Trisubstituted-imidazoles induce apoptosis in human breast cancer cells by targeting the oncogenic PI3K/Akt/mTOR signaling pathway

Article abstract of DOI:10.1371/journal.pone.0153155

Overactivation of PI3K/Akt/mTOR is linked with carcinogenesis and serves a potential molecular therapeutic target in treatment of various cancers. Herein, we report the synthesis of trisubstituted-imidazoles and identified 2-chloro-3-(4, 5-diphenyl-1H-imidazol-2-yl) pyridine (CIP) as lead cytotoxic agent. Na?ve Base classifier model of in silico target prediction revealed that CIP targets RAC-beta serine/threonine-protein kinase which comprises the Akt. Furthermore, CIP downregulated the phosphorylation of Akt, PDK and mTOR proteins and decreased expression of cyclin D1, Bcl-2, survivin, VEGF, procaspase-3 and increased cleavage of PARP. In addition, CIP significantly downregulated the CXCL12 induced motility of breast cancer cells and molecular docking calculations revealed that all compounds bind to Akt2 kinase with high docking scores compared to the library of previously reported Akt2 inhibitors. In summary, we report the synthesis and biological evaluation of imidazoles that induce apoptosis in breast cancer cells by negatively regulating PI3K/Akt/mTOR signaling pathway. Copyright:

Full text of DOI:10.1371/journal.pone.0153155

RESEARCH ARTICLE
Trisubstituted-Imidazoles Induce Apoptosis
in Human Breast Cancer Cells by Targeting
the Oncogenic PI3K/Akt/mTOR Signaling
Pathway
1
☯
2☯
3☯
4
Chakrabhavi Dhananjaya Mohan , V. Srinivasa , Shobith Rangappa , Lewis Mervin ,
5
4,6
4,7
8
8
Surender Mohan , Shardul Paricharak , Sefer Baday , Feng Li , Muthu K. Shanmugam ,
9
9
9
4
Arunachalam Chinnathambi , M. E. Zayed , Sulaiman Ali Alharbi , Andreas Bender ,
8
,9,10
2
1
a11111
Gautam Sethi
, Basappa *, Kanchugarakoppal S. Rangappa *
1
2
Department of Studies in Chemistry, Manasagangotri, University of Mysore, Mysore 570006, India,
Laboratory of Chemical Biology, Department of Chemistry, Bangalore University, Central College Campus,
Palace Road, Bangalore 560001, India, 3 Frontier Research Center for Post-Genome Science and
Technology, Hokkaido University, Sapporo 060–0808, Japan, 4 Centre for Molecular Informatics,
Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge, United Kingdom,
5
Laboratory of Molecular Biology and Genetic Engineering, School of Biotechnology, Jawaharlal Nehru
University, New Delhi 110067, India, 6 Division of Medicinal Chemistry, Leiden Academic Centre for Drug
Research, Leiden University, P.O. Box 9502, 2300 RA Leiden, The Netherlands, 7 Applied Informatics
Department, Informatics Institute, Istanbul Technical University, 34469, Istanbul, Turkey, 8 Department of
Pharmacology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore,
OPEN ACCESS
Citation: Mohan CD, Srinivasa V, Rangappa S,
Mervin L, Mohan S, Paricharak S, et al. (2016)
Trisubstituted-Imidazoles Induce Apoptosis in Human
Breast Cancer Cells by Targeting the Oncogenic
PI3K/Akt/mTOR Signaling Pathway. PLoS ONE
9
Department of Botany and Microbiology, College of Science, King Saud University, Riyadh -11451,
Kingdom of Saudi Arabia, 10 School of Biomedical Sciences, CHIRI Biosciences Research Precinct, Curtin
University, Western Australia 6009, Australia
11(4): e0153155. doi:10.1371/journal.pone.0153155
☯
These authors contributed equally to this work.
*
salundibasappa@gmail.com (B); rangappaks@yahoo.com (KSR)
Editor: Luis Eduardo M Quintas, Universidade
Federal do Rio de Janeiro, BRAZIL
Received: August 21, 2015
Accepted: March 24, 2016
Published: April 20, 2016
Abstract
Overactivation of PI3K/Akt/mTOR is linked with carcinogenesis and serves a potential
molecular therapeutic target in treatment of various cancers. Herein, we report the synthesis
of trisubstituted-imidazoles and identified 2-chloro-3-(4, 5-diphenyl-1H-imidazol-2-yl) pyri-
Copyright: © 2016 Mohan et al. This is an open
access article distributed under the terms of the
Creative Commons Attribution License, which permits dine (CIP) as lead cytotoxic agent. Naïve Base classifier model of in silico target prediction
unrestricted use, distribution, and reproduction in any
revealed that CIP targets RAC-beta serine/threonine-protein kinase which comprises the
medium, provided the original author and source are
Akt. Furthermore, CIP downregulated the phosphorylation of Akt, PDK and mTOR proteins
credited.
and decreased expression of cyclin D1, Bcl-2, survivin, VEGF, procaspase-3 and increased
Data Availability Statement: All relevant data are
within the paper and its Supporting Information files.
cleavage of PARP. In addition, CIP significantly downregulated the CXCL12 induced motil-
ity of breast cancer cells and molecular docking calculations revealed that all compounds
Funding: This research was supported by University
Grants Commission (41-257-2012-SR), Vision Group
bind to Akt2 kinase with high docking scores compared to the library of previously reported
Science and Technology, and Department of Science
and Technology (NO. SR/FT/LS-142/2012) to
Akt2 inhibitors. In summary, we report the synthesis and biological evaluation of imidazoles
that induce apoptosis in breast cancer cells by negatively regulating PI3K/Akt/mTOR signal-
ing pathway.
Basappa. KSR thanks Department of Science and
Technology Indo-Korea [INT/Indo-Korea/122/2011-12]
and Institution of Excellence, University of Mysore for
financial support and instrumentation facility. CDM
thanks the University of Mysore for Department of
Science and Technology-Promotion of University
PLOS ONE | DOI:10.1371/journal.pone.0153155 April 20, 2016
1 / 15

