New dimeric carbazole-benzimidazole mixed ligands for the stabilization of human telomeric G-quadruplex DNA and as telomerase inhibitors. A remarkable influence of the spacer

Article abstract of DOI:10.1039/c5ob00675a

The development of G-quadruplex (G4) DNA binding small molecules has become an important strategy for selectively targeting cancer cells. Herein, we report the design and evolution of a new kind of carbazole-based benzimidazole dimers for their efficient telomerase inhibition activity. Spectroscopic titrations reveal the ligands high affinity toward the G4 DNA with significantly higher selectivity over duplex-DNA. The electrophoretic mobility shift assay shows that the ligands efficiently promote the formation of G4 DNA even at a lower concentration of the stabilizing K⁺ ions. The TRAP-LIG assay demonstrates the ligand's potential telomerase inhibition activity and also establishes that the activity proceeds via G4 DNA stabilization. An efficient nuclear internalization of the ligands in several common cancer cells (HeLa, HT1080, and A549) also enabled differentiation between normal HFF cells in co-cultures of cancer and normal ones. The ligands induce significant apoptotic response and antiproliferative activity toward cancer cells selectively when compared to the normal cells.

Full text of DOI:10.1039/c5ob00675a

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New Dimeric Carbazole-Benzimidazole Mixed Ligands for the
Stabilization of Human Telomeric G-Quadruplex DNA and as
Telomerase Inhibitors. Remarkable Influence of Spacer†
Received 00th January 20xx,
Accepted 00th January 20xx
Basudeb Maji,a Krishan Kumar,a K. Muniyappac and Santanu Bhattacharya*a,b
DOI: 10.1039/x0xx00000x
The development of G-quadruplex (G4) DNA binding small molecules has become an important strategy for targeting
cancer cells selectively. Herein, we report the design and evolution of a new kind of carbazole-based benzimidazole dimers
for their efficient telomerase inhibition activity. Spectroscopic titrations reveal ligand’s high affinity toward the G4 DNA
with significantly higher selectivity over duplex-DNA. Electrophoretic mobility shift assay shows that the ligands efficiently
promote the formation of G4 DNA even at a lower concentration of the stabilizing K+ ions. TRAP-LIG assay demonstrates
the ligand’s potential telomerase inhibition activity and also establish that the activity goes via G4 DNA stabilization. An
efficient nuclear internalization of the ligands in several common cancer cells (HeLa, HT1080, and A549) also enabled in
differentiating from normal HFF cells in co-cultures of cancer and normal ones. The ligands induce significant apoptotic
response and antiproliferative activity toward cancer cells selectively, when compared to the normal cells.
www.rsc.org/
through reverse transcriptase mechanism using its own RNA
template present in the hTR (human telomerase RNA)
component. The up-regulation of telomere by the
overexpressed telomerase maintains the overhang throughout
the generations leading to an uncontrolled proliferation.7
Introduction
The telomere plays an important role in the chromosomal
protection, genomic stabilization, and cellular ageing.1
Telomere in mammalian cells consists of a tandem repeat of
the guanine-rich hexameric (5′-TTAGGG-3′) sequences even up
to 5–15 kb length from the extreme 3′-end. The single-
stranded overhang is susceptible toward degradation through
different cellular mechanisms.2 To protect the telomeric end,
cell adopts certain protecting mechanisms like end-capping
and T/D-loop formation in the presence of few telomere
binding proteins.3 During chromosomal duplication, the
extreme end of the telomere cannot be copied due to the
presence of Okazaki fragments, known as ‘end-replication
problem’.4 Thus, upon every cell division, the telomere length
decreases and when it reaches a critical length, it promotes
genomic instability eventually leading to cell cycle arrest
through ‘end-to-end fusion’.5 To overcome the genomic
problem, cells sometimes overexpress the telomere repairing
protein telomerase, even up to 85-90%.6 Telomerase
maintains the telomere length by adding repetitive nucleotides
However, there is hardly any evidence on the detection of
telomerase in the most somatic cells.6 Thus, the presence of
overexpressed telomerase only in cancer cells offers
a
platform to discriminate them from the normal somatic cells.
Consequently, an effective inhibition of telomerase activity is a
viable strategy for the design and synthesis of an anticancer
agent.
Unlike the duplex DNA which exists essentially throughout the
genome, the telomeric G4 DNA emerges from the single-
stranded 3′-overhang in the telomere. The biological
importance of the G4 DNA was revealed when G4 DNA
mediated telomerase inhibition was invented.8 Telomerase,
which acts only on the single-stranded DNA overhang, was
found to be totally inactive upon folding into secondary
tetraplex structures like the G-quadruplex. Design of ligands
which can stabilize and significantly shift the equilibrium
toward the G4 DNA structure and thereby inhibit the telomere
recognition has become a popular and promising strategy in
oncology.9-11 It should be noted that, ligands with poor
discrimination ability between duplex and G-quadruplex DNA
may result in undesirable cytotoxic events which is considered
as the side effect in DNA targeted chemotherapy. Thus, the
development of efficient G4 DNA binding molecules with high
selectivity over the double-stranded DNA is of keen interest to
achieve desirable chemotherapeutic outcomes.12,13
a.Department of Organic Chemistry, Indian Institute of Science, Bangalore 560 012,
India, Fax: (+91) 80-2360 0529; Tel: (+91)-80-2293 2664; E-mail:
sb@orgchem.iisc.ernet.in
b.Honorary Professor, Jawaharlal Nehru Centre for Advanced Scientific Research
Bangalore, Jakkur 560 064, India
c.Department of Biochemistry, Indian Institute of Science, Bangalore 560 012, India.
†Electronic Supplementary Information (ESI) available: For additional synthetic
procedure, characterization, spectroscopic data, cellular data, computational and
experimental methods, See DOI: 10.1039/x0xx00000x
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DNA.18,19 The carbazoles and bis-benzimidazoles on their own
have been also reported to show significant biological
activities including cell permeability.20-22 Therefore, we have
chosen have new ligands based on benzimidazole-carbazole
mixed core.
Herein, we introduce six novel carbazole based benzimidazole
derivatives which contain two monomers and the other four
are dimeric molecules having different types of linkers that
connect the individual monomeric units (Fig. 1). Each new
ligand was examined toward its G4 DNA binding efficiency
using different spectroscopic, elctrophoretic, enzymatic, and
cellular studies. Finally the nature of DNA-ligand interactions
was further investigated with the help of molecular modelling
studies. All the ligands showed high G4 DNA binding affinity
with significant selectivity over the duplex DNA. The ligands
also demonstrated potential telomerase inhibition ability and
cancer cell selective toxicity. Importantly, the dimeric ligands
demonstrated significantly higher G4 DNA binding and
stabilization capacity compared to their monomeric
counterparts and their activity is found to rely on the nature of
linker chain considerably.
