (J) in hertz (Hz). 13C-NMR spectra were
recorded on a Bruker DPX 250 (63 MHz) or AV300 (75 MHz) spec-
trometer (Bruker). ESI-MS was performed on a Fisons Instruments
VG Platform II (Manchester, UK). MALDI high resolution mass spec-
tra (HRMS) was performed on a LTQ Orbitrap XL (Thermo Fisher
2.2. Biological evaluation
2.2.1. Materials
Ammonium hydrogen carbonate, calcium ionophore A23187,
BWA-4C, DMSO, formic acid, hyperforin, trypan blue solution,
phosphatidylcholine (egg yolk), diphenylpicrylhydrazyl (DPPH),
FeCl3, Ferene-S, glutathione (GSH), lipopolysaccharides, N-
ethylmaleimide (NEM), N-acetylcysteine (NAC), NDGA, MK886,
sodium arsenite, fMLP, glucose-6-phosphate dehydrogenase
(G6PD), and MEM non-essential amino acids were purchased
from Sigma-Aldrich (Taufkirchen, Germany). Alpha-cyano-
hydroxycinnamic acid was purchased from Bruker Daltonics (Bre-
men, Germany). AA (peroxide free), Rev-5901 and Zileuton were
purchased from Cayman Chemical (Ann Arbor, USA). Tween-20,
and EDTA were purchased from AppliChem (Darmstadt, Germany).
PierceTM Chymotrypsin Protease (TLCK treated), MS Grade was pur-
chased from Thermo Fisher Scientific (Bremen, Germany). NaCl,
NADP, glucose 6-phosphate, acetonitrile (HPLC grade) were
purchased from Carl Roth (Karlsruhe, Germany). Alamethicin,
D-saccharic acid, and uridine 50-diphospho-glucuronic acid
(UDPGA) were purchased from Santa Cruz Biotechnology (Heidel-
berg, Germany). Methanol (HPLC Grade) was purchased from
VWR International (Darmstadt, Germany). Trifluoroacetic acid
(TFA) was purchased from Merck (Darmstadt, Germany) and Carl
Roth. RPMI 1660, DMEM medium (high glucose and glutamine),
sodium pyruvate (100 mM), Penicillin and streptomycin, rat
(Sprague Dawley) microsomes and phosphate-buffered saline
(PBS) pH 7.4 were purchased from Gibco/Invitrogen (Paisley, UK).
Fetal calf serum (FCS) was purchased from Biochrom AG (Berlin,
Germany). Lymphocyte Separation Medium was purchased from
Lonza Group (Basel, Switzerland). Human whole blood was
obtained from healthy volunteers with informed consent. Fresh
blood cell concentrates were kindly provided by Städtische
Kliniken Frankfurt-Höchst and german red cross blood donor
OH
O
S
N
S
N
NH
N
Cl
Cl
ST-1083
ST-1905
IC50 [r5-LO]: 0.03 µM
IC50 [PMNL]: 0.68 µM
IC50 [r5-LO]: 0.04 µM
IC50 [PMNL]: 1.78 µM
OH
CH3
OH
CH3
H3C
H3C
H3C
H3C
S
N
S
N
NH
NH
ST-1853
ST-1906
Cl
Cl
IC50 [r5-LO]: 0.57 µM
IC50 [PMNL]: 0.05 µM
IC50 [r5-LO]: 0.33 µM
IC50 [PMNL]: 0.05 µM
service Baden-Württemberg
with informed consent. ML 3000 was kindly provided by Merckle
(Blaubeuren, Germany).
ꢁ
Hessen (Frankfurt, Germany)
Fig. 1. Investigated 2-aminothiazole 5-LO inhibitors and corresponding IC50 values
[19]. r5-LO: purified recombinant 5-LO; PMNL: polymorphonuclear leukocytes, for
ST-1905 cf materials section and 3.6.

54
S.B.M. Kretschmer et al. / Biochemical Pharmacology 123 (2017) 52–62
2.2.2. Diphenylpicrylhydrazyl (DPPH) antioxidant assay
5Z,8E,10E-heptadecatrienoic acid and 12-H(P)ETE, respectively.
All values, of at least three independent measurements, were nor-
malized to vehicle controls and either IC50 values and 95% confi-
dence intervals or means SEM were calculated. BWA-4C (5-LO),
NDGA (12-LO, 15-LO1) and acetylsalicylic acid (COX-1) were used
as control.
Radical scavenging activity of the compounds was assessed by
reduction of DPPH [21,22]. Compounds or vehicle control (DMSO)
were incubated with 5 nmol DPPH in acetate buffered methanol
(0.1 M acetate buffer pH 5.5 and methanol 40/60 V/V). After
30 min gentle shaking in the dark, absorbance was measured at
517 nm, using a microplate reader (Infinite M200, Tecan Group
Ltd, Crailsheim, Germany). Each compound was tested indepen-
dently three times. Compound absorbance values were normalized
2.2.6. Preparation of 100,000ꢀg PMNL homogenate supernatants and
measurement of 5-LO product formation
to vehicle controls. Ascorbic acid and NDGA (100
control.
l
M) were used as
Freshly isolated PMNL were resuspended in PG containing
1 mM EDTA and sonicated 3 times for 10 s. PMNL homogenates
were then centrifuged (100,000ꢀg) for 70 min at 4 °C to obtain
100,000ꢀg supernatants (S100). S100 corresponding to 7.5 ꢀ 106
PMNL were then added to 1 ml PBS pH 7.4 containing 1 mM EDTA,
and 1 mM ATP and preincubated with test compounds, and if sta-
2.2.3. Ferene-S assay
Iron reduction from Fe3+ to Fe2+ was measured by the specific
iron(II) colorimetric detector Ferene-S [23]. Compounds or vehicle
control (DMSO) were incubated with 50
l
M FeCl3 and 0.5 mM
ted phosphatidylcholine (20
prewarming at 37 °C, the reaction was started with the addition
of 20 M AA and 2 mM CaCl2. After 10 min the reaction was
stopped by 1 ml methanol and the formed 5-LO products were
analyzed by HPLC/UV as described for intact cells. Data of at least
three independent measurements were expressed as percentage of
lg/ml) for 15 min at 4 °C. After 30 s
Ferene-S in 0.15 M NaCl with 1% Tween-20 for 1 h at 37 °C in the
dark. Absorbance was measured at 594 nm, using a microplate
reader (Infinite M200, Tecan Group Ltd, Crailsheim, Germany).
