The immunomodulation potential of the synthetic derivatives of benzothiazoles: Implications in immune system disorders through in vitro and in silico studies

Article abstract of DOI:10.1016/j.bioorg.2015.11.004

Benzothiazole and its natural or synthetic derivatives have been used as precursors for several pharmacological agents for neuroprotective, anti-bacterial, and anti-allergic activities. The objecctive of the present study was to evaluate effects of benzot

Full text of DOI:10.1016/j.bioorg.2015.11.004

Accepted Manuscript
The Immunomodulation Potential of the Synthetic Derivatives of Benzothia-
zoles: Implications in Immune System Disorders through in vitro and in silico
Studies
Khalid Mohammed Khan, Mohammad A. Mesaik, Omer M. Abdalla, Fazal
Rahim, Samreen Soomro, Sobia A. Halim, Ghulam Mustafa, Nida Ambreen,
Ahmad Shukralla Khalid, Muhammad Taha, Shahnaz Perveen, Muhammad
Tanveer Alam, Abdul Hameed, Zaheer Ul-Haq, Hayat Ullah, Zia Ur Rehman,
Rafat Ali Siddiqui, Wolfgang Voelter
PII:
S0045-2068(15)30035-3
DOI:
http://dx.doi.org/10.1016/j.bioorg.2015.11.004
YBIOO 1858
Reference:
Bioorganic Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
18 September 2015
12 November 2015
17 November 2015
Please cite this article as: K.M. Khan, M.A. Mesaik, O.M. Abdalla, F. Rahim, S. Soomro, S.A. Halim, G. Mustafa,
N. Ambreen, A.S. Khalid, M. Taha, S. Perveen, M.T. Alam, A. Hameed, Z. Ul-Haq, H. Ullah, Z.U. Rehman, R.A.
Siddiqui, W. Voelter, The Immunomodulation Potential of the Synthetic Derivatives of Benzothiazoles:Implications
in Immune System Disorders through in vitro and in silico Studies, Bioorganic Chemistry (2015), doi: http://
dx.doi.org/10.1016/j.bioorg.2015.11.004
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The Immunomodulation Potential of the Synthetic Derivatives of Benzothiazoles:
Implications in Immune System Disorders through in vitro and in silico Studies
Khalid Mohammed Khana Mohammad A. Mesaikb,c Omer M. Abdallac, Fazal Rahima,d,
Samreen Soomroc, Sobia A. Halimc, Ghulam Mustafac,e, Nida Ambreena, Ahmad Shukralla
Khalidc, Muhammad Tahaa, Shahnaz Perveenf, Muhammad Tanveer Alama, Abdul Hameed,a
Zaheer Ul-Haqc,e, Hayat Ullahd, Zia Ur Rehmand, Rafat Ali Siddiquig and Wolfgang Voelterh
aH. E. J. Research Institute of Chemistry, International Center for Chemical and Biological
b
Sciences, University of Karachi, Karachi-75270, Pakistan, Faculty of Pharmacy, Universiti
c
Kebangsaan Malaysia, Jalan Raja Muda Abdul Aziz, 50300, Kuala Lumpur, Malaysia, Dr.
Panjwani Center for Molecular Medicine and Drug Research, International Center for Chemical
d
and Biological Sciences, University of Karachi, Karachi-75270, Pakistan, Department of
e
Chemistry. Hazara University, Mansehra, Khyber Pakhtunkhwa, Pakistan, Heidelberg Institute
for Theoretical Studies, (HITS), GmbH, Schloss-Wolfsbrunnenweg 35, 69118 Heidelberg,
f
Germany, PCSIR Laboratories Complex, Karachi, Shahrah-e-Dr. Salimuzzaman Siddiqui,
g
Karachi-75280, Pakistan, Agriculture Research Station, Virginia State University, Petersburg,
VA 23806, USA, hInterfakultäres Institut für Biochemie, Universität Tübingen, Hoppe-Seyler-Str.
4, 72076 Tübingen, Germany
Abstract
Benzothiazole and its natural or synthetic derivatives have been used as precursors for several
pharmacological agents for neuroprotective, anti-bacterial, and anti-allergic activities. The
objecctive of the present study was to evaluate effects of Benzothiazole analogs (compounds 1-
26) for their immunomodulatory activities. Eight compounds (2, 4, 5, 8-10, 12, and 18) showed
potent inhibitory activity on PHA-activated peripheral blood mononuclear cells (PBMCs) with
IC50 ranging from 3.7 to 11.9 µM compared to that of the standard drug, prednisolone < 1.5 µM.
Some compounds (2, 4, 8, and 18) were also found to have potent inhibitory activities on the
production of IL-2 on PHA/PMA-stimulated PBMCs with IC50 values ranging between < 4.0 –
12.8 µM. The binding interaction of these compounds was performed through silico molecular
docking. Compounds 2, 8, 9, and 10 significantly suppressed oxidative burst ROS production in
phagocytes with IC50 values between < 4.0 and 15.2 μM. The lipopolysaccharide (LPS)-induced
*Corresponding authors: E-mail: hassaan2@super.net.pk; khalid.khan@iccs.edu; mmesaik@hotmail.com
1