Another substantial advancement in the ligand design strategy
was reported via the use of dimeric forms of ligands.23-29 In
particular, the dimeric ligands derived from 1,3-phenylene-bis-
benzimidazole containing suitable spacers were distinctly
superior compared to their corresponding monomeric forms in
terms of their G4 DNA binding affinity, thermal stability, and
telomerase inhibition activities.29 Interestingly, the activity of
the dimeric ligands was also reported to be highly dependent
on the spacer length where ligands with a hexaethylene glycol
type spacer showed the maximal activity among all the ligands
studied in the series. Till date, only a couple of other reports
on the dimeric G4-ligands are known among which the dimers
based on acridine, quinoacridine, BRACO19, and macrocyclic
hexaoxazoles show quite promising potential toward clinical
applications.23-29 These reports reveal the importance of the
dimeric ligands in terms of their substantially higher activity
than their monomeric counterparts. Moreover, the reports
also emphasize the significance of linker length and their
chemical nature in the ligand’s solubility, affinity, and
selectivity over other relevant DNA structures.23-29 The activity
of the dimeric ligands were found to be highly dependent on
the spacer length. It has been observed that, the length of the
spacer should be optimal so that the two binding moieties can
efficiently interact with their preferable binding sites.
Moreover, incorporation of long hydrophobic methylene
based spacer may actually decrease the ligand’s activity while
oxyethylene based spacer shows higher water solubility and
enhanced DNA binding activity.23-29 Thus, the design of dimeric
ligands targeting the G4 DNA is worthwhile in the
development of potential anticancer drugs for future (ESI, Fig.
S1).
1: R = CH3 (M1)
2: R = CH2-CH2-OH (M2)
O
O
O
O
O
3: R = CH3 (D1)
4: R = CH2-CH2-OH (D2)
N
O
N
N
N
O
5: R = CH3 (D3)
6: R = CH2-CH2-OH (D4)
Fig. 1. Molecular structures of the ligands used in the
investigation.
RESULTS AND DISCUSSION
Based on the rationale presented above, we have now
synthesized a series of ligands having a carbazole core inserted
between two benzimidazole units to form symmetric, planar
Considerations in the Ligand. Recently, there has been a
significant progress in the molecular design of synthetic pharmacophores with two piperazine moieties at the termini.
benzimidazole based systems for the switchover of nucleic acid
recognition from the duplex to the G4 DNA.14 Benzimidazole
based Hoechst molecules were developed long time ago which
have been utilized extensively to target the duplex DNA
through minor groove.15 Thereafter, the efforts were directed
toward the structural modifications to enable their selective
targeting of higher order DNA structure like the G-quadruplex.
For instance, benzimidazoles linked with one central aromatic
ring, like phenyl or pyridine moiety have been demonstrated
for their preferential G-quadruplex DNA binding
properties.16,17 Recently, it has been revealed that the
replacement of the mono-nuclear aromatic ring with extended
planar scaffolds like tri-nuclear heteroaromatic moieties, e.g.,
carbazole, phenanthroline etc. enhances the potential of the
resulting ligands significantly toward the G-quadruplex
The resulting ligands should bind DNA efficiently via stacking
as well as H-bonding interactions whereas the end piperazine
moieties in them should exist in their protonated forms at
physiological pH to offer favorable electrostatic interactions
with DNA.
It is always a challenging task to specifically target G4 DNA
over the predominant DNA form like duplex DNA. The duplex
DNA binder ligand Hoechst 33258 has characteristic crescent-
shape with concave interacting surface of ~145° (ESI, Fig. S1A).
To minimize the duplex DNA affinity, the present set of ligands
was engineered with significantly higher angularity of ~100°
(ESI, Fig. S1B) which can not only ‘discourage’ the Hoechst-like
ds-DNA minor groove binding interaction but may also render
the molecular shape favorable toward the dimension of a G-
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tetrad (ESI, Fig. S1D). The similarity in the dimension of the Scheme 2.a
ligand and that of the G-tetrad predicts a favorable π-stacking
interaction over the comparatively smaller Watson-Crick base
pairs present in the duplex DNA (ESI, Fig. S1C, D). Taken
together, these new molecules should possess not only
adequate affinity toward the G4 DNA but also significant
selectivity over the duplex DNA.
Two monomeric molecules
1 (M1) and 2 (M2) having free
carbazole-N core were synthesized. The corresponding dimeric
molecules (D1, D2, D3, and D4) possess two different types of
spacers that connect the ‘monomers’ via two carbazole-N ends
(ESI, Fig. S2). Following the previous reports on the design of
dimeric molecules, a hexaethylene glycol based spacer was
chosen since the same demonstrated better efficiency toward
the G4 DNA stabilization compared to their monomeric
counterparts. On the other hand, we have also investigated
the effect of insertion of piperazine in the spacer. It may be
noted that the hexaethylene glycol based spacer serves as a
neutral spacer in the dimeric ligands while the linker having
piperazine moieties provides additional sites of possible
protonation at physiological pH.
aReagents, conditions and yield. a) PPh3, Br2, acetonitrile, 0 °C →
rt, 48 h, 70%; b) carbazole, TBAI (cat.), 50% NaOH, THF-H2O, 24 h,
55%; c) POCl3-DMF, ZnCl2, 110 °C, 24 h, 65%. d) 4-(4-
methylpiperazin-1-yl)benzene-1,2-diamine (9) or 2-(4-(3,4-
diaminophenyl)piperazin-1-yl)ethanol (10), Na2S2O5, EtOH, 80 °C,
12 h, 55-60%.
Scheme 3.a
Scheme1.a
aReagents, conditions and yield. a) K2CO3, dry DMF, 110 °C, 24
h, 89%; b) Pd/C-H2, EtOH, rt, 24 h, 100%; c) NaH, NBS, dry
DMF, rt, 6 h, 85%; d) (i) KH, THF, 0 °C, (ii) n-BuLi, -78 °C → 25
°C, (iii) DMF, -78 °C → 25 °C, (iv) 1 M H3PO4, 27%; e) 4-(4-
methylpiperazin-1-yl)benzene-1,2-diamine (9) or 2-(4-(3,4-
diaminophenyl)piperazin-1-yl) ethanol (10), Na2S2O5, EtOH,
80 °C, 12 h, 55-65%.
aReagents, conditions and yield. a) NaOH, THF-H2O, p-TsCl, 0 °C →
rt, 6 h, 100%; b) 1, 4-dibromobutane, TBAI, 50% NaOH, rt, 12 h,
72%; c) piperazine, K2CO3, acetonitrile, rt, 6 h, 89%; d) K2CO3,
acetone, 60 °C, 6 h, 70%; e) ZnCl2, DMF-POCl3, 100 °C, 48 h, 50%; f)
4-(4-methylpiperazin-1-yl)benzene-1,2-diamine (9) or 2-(4-(3,4-
diamino phenyl)piperazin-1-yl)ethanol (10), Na2S2O5, EtOH, 80 °C,
12 h, 45-55%.