After baseline correction with vehicle controls, absorbance values
l
were normalized to maximal iron reduction (NDGA 100
compound was tested independently three times. Ascorbic acid
(100 M) was used as control.
lM). Each
control (DMSO). Hyperforin (10 lM) was used as control.
l
2.2.7. Expression and purification of r5-LO
2.2.4. Isolation of intact PMNL and platelets
Recombinant 5-LO was purified as described by [19]. In brief,
the recombinant protein (5-LO wt or cysteine mutants by [28])
was expressed and purified from a 1 L culture (E. coli BL21(DE3))
using a ATP affinity chromatography (5 ml ATP-agarose column)
and anion exchange chromatography (ResourceQ 1 ml IEX column,
GE Healthcare, Uppsala, Sweden) in an ÄKTA Xpress system (GE
Healthcare).
Human PMNL and platelets were freshly isolated from leuko-
cyte concentrates. PMNL and platelets were isolated according to
[24] and [25]. In brief, PMNL were immediately isolated by dextran
sedimentation, centrifugation on lymphocyte separation medium,
and hypotonic lysis of erythrocytes. Cells (purity of >96–97%) were
finally resuspended in phosphate-buffered saline (PBS), pH 7.4
containing 1 mg/ml glucose (PG). For isolation of human platelets,
platelet rich plasma was withdrawn and mixed with PBS buffer pH
5.9 (1:1). After centrifugation (1849ꢀg, 15 min, room temperature
(RT)) cell pellets were resuspended in 50 ml PBS pH 5.9/NaCl (0.9%)
(1:1) and were centrifuged again (1849ꢀg, 10 min, RT). Finally, pla-
telets were resuspended in PG.
2.2.8. Determination of 5-LO product formation in r5-LO
Test compounds or vehicle control were diluted in 1 ml reaction
mix (3
bation time at 4 °C. After 30 s pre-warming, the reaction was
started by addition of 2 mM CaCl2 and 20 M AA. After 10 min at
lg r5-LO, PBS, 1 mM EDTA, 1 mM ATP) for 15 min preincu-
l
37 °C 5-LO product formation was stopped by 1 ml methanol.
The formed metabolites (all-trans LTB4, 5-H(P)ETE) were analyzed
by HPLC/UV as described for intact cells. Data were normalized to
vehicle control (DMSO) and means SEM or IC50 values and 95%
confidence intervals of at least three independent measurements
were calculated.
2.2.5. Determination of 5-LO, 15-LO, COX and 12-LO product
formation in intact cells
Intact cell assays were performed as described in [19,20]. In
brief, for 5-LO and 15-LO1 assay, freshly isolated PMNL (5 ꢀ 106
or 7.5 ꢀ 106, if stated) were resuspended in PG and 1 mM CaCl2,
preincubated with test compounds or vehicle control for 15 min
at 37 °C and stimulated by the addition of calcium ionophore
2.2.9. In vitro human whole blood assay
A23187 (2.5
preincubated with 300 mM sodium chloride or 10
arsenite 3 min prior to addition of exogenous AA (20
l
M) and exogenous AA (as indicated, 0–30
M sodium
M). After
l
M), or
In vitro whole blood assays were performed as described by
[29]. In brief, heparinized venous blood was prewarmed at 37 °C
for 30 min, preincubated for 30/120 min with the test compounds
l
l
10 min at 37 °C, the reaction was stopped by the addition of
methanol (1 ml). To affect thiol levels (cf Section 3.5) either
and stimulated by addition of 20
(final concentration, previously dissolved in 50
l
M calcium ionophore A23187
l
l autologous
50
lM N-ethylmaleimide (NEM) or 5 mM N-acetylcysteine (NAC)
plasma). After 15 min the reaction was stopped on ice. In case of
was added 20 min prior to compound preincubation.
fMLP stimulation after 30 min prewarming at 37 °C, lipopolysac-
For cyclooxygenase (COX-)1 and 12-LO product formation, 108
freshly isolated platelets were resuspended in 1 ml PG containing
1 mM CaCl2 and subsequently preincubated with test compounds
or vehicle control for 15 min at 37 °C. The reaction was started
charides (10
were preincubated for 15 min. 5-LO reaction was started by addi-
tion of 1 M fMLP and stopped after 1 h as described above. Plasma
lg/ml) were added. After 15 min test compounds
l
supernatants were taken after centrifugation at 9391ꢀg. Liquid-
liquid extraction and LC-MS/MS analysis were performed accord-
ing to [25]. Data were normalized to vehicle control (DMSO) and
means SEM or IC50 values and 95% confidence intervals of at least
three independent measurements were calculated.
with the addition of 10 lM AA and stopped after 10 min at 37 °C
by addition of 1 ml methanol.
For solid phase extraction, Prostaglandin B1 (200 ng) was added
as internal standard, as well as HCl (30 ll, 1 N) and PBS (500 ll).
HPLC analysis (as described by [26,27]) comprised LTB4,
its all-trans isomers, and 5-hydro(pero)xy-6,8,11,14(E,Z,Z,Z)-
eicosatetraenoic acid (5-H(P)ETE) for 5-LO product formation.