Imidazoles Abrogate Oncogenic PI3K/Akt/mTOR Signaling Pathway
Research and Scientific Excellence (DST-PURSE)
Research Associate fellowship. This work was
supported by NUHS Bench-to-Bedside-to-Product
Introduction
PI3K/Akt/mTOR pathway is a major signaling cascade which operates downstream to the
grant to GS. Deanship of Scientific Research, College receptor tyrosine kinases such as epidermal growth factor receptor (EGFR), platelet-derived
of Science Research Centre, King Saud University,
growth factor receptor (PDGFR) and insulin-like growth factor-1 receptor (IGF-1R) [1–3]. The
relay of signals from the aforementioned growth factor receptors leads to the activation of Phos-
phoinositide 3-kinase (PI3K) which catalyses the generation of phosphatidylinositol-3,4,5-tri-
phosphate (PIP3) from phosphatidylinositol-4,5-biphosphate [4]. In turn, PIP3 interacts with
Kingdom of Saudi Arabia is also acknowledged. SP
thanks the Netherlands Organisation for Scientific
Research (NWO, grant number NWO-017.009-065)
and the Prins Bernhard Cultuurfonds for funding. The
funders had no role in study design, data collection
and analysis, decision to publish, or preparation of
the manuscript.
pleckstrin homology (PH) domains of phosphoinositide-dependent kinase (PDK) and Akt
(Also called as Protein Kinase B) and activates them [5]. In addition, PDK contributes to activa-
tion of Akt. Among these two master kinases, Akt acts on wide array of substrates which are
involved in regulation of apoptosis, cell cycle, transcription and translation [6, 7]. Therefore, the
PI3K/Akt/mTOR pathway plays a central role in regulation of the cell proliferation, survival,
migration, angiogenesis and metabolism and extensively contributes to oncogenesis [8–11].
Overactivation of PI3K/Akt/mTOR pathway has been reported in many types of cancers offer-
ing a unique therapeutic target to design novel heterocycles against malignancies [12–15].
Imidazole-based compounds have been extensively studied and have been reported to pos-
sess good anticancer activity in various types of cancer cells [16, 17]. Previously, we reported
the synthesis and antiproliferative activity of imidazole derivatives in various tumor models
Competing Interests: The authors have declared
that no competing interests exist.
[18–20]. Studies have suggested that imidazole conjugates possess good PI3K inhibitory activ-
ity [21]. SB203580 is a pyridinyl imidazole reported to effectively inhibit PI3K indicate that
imidazole-based compounds are pharmacologically important scaffolds to target different type
of cancers [21]. Therefore, in continuation our effort to synthesize and explore the various
pharmacological properties of heterocycles [22–29], in the present study, we synthesized a
series of trisubstituted imidazole derivatives and evaluated their inhibitory efficacy against
PI3K/Akt/mTOR signaling pathway in breast cancer cell lines. An in silico target prediction
revealed that new compounds target RAC-beta serine/threonine-protein kinase which com-
prise the Akt and abrogates PI3K/Akt/mTOR pathway. The lead compound, 2-chloro-3-(4,
5
-diphenyl-1H-imidazol-2-yl) pyridine (CIP) suppressed the proliferation of breast cancer
cells, decreased the phosphorylation of PDK, Akt, mTOR, downregulated the cellular invasion
and activated caspases and cleaved PARP to induce apoptosis.
Materials and Methods
All solvents used were of analytical grade and reagents used were purchased from Sigma-
1
13
Aldrich. H and C NMR spectra were recorded on a Agilent (400 MHz) spectrometer in
CDCl or DMSO-d as solvent, using TMS as an internal standard and chemical shifts are
3
6
expressed as ppm. Mass and purity were recorded on a LC-MSD-Trap-XCT. High resolution
mass spectra were determined on a Bruker Daltonics instrument. The progress of the reaction
was monitored by TLC pre-coated silica gel G plates. The breast cancer MCF-7 and MDA-MB-
2
31 cell lines were obtained from ATCC (Manassas, VA, USA).
General procedure for the synthesis of the trisubstituted imidazoles
The reaction mixture of benzil (1.0 mmol), aldehydes (1.0 mmol), NH OAc (4.0 mmol) and a
4
metal halide catalyst (0.10 mmol), followed by EtOH (4 ml) was stirred and gently refluxed for
5
h (Fig 1A). The completion of reaction was monitored by TLC and 4 ml of water was added
at the end of reaction. The solid was filtered, washed with a mixture of cold EtOH/H O (1:1,
2
v:v). The crude product was recrystallized from acetone/water 9:1.
1
2
-(3, 4-Dimethoxyphenyl)-4, 5-diphenyl-1H-imidazole (1). H NMR (CDCl , 400 MHz)
3
13
δ: 12.454 (s, NH), 6.878–7.631 (m, 13H), 3.961 (s, 6H). C NMR: 149.7, 148.9, 147.9, 136.7,
PLOS ONE | DOI:10.1371/journal.pone.0153155 April 20, 2016
2 / 15

Imidazoles Abrogate Oncogenic PI3K/Akt/mTOR Signaling Pathway
Fig 1. A, Schematic representation for the synthesis of imidazole based small molecules. B, Breast cancer
4
cells (2.5 X 10 /mL, MCF-7 & MDA-MB-231) were plated in triplicate, treated with indicated concentrations (0,
10, 25 and 50 μM) of CIP, and then subjected to MTT assay after 24, 48 and 72 h to analyse proliferation of
cells. CIP suppresses the viability of various breast cancer cell lines in a dose- and time-dependent manner.
doi:10.1371/journal.pone.0153155.g001
1
34.9, 131.4, 131.3, 129.9, 129.3, 129.1, 128.2, 126.6, 126.4, 123.7, 122.1, 113.2, 111.7, 55.7, 55.6.
+
(
MM—ES + APCI) m/z: 357.12 [M+H] . MP 218–219°C
-Bromo-5-(4, 5-diphenyl-1H-imidazol-2-yl) pyridine (2). H NMR (CDCl , 400 MHz)
1
2
3
δ: 13.205(s, NH), 8.777–8.761 (d, J = 4 Hz, 1H), 8.141–8.111 (dd, J = 4 Hz, J = 4 Hz, 1H),
1
2
13
7
1
.483–7.318 (m, 5H), 7.314–7.241 (m, 6H). C NMR: 148.9, 145.3, 141.9, 134.9, 130.4, 130. 5,
+
29.9, 129. 5, 129.1, 128.2, 126. 9, 126. 3, 123.7, 122.2. (MM—ES + APCI) m/z: 377.04 [M+H] ,
HRMS (ESI–TOF) Calculated for C H N Br [M + Na], 398.0263: found 398.0266. MP 248–
20 14 3
2
49°C
-(4-(Methylsulfonyl) phenyl)-4, 5-diphenyl-1H-imidazole (3). H NMR (CDCl , 400
1
2
3
MHz) δ: 13.092 (s, NH), 8.391–8.371 (d, J = 8 Hz, 2H), 8.095–8.075 (d, J = 8 Hz, 2H), 7.596–
1
3
7
1
.377 (m, 10H), 3.391 (s, 3H). C NMR: 147.6, 146.9, 131.8, 131.7, 129.8, 129.7, 129.5, 128.9,
+
26.9, 126.8 123.7, 122.1, 113.2, 111.7, 55.4. (MM-ES+APCI) m/z: 375.11 [M+H] , HRMS
(
ESI–TOF) Calculated for C H N O S [M+Na], 397.0981: found 397.0983. MP 256–257°C
22 18 2 2
1
2
-(4-Butoxyphenyl)-4, 5-diphenyl-1H-imidazole (4). H NMR (CDCl , 400 MHz) δ:
3
1
3
1
3.20 (s, NH), 8.095–8.075 (d, J = 8 Hz, 2H), 7.923–7.765 (m, 2H) 7.483–7.324 (m, 10H),
.941–3.867 (m, 2H), 2.171–1.322 (m, 6H). C NMR: 146.9, 137.6, 133.7, 129.5, 129.5, 129.4,
13
28.9, 126.5, 126.0, 124.7 122.7, 114.7, 65.6, 31.8, 24.1, 17.18. (MM—ES + APCI) m/z: 369.17
+
[
M+H] . MP 229–230°C
1
3
-(4, 5-Diphenyl-1H-imidazol-2-yl)-1H-indole (5). H NMR (CDCl , 400 MHz) δ: 12.4
3
(
7
1
s, 1H), 11.404 (s, 1H), 8.461–8.441 (d, J = 8 Hz, 1H), 8.016–8.006 (d, J = 4 Hz, 1H), 7.587–
13
.128 (m, 13H). C NMR: 144.1, 136.7, 128.9, 128.5, 127.6, 125.1, 124.5, 122.4, 121.9, 120.4,
+
12.1, 106.9. (MM-ES+APCI) m/z: 336.47 [M+H] . MP 290–291°C
1
3-(4, 5-Diphenyl-1H-imidazol-2-yl)-2-methyl-1H-indole (6). H NMR (CDCl , 400
3
MHz) δ: 11.553 (s, 1H, NH), 10.07 (s, 1H, NH), 8.147–7.484 (m, 4H), 7.419–7.102 (m, 10H),
1
3
2
.214 (s, 3H). C NMR: 143.9, 136.7, 136.4, 134.98, 132.6, 128.8, 128.5, 127.6, 126.8, 126.4,
PLOS ONE | DOI:10.1371/journal.pone.0153155 April 20, 2016
3 / 15