nitrites in murine macrophages cell line J774 were found to be inhibited by compounds 4, 8, 9,
and 18 at a concentration of 25µg/mL by 56%, 91%, 58%, and 78%, respectively. Furthermore,
compounds 5, 8, 12, and 18 showed significant (P<0.05) suppressive activity on Th-2 cytokine,
interleukin 4 (IL-4) with an IC50 range of < 4.0 to 40.3 μM. Interestingly compound 4 has shown
a selective inhibitory activity on IL-2 and T cell proliferation (naïve T cell proliferation stage)
rather than on IL-4 cytokine, while compound 12 displayed an interference with T-cell
proliferation and IL-4 generation. Moreover compound 8 and 18 exert non-selective inhibition
on both IL-2 and IL-4 cytokines, indicating a better interference with stage leading to humoral
immune response and hence possible application in autoimmune diseases.
Keywords: Benzothiazoles, T-cell proliferation, cytokines, molecular docking, ROS and Nitric
oxide.
1.
Introduction
Benzothiazole derivatives, natural or synthetic, exhibit a variety of biological activities and,
therefore, have been used as precursors for pharmacological agents for neuroprotective, anti-
bacterial, anti-allergic, immunosuppressive, and antiviral [1-6]. [18,19]. In addition,
benzothiazole and its derivatives are powerful antitumor agents. For example,
phenylbenzothiazoles, which have been synthesized as pro-drugs, were chosen for clinical
evaluation for anti-cancer activity [7-9]. A few other derivatives of benzothiazoles, especially
quinol and 2-(4-aminophenyl), have shown anti-cancer activities both, in in vivo and in vitro
systems [10-13]. Recently, a fluorinated 2-arylbenzothiazole was found to have selective and
potent inhibitory activity against colon, lung, and breast cancer cells [14]. In contrast, some
amino benzothiazoles have been shown to possess mutagenic potency depending on metabolic
activation [15] or to induce DNA damage [16]. For example, 2-p-tolyl-benzothiazoles and 2-m-
tolyl-benzothiazoles exibited mutagenic activity in the T100 strain of Salmonella [17]. At
cellular levels, benzothizole inhibit muscarinic receptor activation, lymphocyte-specific protein
tyrosine kinase (lck) and calmodulin (CaM) activities [20]. In continuation of our ongoing
research on the chemistry and bioactivity of new heterocyclic compounds, we reported recently
β-glucuronidase inhibitory activity of benzothiazole analogs [21].
2

The presented work describes re-synthesis and evaluation of the immunomodulatory potential of
a series of benzothiazoles (1-26), with special emphasis on their effect on the naïve Th cells and
Th-2 cytokine (IL-4). We also studied their effects on phytohemagglutinin (PHA)-induced
proliferation of T-cells and on IL-2 production in order to evaluate their therapeutic potential on
immune modulation, because the mediators of immune response, when overproduced, can cause
serious damage including inflammation and injury [22]. With molecular docking protocols, the
binding patterns of IL-2 inhibitors at the IL-2 receptor alpha (IL-2Rα) binding site [23,24] is
demonstrated. Using the GOLD docking program, the active IL-2 inhibitors were docked at the
ligand binding site of IL-2 protein and the mode of interaction was determined [25].
Furthermore, the effect of these compounds on the production of the short-lived nitric oxide
radical, [26], and the production of reactive oxygen species, in human peripheral blood
phagocytes [27], was determined. Additionally, the effect of these compounds on IL-4
production and their potential cytotoxic effects for normal cell lines were evaluated.
2. Results and Discussion
2.1. Chemistry
Re-synthesis of the benzothiazoles derivatives 1-26 was carried out by reacting 2-
aminothiophenol with different aromatic aldehydes in N, N-dimethylformamide (DMF) as
reported previously [21]. In this reaction, sodium metabisulfite (Na2S2O5) was added to a stirred
solution of 2-aminothiophenol (3.12 mmol) and a suitably-substituted aromatic aldehyde (3.16
mmol) in DMF. The resulting reaction solution was refluxed for 2 h. The progress of reaction
was monitored by TLC. After completion, the reaction was allowed to cool to room temperature.
After addition of water (30 mL), the benzothiazole derivatives 1-26 (Scheme-1) precipitated, and
were collected by filtration. Re-crystallization from methanol yielded pure products (Table-1).
The structures of compounds 1-26 were confirmed by different spectroscopic methods including
1H NMR and EI mass spectroscopy and found to match with the data as described previously
[21].
Insert Scheme-1 Here
Insert Table-1 Here
3