Initially, upon reaction with NBS, carbazole was converted to
3,6-dibromo carbazole (12) which was transformed following a
reported procedure30 in presence of n-BuLi to get the 3,6-
diformyl carbazole (13). The dialdehyde (13) was then coupled
with two different types of freshly prepared diamines (9 or 10)
to afford the corresponding benzimidazole derivatives 1 (M1)
and 2 (M2) through Na2S2O5 mediated oxidative cyclizations
(Scheme 1).16
The monomers (1 and 2) were then individually joined with each
other via two different kinds of linker, one having a neutral
hexaethylene glycol based unit and the other one having both
oxyethylene and protonatable piperazine moiety in between such a
spacer to furnish the dimeric molecules 3 (D1), 4 (D2), 5 (D3), and 6
(D4) respectively (Scheme 2 and 3). All the new compounds as well
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as their reaction intermediates were adequately characterized by
IR, 1H, 13C NMR, mass spectrometry and elemental analysis as given
in the experimental section.
their intrinsic fluorescence. The enhancement in the
fluorescence intensity was more prominent in the case of the
G4 DNA formed in the presence of K+ ions than Na+. This may
be due to better association of the ligands with the K+-
stabilized G4 DNA (ESI, Fig. S8A, C). Among these ligands, the
ones having hydroxyethyl substituted piperazines at the
UV-vis Absorption Spectral Titrations. The affinity of each ligand
toward the preformed G4 DNA was investigated first using UV-vis
absorption spectroscopy. Each ligand solution was titrated against
progressively increasing concentrations of the G4 DNA in 10 mM
Tris-HCl (pH 7.4) containing 0.1 M NaCl/KCl and 0.1 mM EDTA. All
the titrations showed strong hypochromism in the ligand
absorption band suggesting the interaction between the
chromophoric ligand with the nucleobases in the G4 DNA (ESI, Fig.
S3-S5).
termini
(M2, D2, and D4) showed higher fluorescence
enhancements than the methyl substituted piperazines (M1
,
D1, and D3).
2 µM
C
80
A
+
G4 DNA (K )
60
0 µM
40
+
Table 1. Dissociation constants (Kd) of the ligands with the
preformed Hum21 G4 DNA.a
Ligand D4
20
0
G4 DNA ds-DNA
Entry
Ligand
Dissociation
Hypochromicity
n
constant, Kd (µM) (%) (Hum21 G4
400
450
500
550
600
(Hum21 G4 DNA)a
3.30 ± 0.2
DNA)a
Wavelength (nm)
Ligand
B
+ Hum21
+ CT DNA + Telo
1
2
3
4
5
6
M1
D1
D3
M2
D2
D4
17
26
17
40
37
43
2.9
2.0
2.4
2.5
1.9
2.0
0.10 ± 0.001
0.05 ± 0.001
0.63 ± 0.04
D4
0.13 ± 0.01
0.01 ± 0.001
G4 DNA-Ligand
complex
aBinding assays were performed with the preformed Hum21 G4
DNA in 10 mM Tris-HCl (pH 7.4) having 0.1 M NaCl/KCl and 0.1
mM EDTA at 25 °C. Binding assays were performed with 21-mer
telomeric duplex DNA in 10 mM Tris-HCl (pH 7.4) having 40 mM
NaCl and 0.1 mM EDTA. The notation ‘n’ represents the binding
stoichiometry of the ligands with the G4 DNA.
Fig. 2. (A) Fluorescence titrations of 0.8 µM ligand D4 in 10
mM Tris-HCl (pH 7.4) having 0.1 mM EDTA and 0.1 M KCl with
the preformed Hum21 G4 DNA. (B) Photographic fluorescence
images of ligand D4 (20 µM) and ligand-DNA complexes (with
40 µM of Hum21, CT DNA and Telo ds DNA) under UV light
(>365 nm) in 10 mM Tris-HCl (pH 7.4) and 0.1 mM EDTA
having either 0.1 M KCl (for the G4 DNA) or 0.04 M NaCl (for
the CT DNA and the Telo ds DNA) and (C) a schematic
representation of the process.
b
The interaction between the ground states of the conjugated π-
system of the ligand and the nucleobases in DNA probably
decreased the extent of π-π* transition. The absorption data have
been converted into Scatchard plot to determine the binding
constant of each ligand with the G4 DNA following a reported
protocol.18 Similarly, each ligand solution was titrated with duplex
DNA (CT DNA and the telomeric duplex DNA) which showed
practically negligible extent of hypochromism revealing the high
selectivity of the ligands toward the G4 DNA over the duplex DNA
(ESI, Fig. S3-S5). The binding affinity of each ligand toward both
forms of DNA has been summarized in Table 1.
The dissociation constants calculated from the
fluorescence spectral data31 (ESI, Table S1) reveal that, the
dimeric ligands possess significantly higher binding affinity
than their monomeric entity while the dimers containing
piperazine in the linker (D3 and D4) were found to be even
superior to the corresponding ligands having hexaethylene
glycol linkers (D1 and D2). Ligand’s efficient light-up probe
behavior selectively for the G4 DNA as demonstrated from
their fluorescence images (Fig. 2B and ESI, Fig. S9) further
exemplify the superior binding affinities of dimeric ligand D4
toward the G4 DNA.
Fluorescence Emission Spectral Titrations. All the ligands
being aromatic with a π-conjugated skeleton showed intrinsic
fluorescence emission property. Upon excitation at the
molecular absorption maxima (~320 or 350 nm) in buffer, the
ligands showed very low emission which could be possibly due
to the collisional quenching of the excited singlet state by
polar water molecules. Interestingly, addition of the
preformed G4 DNA resulted in the increment in the
fluorescence intensity even up to 5-fold followed by saturation
(Fig. 2A and ESI, Fig. S6-S8). The ligands due to the association
with the G4 DNA earned a hydrophobic microenvironment to
hide from the aqueous media which resulted in the recovery of
Fluorescent Intercalator Displacement Assay. The G4 DNA-FID
assays were undertaken to investigate each ligand’s relative binding
affinity toward the G4 DNA and selectivity over the duplex DNA.
Thiazole orange (TO) which is
a standard G4 DNA binding
fluorophore has been chosen for the displacement assays.32 The
extent of TO displacement ability of a ligand from a preformed TO-
G4 DNA complex corresponds to the decrease in the fluorescence
intensity due to TO upon binding (Fig. 3). All the ligands showed
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efficient TO displacement ability with significantly lower DC50 loops and a central diagonal loop known as the antiparallel G-
(ligand concentrations for the displacement of 50% of the bound TO quadruplex.35
from DNA) values (ESI, Fig. S10, Table 2) indicating their strong G- The CD titrations of the preformed G4 DNA with the ligands
tetrad affinity. The ligands also exhibited high G4 DNA selectivity show that all the ligands can stabilize the G4 DNA in their
over the duplex DNA (CT DNA and Telo ds DNA).
native form. It is believed that the telomeric G4 DNA remains
in equilibrium with the single-stranded form. So it would be
interesting to investigate whether the ligands have any
significant effect in the formation of the G4 structure. We
prepared the G4 DNA in 0.1 M KCl buffer along with the
ligands in different concentrations. The CD spectra showed a
gradual transformation in the CD signature with increasing
Ligand
ligand concentration (Fig.
