N-fused imidazoles as novel anticancer agents that inhibit catalytic activity of topoisomerase IIα and induce apoptosis in G1/S phase

Article abstract of DOI:10.1021/jm200235u

On the basis of structures of known topoisomerase II catalytic inhibitors and initial molecular docking studies, bicyclic N-fused aminoimidazoles were predicted as potential topoisomerase II inihibitors. They were synthesized by multicomponent reactions a

Full text of DOI:10.1021/jm200235u

ARTICLE
pubs.acs.org/jmc
N-Fused Imidazoles As Novel Anticancer Agents That Inhibit Catalytic
Activity of Topoisomerase IIr and Induce Apoptosis in G1/S Phase
Ashish T. Baviskar,† Chetna Madaan,† Ranjan Preet,‡ Purusottam Mohapatra,‡ Vaibhav Jain,† Amit Agarwal,†
Sankar K. Guchhait,*,† Chanakya N. Kundu,*,‡ Uttam C. Banerjee,† and Prasad V. Bharatam†
†National Institute of Pharmaceutical Education and Research (NIPER), Sector 67, SAS Nagar (Mohali), Punjab-160062, India
‡School of Biotechnology, KIIT University, Campus-11, Patia, Bhubaneswar, Orissa-751024, India
S Supporting Information
b
ABSTRACT: On the basis of structures of known topoisome-
rase II catalytic inhibitors and initial molecular docking studies,
bicyclic N-fused aminoimidazoles were predicted as potential
topoisomerase II inihibitors. They were synthesized by multi-
component reactions and evaluated against human topoisome-
rase IIR (hTopoIIR) in decatenation, relaxation, cleavage
complex, and DNA intercalation in vitro assays. Among 31
compounds of eight different bicyclic scaffolds, it was found that imidazopyridine, imidazopyrazole, and imidazopyrazine with
suitable substituents exhibited potent inhibition of catalytic activity of hTopoIIR while not showing DNA intercalation. Molecular
docking studies and molecular dynamics (MD) simulation analysis, ATPase-kinetics and ATP-dependent plasmid relaxation assay
revealed the catalytic mode of inhibition of the title compounds plausibly by blocking the ATP-binding site. N-Fused
aminoimidazoles showed potent anticancer activities in kidney and breast cancer cell lines, low toxicity to normal cells, relatively
higher potency compared to etoposide and 5-fluorouracil in kidney cancer cell lines, and potent inhibition in cell migration. These
compounds were found to exert apoptotic effect in G1/S phase.
’ INTRODUCTION
quiescent cell populations.20ꢀ22 However, the enzymological
differences between topoisomerase IIR and β are subtle and a
little is known about the physiological role of β-isoform.23,24
Drugs or agents acting on topoisomerase II based on their
mode of action are classified into two: topoisomerase II
poison and topoisomerase II catalytic inhibitor.25 Topoi-
somerase II poisons stabilize the cleavable complex of topoi-
somerase II with DNA by forming the covalent ternary
complex of drug/agent-cleaved G segment DNAꢀtopo II, thus
block religation, and enzyme release and induce apoptosis.
Topoisomerase II catalytic inhibitors act at any of the other
steps of catalytic cycle than the poisons do. Several topo II
poisons such as doxorubicin, etoposide, amsacrine, mitoxan-
trone, and ellipticine are currently widely used in clinical
treatment of cancer. However, despite their importance in
cancer chemotherapy, considerable evidence indicates that
topoisomerase II poisons, related to the level of enzyme-
associated DNA breaks versus recombination-repair path-
ways for induction of apoptosis, may trigger chromosomal
translocations that lead to specific leukemias.26,27 Thus, in
recent years, the development of topo II catalytic inhibitors
modulating the cytotoxic effects of topo II poisons and
overcoming the multidrug resistance (MDR) has gained
importance.28ꢀ30 Topoisomerase II catalytic inhibitors are
DNA topoisomerase (topo) has been recognized as an important
target in anticancer drug discovery.1ꢀ5 About 50% of antitumoral
treatment regimens rely on the use of at least one drug that inhibits
topoisomerases.6,7 Several clinically important antitumoral drugs
such as doxorubicin and daunorubicin (anthracycline class), etopo-
side and teniposide (epipodophyllotoxin class), mitoxanthrone,
amonafide, and amsacrine target the type II topoisomerase.8,9
Camptothecin, a clinically important antitumoral drug, and its
derivatives target the type I topoisomerase.10,11 The development
of dual topoisomerase I and II inhibitors as anticancer drugs is also
recently emerging.12
Topoisomerases (I and II) are essential nuclear enzymes that
maintain the topological changes of DNA and play key role in
transcription, replication, and chromosome segregation.13ꢀ16 To-
poisomerase I catalyzes the relaxation of superhelical DNA by
transiently making a break in the single strand and allowing either
controlled rotation of the double helix about the nick or passage of
other strand through the nick before resealing it.17,18 The topoi-
somerase II creates double-stranded DNA breaks in concert and
allow the passage of a second double-stranded DNA through the
transiently broken duplex.19 Human topoisomerase II exists in two
isoforms, R (alpha) and β (beta). While the β isozyme expression
remains relatively constant and at relatively low level throughout
the cell cycle, R isozyme increases its expression throughout S
phase and peaks with 2ꢀ3-fold at G2/M phase of mitosis and its
concentration is even higher in rapidly proliferating tissues than in
Received: March 1, 2011
Published: June 06, 2011
r
2011 American Chemical Society
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Figure 1. Design of 2-aryl-N-fused imidazoles as potential topo II inhibitors.
now being clinically used, for example, aclarubicin and 4,4-1,2-
(ethanediyl)bis(1-isobutoxycarbonyloxymethyl-2,6-piperazine-
dione) (MST 16)31 as antineoplastic agents, (S)-(+)-1,2 bis(3,5-
dioxopiperazinyl)propane (ICRF 187)32 as cardioprotectors, and
suramin and novobiocin as modulators to increase the efficacy of
other drugs.28 In addition, topoisomerase II inhibitors such as
etoposide, salvicine, and bis-indolylmethane have been found to
possess the ability to regulate the expression of genes involved in
invasion,27 metastasis,33,34 and angiogenesis.35
DNA intercalative or nonintercalative topoisomerase II
poisons have been found to possess, in general, some common
pharmacophoric features such as planar or semiplanar poly-
cyclic ring (as major groove binder to DNA), a suitable number
of hydrogen bond donors and acceptors, and a side chain or ring
(as minor groove binder to DNA).36 However, topoisomerase
II catalytic inhibitors regardless of intercalation-ability/non-
ability to DNA are structurally unrelated. Various biaryls such as
2-arylbenzimidazole, benzoxazole, and benzthiazole37,38 are
known as topoisomerase II inhibitors. Bicyclic N-fused imida-
zole, a class of heterocycles represented by marketed drugs,
such as zolpidem (hypnotic) and zolimidine (antiulcer), pos-
sess structural resemblance to these antitopoisomerase II
biaryls (Figure 1).39,40 As a part of our program aimed at
design, synthesis, and bioevaluation of topo-II targeting antic-
ancer agents, we considered that bicyclic N-fused aminoimida-
zole derivatives could act as potential inhibitors of this enzyme.
Recently, we have filed a patent application for the same.41 The
initial molecular docking study of these compounds using FlexX
program with ATPase domain of hTopoIIR supported their
possible inhibition activity.
In this article, we present the synthesis and biological studies
of novel bicyclic N-fused imidazole derivatives. The bioevalua-
tion of these compounds against hTopoIIR was carried out to
study decatenation, relaxation, cleavage complex, and DNA
intercalation processes. In vitro assays revealed that imidazopyr-
idine, imidazopyrazole, and imidazopyrazine derivatives exhib-
ited potent DNA nonintercalating catalytic inhibition of the
enzyme. Being nonintercalating to DNA, these compounds are
more specific for binding to the hTopoIIR. Molecular modeling
studies and in vitro binding assays revealed that N-fused ami-
noimidazoles exerted inhibitory effect possibly by blocking the
ATP-biding site of hTopoIIR. These derivatives showed potent
anticancer activities in kidney and breast cancer cell lines, low
toxicity to normal cells, relatively higher potency compared to
etoposide and 5-fluorouracil in kidney cancer cell lines, and
potent inhibition in cell migration. The compounds were found
to exert apoptotic effect in G1/S phase.
