Inhibition of secreted phospholipases A2 by 2-oxoamides based on α-amino acids: Synthesis, in vitro evaluation and molecular docking calculations

Article abstract of DOI:10.1016/j.bmc.2010.12.030

Group IIA secreted phospholipase A₂ (GIIA sPLA₂) is a member of the mammalian sPLA₂ enzyme family and is associated with various inflammatory conditions. In this study, the synthesis of 2-oxoamides based on α-amino acids a

Full text of DOI:10.1016/j.bmc.2010.12.030

Bioorganic & Medicinal Chemistry 19 (2011) 735–743
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Inhibition of secreted phospholipases A2 by 2-oxoamides based on a-amino
acids: Synthesis, in vitro evaluation and molecular docking calculations
Varnavas D. Mouchlis a, Victoria Magrioti a, Efrosini Barbayianni a, Nathan Cermak b, Rob C. Oslund b,
Thomas M. Mavromoustakos a, Michael H. Gelb b, George Kokotos a,
⇑
a Laboratory of Organic Chemistry, Department of Chemistry, University of Athens, Panepistimiopolis, Athens 15771, Greece
b Department of Chemistry and Biochemistry, University of Washington, Seattle, WA 98195, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Group IIA secreted phospholipase A2 (GIIA sPLA2) is a member of the mammalian sPLA2 enzyme family
and is associated with various inflammatory conditions. In this study, the synthesis of 2-oxoamides based
on a-amino acids and the in vitro evaluation against three secreted sPLA2s (GIIA, GV and GX) are described.
The long chain 2-oxoamide GK126 based on the amino acid (S)-leucine displayed inhibition of human and
mouse GIIA sPLA2s (IC50 300 nM and 180 nM, respectively). It also inhibited human GV sPLA2 with similar
potency, while it did not inhibit human GX sPLA2. The elucidation of the stereoelectronic characteristics that
affect the in vitro activity of these compounds was achieved by using a combination of simulated annealing
to sample low-energy conformations before the docking procedure, and molecular docking calculations.
Ó 2011 Elsevier Ltd. All rights reserved.
Received 20 September 2010
Revised 7 December 2010
Accepted 13 December 2010
Available online 16 December 2010
Keywords:
GOLD
Molecular docking
2-Oxoamides
Phospholipase A2
Simulated annealing
1. Introduction
enzyme reveals a highly conserved Ca2+-binding loop and a catalytic
dyad consisting of His47/Asp91.7,8 The substrate hydrolysis
Phospholipase A2 (PLA2) is a superfamily of enzymes which are
characterized by their ability to catalyze the hydrolysis of the sn-2
ester bond of glycerophospholipids releasing free fatty acids, includ-
ing arachidonic acid, and lysophospholipids.1 Arachidonic acid is
supplied to the downstream cyclooxygenases COX-1 and COX-2,
and to the 5-lipoxygenase for the production of eicosanoids,2 while
lysophospholipids, such as lysophosphatidic acid and lysophospha-
tidylcholine, and their metabolites, such as platelet activating factor,
are potent bioactive mediators, acting through their cognate G-pro-
tein-coupled receptors.
Cytosolic PLA2 (GIVA cPLA2) plays an important role in inflam-
mation. Various studies on transgenic mice lacking GIVA cPLA2
showed a 90% reduction in the production of prostaglandins and
leukotrienes.3,4 The biochemistry and the role of sPLA2 enzymes
have been recently reviewed by Lambeau and Gelb.5 The 10 known
mammalian sPLA2s are the groups: IB, IIA, IIC, IID, IIE, IIF, III, V, X
and XIIA. Among the members of the mammalian sPLA2 enzymes,
the GIIA sPLA2 is an interesting anti-inflammatory drug target be-
cause of its potential role in a number of different inflammatory
diseases.5
proceeds through the activation of a water molecule by the catalytic
histidine and subsequent attack of the sn-2 ester carbonyl carbon.
Besides this histidine, there is an aspartate residue (Asp48), which
together with the other residues of the Ca2+-binding loop (Gly29,
Gly31 and His27), act as a ligand cage for the calcium ion. The crystal
structure of GIIA sPLA2 enzyme has also defined a conserved active
site with a hydrophobic region lined near the N-terminal helix.7,8
Since PLA2 enzymes have been associated with various inflam-
matory diseases, the elucidation of their biological roles through
the use of small synthetic inhibitors is of high importance. Most re-
cently, Magrioti and Kokotos reviewed the various classes of PLA2
inhibitors discussing their potential role as new anti-inflammatory
agents.9 The design of novel GIIA sPLA2 inhibitors may be accom-
plished using computational methods which are greatly aided by
the availability of a number of GIIA sPLA2 crystal structures with
or without a ligand bound in the active site. Among the various
reported crystal structures of the enzyme deposited in the RCSB
protein data bank are the crystal structures with PDB IDs:
1DB4,10 1DB5,10 1J1A11 and 1KVO.12 However, the optimization
of the activity of the GIIA sPLA2 inhibitors, requires the better
understanding of the receptor–ligand interactions and the devel-
opment of a strategy to predict the inhibitory activity of new mol-
ecules. Techniques based on the field of computational chemistry
are very helpful in this case. Previous computational research
works on the GIIA sPLA2 inhibitors include molecular docking cal-
GIIA sPLA2 is a low molecularweight enzyme (14 kDa) with seven
disulfide bonds, and was cloned in 1989.6 The crystal structure of the
⇑
Corresponding author. Tel.: +30 2107274462; fax: +30 2107274761.
E-mail address: gkokotos@chem.uoa.gr (G. Kokotos).
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.12.030

736
V. D. Mouchlis et al. / Bioorg. Med. Chem. 19 (2011) 735–743
culations,13 molecular dynamics simulations (MD)14 and three-
dimensional quantitative structure–activity relationship (3D-
QSAR) methodologies.15,16 Among these techniques molecular
docking is a powerful technique, which contributes to the under-
standing of the stereoelectronic factors that affect the inhibitory
activity of small molecules against a target receptor.17–19
O
OH
( )13
O
a
b
H2N
HN
OMe
OMe
HN
O
2a
1a
Recently, a novel class of GIVA cPLA2 inhibitors, the 2-oxoamides
O
O
OH
( )13
O
(Fig. 1), has been developed.20–27 Long chain 2-oxoamides based on
c
HN
d- and
c-amino acids (m = 2 and m = 1, respectively) are potent
OH
OH
( )13
inhibitors of the GIVA cPLA2 enzyme showing interesting in vivo
anti-inflammatory and analgesic activity.21,25 Notably, such 2-
oxoamides containing a free carboxyl group do not inhibit the other
major intracellular PLA2 enzyme, the Ca+2-independent GVIA
iPLA2,24 although a cross-reactivity might be expected because both
GIVA cPLA2 and GVIA iPLA2 enzymes are serine hydrolases and
share a common catalytic mechanism. However, a few 2-oxoamides
displayed some inhibition, although weak, of GV sPLA2.25
O
GK126
O
3a
Scheme 1. Reagents and conditions: (a) CH3(CH2)13CHOHCOOH, Et3N, WSCI, HOBt,
CH2Cl2; (b) 1 N NaOH, dioxane/H2O 9:1; (c) NaOCl, AcNH-TEMPO, NaBr, NaHCO3,
EtOAc/PhCH3/H2O 3:3:0.5, 0 °C.