Imidazoles Abrogate Oncogenic PI3K/Akt/mTOR Signaling Pathway
1
25.69, 125.31, 124.7, 122.6, 121.8, 120.7, 110.6, 102.8, 14.71. (MM-ES+APCI) m/z: 350.16
+
[
M+H] . MP 298–299°C.
1
4
, 5-Diphenyl-2-(thiophen-2-yl)-1H-imidazole (7). H NMR (CDCl , 400 MHz) δ: 12.79
3
(
(
(
s, 1H, NH), 7.15–7.13 (dd, J = 4 Hz, J = 4 Hz, 1H), 7.69–7.68 (d, J = 4 Hz, 1H), 7.51–7.25
1 2
1
3
m, 11H). C NMR: 141.5, 136.6, 134.7, 133.9, 130.8, 128.6, 128.1, 127.8, 127.0, 126.5, 124.1.
+
MM-ES+APCI) m/z: 303.37 [M+H] . MP 255–256°C
1
4, 5-Diphenyl-2-(thiophen-3-yl)-1H-imidazole (8). H NMR (CDCl , 400 MHz) δ:
3
1
1
1
3
2.63 (s, 1H, NH), 8.04–8.02 (dd, J = 4 Hz, J = 4 Hz, 1H), 7.71–7.68 (dd, J = 4Hz, J = 4 Hz,
1 2 1 2
1
3
H), 7.65–7.63 (dd, J = 4 Hz, J = 4 Hz, 1H), 7.31–7.53 (m, 10H), C NMR: 121.8, 124.2,
1
2
26.5, 127.0, 127.7, 128.1, 128.4, 130.8, 132.5, 134.7, 136.6, 142.7 (MM—ES + APCI) m/z:
+
03.37 [M+H] . MP 257–259°C
1
2-Chloro-3-(4, 5-diphenyl-1H-imidazol-2-yl) pyridine (9, CIP). H NMR (CDCl , 400
3
MHz) δ: 12.591 (1H, NH), 8.841–8.811 (dd, J = 4 Hz, J = 4 Hz, 1H), 8.390–8.370 (dd, J = 4
1
2
1
13
Hz, J = 4 Hz, 1H), 7.567–7.560 (m, 4H), 7.406–7.307 (m, 7H). C NMR: 148.7, 140.1, 139.9,
2
1
33.9, 130.4, 131. 0, 129.9, 122.2, 129. 5, 129.1, 128.2, 126. 9, 126. 3, 123.7, (MM-ES+APCI)
+
m/z: 332.09 [M+H] , HRMS (ESI–TOF) Calculated for C H ClN [M + Na], 354.0768:
20
14
3
found 354.0766. MP 228–229°C
-(4-Chloro-1, 3-dimethyl-2, 5-dihydro-1H-pyrazol-5-yl)-4, 5-diphenyl-1H-imidazole
2
1
(
10). H NMR (CDCl , 400 MHz) δ: 12.591(1H, NH), 7.571–7551 (m, 4H), 7.352–7.283 (m,
3
H), 4.348 (s, 1H), 3.829 (s, 3H), 2.609 (s, 3H), 2.254 (s, 1H). C NMR: 149.7, 140.3, 139.9,
13
6
1
3
3
34.9, 130.4, 131. 0, 129.9, 129. 5, 129.1, 128.2, 126. 9, 76. 3, 43.7, 12.2, (MM-ES+APCI) m/z:
+
51.17 [M+H] , HRMS (ESI–TOF) Calculated for C H ClN [M+Na], 373.1190: found
20
19
4
73.1193. MP 218–219°C.
-(Naphthalen-2-yl)-4, 5-diphenyl-1H-imidazole (11). H NMR (CDCl , 400 MHz) δ:
1
2
3
1
7
1
3
2.71 (s, NH), 8.69 (s, 1H), 8.26–8.24 (d, J = 8 Hz, 2H), 7.967–7.501 (m, 7H), 7.498–7.284 (m,
H). C NMR: 145.4, 137.9, 137.0, 135.2, 133.5, 130.9, 130.2, 129.4, 128.7, 128.4, 128.2,
1
3
28.0, 127.7, 127.5, 127.3, 127.0, 126.5, 126.4, 126.3, 125.9, 125.0 (MM—ES + APCI) m/z:
+
47.20 [M+H] . MP 273–275°C.
Pharmacology
MTT Assay. The antiproliferative effect of newly synthesized compounds against breast
cancer cells was determined by the MTT dye uptake method as described previously [30]. The
breast cancer MCF-7 and MDA-MB-231 cell lines were obtained from ATCC (Manassas, VA,
4
USA). Briefly, breast cancer cells (2.5 X 10 /ml) were incubated in triplicate in a 96-well plate,
in the presence of varying compound concentrations at a volume of 0.2 ml, for different time
intervals at 37°C. Thereafter, a 20 μl MTT solution (5 mg/ml in PBS) was added to each well.
After a 2 h incubation at 37°C, a 0.1 ml lysis buffer (20% SDS, 50% dimethylformamide) was
added; incubation was performed for 1 h at 37°C, and the optical density (OD) at 570 nm was
measured by Tecan plate reader. 0.01% DMSO was used as the negative control and 0.01%
MTT was used as a control agent.
Flow cytometric analysis. Flow cytometric analysis was performed to determine whether
CIP can induce apoptosis of tumor cells as described earlier [31]. Briefly, MDA-MB-231 breast
5
cancer cells (5 x 10 ) were plated in petri dish and 24 h later the cells were exposed to com-
pound CIP (50 μM) for 0, 24, 36 and 48 h. Thereafter cells were washed, fixed with 70% etha-
nol, and incubated for 30 min at 37°C with 0.1% RNase A in PBS. Cells were washed again,
resuspended, and stained with PBS containing 25 μg/ml propidium iodide (PI) for 15 min at
room temperature. The cell cycle distribution across the various phases was analyzed with a
CyAn ADP flow cytometer (Dako Cytomation).
PLOS ONE | DOI:10.1371/journal.pone.0153155 April 20, 2016
4 / 15

Imidazoles Abrogate Oncogenic PI3K/Akt/mTOR Signaling Pathway
5
Caspase 3/7 activity assay. MDA-MB-231 cells (5 x 10 ) were plated in 6-well plates and
allowed to adhere for 24 h. The cells were then exposed to CIP (50 μM) for 0, 24, 36 and 48 h.
1
Thereafter, the Caspase-3/7 activities were determined by a Caspase-Glo 3/7 assay kit (Pro-
mega, Madison, USA) according to the manufacturer’s instructions.
Target prediction-based enrichment of Imidazole series. In order to understand the
mode-of-action of trisubstituted imidazoles towards the inhibition of human breast cancer
cells, we applied an in silico target prediction tool, which was developed using the Bernoulli
Naïve Bayes (BNB) algorithm, as implemented in Scikit-learn [32]. This method has been vali-
dated previously by Mervin et al [33]. Active compounds were extracted from ChEMBL [34],
using pChEMBL values larger than 5 (10 μM) and a confidence score cut-off of 5 to define
activity. Inactive data points were extracted from PubChem [35] for compounds declared inac-
tive from screens stored within BioAssay [36]. The BNB model was trained on these com-
pounds using the 2048 bit Morgan fingerprints, with a radius of 2, generated by RDKit [37].
Stringent class-specific thresholds were applied to binarize predictions for the Imidazole series.
In order to improve the statistical significance of this analysis, predictions for the imidazole
sets are compared to predictions generated by chance for 1,000 sets of randomly selected com-
pounds from the PubChem repository. An Average Ratio enrichment metric is calculated from
this analysis (Eq 1), which reflects the average of the series of predictions in the background
(
Ri), divided by the frequency of predictions in the imidazole series (Fimidazole). A lower score
represents targets that are more enriched, and hence found to be significant for the imidazole
series.
X
1
1;000
i¼1
Ri
Average Ratio ¼
ð1Þ
1
; 000
Fimidazole
Western blotting. Western blotting analysis was performed as described earlier [38].
Briefly, For detection of various proteins, CIP-treated whole-cell extracts were lysed in a lysis
buffer (20 mM Tris (pH 7.4), 250 mM NaCl, 2 mM EDTA (pH 8.0), 0.1% TritonX-100, 0.01
mg/ml aprotinin, 0.005 mg/ml leupeptin, 0.4 mM PMSF,and 4 mM NaVO ). Lysates were
4
then spun at 14,000 rpm for 10 mins to remove insoluble material, and then resolved on a
7
.5% SDS gel. After electrophoresis, the proteins were electrotransferred to a nitrocellulose
membrane, blocked with 5% non-fat milk, and probed with various antibodies (1:1000) over-
night at 4°C. The blot was washed, exposed to HRP-conjugated secondary antibodies for 1 h,
and finally examined by chemiluminescence (ECL; GE Healthcare, Little Chalfont, Bucking-
hamshire, UK).
Invasion Assay. A BD Biocoat™ Matrigel™ invasion chamber with 8-μm pores in the light-
tight polyethylene terephthalate membrane and was coated with a reconstituted basement
5
membrane gel (BD Biosciences). 2 X 10 cells were suspended in serum-free DMEM and
seeded into the Matrigel transwell chambers. The cells were incubated with CIP for 8 h. After
incubation, the outer surfaces of the transwell chambers were wiped with cotton swabs, and the
invading cells were fixed and stained with crystal violet solution. The invading cells were then
counted in five randomly selected areas under microscopic observation.
Molecular docking analysis. The trisubstituted imidazoles were docked to the crystal
structure of human RAC-Beta Serine/Threonine-Protein Kinase (Akt2) complexed with an
inhibitor (PDB: 2JDR) [39] using MOE [40]. Protonation states of amino acids were assigned
using protonate3D in MOE [41]. Ligands were ionized at physiological pH using MOE. The
binding site was defined by the position of the co-crystalized ligand. Docking calculations were
carried out using MOE’s induced fit protocol that treats amino acid side-chains near the bind-
ing site as flexible.
PLOS ONE | DOI:10.1371/journal.pone.0153155 April 20, 2016
5 / 15

