In vitro and in silico exploration of IL-2 inhibition by small drug-like molecules

Article abstract of DOI:10.1007/s00044-013-0564-x

Interleukin-2 (IL-2) is an immunoregulatory cytokine produced by T lymphocytes in response to antigen. It is a potent growth and differentiation factor for several cell-types and is structurally related to the four-helix bundle family of cytokines. Here,

Full text of DOI:10.1007/s00044-013-0564-x

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-013-0564-x
ORIGINAL RESEARCH
In vitro and in silico exploration of IL-2 inhibition
by small drug-like molecules
•
•
•
Saima Kalsoom Umer Rashid Awais Shaukat Omer Mohamed Abdalla
•
•
Khalida Hussain Waqasuddin Khan Samina Nazir Mohammad Ahmad Mesaik
•
•
•
•
Zaheer-ul-Haq Farzana Latif Ansari
Received: 16 November 2012 / Accepted: 27 February 2013
ꢀ Springer Science+Business Media New York 2013
Abstract Interleukin-2 (IL-2) is an immunoregulatory
cytokine produced by T lymphocytes in response to anti-
gen. It is a potent growth and differentiation factor for
several cell-types and is structurally related to the four-
helix bundle family of cytokines. Here, we report IL-2
inhibitory potential and computational studies on different
series of chalcones, benzothiazepines, semicarbazones, and
dihydropyrimidines. These compounds were synthesized in
wet lab and were then tested for their potency as IL-2
inhibitors through in vitro T cell proliferation, IL-2 cyto-
kine production as well as their effect on oxidative burst.
Compounds that showed significant suppressive activity
were further evaluated for their cytotoxicity on normal two
cell lines. Most of the chalcones were found to have a
powerful inhibitory effect on T-lymphocytes proliferation
and cytokine production. Among the aza heterocycles
benzothiazepines, benzoxazepines, and benzodiazepinones
were found to be the strongest IL-2 inhibitors. Molecular
docking and MD simulation studies were carried out to
correlate experimental and theoretical results whereby a
good correlation was observed which indicated that com-
putational studies could provide an alternate tool for the
identification and designing of more potent IL-2 inhibitors.
Keywords IL-2 inhibitors ꢀ Chalcones and heterocycles ꢀ
Molecular docking ꢀ MD simulation
Introduction
Cytokines are small signaling protein molecules that are
secreted by various cells and are used extensively in inter-
cellular communication. The term ‘‘cytokine’’ has been used
to refer immunomodulating agents, such as interleukins and
interferons. Interleukin-2 (IL-2), which is one of the first
cytokines identified, acts at the heart of the immune
response (Smith, 1988). Mature IL-2, a secreted glycopro-
tein of 133 amino acids (15.5 kDa), is a single chain poly-
peptide produced by T cells in response to immune stimuli
mediated by the T-cell receptor (TCR) and major histo-
compatibility complexes (MHC) I and II (Nelson and
Willerford, 1998). Since IL-2 is central in immune and non-
immune mechanisms of resistance to disease, it offers a
large potential for therapeutic applications. Perhaps the best
characterized of these is the use of IL-2 in the treatment of
malignant neoplasms (Murakawa et al., 1985). IL-2 has been
suggested to participate in the pathogenesis of a variety
of diseases. Chronic active hepatitis (Mule et al., 1984),
juvenile diabetes (Saxena et al., 1986), rheumatoid arthritis
(Zielasek et al., 1990) multiple sclerosis (Phadke et al.,
Electronic supplementary material The online version of this
article (doi:10.1007/s00044-013-0564-x) contains supplementary
material, which is available to authorized users.
S. Kalsoom ꢀ A. Shaukat ꢀ F. L. Ansari (&)
Department of Chemistry, Quaid-i-Azam University,
Islamabad 45320, Pakistan
e-mail: fla_qau@yahoo.com
U. Rashid
Department of Chemistry, Hazara University,
Mansehra 21120, Pakistan
O. M. Abdalla ꢀ K. Hussain ꢀ W. Khan ꢀ
M. A. Mesaik ꢀ Zaheer-ul-Haq
Dr. Panjwani Center for Molecular Medicine and Drug Research,
International Center for Chemical and Biological Sciences,
University of Karachi, Karachi 75270, Pakistan
S. Nazir
Nanoscience and Catalysis Division, National Center
for Physics, Quaid-i-Azam University,
Islamabad 44000, Pakistan
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Med Chem Res
1986), allograft rejection (Trotter et al., 1988), and lupus
erythematous (Kirkman et al., 1985) are among those dis-
eases associated with aberrant IL-2 function. Certain
immunodeficiencies have been directly correlated with a defect
in the ability of T cells to secrete IL-2 (Pahwa et al., 1989).
Most of the immunosuppressant drugs used in clinic
today include agents that act at various points in the TCR-
signaling pathway to block IL-2 production. These drugs
include cyclosporin A (CsA), rapamycin FK506, and anti-
IL-2Ra antibodies (anti-Tac) (Brown and Schreiber, 1996;
Wiederrecht et al., 1995; Waldmann and O’Shea, 1998;
Michael and David, 2005). However, these drugs exhibit a
plethora of unwanted side effects and significant toxicities
associated with them (Tilley et al., 1997).
molecules. Organic synthesis has contributed substantially
to the discovery of biologically active small molecules. In
the development of new drugs, it is convenient to have a
range of diverse chemical structures with the desired
pharmacological activity.