acid, respectively, inhibited GIIA sPLA2 in the micromolar range
(IC50 4.20–11.67 lM). At 16.6 lM concentration, none of them
The aim of this work was to identify 2-oxoamides able to inhibit
sPLA2s. In the present report, the synthesis of new 2-oxoamides
showed any significant inhibition of GV sPLA2 and GX sPLA2. It
seems that for the inhibition of GIIA sPLA2, the optimum distance
between the 2-oxoamide group and the carboxyl group corre-
sponds to one carbon atom. Increasing the length leads to less po-
tent inhibitors. Comparing the results for GK111 and AX115, it was
obvious that a free carboxyl group was necessary for the inhibition
of GIIA sPLA2. The introduction of a side chain corresponding to
leucine increased the inhibitory activity for GIIA sPLA2 by an order
of magnitude. GK126 inhibited GIIA sPLA2 with an IC50 value of
based on
a-amino acids and the in vitro evaluation against three
human sPLA2s (GIIA, GV and GX sPLA2) are described. To under-
stand the binding mode of 2-oxoamides to GIIA sPLA2, molecular
docking calculations were performed.
2. Results and discussion
2.1. Synthesis of inhibitors
0.30
vel (IC50 0.44
l
M. This 2-oxoamide also inhibited GV sPLA2 at the same le-
M), while it did not show any inhibition of GX sPLA2.
The synthesis of the new 2-oxoamides is depicted in Schemes 1
and 2. The methyl ester of (S)-leucine (1a) was coupled with
2-hydroxyhexadecanoic acid using 1-(3-dimethylaminopropyl)-3-
ethyl carbodiimide (WSCI) as a condensing agent in the presence
of 1-hydroxybenzotriazole (HOBt) (Scheme 1). After saponification
of compound 2a, oxidation of the 2-hydroxyamide 3a was carried
out by the NaOCl/AcNH-TEMPO method21 leading to the target
compound GK126. Starting from the ethyl ester of (R)-leucine
(1b), compound GK145, the enantiomer of GK126 was prepared.
Compound GK144 was synthesized starting from the ethyl ester
of (S)-leucine by coupling with hexadecanoic acid and subsequent
saponification.
Compounds GK111, GK112, GK122 and GK141 were prepared
by coupling 2-hydroxyhexadecanoic acid with tert-butyl glycinate
(4a), b-alaninate (4b), d-aminovalerate (4c) and (S)-phenylalani-
nate (4d), respectively (Scheme 2). Oxidation of 2-hydroxyamides
5a–d was carried out by the Dess–Martin method,28 followed by
treatment of compounds 6a–d with trifluoroacetic acid, to afford
the target compounds. The synthesis of AX115 is described
elsewhere.26
l
The inhibitory activity of GK126 was also measured against the
corresponding mouse enzymes. Similar IC50 values for mouse GIIA
sPLA2 and GV sPLA2 were found (0.18 lM and 2.60 lM, respec-
tively). Compound GK145, based on (R)-leucine, displayed six
times lower potency for GIIA sPLA2 and four times lower potency
for GV sPLA2, indicating that the (S)-configuration of the a-amino
acid makes more favorable contacts with the enzyme active site.
Replacement of either the 2-oxoamide functionality of GK126 by
an amide functionality (GK144) or the side chain by a chain corre-
sponding to phenylalanine (GK141) led to inactive compounds. The
most potent inhibitor against sPLA2 (GK126) did not present any
significant inhibition against cPLA2 (not higher than 12% inhibition
at 1 lV). Thus, we identified a 2-oxoamide based on the natural a-
amino acid leucine, which displayed submicromolar inhibition of
GIIA sPLA2.
2.3. Molecular docking calculations
The 2-oxoamide inhibitors were initially designed to interact
with the hydroxyl group of the active site serine of GIVA cPLA2.20
Recently, the location of the 2-oxoamide inhibitor AX007 within
the active site of GIVA cPLA2 was determined by a combination of
molecular dynamics and deuterium exchange mass spectrometry.30
However, the binding mode of 2-oxoamides with GIIA sPLA2 is not
obvious because GIIA sPLA2 is not a serine hydrolase and it utilizes a
different catalytic mechanism than GIVA cPLA2. Thus, to under-
stand how the 2-oxoamides interact with GIIA sPLA2, we decided
to perform molecular docking calculations. Four 2-oxoamide inhib-
2.2. In vitro inhibition of GIIA sPLA2, GV sPLA2 and GX sPLA2
The activity of compounds GK111, GK112, GK122, GK126,
GK141, GK144, GK145, and AX115 was studied against three
different human enzymes using a continuous fluorimetric assay
described previously.29 The results for GIIA sPLA2, GV sPLA2 and
GX sPLA2 are summarized in Table 1. 2-Oxoamides GK111,
GK112 and GK122, based on glycine, b-alanine and d-aminovaleric
itors based on
a-amino acids, GK126, GK145, GK111 and GK141,
were selected for this study. The molecular docking calculations
were performed using the genetic docking algorithm GOLD 4.1.31–33
The active site of GIIA sPLA2 consists of a hydrophilic region and
a hydrophobic region. The hydrophilic region, where catalytic
activity occurs, is formed by the residues His47 and Asp91, and
by the Ca2+-binding loop. The Ca2+-binding loop consists of the
residues Gly29, Gly31, His27 and Asp48. The calcium ion is
O
O
H
N
( )n
( )m
OH
R
O
n = 8-12, m = 1,2, R = short alkyl chain
Figure 1. General structure of 2-oxoamide inhibitors of GIVA cPLA2.

V. D. Mouchlis et al. / Bioorg. Med. Chem. 19 (2011) 735–743
737
OH
( )13
a
b
( )n
( )n
H2N
OBut
HN
OBut
R
O
O
4a-d
O
R
5a d
-
O
O
( )n
( )n
OBut
HN
OH
c
HN
( )13
( )13
O
R
O
O
R
O
6a-d
4 6
-
R
n
GK111,
R = H, n = 0
a
b
c
d
H
0
1
3
GK112, R = H, n = 1
H
H
GK122,
R = H, n = 3
GK141, R = CH2C6H5, n = 0
CH2C6H5
0
Scheme 2. Reagents and conditions: (a) CH3(CH2)13CHOHCOOH, Et3N, WSCI, HOBt, CH2Cl2; (b) Dess–Martin reagent, CH2Cl2; (c) 50% TFA/CH2Cl2.