Imidazoles Abrogate Oncogenic PI3K/Akt/mTOR Signaling Pathway
Results and Discussion
Chemical synthesis of trisubstituted imidazoles
We previously reported the microwave assisted synthesis and crystal structure studies of
2
-butyl-4-chloro-imidazole-5-carbaldehyde [19, 20, 42]. In addition, solvent free and micro-
wave-assisted synthesis of 4,5-disubstituted imidazoles have been reported using 1,2-diketones
and urotropine in the presence of ammonium acetate [43]. In this report, we prepared 1,2,4-tri-
substituted-1H-imidazoles by heating a mixture of benzil, an aldehyde and ammonium acetate
using ethanol as solvent. The obtained imidazoles (3a-k) were characterized by melting point,
1
13
1
LC-MS, H NMR, and C NMR analysis. The appearance of a H NMR peak at δ value of 12.5–
2.7 for the HN-proton of imidazole confirms the ring formation. Detailed physical parameters
1
of all the compounds synthesized is provided as supplemental information (S1 Table).
Trisubstituted imidazoles elicit an antiproliferative effect against human
breast cancer cells
Imidazole derivatives are known for their antiproliferative effect towards various cancer models.
We initially evaluated the effect of novel trisubstituted imidazoles against MDA-MB-231 cells
using MTT assay as described earlier [44, 45]. Among the newly synthesized structures, com-
pound 1, 2 and CIP displayed antiproliferative effect with the IC values of 21.1, 17.8 and
50
2
4.1 μM respectively. Further investigation of the compound 1, 2 and CIP against mammary epi-
thelial cells (MCF-10A) revealed that, CIP selectively induce cytotoxicity in breast cancer cells
and did not exhibit significant cytotoxicity against normal cells. To confirm the antiproliferative
potential of the trisubstituted imidazoles, we further investigated the effect of compounds on
hepatocellular carcinoma (HepG2) and normal (LO2) cells and found CIP as a lead anticancer
compound with minimal cytotoxicity towards non-cancerous cells. The cytotoxicity profile of all
the compounds is provided as supplementary information (S1 Table). Thereafter, we investigated
the effect of CIP on MCF-7 and MDA-MB-231 breast cancer cells at different doses (0, 10, 25 &
5
0 μM) and time-points (24, 48 & 72 h). Upon treatment with CIP, we observed a significant
decline in the viability of both the cell lines in a dose and time dependent manner (Fig 1B).
Investigation of structure-activity relationship (SAR) of the tested molecules suggests that
introducing small sized and electron withdrawing atom containing heterocyclic groups at the
position 2 in the imidazole ring would favour the specificity for cancer cells. Molecules having
these SAR features better match the polarity of the Akt2 binding surface, since polar region in
the Akt2 binding pocket is small.
CIP significantly accumulates MDA-MB-231 cells in sub-G1 phase and
increases the activity of executioner caspases
We next investigated the potential of CIP to induce apoptosis in breast cancer MDA-MB-231
cells. We first observed that CIP induced significant apoptosis in a time dependent manner as
evidenced by increased accumulation of cells in Sub-G1 phase of the cell cycle (Fig 2A). We
further noted that exposure of the CIP also caused a significant increase in the activity of effec-
tor caspases -3 and -7 (Fig 2B), thereby indicating the apoptotic potential of CIP against breast
cancer cells.
In silico mode-of-action analysis for CIP that inhibits the growth of
human breast cancer cells
In order to understand the molecular mechanism of CIP responsible for the antiproliferative
effect in human breast cancer cells, we carried out an in silico target prediction analysis.
PLOS ONE | DOI:10.1371/journal.pone.0153155 April 20, 2016
6 / 15

Imidazoles Abrogate Oncogenic PI3K/Akt/mTOR Signaling Pathway
Fig 2. A, The analysis of cell cycle distribution following treatment with CIP was performed using flow
cytometry. MDA-MB-231 cells were exposed to compound CIP (50 μM) for indicated time intervals (0, 24, 36
and 48 h), after which the cells were harvested and stained with PI. The cell distribution across the various
phases of the cell cycle was analyzed with a flow cytometer. We observed that CIP induced significant
apoptosis in a time dependent manner as evidenced by increased accumulation of cells in Sub-G1 phase of
the cell cycle. B, MDA-MB-231 cells were exposed to compound CIP (50 μM) at indicated time intervals (0,
2
4, 36 and 48 h), after which they were harvested and caspase3/7 activity was measured using Caspase-
1
Glo 3/7 assay kit. We found that treatment of MDA-MB-231 cells with CIP caused the significant increase in
the caspases-3/7 activity. * for p<0.05, ** for p<0.005.
doi:10.1371/journal.pone.0153155.g002
Enrichment for the targets from the in silico target prediction analysis are shown in supple-
mentary table (S2 Table). The top 20 targets from the 1,080 models are shown. “RAC-Beta Ser-
nd
ine/Threonine-Protein Kinase” (Ranked 2 ) and “RAC-Alpha Serine/Threonine-Protein
th
Kinase” (Ranked 8 ), comprise the Akt protein, relevant for this study due to its involvement
in the PI3K/Akt/mTOR pathway, and are highly enriched in this analysis.
CIP downregulates phosphorylation of PDK, Akt and mTOR in breast
cancer cells
Based on the in silico prediction, we next analysed whether CIP modulates phosphorylation of
Akt, PDK and mTOR in MDA-MB-231 cells using western blotting as described previously
[46, 47]. As observed by western blot analysis, we found that, CIP downregulated the phos-
phorylation of Akt, PDK and mTOR in a dose-dependent manner (Fig 3) and at the same time
Akt, PDK and mTOR protein expression remained unaltered.
PLOS ONE | DOI:10.1371/journal.pone.0153155 April 20, 2016
7 / 15