4 and ESI, Fig. S12). The
characteristic positive peaks at 293, 268, and 250 nm merged
together to 263 nm with a shift of the trough from 235 nm to
242 nm which is a specific CD signature due to the parallel G4
DNA formed by the human telomere sequences.18
TO
TO-G4 DNA
Ligand-G4 DNA
B
Fig. 3. A schematic representation of the G4 DNA-FID assay by
a G4 DNA binding ligand.
A
20
15
10
5
4
ꢀ
M Hum (KCl)
21
+ M2 (r = 2.5)
+ M2 (r = 5)
+ M2 (r = 7.5)
Table 2. DC50 values (ligand concentrations for the
displacement of 50% of the bound TO from DNA) of the ligands
as determined from fluorescent intercalator displacement
assays.a
Hybrid G4 DNA
Ligand
Ligand
DC50 (µM)
Ligand
D3-G4 (K+)
DC50 (µM)
0.31 ± 0.01
0.27 ± 0.01
> 3.0
0
M1-G4 (K+) 1.27 ± 0.09
M2-G4 (K+) 0.83 ± 0.03
D1-G4 (K+) 0.46 ± 0.03
D2-G4 (K+) 0.35 ± 0.02
D4-G4 (K+)
250
300
350
D4-CT DNA (K+)
D4-Telo ds (K+)
Parallel
G4 DNA
Wavelength (nm)
> 3.0
Fig. 4. (A) Formation of the G4 DNA in presence of M2 in 10
mM Tris-HCl (pH 7.4) containing 0.1 M KCl and 0.1 mM EDTA
and (B) a schematic representation of the process.
aThe G4 DNA-FID assays have been performed in 10 mM-Tris-
HCl buffer (pH 7.4) containing 0.1 M KCl and 0.1 mM EDTA.
Results are average of two independent experiments.
There are only a handful of reports involving organic small
molecules that induce topological transformations under the
physiological conditions.16,18,27,34,36 Though the existence of a
telomeric parallel G4 DNA structure in K+ solution is rather
unusual, such an organization may nevertheless possess much
higher stability than the probable mixed hybrid G4 structure.
The loops around the G-tetrads of K+-stabilized G4 DNA in
solution state may inhibit the ready access of the planar
ligands to the end-tetrads. On the other hand K+-stabilized G4
DNA has also been found to exist in parallel structure in the
crystalline state (PDB 1KF1) which has only sidewise loops.35
Thus, the end-tetrads in 1KF1 may be entirely accessible to the
tetrad targeting planar ligands. The presence of favorable
binding sites may allow topological transformation of the G4
DNA in the presence of ligands. The presence of ligand may
further guide the telomere to fold in the thermodynamically
more stable form of G4 DNA and justify the G4 DNA targeted
approach for drug design.
Circular Dichroism Spectroscopy. The DNA-ligand interactions
were further probed using circular dichroism (CD)
spectroscopy. The preformed G4 DNA was titrated against
increasing ligand concentrations (prepared in the KCl or NaCl
buffer of pH 7.4). All the titrations showed enhancement in the
CD intensity followed by saturation resembling the
stabilization of the G4 DNA by the ligands (ESI, Fig. S11).
Ligand Induced G4 DNA Structural Alteration. The
polymorphic nature of human telomeric G-rich sequence in the
presence of different monovalent ions like Na+, K+, NH4+ and Li+
have been extensively studied and established either from
NMR or from solved crystal structures.33 The K+-stabilized
mixed hybrid structure has the highest stability whereas Na+
can stabilize to a considerably lower extent.34 The crystal
structure and NMR solved structures suggest that in the
presence of K+ ions the sequence d[5′-AG3(T2AG3)3-3′] adopts
an intramolecular mixed hybrid structure whereas in the
presence of Na+ ions it forms an intramolecular G-tetraplex
stabilized by three stacked G-tetrads connected by two lateral
Thermal Denaturation Study. The thermal denaturation of
DNA has been monitored by following the temperature-
dependent changes in CD spectra. CD spectroscopy provides
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1.2
1.0
0.8
0.6
0.4
0.2
0.0
1.2
A
Hum (KCl)
Hum (KCl)
B
21
21
M2 (r = 7.5)
D2 (r = 5)
D4 (r = 2)
M2 (r = 5)
D2 (r = 5)
D4 (r = 5)
1.0
0.8
0.6
0.4
0.2
0.0
30
40
50
60
70
90
100
(o 80
C)
30
40
50
60
70
90
100
(o 80
Temperature
Temperature
C
)
C
D4
D
D4
D3
D2
D1
M2
M1
D3
D2
D1
M2
M1
In presence
Preformed
In presence
Preformed
0
5
10
15
20
25
0
5
10
15
20
Fig. 5. Melting profiles of (A) G4 DNA incubated with various ligands (M2, D2 and D4) as monitored at a wavelength of 293 nm and (B)
the G4 DNA formed in the presence of ligands (M2, D2 and D4) of specified concentration as monitored at a wavelength of 263 nm.
Relative elevation in the G4 DNA melting temperature upon ligand incubation with either preformed G4 DNA or G4 DNA formed in the
presence of ligand in 10 mM Tris-HCl (pH 7.4) containing 0.1 mM EDTA and (C) 0.1 M NaCl or (D) 0.1 M KCl. The° results are average of
two independent experiments and are within ± 0.5 °C of each other.
characteristic signature of the nucleobases in the DNA nature of the lateral loops in 2HY9 keeps the G-tetrads
sequence as well as the conformation it adopts. Any change in accessible to the planar ligands. Thus, all the ligands showed
the DNA conformation, affects the position and amplitudes of better affinity toward the K+ ion stabilized G4 DNA form than
the CD signal.37 The diverse CD signatures of the G4 DNA the one stabilized by the Na+ ions. The higher denaturation
formed in the presence of different stabilizing ions originate temperatures of the G4 DNA in the presence of ligands were
from the nature of folding and orientation of the nucleobases most likely due to the topological transformation from the
flanked from the glycosidic bonds. Both the G4 DNA structures hybrid to more stable parallel structure (Fig. 4). The high
formed in Na+ and K+ ion environment afford a prominent thermal stability demonstrated the potency of the ligands as
characteristic peak at 293 nm whereas the G-quadruplexes efficient G4 DNA binding and stabilizing agents. The melting
formed in the presence of ligand (in KCl buffer) showed a peak curves of the preformed G4 DNA incubated with each ligand
at 263 nm.