Table 1
In vitro inhibition of the enzymatic activity of human GIIA, GV and GX sPLA2s by 2-oxoamides
Code
Structure
IC50 (lM)
hGIIA sPLA2
4.20 0.20
hGV sPLA2
hGX sPLA2
O
O
H
N
GK111
GK112
GK122
AX115
43%a
23%a
18%a
7%a
( )13
OH
O
O
H
N
OH
5.60 0.25
11.67 0.50
3%a
47%a
49%a
9%a
( )13
O
O
O
O
O
O
H
N
OH
( )13
O
O
O
O
H
N
22%a
( )13
OMe
OH
O
H
N
( )13
0.30 0.06
0.44 0.04
50%a
GK126
GK145
GK144
(0.18 0.04)b
(2.60 0.33)c
(58%a)d
O
O
O
H
N
( )13
OH
OH
OH
1.75 0.07
1.50 0.23
>1.66
>1.66
O
O
O
H
N
( )13
>1.66
>1.66
O
O
H
N
( )13
GK141
>1.66
>1.66
>1.66
a
b
c
At 16.6 lM concentration.
For mouse GIIA sPLA2.
For mouse GV sPLA2.
For mouse GX sPLA2.
d
hepta-coordinated with a pentagonal bipyramidal geometry pro-
viding two binding sites for the substrate or the inhibitor.34 The
hydrophobic region, which binds the fatty acid tails of the sub-
strate, is formed by aliphatic and aromatic residues within or
closed to the N-terminal helix, including Leu2, Phe5, Ile9, Ala17,
Ala18, Tyr21 and Phe98.

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V. D. Mouchlis et al. / Bioorg. Med. Chem. 19 (2011) 735–743
Four GIIA sPLA2 inhibitor X-ray structures have been selected in
lowest GOLDscore Fitness was obtained for inhibitor GK141, which
also showed the lowest inhibition against GIIA sPLA2.
order to test if GOLD 4.1 is able to reproduce experimental crystallo-
graphic data (see Table 1 in Supplementary data).10–12 For each
inhibitor, the best score pose has been selected to compare with
the crystallographic one. The RMSD values after the superimposi-
tion of the crystallographic conformation and the conformation
predicted by GOLD are smaller or equal to 1.0 Å, indicating that
the two conformations are almost identical. Thus, the main inter-
actions of each inhibitor with the GIIA sPLA2 active site, reported
Using GOLD, the inhibitor–enzyme complexes were calculated
and, it was possible to understand the main stereoelectronic char-
acteristics that affect the inhibition of these compounds. Figure 2
presents the binding of the inhibitor GK126 in the GIIA sPLA2 ac-
tive site. The 2-oxoamide group participates in two hydrogen
bonds with Gly29. The 2-carbonyl group participates in a hydrogen
bond with the N–H of Gly29 (C@OÁ Á ÁH–N 2.50 Å, OÁ Á ÁN 3.30 Å) and
the N–H of the 2-oxoamide group forms a hydrogen bond with the
carbonyl group of Gly29 (N–HÁ Á ÁO@C 1.65 Å, NÁ Á ÁO 2.65). The 2-
carbonyl group also interacts with the calcium ion (NHCOCOÁ Á ÁCa2+
2.40 Å). The carboxylate group of the inhibitor interacts with the
calcium ion (COOÀÁ Á ÁCa2+ 2.87 Å) and is involved in a hydrogen
bond with Lys62 through a water molecule placed near the hydro-
philic region of the active site (OÁ Á ÁH–OH 1.94 Å, OÁ Á ÁO 2.90 Å and
H2OÁ Á ÁH–N 1.89 Å, OÁ Á ÁN 2.89 Å). The side chain of (S)-leucine is in
a suitable orientation to interact with Leu2 of the active site in ali-
phatic/aliphatic interactions. The long aliphatic 2-oxoacyl chain is
accommodated in the hydrophobic region of the active site and
participates in aliphatic/aliphatic and in aliphatic/aromatic inter-
actions with residues Val3, Phe5, His6, Ile9, Ala17 and Phe98.
As aforementioned, the inhibitor GK145 is the (R)-enantiomer
of the inhibitor GK126. According to the GK145-GIIA sPLA2
complex calculated by GOLD (Fig. 3) the amide carbonyl of the 2-
oxoamide group interacts with the calcium ion (NHCOÁ Á ÁCa2+
2.30 Å). With regards to the inhibitor GK145, the 2-oxoamide
group is flipped in comparison with that of GK126. As a result,
the two hydrogen bonds with Gly29, in which participates the 2-
oxoamide functionality of GK126, are not observed upon binding
of GK145. This may be the reason that the inhibitor GK145 gener-
ates a lower GOLDscore Fitness and a higher IC50 value against GIIA
sPLA2. On the other hand, the carboxylate group of the inhibitor
interacts with the calcium ion (COOÀÁ Á ÁCa2+ 2.65 Å), but does not
participate in the hydrogen bond with Lys62 through the water
molecule. Instead of the hydrogen bond with Lys62, a hydrogen
bond of the carboxylate group with a water molecule near the
hydrophilic region of the active site is observed. The side chain of
(R)-leucine is placed near Leu2 of the active site and interacts in
a similar mode as the (S)-leucine of inhibitor GK126. The long ali-
phatic 2-oxoacyl chain is accommodated in the hydrophobic region
and interacts with the residues Val3, Phe5, His6, Ile9, Ala17 and
Phe98 in a similar mode as the one of the enantiomeric GK126.
in the literature,10–12 were reproduced by GOLD
.
Six inhibitors (Table 2) have been chosen for the molecular
docking studies since they possess a wide variety of biological
activity. The high flexibility of the molecules imposed the use of
a conformational sampling in an attempt to obtain low-energy
conformers and to avoid false positives.35 For this purpose the sim-
ulated annealing module in the SYBYL 8.0 molecular modeling pack-
age36 was used in order to generate 100 annealed structures for
each inhibitor, before the docking procedure. The annealed struc-
tures have subsequently been docked in the enzyme active site
using GOLD. For each inhibitor the best score pose was chosen as
the one that represents better the putative bioactive conformation.
The docking results are summarized in Table 2. A good correlation
between the IC50 values and the GOLDscore Fitness values
(r2 = 0.798, N = 5) was observed. Five inhibitors with an experimen-
tally determined IC50 value have been used for the correlation pro-
cedure. However, the present study has focused on 2-oxoamides
which are based on
a-amino acids (GK126, GK145, GK111 and
GK141). The most active inhibitor GK126 is scored with the highest
GOLDscore Fitness. The inhibitor GK145, which is the (R)-enantio-
mer of GK126, shows both a higher IC50 value against GIIA sPLA2
and a lower GOLDscore Fitness than the inhibitor GK126. The
GK111 inhibitor displays a higher IC50 value and a lower GOLD-
score Fitness than those of the inhibitors GK126 and GK145. The
Table 2
The GOLDscore Fitness for the GIIA sPLA2 2-oxoamide inhibitors based on the
amino acids
a-
Compound
Structure
O
IC50
(
l
V)
Gsc. Fit.
O
O
O
H
N
GK126
(S)-Leu
( )13
OH
OH
OH
0.30 0.06
1.75 0.07
85.91
The inhibitor GK111 is based on the a-amino acid glycine. The
binding of GK111 in the GIIA sPLA2 active site (Fig. 4) reveals an
O
O
H
N
( )13
GK145
(R)-Leu
84.91
O
O
H
N
GK111
Gly
4.20 0.20
5.60 0.25
11.67 0.50
78.89
77.33
75.69
( )13
O
O
O
O
O
H
N
OH
GK112
GK122
( )13
O
O
H
N
OH
( )13
O
O
O
H
N
( )13
OH
GK141
(S)-Phe
>1.66
73.09
Figure 2. The binding mode of the inhibitor GK126 in the GIIA sPLA2 active site as
calculated using GOLD
.