Chemistry. Multicomponent reactions (MCRs) offer the
elegant performance of multiple reactions into a sequence in
one reaction step without isolating the intermediates, atom
economy, structural complexity of products (structural economy),
bond-forming efficiency (bond-forming economy), convergent
synthesis, and the feasibility of introducing maximum chemical
diversity elements in one chemical event. They are being
increasingly tailored and fine-tuned for synthesizing various
heterocyclic scaffolds which are particularly useful in the pre-
paration of diverse libraries of bioactive molecules. As a part of
our research program for the preparation of therapeutically
relevant heterocyclic compounds, we have recently published
several novel and efficient methods for the multicomponent
reactions42 including the GroebkeꢀBlackburnꢀBienaymꢀe mul-
ticomponent reaction (GBB MCR) of aldehyde, amine and
isocyanide toward the syntheses of N-fused imidazoles and their
derivatives.43ꢀ45 Keeping in view the potential structural features
of N-fused 2-aryl-3-aminoimidazole for exhibiting topoisomerase
II inhibition activity, we synthesized a series of investigational
compounds with the diverse N-fused imidazole scaffolds with
different aryl-substitution at 2-position, moieties linked to amine
at 3-position and substitution linked to fused nonimidazole ring
(compounds 1ꢀ31, Figure 2). They were prepared following our
developed GBB MCR methods and the subsequent known
transformations (Scheme 1). The bicyclic N-fused alkylaminoi-
midazo-pyridine, pyrimidine, pyrazine, and pyrazole (compounds
1ꢀ24) were synthesized by ZrCl4-catalyzed MCR of correspond-
ing heterocyclic amidine, aldehyde, and isocyanide in PEG-400 at
50 °C (method A) or at 140 °C in case of microwave-mediated
dielectric heating process (method B) in good to excellent yields.
Tricyclic N-fused imidazobenzimidazole (compound 25) was
prepared by the microwave-promoted dielectric heating process
(method B), which was found to be successful compared to the
conventional heating method. Free amine-containing N-fused
imidazoles (compounds 26ꢀ30) were prepared by a domino
process of microwave-assisted ZrCl4-catalyzed MCR and subse-
quent de-tert-butylation promoted by HBF4 (method C). The
GBB MCR and subsequent deisooctylation (method D) afforded
the preparation of free amine-containing N-fused pyrazole
(compound 31). All the products were identified by 1H and 13
C
NMR and IR spectroscopies and confirmed by CHN analysis or
HRMS spectroscopy.
’ BIOLOGICAL STUDIES, RESULTS AND DISCUSSION
Inhibition Studies of Topoisomerase IIr. Several in vitro
experiments such as decatenation, relaxation, cleavage complex,
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Figure 2. Compounds (1ꢀ24) were synthesized either following method A or method B; compound 25 was prepared following method B; compounds
(26ꢀ30) were made by method C and compound 31 by method D.
and DNA intercalation assays and ATPase-kinetics were per-
formed for the bioevaluation of synthesized N-fused imidazole
derivatives for their ability to inhibit human DNA topoisomerase
IIR (hTopoIIR) and for the investigation of mode of action. A
screening kit purchased from TopoGEN, Inc. (Columbus, OH)
was utilized. The screening protocols as mentioned in the supplier
manual and reported in literature were followed with modification.
The concentrations of the reagents were varied according to
requirement of our experiments (see Experimental Section).
ATP-dependent decatenation assay, a specific assay for the
topoisomerase II inhibition activity, was performed with inves-
tigational compounds using kinetoplast DNA (kDNA) as a
substrate. Etoposide, a known topoisomerase II-inhibiting antic-
ancer drug, was used as standard. The results are presented in
Figure 3A. The decatenation of kDNA by catalytic activity of
topoisomerase II forms different decatenation products: nicked
(Nck), relaxed (Rel), and supercoiled (SC) DNA. The catenated
kDNA appears in agarose gel at the top and cannot enter into the
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Scheme 1. Methods of Preparation of Investigational Com-
pounds As Potential hTopoIIr Inhibitors
Topo II catalytic inhibitors are of two types: DNA intercala-
tors and nonintercalators.46 To determine the intercalation/
nonintercalation ability of potent compounds, DNA intercala-
tion assay using negatively supercoiled small circular plasmid
DNA (isolated from Escherichia coli) as substrate was
performed.47 In this assay, 250 ng of plasmid was incubated with
ethidium bromide (1 μg/mL), standard drug (etoposide), and
investigational compounds. Samples were then loaded in 1%
agarose gel, and electrophoresis was run without ethidium
bromide. DNA intercalators change the degree of supercoiling
or mass of the plasmid DNA, and this change can be detected by
observing the retardation in migration during electrophoresis. By
performing this assay, it was found that in the presence of
intercalative agent (ethidium bromide), there was retardation
in the migration of the DNA (Figure 4B), whereas, in the case of
etoposide (DNA nonintercalator) and compounds tested, there
was no such retardation. This result indicates that the tested
compounds are DNA nonintercalators.
From the decatenation, relaxation, stabilization of cleavage
complex, and DNA intercalation assays, it is revealed that the
investigational compounds (5, 16, 23, 29, and 31) act as DNA
nonintercalating hTopoIIR catalytic inhibitors. For the measure-
ment of IC50 value, a study of decatenation assay at various
concentrations ranging from 10 to 100 μM for the compound 5
(as a representative) was performed. From the plot it was found
that compound 5 had IC50 value of 52.66 μM (Figure S1,
Supporting Information).
StructureꢀActivity Relationship Study. Upon the basis of
the results of topoisomerase II inhibition exhibited by N-fused
imidazoles in decatenation and relaxation assays, we synthesized
a series of relevant compounds to find out the structureꢀactivity
relationship and the pharmacophoric importance of scaffold,
moieties/substituents, and their positions (Figure 2). N-Fused
imidazoles without aryl-substitution at 2-position (compounds 6,
11, and 22) did not exhibit any inhibition activity of hTopoIIR.
The presence of naphthalene moiety at 2-position in imidazo-
pyridine (compound 7) showed good inhibitory activity, while
the presence of N,N-dimethylphenyl (compound 4) showed
moderate activity. In addition to bicyclic N-fused imidazoles,
imidazo-benzimidazole (compound 25), which is a tricyclic
molecule, was also found to be considerably active against
topoisomerase II. N-Fused imidazoles containing ꢀNH2 without
tert-butyl group at 3-position (compounds 26ꢀ31), in compar-
ison to their 3-tert-butylamine-containing analogues (except
compound 30), showed lower inhibition activity. The com-
pounds 10, 14, 18, 24, and 28 showed poor activity, and
compounds 6, 8, 13, 15, 17, 20, and 27 did not show any
inhibition.
All together, suitable combination of nonimidazole scaffold
(pyridine, pyrazine, or pyrazole) and its substitution, relevant
aryl-substitution at 2-position, and tert-butyl-/free-amine at 3-
position is crucial for exhibiting inhibitory activity of hTopoIIR.
Molecular Docking Analysis. As potent topoisomerase II
inhibitors showed catalytic inhibition of the enzyme and noninter-
calation with DNA, we were interested in identifying the potential
binding of N-fused imidazole derivatives to the ATPase domain of
hTopoIIR in the active site by molecular docking simulation using
the FlexX program48 incorporated in SYBYL 7.1.49 To validate the
molecular dockingprotocol, ATP andAMPPNP (bound ligand, 50-
adenylyl-β,γ-imidodiphosphate) were initially docked into the
crystal structure of ATPase domain of hTopoIIR.50 The docked
AMPPNP found to have similar binding pose as compared to the
gel because of its overall size while other decatenated products
move into. This characteristic pattern was also observed in our
experiment. Etoposide showed partial or moderate decatenation
because of its known reversible inhibition of topoisomerase
IIR.30 Almost no decatenation activity was observed in the
presence of compounds 5, 9, 16, 23, and 30. Partial or moderate
inhibition of topoisomerase IIR was shown by compounds 12,
26, 29, and 31, while compounds 2, 11, 19, 21, and 22 did not
show any inhibition. Quantification of decatenation products
formed (Nck, Rel, and SC) was done by densitometric data
obtained using QuantityOne (BioRad), and the results were
compared with etoposide (Figure 3C). In comparison to etopo-
side, compounds 5, 9, 16, 23, and 30 were found to show higher
inhibition of topoisomerase IIR whereas compounds 12, 26, 29,
and 31 exhibited considerable inhibition activity. To confirm
their topoisomerase IIR inhibitory potential, relaxation assay
using etoposide as positive control was then performed. In this
assay, topoisomerase II converts the negatively supercoiled form
of substrate pRYG plasmid DNA into relaxed topoisomers that
migrate more slowly. Results of our experiment as shown in
Figure 3B also reveal the same observation. The products formed
(relaxed topoisomers) were quantified, and the results were
compared with etoposide (Figure 3D). It was observed that in
comparison to etoposide, compounds 5, 9, 16, 23, and 30 were
found to be more potent topoisomerase IIR inhibitors and
compounds 12, 26, 29, and 31 exhibited moderate topoisome-
rase II inhibition. This result of inhibition-potency was found to
be consistent with decatenation assay.
Furthermore, to gain insight into the mode of action of
investigational potent compounds as topoisomerase II poisons
or catalytic inhibitors, cleavage complex assay was performed.
Topoisomerase II poisons act by stabilizing enzymeꢀDNA clea-
vage complex and thus lead to the formation of linear DNA. In
cleavage complex assay, initially the relaxation of the plasmid
DNA (pBR322) was allowed to start by addition of topo II and
then the compound was added. The gel was run in the presence
of ethidium bromide in order to clearly visualize the linear form
of the DNA (Figure 4A). In contrast to etoposide (topo II
poison), the linear form of the DNA was not visible in the case
of investigational compounds (5, 16, 23, 29, and 31). From
these results it can be concluded that tested compounds did not
act as classical topoisomerase II poisons even at 200 μM
concentration. In addition, no linear band was observed when
etoposide was preincubated with compound 5, which revealed
its antagonizing effect on topoisomerase II poison. These
observations indicate that these N-fused imidazoles act as
topoisomerase IIR catalytic inhibitors.
