Structure-based discovery of inhibitors of microsomal prostaglandin E 2 synthase-1, 5-lipoxygenase and 5-lipoxygenase-activating protein: Promising hits for the development of new anti-inflammatory agents

Article abstract of DOI:10.1021/jm101238d

Microsomal prostaglandin E₂ synthase (mPGES)-1 catalyzes the transformation of PGH₂ to PGE₂ that is involved in several pathologies like fever, pain, and inflammatory disorders. To identify novel mPGES-1 inhibitors, we used in silico screening to rapidly direct the synthesis, based on the copper-catalyzed 3 + 2 Huisgen's reaction (click chemistry), of potential inhibitors. We designed 26 new triazole-based compounds in accordance with the pocket binding requirements of human mPGES-1. Docking results, in agreement with ligand efficiency values, suggested the synthesis of 15 compounds that at least in theory were shown to be more efficient in inhibiting mPGES-1. Biological evaluation of these selected compounds has disclosed three new potential anti-inflammatory drugs: (I) compound 4 displaying selectivity for mPGES-1 with an IC₅₀ value of 3.2 μ-M, (II) compound 20 that dually inhibits 5-lipoxygenase and mPGES-1, and (III) compound 7 apparently acting as 5-lipoxygenase-activating protein inhibitor (IC₅₀ = 0.4 μ-M).

Full text of DOI:10.1021/jm101238d

ARTICLE
pubs.acs.org/jmc
Structure-Based Discovery of Inhibitors of Microsomal Prostaglandin
E2 Synthase-1, 5-Lipoxygenase and 5-Lipoxygenase-Activating
Protein: Promising Hits for the Development of New
Anti-inflammatory Agents
Rosa De Simone,† Maria Giovanna Chini,† Ines Bruno,*,† Raffaele Riccio,† Daniela Mueller,‡ Oliver Werz,‡
and Giuseppe Bifulco*,†
†Department of Pharmaceutical Sciences, University of Salerno, Via Ponte Don Melillo, 84084 Fisciano (SA), Italy
‡Department of Pharmaceutical Analytics, Pharmaceutical Institute, University of Tuebingen, Auf der Morgenstelle 8,
D-72076 Tuebingen, Germany
S Supporting Information
b
ABSTRACT: Microsomal prostaglandin E2 synthase (mPGES)-1
catalyzes the transformation of PGH2 to PGE2 that is involved in
several pathologies like fever, pain, and inflammatory disorders. To
identify novel mPGES-1 inhibitors, we used in silico screening to
rapidly direct the synthesis, based on the copper-catalyzed 3 þ 2
Huisgen's reaction (click chemistry), of potential inhibitors. We
designed 26 new triazole-based compounds in accordance with the
pocket binding requirements of human mPGES-1. Docking results,
in agreement with ligand efficiency values, suggested the synthesis of
15 compounds that at least in theory were shown to be more
efficient in inhibiting mPGES-1. Biological evaluation of these
selected compounds has disclosed three new potential anti-inflam-
matory drugs: (I) compound 4 displaying selectivity for mPGES-1
with an IC50 value of 3.2 μM, (II) compound 20 that dually
inhibits 5-lipoxygenase and mPGES-1, and (III) compound 7
apparently acting as 5-lipoxygenase-activating protein inhibitor
(IC50 = 0.4 μM).