V. D. Mouchlis et al. / Bioorg. Med. Chem. 19 (2011) 735–743
739
Figure 3. The binding mode of the inhibitor GK145 in the GIIA sPLA2 active site as
calculated using GOLD
Figure 5. The binding mode of the inhibitor GK141 in the GIIA sPLA2 active site as
calculated using GOLD
.
.
interaction of the 2-carbonyl group of the inhibitor with the cal-
cium ion (NHCOCOÁ Á ÁCa2+ 2.70 Å). The carboxylate group interacts
with the calcium ion (COOÀÁ Á ÁCa2+ 2.90 Å) and participates in two
hydrogen bonds with Gly31 (COOÀÁ Á ÁH–N 2.40 Å, OÁ Á ÁN 3.40 Å) and
Lys62 through a water molecule (OÁ Á ÁH–OH 1.91 Å, OÁ Á ÁO 2.89 Å
and H2OÁ Á ÁH–N 1.89 Å, OÁ Á ÁN 2.89 Å). In comparison with the bind-
ing of the inhibitor GK126, the two hydrogen bonds of the 2-oxoa-
mide group with Gly29 are not observed. The lack of the (S)-leucine
side chain, which interacts with Leu2 of the active site, also re-
duces the GOLDscore Fitness and increases the IC50 value against
GIIA sPLA2. The long aliphatic 2-oxoacyl chain interacts with the
residues Phe5, His6, Ile9, Ala17 and Phe98.
other hand, the region around the side chain of (S)-phenylalanine
seems to be narrow and perhaps the phenyl ring clashes with the
aforementioned residues; this may be the reason that GK141 gives
the lowest GOLDscore Fitness among the studied 2-oxoamide
inhibitors. The long aliphatic 2-oxoacyl chain participates in
aliphatic/aliphatic and aliphatic/aromatic interactions with resi-
dues including Phe5, His6, Ile9 and Phe98.
Based on the molecular docking studies, it was possible to
understand the structural characteristics that contribute in the
enhancement of the inhibitory activity of the 2-oxoamide GK126
(Fig. 6). The 2-oxoamide group is essential for the binding because
it participates in hydrogen bonds with Gly29 and interacts with the
calcium ion. The (R)-enantiomer displays a lower GOLDscore and a
higher IC50 value, because the 2-oxoamide functionality is flipped
and does not engage in hydrogen bonding with Gly29. The carbox-
ylate group is also essential for binding because interacts with the
calcium ion and participates in a hydrogen bond with Lys62
through a water molecule placed near the hydrophilic region of
the active site. The (S)-leucine side chain contributes to the tight
binding of GK126 by interacting with the side chain of the active
site Leu2. The inhibitor GK111 lacks the (S)-leucine side chain
and as a consequence its inhibitory activity is decreased. The long
2-oxoacyl aliphatic chain contributes to the tight binding by inter-
acting with the hydrophobic region of the active site (Val3, Phe5,
His6, Ile9, Ala17 and Phe98).
The binding of the inhibitor GK141 (Fig. 5), which is based on the
a-amino acid (S)-phenylalanine, indicates a hydrogen bond of the
N–H of the 2-oxoamide group with Gly29 (N–HÁ Á ÁO 1.97 Å, NÁ Á ÁO
2.98 Å). The 2-carbonyl group of the inhibitor interacts with the cal-
cium ion (NHCOCOÁ Á ÁCa2+ 2.60 Å). The carboxylate group interacts
with the calcium ion (COOÀÁ Á ÁCa2+ 2.64 Å) and participates in two
hydrogen bonds with Gyl31 (COOÀÁ Á ÁH–N 1.67 Å, OÁ Á ÁN 2.60 Å)
and with Lys62 through a water molecule (OÁ Á ÁH–OH 1.60 Å,
OÁ Á ÁO 2.56 Å and H2OÁ Á ÁH–N 1.89 Å, OÁ Á ÁN 2.89 Å). The 2-carbonyl
group of GK141 does not participate in the hydrogen bond with
Gly29 as the one of the inhibitor GK126. The side chain of (S)-Phe
seems to participate in aromatic/aliphatic and aromatic (p–p)
stacking interactions with Leu2, Phe5, Tyr51 and His47. On the
A variety of sPLA2 inhibitors have been reported in the litera-
ture.5,9,37 Gelb et al. have studied various inhibitors for their selec-
tivity over the complete set of human and mouse sPLA2s (groups I,
Figure 4. The binding mode of the inhibitor GK111 in the GIIA sPLA2 active site as
Figure 6. The pharmacophore segments of the inhibitor GK126 and their interac-
calculated using GOLD
.
tions with the surrounding amino acids and the calcium ion of the active site.

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V. D. Mouchlis et al. / Bioorg. Med. Chem. 19 (2011) 735–743
II, V, X, and XIIA sPLA2).38 They have reported a highly potent and
selective indole-based inhibitor of GX sPLA2, specific inhibitors for
GIIA and GIIE sPLA2 and a substituted 6,7-benzoindole that inhibits
nearly all human and mouse sPLA2s in the low nanomolar range.39
In recent years, interest in sPLA2 inhibitors has increased because
they seem to play an important role in the prevention of athero-
sclerotic cardiovascular disease.40,41 In the present work, we dem-
onstrate that a new 2-oxoamide is able to inhibit human GIIA
sPLA2 in the submicromolar range and the mode of its interaction
with the GIIA sPLA2 has been studied using molecular docking.
These data indicate that this compound constitutes a new lead
for the development of new GIIA sPLA2 inhibitors.
were added at 0 °C. The reaction mixture was stirred for 1 h at
0 °C and overnight at room temperature. The solvent was evapo-
rated under reduced pressure and EtOAc (20 mL) was added. The
organic layer was washed consecutively with brine, 1 N HCl, brine,
5% NaHCO3, and brine, dried over Na2SO4 and evaporated under
reduced pressure. The residue was purified by column chromatog-
raphy using CH2Cl2/MeOH 99:1 as eluent.
4.2.1.1. (2S)-Methyl 2-(2-hydroxyhexadecanamido)-4-methyl-
pentanoate (2a). Yield 79%; white solid; mp 47–49 °C; 1H NMR
(200 MHz, CDCl3): d 7.02–6.82 (m, 1H), 4.70–4.55 (m, 1H), 4.20–
4.10 (m, 1H), 3.73 (s, 3H), 3.07 (s, 1H), 1.81–1.53 (m, 5H), 1.39–
1.24 (m, 24H), 0.89 (m, 9H); 13C NMR (50 MHz, CDCl3): d 174.15,
173.91, 72.05, 52.30, 50.27, 41.40, 34.74, 34.64, 31.89, 29.66,
29.63, 29.54, 29.36, 29.33, 24.82, 22.80, 22.66, 21.76, 21.67, 14.09.