Our research group has been engaged in target-based
drug designing and has reported the synthesis of diverse
classes of small drug-like compounds and quite a few have
been identified as potential candidates for the inhibition of
enzymes such as urease, cholinesterase etc. Moreover, the
leads identified in different bioassays were subjected to
molecular modeling and docking studies and an attempt
was made to rationalize the results obtained in different
bioassays. Based on these in silico studies, some predic-
tions for the synthesis of new enzyme inhibitors with
activity were also made (Ansari et al., 2011; Nawaz et al.,
2008; Zaheer-ul-Haq et al., 2010).
The first small molecule shown to inhibit the IL-2:IL-
2Ra interaction was reported by Roche (Waldmann and
O’Shea, 1998). Compound a, was an enantiomer-specific,
competitive inhibitor of IL-2Ra with an IC50 = 6 lM.
In the present study, we screened a library of 50 syn-
thetic compounds belonging to six different classes both
cyclic and alicyclic e.g., chalcones, oxazolines, pyrazo-
lines, dihydropyrimidines, benzothiazepines, and semicar-
bazones which were synthesized and tested for their
behavior as IL-2 inhibitors. Due to the immense biological
applications of these compounds, herein we report a
comparative study of IL-2 inhibitory potential of the these
fifty compounds by conducting molecular docking and MD
simulation studies using Molecular Operating Environment
software (MOE 2011.10). These studies were carried out to
develop a deeper understanding of inhibitory behavior of
these compounds. Such information can be quite valuable
in search of new immunomodulatory compounds.
O
C
C
H2N
HN
HN
N
O
O
CH3
(a)
A fragment-minded approach was used to evolve com-
pound a into a more potent and drug-like inhibitor.
Braisted et al. (2003) reported a compound (b, IC50
=
6 lM) that binds to IL-2, preventing its association with
IL-2Ra .
N
NH
O
H
Cl
Cl
N
N
NH2
NH
N
H
Results and discussion
O
(b)
Chemistry
Optimization of compound b included the introduction
Chalcones 1–14 (Scheme 1) (Ansari et al., 2005; Ahsan Ullah
et al., 2007) were synthesized by Claisen Schmidt conden-
sation of different substituted aldehydes and different ace-
tophenones. These chalcones were transformed into different
classes of 5- or 7-member heterocycles by an [3 ? 2] or
[4 ? 3] annulations, respectively. The epoxide 15 was syn-
thesized by the reaction of a chalcone with hydrogen peroxide
(Kee and Gavriilidis, 2007). Oxazolines (16–17) were syn-
thesized by refluxing the corresponding chalcones with
hydroxylamine as reported in literature earlier (Chandrase-
khar et al., 2002). Following the same protocol, pyrazolines
(18–20) were prepared by the reaction of chalcones with
phenylhydrazine (Sivakumar et al., 2010; Baseer et al., 2013).
2,3-Dihydrobenzothiazepines (21–26) were synthesized in
solid phase earlier by the reaction of different chalcones
with O-aminothiophenol (Ansari et al., 2008a, b; Levai and
of a furanoic acid fragment onto the dichlorophenyl ring
(c, IC50 = 0.060 lM) which offered a significant increase
in activity.
HOOC
O
Me
N
N
O
O
H
Cl
Cl
N
N
NH2
NH
N
H
O
(c)
In our recent paper, we isolated natural products from
Lindelofia stylosa which has shown ability to modulate the
immune response (Mesaik et al., 2012).
In both lead identification and lead optimization pro-
cesses there is an acute need for new organic small
123

Med Chem Res
Scheme 1 Synthesis of
chalcones and the derived
azaheterocycles
R5
R8
O
R1
R4
O
R6
R7
R2
R3
H
CH3
+
R1
R4
N
R1
R4
O
R5
R8
S
R5
R1
O
R5
R2
R3
R2
R3
R6
R7
R2
R3
R6
R7
R6
H2O2
O
NH2
SH
R7
R8
R8
R4
(15)
(1-14)
(21-26)
Ph-NH-NH2
NH2OH
Ph
R1
N
N
R5
LiAlH4
R2
R3
R6
R7
R1
R4
N
O
R5
R2
R3
R6
R7
R4
R8
R1 HN
S
(18-20)
R5
R2
R3
R8
R6
R7
(16-17)
R4
R8
(27-29)
No.
R1
H
R2
OMe
H
R3
R4
R5
R6
Br
Cl
H
R7
H
1
OMe
H
OMe
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
OH
H
H
H
OMe
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
2
NH2
H
H
3
H
H
F
4
H
H
H
H
Me
H
5
H
OH
OH
H
H
NO2
6
H
H
H
H
H
Cl
H
H
H
H
7
NH2
NH2
H
H
OMe
8
H
H
Br
H
9
H
NH2
NH2
NH2
H
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
H
H
H
H
H
Me
H
H
OH
H
H
NH2
H
NO2
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
OH
OH
H
H
OMe
H
H
H
H
H
OMe
F
H
H
H
H
H
H
H
H
H
H
Br
H
H
OH
OH
OH
OH
OH
OH
OH
H
H
OH
OH
OH
H
H
OMe
Br
H
H
NMe2
F
H
H
H
Cl
H
H
NO2
OMe
F
OH
OH
OH
H
H
H
H
H
Cl
Szikszai, 2008). Tetrahydrobenzothiazepines (27–29) were
synthesized by the reaction of 2,3-dihydrobenzothiazepines
with lithium aluminium hydride. A diastereomeric mixture
of tetrahydrobenzothiazepines was obtained which was
separated through fractional crystallization in each case. Two
diastereomers were obtained in different yield (Ansari et al.,
2005). XRD data of compound 29 has earlier been reported by
our group (Parvez et al., 2003).