’ INTRODUCTION
Recently, great attention has been focused on the microsomal
prostaglandin E2 synthase (mPGES)-1 enzymeresponsible for the
conversion of the COX-derived unstable peroxide PGH2 into
PGE2; this enzyme is overexpressed in several inflammatory
disorders5 as well as in many human tumors.6-8 Inhibition of
microsomal prostaglandin E2 synthase-1 (mPGES-1) has been
proposed as a more promising approach for the development of
safer drugs in inflammatory disorders,9,10 devoid of classical
NSAID side effects, as this inducible enzyme affects the biosynth-
esis of massive PGE2 generation as a response to inflammatory
stimuli.11 mPGES-1 is a glutathione (GSH)-dependent trans-
membrane enzyme belonging to the MAPEG (membrane-asso-
ciated proteins involved in eicosanoid and glutathione metabolism)
family. This protein family consists of membrane-bound proteins,
with diverse functions, like leukotriene C4 synthase (LTCS),
microsomal glutathione transferase-1 (MGST-1), and 5-lipoxy-
genase-activating protein (FLAP). Among the three isoforms so
far identified for prostaglandin E synthase (PGES), it is mPGES-1,
Nonsteroidal anti-inflammatory drugs (NSAIDs) represent so
far the pivot of inflammation therapy as a consequence of their
potent effect in the suppression of prostaglandins (PGs), pro-
minent bioactive mediators involved in key physiological
functions1 and also implicated in several pathologic conditions
like inflammation and tumorigenesis.2 However, especially for
long-term treatments—like those required for chronic patholo-
gies such as rheumatoid arthritis—their use comprises severe
side effects; in particular, NSAIDs are well-known to be endowed
with relevant gastric toxicity3 due to the efficient suppression of
constitutively generated PGE2 involving the cyclooxygenase
(COX)-1 pathway with gastro-protection functions. Not long
ago, the introduction of coxibs in therapy was initially considered
as a solution of all of the problems connected with the use of
NSAIDs, as these selective COX-2 inhibitors were shown to
exhibit potent anti-inflammatory activity without causing signifi-
cant gastrointestinal injury. Unfortunately, various clinical evi-
dence indicated their implication in serious cardiovascular
accidents.4 In this perspective, there is an ever growing need
for the research of safer anti-inflammatory drugs.
Received: July 22, 2010
Published: February 16, 2011
r
2011 American Chemical Society
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Chart 1. Chemical Structures of Compounds 1-26 Utilized for Molecular Docking Studies
functionally coupled with COX-2, that seems to be the isoform
primarily involved in pathologies.11
in literature; therefore, the discovery of potent inhibitors of this
interesting target would be of great relevance for the develop-
ment of a new generation of anti-inflammatory agents with
potentially safer profiles.
By means of an in silico screening, we describe here the
development of fast synthetically accessible triazole-based com-
pounds,13-19 representing innovative scaffolds in this area as
potential mPGES-1 inhibitors. In the course of our screening,
we discovered a very promising dual inhibitor of mPGES-1
and 5-LO, and we identified other compounds of interest that
may provide precious guidelines for the drug development
process.
Even though Jegersch€old et al.12 have recently elucidated the
electron crystallographic structure of closed conformation of
mPGES-1, the open form of the protein constitutes a model for
the productive enzyme. Therefore, the absence of the three-
dimensional (3D) X-ray crystal structure of the open mPGES-1
conformation with a substrate or an inhibitor bound has repre-
sented a major difficulty for the rational design of new specific
inhibitors, making the classical receptor-based approach quite
challenging. In fact, despite many efforts spent in this area, only
very few effective in vivo mPGES-1 inhibitors have been reported
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’ RESULTS AND DISCUSSION
structure solved by Holm et al. in 200634 in which significant
amino acid conservation in comparison to mPGES-135 (38% of
homology sequence) can be recognized. Recently, the structure-
based drug design targeting mPGES-1 was facilitated by the work
of Hamza et al.,29,30 who have described the PGH2 binding to the
mPGES-1-GSH complex. More precisely, as also demonstrated
by site direct mutagenesis, the natural ligand at the interface of
Molecular Docking Studies. The lack of a 3D X-ray crystal
structure of open mPGES-1 conformation has stimulated many
efforts for identifying the key characteristics of mPGES-1 in-
hibitors, based on quantum mechanic (QM) calculations,20
structure-activity relationship (SAR)21,22 and 3D quantitative
structure-activity relationship (3D-QSAR) analysis,23-26 multi-
step ligand-based strategy,27 high-throughput screening (HTS),28
molecular modeling and dynamics simulation,29 and site-direct
mutagenesis studies.30 As reported by Friesen et al.,10 these efforts
have led to the identification of several classes of mPGES-1 inhib-
itors: fatty acids and PGH2 analogues,31 indole and 43 (MK-886,
Chart 2) analogues,32 phenantrene imidazoles,28 nonacidic agents,27
and other inhibitors. Considering the well-known characteristic
of indole-based agents—the simultaneous contributions to the
inhibitory activity on mPGES-1 of hydrophobic and electrostatic
effects—and the ring size of fatty acids and PGH2 analogues as
starting point, we designed new triazole nucleus templates as
potential scaffolds for anti-inflammatory drugs. We designed a
small set of compounds (Chart 1) decorating a disubstituted
triazole ring, taking into account both the synthetic accessibility
and the compatibility of R1 and R2 groups with the binding
requirements of the pocket situated in the region at the interface
of the two mPGES-1 subunits. In particular, we gradually
increased the length, size, and hydro- and lipophilicity of R1
and R2 with the aim to optimize their chemophysical properties.