2.2.
Effect on T-Cell Proliferation
Efficacy of benzothiazoles on T-cells was tested using the T-cell proliferation assay. Eight
compounds, 2, 4, 5, 8-10, 12, and 18, showed significant suppressive activities (P < 0.005)
compared to that of the standard drug prednisolone (IC50 = < 1.5 µM) [28] with IC50 values of
11.5 ± 1.2, 10.1 ± 0.4, 11.9 ± 0.9, 4.5 ± 0.4, 4.9 ± 0.1, 4.1 ± 0.4, 3.7 ± 0.4, and 7.5 ± 1.5 µM,
respectively (Figure-1). The inhibitory activity on T-cell immune response of these compounds
might be useful as lead molecules for the development of drugs against various immune
disorders.
Insert Figure-1 Here
2.3. Effect on Th cells (Naïve) IL-2 cytokine
Compounds that showed inhibitory activity on the proliferation of T-cells were also tested for
their effect on the production of IL-2 by T-cells because IL-2 acts as a growth factor for T-cells.
Compounds 2, 4, 5, 8, and 18 were found to suppress IL-2 production significantly (p < 0.005, <
0.05), when compared to that of the mock control (activated cells without addition of
compounds). Four compounds, 2, 4, 8, and 18, were found to have potent inhibitory activity with
IC50 values of < 4.0, 12.8 ± 3.1, < 4.0, and 4.2 ± 0.4 µM, respectively. Compound 5 had a
slightly lower level of inhibition as compared to the afore-mentioned compounds with IC50 of 26
± 3.5 µM (Figure-2).
Insert Figure-2 Here
2.4. Effect on ROS generation
Compounds 1-26 were screened for immunomodulatory ROS activity. Out of twenty-six
screened compounds, only four (2, 8-10) showed significant inhibitory activity on t ROS
generation with IC50 values of 7.4 ± 2.3, < 4.0, 15.2 ± 0.8, and 4.5 ± 0.4 μM, respectively. These
values were superior to the standard Ibuprofen (IC50 = 57.2 ± 5.8 μM). The compounds 13, 14,
20, and 24 showed moderate inhibitory activity on ROS generation with IC50 values of 45.6 ±
4

3.7, 46.4 ± 4.7, 73 ± 0.5, and 66.3 ± 11.5 μM, respectively (Table-2). The remaining compounds
were found to be inactive, exhibiting less than 50% of inhibition. These results demonstrate that
the compounds could potentially exert an inhibitory effect on the innate immune response.
2.5.
Effect on NO production
The effect of these compounds on nitric oxide accumulation by stimulated macrophages was also
determined. Lipopolysaccharide (LPS)-treated J774.2 mice macrophages are widely used to
study the mechanisms of nitric oxide synthase (NOS-2) induction. Data shown in Table 2 and
Figure 3 indicates that out of twenty-six compounds tested, four of them, compounds 4, 8, 9, and
18 showed very potent inhibition at 25 mM concentration on NO production with percentages of
inhibition of 56.1 ± 0.6, 91.1 ± 0.7, 58.5 ± 3.3, and 78.1 ± 1.6, respectively (Table-2 and Figure-
3). Furthermore, the IC50 values for compounds 4, 8, 9, and 18 were found to be 34.8 ± 4.0, < 20,
51.8 ± 8.6, and 126 ± 16.6 μM, respectively. If NO is overproduced it reacts with superoxide and
gives rise to the very toxic radical peroxynitrite which may play a role in many diseases with an
autoimmune etiology. So, the suppression of NO could be a suitable target toward inflammatory
diseases.
Insert Table-2 Here
Insert Figure-3 Here2.6.
Effect on IL-4 cytokine
In order to study the target specificity of the compounds that inhibit the T-cell proliferation, the
Th-2 cytokine (IL-4) production was determined which act as a growth supporting factor for B
cells proliferation beside IL-2. Only compound 12 showed potent inhibitory activity with an IC50
value of < 4.0 µM, whereas compound 5, 8, and 18 had moderate inhibitory activities with IC50
values of 39.2 ± 2.6, 40.3 ± 2.5, and 31 ± 0.4 µM, respectively (Figure-4). Although compounds
2, 4, 5, 8, and 18 suppress production of IL-2, not a similar inhibitory activity for the IL-4
production was observed. This effect reflects that these compounds specifically inhibit the Th
cells (Naïve) IL-2 cytokine rather than Th-2 cytokines (IL-4 in particular).
Insert Figure-4 Here
5