15-LO1 product formation included 15-hydro(pero)xy-5,8,11,13
(Z,Z,Z,E)-eicosatetraenoic acid (15-H(P)ETE), COX-1 and 12-LO pro-
duct formation in human platelets included 12-(S)-hydroxy-
2.2.10. Sample preparation for MALDI-MS
Glutathione (GSH, 1 mM, final concentration) was diluted in
PBS buffer pH 7.4 together with the compound ST-1083, ST-1905,
ST-1906 (each 100
lM), or ST-1853 (300 lM) in a total volume
of 2 l. For quantitative measurements, a concentration series from
l

S.B.M. Kretschmer et al. / Biochemical Pharmacology 123 (2017) 52–62
55
1 to 50
l
M of ST-1083 and ST-1905 was prepared. The incubation
modifications: none and variable modifications (Table 1). MS/MS
database searches were also performed after conversion to mgf
files with the following parameters using the MASCOT MS/MS
Ion search tool: enzyme for proteolysis: trypsin; missed cleavages
sites: up to one; if chymotrypsin was used then: enzyme for prote-
olysis: chymotrypsin and missed cleavage sites up to two; fixed
modifications: the particular substance meant to bind on the pep-
tide or none for the peptide structure verification; Peptide mass
tolerance: 50 ppm; MS/MS-ion mass tolerance: 0.5 Da; peptide
charge: +1.
was performed directly on a Thermo Scientific 384-well MALDI-MS
target (Thermo Fisher Scientific, Dreieich, Germany) in a humidity
chamber especially for MALDI-MS targets for 30 min at 37 °C with
saturated humidity. After 30 min incubation time, the reaction was
stopped by the addition of
1 ll of 3 mg/ml alpha-cyano-
hydroxycinnamic acid in a mixture of acetonitrile/water/trifluoroa
cetic acid (TFA) (70/30/0.1 V/V). The incubation of purified 5-LO
was performed following the same procedure and compound con-
centration (100 and 300
l in a total volume of 2
chymotrypsin or a mixture of both proteases with an enzyme con-
centration of 12.5 ng/ l in 25 mM NH4HCO3 was added to the dro-
lM). Final 5-LO concentration was 150 ng/
l
l
l. After 30 min incubation, 1 l of trypsin,
l
2.2.12. Cell culture
l
The human leukemic monocyte cells U937 and the human liver
cancer cell line Hep G2 were purchased from Deutsche Sammlung
für Mikroorganismen und Zellkulturen (DSMZ, Brunswick, Ger-
many). U973 cells were maintained in RPMI 1660. Complete cul-
plets on the MALDI-target. Enzymatic digestion was performed for
1 h and stopped by the addition of the alpha-cyano-
hydroxycinnamic acid with the same concentration as used for
the GSH-binding experiment. After crystallization of the matrix, a
chilled five percent formic acid solution was used to wash the sam-
ture media contained 10% FCS, 100
100 U/ml penicillin. Hep G2 cells were maintained in DMEM, con-
taining 10% FCS, 100 g/ml streptomycin, 100 U/ml penicillin,
lg/ml streptomycin and
ple spots. For sample washing, 2
ll of the formic acid solution were
l
used to coat the matrix crystals and were immediately removed to
wash away salt contaminants due to high salt concentration in PBS
1 ꢀ MEM non-essential amino acids and 1 mM sodium pyruvate.
Cells were cultured at 37 °C in a humidified atmosphere containing
5% CO2 and discarded after passage 35.
buffer. After sample washing,
1 ll of acetonitrile/water/TFA
(80/20/0.1 V/V) was used to recrystallize the matrix for a more
homogeneous crystal size distribution.
2.2.13. Cell viability (water soluble tetrazolium-1 (WST-1)) assay
Cell viability was assessed using a WST-1 assay (Roche Diagnos-
tics GmbH, Mannheim, Germany). U937 cells were seeded in flat-
bottomed 96-well plates at a density of 104 cells per well. After
treatment of cells with test compounds or vehicle (DMSO) for
48 h in presence of 10% FCS, cell viability was assessed according
to the distributor’s protocol using a microplate reader (Infinite
M200, Tecan Group Ltd, Crailsheim, Germany). Absorbance values
were measured after 48 h, baseline corrected with the absorbance
of culture medium plus WST-1 in the absence of cells and normal-
ized to vehicle control (DMSO). Hep G2 cells were seeded in flat-
bottomed 96-well plates at a density of 3 ꢀ 104 cells per well for
48 h in presence of 10% FCS. After 24 h culture medium and test
compounds were refreshed. Analysis was carried out as stated for
U937 cells. Each compound was tested independently at least three
2.2.11. MALDI-MS-measurement and data-analysis
All MALDI-MS spectra were recorded with a MALDI LTQ-
Orbitrap XL (Thermo Fisher Scientific). For GSH-binding, all mea-
surements were recorded from m/z: 400.00 – 800.00 with a resolu-
tion of 60 000 and 50 subspectra were accumulated for data-
analysis. The peptide mass fingerprint measurements of the 5-LO
digests were recorded from m/z: 800.00 – 4000.00 with a resolu-
tion of 30 000 and 30 subspectra were accumulated for database
search. MS/MS spectra were obtained using collision-induced dis-
sociation fragmentation in combination with the ion-trap mass
analyzer of the MALDI LTQ-Orbitrap XL. 100 subspectra were accu-
mulated for analysis. The isolation width for MS/MS analysis of the
GSH-bound substances was 0.5 Da and 0.7 Da for the MS/MS-
analysis of the (chymo-)tryptic peptides after digestion. The
laser-energy of the MALDI ion source was adjusted as needed for
optimal measurement results and kept constant. All measurements
were performed as quintuplicates for a reproducible MS and MS/
MS analysis. For the qualitative analysis of the GSH-binding, the
obtained high-resolution mass spectra were compared with a the-
oretical spectrum generated by the XcaliburTM Qual browser version
2.0.4 software. For a more specific and accurate evidence of the
GSH-binding, the MS/MS spectra were analyzed. A neutral loss of
129.04 Da indicated glutamic acid loss, therefore making a thiol
binding more probable. Deisotoping of the obtained spectra and
converting the raw-files to mascot generic files (mgf) for further
data-analysis and database search were performed using an in-
house programmed KNIMEÒ-workflow. All quantitative GSH-
binding measurements were analyzed by a KNIMEÒ workflow with
automatical calculation of the relative intensity using the matrix
signal m/z: 419.32 as internal standard. The peak lists of the pep-
tide mass fingerprint analysis were subsequently submitted to
the MASCOT-database search (Mascot version 2.4). Parameters
for the database search were: peptide mass tolerance: 10 ppm,
enzyme for proteolysis: trypsin, missed enzyme cleavage sites:
maximum 1, fixed modifications: none, variable modifications
(Table 1).