To identify the key structural features necessary for mPGES-1
inhibition, we performed an in silico screening by molecular
docking using AutoDock 3.0.5 software33 of a small set of
molecules. For our docking calculation, we used the MGST-1
Table 1. List of the Corresponding Amino Acids Present in
Both mPGES-1 and MGST-1 Catalytic Sites
mPGES-1
MGST-1
Arg110
Arg70
Arg113
Arg73
Asn74
Tyr117
Leu121
Arg126
His72
Asn77
Tyr120
Leu124
Arg129
His75
Lys26
Lys25
Glu66
His113
Arg73
Glu69
His116
Leu76
Pro125
Ala132
Arg72
Arg122
Thr129
Leu69
Ile125
Thr78
Tyr130
Asn128
Asn81
Phe133
Figure 1. Calculated affinities and ligand efficiency of compounds 1-26 for MGST-1.
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Figure 2. Three-dimensional model of interactions of 4 (A) and 20 (B) with the MGST-1 binding site. The protein is represented by secondary
structure, by CPK, and by lines colored by atom type (C, gray; polar H, sky blue; N, blue; and O, red). Compound 4 (A) is depicted by sticks and balls (by
atom type: C, yellow; O, red; and N, blue). Compound 20 (B) is depicted by sticks and balls (by atom type: C, blue; O, red; N, dark blue; and S, yellow).
Scheme 1. Synthetic Strategy for the Synthesis of Compounds 4, 6-8, 11, 14, 15, 17, 18, 20-22, 24, and 26a
a Reagents and conditions: (a) CuSO4, sodium ascorbate, H2O/t-BuOH 1:1, room temperature, overnight. (b) CuSO4, Cu(0), NaN3, t-BuOH/H2O
1:1, microwaves, 30 min. (c) CuI, 2,6-lutidine, CHCl3 dry, 12 h, 0 °C. (d) C6H5COCl, DMF dry, reflux, overnight. (e) RB(OH)2 (a-i), CsF,
Pd(dppf)Cl2, H2O/THF 1:1, microwaves, 20-30 min.
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Scheme 2. Synthetic Strategy for the Synthesis of Compound 23a
a Reagents and conditions: (a) CuI, DIPEA, NBS, THF, room temperature, 4 h. (b) CsF, Pd(dppf)Cl2, H2O/THF 1:1, microwaves, 30 min.
Table 2. Effect of Test Compounds on the Activity of mPGES-1a
cation-π interaction with Lys67, Arg72, and Arg196 for 4 and with
Lys67 and Arg196 for 20. The above in silico results suggested the
synthesis of the molecules 4, 6-8, 11, 14, 15, 17, 18, 20-24,
and 26, all within the lowest free energy of binding and a good
mPGES-1 activity
compound
IC 50 (μM)
remaining activity at 30 μM (%)
ligand efficiency (Ebinding < 9 kcal/mol and ΔG/NHA deeper
than -0.24 kcal/mol) as the starting point for obtaining
preliminary experimental results. The evaluation of the bioac-
tivity of this small set of compounds might be helpful for the
comprehension of the key features of new mPGES-1 triazole-
based inhibitors.