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Figure 3. hTopoIIR-inhibitory effect of N-fused imidazoles in decatenation and relaxation agarose gel electrophoresis assays. (A) Decatenation assay:
kDNA (intact kinetoplast DNA) was used as substrate, decatenation products formed were Nck (nicked), Rel (relaxed), and SC (supercoiled) DNA.
kDNA was treated with hTopoIIR in presence of either 100 μM etoposide or N-fused imidazoles. (B) Relaxation assay: Negatively supercoiled pRYG
plasmid DNA was used as substrate and products formed were its relaxed topoisomers. pRYG DNA was treated with hTopoIIR (without ethidium
bromide) in the presence of either 100 μM etoposide or investigational compounds. (C) Quantification of product formed in kDNA decatenation assay.
(D) Quantification of product formed in pRYG relaxation assay.
cocrystallized ligand with a root mean square deviation (rmsd) of
about 1.55 Å. Moreover, superimposition of docked ATP and
cocrystallized AMPPNP suggested that ATP also occupies the
binding pocket in a similar fashion (Figure 5) and making 3 point
contacts with Mg as well as forming key interactions with the
residues of ATPase domain similar to that of bound AMPPNP
(Figure 6), thus validating the adopted docking methodology. This
also suggested that docking protocol is reliable enough to carry out
further binding mode analysis of investigational ligands.
In detail, the binding model of ATP with ATPase domain of
hTopoIIR (Figure 6) revealed that the amino group of adenine
moiety showed hydrogen bonding with the side chain carbonyl
oxygen of Asn120 and the two hydroxyl groups of ribose unit
formed hydrogen bonding interaction with the side and main chain
of Ser149, respectively. Interestingly, the oxygen atoms in the three
phosphate group were involved in several interactions with the
ATPase domain, viz: hydrogen bonding with the main chain
nitrogen atom of the residues Arg162, Asn163, Gly164, Tyr165,
Gly166, and Ala167 (Walker A consensus motif). In addition, these
phosphate groups also showed hydrogen bonding interactions with
the side chains of residues Asn91, Ser148, Asn150, Lys168, and
Gln376. Consistent with the interactions shown by the bound
ligand AMPPNP with the crystal structure of ATPase domain, the
docking model of ATP also showed that phosphate group forms a
salt bridge with the highly conserved Lys378 from the QTK-loop
(Gln376-Thr377-Lys378) of the transducer domain.
The highly conserved Walker A consensus ATP binding site of
hTopoIIR is composed of residues 161ꢀ166 (GXXGXG)51 and
is important for the binding of ATP as well as for the catalytic
inhibitors acting on ATPase domain. Wessel et al. performed site-
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Figure 4. (A) Stabilization of cleavage complex assay: After treatment with hTopoIIR, negatively supercoiled plasmid was incubated with either 100 μM
etoposide or investigational compounds or both (for testing antagonizing effect, last 3 lanes). The agarose gel was run in excess of ethidium bromide to
check the formation of linear (Lin) band. In contrast to etoposide, a linear band was not observed in any of the tested compounds. Compound 5 was
found to antagonize the effect of etoposide as topoisomerase II poison. (B) DNA intercalation assay: Negatively supercoiled plasmid was incubated with
1 μg/mL ethidium bromide or 100 μM etoposide or investigational compounds. Agarose gel electrophoresis (without ethidium bromide) was run,
which clearly showed retardation in the migration of ethidium bromide (intercalator)-treated DNA, whereas no retardation was observed in DNA
treated with investigational compounds.
Figure 5. Superimposition of cocrystallized AMPPNP (magenta), best
docked pose of ATP (green) and compound 5 (cyan) in the hTopoIIR
Figure 6. Binding model of ATP as revealed from FlexX docking in
hTopoIIR ATP binding site. The blue dashed lines represent hydrogen
bonds and magenta sphere represents Mg cation. H-bond distances (in Å)
between heteroatoms of ligand and amino acid residues are as follows: Asn91
(2.71), Asn120 (3.37), Ser148 (2.53), Ser149 (2.87), Asn150 (3.12),
Arg162 (3.12), Asn163 (2.78), Gly164 (3.01), Tyr165 (2.59), Gly166
(2.59), Ala167 (2.98), Lys168 (2.72), Gln376 (2.93), and Lys378 (2.95).
ATP binding site. The magenta sphere represents Mg cation showing
octahedral coordination geometry.
directed mutagenesis studies and found that the Arg162Gln and
Tyr165Ser mutations (in the ATP binding site) lead to drug
resistance.52,53 Moreover, the importance of residue Gly164 was
established by Skouboe et al.54 The results of their study revealed
that ATP binding was inhibited by mutation of residue Gly164 to
Ile. The docking model of ATP as discussed above also showed
that the residues in Walker A motif are crucial for the binding of
ATP, thus suggesting the reliability of docking methodology.
Further studies demonstrated that N-fused imidazoles might
bind at the ATP site of hTopoIIR ATPase domain. The super-
imposition of the cocrystallized AMPPNP, docked ATP, and
most active compound 5, which is also a best scored (ꢀ35.45)
compound according to the docking results (Table 1), indicate
that the benzene ring and para carboxylic group overlap with the
phosphate groups of AMPPNP and ATP in the ATP binding site
(Figure 5). This observation implies that the substitution at
2-position of N-fused imidazole ring is essential for the compe-
titive binding with ATP. This is also consistent with the experi-
mental results and SAR study because the compounds 6 and 11
lacking substitution at 2-position of N-fused imidazole ring were
found to be inactive as catalytic inhibitors.
compounds 6 and 11 (inactive). As indicated in the interaction
model between most active compound 5 and ATPase domain
(Figure 7), the carboxylic group at para position of benzene ring
formed multiple hydrogen bonding interactions with the back-
bone nitrogen atom of the residues Arg162, Asn163, Gly164,
Tyr165, and Gly166. The same carboxylic acid group also formed
hydrogen bonding interactions with the side chain of residues
Gln376 and Lys378. In addition, imidazole nitrogen of compound
5 showed hydrogen bond with the side chain of Ser148 and
backbone nitrogen atom of Ser149, respectively. Most notably, the
benzene ring substituted at 2-position in compound 5 showed an
effective π interaction with the magnesium cation (magenta
sphere in Figure 7), and the 3-substituted tert-butyl group of
compound 5 was involved in the hydrophobic interaction with the
Asn91, Arg98, and Asp94. Moreover, the same tert-butyl group
may also be involved in weakCH O type ofinteractionwith the
3 3 3
side chain of residues Asn91 and Asp94, which may give extra
stability to the enzymeꢀligand interactions. Additionally, pyridine
ring fused with imidazole also showed hydrophobic interaction
with residues Ile141 and Phe142. The docking results for other
active compounds (23, 30, and 31) also predicted favorable
Table 1 demonstrates the result of the molecular docking
along with hydrogen bonding and metal ion interactions of
compounds 5, 23, 30, and 31 (potent topoisomerase II
inhibitors), compounds 26 and 29 (moderately active), and
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Table 1. FlexX Docking Results for Some of the Synthesized Compounds and Substrate ATP in Presence of Two Conserved Water
Molecules in hTopoIIr ATP Binding Site
hydrogen bonding interactions
compd
experimental activity
substrate
FlexX score
backbone
side chain
interaction with Mg cation
ionic interaction
ATP (ꢀ4)a
ꢀ84.06
Ser149, Arg162, Asn163, Gly164,
Tyr165, Gly166, Ala167
Ser149, Arg162, Asn163,
Gly164, Tyr 165, Gly166
Ser149, Gly164, Ala167
Arg162, Asn163, Gly164
Asn91
Asn91, Asn120, Ser148, Ser149,
Asn150, Lys168, Gln376, Lys378
Gln376, Lys378
5 (ꢀ1)a
good
ꢀ35.45
π interaction
31
23
30
29
26
11
6
good
ꢀ23.53
ꢀ22.73
ꢀ20.20
ꢀ11.50
ꢀ10.86
ꢀ10.23
ꢀ8.78
Asn91, Ser148, Ser149, Asn150 (4)
Asn91, Ser148 (2), Ser149, Asn150
Asp94, Ser148, Asn150 (2)
Asn150
ionic interaction
ionic interaction
π interaction
π interaction
π interaction
no interaction
no interaction
good
good
moderate
moderate
not active
not active
Asn91
Asn91
Asn150, Ser148
Asn95, Ser149
Ser149
a Values in bracket indicates the total charge on the compound, while other compounds are considered as neutral.