showed hysteresis in the reverse scan (0.5 °C/min) possibly
The thermal denaturation study has therefore been due to the formation of G4 DNA structure in a kinetically
followed at the wavelength of either 293 nm or 263 nm slower process. However, the G4 DNA formed in the presence
according to the G4 DNA-ligand complex CD signature under of each ligand in KCl buffer did not show any hysteresis in the
investigation. The melting studies showed an elevation in the reverse melting scan possibly due the formation of
denaturation temperature of the G4 DNA upon complexation thermodynamically and kinetically more stable parallel G4
with the ligands (Fig. 5A, B and ESI, Fig. S13, S14). Interestingly, DNA structure (ESI, Fig. S13C).
the present set of ligands showed greater stability toward the Electrophoretic Mobility Shift Assay (EMSA). The contribution
K+-stabilized G4 DNA than the G4 DNA stabilized by Na+ ions of the ligands toward the formation of the G4 DNA was further
(Fig. 5C, D and ESI, Table S2, S3). Moreover, the stability of the investigated by electrophoretic mobility shift assay (EMSA).
G4 DNA formed in the presence of a ligand is significantly We have used
higher than the ligand-DNA complex formed upon incubation oligonucleotide having two stretches of GGG sequence d[5′-
with the preformed G4 DNA (Fig. 5C, D and ESI, Table S2, S3). GGGTTAGGG-3′] where two such strands may associate to
a
9-mer (Hum9) human telomeric
The Na+-stabilized G4 DNA (PDB 143D) has mixed form an intermolecular dimeric G4 DNA structure via
antiparallel and parallel stranded intramolecular structure with Hoogsteen H-bonding among the guanine bases (Fig. 6A).38,39
three G-tetrads connected by one diagonal and two lateral Additionally, such four DNA strands can also combine together
(edgewise) TTA loops which makes the G-tetrads inaccessible and form a tetrameric inter-molecular G4 DNA structure (Fig.
to the planar ligands.35 On the other hand, K+-stabilized G4 6A). To probe whether such DNA structures are formed in the
DNA (PDB 2HY9) has two lateral and one sidewise loops.35 presence of ligands or not, we performed experiments taking
Unlike possessing a rigid diagonal loop as in 143D, the dynamic 10 µM of DNA along with different ligand concentrations in 10
6 | J. Name., 2012, 00, 1-3
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A
ARTICLE
Thymine
= Ligand
Adenine
ss-DNA
Dimeric G4 DNA Tetrameric G4 DNA
C
1
2
3
4
5
6
7
8
9 10
Higher order form
B
Tetrameric G4 DNA
Dimeric G4 DNA
ss-DNA
Fig. 6. (A) A schematic representation of the possible way of G4 DNA formation from the Hum9 DNA d[5′-GGGTTAGGG-3′].
Electrophoretic mobility shift assay of 5′-GGGTTAGGG-3′ (Hum9) DNA with increasing concentration of ligands. (B) lane 1: DNA alone
without KCl, lanes 2-8: DNA + M2 (r = 0, 1, 2, 4, 6, 8, 10) in 50 mM KCl. (C) lane 1: DNA alone without KCl, lanes 2-10: DNA + D4 (r = 0, 1,
2, 3, 4, 5, 6, 8, 10) in 50 mM KCl.
[Ligand]
A
B
C
D
–Ve
+Ve
+Ve–Ve
c
+Ve
+Ve
c
1.2
E
F
ꢁ
T
vs. IC plot
50
m
1.0
0.5
0.0
M2
Linear fit
1.0
0.8
0.6
0.4
0.2
D2
D4
D3
4
6
8
10
12
14
16
0
1
2
3
4
5
ꢁ
OC
)
m (
T
[Ligand], ꢀM
Fig. 7. TRAP-LIG assay for ligand (A) M2, (B) D2, (C) D4, and (D) D3. Lane +ve: positive control; lane –ve: negative control; lane c: the
final concentration of the solvent used for the dilutions and other lanes show the effect of increasing ligand concentrations in the
range from 0.01 µM to 5 µM. (E) Telomerase activity against ligand concentration evaluated from TRAP-LIG assay measured by UVI-
Tech gel documentation station using UVI-Band Map software (version 97.04). (F) Plot of thermal stabilization against IC50 values
obtained from DNA denaturing experiments and TRAP-LIG assay.
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mM Tris-HCl buffer (pH 7.4) containing 50 mM KCl and 0.1 mM the short-term cell viability assay (Fig. 8C, D). This could be
EDTA. We observed that with increasing ligand concentration, attributed to the cytotoxic events imparted by the telomerase
there was a sharp transition from the band of higher mobility inhibition due to the ligand mediated G4 DNA stabilization.
to a band of lower mobility for both of the ligand M2 (Fig. 6B)
HeLa Cells/M2 HeLa Cells/D2 HeLa Cells/D4 HT1080/D4 HFF/D4
A
120
and D4 (Fig. 6C). The band with higher mobility corresponds to
the single-stranded DNA whereas the less mobile one
100
resembles the formation of an intermolecular dimeric G4 DNA
of higher molecular weight.38,39 Ligand M2 showed a transition
80
of the ss-DNA to the dimeric G4 DNA whereas D4 also
generated a band corresponding to the tetrameric G4 DNA
along with higher order structure (Fig. 6C).39 The existence of a
higher order band may be attributed to the association of
individual G4 DNA in the presence of high concentration of the
strong DNA binder like D4. The experiment therefore revealed
the potential of such ligands as G-quadruplex promoter and
stabilizer even at a significantly lower K+ concentration than
the one under more favorable physiological environment.
Telomerase Inhibition Assay (TRAP-LIG assay). The over-
expressed telomerase maintains the telomere length
throughout generations and this is one mechanism how the
cells become immortal. Thus, inhibition of telomerase activity
through G4 DNA stabilization has become a viable strategy in
oncology. We have tested the present set of ligands for their
60
40
20
0
5 µM
10 µM
15 µM
20 µM
B
HFF
HFF + M2
HFF + D4
HeLa
HeLa + M2
Hela + D4
anti-telomerase activity through
assay.40,41
a
standard TRAP-LIG
HeLa Cells
A549 Cells
All the ligands were found to be highly potent telomerase
inhibitors (Fig. 7A-D). The dimeric ligands were invariably
found to be more efficient than the corresponding monomeric
ligand. The IC50 values showed a linear relationship with the
extent of G4 DNA stabilizing abilities of the ligands (Fig. 7E, F).
This manifests an indirect but strong evidence of telomerase
inhibition mechanism through the G4 DNA stabilization.