123

Med Chem Res
Chalcones30–35 (Scheme 2) were synthesizedbythe same
strategy leading to ferrocenylchalcones (Wu et al., 2002) and
biphenylchalcones (Thirunarayanan and Vanangamudi, 2007)
using either acetylferrocenyl or acetylbiphenyl and different
substituted arylaldehydes. Compound 36 was obtained by
the reaction of acetophenone with 2-thiophenecarboxalde
hyde while chalcone 37 was a result of condensation of
3-hydroxyacetophenone with 2-pyridinecarboxaldehyde (Ahsan
Ullah et al., 2007).
crotonic acid. Naphthodiazepinone 43 was the product of
the reaction of 2,3-diamino naphthalene with crotonic acid.
3,4-Dihydropyrimidine-2-ones (44–46, Scheme 4) were
synthesized by a modified multicomponent strategy devel-
oped by Biginelli by condensation of different arylalde-
hydes, urea, and dibenzoylmethane (Akbas and Furgan,
2008).
Synthesis of semicarbazones 47 and 48 was carried out
by the reaction of different carbonyl compounds with
semicarbazides. Semicarbazones 49 and 50 were prepared
by the reaction of exocyclic carbonyl group of isatin and
benzodiazepinone with semicarbazide, respectively, as
reported earlier by our group (Jafri et al., 2012)
(Scheme 5).
Benzodiazepinone 38–40 (Scheme 3) were synthesized
by the reaction of o-phenylenediamine or 4-nitropheny-
lenediamine with crotonic acid as reported earlier by our
group (Jafri et al., 2012), while benzodiazepinone 42 was
prepared by the reaction of 2,3-diaminopyridine and
Scheme 2 Synthesis of
chalcones derived from acetyl
compounds
O
O
O
Me
H
Ring A
Ring A
+
Ring B
Ring B
(30-37)
No.
30
31
32
33
34
35
36
37
Ring A
Ring B
2-Ferrocenyl
2-Ferrocenyl
2-Ferrocenyl
Biphenyl
2-NO2Ph
4- NO2Ph
5- NO2Ph
2-OHPh
Biphenyl
3-OHPh
Biphenyl
3-OMePh
2-Thienyl
2-Pyridyl
Phenyl
3-OHPh
R2
NH2
Scheme 3 Synthesis of
heteroazepinones
H
N
O
NH2
NH2
O
H
N
R2
H
R1 R3
R4
C
C
COOH
R4
CH3
R4
CH3
N
H
R1
R3
CH3
(43)
(38-42)
No.
38
39
40
41
42
43
R1
CH
CH
CH
CH
N
R2
H
R3
NH
NH
NH
O
R4
H
NH2
NO2
H
H
Me
H
H
NH
-
H
-
-
H
R2
Scheme 4 Synthesis of 3,4-
dihydroyrimidines (44–46)
R1
R2
O
R1
O
O
Ph
NH
O
SnCl2.2H2O
Ph
+
+
H2N
NH2
Ph
N
H
ACN, ))))
CHO
O
Ph
(44-46)
No.
44
R1
R2
H
H
45
OMe
H
OH
46
OMe
123

Med Chem Res
H
N
O
Scheme 5 Synthesis of
semicarbazones (47–50)
O
H
N
O2N
Ph
N
O
H
N
N
HN Ph
O
H
N
H
N
N
H
O2N
H
H
N
H
N
HN
N
N
H
NH2
Ph
N
H
N
H
O
(50)
(49)
R1
O
R2
R1
H
N
H
N
R2
N
Ph
O
(47-48)
No.
47
R1
R2
3-OHPh
Me
H
48
4-OMePh
Table 1 Effect of organic compounds (1–50) on ROS generation
In vitro studies
No.
IC50 (lm/mL)
The synthesized compounds 1–50 were tested for their
potency as IL-2 inhibitors through in vitro T cell prolifer-
ation and IL-2 cytokine production assay as well as their
effect on oxidative burst. Compounds that showed signifi-
cant suppressive effect were further evaluated for their
cytotoxicity on normal two cell lines. Most of the chal-
cones were found to have a powerful inhibitory effect on
lymphocyte proliferation and cytokine production.
3
65.1 34.6
31.0 8.9
24.7 3.8
2.9 0.5
52.1 12.9
24.4 18.8
44.1 9.8
12.4 1.3
4.8 0.1
17.0 7.7
6.9 0.5
0.2 0.0
36.3 0.1
15.8 2.
72.2 9.8
21.2 2.7
4.5 0.6
12.4 2.9
2.3 0.7
8.0 0.8
5.3 0.5
4
5
6
7
9
10
11
12
13
17
26
31
32
36
37
39
40
41
47
48
Effect of compounds on generation of reactive oxygen
species (ROS)
The overproduction of ROS by phagocytes causes oxida-
tive damage to membrane lipids, DNA, proteins, and
lipoproteins. These reactions have functional conse-
quences, which may be deleterious to cells and tissues.