2.7. Cytotoxicity
To determine the cytotoxic effect of those compounds on normal cells, the MTT assay was
performedusing the normal mouse fibroblast cell line 3T3 NIH. We observed that except 18 all
of the tested compounds 2, 4, 5, 8, 10, 14, and 24 did not suppress the growth of 3T3 cells even
at the highest concentration (100 μM). However, mild cytotoxicity was shown with compounds 9
and 13. Compound 18 showed a cytotoxic activity with an IC50 value of 40.1 ± 5.0 µM.
Therefore, we assume that benzothiazole derivatives can act as anti-inflammatory lead
molecules.
2.8.
Molecular Docking Studies of IL-2 Inhibitors
Molecular docking is the most widely used computational tool to identify protein-ligand
interactions [29] and gain insights into the molecular mechanism of protein-ligand binding, e.g.
to determine the binding orientation of active IL-2 inhibitors into the ligand binding site. The
cytokine IL-2 binding site is a heterotrimeric receptor complex consisting of α, β and γ chains
(IL-2Rα, IL-2Rβ and IL-2Rγ). Reported studies suggest that antibodies that block IL-2-IL-2Rα
binding have effective clinical implication [30, 31]. Therefore, the new IL-2 inhibitors were
targeted against the IL-2Rα binding site which is dissected into two distinct sub-sites; the first is
relatively fixed and includes the center of helix B and the A′-B loop and the other is highly
mobile which comprises the C-terminus of helix B, the loop connecting helices A and A′, and the
loop connecting helix B to helix C [35]. Prior to the docking of compounds 2,4,5,8, and 18, the
co-crystallized compound (phenylalanine methyl ester derivative) was successfully re-docked
into the ligand binding site of the protein with a RMSD value of 1.2Å. The results are displayed
in supporting information. The re-docking analysis suggested that the Swiss Dock docking server
was able to predict the known binding conformation accurately and can be used to elucidate the
binding patterns of our synthesized compounds. The docked binding modes of compounds 2, 4,
5, 8, and 18 revealed that all these compounds fit into the cavity formed between the A and A′
helix and B helix (Fig 5a). The binding site residues lying within 5 Å for all five active
compounds were found to be Lys32, Pro31, Arg35, Meth36 (A’ helix) and the hydrophobic
residues Val66 and Leu69 (residing on B helix). The dock poses of the active compounds
reflected that Arg35 and Lys32 are important for hydrogen bonding with the benzothiazole ring
and the substituted phenyl ring containing a hydroxyl or methoxy group. Moreover, two
important amino acid residues from the B helix (Val66 and Leu69) showed hydrophobic
6

interactions with the aromatic ring of the benzothiazol skeleton (Fig 5b). Additional π-π
interactions exist between the aromatic rings of the ligands Phe39 C-terminus of the A′ helix.
These interactions were expected to be clearly observed during molecular dynamics simulation
due to possible side chain flipping and reorientation of the ligand in the binding pocket. These
adaptable changes may help in stabilization of these compounds by providing π-π interactions.
The docked binding modes of five active compounds and side chain residues interacting with
ligand 2 are shown in Figures 5a-c.
Insert Figure-5a Here
Insert Figure-5b Here
Insert Figure-5c Here
2.9.
Molecular Dynamics Simulation:
To further validate and confirm the binding mode of ligands predicted by Swiss Dock, molecular
dynamics (MD) simulation studies were carried out in aqueous medium. A total of 15 ns MD
simulations were performed in an explicit solvent model. Results were analyzed to check the
overall structural stability by using root mean square deviation (RMSD) and the energy output.
The structure seems to be stable after 2 ns of unrestrained equilibration. RMSD for all backbone
atoms was calculated by using a minimized structure as a reference point and it showed RMSD
convergence around 1.75 Å with slight fluctuations, which were expected due to highly mobile
loop regions discussed earlier. Fluctuation for each residue was carried out using the B-factor
analysis which showed major flexibility for the residues forming loop regions, whereas ligand
binding region seemed to be stable. The energy output was calculated using perl script which
showed an increase in the potential energy during heating, but after that it was decreased during
equilibration and remained stable during production run. Trajectories were visualized by using
VMD software (Figure-6).
Insert Figure-6 Here
The binding mode of ligands was analyzed for entire trajectories which showed no difference
from initial conformation and remained stable in the binding pocket as identified by Swiss Dock.
The superposition of starting structure with last snapshot of 15 ns simulation was performed to
see any dynamic change adopted by the ligand during simulation. Although the ligand tries to
7

adopt different conformations, it returns back to the original position. This may be due to its
strong hydrophobic interaction between V66 and L69 residues with the benzothiazole skeleton.
At the some point aromatic interactions are visible between F39, which flips closer to the
benzene ring of the ligand (Figure-6). From MD simulation studies, we observed stable
interactions between ligand and its binding pocket residues which was maintained throughout the
simulation. A further detailed MD simulation study is beyond the scope of this manuscript.
Insert Table-3 Here
3.
Conclusion:
All twenty six benzothiazoles were tested on various assays and it was found that some
derivatives show potent inhibitory potential for the generation of IL-2, IL-4, ROS, and NOS.
Eight compounds, 2, 4, 5, 8-10, 12, and 18, exhibited dose-dependent suppressive activities on
PHA-activated T-cell proliferation.. However, only compounds 2, 4, 5, 8, and 18 were found to
suppress IL-2 .production, whereas compound 12 showed potent inhibitory activity on IL-4
generation beside T cell proliferation inhibition. Compounds 2, 8-10 showed a potent inhibitory
activity on ROS formation compared to that of the standard control (ibuprofen). Compounds 4,
8, 9, and 18 were also acted as highly potent inhibitors for NO generation as well. IL-2 functions
as a master regulator to direct for cell fate through priming for differentiation and maintaining
the differentiated state. One of tragic effects of IL-2 is promotion of inflammatory responses
which is mainly maintained through the generation of Th1 and Th2 effector cells. These effects
could be reduced through selective inhibition. From these results we can observe compounds 4
and 12 to exert selective inhibition on IL-2, IL-4, respectively. Additionally compound 8 and 18
exert non-selective inhibition on IL-2 as well as on IL-4 cytokines indicating a better interference
with stage leading to humoral immune response. While compound 4 found effecting merely the
naïve T cell proliferation stage. Consequently, in silico studies were performed to recognize the
binding mode of these compounds. The planned scaffold of immunomodulatory agents deals
with the possibility of further beneficial modifications that could give rise to lead molecules with
improved inhibitory activity and selectivity. Cytotoxicity of the above identified
8