Table 2. Averaged Binding Free Energy Results for
EnzymeꢀLigand (Compound 5) Complex along with
Its Different Energy Components
energy
termsa (kcal/mol)
energy
energy
termsa
(kcal/mol)
termsa
(kcal/mol)
ΔEELE
ΔEVDW
ΔEGAS
ꢀ253.50
ꢀ36.21
ꢀ289.71
ΔPBSUR
ΔPBCAL
ΔPBSOL
ΔPBELE
ΔPBTOT
ꢀ5.54
231.52
225.99
ꢀ21.98
ꢀ63.72
ΔGBSUR
ΔGB
ꢀ5.54
227.00
221.46
ꢀ26.50
ꢀ68.25
ΔGBSOL
ΔGBELE
ΔGBTOT
a The meaning of the different terms in this table is as follows: ELE =
electrostatic energy as calculated by the MM force field; VDW = van der
Waals contribution from MM; INT = internal energy arising from bond,
angle and dihedral terms in the MM force field (this term always
amounts to zero in the single trajectory approach); GAS = total gas
phase energy (sum of ELE, VDW and INT); PBSUR/GBSUR =
nonpolar contribution to the solvation free energy calculated by an
empirical model; PBCAL/GB = the electrostatic contribution to the
solvation free energy calculated by PB or GB respectively; PBSOL/
GBSOL = sum of nonpolar and polar contributions to solvation;
PBELE/GBELE = sum of the electrostatic solvation free energy and
MM electrostatic energy; PBTOT/GBTOT = final estimated binding
free energy calculated from the terms above (kcal/mol).
Figure 7. Binding model of active compound 5 (cyan) as revealed from
FlexX docking in hTopoIIR ATP binding site. The blue dashed lines
represent hydrogen bonds and magenta sphere represents Mg cation.
H-bond distances (in Å) between heteroatoms of ligand and amino acid
residues are as follows: Ser148 (3.33), Ser149 (2.56), Arg162 (2.83),
Asn163 (2.25), Gly164 (3.03), Tyr165 (2.99), Gly166 (3.09), Gln376
(2.98), and Lys378 (3.43).
interactions between the compounds and key residues at the
ATPase domain, similar to that observed in the interaction model
of compound 5 (Table 1).
bonding interactions with the key residues of the ATPase
domain as shown by ATP (Table 1). Therefore, these com-
pounds can effectively compete with the ATP for occupying the
ATP-binding pocket of hTopoIIR.
Thus, molecular modeling studies performed on a small
number of N-fused imidazole derivatives from our chemical
archives suggest that some compounds are catalytic inhibitors
acting via occupying the ATP binding pocket of ATPase
domain of hTopoIIR and making favorable interactions with
its key residues.
Molecular Dynamics Simulation Analyses. The molecu-
lar docking analyses of compound 5 in the active site of
ATPase domain of hTopoIIR provided a pose which explains
most of the enzymeꢀligand (inhibitor) interactions. To
validate that these interactions are realistic, stability of the
From the detailed analysis of active compounds binding
model, it was found that all potent and moderately active
compounds showed an effective interaction with the magne-
sium, either via π interaction (5, 26, 29, and 30) or ionic
interaction (23 and 31) (Table 1). This type of interaction was
missing in case of the inactive compounds (6 and 11), as they
lack benzene ring at 2-position of N-fused imidazole system.
These observations are consistent with the results for the
docking of pyranonaphthoquinones into the ATP binding
pocket of hTopoIIR, reported by Jimenez-Alonso et al.30 More-
over, the inactive compounds (6 and 11) showed less favorable
docking score and missed the key interactions shown by the
active compounds and ATP with the residues of ATPase
domain. The binding model of the active compounds (5, 23,
30, and 31) also revealed that they share common hydrogen
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Figure 8. Concentration-dependent inhibition of topoisomerase IIRꢀATPase activity by compound 5. (A) LineweaverꢀBurk plot of the ATPase
activity assay. (B) Slopes of the respective lines against different concentrations of compound 5.
Figure 9. Inhibition of topoisomerase activity by compound 5 in HEK 293 cells. Reaction was carried out according to the protocol described in
Experimental Section. Image shown here is representative of three independent experiments.
enzymeꢀligand complex was assessed by MD simulations
implementing AMBER 10 software.55
The energy vs time plot showed an unperturbed energy
profile during the production run, confirming the stability of
complex (Figure S3, Supporting Information). The estimated
fluctuation in the rmsd of the enzymeꢀligand complex during
the equilibration to production run is less than 1 Å, again
confirming the stability of complex (Figure S4, Supporting
Information). The hydrogen bond analyses confirmed the
persistence of hydrogen bonding interactions of ligand
(compound 5) with residues Arg162, Asn163, and Lys378, while
hydrogen bonding with residues Ser149, Gly164, and Gln376 are
less consistent (Figure S5a,b, Supporting Information).
The prediction of binding affinity of ligands was per-
formed on the basis of binding free energy calculations using
Figure 10. ATP reverts the topoisomerase inhibition of compound 5.
The image shown here is representative of three independent
experiments.
molecular mechanics PoissonꢀBoltzmann surface area
(MM-PBSA) and molecular mechanics generalized Born
surface area (MM-GBSA) protocol.56,57 In the present study,
ꢀ68.25 kcal/mol, respectively. ΔPBTOT accounts oftotal relative
we estimated the different components of interaction energy
binding free energy which equal to ΔEGAS + ΔPBSOL which shows
that most of the contribution to the binding free energy comes
that contribute to binding, these include van der Waals,
electrostatic, polar solvation, and nonpolar solvation inter-
from the electrostatic component, whereas van der Waals con-
action energies. Table 2 shows the averaged binding free
tribution is comparatively less. This is due to the fact that ligand
energy results for enzymeꢀligand complex along with their
(compound 5) has negatively charged carboxylic group at para
position of benzene, which is forming favorable hydrogen bonding
and electrostatic interactions with the backbone nitrogen atom of
Walker A consensus motif residues.
different energy components (see Supporting Information
for more detail). The binding free energy calculated by the
above two methodologies, ΔGMM/PBSA and ΔGMM/GBSA for
enzymeꢀligand (compound 5) complex are ꢀ63.72 and
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Figure 11. Clonogenic cell survival after treatment with indicated compounds of various concentrations for 48 h. Dotted lines represent the % survival
of HEK-293 cells, and bold lines represent % survival of Vero cells, respectively. Data represented here are mean of three independent experiments, and
values are mean ( SD.
ATPase Assay. Molecular docking studies of N-fused aminoi-
midazole (compound 5) as mentioned above suggested their
catalytic mode of inhibition, plausibly via blocking the ATP-
binding site of topoisomerase. To prove this observation, an in
vitro ATPase assay for DNA dependent ATP hydrolysis of
hTopoIIR was performed using compound 5. Different concen-
trations of ATP (200, 280, 360, 440, and 520 μM) and 6 units of
topoisomerase II were incubated with compound 5 of a fixed
concentration 0, 50, 100, and 150 μM, respectively. Lineweaverꢀ
Burk plot of the ATPase assay (Figure 8A) shows decrease in Km
while no change in Vmax. This indicates that compound 5 is a
competitive inhibitor of ATP.58,59 Calculated Ki value for com-
pound 5 was 75 μM (Figure 8B).
Inhibition of Topoisomerase Activity in HEK 293 Cells. A
DNA unwinding assay was performed using compound 5, HEK
293 nuclear lysate, and plasmid DNA following the procedure
as described in the Experimental Section. For nuclear cell lysate
without compound 5, supercoiled plasmid was relaxed or linear
and thus did not enter into the 0.9% agrarose gel (Figure 9).
This may be because of topoisomerase activity in nuclear lysate
of HEK 293 cells. However, in the presence of compound 5
with increasing concentrations, the supercolied DNA topo-
isomer was found to be more and at 35 μM concentration good
inhibition (compare lane 1 with 6) of topoisomerase activity
was found (Figure 9).
relaxation assay of topoisomerase activity was performed
using HEK 293 nuclear lysate, compound 5, and ATP of
various concentrations. Figure 10 illustrates that in the ab-
sence of compound 5, topoisomerase effectively relaxes the
supercoiled form of the plasmid (lane 2), but in the presence
of compound 5 and etoposide (positive control), topoisome-
rase activity was inhibited and a more supercoiled form of the
plasmid was observed (lane 3 and 4, respectively). Interest-
ingly, when a fixed concentration of compound 5 and increas-
ing doses of ATP were added to the reaction mixture,
increasing amount of the relaxed form of the plasmid was
observed. Thus, it appears that ATP with higher concentra-
tions displaces compound 5 from the active site of topoi-
somerase (lane 5 to 9). This implies that ATP and compound
5 are competitive inhibitors of topoisomerase ATP binding
domain which is in accordance with in vitro hTopoIIR-
ATPase study.
Anticancer Activities of Compounds 5, 16, 23, 29, and
31. Clonogenic Cell Survival Assay of Kidney Cancer Cells (HEK
293). The clonogenic cell survival assay is a long-term cell viability
assay that determines the ability of a single cell to proliferate
indefinitely, thereby retaining its reproductive ability to form a
colony or a clone. This assay was done to investigate the
anticolony-forming potential of N-fused imidazoles 5, 16, 23,
29, and 31 in kidney cancer cell lines (HEK 293) as compared to
normal kidney epithelial cells (Vero). Each compound showed
colony formation inhibition in a concentration-dependent
ATP Dependent Plasmid Relaxation Assay. In addition to
in vitro ATPase kinetic studies, an ATP dependent plasmid
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Figure 12. MTT cell survival assay. Cells were treated with investigational compounds and etoposide of various concentrations for 48 h and the assay
was carried out according to the protocol as described in Experimental Section. The sign —O—, ---4---, , and ---9--- represent for Vero cells/
2
3 3 3 3 3 3
investigational compound, Vero/etoposide, HEK 293/etoposide, and HEK 293/investigational compound, respectively. Bar diagram represents the
effect of compound 5 (12 μM) on Vero and HEK 293 cell survival with time. Data represented here are the mean of three independent experiments and
values are mean ( SD.
manner (Figure 11). HEK 293 cells were more susceptible than
Vero cells to the tested compounds.
a separate set of experiments was performed by treating the
cells with compound 5 of a fixed concentration (12 μM) for
different time intervals. It was noticed that more than 50%
HEK 293 cells died after 48 h exposure of compound without
considerably affecting the Vero cells (Figure 12, bar diagram).