times; means SEM were calculated. 100 lM Zileuton (cell viabil-
ity U937: 88.98 3.92%, Hep G2: 101.60 2.55%) and Rev-5901
(3.24 1.94%, 45.16 7.83%) were used as control.
2.2.14. In vitro metabolism
For phase I metabolism, test compounds (10
lM) were diluted
in phosphate buffer (0.1 M, pH 7.4) together with 50
ll NADPH-
regenerating system (30 mM glucose-6-phosphate, 4 U/ml
glucose-6-phosphate dehydrogenase, 10 mM NADP, 30 mM
MgCl2). After 5 min preincubation time at 37 °C the reaction was
started by the addition of 13 ll rat liver microsome mix (20 mg
protein/ml in 0.1 M phosphate buffer). The incubation was per-
formed in a shaking water bath at 37 °C and subsequently stopped
by the addition of 500 ll ice-cold methanol at 0, 30 and 60 min.
The samples were centrifuged at 10,000ꢀg for 5 min at 4 °C. The
supernatants were analyzed and quantified by HPLC. Control sam-
ples were performed to ensure stability of the compounds in the
reaction mixture. Controls comprise neglected NADPH, test com-
pounds (to determine the baseline) and heat inactivated micro-
somes. The amounts of the test compounds were quantified by
an external calibration curve and normalized to the starting
(0 min) value. Phase II metabolism (glucuronidation) was deter-
mined similarly in phosphate buffer (0.1 M, pH 7.4) containing
30 mM MgCl2. At variance with the terms of phase I metabolism
the NADPH-regenerating system was exchanged for 5 mM uridine
Oxidized modifications were considered as a possible modifica-
tion due to oxidation during the reaction. If chymotrypsin was
used for proteolysis the database search parameters were as fol-
lows: peptide mass tolerance: 10 ppm, enzyme for proteolysis:
chymotrypsin, missed enzyme cleavage sites: maximum 2, fixed
50-diphospho-glucuronic acid (UDPGA), alamethicin (25
lg/ml),
and saccharic acid 1,4 lactone (5 mM). Determination of remaining
parent compound is conducted as described above. Each

56
S.B.M. Kretschmer et al. / Biochemical Pharmacology 123 (2017) 52–62
Table 1
Parameters for MASCOT database search.
Modification name
Mass shift
(monoisotopic/average)
Oxidation possible
(bound to 5-LO)
Mass shift of oxidized substance
(monoisotopic/average)
Amino acid specificity
for database search
ST-1083, ST-1905
ST-1853
ST-1906
300.01/300.76
316.19/316.46
376.02/377.29
Yes
Yes
Yes
297.99/298.74
314.14/314.44
374.00/377.27
C, H, K
C, H, K
C, H, K
compound was tested independently at least three times and
means SEM were calculated. For phase metabolism 7-
ethoxycoumarin (remaining compound: 52.67 1.45% after
60 min) and 7-hydroxycoumarin for phase II metabolism (below
limit of detection after 60 min) were used as control compounds.
mechanism of 5-LO inhibition, we further investigated the capabil-
ities of iron reduction using a Ferene-S assay. In line with the rad-
ical scavenging activities, the compounds showed moderate iron
I
reducing properties at 10 lM. Notably, both lead compounds
showed a stronger reduction of Fe3+ than the parent compound
ST-1083. As expected, ST-1905 displayed no reducing activities,
since the compound is already in an oxidized state.
2.3. Statistics
For single measuring points, means SEM were calculated. IC50
values and 95% confidence intervals (CI) were determined using a
nonlinear fit (log (inhibitor) vs. normalized response – variable
slope) by GraphPad Prism software version 7.00 for Windows
(GraphPad Software, La Jolla, USA). For this, five to eight increasing
concentrations of each compound were measured in three to eight
independent experiments. Absolute values were normalized to
vehicle controls. For comparison of means, unpaired, two-tailed
3.2. ST-1083 and ST-1905 but not the lead compounds show covalent
binding to 5-LO
Previous work from our lab described several 5-LO inhibitors,
like U73122 [28], nitro fatty acids [30], as well as certain quinones
(submitted data, Maucher et al.) to bind covalently to 5-LO by
nucleophilic attack of cysteine residues. Especially the cysteines
159, 300, 416, and 418 (located in the substrate entry channel of
5-LO) are prone to be potential targets for inhibition. As the com-
pounds are capable of oxidation, we next investigated covalent
binding by the compounds via MS analysis. For ST-1083 and ST-
1905 binding both to a peptide containing C159 (Fig. 3A) and a
peptide containing C416 and C418 (Fig. 3B) could be observed.
To further distinguish both cysteines, the experiment was re-run,
using the r5-LO C416A mutant. Here, too, the binding of ST-1083
and ST-1905 was detected (data not shown). On the contrary, both
lead compounds bearing the 2,6-dimethyl substitution of the
aminophenolic moiety (ST-1853, ST-1906) showed no binding to
r5-LO.
t-tests were performed.
significant.