Thus, the inhibition of ROS production is a popular target
for the attenuation of many inflammatory diseases (Shen
et al., 2002). In this study, we have tested 46 compounds
out of 50 in the chemiluminescence assay. Five compounds
(6, 12, 26, 39, and 41,) were found to be very potent
inhibitors with an IC50 value of approximately 5 or less
than 5 lg/mL. Compounds 11, 17, 32, 47, and 40 showed
an IC50 value of 12.4, 6.9, 15.8, 8.0, and 12.4, respectively.
The results are summarized in Table 1.
Results are expressed as mean SD of IC50 in lg/mL for triplicate
runs of each parameter
Effect of compounds on T cell proliferation and IL-2
inhibition
compounds tested, 21 compounds showed an interference
with the PHA stimulated proliferation with an IC50 less
than 20 lg/mL (Table 2).
Activated T cells play a central role in the regulation of
immunity, and once activated, T cells may act as direct
cytotoxic effectors or provide help for other cells that are
important in the effector phase of the immune response.
Here, T cells were activated in vitro by phytohemaggluti-
nin (PHA) in the presence of three concentrations (0.5,
5.0 and 50 lg/mL) of each compound. Out of the 37
As a growth factor for T cell, 23 compounds were
subjected to screen their effect on IL-2 production. We
found that the production of IL-2 by peripheral blood
monocytes was very weak upon PHA activation. In order to
achieve a better and higher production level of IL-2, T cells
were activated by a combination of PHA and phorbol-
myristate acetate (PMA) as it has been reported that PMA’s
123

Med Chem Res
Table 2 Effect of compounds on T-cell proliferation and IL-2
inhibition
Table 3 Cytotoxicity effect of synthesized compounds on cancer cell
lines
No.
T-cell proliferation IC50 (lg/mL)
IL-2 inhibition
No.
Cytotoxicity
3T3L-1 cells
CC-1 cells
3
15.7 0.16
7.8 0.67
0.7 0.02
2.1 0.03
17.0 3.04
\0.5
20.7 0.6
15.6 0.2
1.5 0.0
1.4 0.2
15.1 0.2
2.5 0.1
31.8 0.3
15.1 0.2
14.2 0.1
16.1 1.6
19.7 0.4
–
4
5
3.4 0.05
5.3 0.3
9.5 0.7
16 0.35
20\
7.0 0.3
15.6 2.4
16.7 0.69
[20
5
6
6
9
8
26
37
9
20\
10
11
12
13
15
17
26
31
32
36
37
39
41
44
45
46
48
49
3.0 1.3
3.2 2.7
8.2 3.3
1.9 0.0
11.0 0.91
21.9 9.06
15.0 2.8
0.749 0.4
9.77 0.2
10.1 2.03
1.2 0.04
4.4 1.2
6.8 0.4
3.3 1.0
11.1 0.3
8.2 0.6
10.5 4.6
25.5 4.5
mouse embryo fibroblast and CC-1 rat Wistar hepatocyte
cell-lines. Cells were incubated for 48 h with three con-
centrations of each compound. The activity of compounds
26 and 37 in exerting suppressive effect in vitro on T-cell
proliferation and IL-2 production is clearly independent of
any cytotoxic action as shown in Table 3. The toxicity or
the IC50 of these two compounds on 3T3-L1 and CC-1 was
found to be more than or nearly equal to the highest dose
tested (20 lg/mL). The activity of compounds 5, 6, and 9
seems to be due to their cytotoxic action as their IC50 value
for the growth of these cell lines ranges from 3.4 to16.7 lg/
mL (Table 3).
2.3 0.2
13.5 0.0
15.2 0.0
26.7 0.8
5.0 0.5
13.0 0.3
14.9 1.7
–
–
In silico studies
–
37.3 6.1
–
Molecular docking
Results are expressed as mean SD of IC50 in lg/mL for triplicate
runs of each parameter
The binding modes of the compounds were predicted via
molecular docking technique using MOE software, which
revealed that all the compounds were accommodated well
in the ligand binding site of IL-2. Initially, MOE docking
performance was assessed by re-docking co-crystallized
structure from PDB ID 1M48. The co-crystallized ligand,
namely FRG (1) was docked into the active site of IL-2 to
insure that the docking program regenerates the bound
conformation correctly. The root-mean square deviation
(RMSD) of the docked conformation to the experimental
conformation was around 1.0.
mitogenic effect is additive to that of PHA, suggesting that
PHA and PMA induce IL-2 receptor gene expression
through different, complementary mechanisms. It was
observed that when T cells were forced to produce high
concentration of IL-2 using a combination of PHA and
PMA, some compounds showed suppressive activity
against T cells. Dihydropyrimidines failed to suppress the
production of IL-2, where they showed IC50 [ 50 lg/mL
(Table 2). 13 compounds (3, 4, 8, 10, 11, 12, 13, 15, 31, 32,
36, 39, and 41) showed a suppressive activity with IC50
between 10 and 35 lg/mL. In addition five compounds (5,
6, 9, 26 and 37) showed an IC50value of 5 or \ 5 lg/mL as
presented in Table 3.