immunosuppressive compounds was excluded by MTT, as assayed by the murine fibroblast cell
line 3T3 of NIH. Therefore, we anticipate that the benzothiazole derivatives which were
evaluated in the present study, can act as valuable anti-inflammatory/immunosuppressive lead
agents. However, the exact mode of action of these compounds and the detail of activities
deserve detail investigations which are in progress.
4.
Experimental Section
4.1.
Materials and Methods
All reagents and solvents were purchased from E. Merck, Germany and were of analytical grade.
NMR experiments were performed on an Avance Bruker AM 300 MHz machine. CHN analyses
were carried out on a Carlo Erba Strumentazione-Mod-1106, Milano, Italy. Ultraviolet (UV)
spectra were recorded on a Perkin-Elmer Lambda-5 UV/VIS spectrophotometer in MeOH and
infrared (IR) spectra was recorded on a JASCO IR-A-302 spectrometer as KBr discs. Electron
impact mass spectra (EI MS) were obtained on a Finnigan MAT-311A (Germany) mass
spectrometer. Thin-layer chromatography (TLC) was performed on pre-coated silica gel
aluminum plates (Kieselgel 60, 254, E. Merck, Germany). Chromatograms were visualized either
by UV at 254 and 365 nm or iodine vapors.
4.2. General procedure for the synthesis of compounds 1-26
In a typical reaction, benzothiazoles 1-26 were re-synthesized by dissolving 2-aminothiophenol
(3.12 mmol) and different aromatic aldehydes (3.16 mmol) in DMF (10 mL). Sodium
metabisulfite (Na2S2O5, 0.61 g) was also added to the above-mentioned solution with continuous
stirring. The resulting reaction mixture was refluxed for 2 h and the progress of the reaction was
monitored by TLC analysis. After completion of the reaction, it was allowed to cool to room
temperature and water (30 mL) was added. The solid precipitated benzothiazoles (1-26) were
collected on a filter and obtained in high yields. Recrystallization from methanol yielded pure
products and their physical and spectroscopic data matched satisfactorily with previously
reported literature values [21].
4.3.
T-cell proliferation assay
Fresh venous blood from healthy donors was mixed with an equal volume of incomplete RPMI-
1640 media (Mediatech Inc., Herndon, VA, USA) containing 2 mM L-glutamine and 1%
9

penicillin/streptomycin. The diluted blood was then layered onto lymphocyte separation medium
º
(MP Biomedicals, Inc., Ohio, USA) and centrifuged at 400 g for 20 min at 25 C. The
mononuclear cell layer was collected, washed with RPMI-1640 and centrifuged at 300 g for 10
º
min at 4 C. The peripheral mononuclear blood cells (PBMNCs) were re-suspended in RPMI-
1640 complete medium containing 10% fetal bovine serum (PAA laboratories GmbH, Pasching,
Austria). In a 96-well round-bottomed plate (Iwaki, Scitech. DIV., Ashai Techno Glass, Japan),
50 µL of cell suspension (2.5 × 106 cell/mL), 50 µL of phytohemagglutinin (PHA) for a final
concentration of 5 mM, 50 µL of supplemented RPMI-1640 along with 50 µL of test compounds
in a final concentration of 0.5, 5 and 50 mM (in triplicates) were added to the culture mixture.
°
Plates were then incubated at 37 C in a humidified atmosphere of 5% CO2 in air for 72 h. To
each well, 0.5 µCi [methyl-3H]-thymidine (Amersham Place Little Chalfont, Buckinghamshire,
UK) was added for an additional 18 h. Cells were harvested using a cell harvester (Inotech,
Dottikon, Switzerland), and the level of radioactive [methyl-3H]-thymidine incorporated in
newly synthesized DNA was measured using a liquid scintillation counter (Beckman coulter, LS
6500, Fullerton, CA, USA).
4.4.
Interleukin-2 (IL-2) assay
The PBMCs were cultured in a 96-well flat-bottomed plate (1.0 x 105cell/well) in the presence or
absence of three concentrations of test compounds (1.0, 5.0, and 20 mM) with
phytohemagglutinin (PHA) and phorbol myristate acetate (PMA) in a final concentration of 5
°
mM and 20 mM, respectively. After an incubation period of 18 h at 37 C in a humidified
atmosphere of 5% CO2 in air, the supernatant was collected for IL-2 determination. Interleukin-2
levels were measured using an enzyme-linked immunosorbent assay (ELISA; R&D Systems,
Minneapolis, MN, USA). The assay was performed according to the manufacturer’s instructions.
Briefly, a 96-well flat-bottomed ELISA plate was coated with 4.0 mM mouse anti-human IL-2 in
PBS, pH 7.4. Then, re-combinant human IL-2 standards and culture supernatant samples were
added and incubated for 2 h followed by washing steps and finally addition of biotinylated goat
anti-human IL-2. After an incubation period of 2 h at room temperature, the plates were again
washed and streptavidin-conjugated horseradish peroxidase was added to each well and the
plates were incubated for additional 20 min at room temperature. After final washing, an enzyme
substrate solution of H2O2 and tetramethylbenzidine (1:1 v/v) was added, and the color was
allowed to develop at room temperature in the dark. The plates were then read at 450 nm in a
10

plate reader (DIAReader GMBH, Wiener, Neudorf, Austria). The results were analyzed using the
Microsoft Excel Program.
11