Imidazoles Abrogate Oncogenic PI3K/Akt/mTOR Signaling Pathway
Fig 3. MDA-MB-231 cells were treated with CIP at indicated concentrations (0, 10, 15, 25 and 50 μM) for
8
h and levels of phospho-PDK, phospho-Akt, phospho-mTOR and PDK, Akt, mTOR was analysed
using western blotting. We found that, CIP downregulated the phosphorylation of Akt, PDK and mTOR in a
dose-dependent manner without any change in the expression levels of Akt, PDK and mTOR proteins.
doi:10.1371/journal.pone.0153155.g003
CIP induces apoptosis of human breast cancer cells
Cell shrinkage, formation of apoptotic bodies, activation of caspases, and cleavage of chromo-
somal DNA mediated by caspase dependent nucleases are the major events in the cells undergoing
apoptosis [48]. In the event of cell committed to apoptosis, cascade of reactions occurs ultimately
leading to the activation of caspase-3 which further cleaves full length PARP into fragments [49,
50]. Therefore, we first investigated the effect of CIP on procaspase-3 and found the activation of
procaspase-3 in a time time-dependent manner (Fig 4A). Similarly, we observed the subsequent
decrease in full length PARP with increase in cleaved fragment. We further determined the effect
of CIP on the expression of various proliferative and survival proteins such as cyclin D1, VEGF,
survivin and Bcl-2 in MDA-MB-231 cells. Fig 4B demonstrates a substantial decline in the expres-
sion of cyclin D1, VEGF, survivin and Bcl-2 proteins in the time-dependent manner.
CIP suppresses CXCL12-induced MDA-MB-231 Cell invasion
Akt-targeted gene products are known to be involved in motility of cancer cells [5]. Therefore,
we evaluated potential of CIP to modulate on cellular invasion according to the method
described previously [51]. In addition, several studies have demonstrated the critical role and
interaction of CXCL12 with CXCR4 in cancer cells which contributes largely to tumor metasta-
sis [52]. Fig 5 indicates the movement of the cells in the presence and absence of CIP and
CXCL12. CIP was found to significantly suppress the invasion of MDA-MB-231 cells by nearly
5
0%, both in the presence and absence of CXCL12, compared with the respective controls, sug-
gesting that CIP interferes with invasive ability of breast cancer cells.
Molecular docking analysis
Based on the in silico and in vitro results, we investigated the binding of the imidazole series
compounds to the RAC-Beta Serine/Threonine-Protein Kinase (Akt2 kinase) using MOE
PLOS ONE | DOI:10.1371/journal.pone.0153155 April 20, 2016
8 / 15

Imidazoles Abrogate Oncogenic PI3K/Akt/mTOR Signaling Pathway
Fig 4. A and B, MDA-MB-231 cells were treated with CIP (25 μM) for indicated time intervals (0, 12, 24,
3
2
6 and 48 h) and expression of apoptotic markers (PARP, Procaspase-3), antiapoptotic proteins (Bcl-
, Survivin, VEGF), cell cycle regulator (Cyclin D1) was profiled using western blot analysis. We
observed the significant decline in the expression of PARP, procaspase-3, cyclin D1, VEGF, survivin and Bcl-
2
proteins in the time-dependent manner without alteration in the levels of beta actin.
doi:10.1371/journal.pone.0153155.g004
platform. Our docking calculations revealed that trisubstituted imidazoles bind with high
docking scores:the average of the MOE scores for the compounds is found to be -8.38. To vali-
date our in silico docking results, we have docked previously reported compounds that have
low micro molar affinity for Akt2 Kinase (92 molecules having IC50 value of 1–50 μM extracted
from ChEMBL database) using the same docking protocol. The average of the docking scores
for this data is -8.62 which is very close to the average score for the imidazole series.
Docking of the trisubstituted imidazoles reveal common binding modes as shown in Fig 6.
Phenyl moieties at positions 4 and 5 perfectly fit in the hydrophobic profile of the surface of
the deep binding pocket (Fig 6A and 6B). Fragments at position 2, which contain electronega-
tive atoms, match to the polar surface of the binding pocket (Fig 6A and 6B).
Statistical analysis
ꢀ
ꢀ
Student t-test was used to analyze the data. for p<0.05 was considered statistical significant.
ꢀ
ꢀ
for p<0.05; for p<0.005.
PLOS ONE | DOI:10.1371/journal.pone.0153155 April 20, 2016
9 / 15

Imidazoles Abrogate Oncogenic PI3K/Akt/mTOR Signaling Pathway
Fig 5. MDA-MB-231 cells were suspended in serum-free DMEM and seeded into the Matrigel transwell
chambers and were incubated with CIP (25 μM) for 8 h. The invading cells were fixed and stained with
crystal violet solution and invaded cells were counted in five randomly selected areas. CIP significantly
suppressed the cell invasion both in the presence and absence of CXCL12 suggesting that CIP interferes
with invasive potential of breast cancer cells.
doi:10.1371/journal.pone.0153155.g005
Discussion
The present report demonstrates the anticancer activity of trisubstituted imidazole that targets
PI3K/Akt/mTOR pathway. CIP, the lead molecule among the synthesized imidazole deriva-
tives supressed the proliferation of breast cancer (MDA-MB-231, MCF-7) and hepatocellular
carcinoma (HepG2) cells and did not induce significant cytotoxicity against normal (MCF-
1
0A, LO2) cells. CIP caused the accumulation of breast cancer cells in Sub-G1 phase, increased
the executioner caspase-3/7 activity, downregulated the phosphorylation of PDK, Akt and
Fig 6. In silico molecular docking studies of trisubstituted imidazoles against kinase domain of Akt2:
Common binding poses of trisubstituted imidazoles towards the Akt2 kinase domain. The molecular surface
of the protein is represented based on the surface polarity; green, pink and red colours show hydrophobic,
polar and solvent exposed regions, respectively. For the sake of better visualisation of the binding pocket
surface of Akt2, molecular surface was rendered in two panels (A and B) in which ligands were rotated 180
degrees.
doi:10.1371/journal.pone.0153155.g006
PLOS ONE | DOI:10.1371/journal.pone.0153155 April 20, 2016
10 / 15

Imidazoles Abrogate Oncogenic PI3K/Akt/mTOR Signaling Pathway
mTOR in breast cancer cells. Further, it modulated the levels of various proteins involved in
cell cycle regulation (Cyclin D1), angiogenesis (VEGF), apoptosis (Caspase-3, PARP), and sur-
vival (Survivin, Bcl-2). Further, CIP inhibited the cell invasion indicating its inhibitory role in
cell motility.
One of the major events in apoptosis is the activation of caspases thereby cleavage of PARP
and activation of caspase-activated deoxyribonuclease (CAD). In the event of cell undergoing
apoptosis, caspase-3 activates CAD, and CAD in turn degrades genomic DNA into oligomers
[53, 54]. The cells with lesser DNA content are often termed as hypodiploid cells which are
detected as Sub-G1 cell population. The lead compound CIP caused the accumulation of cells
in Sub-G1 phase demonstrating that CIP induce caspase mediated apoptosis. These results
were confirmed and correlated by the cleavage of PARP, a prominent DNA repair enzyme and
activation of executioner caspase-3 and caspase-7.
We performed cheminformatics-based target enrichment studies to predict the probable
molecular therapeutic target of the lead compound and found a number of targets to be
enriched, including Akt protein, and other proteins belonging to the MAPK pathway.
Although, the results of the study presented multiple targets, we analysed the effect of CIP on
PI3K/Akt/mTOR signalling pathway, because, previous studies have highlighted imidazole
derivatives as inhibitors of PI3K/Akt/mTOR proteins [55–57]. In silico predicted target was
experimentally validated by in vitro investigation in breast cancer cell lines. Several studies
have showed that Akt pathway plays pivotal role in cell survival, growth and proliferation [58].
In order to demonstrate the effect of CIP on Akt signalling pathway, we investigated the effect
of CIP on the phosphorylation of Akt pathway proteins including PDK1 (Ser-241), Akt (Ser-
4
73) and mTOR. We observed the downregulation in the phosphorylation of proteins involved
in Akt signalling cascade. Therefore, it is evident that, CIP mediates its anticancer activity at
least partly via blockade of PI3K/Akt/mTOR signalling pathway. This finding opens an avenue
for the development of novel trisubstituted imidazole based small molecules as therapeutic
agents that target PI3K/Akt/mTOR signalling pathway in human diseases.
Conclusion
In conclusion, we synthesized trisubstituted imidazoles, identified the bioactive cytotoxic lead
structure, predicted the likely target and demonstrated in vitro efficacy of lead compound to
abrogate the activation of the PI3K/Akt/mTOR pathway in human breast cancer cells. There-
fore, designing of CIP-based small molecule inhibitors to abrogate PI3K/Akt/mTOR pathway
may serve as an effective therapeutic strategy to fight against various types of cancer.
Supporting Information
S1 Table. Physical parameters and cytotoxicity profile of newly synthesized compounds.
(
DOC)
S2 Table. Top 20 ranked targets for the imidazole series.
DOC)
(
Acknowledgments
This research was supported by University Grants Commission (41-257-2012-SR), Vision
Group Science and Technology, Department of Science and Technology (NO. SR/FT/LS-142/
2
012) to Basappa. KSR thanks Department of Science and Technology Indo-Korea [INT/Indo-
Korea/122/2011-12] and Institution of Excellence, University of Mysore for financial support
and instrumentation facility. CDM thanks the University of Mysore for Department of Science
PLOS ONE | DOI:10.1371/journal.pone.0153155 April 20, 2016
11 / 15