The quality level of the docking complex was similar to
that of the original crystal structure. This result suggested
that methods applied for the docking simulation was suf-
ficient for further analysis and could be accepted for virtual
screening of IL-2 inhibitors. The important contact residues
for compound 1 were Lys35, Arg38, Met39, and Phe42.
Compound 1 can be divided into two components: a
hydrophilic fragment that contained a piperidyl guanidine
and a hydrophobic fragment containing a biaryl alkyne.
The piperidyl guanidine moiety formed a two arene-cation
bridge with Phe42 and Tyr45 and a side-chain donor
linkage with carboxylate of Glu62. On the other hand
the biaryl alkyne was well placed in the narrow channel
Cytotoxicity effect
In order to verify the suppressive activity of compounds
that showed extreme potency against T-cell proliferation
and IL-2 production, 5, 6, 9, 26, and 37 were tested for
their cytotoxic effects. For this purpose, a set of two
cytotoxicity experiments was developed using 3T3-L1
123

Med Chem Res
Fig. 1 Modeled mode of binding of FRG (1) in 1M48 active site. a 3D, b 2D
Fig. 2 Modeled mode of binding of chalcone 6 in 1M48 active site
Fig. 3 Modeled mode of binding of compound 5 in the active site of
1M48
created by the hydrophobic side chains of Arg38, Met39,
Phe42, Leu72, and Lys76 (Fig. 1).
with Leu72, Arg38 and also side chain acceptor interac-
˚
tions (2.74 A) with Arg38 (Fig. 2).
All the compounds were docked into the active site of
IL-2 using the above docking method to determine their
most suitable docking conformations. The Active Site
Finder tool of MOE was used to dock the reference and test
compounds into the receptor binding site of IL-2. All the
ligands that adopted almost identical position at the surface
of the IL-2 protein comprise a hydrophobic cage of Arg38,
Thr41, Phe42, Phe44, Lys43, Tyr45, Glu68, and Leu72.
The binding pattern of the most active compounds
showed that they were involved in hydrogen bonding with
three amino acids (Arg38, Thr41, Val69). The hydroxyl
group of the most active compound i.e., clacone 6
(IC50 = 1.4 lM) was involved in arene–cation interactions
The putative binding mode of compound 5 (IC50 =
1.5 lM) is shown in Fig. 3. The 4-NO2 aryl ring interacted
with Arg38 forming arene-H bond, while the oxygen of
carbonyl group formed a hydrogen bonding with Arg38 as
˚
hydrogen-bond donor (HBD) at a distance of 2.77 A.
Besides molecular docking and MD simulations, another
tool helpful in drug designing is the prediction of properties
of drug-like molecules using the Lipinski’s rule of five
(Ro5) which states four characteristics, H-bond donors and
acceptors, molecular weight, and log P that are usually
needed to make molecules more drug-like or orally active
(Lipinski, 2000). For example, poor absorption or perme-
ation of a drug is more likely when:
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Med Chem Res
•
•
•
•
There are more than 5 hydrogen-bond donors.
There are more than 10 hydrogen-bond acceptors.
Molecular weight is greater than 500.
Log P is greater than 5.
The amino acid residues that were involved in hydrogen
bonding interactions (Arg38, Thr41 and Val69) had very less
˚
fluctuation (less than 0.9 A), since they were involved in
hydrogen bonding interactions. The carbonyl oxygen atom of
Val69 formed a strong hydrogen bonding interaction with the
hydroxyl group of chalcone 6. As indicated in docking
studies, the hydrogen bonding interaction became stronger
during 2,400–2,600 ps. However, it attained an average
Furthermore, molecules with a polar surface area of
˚
greater than 140 A are usually believed to be poor at per-
meating cell membranes. Keeping in view the significance
of these descriptors, the molecular descriptors of all the
synthesized compounds as well as for the known IL-2
inhibitors were calculated and all the compounds were
found to comply with the Ro5 cut-off limits (Figure S-1 in
Supplementary data for known IL-2 inhibitors).
˚
interatomic distance around 3 A. The Oc atom of Thr41
formed a hydrogen bond with the hydroxyl group of chalcone
6. This hydrogen bonding interaction weakened more than
˚
5 A during the same time when Val69 interaction was strong,
making the chalcone-specific toward the active site region.
During the whole simulation run time, Thr41 was no longer
involved with the ligand. The behavior of Ne2 of Arg38 in the
sense of hydrogen bonding interaction with the carbonyl
group of chalcone remained fairly the same as that of Thr41.
But it became very weak till 1,250 ps, afterward the plot
showed the same trend as observed with the Thr41. It might
be suggested that any charged moiety should be present at that
site to form a strong interaction with Arg38 (Fig. 5).
MD simulation studies
MD simulation studies were carried out on chalcone 6 iden-
tified as the lead of the library with IC50 = 1.5 lg/mL. We
initiated our analysis of the MD results by plotting pressure,
density, temperature, total energy, kinetic energy, and poten-
tial energy graphs from the output files. It was noticed that the
temperature gradually increased and equilibrated reasonable
by around 300 K. Similarly, changes in the pressure, kinetic
energy, and potential energy were also stabilized at equili-
brated values. Mass-weighted RMSD (only polypeptide
backbone) calculated as a function of time indicated the sta-
bility of protein atoms during simulation. The system was
Conclusion
A 50 membered library of small drug-like molecules both
acyclic and cyclic were synthesized and their in vitro IL-2
potential was evaluated. Chalcones 5 and 6 were found to
be the lead structures with IC50 values 1.5 and 1.4 lg/mL,
respectively. In silico molecular docking and MD simula-
tion studies also supported the results of in vitro studies.