4
3.2
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
12.0 ( 3.7**
89.0 ( 2.9
60.1 ( 4.3**
78.2 ( 12.8
76.1 ( 7.9
73.6 ( 8.0
96.8 ( 0.5
91.9 ( 8.1
87.9 ( 8.4
59.8 ( 7.2**
85.0 ( 9.3
90.0 ( 6.8
72.1 ( 4.2*
98.2 ( 7.3
88.7 ( 2.7
6
7
8
11
14
15
17
18
20
21
22
23
24
26
Chemistry. For the synthesis of compounds 4, 6-8, 11, 14,
15, 17, 18, 20-22, 24, and 26, we utilized the synthetic procedure
outlined in Scheme 1. Except for 11, we took advantage of 1,3-
dipolar cycloaddition reaction (click reaction) to generate the
triazole intermediates 30, 31, 35-37, and 39 that were succes-
sively subjected to the Suzuki cross-coupling reaction with the
appropriate commercially available boronic acids a-i. The tria-
zoles intermediates were generated through the condensation
between the appropriate terminal alkynes and the azides. In more
detail, when we started from the commercially available azide 27,
the traditional protocol for 1,3-dipolar cycloaddition at room
temperature for 18 h in the presence of CuSO4 as catalyst and
sodium ascorbate in a mixture of tert-butanol/water (t-BuOH/
H2O) 1:1 was used (Scheme 1a).40 On the contrary, when the
azides were generated in situ with sodium azide starting from the
corresponding halides, the microwave irradiation technique pro-
vided a faster way to obtain the desired triazole intermediates 35-
37 (Scheme 1b) in a one-pot reaction.41
The synthesis of intermediate 39 required a different proce-
dure because of the presence of the sulfonyl functionality, which
is a strong electron-withdrawing group that could negatively
affect the reaction outcome and favor a rapid conversion of the
Cu-containing transition state into a variety of byproduct rather
than the desired cycloadduct.42,43 However, we were able to
obtain the desired sulfonyl triazole intermediate 39, carrying out
the cycloaddition between 1-bromo-4-ethynylbenzene 29 and
4-carboxybenzenesulfonazide 38 in dry chloroform at 0 °C in
presence of 2,6-lutidine as base (Scheme 1c).44
The triazole intermediates 31, 35-37, and 39 were finally
subjected to the Suzuki cross-coupling reaction with the appro-
priate boronic acids a-i following the experimental conditions
previously optimized by us,45 providing for the use of [1,10-bis-
(diphenylphosphino)ferrocene]dichloropalladium(II) (Pd(dppf)-
Cl2) as a catalyst and CsF as a base in a mixture of tetrahydrofur-
an/water (THF/H2O) 1:1, under microwave irradiation; the
desired final compounds 4, 6-8, 14, 15, 17, 18, 20-22, 24, and
26 (Chart 1) were obtained in satisfactory yield. Compound 11
was instead obtained submitting the triazole 30 to direct acyla-
tion with benzoyl chloride in N,N-dimethylformamide (DMF) as
the solvent (Scheme 1).
a Data are given as means ( SEs, n = 4-6. *p < 0.05, and **p < 0.01.
each mPGES-1 monomer establishes a strong salt bridge be-
tween its carboxylate group and the highly conserved Arg110 in
the MAPEG family and interacts with Arg70, Asn74, Arg73,
Glu77, Tyr117, Leu121, Arg122, Arg126, Thr129, Arg110,
His72, Lys26, Leu69, and Ile125. Taking into account the
considerations above, we referred to the sequence alignment of
these two MAPEG super family members for the rationalization
of the small molecules binding mode (Table 1).29 The theoretical
affinities of compounds 1-26 calculated by docking, reported as
the most favorable MGST-1 free energy of binding together with
the ligand efficiency36-39 (binding energy for heavy atom mole-
cular ΔG/NHA), are shown in Figure 1.
The data shown in Figure 1 indicate the best calculated
affinities for compounds presenting one H-bond acceptor group
and a lipophilic substituent of adequate dimensions.39 For the
sake of simplicity, we report the most promising candidates
derived from the in silico screening, 4 and 20 (Figure 2), to trace
the features of new potential anti-inflammatory drugs.