Anal. Calcd for C23H45NO4: C, 69.13; H, 11.35; N, 3.51. Found: C,
68.99; H, 11.48; N, 3.59.
3. Conclusion
2-Oxoamide derivatives based on a-amino acids have been syn-
thesized and tested for their in vitro inhibitory activity against
three human sPLA2s (GIIA, GV and GX). Compound GK126, which
is based on (S)-leucine, displayed inhibition of human and mouse
GIIA sPLA2 (IC50 300 nM and 180 nM, respectively). It also inhibited
the human GV sPLA2 with similar potency, while it did not display
any measurable inhibition of GX sPLA2. Using a combination of
simulated annealing and molecular docking calculations, it was
possible to explore the inhibitor–enzyme complexes and rational-
ize the stereoelectronic characteristics that affect the inhibition
potency of these compounds. The annealed structures resulted
from the investigation of the conformational space of each inhibi-
tor were docked in the enzyme active site. Inhibitor GK126, which
is the most active compound among the inhibitors tested, is scored
with the highest GOLDscore Fitness. The 2-oxoamide functionality
is essential for the binding by interacting with the calcium ion and
by participating in two hydrogen bonds with Gly29.
4.2.1.2. (2R)-Ethyl 2-(2-hydroxyhexadecanamido)-4-methyl-
pentanoate (2b). Yield 64%; white solid; mp 34–35 °C; 1H NMR
(200 MHz, CDCl3): d 7.00–6.90 (m, 1H), 4.60–4.55 (m, 1H), 4.15–
4.00 (m, 3H), 1.78–1.58 (m, 6H), 1.41–1.22 (m, 27H), 0.90 (m,
9H); 13C NMR (50 MHz, CDCl3): d 174.27, 173.27, 72.08, 61.37,
50.37, 41.44, 34.74, 34.64, 31.86, 29.63, 29.52, 29.37, 29.29,
24.88, 24.83, 22.79, 22.62, 21.80, 21.71, 14.03. Anal. Calcd for
C24H47NO4: C, 69.69; H, 11.45; N, 3.39. Found: C, 69.57; H, 11.56;
N, 3.44.
4.2.1.3.
(S)-Ethyl 4-methyl-2-palmitamidopentanoate. Yield
79%; white solid; mp 51–52 °C; 1H NMR (200 MHz, CDCl3): d 6.00
(d, J = 8.4 Hz, 1H), 4.67–4.52 (m, 1H), 4.15 (q, J = 7.1 Hz, 2H), 2.18
(t, J = 7.5 Hz, 2H), 1.57 (m, 5H), 1.24 (m, 27H), 1.00–0.85 (m, 9H);
13C NMR (50 MHz, CDCl3): d 173.25, 172.83, 61.12, 50.51, 41.71,
36.47, 31.83, 29.59, 29.56, 29.51, 29.41, 29.26, 29.14, 25.54,
24.80, 22.72, 22.58, 21.91, 14.03, 14.00. Anal. Calcd for
4. Experimental
4.1. Chemistry
C24H47NO3: C, 72.49; H, 11.91; N, 3.52. Found: C, 72.34; H, 12.09;
N, 3.41.
Melting points were determined on a Buchi 530 apparatus and
are uncorrected. Specific rotations were measured on a Perkin–El-
mer 343 polarimeter using a 10 cm cell. NMR spectra were re-
corded on a Varian Mercury spectrometer. 1H and 13C NMR
spectra were recorded at 200 MHz and 50 MHz, respectively in
CDCl3 or as specified. Chemical shifts are given in ppm, and peak
multiplicities are described as follows: s, singlet, d, doublet, t, trip-
let and m, multiplet. Electron spray ionization (ESI) mass spectra
were recorded on a Finnigan, Surveyor MSQ Plus spectrometer.
TLC plates (Silica Gel 60 F254) and Silica Gel 60 (70–230 or 230–
400 mesh) for column chromatography were purchased from
Merck. Spots were visualised with UV light and/or phosphomolyb-
dic acid and/or ninhydrin, both in EtOH. Dichloromethane was
dried by standard procedures and stored over molecular sieves.
All other solvents and chemicals were reagent grade and used
without further purification.
4.2.1.4. (2S)-tert-Butyl 2-(2-hydroxyhexadecanamido)-3-phe-
nylpropanoate (5d). Yield 57%; white solid; mp 55–56 °C; 1H
NMR (200 MHz, CDCl3): d 7.30–7.20 (m, 5H, Ar), 4.80–4.68 (m,
1H), 4.07–4.01 (m, 1H), 3.15–2.97 (m, 2H), 1.80–1.46 (m, 2H),
1.37 (s, 9H), 1.23 (s, 24H), 0.84 (t, J = 6.7 Hz, 3H); 13C NMR
(50 MHz, CDCl3): d 173.60, 170.63, 135.78, 129.09, 128.03, 126.61,
82.13, 71.74, 52.90, 37.87, 34.47, 31.59, 29.37, 29.22, 29.12, 29.04,
27.57, 24.61, 22.36, 13.80; MS (ESI) m/z (%): (48) 476.3 (M+Y)+,
t
420.3 (100) (MÀ Bu+H+H)+. Anal. Calcd for C29H49NO4: C, 73.22;
H, 10.38; N, 2.94. Found: C, 73.05; H, 10.46; N, 3.04.
4.2.2. General method for the saponification of 2-hydroxya
mides
To a stirred solution of a 2-hydroxyamide ester (1.0 mmol) in a
mixture of dioxane–H2O (9:1, 10 mL) was added 1 N NaOH (1.1 mL,
1.1 mmol), and the mixture was stirred for 12 h at room tempera-
ture. The organic solvent was evaporated under reduced pressure,
and H2O (5 mL) was added. The aqueous layer was washed with
EtOAc, acidified with 1 N HCl, and extracted with EtOAc
(3 Â 6 mL). The combined organic layers were washed with brine,
dried over Na2SO4, and evaporated under reduced pressure.
The synthesis of inhibitor AX115 and of compounds 5a–c and
6a–c has been described elsewhere.26
4.2. Synthesis of the 2-oxoamide inhibitors
4.2.1. General method for the coupling of 2-hydroxyhexadeca
noic acid with amino components
4.2.2.1. (2S)-2-(2-Hydroxyhexadecanamido)-4-methylpentanoic
acid (3a). Yield 89%; white solid; 1H NMR (200 MHz, CDCl3): d 7.80
(br s, 1H), 7.40–7.30 (m, 1H), 4.44 (m, 1H), 4.08 (m, 1H), 1.80–1.50
(m, 6H), 1.45–1.10 (m, 24H), 0.89 (m, 9H); 13C NMR (50 MHz,
CDCl3): d 176.59, 175.40, 72.19, 71.98, 51.21, 40.56, 40.39, 34.47,
31.95, 29.74, 29.68, 29.48, 29.37, 25.27, 25.15, 24.97, 23.02,
To a stirred solution of 2-hydroxyhexadecanoic acid (1.0 mmol)
and hydrochloride amino component (1.0 mmol) in CH2Cl2
(10 mL), Et3N (0.3 mL, 2.2 mmol) and subsequently 1-(3-dimethyl-
aminopropyl)-3-ethyl carbodiimide hydrochloride (WSCI) (0.21 g,
1.1 mmol) and 1-hydroxybenzotriazole (HOBt) (0.14 g, 1.0 mmol)

V. D. Mouchlis et al. / Bioorg. Med. Chem. 19 (2011) 735–743
741
22.68, 21.44, 21.26, 14.10. Anal. Calcd for C22H43NO4: C, 68.53; H,
11.24; N, 3.63. Found: C, 68.41; H, 11.42; N, 3.51.