All these compounds were found to obey Ro5 and it is
anticipated that these compounds may be potential candi-
dates for developing new immunomodulating agents.
˚
converged at an average value of 1.64 A. The black curve
shows that a plateau is formed till 7,500 ps indicating the
presence of a precise conformation followed by an increment
in the RMSD value. This rise could be augmented by a change
in the conformation which eventually came down at the end of
simulation run time (Fig. 4a), while mass-weighted RMSF
calculated as a functionof time demonstratedthefluctuation of
amino acid residues (Fig. 4b).
Fig. 4 a Mass-weighted RMSD (only polypeptide backbone) calculated as a function of time. b Mass-weighted RMSF (only polypeptide
backbone) calculated as a function of time
123

Med Chem Res
Fig. 5 Hydrogen bonding interactions between a carbonyl oxygen atom of Val69. b Oc atom of Thr41 with the hydroxyl group of chalcone 6
and c Ne2 of Arg38 with the carbonyl of chalcones, plotted as a function of time
Materials and methods
Characterization data of yet unreported heteroazepinones
41–43 has been given below.
Chemistry
2-Methyl-2,3-dihydrobenzo[b][1,4]oxazepin-
4(5H)-one (41)
Synthesis and characterization of chalcones 1, 9, 10, 11, and
13 (Ansari et al., 2005) and chalcones 3–6, 12, 14, 36, and 37
(Ahsan Ullah et al., 2007; Ansari et al., 2009) have been
reported by our group. Furthermore, synthesis and charac-
terization of pyrazolines 20 (Baseer et al., 2013), 2,3-dihy-
dro-1,5-benzothiazepines 21–26 (Ansari et al., 2008a, b), and
tetrahydrobenzothiazepines 27–29 (Ansari et al., 2005; Par-
vez et al., 2003) have also been reported by our group.
Heteroazepinones 38–40, semicarbazones 47–50 (Jafri et al.,
2012) and dihydropyrimidines 41–46 (Akbas and Furgan,
2008) have also been reported by our group.
1
Yield 70 %, mp. 222 ꢁC, HNMR (CDCl3): d 7.28–7.89
(m, 4H, aryl-H), 8.75 (bs, NH), 3.75 (m, 1H, J4,3a = 4.2 Hz,
J4,3b = 4.2 Hz, J4,3b = 7.2 Hz, J4,Me = 6.0 Hz, H-4), 2.42
(dd, 1H, J3a,3b = 12.6 Hz, J3a,4 = 4.4 Hz, H-3a), 2.17 (dd,
J3b,3a = 12.8 Hz, J3b,4 = 7.2 Hz, H-3b), 1.37 (d, J4,Me
=
6.0 Hz, Me). 13C NMR (75 MHz, DMSO-d6): 20.4 (Me),
43.3 (CH2), 55.4 (CH), 108–124 (Ar–C), 172.0 (C=O),
GC–MS (m/z): 177 (M?).
2-Amino chalcones 2, 7, 8 (Yang et al., 2000), chalcone
epoxide 15 (Kee and Gavriilidis, 2007), oxazolines 16, 17
(Sharma and Sharma, 2010), pyrazolines 18, 19 (Sivaku-
mar et al., 2010), ferrocenyl chalcones 30–32 (Wu et al.,
2002) and biphenyl chalcones 33–35 (Thirunarayanan and
Vanangamudi, 2007) have been synthesized in the lab and
their physical constants agreed with the reported data.
4-Methyl-4,5-dihydro-1H-pyrido[2,3-b][1,4]
diazepin-2(3H)-one (42)
Yield 60 %, mp. 194 ꢁC, 1H NMR (CDCl3): d 7.94 (d, 1H,
J = 8.3 Hz, H-9), 6.66 (dd, 1H, J = 2.5 Hz, H-8), 7.95 (d,
J = 2.5 Hz, H-7), 3.02 (m, 1H, J = 4.2 Hz, J = 7.2 Hz,
J = 6.0 Hz, H-4), 2.49 (dd, 1H, J = 12.8 Hz, J = 4.2 Hz
123

Med Chem Res
H-3a), 2.22 (dd, 1H, J = 12.8 Hz, H-3b). 13C NMR
(75 MHz, DMSO-d6): 21.0 (Me), 42.7 (CH2), 53.0 (CH),
106–142 (Ar–C), 170.0 (C=O), GC–MS (m/z): 177 (M?).
CO2 in air, supernatant was collected for IL-2 determina-
tion. IL-2 levels were measured using enzyme-linked
immunosorbent assay (ELISA) development kit (R&D
systems, Minneapolis, MN, USA). 96-well ELISA plates
were coated with 4.0 lg/ml mouse anti-human IL-2, in
phosphate buffered saline (PBS) at 7.4 pH. Then, 100 lL
recombinant human IL-2 standards (R&D systems, Min-
neapolis, MN, USA) and culture supernatants samples were
added. The plates were incubated for 2 h. at room tem-
perature and washed three times with PBS, followed by the
addition of 100 lL/well of biotinylated goat anti-human
IL-2 and further incubated for 2 h at room temperature.