From the analysis of the results, both compounds disclosed a
similar binding mode at the interface of the target monomer. Our
proposed poses are in agreement with the model reported by
Hamza et al.29 In fact, the compounds interact with residues
considered critical for PGH2 binding, such as the hydrogen
bonds with the carboxy group in 4 and 20 with the highly
conserved Arg113 in MGST-1 (Arg110mPGES-1), guaranteeing, at
least in theory, the enzyme binding specificity, as well as van der
Waals and other interactions with residues of the active site—the
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Figure 3. Inhibition of mPGES-1. (A) Concentration-response curves of compounds 4 and 20 for inhibition of mPGES-1 activity in microsomal
preparations of IL-1β-stimulated A549 cells. (B) Reversibility of mPGES-1 inhibition by compound 4. Wash-out experiments were performed as
described in the text. Data are given as means ( SEs, n = 4-5; **p < 0.01.
Finally, to synthesize compound 23, we took advantage of a
multicomponent one-pot reaction between phenyl-1-propyne
40 and azidomethyl phenyl sulfide 27 in the presence of CuI-
NBS (N-bromosuccinimide) and diisopropylethylamine (DIP-
EA); this step provided the desired 1,4,5 trisubstitued-1,2,3-
triazole 41 bearing an iodine atom at the C-5 position (Scheme 2).46
In the last step, the triazole intermediate 41 was subjected to the
Suzuki cross-coupling reaction with 4-formyl-phenyl-boronic
acid 42, affording compound 23 in good yield.
(Figure 3A) revealed an IC50 value of 3.2 μM and indicated an
almost complete inhibition of mPGES-1 activity at 30 μM.
In contrast to 4, compound 20 failed to entirely suppress
mPGES-1 activity, and the concentration-response curve see-
mingly reached a plateau with maximum inhibition of approxi-
mately 40% at the highest concentration (Figure 3A). Similarly,
for compounds 7 and 23, the maximal inhibition at 30 μM was
less than 40%, and thus, IC50 could not be obtained. Wash-out
experiments suggest a reversible mode of action of compound 4,
as 10-fold dilution of the incubation mixture reversed mPGES-1
inhibition by 4 (Figure 3B).
Previous studies on acidic mPGES-1 inhibitors showed that
such compounds often interact also with other enzymes within
the arachidonic acid cascade, such as 5-LO or FLAP. In fact,
interference with 5-LO or FLAP, the key enzymes in the for-
mation of leukotrienes (LTs) from arachidonic acid, is consid-
ered a valuable characteristic of a given mPGES-1 inhibitor,
because dual suppression of PGE2 and LT formation might be
superior over single interference in terms of higher anti-inflam-
matory efficacy as well as in terms of reduced side effects.48 Thus,
we further analyzed the test compounds (30 μM, each) for
inhibition of 5-LO activity in a cell-free assay using isolated
human recombinant 5-LO as the enzyme source. The well-
recognized 5-LO inhibitor (E)-N-hydroxy-N-(3-(3-phenoxy-
phenyl)-allyl)acetamide (BWA4C)49 was used as a positive
control, and DMSO (0.3%, v/v) was used as a vehicle control.
Intriguingly, among the test compounds, 20 was the most active
derivative with IC50 = 0.8 μM, followed by 21, 4, 17, and 7
(Table 3, IC50 = 4.1, 6.7, 8.8, and 27 μM, respectively), which all
inhibited 5-LO activity in a concentration-dependent manner
(Figure 4A). 5-LO inhibition was reversible, as demonstrated by
wash-out experiments (not shown). Also, 6, 14, 23, and 26
significantly inhibited 5-LO at a concentration of 30 μM, but the
Before submitting the test compounds 4, 6-8, 14, 15, 17, 18,
20-24, and 26 synthetically obtained to the biological assays,
their purities (>95%) were verified by Agilent Technologies 1200
series high-performance liquid chromatography (HPLC) with
ultraviolet (UV) detection at 280 nm (method: Jupiter C-18
column, 250 mm ꢀ 4.60 mm, 5 μm, 300 Å; 1.0 mL/min flow rate;
5-100% in 30 min of 0.1% TFA/CH3CN-0.1% TFA/H2O).