Cell Survival Activity of Compound 5 and 5-Fluorouracil in
Cancer Cell Lines. To check the relative potency of anticancer
activities of investigational N-fused imidazoles with a clinically
used anticancer drug 5-fluorouracil,61 an experiment using
kidney cancer cell line was performed (Figure 13A). Cells were
treated separately with different compounds and 5-fluorouracil of
various concentrations for 48 h, and then cell survival was
measured by MTT assay. All the compounds showed a char-
acteristic cell survival profile. LC50 for compound 5, 16, 23, 29,
and 31 were found to be 14, 10, 15, 17, and 27 μM, respectively.
However, 5-fluorouracil caused 50% cell death at 30 μM
concentration.
LC50 (the concentration required to kill half the cells in
the cell culture) exhibited by compounds 5, 16, 23, 29,
and 31 were 30, 20, 15, 16, and 25 μM, respectively, in
HEK 293 cells and 98, 25, 22, 28, and 62 μM, respectively, in
Vero cells (Figure 11). From these data it appears that all the
tested compounds offered more anticolony-forming ability
in kidney cancer cells compared to normal kidney epithe-
lial cells.
MTT Cell Survival Assay of Kidney Cancer Cells (HEK 293).
To investigate cytotoxicity of compounds (5, 16, 23, 29, and
31) in both HEK 293 and Vero cells, an MTT assay was carried
out.60 Cell survival was measured after treatment of cells with
the investigational compounds for 48 h. With increasing
concentration of the compounds, the cell viability of HEK
293 significantly decreased in comparison to Vero cells.
Compound 5, 16, 23, 29, and 31 killed 50% HEK 293 cells
(LC50) at 12, 10, 15, 17, and 25 μM, respectively. However,
any compound of even 100 μM concentration did not cause
50% cell death in Vero cells (Figure 12). Etoposide showed
LC50 at 90 μM in HEK 293 cells while it exerted similar effect
in Vero cells. To investigate relative survival of cells with time,
To investigate further the cytotoxic effect of compound 5
on another cell line such as breast cancer, a MTT assay was
performed using breast cancer (MCF 7) cells and normal
breast epithelial cells (MCF 10A). Figure 13B illustrates the
anticell survival potential of compound 5 on MCF 7 and MCF
10A cells. Compound 5 caused 50% cell death at 15 μM in
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Figure 13. Effect of compound 5 on cell survival and migration in anchorage dependent manner. (A) Comparison of anticell survival activity of
compounds (5, 16, 23, 29, and 31) with 5-fluorouracil in HEK 293 cells by cell survival assay. (B) Graphical presentation for the effect of compound 5 on
breast cancer cells (MCF 7) as compared to normal breast epithelial cells (MCF 10A). Data represented here are the mean of three independent
experiments and values are mean ( SD. (C) Effect of compound 5 on cell migration in HEK 293 cells. After making the wound with a sterile micro tip,
the cells were cultured in absence and presence of compound 5 (25 μM). Results shown here are the best of three independent experiments.
MCF 7 cells, whereas 75% cell survival was noted at 100 μM
in MCF 10A (p < 0.05) (Figure 13B).
cleaved PARP product (86 KD) was higher in the treated cells
than untreated cells and increased with increasing concentra-
tions of the drug. As expected, the expression of β-actin, which
served as a loading control, remained unaltered. These relative
expressions of relevant apoptotic markers including the in-
creasing Bax/Bcl-XL ratio, Caspase-3, and PARP cleavage
clearly indicate that compound 5 causes apoptosis in can-
cer cells.
Regulation of Cell Cycle by Compound 5 in Kidney Cancer
Cells. To check the regulation of cell cycle profile and to
understand the mechanism by which compound 5 can induce
apoptosis, we analyzed the kidney cancer cells after treatment
with compound using fluorescence activated cell sorter (FACS)
by staining with propidium iodide. Figure 14D demonstrates
the cell cycle profile of HEK 293 cells after exposure to
compound 5 for 48 h. A sharp dose-dependent increase in
apoptosis (Sub G0) was observed with the drug. At 15, 25, and
35 μM concentrations, the percentage of apoptotic nuclei were
noted to be 24.61 ( 1.5, 27.46 ( 1.5, and 49.29 ( 1.0,
respectively (p < 0.05). It was observed that the percentage of
G1-DNA population was significantly decreased with increas-
ing concentrations of compounds 5 (approximately 21.52% at
35 μM in contrast to 61.7% in untreated cells). It was also
noticed that S phase DNA population decreased (less than
10%) in comparison to untreated cells. However, no significant
change was noted in G2/M phase. A histographical presenta-
tion of cell cycle profile of HEK 293 after treatment with
compound 5 is provided (Figure 14E). Thus, from the above
Anti-Cell Migratory Activity of Compound 5 in HEK 293 Cells.
To examine the inhibitory effect of compound 5 on anchorage-
dependent cell migration, a wound healing assay was performed
according to protocol described in Experimental Section. The
gap of the treated cells increased with time in contrast to the
untreated cells (Figure 13C). In untreated cells, the gap was
completely filled at 24 h. Thus, this assay in conjunction with
the assays as discussed above (clonogenic and MTT cell
survival) reveals that compound 5 possesses the inhibitory
activities of colony formation, viability, and migration of cancer
cells (Figure 11ꢀ13).
Apoptosis by Compound 5 in HEK 293 Cells. Apoptosis of
HEK 293 cells were determined after 48 h of treatment with
compound 5 using 40-6-diamidino-2-phenylindole (DAPI) nu-
clear staining method (Figure 14A). It was observed that
apoptotic nuclei increased with increasing concentrations of
compound 5 (Figure 14B). It was noticed that at 15, 25, and
35 μM concentrations, compound 5 caused approximately 20,
40, and 80% apoptosis, respectively. To further confirm the
apoptotic effect of this compound in HEK 293 cells, a study of
protein levels of some apoptotic markers such as Caspase-3,
Bax, Bcl-XL, and PARP cleavage was performed by Western
blot analysis (Figure 14C). The expression of Bcl-XL was
reduced when cells were exposed to compound 5 of increasing
concentrations (0, 15, and 25 μM). In contrast to Bcl-XL, the
expression of Bax and Caspase-3 was increased. The level of
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Figure 14. Regulation of cell cycle profile and apoptosis caused by compound 5 in HEK 293 cells. Cells were treated for 48 h with different
concentrations of compound 5, and then experiments were carried out according to the method as described in Experimental Section. (A) DAPI nuclear
staining. Images are one of the three replicates of the individual experiments. Images 1, 2, 3, and 4 represent cells treated with compound 5 of 0, 15, 25,
and 35 μM, respectively. (B) A graphical presentation of apoptotic nuclei of (A) (P < 0.05). (C) Western blot analysis of whole cell extracts. Lower panel
(β-actin) represent equal loading of each lane. (D) Tabular form of percentage of DNA in each phase of cell cycle, and (E) graphical presentations of
FACS analysis of (D). Here the data presented are the best of three independent experiments.
results it appears that cancer cells undergo significant apoptosis
in G1/S boundary phase of cell cycle.
Compared to etoposide and 5-fluorouracil, these compounds were
found to be more potent in anticancer activity (kidney cancer cells).
Compound 5, chosen as a representative, was found to be potent
anticancer agent in breast cancer cells (MCF 7) while relatively
less toxic to normal cells (MCF 10A). It showed also the anticell
migration. DAPI nuclear staining and the study of expression level
of apoptotic marker proteins revealed that compound 5 caused
programmed cell death, and the FACS analysis indicated that the
apoptosis occurred in G1-S boundary phase of cell cycle. The specific
topoisomerase IIR catalytic inhibition activity via ATPase domain-
binding, inhibition of topoisomerase in cancer cells, apoptotic affect
in G1/S boundary phase, known increased expression of topoisome-
rase IIR enzyme in G1/S phase, and the molecular docking studies of
potent N-fused aminoimidazoles indicate that these novel com-
pounds exerted the anticancer activity by programmed cell death via
catalytic ATPase domain-inhibition of topoisomerase IIR. These
results will aid in the rational design of novel agents that target the
catalytic inhibition of hTopoIIR and will provide insights into the
discovery of novel anticancer agents.