Plates were again washed and 100 lL/well of streptavidin
conjugated horse reddish peroxidase was added and incu-
bated for additional 20 min at room temperature. After
three final washes, 100 lL/well of the enzyme substrate
solution of H2O2 and tetramethylbenzidine (1:1 v/v) was
added and the color was allowed to develop at room tem-
perature in the dark. Plates were then read at 450 nm in a
plate reader (DIAReader GMBH, Wr. Neudorf, Austria).
Results were analyzed using Microsoft Excel software.
4-Methyl-4,5-dihydro-1H-naphtho[2,3-b][1,4]diazepin-
2(3H)-one (43)
1
Yield 60 %, mp. 240 ꢁC, HNMR (CDCl3): d 6.5–7.8 (m,
aryl-H), 8.0 (s, 1H, NH), 3.02 (m, 1H, J4,3a = 4.2 Hz,
J4,3b = 7.2 Hz, J4 = 6.0 Hz, H-4), 2.55(dd, 1H, J3a,3b
12.8 Hz, J3a,4 = 4.2 Hz, H-3a), 2.3 (dd, 1H, J3b,3a
=
=
12.8 Hz, J3b,4 = 7.2 Hz, H-3b), 1.10 (d, 3H, J4,Me = 6.0 Hz,
Me). 13C NMR (75 MHz, DMSO-d6): d 20.0(CH3), 42.7
(CH2), 53.0 (CH), 108–126 (Ar–C), 170.7 (C=O) GC–MS
(m/z): 226 (M?).
Biologic activities
T-cell proliferation assay
Fresh venous blood from healthy donor was mixed with
equal volume of RPMI-1640 incomplete medium (Media-
tech Inc., Herndon, VA, USA) containing 2 mM L-gluta-
mine. The diluted blood was then layered onto lymphocyte
separation medium (MP Biomedicals, Inc., Ohio, USA),
and centrifuged for 20 min at 25 ꢁC. The mononuclear cell
layer was collected, washed with incomplete RPMI-1640
and centrifuged for 10 min and at 4 ꢁC. The peripheral
blood mononuclear cells (PBMNCs) were resuspended in
RPMI-1640 containing 10 % fetal bovine serum of PAA
laboratories GmbH, Pasching, Austria. In a 96-well plate
(IWAKI, SciTech. DIV., Ashai Techno glass, Japan),
50 lL of cell suspension (2.5 9 106 cell/mL), 50 lL of
PHA with a final concentration of 5 lg/mL, 50 lL sup-
plemented RPMI-1640, and 50 lL of test compounds in a
final concentration of 0.5, 5, and 50 lg/mL in triplicate
were added. Plates were then incubated at 37 ꢁC in a
humidified atmosphere of 5 % CO2 in air for 72 h. To each
well, 0.5 lCi [methyl-3H]-thymidine (Amersham Place
Little Chalfont, Buckinghamshire, UK) was added for
additional 18 h. Cells were harvested using Inotech cell
harvester (Dottikon, Switzerland), and radioactive thymi-
dine incorporation level was measured by Beckman coulter
liquid scintillation counter (LS 6500, Fullerton, CA, USA).
Chemiluminscence assay (ROS)
In this assay, formation of the ROS in whole blood during
the oxidative burst was measured by the luminol-enhanced
chemiluminescence assay procedure (Waldmann and
O’Shea, 1998; Michael and David, 2005). In brief, three
concentrations of each compound (1.0, 10 and 100 lg/mL)
were prepared in 25 lL of Hank’s Buffered Salt Solution
(HBSS2?) in 96-well white flat-bottomed plate for a final
incubation volume of 100 lL. Then, 25 lL of whole blood
diluted 1:50 in suspension of HBSS2? with calcium chlo-
ride and magnesium sulfate from Sigma (St. Louis, USA)
was added. Positive, negative control, and blank wells were
included in the assay. Cells and compounds were incubated
for 30 min at 37 ꢁC, then 25 lL luminol (3-amin-
ophthalhydrazide) purchased from Research Organics
(Cleveland, OH, USA), was added into each well and
25 lL serum opsonized zymosan (Saccharomyces cerevi-
siae origin) purchased from Fluka, (Buchs, Switzerland)
was added except for negative and blank wells. The ROS
chemiluminescence kinetic was monitored with lumino-
meter from Lab Systems Luminoskan, (Helsinki, Finland)
for 50 min in the repeated scan mode. Peak and total
integral chemiluminescence reading were expressed in the
relative light unit (RLU).
IL-2 assay
The PBMNCs were cultured in a 96-well flat-bottomed
plate (1.0 9 105 cell/well) in the presence or absence
of three concentrations of test compounds (0.5, 5, and
50 lg/mL), and phytohemagglutinin PHA/PMA in a final
concentration of 5 and 20 ng/mL. After an incubation
period of 18 h at 37 ꢁC in a humidified atmosphere of 5 %
Cytotoxicity assays using 3T3-L1 and CC-1 cell-lines
and MTT
In vitro cytotoxicity assays were performed as described by
Scholz et al. (1999) using the 3T3-L1 mouse embryo
123

Med Chem Res
Molecular dynamics simulation method
fibroblast cell line (American Type Culture Collection
(ATCC), Manassas, VA 20108, USA), and CC-1 cells, a rat
Wistar hepatocyte cell line (European Collection of Cell
Cultures, Salisbury, UK). The CC-1 cells were suspended
in Minimum Essential Medium Eagle (MEM) sup-
plemented with 10 % FBS, 2 mM glutamine, 1 %
non-essential amino acids and, 20 mM HEPES, while the
3T3-L1 cells were suspended in Dulbecco’s Modified
Eagle’s Medium (DMEM) formulated with 10 % FBS.