(30 mL) was added. The organic phase was washed with saturated
aqueous NaHCO3 (20 mL) containing Na2S2O3 (1.5 g, 9.5 mmol),
H2O (20 mL), dried over Na2SO4 and the organic solvent was evap-
orated under reduced pressure. The residue was purified by col-
umn chromatography using petroleum ether (bp 40–60 °C)/EtOAc
9:1 as eluent.
4.2.2.2. (2R)-2-(2-Hydroxyhexadecanamido)-4-methylpentanoic
acid (3b). Yield 54%; white solid; 1H NMR (200 MHz, CDCl3): d 7.80
(br s, 1H), 7.40–7.30 (m, 1H), 4.46 (m, 1H), 4.07 (m, 1H), 1.80–1.50
(m, 6H), 1.45–1.10 (m, 24H), 0.89 (m, 9H); 13C NMR (50 MHz,
CDCl3): d 176.57, 175.38, 72.20, 71.98, 51.15, 40.55, 40.37, 34.45,
31.92, 29.73, 29.67, 29.47, 29.36, 25.26, 25.13, 24.96, 23.02,
22.67, 21.43, 21.25, 14.09. Anal. Calcd for C22H43NO4: C, 68.53; H,
11.24; N, 3.63. Found: 68.43; H, 11.32; N, 3.51.
4.2.4.1. (S)-tert-Butyl 2-(2-oxohexadecanamido)-3-phenylpro-
panoate (6d). Yield 63%; white solid; mp 46–47 °C; [a]D = +29.0 (c
1, CH2Cl2); 1H NMR (200 MHz, CDCl3): d 7.34 (d, J = 8.4 Hz, 1H)
7.30–7.08 (m, 5H Ar), 4.72–4.62 (m, 1H), 3.08 (d, J = 6.3 Hz, 2H),
2.84 (t, J = 7.3 Hz, 2H), 1.62–1.45 (m, 2H), 1.37 (s, 9H), 1.23 (s,
22H), 0.84 (t, J = 6.7 Hz, 3H); 13C NMR (50 MHz, CDCl3): d 198.23,
169.48, 159.45, 135.61, 129.25, 128.35, 126.97, 82.45, 53.49,
37.96, 36.56, 31.81, 29.54, 29.48, 29.32, 29.25, 29.21, 28.93,
27.77, 23.06, 22.58, 14.01; MS (ESI) m/z (%): 472.5 (MÀY)À. Anal.
Calcd for C29H47NO4: C, 73.53; H, 10.00; N, 2.96. Found: C, 73.41;
H, 10.16; N, 3.09.
4.2.2.3. (S)-4-Methyl-2-palmitamidopentanoic acid (GK144). Yield:
100%; white solid; mp 93–95 °C; 1H NMR (200 MHz, CDCl3): d
10.46 (s, 1H), 6.22 (d, J = 8.2 Hz, 1H), 4.70–4.55 (m, 1H), 2.24 (t,
J = 7.6 Hz, 2H), 1.80–1.45 (m, 5H), 1.40–1.10 (m, 24H), 1.00–0.85
(m, 9H); 13C NMR (50 MHz, CDCl3) d 176.47, 174.23, 50.83, 41.24,
36.44, 31.89, 29.66, 29.48, 29.32, 29.18, 25.63, 24.86, 22.79,
22.64, 21.85, 14.06. Anal. Calcd for C22H43NO3: C, 71.50; H, 11.73;
N, 3.79. Found: C, 71.31; H, 11.89; N, 3.70.
4.2.5. General method for the cleavage of tert-butyl ester
A solution of the tert-butyl ester derivative (1 mmol) in 50%
TFA/CH2Cl2 (0.5 M) was stirred for 1 h at room temperature. The
organic solvent was evaporated under reduced pressure. The resi-
due was purified by recrystallization [EtOAc/petroleum ether (bp
40–60 °C)].
4.2.3. General method for the oxidation of 2-hydroxyamides
(Method A)
To a solution of 2-hydroxyamide (1.0 mmol) in a mixture of tol-
uene (3 mL) and EtOAc (3 mL),
a solution of NaBr (0.11 g,
1.1 mmol) in water (0.5 mL) was added followed by AcNH-TEMPO
(2.2 mg, 0.01 mmol). To the resulting biphasic system, which was
cooled at 0 °C, an aqueous solution of 0.35 M NaOCl (3.1 mL,
1.1 mmol) containing NaHCO3 (0.25 g, 3 mmol) was added drop-
wise under vigorous stirring, at 0 °C over a period of 1 h. After
the mixture had been stirred for a further 15 min at 0 °C, EtOAc
(10 mL) and H2O (10 mL) were added. The aqueous layer was sep-
arated and washed with EtOAc (2 Â 10 mL). The combined organic
layers were washed consecutively with 5% aqueous citric acid
(10 mL) containing KI (0.04 g), 10% aqueous Na2S2O3 (10 mL), and
brine and dried over Na2SO4. The solvents were evaporated under
reduced pressure and the residue was purified by column chroma-
tography using petroleum ether (bp 40–60 °C)/EtOAc 2:8 as eluent.
4.2.5.1. 2-(2-Oxohexadecanamido)acetic acid (GK111). Yield
90%; white solid; 1H NMR (200 MHz, CDCl3): d 5.00 (m, 1H), 3.95
(d, J = 4.0 Hz, 2H), 2.85 (t, J = 7.0 Hz, 2H), 1.80–1.50 (m, 4H),
1.40–1.15 (m, 20H), 0.90 (m, 3H); 13C NMR (50 MHz, CD3OD): d
197.85, 172.79, 161.34, 40.44, 40.30, 38.31, 36.58, 31.91, 29.61,
29.44, 29.37, 29.32, 29.01, 23.20, 23.07, 22.57, 13.30; MS (ESI) m/
z (%): 326.4 (M-Y)-. Anal. Calcd for C18H33NO4: C, 66.02; H, 10.16;
N, 4.28. Found: C, 65.89; H, 10.34; N, 4.15.
4.2.5.2. 3-(2-Oxohexadecanamido)propanoic acid (GK112). Yield
75%; white solid; 1H NMR (200 MHz, CDCl3): d 4.90 (b, 1H), 3.48
(t, J = 7.0 Hz, 2H), 2.82 (t, J = 7.2 Hz, 2H), 2.54 (t, J = 7.0 Hz, 2H),
1.62–1.55 (m, 2H), 1.50–1.05 (m, 22H), 0.88 (t, J = 7.0 Hz, 3H);
13C NMR (50 MHz, CDCl3) d 198.32, 173.91, 160.26, 38.17, 36.59,
34.94, 33.35, 32.98, 31.91, 29.61, 29.42, 29.32, 29.02, 23.36,
23.11, 22.57, 13.29; MS (ESI) m/z (%): 340.3 (100) [MÀH]À. Anal.