Imidazoles Abrogate Oncogenic PI3K/Akt/mTOR Signaling Pathway
and Technology-Promotion of University Research and Scientific Excellence (DST-PURSE)
Research Associate fellowship. This work was supported by NUHS Bench-to-Bedside-To-
Product grant to GS. Deanship of Scientific Research, College of Science Research Centre, King
Saud University, Kingdom of Saudi Arabia is also acknowledged. SP thanks the Netherlands
Organisation for Scientific Research (NWO, grant number NWO-017.009–065) and the Prins
Bernhard Cultuurfonds for funding.
Author Contributions
Conceived and designed the experiments: KSR B AB GS. Performed the experiments: CDM VS
SR LM SP SB FL MKS SM AC MZ SAA. Analyzed the data: KSR B AB GS CDM. Contributed
reagents/materials/analysis tools: KSR B AB GS. Wrote the paper: KSR B AB GS CDM.
References
1.
2.
3.
Ludwig JA, Lamhamedi-Cherradi S-E, Lee H-Y, Naing A, Benjamin R. Dual Targeting of the Insulin-
Like Growth Factor and Collateral Pathways in Cancer: Combating Drug Resistance. Cancers. 2011; 3
(
3):3029–54. doi: 10.3390/cancers3033029. PMC3759185. PMID: 24212944
Arcaro A, Guerreiro AS. The Phosphoinositide 3-Kinase Pathway in Human Cancer: Genetic Alter-
ations and Therapeutic Implications. Current Genomics. 2007; 8(5):271–306. doi: 10.2174/
138920207782446160. PMC2652403. PMID: 19384426
Makinoshima H, Takita M, Saruwatari K, Umemura S, Obata Y, Ishii G, et al. Signaling through the
Phosphatidylinositol 3-Kinase (PI3K)/Mammalian Target of Rapamycin (mTOR) Axis Is Responsible
for Aerobic Glycolysis mediated by Glucose Transporter in Epidermal Growth Factor Receptor (EGFR)-
mutated Lung Adenocarcinoma. Journal of Biological Chemistry. 2015; 290(28):17495–504. doi: 10.
1074/jbc.M115.660498 PMID: 26023239
4
.
.
Keane KN, Cruzat VF, Carlessi R, de Bittencourt PIH, Newsholme P. Molecular Events Linking Oxida-
tive Stress and Inflammation to Insulin Resistance and β-Cell Dysfunction. Oxidative Medicine and Cel-
lular Longevity. 2015; 2015:15. doi: 10.1155/2015/181643
5
LoPiccolo J, Blumenthal GM, Bernstein WB, Dennis PA. Targeting the PI3K/Akt/mTOR pathway: effec-
tive combinations and clinical considerations. Drug resistance updates: reviews and commentaries in
antimicrobial and anticancer chemotherapy. 2008; 11(1–2):32–50. Epub 2008/01/02. doi: 10.1016/j.
drup.2007.11.003 PMID: 18166498; PubMed Central PMCID: PMCPmc2442829.
6.
Amaravadi R, Thompson CB. The survival kinases Akt and Pim as potential pharmacological targets.
Journal of Clinical Investigation. 2005; 115(10):2618–24. doi: 10.1172/JCI26273. PMC1236693. PMID:
1
6200194
Datta SR, Brunet A, Greenberg ME. Cellular survival: a play in three Akts. Genes & development. 1999;
3(22):2905–27. Epub 1999/12/02. PMID: 10579998.
7
.
.
1
8
Martelli AM, Evangelisti C, Chiarini F, Grimaldi C, Manzoli L, McCubrey JA. Targeting the PI3K/AKT/
mTOR signaling network in acute myelogenous leukemia. Expert Opinion on Investigational Drugs.
2009; 18(9):1333–49. doi: 10.1517/14728220903136775 PMID: 19678801.
9.
Brader S, Eccles SA. Phosphoinositide 3-kinase signalling pathways in tumor progression, invasion
and angiogenesis. Tumori. 2004; 90(1):2–8. PMID: 15143962
1
1
1
1
1
0. Whittaker S, Marais R, Zhu AX. The role of signaling pathways in the development and treatment of
hepatocellular carcinoma. Oncogene. 2010; 29(36):4989–5005. doi: 10.1038/onc.2010.236 PMID:
20639898
1. Taniguchi CM, Kondo T, Sajan M, Luo J, Bronson R, Asano T, et al. Divergent regulation of hepatic glu-
cose and lipid metabolism by phosphoinositide 3-kinase via Akt and PKCλ/ζ. Cell Metabolism. 2006; 3
(
5):343–53. doi: 10.1016/j.cmet.2006.04.005 PMID: 16679292
2. Yap TA, Garrett MD, Walton MI, Raynaud F, de Bono JS, Workman P. Targeting the PI3K–AKT–mTOR
pathway: progress, pitfalls, and promises. Current Opinion in Pharmacology. 2008; 8(4):393–412.
PMID: 18721898
3. McAuliffe PF, Meric-Bernstam F, Mills GB, Gonzalez-Angulo AM. Deciphering the Role of PI3K/Akt/
mTOR Pathway in Breast Cancer Biology and Pathogenesis. Clinical Breast Cancer. 2010; 10, Supple-
ment 3:S59–S65. doi: 10.3816/CBC.2010.s.013
4. Ching CB, Hansel DE. Expanding therapeutic targets in bladder cancer: the PI3K/Akt/mTOR pathway.
Lab Invest. 2010; 90(10):1406–14. doi: 10.1038/labinvest.2010.133 PMID: 20661228
PLOS ONE | DOI:10.1371/journal.pone.0153155 April 20, 2016
12 / 15