4.5. Chemiluminscence assay (ROS)
Formation of reactive oxygen species (ROS) in whole blood during the oxidative burst was
determined by the luminol-enhanced chemiluminscence assay [32]. In brief, three concentrations
of each compound (1.0, 10, and 100 mM) were prepared in 25 µL of Hank's balanced salt
solution containing calcium chloride and magnesium sulfate (HBSS++) in a half area 96-well
white flat-bottomed plate (Sigma Aldrich, Steinheim, Germany) for a final volume of 100 µL.
Then 25 µL of whole blood diluted 1:50 in a suspension of HBSS++ was added. Positive
(zymosan-activated cells), negative controls (non-activated cells) and blank wells were included.
º
Cells and compounds were incubated for 30 min at 37 C, then 25 µL luminol (3-
aminophthalhydrazide; Research Organics Cleveland, Ohio, USA), was added into each well
followed by addition of 25 µL of serum-opsonized zymosan (Saccharomyces cerevisiae origin;
Fluka, Buchs, Switzerland) except for negative and blank wells. The ROS chemiluminescence
kinetic was monitored with a luminometer from Labsystems (Helsinki, Finland), for 50 minutes
in the repeated scan mode. Peak and total integral chemiluminescence readings were expressed
in the relative light unit.
4.6.
Nitrite concentration in Mouse Macrophage Culture Medium
The mouse macrophage cell line J774.2, obtained from ECACC, Salisbury, Wiltshire (UK), was
cultured in T75 flasks in Dulbecco's Modified Eagle Medium (DMEM) containing 10% fetal
°
bovine serum, supplemented with 1% streptomycin/penicillin. Flasks were kept at 37 C in the
atmosphere of humidified air containing 5% CO2. Cells were then seeded in a 24-well plate (106
cells/mL), and nitric oxide synthase (NOS2) in the macrophages was induced by the addition of
20 mM E. coli lipopolysaccharide (LPS) (DIFCO Laboratories, Michigon, USA). The test
compounds were added at a concentration of 25 mM soon after LPS stimulation and incubated at
°
37 C in 5% CO2. The cell culture supernatant was collected after 24 h. Nitrite accumulation in
the J774.2 cell culture supernatants was determined using the Griess method [33], where 50 µL
of 1% sulphanilamide in 2.5% phosphoric acid, followed by 50 µL of 0.1% (1-naphtyl)
ethylenediamine in 2.5% phosphoric acid were added to 50 µL culture medium. After 10 minutes
of incubation at room temperature, the absorbance at 550 nm was read. Micromolar
concentrations of nitrite were calculated from a standard curve constructed with sodium nitrite as
reference compound. The results are expressed as means ± SD of three experiments.
12

4.7.
Interleukin-4 (IL-4) assay
The PBMNCs were cultured in a 96-well flat-bottomed plate (2.0 x 106cell/mL) in the presence
or absence of three concentrations of test compounds (1.0, 5.0, and 25 mM) and
phytohemagglutinin (PHA) in a final concentration of 7.5 mM. After an incubation period of 18
º
h at 37 C in 5% CO2, the supernatant was collected for IL-4 determination. Interleukin-4 levels
were determined by using an enzyme-linked immunosorbent assay (ELISA) kit (Diaclone,
France). The assay was performed according to the manufacturer’s instructions. Briefly, in a 96-
well ELISA, coated with monoclonal mouse anti-human IL-4, 100 µl recombinant human IL-4
standards and culture supernatant samples were added. The plates were incubated for 2 h at room
temperature and washed three times with washing buffer, followed by the addition of
biotinylated goat anti-human IL-4 and further incubated for 1 h at room temperature. Then, the
plates were again washed, and streptavidin-conjugated horseradish peroxidase was added and
incubated for an additional 20 min at room temperature. After three final washings, the enzyme
substrate solution was added and incubated for 12 to 15 min to allow color development at room
temperature in the dark. Then stop solution was added and the plates were read at 450 nm in a
plate reader (DIAReader GMBH, Wiener, Neudorf, Austria). The results were analyzed using
Microsoft Excel Program.
4.8.
Cytotoxicity assessment using MTT assay
In vitro cytotoxicity assays were performed as described previously [34] using the 3T3 NIH
mouse embryo fibroblast cell line (American Type Culture Collection ‘ATCC, Manassas, VA
20108, USA). The 3T3-NIH cells were suspended in Dulbecco's Modified Eagle's Medium
(DMEM) formulated with 10% FBS. In flat-bottomed plates, the cells were plated at a

concentration of 6 × 104 cells/mL and incubated for 24 h at 37 C and 5% CO2 environment.
After removal of media, the cells were challenged with three different concentrations (0.5, 5.0,

and 25 mM) of compounds in triplicates and then further incubated for 48 h at 37 C in a CO2
incubator. Cell viability was assessed by exposure of 0.5 mM of MTT (3-(4,5-dimethylthiazol-2-
yl)-2,5-diphenyltetrazolium bromide) for 4 h followed by the removal of the supernatant and
addition of DMSO to solubilize the formazan complex. The plates were read at 540 nm after one
minute shaking and readings were processed using MS Excel software Program. The results were
expressed as means ± SD of three experiments.
13