Analysis of the Bioactivity. To assess the ability of the
selected compounds 4, 6-8, 11, 14, 15, 17, 18, 20-24, and
26 to interfere with the activity of mPGES-1, a cell-free assay
using the microsomal fractions of interleukin-1β (IL-1β)-stimu-
lated A549 cells (as source for mPGES-1) was applied. In a first
screening round, all compounds, solubilized in dimethyl sulf-
oxide (DMSO), were tested at concentrations of 30 μM. Because
of the limited solubility of the test compounds in aqueous assay
buffers, concentrations >30 μM could not be tested. The
mPGES-1 inhibitor compound 43 (IC50 = 2.4 μM)47 was used
as a reference control, and DMSO (0.3%, v/v) was used as a
vehicle control. As shown in Table 2, compounds 4, 7, 20, and 23
significantly inhibited mPGES-1 activity, whereas all other
derivatives were not significantly active at a concentration of
30 μM. Interestingly, these data confirm the results from the
docking studies favoring 4 and 20 as mPGES-1 inhibitors. More
detailed analysis of 4 in concentration-response studies
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Figure 4. Inhibition of 5-LO. (A) Concentration-response studies for purified recombinant 5-LO. (B) Concentration-response studies for 5-LO
product formation in intact human neutrophils. Data are given as means ( SEs, n = 4-5.
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Table 3. Effect of Test Compounds on the Activity of 5-LO in Cell-Free and Cell-Based (Intact Neutrophils) Assaysa
5-LO activity; cell-free
remaining activity at 30 μM (%)
5-LO activity; intact neutrophils
compound
IC 50 (μM)
IC 50 (μM)
remaining activity at 30 μM (%)
4
6.7
>30
27
20.0 ( 0.9**
62.3 ( 1.4**
48.8 ( 0.4**
82.9 ( 0.9
9.2
0.9
20.1 ( 11.0**
34.8 ( 7.0**b
1.1 ( 0.3**b
85.7 ( 3.5
6
7
0.4
8
>30
>30
>30
>30
8.8
>30
9.3
11
14
15
17
18
20
21
22
23
24
26
80.8 ( 5.3
14.6 ( 5.2**
70.2 ( 8.5
58.4 ( 12.7*
77.4 ( 0.9
>30
>30
>30
>30
0.6
79.9 ( 10.7
52.7 ( 15.0*
92.1 ( 8.1
3.5 ( 2.5**b
21.2 ( 4.3**b
84.5 ( 3.4
17.3 ( 2.5**b
22.0 ( 2.6**b
70.1 ( 9.0
10.1 ( 4.6**
82.5 ( 4.4
>30
0.8
13.6 ( 2.8**b
5.1 ( 0.8**
78.4 ( 10.3
57.3 ( 1.2**
60.7 ( 10.0
59.2 ( 6.4*
4.1
2.8
>30
>30
>30
>30
>30
6.0
1.7
>30
a Data are given as means ( SEs, n = 4-6. *p < 0.05, and **p < 0.01. b Remaining activity at 10 μM.
Chart 2. Chemical Structures of 43 and 44
magnitude of inhibition did not exceed 50% (Table 3), and thus,
IC50 values could not be determined.
Because FLAP inhibitors do not inhibit 5-LO activity in cell-free
assays but only LT formation in intact cells,50 we assessed a
potential inhibitory effect on FLAP in human neutrophils activated
by ionophore A23187. Compound 43 (IC50 for FLAP in neutro-
phils approximately 70 nM)51 served as the control, and DMSO
(0.3%, v/v) was used as the vehicle control. Compounds 4, 6, 7,
11, 20, 21, 23, and 24 reduced 5-LO product formation at 30 μM
by more than 50% in a concentration-related manner with IC50
values in the range of 0.4-9.3 μM (Table 3 and Figure 4B). For 4
and 20, the IC50 values were determined at 8.8 and 0.6 μM,
respectively, which fits well with the activities in cell-free 5-LO
assays, and also, 21 was similarly efficient (IC50 = 2.8 μM) as for
isolated 5-LO.