500
500
400
300
200
100
0
C
D
Untreated
cells
400
300
200
100
0
Untreated
cells
M2
D2
D4
M2
D2
D4
0
5
10
15
20
0
5
10
15
20
Days
Days
In vitro Cytotoxicity. The cell viabilities of ligand (M2, D2, and Fig. 8. Effect of ligands (M2, D2, and D4) on the cell viability
D4) treated cancer cells and normal cells were investigated in a after (A, B) short-term (72 h) and (C, D) long-term (15 days)
MTT based short-term (72 h) cell viability assay. The ligand (5, exposure to different cancer and normal cells. The data
10, 15, and 20 µM) treated HeLa (Human cervical cancer), represented are based on triplicates of each concentration
HT1080 (human fibrosarcoma), and A549 (human lung treatment from at least three independent experiments.
adenocarcinoma) cells showed a moderate decrease in the cell
viabilities. The relatively higher IC50 values (~20 µM) in a short- Apoptosis Assay. The mode of cytotoxicity induced by the
term assay indeed reveal the characteristic of a G-quadruplex ligand treatments was investigated using Annexin V-FITC and
selective DNA binding ligand over duplex DNA. On the other PI dual staining assay.43 HeLa cells, treated with 20 µM of each
hand, the similar experiments performed in telomerase of the ligand (M2
negative primary HFF (human foreskin fibroblast) cells42 did of significant apoptotic population in accordance with their
not show any noticeable loss in cell viability (Fig. 8A and ESI, activity observed in MTT cell viability assays. The ligands M2
, D2, and D4) for 48 h, showed the presence
,
Fig. S15). The bright field images of ligand treated cells D2, and D4 exhibited ~9%, ~15%, and ~35% of early apoptotic
corroborated the observations of cell viability assays where populations after 48 h respectively (Fig. 9 A-D).
the differences in cellular morphology were clearly
The effect of the ligand activity leading to cell death
distinguishable in comparison with that of untreated cells, induced by apoptosis was also examined by PI nuclear
while no such events were observed in the ligand treated counterstaining under fluorescence microscopy. The HeLa cells
normal HFF cells (Fig. 8B).
incubated with each ligand (48 h) showed the presence of
The G4 DNA binding ligands which target telomerase condensed and fragmented nuclei, which are characteristic of
inhibition pathway, require a sufficient time lag (≥8 days) to the apoptotic cells (Fig. 9 E-J and ESI, Fig. S16). On the other
exhibit their activity for telomere shortening to occur.9 hand, normal cells treated with each of the ligands did not
Therefore, the long-term (15 days) cytotoxicity experiments exhibit any noticeable change in the nuclear morphology
were also performed with a sub-cytotoxic ligand concentration under the similar conditions (ESI, Fig. S17). This observation
(5 µM). A significant cell growth inhibition was observed in seems to be quite evident for the cancer cell specific toxicity
ligand treated cells in accord with their activity as observed in induced by apoptosis upon treatment with a ligand.
8 | J. Name., 2012, 00, 1-3
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ARTICLE
E
H
A
B
~9%
F
I
D
C
G
J
~15%
~35%
Fig. 9. Representative dot plots for Annexin-V and PI staining of untreated HeLa cells (A) and after incubation with ligands M2 (B), D2
(C), and D4 (D). The lower right quadrant represents early apoptotic cell population originated due to the Annexin V-FITC staining only.
Representative bright field and fluorescence microscopic images of HeLa cells using PI as a nuclear counterstain. (E) Untreated HeLa
cells and (H) cells treated with 20 µM of the ligand D4 for 48 h. Panel E-G and H-J (top to bottom) represent bright field, PI nuclear
counterstain (red) and overlay of the previous two images.
Cellular Internalization. The cellular internalization of G4 DNA We also looked at the cellular internalization of ligands in a co-
binding ligands was analyzed by means of fluorescence culture environment of cancer (HT1080) and primary HFF
microscopy in cancer cell lines (HT1080 and HeLa) and normal cells.44 The mixed cells in culture were incubated with ligand,
cell line (HFF) as well. The HeLa and HT1080 cells showed a D4 and observed under fluorescence microscopy. The bright
prominent nuclear localization of the ligands after 24 h of field images of HT1080 and HFF cells helped the analysis to be
incubation (Fig. 10 and ESI, Fig. S18) where PI (propidium clearer because of their apparently distinct morphologies.
iodide) served as a nuclear counterstain. On the other hand, Interestingly, the nuclear distribution of ligand was more
none of the ligands showed apparently distinguished nuclear distinguished in HT1080 cells while HFF cells showed a
entry in normal HFF cells as it was observed in the cancer cells significant cytoplasmic distribution. The ligand distribution
(Fig. 11A, B).
pattern in co-culture is highlighted with help of arrows (Fig.
11D). The observation made under co-culture experiments
were nearly similar to that of cellular internalization pattern as
shown in individually cultured cancer and normal cells.
A
B
Fig. 10. Representative fluorescence microscopic images depicting cellular internalization of the ligand (A) M2 and (B) D4 in HT1080
cells at a concentration of 10 µM after 24 h. PI served as a nuclear counterstain. Panels A and B represent (left to right) bright field,
ligand fluorescence (blue), PI nuclear counterstain (red) and overlay of ligand and PI fluorescence. Scale bar = 20 µm.
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toward the groove interacting with the anionic phosphate
backbone. For dimer, one arm of the ligand interacts in the
same fashion like the monomer targeting the end G-tetrad
whereas the other arm interacts with the groove created by
the T17-T18-A19 nucleobases (Fig. 12B and ESI, Fig. S19B). The
proximity of the linker piperazine with G15 phosphate residue
and dual nature of the G-quadruplex recognition46 enhanced
the DNA-ligand interaction to a significant extent. The MD
simulation results showed that the dimeric molecule interacts
with 1KF1 more efficiently than the corresponding monomer.
A
B
D
C
Experimental methods
Synthetic procedures
Fig. 11
depicting (A) HFF cells after incubation with 10 µM of D4 for
24 and (B) HT1080 and HFF co-cultured cells after
.
Representative fluorescence microscopic images 3,6-Bis-(6-(4-methylpiperazin-1-yl)-1H-benzo[d]imidazol-2-yl)-
9H-carbazole (1, M1). Freshly prepared (4-(4-methyl piperazin-
1-yl)benzene-1, 2-diamine) (185 mg, 0.897 mmol) and the
h
9
incubation with 10 µM of D4 for 24 h; Red arrows indicate
HT1080 cells and white arrows indicate HFF ones. Panels A
and B (from left to right) represent bright field and ligand
fluorescence (blue) respectively. Scale bar = 20 µm.
dialdehyde 13 (100 mg, 0.448 mmol) were taken with 30 mL of
ethanol. An aqueous solution of Na2S2O5 (90 mg, 0.47 mmol)
was added to the mixture and refluxed for 12 h. The reaction
mixture was then cooled, filtered and evaporated to dryness
under reduced pressure. The crude mass was then dissolved in
methanol and precipitated by addition of ethyl acetate to it.