4.9. Molecular Docking
For investigation of binding modes of IL-2 inhibitors and their interactions at molecular level,
docking and all atomic molecular dynamics (MD) simulations were performed. For docking
calculations, the crystal structure of interleukin-2 in complex with phenylalanine methyl ester
(PDB entry code 1M48) was retrieved from the RCSB Brookhaven Protein Data Bank [35,36].
In the crystal structure of 1M48, residues forming the BC loop (75-80) were missing which were
modeled by transferring coordinates from IL-2 PDB structure (3INK) using PYMOL software.
The modeled structure was run for short minimization using the AMBER12 software package
which was then used as starting structure for docking studies. All water molecules were removed
from the original PDB crystal structure and hydrogen atoms were added by default program in
tleap during minimization. Lignad structures were constructed by using the ChemDraw program
and converted into 3D using babel 2.2. Geometry optimizations for the 3D structures were
performed using Tripos force field with a distance-dependent dielectric and the Powell conjugate
gradient algorithm [37]. Gasteiger-Hückel charges were used for the ligands. Online web server
Swiss Dock was used for docking studies of experimentally-determined active compounds (2, 4,
5, 8, and 18) which a separate pdb file for protein and mol2 files for ligands were uploaded and
default parameters were selected to perform docking on the whole protein. After the docking
assay was completed a zip file was generated for showing various binding modes. UCSF
Chimera was used for visualization of binding modes.
4.10.
Molecular Dynamics Simulations
On basis of highest ranking binding mode (BMs) of ligands with IL-2, generated by Swiss Dock,
we selected ligand 2 for further confirmation of active site residue interactions using explicit
solvent molecular dynamics simulations. The docked complex was prepared in tleap package in
AMBER 12 (Amber generalized force field for organic molecules) using FF99SB and GAFF for
protein-ligand complex, respectively. Before using tleap all the parameters for ligand were
prepared in antechamber program given in AMBER package. The system was solvated by using
a TIP3P octahedral 10 Å water box which added a total of 7014 water residues, making up a total
system of 23220 atoms. Energy minimization was performed in two stages: firstly by applying a
positional restrain weight of 500 kcal/mol·Å2 on all atoms except for hydrogen (1000 steps of
steepest descent minimization and 4000 steps of conjugate gradient methods), second by step
14

minimization (10,000 steps), done by simply releasing restrain weight from all atoms. The
heating was perform by slowly increasing temperature from 10 to 300 K using NVT ensemble in
25 ps with position restrain applied on all heavy atoms (50 kcal/mol·Å2 ). During heating
integration the time step was set to 1 fs and Langevin dynamics collision frequency γ = 1.
Heating was followed by 200 ps equilibration under NPT ensemble with slowly decreasing the
force of restrain on heavy atoms from 50 kcal/mol·Å2 to 0 in 8 steps. During equilibration,
temperature and pressure were kept constant at 300 K and 1.0 bar by applying Langevin
thermostat and isotropic position rescaling, respectively, at 1ps relaxation time. Finally, all the
restrains were removed during 2 ns equilibration with SHAKE algorithm to constrain all the
bonds involving hydrogen atoms and integration time was increased to 2 fs. The MD simulation
was performed by keeping particle mesh ewald (PME) on, and non-bonded interactions were
truncated at a cut off value of 10 Å.
Acknowledgement:
The authors are thankful to the Higher Education Commission (HEC) Pakistan (Projects No. 20-
2073) for financial support and to DAAD (Deutscher Akademischer Austauschdienst), Bonn,
(Germany) for fellowship grant to Prof. Dr. Khalid M. Khan.
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19

Captions:
Scheme-1: Synthesis of Benzothiazole derivatives 1-26
Figure-1: Effect of compounds 2, 4, 5, 8-10, 12, and 18 on T-cell proliferation. PBMNCs were
stimulated with 5 µg PHA in the presence of three concentrations of test compounds or without
addition of compounds (+ve). Cell proliferation was assessed as indicated in the section
Materials and Methods. The figure represents compounds that show potent activity against T-cell
proliferation the bars indicate mean radioactivity (CPM) ± SD of triplicate measurements.
Significance difference was calculated in comparison with the PHA positive control (+ ve), P <
0.005**, P < 0.05*. -ve: control cells without PHA or compound.
Figure-2: Concentration-response graph for IL-2 production by PBMNCs. The bars represent
the effect of compounds 2, 4, 5, 8, and 18 on IL-2 production by PHA/PMA-stimulated PBMCs
after an incubation of 18 h. Data are mean values ± S.D (n=3) of produced IL-2 in pg/mL.
P<0.05*, P< 0.005**
Figure-3: Concentration-response curve for benzothiazoles 4, 8, 9, and 18 on NO production by
macrophages-stimulated with LPS in the presence of increasing concentrations of the
compounds. Results shown are the mean ± SD of triplicate measurements expressed as
percentage of stimulated control in the absence of compounds.
Figure-4: Effect of benzothiazoles on the production of IL-4 using human peripheral blood mononuclear
cells (PMBCs). The graph represents the effect of seven benzothiazoles on PHA-stimulated IL-4
production after 18 h incubation. Each bar represents a mean of triplicate readings. + C: cells in the
presence of PHA, - C: cells without PHA.
Figure-5a: Binding orientation of ligand-2, docked in the binding cavity between A’ and B
helices. Top 9 clusters generated by SwissDock are shown in this figure where the
benzothiazole skeleton is found to be rigid and shows the same orientation, but only a slight
difference in the orientation of the R group, connected to the skeleton by a rotatable bond, is
observed.
Figure-5b: Binding of five different ligands superimpose completely on each other. The
benzothiazol skeleton shows binding at the same position, while, the R group has a slightly
different orientation as seen in the previous figure.
20