A
p-value 6 0.05 was considered
3. Results
3.1. Aminophenolic thiazole compounds show redox activity
Since the reported compounds generally could possess antioxi-
dant properties along with the aminophenolic moiety, we firstly
tested the compounds’ radical scavenging activities in a DPPH
assay. Here, by measurement of the reduction of the stable radical
DPPH, the reducing properties of a compound can be estimated. In
comparison of the two lead compounds ST-1853 and ST-1906, we
assessed the parent compound ST-1083 as well as its oxidized
quinone imine form, ST-1905 (Fig. 1). Except for ST-1905 all
investigated compounds showed moderate reducing properties at
3.3. Compounds show different susceptibility to GSH trapping
In order to assess this reactivity, we similarly incubated the
compounds with GSH for MALDI-MS analysis and screened for
the exact mass of the GSH adduct, as well as the neutral loss of
10 l
M (Fig. 2). Since reduction of Fe3+ to Fe2+ is a relevant
Fig. 2. Radical scavenging (A) and reducing properties (B) of the test compounds A) Compounds or vehicle control (DMSO) were incubated with 5 nmol DPPH for 30 min.
Absorbance was measured at 517 nm and normalized to vehicle control. Ascorbic acid (100 M) and NDGA (100 M) were used as control (ascorbic acid: 3.67 1.23%, NDGA
3.91 1.50%), n = 3. B) Compounds or vehicle control (DMSO) were incubated with 50 M FeCl3 and 0.5 mM Ferene-S as iron(II) specific colorimetric detector. Absorbance
was measured at 594 nm and normalized to NDGA (100 M) control. Ascorbic acid (100 M) was used as control (108.4 21.75%), n = 3. Data are shown as means + SEM.
l
l
l
l
l

S.B.M. Kretschmer et al. / Biochemical Pharmacology 123 (2017) 52–62
57
Fig. 3. MS/MS spectra of modified and unmodified cysteine containing peptides. The matched masses in the MS/MS spectra are marked with the sequence belonging to the
mass. MS/MS spectra were recorded after collision induced dissociation fragmentation in the ion trap mass analyzer. A) MS/MS spectra of peptide [159–165]. 1: unmodified
peptide, 2: peptide modified with ST-1083, 3: peptide modified with ST-1905. For ST-1853 and ST-1906 only the precursor mass and water loss could be detected and it was
therefore no approval of the modified peptide structure. B) MS/MS spectra of peptide [415–421]. 1: unmodified peptide, 2: peptide modified with ST-1083, 3: peptide
modified with ST-1905. For ST-1853 the precursor mass of the modified peptide could not be isolated and fragmented. ST-1906 showed no MS/MS spectra.

58
S.B.M. Kretschmer et al. / Biochemical Pharmacology 123 (2017) 52–62
129 Da of glutamic acid. Under these conditions, all investigated
compounds are capable of binding GSH (data not shown). For a
quantitative analysis of this GSH binding, we further gathered rel-
ative intensity data for ST-1083 and ST-1905 by using alpha-
cyano-hydroxycinnamic acid as an internal standard. Thereby it
became evident that reactivity is driven by the quinone imine,
since the relative intensity of GSH binding of ST-1905, the oxidized
derivative, was markedly higher than the corresponding hydro-
quinone amine ST-1083 (Fig. 4).
100
80
60
40
20
0
r5-LO wt
r5-LO 4C
r5-LO C159S
r5-LO C300S
r5-LO C416S
r5-LO C418S
M
-1
M
M
µ
µ
µM
µ
µM
6
3.4. Potency of ST-1083 and ST-1905, but not of the lead compounds is
impaired for 5-LO cysteine mutants
1
.1
.
.
.6
1
0
0
0
0
C
4
3
6
0
8
05
53
A-
0
9
8
9
1
1
1
-
-
-
BW
T
T
T
T
S
S
S
S
To further validate the results of MS analysis, activity based
5-LO assays were conducted using purified recombinant 5-LO
mutants. Here, in line with the detected covalent binding to one
of the cysteines in the entry channel of 5-LO by unsubstituted
aminophenols (cf 3.2), ST-1083 as well as the oxidized form
ST-1905 showed a decreased potency using r5-LO 4C mutant
(r5-LO C159S, C300S, C416S, C418S; cf [31]) (Fig. 5). The potency
of ST-1853 and ST-1906 is hereby not impaired. In case of r5-LO
4C and C416S respectively, potency is not only not declined, but
even increased (Fig. 5). Of interest, compounds with an altered
aminophenolic moiety that cannot oxidize to the respective
quinone imine showed no variation in inhibitory activity
throughout each and every studied r5-LO mutant/wt (data not
shown). This altogether reveals the cysteine dependent inhibition
of unsubstituted aminophenols but not of the lead compounds.
Fig. 5. Inhibition of 5-LO activity of wt and cysteine mutants by the test
compounds. 3 g r5-LO wt or cysteine mutant (C159S, C300S, C416S, C418S, and
combination thereof (4C)) were incubated for 15 min at 4 °C with test compounds
or vehicle control (DMSO) in 1 ml PBS pH 7.4 containing 1 mM EDTA. After pre-
warming for 30 s, 5-LO reaction was started by addition of 20 lM AA and 2 mM
CaCl2. After 10 min the reaction was stopped by the addition of 1 ml methanol. 5-LO
product formation was analyzed via HPLC/UV after solid-phase extraction. Data
were normalized to respective vehicle controls and are shown as means + SEM.