Imidazoles Abrogate Oncogenic PI3K/Akt/mTOR Signaling Pathway
1
1
5. Liu P, Cheng H, Roberts TM, Zhao JJ. Targeting the phosphoinositide 3-kinase pathway in cancer. Nat
Rev Drug Discov. 2009; 8(8):627–44. doi: 10.1038/nrd2926 PMID: 19644473
6. Rademaker-Lakhai JM, van den Bongard D, Pluim D, Beijnen JH, Schellens JHM. A Phase I and Phar-
macological Study with Imidazolium-trans-DMSO-imidazole-tetrachlororuthenate, a Novel Ruthenium
Anticancer Agent. Clinical Cancer Research. 2004; 10(11):3717–27. doi: 10.1158/1078-0432.ccr-03-
0746 PMID: 15173078
1
7. Baviskar AT, Madaan C, Preet R, Mohapatra P, Jain V, Agarwal A, et al. N-Fused Imidazoles As Novel
Anticancer Agents That Inhibit Catalytic Activity of Topoisomerase IIα and Induce Apoptosis in G1/S
Phase. Journal of Medicinal Chemistry. 2011; 54(14):5013–30. doi: 10.1021/jm200235u PMID:
21644529
1
8. Thimmegowda N, Swamy SN, Kumar CA, Kumar YS, Chandrappa S, Yip GW, et al. Synthesis, charac-
terization and evaluation of benzimidazole derivative and its precursors as inhibitors of MDA-MB-231
human breast cancer cell proliferation. Bioorganic & medicinal chemistry letters. 2008; 18(1):432–5.
1
9. Kumar CA, Jayarama S, Basappa, Salimath BP, Rangappa KS. Pro-apoptotic activity of imidazole
derivatives mediated by up-regulation of Bax and activation of CAD in Ehrlich Ascites Tumor cells.
Investigational new drugs. 2007; 25(4):343–50. Epub 2007/03/21. doi: 10.1007/s10637-006-9033-4
PMID: 17372679.
2
0. Kumar CA, Swamy SN, Gaonkar SL, Basappa, Salimath BP, Rangappa KS. N-Substituted-2-butyl-5-
chloro-3H-imidazole-4-carbaldehyde derivatives as anti-tumor agents against Ehrlich ascites tumor
cells in vivo. Medicinal chemistry (Shariqah (United Arab Emirates)). 2007; 3(3):269–76. Epub 2007/
05/17. PMID: 17504198.
2
2
1. Zhao S, Konopleva M, Cabreira-Hansen M, Xie Z, Hu W, Milella M, et al. Inhibition of phosphatidylinosi-
tol 3-kinase dephosphorylates BAD and promotes apoptosis in myeloid leukemias. Leukemia. 2004; 18
(
2):267–75. PMID: 14628071
2. Kannaiyan R, Manu K, Chen L, Li F, Rajendran P, Subramaniam A, et al. Celastrol inhibits tumor cell
proliferation and promotes apoptosis through the activation of c-Jun N-terminal kinase and suppression
of PI3 K/Akt signaling pathways. Apoptosis. 2011; 16(10):1028–41. doi: 10.1007/s10495-011-0629-6
PMID: 21786165
23. Sugahara K, Thimmaiah KN, Bid HK, Houghton PJ, Rangappa KS. Anti-tumor activity of a novel HS-
mimetic-vascular endothelial growth factor binding small molecule. PloS one. 2012; 7(8).
24. Bharathkumar H, Mohan CD, Rangappa S, Kang T, Keerthy H, Fuchs JE, et al. Screening of quinoline,
1, 3-benzoxazine, and 1, 3-oxazine-based small molecules against isolated methionyl-tRNA synthe-
tase and A549 and HCT116 cancer cells including an in silico binding mode analysis. Organic & Biomo-
lecular Chemistry. 2015.
2
5. Fongmoon D, Shetty AK, Basappa, Yamada S, Sugiura M, Kongtawelert P, et al. Chondroitinase-medi-
ated degradation of rare 3-O-sulfated glucuronic acid in functional oversulfated chondroitin sulfate K
and E. The Journal of biological chemistry. 2007; 282(51):36895–904. Epub 2007/10/24. doi: 10.1074/
jbc.M707082200 PMID: 17951579.
2
2
2
6. Mantelingu K, Sadashiva M, Rangappa K. A simple and efficient method for the synthesis of 1, 2-benzi-
soxazoles: a series of its potent acetylcholinesterase inhibitors. Indian journal of chemistry Sect B:
Organic chemistry, including medical chemistry. 2004; 43(9):1954–7.
7. Anusha S, Anandakumar B, Mohan CD, Nagabhushana G, Priya B, Rangappa KS. Preparation and
use of combustion-derived Bi 2 O 3 for the synthesis of heterocycles with anti-cancer properties by
Suzuki-coupling reactions. RSC Advances. 2014; 4(94):52181–8.
8. Sadashiva MP, Basappa S, Nanjundaswamy S, Li F, Manu KA, Sengottuvelan M, et al. Anti-cancer
activity of novel dibenzo[b,f]azepine tethered isoxazoline derivatives. BMC chemical biology. 2012;
12:5. Epub 2012/10/05. doi: 10.1186/1472-6769-12-5 PMID: 23033888; PubMed Central PMCID:
PMCPmc3554437.
2
3
3
9. Priya BS, Anil Kumar C, Nanjunda Swamy S, Basappa, Naveen S, Shashidhara Prasad J, et al. 2-(2-
(
2-Ethoxybenzoylamino)-4-chlorophenoxy)-N-(2-ethoxybenzoyl)benzamine inhibits EAT cell induced
angiogenesis by down regulation of VEGF secretion. Bioorg Med Chem Lett. 2007; 17(10):2775–80.
Epub 2007/03/23. doi: 10.1016/j.bmcl.2007.02.074 PMID: 17376684.
0. Ashwini N, Garg M, Mohan CD, Fuchs JE, Rangappa S, Anusha S, et al. Synthesis of 1,2-benzisoxa-
zole tethered 1,2,3-triazoles that exhibit anticancer activity in acute myeloid leukemia cell lines by inhib-
iting histone deacetylases, and inducing p21 and tubulin acetylation. Bioorg Med Chem. 2015; 23
(
18):6157–65. Epub 2015/08/25. doi: 10.1016/j.bmc.2015.07.069 PMID: 26299825.
1. Anusha S, Mohan CD, Ananda H, Baburajeev CP, Rangappa S, Mathai J, et al. Adamantyl-tethered-
biphenylic compounds induce apoptosis in cancer cells by targeting Bcl homologs. Bioorganic & Medic-
inal Chemistry Letters. 2016; 26(3):1056–60. doi: 10.1016/j.bmcl.2015.12.026
PLOS ONE | DOI:10.1371/journal.pone.0153155 April 20, 2016
13 / 15