Figure 5c: Superimposed structures of 1M48 with the crystallized ligand FRG (in pink) and
docked compound 2 (yellow). The binding site residues interacting with compound 2 (in yellow)
within 5 Å distance are shown in magnified area. Main residues from A’ (K32, P31, R35, and
M36) and B helices (V66 and L69) show interactions with the ligand. All figures were prepared
using UCSF Chimera and Pymol software.
Figure-6: RMSD graph shows structural convergence after 1ns equilibration using a minimized
structure as a reference point.
Scheme-1:
21

Figure-1:
25000
20000
15000
10000
5000
50 µg/mL
5.0 µg/mL
0.5 µg/mL
*
*
*
*
*
*
**
**
**
**
**
**
**
**
**
**
**
**
**
**
**
**
0
2
4
5
8
9
10
12
18
+ ve
- ve
Figure-2:
20 µg/mL
5.0 µg/mL
1.0 µg/mL
1400
1200
1000
800
600
400
200
0
*
**
**
**
**
**
**
**
**
**
**
**
**
2
4
5
8
18
+ ve control
22

Figure-3:
120
4
9
100
80
60
40
20
0
18
8
25
5
0.5
Figure-4:
25 µg/mL 5.0 µg/mL 1.0 µg/mL
60
50
40
30
20
10
0
4
5
8
9
10
12
18
+ C
- C
23

Figure-5a:
24

Figure-5b:
25

Figure-5c:
26

Figure-6:
27

Basic Skeleton of Benzothiazole
Table-1: Structure of the Benzothiazoles (1-26) that were evaluated for anti-inflammatory
activity
Comp.
No.
Comp.
No.
Comp.
No.
R
R
R
OH
OH
OH
2'
6'
5'
6'
5'
6'
5'
1
2
3
4
5
10
11
12
13
14
19
20
21
22
23
N
OEt
4'
4'
4'
HO
6'
5'
Cl
6'
2'
3'
6'
5'
3'
5'
4'
MeO
N
Cl
1'
8'
7'
6'
2'
3'
6'
5'
2'
6'
5'
3'
4'
OMe
OH
Br
5'
OMe
2'
3'
6'
5'
2'
3'
6'
5'
6'
5'
3'
O H
2'
OEt
OH
N
6'
5'
2'
3'
F
6'
6'
5'
5'
4'
3'
OH
4'
1'
OH
S
2'
3'
6'
5'
6'
5'
6
7
15
16
24
25
3'
3'
4'
Cl
4'
Me
Me
OH
2'
3'
6'
5'
6'
5'
6'
5'
3'
3'
4'
4'
Cl
28

1'
2'
6'
5'
OH
6'
2'
6'
5'
8
9
17
18
26
Cl
3'
HO
Cl
4'
Cl
4'
1'
OH
2'
6'
6'
5'
3'
OH
HO
OH
OH
Table-2: Effect of benzothiazoles (1-26) on phagocyte oxidative burst activity and nitrite
production
IC50 (µg/mL) for
phagocytes ROS
% of NO inhibited by 25
µg/mL of compounds
Cytotoxicity
IC50 (µg/mL)
Compounds
1
2
>100
1.9 0.6
>100
26.5 0.0
28.9 1.3
20.5 5.9
56.1 0.6
44.5 0.3
35.5 7.4
5.4 2.8
ND
>25
3
ND
4
>100
>25
5
>100
>20
6
34.3 4.4
>100
ND
7
ND
8
<1
91.1 0.7
58.5 3.3
14.3 1.0
-7.4 3.0
-1.5 0.6
32.7 8.3
16.8 0.8
10.8 2.4
20.2 1.5
14.1 3.0
78.1 1.6
11.7 1.7
41.6 4.7
11.5 2.7
>20
9
3.7 0.2
1.1 0.1
>100
11.4 1.8
ND
10
11
12
13
14
15
16
17
18
19
20
21
ND
>100
ND
11.1 0.9
11.8 1.2
>100
11.5 0.0
>25
ND
>100
ND
81.0 4.1
>100
ND
10.4 1.3
ND
>100
15.5 1.0
>100
>25
ND
29