’ CONCLUSIONS
In conclusion, several derivatives of bicyclic N-fused aminoimida-
zoles were synthesized by multicomponent reaction and its tandem
process of dealkylation. The hTopoIIR-inhibitory activity of these
new chemical entities (NCEs) was tested in decatenation, relaxation,
and cleavage complex in vitro assays. DNA intercalation assay was
performed with EtBr as positive control. It revealed that five
compounds, 5, 16, 23, 29, and 31, acted as potent DNA noninterca-
lating topoisomerase IIR catalytic inhibitors. Being nonintercalating
to DNA, these compounds are more specific for binding to the
hTopoIIR. Molecular docking studies using FlexX program incor-
porated in SYBYL 7.1 of potent N-fused aminoimidazoles into the
ATPase domain of hTopoIIR, and the molecular dynamics simula-
tion studies suggested their catalytic mode of inhibition, plausibly via
blocking the ATP-binding site of enzyme. This ATP-competitive
inhibition by N-fused imidazole was also further evident by ATPase
kinetic study and ATP dependent plasmid relaxation assay of nuclear
lysate using compound 5. Clonogenic and MTT cell survival assay of
kidney cancer cells (HEK 293) revealed that these derivatives
showed potent anticancer activity and low toxicity to normal cells.
’ EXPERIMENTAL SECTION
Chemistry. The compounds 1ꢀ24 were prepared following the
reported method A or B as mentioned in Scheme 1.43,44 Compound 25
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was synthesized following the reported method B. The method C
afforded the preparation of compounds 26ꢀ30.45 Compound 31 was
synthesized by reported method D. All the products were identified by
1H and 13C NMR and IR spectroscopies and confirmed by elemental
analysis or HRMS spectrometry. Analytical HPLC was used to deter-
mine the purity of the compounds and the purity was found to be greater
than 95%. All these data are provided in Supporting Information.
Biological Assay. The testing of new chemical entities was per-
formed using a commercially available topoisomerase II drug screening
kit, purified hTopoIIR, and etoposide purchased from TopoGEN, Inc.
(Columbus, OH). A stock solution of etoposide of concentration,
10 μM in DMSO, was prepared and stored at ꢀ20 °C. Synthesized
compounds were also stored at ꢀ20 °C. The assay protocols followed
were same as mentioned in the supplier manual, except the concentra-
tion of the reagents which were varied according to the requirement.
TopoII-Mediated DNA Decatenation Assay. In agarose gel
decatenation assay, kinetoplast DNA (kDNA) was used as a substrate.
All the reactions were assembled on ice in micro centrifuge tubes. Briefly,
2ꢀ4 units of purified human topoisomerase II, 150 ng of catenated
kDNA, and 100 μM drug dissolved in DMSO were combined in freshly
prepared 5ꢁ complete reaction buffer. The reaction mixture was
brought to a final volume of 20 μL with deionized, distilled H2O. For
the standard assay method, the order of addition to the assay was H2O,
5ꢁ assay buffer (buffer A: 0.5 M Tris-HCl (pH 8), 1.50 M sodium
chloride, 100 mM magnesium chloride, 5 mM dithiothreitol, 300 μg of
bovine serum albumin/mL; buffer B: 20 mM ATP in water), kDNA,
followed by either test compound or standard drug and finally topoi-
somerase II. The incubation was run at 37 °C for 30 min. The reaction
was then stopped with 10% SDS, followed by digestion with proteinase
K and further incubated at 37 °C for 15 min. Topological forms of kDNA
were resolved by running 1% agarose gel electrophoresis in Tris-acetate-
EDTA (TAE) buffer containing 0.5 μg/mL ethidium bromide and
further destained with water for 20 min. The bands of kDNA and
decatenated products were visualized by UV trans-illumination, and the
products were quantified with QuantityOne (BioRad).
TopoII-Mediated DNA Relaxation Assay. In relaxation assay
supercoiled plasmid DNA (pRYG) was used as substrate. Reaction
mixture contained freshly prepared 5ꢁ complete buffer ( A: 0.5 M Tris-
HCl (pH 8), 1.50 M sodium chloride, 100 mM magnesium chloride,
5 mM dithiothreitol, 300 μg of bovine serum albumin/mL; buffer B:
20 mM ATP in water), 250 ng of supercoiled plasmid DNA (pRYG),
followed by either test compound or standard drug (100 μM) and finally
topoisomerase II (2ꢀ4 units) in a total of 20 μL. Relaxation was
employed at 37 °C for 30 min and stopped with 10% SDS followed
by digestion with proteinase K and further incubated at 37 °C for 15 min.
Electrophoresis was carried out in a 1% agarose gel in TAE buffer
without ethidium bromide. Further the gel was stained with ethidium
bromide (0.5 μg/mL) for 15 min and destained with water for 20 min
and photographed using BioRad. Quantification of the products was
carried out as mentioned in decatenation assay.
this assay, 250 ng plasmid was incubated at 37 °C for 20 min with 1 μg/
mL ethidium bromide, 100 μM standard drug (etoposide), or with the
investigational compound. Samples were then loaded in 1% agarose gel
and electrophoresis was carried out in TAE buffer. Further the gel was
stained with ethidium bromide (0.5 μg/mL) for 15 min and destained
with water for 20 min and photographed using BioRad.
Molecular Modeling. In the rational drug discovery pipeline,
modeling and informatics play an indispensable role in the identification
of lead compounds and their most plausible mechanisms of action
against particular biological targets.62 With this outlook, we have
performed molecular modeling study on N-fused imidazole derivatives
in order to identify their potential binding with the ATPase domain of
hTopoIIR. To date, the structure of the complete hTopoIIR is unavail-
able and only the crystal structure of the ATPase domain of hTopoIIR
has been reported.50
The 3D structure of the compounds under study were built using
SYBYL 7.1 molecular modeling package,49 installed on a Silicon
Graphics Fuel Workstation running IRIX 6.5. GasteigerꢀH€uckel partial
charges were applied to the atoms in the compound for geometry
optimization. All the structures were then minimized by Powell method
using Tripos force field, with 0.05 kcal/mol energy gradient convergence
criterion. Finally, database of ligands was created in SYBYL.
Enzyme and Ligand Structure Preparation. The X-ray crys-
tallographic structures of the ATPase domain of hTopoIIR (residues
29ꢀ428) bound to AMPPNP (50-adenylyl-β,γ-imidodiphosphate, a
nonhydrolyzable ATP analogue, PDB: 1ZXM) and ADP (PDB:
1ZXN), solved at 1.86 and 2.51 Å resolution respectively are available
in the Protein Data Bank.50 For the purpose of docking in the present
work, the structure 1ZXM was selected because of its high resolution
and better overall B-factor. 1ZXM is a homodimer consisting of two
chains A and B, each having ligand AMPPNP and Mg ion in the ATP
binding pocket. The Mg ion in an active site forms the distorted
octahedral metal-ion coordination shell by making three point con-
tacts with all three phosphates of AMPPNP, with the side chain
carbonyl oxygen of Asn91 and two conserved water molecules (927
and 928).
Before enzyme structure preparation, chain A was extracted along
with the associated AMPPNP and Mg ion, while chain B was removed.
Enzyme structure was prepared by using the Protein Preparation Wizard
tool implemented in Maestro interface (Maestro 9.0, Schr€odinger, LLC,
New York). The right bond orders were assigned, and the hydrogen
atoms were added to the enzyme. The orientation of hydroxyl groups,
amide groups of Asn and Gln, and charge state of His residues were
optimized using the utility protassign. The final step was a restrained
minimization by the impref utility until the average rmsd of the hydrogen
atoms reached 0.3 Å, leaving heavy atoms in place. Magnesium ion was
assigned a charge of +0.9.30 Subsequently, two sets of prepared enzyme
structure were saved, one after removing all the crystallographic water
molecules and another with two conserved water molecules (927 and
928) in the active site while removing all others. Ligands were prepared
using LigPrep module of Maestro interface by generating all possible low
energy ionization, tautomeric, and stereoisomeric states within the range
of pH 7.0 ( 2.0 using OPLS_2005 force field.
In the X-ray crystallographic structures of ATPase domain of
hTopoIIR (PDB id: 1ZXM),50 amino acid residues from 346 to 349
(loop region) and 406 to 428 (C-terminal) are missing in the chain A.
For a successful dynamics run, it is necessary to add the missing residues
of the loop region. Therefore, python script file defining the missing
residues was run using Modeller9v8 program.63 To obtain the complete
amino acid sequence of ATPase domain of hTopoIIR, fasta format file
was downloaded from UniProt (accession no. P11388). The fasta file
was used as an input in the alignment script of Modeller9v8 and an
alignment file was created. Subsequently, the python script file for
adding missing residues was run and the three models with added
Stabilization of the Cleavage Complex. Cleavage complex
assay was performed using supercoiled plasmid DNA (pBR322) follow-
ing the protocol of the relaxation assay as discussed above with
modification that 8 units of hTopoIIR was added 3ꢀ5 min before the
addition of the compound. Reaction mixture was incubated at 37 °C for
20 min and stopped with 10% SDS, followed by digestion with
proteinase K and further incubated at 45 °C for 30 min. After the
addition of the loading buffer, the reaction was heated for 2 min at 70 °C.
Electrophoresis was carried out in a 1% agarose gel in TAE buffer
containing 0.5 μg/mL ethidium bromide, and further the gel was
destained with water for 20 min.