A remarkable and concentration-dependent suppression of
cellular 5-LO product synthesis was found for 7 with IC50 = 0.4
μM and also for 6 and 24 (IC50 = 0.9 and 1.7 μM, respectively),
although these compounds hardly inhibited 5-LO in the cell-free
assay. This suggests that for suppression of 5-LO product forma-
tion in intact cells, 6, 7, and 24 may primarily act at other targets
than the 5-LO enzyme, presumably on FLAP. Such mechanisms
may also be attributed to compounds 11 and 23, although with
lower potencies (IC50 = 9.3 and 6 μM, respectively).
All in all, on the basis of the outcomes of the biological activity
data, 4 is the most efficient inhibitor of mPGES-1, 7 might act as a
FLAP inhibitor, while 20 might be a potent direct 5-LO inhibitor,
besides moderate inhibition of mPGES-1. Hence, we aimed to
rationalize the results through molecular modeling studies. As
preliminarily remarked, it should be put in evidence that com-
pounds 4 and 7, inhibiting the two MAPEG family members,
showed quite similar chemical features; on the contrary, the more
encumbering ligand 20 seems to target no structurally related
MAPEG enzymes. For our calculations, we used the 3D structure
of FLAP in complex with the inhibitor 44 (MK-591, Chart 2)52
solved by Ferguson et al.53 in 2007 [protein data bank (PDB) ID
code 2Q7M]. Because of the lack of crystal structure information
on 5-LO, we used a 15-LO54 (PDB ID code 1LOX) enzyme,
Figure 5. Three-dimensional model of interactions between 7 and
FLAP. The protein is represented by secondary structure, by CPK, and
by lines colored by atom type (by atom type: C, gray; polar H, sky blue;
N, blue; O, red; and S, yellow). Compound 7 is depicted by sticks and
balls (by atom type: C, sky blue; O, red; N, dark blue; S, yellow; and H,
white). Compound 44 is depicted by sticks and balls (C, O, N, S, and Cl
dark pink). The figure highlights similar interactions for both 7 and 44
with arachidonic acid-binding site.
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Figure 6. Three-dimensional model of interactions of 20 and 15-lipoxygenase enzyme active site. The protein is represented by CPK and by lines
colored by atom type (by atom type: C, gray; polar H, sky blue; N, blue; O, red; and S, yellow). Two different conformations (A and B) of the complex are
depicted. Compound 20 is depicted by sticks and balls (by atom type: C, green; O, red; N, dark blue; and S, yellow).
presenting the highest sequence similarity (38% identity with
human 5-LO; see the Supporting Information) among the
dioxygenase family (8-, 9-, 11-, and 12-LO).
most promising mPGES-1 inhibitor 4. In light of the good
qualitative accordance between the results from the biological
assays and the prediction of the molecular docking calculations, a
satisfactory explanation of the putative binding mode for the new
triazole based compounds was provided. Biological assays dis-
closed three different benchmark compounds 4, 7, and 20 as
inhibitors of mPGES-1, FLAP, and 5-LO, respectively. The
future perspectives, in fact, regard the decoration of the triazole
ring with more polar in nature R1 and R2, in order to increase
their solubility in the biological liquids. At the moment, the most
feasible improvement for the development of new mPGES-1
inhibitors consists of generating analogues of 4 with a R2-modi-
fied position. In particular, the fundamental biphenyl portion—
present in the three different compounds 4, 7, and 20—needs a
more appropriate substitution pattern to increase the interaction
efficiency with the polar aminoacids of the catalytic site (e.g.,
Thr33, Arg37, Lys67, and Arg129). In conclusion, our results
prove the efficiency of the triazole group as a new scaffold useful
in the rational design of new promising candidates as anti-
inflammatory drugs and shed light on the chemical decorations
functional for the design of further members belonging to this
new class of inhibitors.