Using flat-bottomed plates, both cell lines were plated at a
concentration of 6 9 104 cells/mL and incubated for 24 h
at 37 ꢁC and 5 % CO2 environment. After removal of
medium, cells were challenged with three different con-
centrations (1.0, 5.0, and 20 lg/mL) of compounds in
triplicate and were then further incubated for 48 h at 37 ꢁC
in CO2 incubator. Following exposure to each compound,
cells viability was assessed using 0.5 mg/mL of MTT
(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro-
mide) for 4 h followed by removal of supernatant and the
addition of DMSO to solubilize the formazan complex.
Plates were read at 540 nm after one minute shaking and
readings were processed using MS Excel software. Results
were expressed as mean SD of triplicate readings.
Molecular dynamics simulations were carried out for IL-2
(PDB entry code 1M48) with the docked inhibitor 6 in an
explicit solvent system using the AMBER 10.0 package.
AMBER03 force field parameters were used to establish
the potential of IL-2 and generalized AMBER force field
(GAFF) parameters were used to generate the potentials of
chalcone 6. To ensure the electroneutrality of the system,
one Na? ion was added in the modeled system followed by
solvation coupled with TIP3P rectangular box around the
solute unit. Overall, the system covered a dimension of
3
˚
70.056 9 77.104 9 90.733 A containing 37, 239 all-
atoms (having 10, 425 water molecules inclusive). The
solvated system was subjected to thorough energy mini-
mizations before MD simulation. First, restrain minimiza-
tion of water molecules was done while holding the solute
fixed (5,000 steps using the steepest descent algorithm
followed by 5,000 steps of conjugate gradient minimiza-
tions of the whole system). This was done to remove the
steric conflicts between a protein and water molecules and
also to relax the system. An unrestrained minimization was
then carried out using the same procedure as for restrained
minimization. Bond lengths involving hydrogen atoms
were constrained with SHAKE (Ryckaert et al., 1977)
Computational studies
2
˚
algorithm with harmonic restraints of 25 kcal/mol A . The
minimized system was first subjected to a gradual tem-
perature increase from 0 to 300 K over 40 ps, and then
equilibrated for 25 ps at 300 K. An unrestrained equili-
bration was also done for 100 ps followed by production
runs of 10 ns. NPT ensemble system that is constant tem-
perature (298 K) and constant pressure (1 atm) were con-
trolled by the Berendsen coupling algorithm with a time
constant for heat-bath coupling of 0.2 ps. The dielectric
constant was set to 1.0. The cut-off distance was set to
Molecular docking method
Docking studies were performed, using the Molecular
Operating Environment by Chemical Computing Group
Inc. (MOE 2011.10) on a Pentium 1.6 GHz workstation,
512 MB memory using the Windows Operating system.
The data of high resolution crystal structure of IL-2 com-
plex with its ligand FRG were obtained from Protein Data
Bank (PDB entry code 1M48) (Arkin et al., 2003). The
study was carried out on B subunit of enzyme protein. The
edited crystal structure after removing water molecules was
imported into MOE and all hydrogen atoms were added to
the structure with their standard geometry followed by their
energy minimization using MOPAC 7.0. The resulting
model was subjected to systematic conformational search
at default parameters with RMS gradient of 0.01 kcal/mol
using Site Finder (Ansari et al., 2011). Enzymes were
searched for their active sites and dummy atoms were
created from the resulting alpha spheres. The backbone and
residues were kept fixed and the energy minimization was
performed. RMSD was used to compare the ligand between
the predicted and its corresponding crystal structure. The
˚
9.0 A. Long-range electrostatic calculations were carried
out by particle mesh Ewald method (Essmann et al., 1995).
The PTRAJ module of AMBER package was used for the
analysis of the subsequent trajectories. VMD (version-
1.8.6) (Humphrey et al., 1996) CHIMERA (Pettersen et al.,
2004) was also used to visualize the snapshots (structure
changes obtained after the MD simulations). The MD
simulation studies were calculated using the MPI SANDER
module of AMBER 10 installed on cluster computing
facility consisting of 10 nodes (equipped with Intelꢂ
Xeonꢂ QuadTM core processor running under openSuSE
LINUX OS).
˚
resulting docked poses with RMSD less than 1.5 A were
Acknowledgments The authors Farzana Latif Ansari, Zaheer-ul-
Haq, and Saima Kalsoom acknowledge the Higher Education Com-
mission, Islamabad, Pakistan for providing financial support to con-
duct this study.
clustered together. The lowest energy minimized pose was
used for further analysis. Rule of five parameters (Ro5) were
calculated by ligand property calculation function of MOE.
123

Med Chem Res
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Lindelofia stylosa. Chem Biol Drug Des 79:290–299
Michael D, David S (2005) Transplant-related immunosuppression: a
review of immunosuppression and pulmonary infections. Proc
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