Repetitive precipitation of the solid afforded the pure product
Molecular Dynamics Simulation. The interaction between
each ligand and the G4 DNA was further investigated with the
help of Molecular Dynamics simulation studies. We performed
studies considering the monomeric ligand M2 and the most
potent dimeric ligand D4. The optimized structures were
converted into PDB using Open Babel software and docked
with parallel propeller-type X-ray resolved G4 DNA crystal
structure (PDB 1KF1) using Autodock 4.0 software. The DNA-
ligand complexes obtained from the docking studies were
taken for the higher level of MD simulation using Amber 9
package software.45 The MD simulations have been performed
as adjudged by TLC (5% MeOH/CHCl3 on pre-coated silica gel).
1
(173 mg, 65%). H
This was isolated as a brownish powder
1
NMR (400 MHz, DMSO-d6): δ ppm 12.59 (br, 1H); 11.76 (br,
1H), 9.0 (s, 2H), 8.22 (d, J = 8, 2H), 7.65 (d, J = 8.4, 4H), 7.48 (s,
2H), 6.95 (s, 2H), 3.15 (s, 6H), 2.56 (s, 4H), 2.29 (s, 4H); 13C
NMR (100 MHz, DMSO-d6): δ ppm 172.12, 152.09, 147.09,
141.13, 124.74, 122.89, 121.8 5, 118.55, 113.73, 111.80, 61.32,
59.20, 57.12, 52.68, 48.94, 21.18, 15.27; IR (KBr): 3422, 2970,
2870, 1635, 1442, 1289, 1241, 1185, 1143, 1009, 963, 897, 815
cm-1; HRMS: m/z = 596.3247, Calcd. = 596.3250 [M+H]+; mp
>300 °C. Anal. (Calcd for C36H37N9) C, 72.58; H, 6.26; N, 21.16;
found: C, 72.31; H, 6.28; N, 21.2.
with
a gradual reduction of the constraint following a
procedure reported earlier (SI).18 The final production run of 6
ns without any constraint retained the DNA-ligand stable
association reflected from the corresponding RMSD plot (ESI,
Fig. S19C).
3,6-Bis-(6-(4-(ethanol-2-yl)-piperazin-1-yl)-1H-benzo[d]imid-
azol-2-yl)-9H-carbazole (2, M2). Freshly prepared (2-(4-(3,4-
diaminophenyl)piperazin-1-yl)ethanol) 10 (106 mg, 0.45 mmol)
and the dialdehyde 13 (50 mg, 0.22 mmol) were taken in 30
mL of ethanol. An aqueous solution of Na2S2O5 (44 mg, 0.23
mmol) was added to the mixture and refluxed for 12 h. The
reaction mixture was then cooled, filtered and evaporated to
dryness under reduced pressure. The crude mass was then
dissolved in methanol and precipitated by the addition of ethyl
acetate to it. The repetitive precipitation afforded a product
which was adjudged as pure by TLC (5% MeOH/CHCl3 on pre-
A
B
coated silica gel). This was isolated as a brownish powder 2 (77
1
mg, 55%). H NMR (400 MHz, DMSO-d6): δ ppm 12.8 (br, 2H),
9.02 (s, 2H), 8.24 (d, J = 8.4, 2H), 7.66 (d, J = 8.4, 2H), 7.49 (t, J
= 8.4, 2H), 7.04 (d, J = 9.2, 2H), 6.97 (d, J = 8.4, 2H), 4.8 (s, 2H),
3.67 (s, 8H), 2.95 (br, 8H), 2.81 (br, 8H); 13C NMR (100 MHz,
DMSO-d6): δ ppm 172.12, 152.09, 147.09, 141.13, 124.74,
122.89, 121.85, 118.55, 113.73, 111.80, 61.32, 59.20, 57.12,
52.68, 48.94, 21.18, 15.27; IR (KBr): 3420, 2972, 2865, 1632,
Fig. 12. MD simulated G4 DNA-ligand complexes for ligand (A)
M2 and (B) D4 with 1KF1.
The monomeric ligand efficiently stacked over the G-tetrad
through the planar aromatic moiety covering three guanine
bases (Fig. 12A). The end-piperazine moiety approached
10 | J. Name., 2012, 00, 1-3
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1445, 1282, 1240, 1182, 1054, 945, 898 cm-1; HRMS: m/z = 1,2-Bis(2-(4-(4-(9H-(3,6-Bis(6-(4-(ethanol-2-yl)piperazin-1-yl) -
656.3462, Calcd. = 656.3461 [M+H]+; mp >300 °C. Anal. (Calcd 1H-benzo[d]imidazol-2-yl)-9H-carbazole)-9-yl)butyl)piperaz-
for C38H41N9O2) C, 69.6; H, 6.3; N, 19.22; found: C, 69.73; H, in-1-yl)ethoxy)ethane (6, D4). Compound
6.33; N, 19.18.
6
synthesized from compound 23 following a similar procedure
has been
1
as described for the synthesis of compound 2. H NMR (400
MHz, CD3OD): δ ppm 12.8 (br), 9.05 (s, 4H), 8.30 (d, J = 8.4,
4H), 7.82 (d, J = 8.4, 4H), 7.47 (d, J = 7.6 4H), 7.04 (s, 4H), 6.96
(d, J = 8, 4H), 4.76 (s, 4H), 4.50 (s, 6H), 3.63 (s, 12H), 3.22 (s,
22H), 2.86-2.50 (m, 36H), 1.83 (s, 4H), 1.54 (s, 4H); 13C NMR
(100 MHz, DMSO-d6): δ ppm 173.8, 152.0, 147.3, 141.4, 139.1,
125.0, 122.5, 121.3, 118.7, 115.9, 114.3, 110.3, 101.1, 69.5,
66.9, 63.5, 62.6, 59.0, 57.0, 56.2, 56.1, 55.4, 52.5, 51.2, 51.0,
48.8, 44.8, 43.1, 42.5, 26.1, 22.4, 21.5, 15.0; IR (KBr): 3450,
2937, 2709, 1633, 1606, 1457, 1380, 1242, 1022, 960, 813, 722
cm-1; MALDI: m/z = 1706.015, Calcd. = 1706.015 [M+H]+; mp
>300 °C. Anal. (Calcd for C98H124N22O6.H2O) C, 68.27; H, 7.37; N,
17.87; found: C, 68.17; H, 7.36; N, 17.91.