Calcd for C19H35NO4: C, 66.83; H, 10.33; N, 4.10. Found: C, 66.68;
H, 10.48; N, 4.19.
4.2.3.1. (S)-4-Methyl-2-(2-oxohexadecanamido)pentanoic acid
(GK126). Yield 29%; yellow oil; 1H NMR (200 MHz, CDCl3): d 7.46
(m, 1H), 4.51 (m, 1H), 2.91 (t, J = 7.0 Hz, 2H), 1.80–1.40 (m, 5H),
1.40–1.10 (m, 22H), 0.90 (m, 9H); 13C NMR (50 MHz, CDCl3): d
198.68, 170.47, 160.36, 51.40, 40.83, 36.87, 31.91, 29.68, 29.49,
29.42, 29.36, 29.08, 24.86, 23.07, 22.92, 22.67, 21.53, 14.09; MS
(ESI) m/z (%): 384.3 (M+Y)+. Anal. Calcd for C22H41NO4: C, 68.89;
H, 10.77; N, 3.65. Found: C, 68.75; H, 10.89; N, 3.75.
4.2.5.3. 5-(2-Oxohexadecanamido)pentanoic acid (GK122). Yield
91%; white solid; mp 103–105 °C; 1H NMR (200 MHz, CDCl3): d
7.06 (t, J = 5.6 Hz, 1H), 3.39–3.26 (m, 2H), 2.91 (t, J = 7.4 Hz, 2H),
2.40 (t, J = 7.0 Hz, 2H), 1.78–1.48 (m, 6H), 1.26 (s, 22H), 0.88 (t,
J = 6.6 Hz, 3H); 13C NMR (50 MHz, CDCl3)
d 199.34, 178.56,
4.2.3.2. (R)-4-Methyl-2-(2-oxohexadecanamido)pentanoic acid
(GK145). Yield 35%; yellow oil; 1H NMR (200 MHz, CDCl3): d 7.38
(m, 1H), 4.56 (m, 1H), 2.91 (t, J = 7.0 Hz, 2H), 1.80–1.45 (m, 5H),
1.40–1.10 (m, 22H), 0.90 (m, 9H); 13C NMR (50 MHz, CDCl3): d
198.58, 177.46, 160.29, 51.18, 40.82, 36.85, 34.74, 31.90, 29.67,
29.47, 29.40, 29.34, 29.05, 28.87, 27.27, 24.85, 23.05, 22.88,
22.66, 21.50, 14.09; MS (ESI) m/z (%): 384.3 (M+Y)+. Anal. Calcd
for C22H41NO4: C, 68.89; H, 10.77; N, 3.65. Found: C, 68.72; H,
10.89; N, 3.76.
160.27, 38.83, 36.73, 33.27, 31.89, 29.62, 29.57, 29.42, 29.32,
29.04, 28.58, 23.16, 22.66, 21.79, 14.09; MS (ESI) m/z (%): 368.3
(100) [MÀH]À. Anal. Calcd for C21H39NO4: C, 68.25; H, 10.64; N,
3.79. Found: C, 68.11; H, 10.80; N, 3.84.
4.2.5.4. (S)-2-(2-Oxohexadecanamido)-3-phenylpropanoic acid
(GK141). Yield 95%; white solid; mp 104–105 °C; [a]D = +8.7 (c 1,
CH3OH); 1H NMR (200 MHz, CD3OD): d 7.31–7.15 (m, 5H Ar),
4.72–4.64 (m, 1H), 3.26–3.23 (m, 1H), 3.12–2.96 (m, 2H), 2.75 (t,
J = 7.0 Hz, 2H), 1.59–1.46 (m, 2H), 1.29 (s, 22H), 0.90 (t, J = 6.7 Hz,
4.2.4. General method for the oxidation of 2-hydroxyamides
(Method B)
3H); 13C NMR (50 MHz, CD3OD):
d 199.14, 174.29, 162.39,
138.14, 130.29, 129.45, 127.88, 54.54, 39.65, 38.04, 37.74, 33.09,
30.78, 30.68, 30.58, 30.50, 30.13, 24.29, 23.75, 14.46; MS (ESI) m/
z (%): 418.2 (100) (M+Y)+. Anal. Calcd for C25H39NO4: C, 71.91; H,
9.41; N, 3.35. Found: C, 71.81; H, 9.53; N, 3.22.
To a solution of 2-hydroxyamide (1.0 mmol) in dry CH2Cl2
(10 mL) Dess–Martin periodinane was added (0.64 g, 1.5 mmol)
and the mixture was stirred for 1 h at room temperature. The
organic solvent was evaporated under reduce pressure and Et2O

742
V. D. Mouchlis et al. / Bioorg. Med. Chem. 19 (2011) 735–743
4.3. In vitro sPLA2 assay
whether any residues in the superimposed protein differ apprecia-
bly in position or conformation from those in the reference site. No
significant differences were observed. Even though all crystal
structures are suitable, the 1DB4 PDB file was chosen for the
molecular docking calculations because this file contains a single
unit of the GIIA sPLA2 enzyme co-crystallized with an indole inhib-
itor. The 1DB4 crystal structure was prepared using the ‘Protein
Preparation Wizard’ panel43 of Schrödinger Suite 2009. Using the
‘Preprocess and analyze structure’ tool, the bond orders were as-
signed, all the hydrogen atoms were added, the calcium ion was
treated in order to have the correct geometry and the correct for-
mal charge (+2), the disulfide bonds were assigned, and all the
water molecules at a distance greater than 5 Å from any het group
were deleted. Using EPIK 2.0,44,45 a prediction of het groups ioniza-
tion and tautomeric states was performed. An optimization of the
hydrogen bonding network was performed using the ‘H-bond
assignment’ tool. Finally, using the ‘Impref utility’ the positions
of the hydrogen atoms were optimized by keeping all the heavy
atoms in place. The prepared structure was saved in PDB file for-
mat and was used as the input file in the docking calculations.
A continuous fluorimetric assay described previously was used
to determine the inhibitory activity of compounds GK111, GK112,
GK122, GK126, GK141, GK144, GK145, and AX115.29
4.3.1. IC50 value determination
IC50 values for compounds GK111, GK112, GK122, GK126 and
GK145 are reported as the mean of duplicate or triplicate analysis
with standard deviations. All other compounds were screened at
single doses and reported as >1.66
lM, or a % inhibition at
16.6 M is given. IC50 values were determined by nonlinear regres-
l
sion analysis of a semi-log plot of percent inhibition versus log of
inhibitor concentration. Curves were fit to a variable slope sigmoi-
dal dose–response curve using the Kaleidagraph software. Five
inhibitor concentrations ranging from 10% to 90% inhibition were
used for each IC50 value determination.