Absolute values of vehicle controls were 2005 171.1 (wt), 1788 80.93 (C159S),
1764 187.9 (C300S), 803 46.61 (C416S), 1629 79.79 (C418S), and 1332 88.28
(4C) ng/ml, n = 3–4.
l
3.6. Inhibitory profile of lead compounds ST-1853 and ST-1906
As we could recently show [19], both compounds exhibit direct
5-LO inhibition as seen by inhibition of r5-LO (IC50 [ST-1853]
3.5. Lead compounds show no dependency on thiol level
= 0.33
(PMNL) this high potency is even improved (IC50 [ST-1853,
ST-1906] = 0.05 M) [19]. These properties are in contrast to the
parent compound ST-1083, and its oxidized form, ST-1905 (IC50
[r5-LO] = 0.04 M (0.02 – 0.07 M 95% CI), IC50 [PMNL] = 1.78
(1.08 – 2.92 M 95% CI)) (Fig. 1). A plausible explanation for this
positive shift of ST-1853 and ST-1906 to higher potencies in intact
cells might be off-target effects concerning 5-LO upstream proteins
(cPLA2, FLAP). Thus, we further evaluated 5-LO product inhibition
at various substrate concentrations, used alternative stimuli and
a FLAP inhibitor assay (Fig. 7). FLAP inhibitors (e.g. MK886 [33])
normally present higher potency at lower substrate concentra-
tions, while at higher exogenous substrate amounts they fail to
fully inhibit 5-LO product formation (cf [34]). Here it was shown
that ST-1853 is neither displaced by higher substrate (AA) concen-
trations nor considerably more potent at low AA concentrations
(Fig. 7A). Furthermore, the potency of ST-1853 and ST-1906 was
not impaired by 5-LO phosphorylation (mediated by hyperosmotic
or genotoxic cell stress) (Fig. 7B, C). In addition, 5-LO activity of
lM, IC50 [ST-1906] = 0.57 lM). In the intact cell system
Since redox activity seems to be important for inhibitory
potency, we next altered the cellular redox tone by increasing or
decreasing the cellular thiol level. For this purpose we first added
N-ethylmaleimide (NEM) to intact PMNL to decrease cellular thiols.
In this case, thiol reactive compounds, like U73122 should show an
increased potency, due to lessened GSH adducts [32]. This behavior
was reflected partly by ST-1083 and its quinone imine analogue
ST-1905 (Fig. 6). Substituted analogues, like ST-1853 and
ST-1906, on the contrary showed no difference in inhibition. Vice
versa, increase of cellular thiols via addition of N-acetylcysteine
(NAC) should impair the potency of thiol reactive compounds,
due to increased thiol trapping. Indeed, the potency of U73122
and ST-1905 was decreased, while the potency of ST-1083 and
the lead compounds ST-1853 and ST-1906 was unaltered.
l
l
l
lM
l
10
ST-1905
MK886-pretreated PMNL was still reduced by 0.03 lM ST-1853
(residual activity of 86.88 4.82% for 7.5 ꢀ 106 PMNL and no inhi-
bition of r5-LO), whereas incubation of two FLAP inhibitors
(MK886 and ML 3000 [34]) showed no further inhibition (Fig. 7D).
To rule out additional inhibition by interference with the
C2-like domain of 5-LO, 100 000 ꢀ g supernatant of PMNL homo-
genates were incubated in absence or presence of phosphatidyl-
choline. Here too, there was no decisive difference in treatment
(data not shown).
In order to screen the compounds in more physiological condi-
tions, we next conducted in vitro human whole blood assays. In
detail, we analyzed 5-LO activity stimulated by calcium ionophore
A23187, to acquire maximal 5-LO activation, compared to the more
physiological stimulus fMLP (Fig. 8). Furthermore, we varied the
preincubation time of the compounds (30 min/ 2 h) in order to
get insight in plasma stability.
ST-1083
8
6
4
2
0
0
10
20
30
40
50
concentration [µM]
Fig. 4. Quantitative MALDI-MS based analysis of GSH binding. Compounds were
incubated with 1 mM GSH for 30 min at 37 °C in a humidity chamber directly on a
384 well MALDI-MS target. The matrix signal m/z: 419.32 was used as internal
standard. Data are displayed as means + SEM (error bars shorter than the height of
the symbol), n = 5.

S.B.M. Kretschmer et al. / Biochemical Pharmacology 123 (2017) 52–62
59
Fig. 6. Influence of thiol level on 5-LO inhibition. 5 ꢀ 106 PMNL were resuspended in PBS glucose (1 mg/ml) and 1 mM CaCl2 and pretreated for 20 min at 37 °C with A) 50
lM
NEM to deplete or B) 5 mM NAC to increase thiol levels. After 15 min incubation time with test compounds or vehicle controls (DMSO), 5-LO reaction was stimulated by
2.5 lM A23187 and 20 lM exogenous AA. After 10 min reaction was stopped by addition of 1 ml methanol. HPLC/UV analysis was performed after solid-phase extraction.
Data were normalized to vehicle controls and are shown as means + SEM. Absolute values of vehicle controls were 18.27 0.82 (with NEM), 126 16.83 (without NEM) and
49.55 1.90 (with NAC), and 166.8 4.17 ng per 1 ꢀ 106 PMNL (without NAC), n = 3.
Fig. 7. Cellular screening assays for 5-LO inhibition. 7.5 ꢀ 106 PMNL were resuspended in PBS glucose (1 mg/ml) and 1 mM CaCl2 and pretreated 15 min at 37 °C with test
compounds or vehicle controls (DMSO). 5-LO reaction was started by A) 2.5 lM A23187 and varying concentrations of exogenous AA, B,C) 2.5 lM A23187 and 20 lM AA or
300 mM NaCl or 10 lM NaAsO2 for 3 min and addition of 20 lM AA, and D) 2.5 lM A23187 and 20 lM AA. After 10 min reaction was stopped by addition of 1 ml methanol.
HPLC/UV analysis was performed after solid-phase extraction. Data were normalized to vehicle controls and are shown as means + SEM, n = 3–7. Absolute values of vehicle
controls were A) 43.92 2.48 (w/o), 25.99 1.42 (1
l
M AA), 50.31 1.81 (3
lM AA), 125.5 5.50 (10
lM AA), 236.5 10 (20
lM AA), 174.5 4.45 (30
l
M AA) ng per 1 ꢀ 106
PMNL, B) 66.38 7.22 (NaAsO2), 70.22 3.30 (NaCl), and 236.5 10.00 (A23187) ng per 1 ꢀ 106 PMNL, C) 270.8 39.74 (NaAsO2), 179.3 22.40 (NaCl), 129.1 9.46 (A23187)
ng per 1 ꢀ 106 PMNL, D) 180.9 20.89 ng per 1 ꢀ 106 PMNL.