DNA Intercalation Assay. Using a plasmid mega kit (Zymo
Research), negatively supercoiled plasmid was isolated from E. coli.
The protocol followed was same as mentioned in the supplier manual. In
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missing residues were generated from alignment information. The best
model was selected on the basis of lowest MolPdf score and rmsd
between the model and crystal structure.
From the enzymeꢀligand complexes, ligand was extracted and saved
as .mol2 file, which was further used as an input in the Antechamber
module of AMBER to generate .prepin and .frcmod files. Partial atomic
charges were calculated by AM1BCC method and charge on ligand
molecule was set to ꢀ1. General Amber Force Field (GAFF) was used to
generate missing parameters of the ligands. Parameters for Mg ion were
taken from the parm99.dat file of AMBER.70 The final processing of the
enzymeꢀligand complex was carried in tleap module of AMBER to
generate parameter topology file of complex. Enzymeꢀligand com-
plexes were immersed in an octahedral water box using the TIP3P
model71 by choosing a 12 Å solvation shell around the complex
structure. To neutralize the system, sufficient numbers of counter
chloride ions were added. Afterward, coordinate (.inpcrd) and para-
meter (.prmtop) files of complex were generated.
The minimization of system was carried in three successive steps: In
the first step, the enzymeꢀligand complex was restrained with a force
constant of 2 kcal/mol/Å2 and only solvent phase was minimized with
500 cycles of each steepest descent and conjugate gradient methods.
This allowed the water molecules to reorganize themselves to eliminate
any bad contacts with the enzyme-ligand complex. In the second step,
the restraint weight was reduced to 1 kcal/mol/Å2 to maintain gradual
decrease and 250 cycles minimization was implied as above. In the third
step, the whole system was minimized for 500 cycles without putting any
restraints by both minimization methods as mentioned above. The
minimized complex structures were subjected to heating under NVT
ensemble for 50 ps where the system was gradually heated from 0 to 310
K by putting restraint with a force constant of 2 kcal/mol/Å2. Subse-
quently, 50 ps of density equilibration with weak restraints of 1 kcal/
mol/Å2 on the complex and 100 ps of density equilibration without any
restraints followed by 500 ps of constant pressure equilibration at 310 K
were performed.
Finally, production phase was performed under NPT ensemble at 310
K and 1 atm pressure for 1.3 ns. Equations of motion were solved using
the Verlet leapfrog algorithm.72 For entire simulation, the integration
step size was taken as 2 fs, and the trajectory snapshots were saved after
each 2 ps. During the MD run, all covalent bonds containing hydrogen
atoms were constrained using SHAKE algorithm.73 A Langevin thermo-
stat and barostat was used for temperature and pressure coupling. The
nonbonded cutoff was kept at 8 Å, and long-range electrostatic interac-
tions were treated by the Particle-Mesh Ewald (PME) method. Ptraj
module of Amber tools, VMD,74 and Xmgrace were used for the analysis
of trajectories. The trajectories obtained from MD were used for binding
free energy calculations. The calculations were performed using MM-
PBSA/GBSA method, implemented in Amber 10.53,54
ATPase Assay. ATP hydrolysis of hTopoIIR was evaluated by
quantifying the inorganic phosphate which was released during the
reaction. Six units of hTopoIIR and supercoiled plasmid DNA (pRYG)
were combined in freshly prepared buffer (10 mM Tris-HCl, pH 7.5,
175 mM KCl, 0.1 mM EDTA, 5 mM MgCl2, 2 mM DTT, 2.5% glycerol).
Then compound 5 along with ATP was added to the reaction mixture
and the resultant solution was incubated at 37 °C. After 20 min of
incubation, reaction was stopped by the addition of malachite green
reagent, and immediately OD was taken at 620 nm on microtiter plate
reader (Thermo Scientific Multiskan Spectrum). For kinetic studies,
same protocol was followed for increasing concentrations of ATP (200,
280, 360, 440, and 520 μM) at each respective fixed concentration of
compound 5 (0, 50, 100, and 150 μM).
Molecular Docking. In the previous molecular docking studies
carried on ATPase domain of hTopoIIR to speculate the probable
binding mode of synthesized compounds, some of the authors have
considered water molecules in the active site of enzyme while perform-
ing docking simulation,64 whereas other research groups have neglected
the role of conserved water molecules.29,30,58,65ꢀ67 Therefore, to under-
stand the atomic level differences in terms of interactions, binding pose,
and binding affinity of docked ligands in two cases, we have performed
docking in two sets of prepared enzyme structure: with two conserved
water molecules in the active site and without any water molecules.
Docking simulation was performed by using three molecular docking
programs FlexX,48 GLIDE,68 and GOLD.69 The comparative docking
results of FlexX, GLIDE, and GOLD molecular docking programs for
some of the synthesized compounds and substrate ATP in presence and
absence of two conserved water molecules in hTopoIIR ATP binding
site are presented in Tables S1 and Table S2, respectively (see
Supporting Information). The results reported in Tables S1 and S2
clearly indicate that FlexX scores can distinguish active/moderate/
inactive ligands. Further, the poses and scores obtained using the two
water molecules in the coordination mode with Mg ion were found to be
more realistic. Hence, the results presented in this article are based on
FlexX software, employing an enzyme structure with two water mol-
ecules included in the active site. FlexX program (version 1.2)48
incorporated in SYBYL 7.149 by Tripos Associates was utilized for
molecular docking. FlexX docking into the ATPase domain of hTopoIIR
active site cavity consisted of three steps: (1) enzyme structure
preparation, (2) constructing the ligand structure and building a ligand
database for multiligand docking process, and (3) defining the receptor
description file (RDF). RDF was generated in order to define the active
site of the enzyme, using all residues located within 6.5 Å from a bound
ligand (AMPPNP). FlexX is a technique for structure-based drug design
based on an incremental construction method. Fragments of the ligand
are automatically placed into the active site using an algorithmic
approach based on a pattern recognition technique called pose cluster-
ing. Each pose of the ligand in the enzyme cavity is scored based on
proteinꢀligand interactions. Finally, the binding energies are estimated
in the form of docking score, poses are ranked, and the top ranking poses
are chosen for analysis.
Initially, ATP and AMPPNP were docked into the ATPase domain of
hTopoIIR. The rationale behind this docking was to validate the docking
protocol by assessing the binding pose and hydrogen bondinginteractions
of these substrates with key residues of the ATPase domain of an enzyme.
After successfully setting up the docking protocol and its validation, some
of the synthesized compounds (potent, moderately active, and inactive)
were docked, which were initially prepared in Maestro using LigPrep
utility, while considering all possible tautomers of the species wherever
possible. Docking simulations of these compounds were carried out using
FlexX multiple ligand dockingmodewith a SYBYL databasefile asa ligand
source. After running FlexX, 30 docked conformers were displayed in a
molecular spread sheetand ranked accordingtotheir FlexX dockingscore.
All the poses were visually inspected, and the most suitable docking pose
was selected on the basis of score and interactions with Mg cation and key
residues of the active site. Similar docking protocols were employed to
perform molecular docking analyses using GLIDE and GOLD software
(Supporting Information)
Cell Culture and Chemicals. The Vero (kidney epithelial cells of
African green monkey), HEK 293 (Kidney cancer cells), and MCF 7
(Breast cancer cells) were maintained in Dulbecco’s Modified Eagle’s
Medium (DMEM) with 1% antibiotic (100 units of penicillin and 10 mg
streptomycin per mL in 0.9% normal saline) and 10% fetal bovine serum
(Himedia, India) in a humidified CO2 incubator in 5% CO2 at 37 °C.
The MCF 10A (normal breast epithelial cells) cells were grown in
Molecular Dynamics Simulation. The best docking pose ob-
tained from FlexX program for compound 5 of the series in complex with
the enzyme structure with two conserved water molecules was saved as
.pdb files for MD study. Full atomistic simulations were performed in
water as an explicit solvent for enzymeꢀligand complexes using AMBER
1055 molecular dynamics software package.
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Journal of Medicinal Chemistry
ARTICLE
DMEM/F-12 (50:50, v/v) medium supplemented with 10% (v/v) fetal
bovine serum (Himedia, India), 100 U/mL of penicillin, 100 μg/mL
streptomycin, 0.5 μg/mL of hydrocortisone, 100 ng/mL of cholera
toxin, 10 μg/mL of insulin, 10 ng/mL of epidermal growth factor, and
1% (w/v) L-glutamine at 37 °C under a humidified CO2 incubator in 5%
CO2 at 37 °C. 5-Fluorouracil, etoposide, and all other chemicals of
analytical grade were purchased from Sigma Chemical Ltd. (St Louis,
MO, USA). Stock solutions (100 mM) of investigational compounds
were prepared in DMSO and stored at ꢀ20 °C. For treatment, the
solution was diluted in DMEM and added to cultures to provide the
desired final concentration. After 60ꢀ70% confluence, the cells were
exposed to different compounds of indicated concentrations at required
time periods.