Imidazoles Abrogate Oncogenic PI3K/Akt/mTOR Signaling Pathway
32. Pedregosa F, Varoquaux G, Gramfort A, Michel V, Thirion B, Grisel O, et al. Scikit-learn: Machine learn-
ing in Python. The Journal of Machine Learning Research. 2011; 12:2825–30.
3
3. Mervin LH, Afzal AM, Drakakis G, Lewis R, Engkvist O, Bender A. Target prediction utilising negative
bioactivity data covering large chemical space. Journal of Cheminformatics. 2015; 7:51. doi: 10.1186/
s13321-015-0098-y. PMC4619454. PMID: 26500705
3
3
4. Gaulton A, Bellis LJ, Bento AP, Chambers J, Davies M, Hersey A, et al. ChEMBL: a large-scale bioac-
tivity database for drug discovery. Nucleic acids research. 2012; 40(Database issue):D1100–7. Epub
2011/09/29. doi: 10.1093/nar/gkr777 PMID: 21948594; PubMed Central PMCID: PMCPmc3245175.
5. Wang Y, Xiao J, Suzek TO, Zhang J, Wang J, Bryant SH. PubChem: a public information system for
analyzing bioactivities of small molecules. Nucleic acids research. 2009; 37(Web Server issue):W623–
33. Epub 2009/06/06. doi: 10.1093/nar/gkp456 PMID: 19498078; PubMed Central PMCID:
PMCPmc2703903.
36. Database resources of the National Center for Biotechnology Information. Nucleic acids research.
2013; 41(Database issue):D8–d20. Epub 2012/11/30. doi: 10.1093/nar/gks1189 PMID: 23193264;
PubMed Central PMCID: PMCPmc3531099.
3
3
7. Landrum GRO-schwro.
8. Rakesh KS, Jagadish S, Vinayaka AC, Hemshekhar M, Paul M, Thushara RM, et al. A new ibuprofen
derivative inhibits platelet aggregation and ROS mediated platelet apoptosis. PLoS One. 2014; 9(9):
e107182. Epub 2014/09/23. doi: 10.1371/journal.pone.0107182 PMID: 25238069; PubMed Central
PMCID: PMCPmc4169656.
39. Davies TG, Verdonk ML, Graham B, Saalau-Bethell S, Hamlett CC, McHardy T, et al. A structural com-
parison of inhibitor binding to PKB, PKA and PKA-PKB chimera. Journal of molecular biology. 2007;
367(3):882–94. Epub 2007/02/06. doi: 10.1016/j.jmb.2007.01.004 PMID: 17275837.
4
4
0. Molecular Operating Environment (MOE), Chemical Computing Group Inc., Montreal, Canada, 2014.
1. Labute P. Protonate3D: assignment of ionization states and hydrogen coordinates to macromolecular
structures. Proteins. 2009; 75(1):187–205. Epub 2008/09/25. doi: 10.1002/prot.22234 PMID:
18814299; PubMed Central PMCID: PMCPmc3056144.
42. Rangappa KS. New cholinesterase inhibitors: synthesis and structure–activity relationship studies of 1,
2
2
‐benzisoxazole series and novel imidazolyl‐d2‐isoxazolines. Journal of physical organic chemistry.
005; 18(8):773–8.
43. Bratulescu G. Synthesis of 4, 5-Substituted Imidazoles by a Fast Condensation of 1, 2-Diketones and
Urotropine in Heterogeneous Medium. Synthesis. 2009;(14: ):2319–20.
44. Kim SW, Kim S-M, Bae H, Nam D, Lee J-H, Lee S-G, et al. Embelin inhibits growth and induces apopto-
sis through the suppression of Akt/mTOR/S6K1 signaling cascades. The Prostate. 2013; 73(3):296–
305. doi: 10.1002/pros.22574 PMID: 22887478
4
4
4
4
5. Bharathkumar H, Mohan CD, Ananda H, Fuchs JE, Li F, Rangappa S, et al. Microwave-assisted syn-
thesis, characterization and cytotoxic studies of novel estrogen receptor α ligands towards human
breast cancer cells. Bioorganic & medicinal chemistry letters. 2015; 25(8):1804–7.
6. Subramaniam A, Shanmugam MK, Ong TH, Li F, Perumal E, Chen L, et al. Emodin inhibits growth and
induces apoptosis in an orthotopic hepatocellular carcinoma model by blocking activation of STAT3.
British Journal of Pharmacology. 2013; 170(4):807–21. doi: 10.1111/bph.12302 PMID: 23848338
7. Neelgundmath M, Dinesh KR, Mohan CD, Li F, Dai X, Siveen KS, et al. Novel synthetic coumarins that
targets NF-κB in Hepatocellular carcinoma. Bioorganic & medicinal chemistry letters. 2015; 25(4):893–
7.
8. Mohan CD, Bharathkumar H, Bulusu KC, Pandey V, Rangappa S, Fuchs JE, et al. Development of a
novel azaspirane that targets the Janus kinase-signal transducer and activator of transcription (STAT)
pathway in hepatocellular carcinoma in vitro and in vivo. The Journal of biological chemistry. 2014; 289
(
49):34296–307. Epub 2014/10/17. doi: 10.1074/jbc.M114.601104 PMID: 25320076; PubMed Central
PMCID: PMCPmc4256360.
4
9. Keerthy HK, Mohan CD, Sivaraman Siveen K, Fuchs JE, Rangappa S, Sundaram MS, et al. Novel syn-
thetic biscoumarins target tumor necrosis factor-alpha in hepatocellular carcinoma in vitro and in vivo.
The Journal of biological chemistry. 2014; 289(46):31879–90. Epub 2014/09/19. doi: 10.1074/jbc.
M114.593855 PMID: 25231984; PubMed Central PMCID: PMCPmc4231667.
5
5
0. Park KR, Nam D, Yun HM, Lee SG, Jang HJ, Sethi G, et al. beta-Caryophyllene oxide inhibits growth
and induces apoptosis through the suppression of PI3K/AKT/mTOR/S6K1 pathways and ROS-medi-
ated MAPKs activation. Cancer letters. 2011; 312(2):178–88. Epub 2011/09/20. PMID: 21924548.
1. Bharathkumar H, Paricharak S, Dinesh K, Siveen KS, Fuchs JE, Rangappa S, et al. Synthesis, biologi-
cal evaluation and in silico and in vitro mode-of-action analysis of novel dihydropyrimidones targeting
PPAR-γ. RSC Advances. 2014; 4(85):45143–6.
PLOS ONE | DOI:10.1371/journal.pone.0153155 April 20, 2016
14 / 15

Imidazoles Abrogate Oncogenic PI3K/Akt/mTOR Signaling Pathway
5
2. Sun Y, Mao X, Fan C, Liu C, Guo A, Guan S, et al. CXCL12-CXCR4 axis promotes the natural selection
of breast cancer cell metastasis. Tumour biology: the journal of the International Society for Oncodeve-
lopmental Biology and Medicine. 2014; 35(8):7765–73. Epub 2014/05/09. doi: 10.1007/s13277-014-
1816-1 PMID: 24810923; PubMed Central PMCID: PMCPmc4158177.
5
5
5
3. Enari M, Sakahira H, Yokoyama H, Okawa K, Iwamatsu A, Nagata S. A caspase-activated DNase that
degrades DNA during apoptosis, and its inhibitor ICAD. Nature. 1998; 391(6662):43–50. Epub 1998/
01/09. doi: 10.1038/34112 PMID: 9422506.
4. Roopashree R, Mohan CD, Swaroop TR, Jagadish S, Raghava B, Balaji KS, et al. Novel synthetic bis-
benzimidazole that targets angiogenesis in Ehrlich ascites carcinoma bearing mice. Bioorg Med Chem
Lett. 2015; 25(12):2589–93. Epub 2015/04/30. doi: 10.1016/j.bmcl.2015.04.010 PMID: 25920563.
5. Kim HM, Kim CS, Lee JH, Jang SJ, Hwang JJ, Ro S, et al. CG0009, a novel glycogen synthase kinase
3
inhibitor, induces cell death through cyclin D1 depletion in breast cancer cells. PLoS One. 2013; 8(4):
e60383. Epub 2013/04/09. doi: 10.1371/journal.pone.0060383 PMID: 23565238; PubMed Central
PMCID: PMCPmc3614906.
56. Siddharth S, Mohapatra P, Preet R, Das D, Satapathy SR, Choudhuri T, et al. Induction of apoptosis by
4
-(3-(tert-butylamino)imidazo[1,2-alpha]pyridine-2-yl) benzoic acid in breast cancer cells via upregula-
tion of PTEN. Oncology research. 2013; 21(1):1–13. Epub 2013/12/18. doi: 10.3727/
96504013x13786659070190 PMID: 24330847.
0
5
7. Lali FV, Hunt AE, Turner SJ, Foxwell BM. The pyridinyl imidazole inhibitor SB203580 blocks phosphoi-
nositide-dependent protein kinase activity, protein kinase B phosphorylation, and retinoblastoma hyper-
phosphorylation in interleukin-2-stimulated T cells independently of p38 mitogen-activated protein
kinase. The Journal of biological chemistry. 2000; 275(10):7395–402. Epub 2000/03/04. PMID:
10702313.
5
8. Manning BD, Cantley LC. AKT/PKB signaling: navigating downstream. Cell. 2007; 129(7):1261–74.
Epub 2007/07/03. doi: 10.1016/j.cell.2007.06.009 PMID: 17604717; PubMed Central PMCID:
PMCPmc2756685.
PLOS ONE | DOI:10.1371/journal.pone.0153155 April 20, 2016
15 / 15