60
S.B.M. Kretschmer et al. / Biochemical Pharmacology 123 (2017) 52–62
Fig. 8. Human whole blood studies. A) 450
ll venous blood was preincubated for 2 h at 37 °C with test compounds or vehicle (DMSO). 5-LO reaction was started by addition
of 20
liquid–liquid extraction. Data were normalized to vehicle controls and are shown as means + SEM. B) 450
lipopolysaccharides, then, for another 15 min test compounds were added. 5-LO reaction was stimulated by addition of 1
l
M A23187, dissolved in autologous plasma. After 15 min, the reaction was stopped on ice. Plasma supernatants were analyzed for LTB4 and 5-HETE by LC–MS/MS after
l venous blood was preincubated for 15 min at 37 °C with 10 g/ml
M fMLP and stopped on ice after 1 h. 5-LO products
M) was used as control (A: 8.64 1.55%,
l
l
l
were measured as described in A). Absolute values of vehicle controls were A) 222.2 22.03 and B) 16.48 1.86 ng/ml. Zileuton (10
l
B: 13.02 1.12% residual activity), n = 3.
ST-1853 and ST-1906 both achieved a high potency with IC50
values of 0.78 M (0.48–1.28 95% CI) and 1.06 M (0.88–1.28
inhibition of 15-LO and COX only started at concentrations outside
of this reasonable safety margin (10 M) (residual activity 15-LO:
l
l
l
95% CI), respectively, when preincubated for 2 h and stimulated
with calcium ionophore A23187 (Fig. 8A). This long preincubation
time had no negative effect on the compounds’ potencies, as a
reduced (30 min) preincubation time resulted in similar inhibitory
activities (data not shown). The high potency is also retained when
LT formation was stimulated by the bacterial chemotactic peptide
46.50 9.21% (ST-1853) and 40.45 12.04% (ST-1906); COX:
76.76 4.38% (ST-1853) and 71.72 2.05% (ST-1906)), whereas
12-LO was not impaired.
3.8. In vitro pharmacokinetic profile of ST-1853 and ST-1906
fMLP (IC50 below 1 lM) (Fig. 8B).
Given that the 5-LO inhibitory potency of both lead compounds
was unaffected by prolonged incubation time in human whole
blood (cf 3.6), we next conducted microsomal stability studies. In
the course of one hour, concerning cytochrome P450 mediated
phase I metabolism, both lead compounds were similarly degraded
and showed a certain degree of metabolism with approximately
70% remaining parent compound (ST-1853: 70.77 1.74%,
ST-1906: 74.17 0.51%). Further on, we validated if the
2,6-dimethyl substitution of the aminophenolic moiety is capable
of reducing phase II metabolism, namely glucuronidation. Indeed,
in comparison with an unsubstituted aminophenol derivative
3.7. Specificity towards other eicosanoid synthesizing enzymes
Since many LO inhibitors did not present a pronounced speci-
ficity with regard to other AA metabolizing enzymes, we next
screened specificity of both lead compounds on LO isoforms 12
and 15 as well as COX (Fig. 9). Here, ST-1853 and ST-1906 did
not show off-target inhibition at total 5-LO product inhibition
(0.3
lM). Indeed, there was an at least 100 fold threshold between
IC50 of 5-LO and 12-LO, 15-LO, and COX inhibition. Moderate
Fig. 9. Specificity of A) ST-1853 and B) ST-1906 towards LO isoforms and COX. For 5- and 15-LO, 5 ꢀ 106 PMNL were resuspended in PBS glucose (1 mg/ml) and 1 mM CaCl2
and pretreated for 15 min at 37 °C with test compounds or vehicle controls (DMSO). LO reaction was started by addition of 2.5 lM A23187 and 20 lM AA. For 12-LO and COX,
108 platelets were resuspended in 1 ml PBS glucose (1 mg/ml) and 1 mM CaCl2 and subsequently preincubated with test compounds for 15 min at 37 °C. 12-LO and COX
reaction was started by addition of 10 lM AA. After 10 min reaction was stopped by addition of 1 ml methanol. HPLC/UV analysis was performed after solid-phase extraction.
Data were normalized to vehicle controls and are shown as means + SEM. Absolute values of vehicle controls were 165.9 21.65 (5-LO), 6.63 0.81 (15-LO) ng per 1 ꢀ 106
PMNL, 1792 312.7 (12-LO), 893 69.84 (COX) ng per 108 platelets. Residual activity of control compounds was below limit of detection (15-LO, NDGA, 100
lM), 5.07 2.75%
(12-LO, NDGA, 100 lM) and 4.64 2.32% (COX, acetylsalicylic acid, 100 lM).

S.B.M. Kretschmer et al. / Biochemical Pharmacology 123 (2017) 52–62
61
(ST-1904) the substitution pattern of ST-1906 successfully slowed
down glucuronidation after 1 h of incubation significantly
(unpaired, two-tailed t-test, p value 0.0022, degrees of freedom:
7). In detail, 95.29 4.71% of the lead compound was unaffected
by glucuronidation, while only 69.84 2.05% of ST-1904 remained
unaltered.
prevented by this substitution pattern. Nonetheless modification
of the reducing milieu of the intact cell via alteration of the thiol
level did not alter the inhibitory potency. An alternative explana-
tion for the higher potency in intact cells could be an enrichment
of compound in intact cells leading to higher concentrations at
the target area. In contrast to the unsubstituted compounds, ST-
1853 and ST-1906 had a higher potency using the purified 4C
and C416S enzyme compared to wt 5-LO. C416 of the 5-LO wt
may therefore feature negative effects on binding of the lead
compounds.