Topoisomerase Inhibition Assay. Topoisomerase assay was
used to assess the ability of compound 5 to inhibit the function of
topoisomerase activity in conversion of negatively supercoiled plasmid
DNA into relaxed/linear DNA.75,76 As topoisomerases are nuclear
proteins, we prepared nuclear cell lysate according to the protocol of
Wiley Intersciences for preparation of mammalian cell nuclear extracts
for assaying topoisomerase activity.77 Confluent dishes (80ꢀ90%) were
treated with either compound 5 of increasing concentrations or 100 μM
etoposide (positive control) for 48 h prior to harvest, and then nuclear
lysate was prepared. The topoisomerase reaction contains 100 μg
nuclear protein, a common plasmid for substrate (FOP FLASHꢀ
TCF/LEF transcription factor, responsible for βꢀcatenin-Wnt-signal-
ing pathway) (1 μg) and reaction buffer (200 mM Tris-Cl, pH-7.5,
100 mM MgCl2, 10 mM ATP, 10 mM EDTA, 10 mM dithiothreitol,
1.5 mM KCl, 300 μg/mL bovine serum albumin). The tubes were
incubated at 37 °C for 30 min, and reaction was stopped by addition of
1% SDS. Then 30 μL of reaction mixture was loaded in each well of 0.9%
horizontal agarose gel electrophoresis containing ethidium bromide.
Electrophoresis was done for 3ꢀ4 h at 10ꢀ15 V. Photographs were
taken using gel documentation system (UVP, Germany). Data pre-
sented here is the best of five experiments.
ATP Dependent Plasmid Relaxation Assay. To assess the
ATP dependent topoisomerase activity of HEK 293 nuclear lysate, a
plasmid based relaxation assay was carried out following the method as
described above in topoisomerase inhibition assay. Instead of using
compound 5 treated nuclear lysate, here compound 5 was added into the
final reaction mixture. The reaction mixture contained 100 μg of protein,
plasmid (FOP FLASH ꢀTCF/LEF) (1 μg), 200 mM Tris-Cl, pH-7.5,
100 mM MgCl2, 10 mM EDTA, 10 mM dithiothreitol, 1.5 mM KCl,
300 μg/mL bovine serum albumin, 35 μM compound 5, and various
concentrations of ATP (0, 20, 50, 100, 150, 200, 250 mM). Etoposide
(100 μM) was used as positive control. Data presented here is the best of
five experiments.
Clonogenic Cell Survival Assay. Exponentially growing HEK
293 cells were trypsinized and diluted to approximately 500ꢀ600 viable
cells per well in 12-well culture plates. After 24 h of incubation, cells were
treated with compounds of varying concentrations for 48 h. The media
was aspirated and replaced with fresh normal growth medium, and cells
were allowed to grow for 5ꢀ6 doubling.78 Media was removed and
plates were air-dried. Colonies were stained with 0.2% crystal violet for
1 h. The excess dye was repeatedly washed with distilled water. Plates
were air-dried, and stained colonies were manually counted. The data
presented is percent survival with respect to control and is the best of
three independent experiments.
cells were treated with 12 μM of compound 5 at different time periods.
After completion of treatment, the media was removed, 100 μL of 0.05%
MTT reagent was added to each well, and it was incubated at 37 °C for
overnight to allow the formation of purple formazan crystals. A 100 μL
of NP-40 detergent solution was added to each well, and the reaction
mixture was incubated in dark for 1 h at room temperature for dissolving
the formazan crystals. Color density was measured spectrophotometri-
cally at 570 nm using microplate reader (Berthold, Germany). Experi-
ments were conducted in triplicates, and the data is presented as percent
survival.
Wound Healing Assay. HEK 293 cells were cultured in 35 mm
tissue culture discs to 90% confluence. A sterile micro tip was used to
create a clean wound in the cell mono layer across the center of the well.
Cell debris was removed by washing the plates with fresh media. The
cells were exposed to compound 5 (0, 15, 25, and 35 μM) and were
allowed to migrate in the medium. The wound was assessed by a
microscope (Nikon, Japan) at 10ꢁ magnification at different time point
(0, 6, 12, and 24 h).
DAPI Staining. Roughly 1 ꢁ 106 HEK 293 cells were seeded in 35
mm tissue culture discs. Cells were treated with compound 5 of
increasing concentrations (0, 15, 25, and 35 μM) for 48 h. After
treatment, the cells were fixed with ice chilled acetone:methanol (1:1)
for 30 min at 4 °C in dark. Fixed cells were washed twice with chilled 1ꢁ
PBS, and then stained with DAPI for 1 h at 4 °C in dark. Stained cells
were washed repeatedly with chilled 1ꢁ PBS to remove the excess DAPI
stain and observed under fluorescence microscope (Nikon, Japan) at
40ꢁ magnification.
FACS Analysis. HEK 293 cells (1 ꢁ 106) were cultured in 60 mm
tissue culture plates. After 70ꢀ80% confluence, cells were exposed to
different concentrations (0, 15, 25, and 35 μM) of compound 5 for 48 h.
After treatment, cells were trypsinized and washed twice with 1ꢁ PBS
containing 0.05% RNase-A. Finally, cells were resuspended in 0.1 mL of
propidium iodide solution (50 μg/mL). Cells were incubated for 30 min
in dark at room temperature. Fluorescence emitted from propidium
iodideꢀDNA complexes was quantified after laser excitation of the
fluorescent dye by Fluorescence-Activated Cell Sorter (FACS) (Becton
and Dickinson, CA). Finally, DNA content of the cells at different phases
of cell cycle was determined by using Cell Quest Software (Becton and
Dickinson, CA).
SDS-PAGE and Western Blot Analysis. HEK 293 cells were
grown on 100 mm tissue culture discs at a density of 1 ꢁ 106 cells per
plate and incubated overnight. Then the cells were exposed to com-
pound 5 (0, 15, 25, and 35 μM) for 48 h prior to harvest. The cell lysate
was prepared by using modified RIPA lysis buffer (50 mM tris, 150 mM
NaCl, 0.5 mM deoxycholate, 1% NP-40, 0.1% SDS, 1 mM Na3VO4,
5 mM EDTA, 1 mM PMSF, 2 mM DTT, 10 mM β-glycerophosphate,
50 mM NAF, 0.5% triton X-100, protease inhibitor cocktail). Protein
estimation was done by Bradford’s method. Proteins were separated in
10% SDS-PAGE and transferred to PVDF membrane. Membranes were
blocked overnight in 10% skimmed milk in 1ꢁ TBS-T (Tris-buffered
saline containing 0.05% of Tween-20) at 4 °C and immunoblotted with
antibodies anti-Bcl-XL, anti-Bax, and anti-PARP. Detection of signals
was done using ECL Western blotting reagent and chemiluminescence
was exposed on Kodak X-Omat films. Antibody anti-β-actin was used as
loading control. All the antibodies were procured from cell signaling
technology, CA, USA.
Statistical Analysis. A two-tailed Student’s t test was employed
where P < 0.05 was considered to be statistically significant.
MTT Cell Survival Assay. Survival of the cells after treatment with
drug was measured using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylte-
trazolium bromide (MTT) cell survival assay. Briefly, 8000ꢀ10000 cells
of HEK 293, Vero, MCF 7, and MCF 10A were plated in triplicate in
different 96-well flat-bottom tissue culture plates, and after 24 h of
seeding, they were exposed to either investigational compounds or
etoposide of different concentrations for 48 h. For time scan analysis, the
’ ASSOCIATED CONTENT
S
Supporting Information. General experimental proce-
b
dure; 1H, 13C, mass, IR, CHN, or HRMS, and melting points for
all compounds, determination of LC50 value of compound 5 for
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Journal of Medicinal Chemistry
ARTICLE
hTopoIIR-decatenation in vitro assay, molecular docking proto-
cols for GLIDE and GOLD, tautomeric preferences and MD
simulation analysis. This material is available free of charge via the
Internet at http://pubs.acs.org.
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’ AUTHOR INFORMATION
Corresponding Author
*For S.K.G.: phone, +91 (0)172 2214683; fax, +91 (0)172
2214692; E-mail, skguchhait@niper.ac.in. For C.N.K.: phone,
+91 (0)674 2725466; fax, + 91 (0)674 2725732; E-mail, cnkundu@
gmail.com.
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We gratefully acknowledge the financial support from DST,
ICMR, and DBT, Government of India, New Delhi, for this
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’ ABBREVIATIONS USED
hTopoIIR Human topoisomerase IIR; MDR multidrug resistance;
MCR multicomponent reaction; kDNA kinetoplast DNA; Nck
nicked; Rel relaxed; SC supercoiled; Lin linear; AMPPNP 50-adeny-
lyl-β,γ-imidodiphosphate; rmsd root-mean-square deviation; MD
molecular dynamics; MM-PBSA molecular mechanics Poissonꢀ
Boltzmann surface area; MM-GBSA molecular mechanics general-
ized Born surface area; FACS fluorescence activated cell sorter;
DAPI 40-6-diamidino-2-phenylindole; NCEs new chemical enti-
ties; RDF receptor description file; TAE Tris-acetate-EDTA;
DMEM Dulbecco’s Modified Eagle’s Medium; MTT 3-(4,5-di-
methylthiazol-2-yl)-2,5-diphenylte-trazolium bromide
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