4.3.2. Assay procedure
To seven wells of a 96-well microtiter plate was added 100
solution A (27 M bovine serum albumin, 50 mM KCl, 1 mM CaCl2,
50 mM Tris–HCl, pH 8.0) followed by the desired concentration of
inhibitor (1 L in DMSO from serial-diluted stock solutions) or 1
of DMSO for control reactions. To the first well was added an addi-
tional 100 L of solution A as a negative control. Solution B was
lL of
l
4.4.2. Preparation of the ligands
l
lL
The crystallographic ligands and the 2-oxoamide ligands were
sketched using the SYBYL 8.0 molecular sketcher.36 All the hydrogen
atoms were added to define the correct ionization and tautomeric
states, and the oxygen atoms of the carboxylate and phosphonate
groups were considered as equivalent with atom type for the oxygen
atoms of O.co2. The molecules were subjected to energy minimiza-
tion using the standard Tripos46 molecular mechanics force field of
the SYBYL 8.0 molecular modeling package, and the Powell47 energy
minimization algorithm with a gradient of 0.01 kcal molÀ1 ÅÀ1
was used for the minimization procedure.
l
prepared by mixing the appropriate amount of sPLA2, depending
on the specific activity, to Solution A. This solution was prepared
immediately prior to each set of assays, to avoid loss of enzymatic
activity due to sticking to the walls of the container. Solution B was
delivered in 100
Quickly after the addition of Solution B, the assay was initiated by
the addition of 100 L of Solution C (4.2 M 1-hexadecanoyl-2-(1-
lL portions to all seven wells except the first well.
l
l
pyrenedecanoyl)-sn-glycero-3-phosphoglycerol (pyrPG, Molecular
Probes) vesicles in assay buffer (50 mM KCl, 1 mM CaCl2, 50 mM
Tris–HCl, pH 8.0)) to all seven wells. The fluorescence (excita-
tion = 342 nm, emission = 395 nm) was read with a microtiter
plate spectrophotometer (Fluorocount, Packard Instruments). Con-
trol reactions without enzyme or inhibitor were run with each as-
say of seven wells and the percent inhibition calculated from the
initial slopes of fluorescence versus time. The amount of enzyme
used per well are as follows: hGIIA, 6 ng; hGV, 12.5 ng; hGX,
17.7 ng; mGIIA, 3.4 ng; mGV, 12.7 ng; mGX, 10.7 ng. All of the re-
combinant mouse and human sPLA2s were prepared as previously
described.38
4.4.3. Simulated annealing method
The investigation of the conformational space of each inhibitor,
using the method of simulated annealing48 was performed. Each
inhibitor was heated at temperature 2000 K for 2000 fs and was
cooled at a temperature 0 K for 10000 fs for 100 cycles. For the sim-
ulation, the Tripos46 standard molecular mechanic force field was
used. The dielectric function was selected to be distance dependent
with a value of 1 for the distance-dependent dielectric constant
and nonbonded cutoffs of 8.0 Å. The annealed structures were min-
imized using the Powell energy minimization algorithm with a
gradient of 0.01 kcal molÀ1 ÅÀ1. For the simulated annealing the
SYBYL 8.036 was used.
4.4. Computational methods
4.4.4. Molecular docking procedure
4.4.1. Preparation of the GIIA sPLA2 crystal structure
For the molecular docking calculation the genetic algorithm
GOLD 4.131 was used along with the standard GOLD scoring function
GOLDscore.49 The active site of the GIIA sPLA2 was set as 6.0 Å
around the native ligand. All the critical amino acids (Leu2, Val3,
Phe5, His6, Ile9, Ala17, Ala18, Tyr21, His27, Gly29, Gly31, Asp48,
Tyr51, Lys62 and Phe98) including the calcium ion and the
His47/Asp91 catalytic dyad were considered in the active site.
For the five water molecules, near the active site, the state ‘Toggle’
was specified, and the orientation of the hydrogen atoms of each
water molecule was automatically optimized using the ‘Spin’ op-
tion. The geometry of the calcium ion was simulated by GOLD as
octahedral with RMSD of 0.465 Å. For highest accuracy of the dock-
ing calculations, long search settings were used for the GOLD GA:
100 dockings with 100,000 GA operations per docking; the algo-
rithm was not allowed to terminate early when the same solution
was produced repeatedly. The GOLD algorithm supports flexible
docking for the ligand, which is not depended on its initial confor-
mation, and provides partial flexibility for the receptor.
The crystal structures of the GIIA sPLA2 enzyme which were
deposited in the RCSB protein data bank with PDB IDs: 1DB4 holo
form 2.20 Å X-ray resolution,10 1DB5 holo form 2.80 Å X-ray reso-
lution,10 1J1A holo form 2.20 Å X-ray resolution,11 1KVO holo form
2.00 Å X-ray resolution12 were downloaded. The objective was to
judge which crystal structure is appropriate for the docking calcu-
lations. This was determined by using the following procedure: (i)
using ‘Superposition panel’ of MAESTRO 9.042 all the crystal structures
were superimposed based on all the backbone atoms including
beta carbons. The crystal structure with PDB ID: 1DB4 was chosen
as a reference structure for the superposition (RMSD between:
1DB4–1DB5: 0.147 Å, 1DB4–1J1A: 0.746 Å, 1DB4–1KVO: 0.487 Å).
No significant structural differences were observed as indicated
by the RMSD values (Fig. 1 in Supplementary data); (ii) the active
site region was examined to determine if the superimposed ligands
can fit into the reference site without steric clashes. No significant
steric clashes were observed; (iii) the active site region of all the
crystal structures, in turn, was examined in order to determine

V. D. Mouchlis et al. / Bioorg. Med. Chem. 19 (2011) 735–743
743
19. Wei, D.; Jiang, X.; Zhou, L.; Chen, J.; Chen, Z.; He, C.; Yang, K.; Liu, Y.; Pei, J.; Lai,
L. J. Med. Chem. 2008, 51, 7882.
Acknowledgments
20. Kokotos, G.; Kotsovolou, S.; Six, D. A.; Constantinou-Kokotou, V.; Beltzner, C. C.;
Dennis, E. A. J. Med. Chem. 2002, 45, 2891.
21. Kokotos, G.; Six, D. A.; Loukas, V.; Smith, T.; Constantinou-Kokotou, V.;
Hadjipavlou-Litina, D.; Kotsovolou, S.; Chiou, A.; Beltzner, C. C.; Dennis, E. A. J.
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J. Pept. Sci. 2005, 11, 431.
23. Yaksh, T. L.; Kokotos, G.; Svensson, C. I.; Stephens, D.; Kokotos, C. G.;
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24. Stephens, D.; Barbayianni, E.; Constantinou-Kokotou, V.; Peristeraki, A.; Six, D.
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25. Six, D. A.; Barbayianni, E.; Loukas, V.; Constantinou-Kokotou, V.; Hadjipavlou-
Litina, D.; Stephens, D.; Wong, A. C.; Magrioti, V.; Moutevelis-Minakakis, P.;
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The project is co-funded by the European Social Fund and
National Resources (EPEAEK II) (G.K.) and by a grant from the
National Institutes of Health (HL36235) (M.H.G.). V. D. Mouchlis
was funded by the Research Promotion Foundation (RPF) (bilateral
agreement CY-SLO/0407/06).
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2010.12.030.
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