Taking into account the considerations reported above for the
MGST-1 enzyme, also in the case of FLAP, the binding speci-
ficity was conferred by the H-bond with the Lys116. In our
proposed pose, 7 (Figure 5) not only interacts with the funda-
mental amino acids but also adopts the equivalent spatial
disposition of the cocrystallized inhibitor,54 maintaining the
same interactions network. Moreover, the phenyl group in R1
forms a π-π stacking with Phe25.
Three different classes of inhibitors can be generally consid-
ered for 5-LO:55 (1) antioxidant agents interfering with the redox
catalytic cycle of the enzyme, (2) iron-chelating agents, and (3)
nonredox type inhibitors, which compete with arachidonic acid
for the binding to the enzyme.50 In our docking studies, we
supposed that 20 acts as nonredox type LO inhibitor. As
described for mPGES-1, the rationalization of the 5-LO binding
mode was obtained considering the fundamental amino acids in
the active site of the enzyme as reported by Charlier et al.56 (see
the Supporting Information), taking into consideration the
specific polar interaction of the carboxylate moiety of arachidonic
acid with Lys4095-L0 (Arg40315-LO).
For compound 20, we obtained two different conformation
families, accounting for two independent high affinity binding
modes (Figure 6A,B). In both of the conformations, the specific
interaction with Arg403 was maintained. In particular, in the first
conformation (Figure 6A), the phenyl group in R1 shows a
cation-π interaction, while in the second conformation
(Figure 6B), the same cation-π interaction with Arg403 was
exerted by the naphtyl group in R2 present in the alternative
conformation. In the latter case, the oxygen atom in R2 forms an
additional H-bond with the positively charged (Arg403) residue.
Even if R1 and R2 are located on the opposite sites of the target
pocket, and the other interactions with the receptor counterpart
remain unmodified and are in accordance with the structural
requirements indicated by Charlier et al.,56 that is, two hydro-
phobic groups, an aromatic ring, and two hydrogen bond
acceptors.
’ ASSOCIATED CONTENT
S
Supporting Information. Docking calculations and their
b
detailed results; detailed procedure for the synthesis of com-
pounds 4, 6-8, 11, 14, 15, 17, 18, 20-24, and 26; HPLC
conditions and nuclear magnetic resonance (NMR) data for the
tested compounds; and protocol of the bioactivity assays. This
material is available free of charge via the Internet at http://pubs.
acs.org.
’ AUTHOR INFORMATION
Corresponding Author
*(I.B.) Tel: þ39 089 969743. Fax: þ39 089 969602. E-mail:
brunoin@unisa.it. (G.B.) Tel: þ39 089 969741. Fax: þ39 089
969602. E-mail: bifulco@unisa.it.
’ ACKNOWLEDGMENT
’ CONCLUSIONS
Financial support by the University of Salerno and by Minis-
tero dell'Istruzione, dell'Universitꢀa e della Ricerca (MIUR),
We have applied a rapid in silico screening on a small set of
triazole derivates for directing the click chemistry synthesis of the
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Journal of Medicinal Chemistry
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PRIN-06, and Aureliasan GmbH (Tuebingen, Germany) is
gratefully acknowledged.
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’ ABBREVIATIONS USED
GSH, tripeptide glutathione; MAPEG, membrane-associated
proteins in eicosanoid and glutathione metabolism; MGST-1,
microsomal glutathione transferase 1; mPGES-1, microsomal
prostaglandin E2 synthase; 5-LO, 5-lipoxygenase; FLAP, 5-lipoxy-
genase-activating protein; COX, cyclooxygenase; PG, prosta-
glandin; LT, leukotriene; LTCS, leukotrien C synthase; IL-1β,
interleukin 1β; PDB, protein data bank; NSAID, nonsteroi-
dal anti-inflammatory drug; QM, quantum mechanic; SAR,
structure-activity relationship; QSAR, quantitative structure-
activity relationship; HTS, high-throughput screening; DMF,
dimethylformamide; THF, tetrahydrofuran; DIPEA, N,N-diiso-
propylethylamine; DMSO, dimethylsulfoxide; NBS, N-bromo-
succinimide
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