3.9. Lead compounds show no cytotoxicity
Lastly, to rule out cytotoxicity, cell viability measurements were
conducted. On this account, WST-1 assays were carried out. Previ-
ously, we could show that it is possible to disarm the observed
cytotoxicity of unsubstituted aminophenols like ST-1083 via intro-
ducing the characteristic 2,6-dimethyl substitution of ST-1853 and
ST-1906 using human leukemic monocytic cells (U937) [19]. To
further confirm this outcome, we studied the cell viability also in
an hepatic cell line, human Hep G2. Here, too, after 48 h of incuba-
tion time, ST-1853 and ST-1906 showed no impairment of cell via-
bility in comparison to vehicle control. In both cell lines the lead
Anti-LT therapy by 5-LO inhibition has been hampered by occur-
ring liver toxicities by Zileuton or the clinical phase II compound
Atreleuton [36,37] However both compounds possess a thiophene
as well as an N-hydroxyurea moiety. These features could be linked
with reactive thiophene intermediates [38,39] and redox cycling
via electron transfer [40] potentially leading to the toxicity stated
above. Indeed, there is no declaration of a class effect concerning
liver toxicity by 5-LO inhibition per se [36]. The new lead com-
pounds represent novel scaffolds with high efficacy in various
in vitro assays. They circumvent bioactivation liabilities by elimi-
nating a metabolic hot spot with the C5 methylated thiazole moiety
[41,42] as well as the above-mentioned 2,6 dialkylation. Taken
together, based on our in vitro data, unsubstituted aminophenolic
derivatives achieve their high potency partly by covalent binding
to C159 and C418 of r5-LO. The poor potency of the compounds
in intact cells could be explained by GSH trapping. This thiol reac-
tivity may also be an explanation for the observed cytotoxicity of
unsubstituted compounds (cf [19]). Nonetheless, the identification
of novel small molecules targeting C159 and C418 of 5-LO may pro-
vide the basis for novel 5-LO drug discovery approaches specifically
targeting these cysteine residues. Here, basic approaches can
already be seen by another study in our group (Maucher et al.,
submitted data). The methylation motif of the lead compounds
ST-1853 and ST-1906 on the other hand led to higher potencies
in intact cells. Here, we could show that the structural motif of
the two lead compounds prevented covalent modification of
r5-LO as well as phase II metabolism (glucuronidation) while
maintaining reducing properties. ST-1853 and ST-1906 displayed
a high potency in physiologically relevant assays together with
high specificity against related enzymes. Adding the moderate
in vitro metabolism and non-cytotoxicity, they present well-
balanced, most favorable characteristics that demand for further
preclinical screening as anti-inflammatory drug candidates.
compounds presented a cell viability >90% at 10 lM.
4. Discussion
Based on the results of our previous SAR study on 2-
aminothiazole 5-LO inhibitors [19], we encountered a potency loss
between r5-LO and intact PMNL for unsubstituted compounds and
a positive shift towards a higher potency in PMNL for compounds
bearing a methylated substitution motif. In the present study we
demonstrated that the high potency of N-phenyl-4-aryl-1,3-
thiazole-2-amine derivatives in the purified r5-LO assay might be
due to covalent binding of the unsubstituted aminophenolic moi-
ety to C159 and C418. We further give an explanation for the
reduced activity in intact cells, as the compounds are capable of
binding GSH. Corresponding previous studies have shown that
compounds with tampered aminophenolic moiety show only a
diminished potency [19]. Further on, compound ST-1905, bearing
the quinone imine moiety, and its reduced equivalent ST-1083 pre-
sented similar potencies at the r5-LO wt (high potency) and 4C
mutant assay (impaired potency) (cf Fig. 5). In line, ST-1905
showed a more pronounced binding of GSH in comparison with
the hydroquinone amine and a lower potency in intact cells.
Together with the previously deduced SAR this suggests that redox
activity is one aspect of the inhibitory mode of action. While the
DPPH assay attests antiradical activity of the compounds, the
Ferene-S assay also approved the capability of reducing Fe3+, there-
with providing the basis of a partly redox active mechanism of
inhibition. Both lead compounds ST-1853 and ST-1906 showed a
higher potency in intact cells than with the purified recombinant
enzyme. This is rather unusual, since intact cells have to be perme-
ated and present metabolizing enzymes as well as off targets. Inhi-
bition of 5-LO upstream enzymes (cPLA2, FLAP) would be a
plausible explanation for this behavior. Yet in addition to a FLAP
inhibitor assay there is no impairment of potency, when analyzing
5-LO product formation with varying AA concentrations or 5-LO
stimuli. Furthermore, ST-1853 and ST-1906 could, like the untypi-
cal 5-LO inhibitor Hyperforin, interfere with the membrane bind-
ing C2-like domain [15]. However, unlike Hyperforin, the potency
Conflict of interests
The authors declare no conflict of interest.
Acknowledgements
We thank Anja Vogel, Astrid Brüggerhoff, and Sven George for
expert technical assistance, Michael Hörnig for mutation of r5-
LO, and Olof Rådmark for the pT3-5-LO plasmid. The study was
supported by Else Kröner-Fresenius-Stiftung (TRIP, Dr. Hans Krön
er-Graduiertenkolleg), LOEWE OSF, Fonds der Chemischen Indus-
trie, LOEWE TMP and Fraunhofer-Projektgruppe für Translationale
Medizin und Pharmakologie (TMP), Deutsche Forschungsgemein-
schaft DFG (SFB 1039, Sachbeihilfe MA-5825/1-1, and INST
208/664-1) and the European COST Actions CA15135 and CM1207.
of ST-1853 is not impaired with the presence of 20 lg/ml phos-
phatidylcholine, indicating a different mechanism of action. Both
lead compounds bear the 2,6-dimethyl substitution pattern which
may add inductive effects to enhance reductive properties of the
compound. As shown for acetaminophen derivatives this substitu-
tion pattern does not impair redox cycling [35]. Yet potentially
toxic protein modification by the oxidized form or certain phase
II metabolism (in this study shown for glucuronidation) can be
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