1,17-bis(9H-(3,6-Bis(6-(4-methylpiperazin-1-yl)-1H-benzo[d]
imidazol-2-yl)-carbazol)-9-yl)-3,6,9,12,15-pentaoxa-heptadec-
ane (3, D1). Compound
compound 17 following a similar procedure as described for
3 has been synthesized from
1
compound 1. H NMR (400 MHz, DMSO-d6): δ ppm 12.87 (br),
9.06 (s, 4H), 8.30 (dd, J1 = 1.2, J2 = 8.8, 4H), 7.84 (dd, J1 = 8.8, J2
= 9.8, 4H), 7.48 (d, J = 7.6, 4H), 7.07 (s, 2H), 6.96 (d, J = 8.4,
8H), 4.90-4.7 (m, 4H), 4.15 (s, 2H), 3.91-3.74 (m, 14H), 3.62-
3.56 (m, 18H), 3.27 (s, 22H), 2.35 (s, 6H); 13C NMR (100 MHz,
DMSO-d6): δ ppm 15.12, 33.29, 43.10, 43.54, 43.99, 48.78,
53.77, 61.32, 68.88, 70.57, 110.54, 113.74, 118.39, 121.95,
121.99, 122.30, 122.48, 122.62, 124.64, 141.26, 141.38,
141.44, 141.58, 146.92, 151.89, 172.32; IR (KBr): 3512, 2937,
2821, 2699, 1636, 1457, 1289, 1240, 1219, 1190, 1145, 1010, G-Quadruplex Formation. Hum21 sequence, d[5′-G3(T2AG3)3-3′]
1024, 964, 897, 813 cm-1; HRMS: m/z = 703.3676, Calcd. = was incubated in 10 mM Tris-HCl (pH 7.4), containing 0.1 M of
703.3671 [M-2CH3]2+; mp >300 oC. Anal. (Calcd for the indicated salt and 0.1 mM EDTA and the mixture was
C84H96N18O5) C, 70.17; H, 6.73; N, 17.54; found: C, 70.05; H, heated at 95 °C for 5 min and cooled slowly to room
6.75, N, 17.58.
temperature over 24 h. The formation of G4 DNA was
confirmed by their circular dichroic spectral signatures and
melting profiles with that reported in literature as well as by
PAGE .26
1,17-bis(9H-(3,6-Bis(6-(4-(ethanol-2-yl)piperazin-1-yl)-1H-
benzo[d]imidazol-2-yl)-9H-carbazole)-9-yl)-3,6,9,12,15-penta-
oxa-heptadecane (4, D2). Compound 4 has been synthesized
from compound 17 following a similar procedure as described Electrophoretic Mobility Shift Assay (EMSA). The 9-mer
for compound 2.
1H NMR (400 MHz, DMSO-d6): δ ppm 12.7 telomeric DNA sequence d[5′-GGGTTAGGG-3′] was labeled
(br), 9.04 (s, 4H), 8.30 (dd, J1 = 1.2, J2 = 8.8, 4H), 7.85 (dd, J1 = with 32P-ATP at the 5′-end and purified by Sephadex G-50
8.8, J2 = 9.8, 4H), 7.49 (d, J = 8.4 4H), 7.09-6.96 (m, 8H), 5.1 (br, column followed by 15% PAGE. The required band was
4H), 4.90 (s, 4H), 4.70 (s, 4H), 4.14 (t, J = 6, 4H), 3.92 (t, J = 5.8, transferred to a centrifuged tube and eluted in TE buffer of pH
4H), 3.74 (m, 12H), 3.63 (m, 10H), 3.03 (s, 24H) 2.93 (s, 8H); 13
C
7.4 to get the labeled oligomer. The labeled DNA was then
NMR (100 MHz, DMSO-d6): δ ppm 162.6, 162.5, 157.2, 152.3, mixed with unlabeled ODN of identical sequence for further
152.1, 135.4, 135.2, 135.1, 133.2, 133.1, 132.6, 132.3, 129.1, experiments. An 20 µL aliquot of 10 µM DNA in 10 mM Tris-HCl
128.9, 124.5, 121.2, 81.1, 79.5, 72.1, 68.9, 66.5, 62.56, 58.6, (pH 7.4) containing 50 mM KCl and 0.1 mM EDTA was mixed
54.3, 53.9, 21.2; IR (KBr): 3402, 3020, 2871, 1633, 1572, 1456, with different concentrations of a given ligand and heated to
1324, 1218, 1054, 835 cm-1; HRMS: m/z = 763.4062, Calcd. = 95 °C for 5 min followed by slow cooling to rt over 24 h. An
763.4064 [M-CH2O]2+; mp >300 oC. Anal. (Calcd for aliquot of 10 µL of this solution was mixed with 2 µL of gel
C88H104N18O9.H2O) C, 67.07; H, 6.78; N, 16.0; found: C, 66.93; H, loading dye (30% glycerol, 0.1% bromophenol blue, and 0.1%
6.8, N, 16.05.
xylene cyanol) and loaded on a 15% polyacrylamide gel and
electrophoresed for 8 h at 120 V at 4 °C in 0.5X running buffer
(pH 7.4) containing 20 mM KCl. Gels were finally dried and
visualized using a phosphorimager.
1,2-Bis(2-(4-(4-(9H-(3,6-Bis(6-(4-methylpiperazin-1-yl)-1H-
benzo[d]imidazol-2-yl)-carbazol)-9-yl)butyl)piperazin-1-yl)-
ethoxy)ethane (5, D3). Compound
from compound 23 following a similar procedure as described Cell Viability Assay. The cells were seeded in 96-well cell
for the synthesis of compound
. 1H NMR (400 MHz, CD3OD): δ culture plates (15 × 103/well) and grown for 24 h for the cells
5 has been synthesized
1
ppm 12.65 (br), 9.05 (s, 4H), 8.30 (t, J = 6.0, 4H), 7.80 (d, J = to adhere. The cells were then exposed to different
8.4, 4H), 7.46 (d, J = 8.4, 4H), 7.04 (s, 4H), 6.94 (d, J = 7.6, 4H), concentrations of ligands or equivalent volume of DMSO
4.47 (s, 4H), 3.17 (s, 22H), 2.64 (s, 20H), 2.34 (s, 24H), 1.82 (s, (0.1%) in the presence of 10% FBS. After 72 h of incubation, 20
4H), 1.50 (s, 4H); 13C NMR (100 MHz, DMSO-d6): δ ppm 171.99, μl of MTT (5 mg/mL) reagent was added to each well and
151.66, 147.28, 141.13, 124.58, 122.40, 121.93, 118.41, incubated for another 4 h. The old medium was then removed
113.58, 110.06, 79.15, 69.48, 67.70, 61.22, 59.71, 56.70, 56.52, and DMSO (200 μl) was added to the wells. The readings were
54.36, 52.27, 51.88, 49.42, 48.57, 44.94, 42.39, 26.14, 22.96, taken at 570 nm on a ELISA plate reader. The results reported
15.10; HRMS: m/z = 1585.9728; Calcd. = 1585.9730 [M+H]+; are based on the triplicates of three independent experiments.
mp >300 °C. Anal. (Cald for C94H116N22O2) C, 71.18; H, 7.37; N, Percentage (%) cell viability was calculated using the formula,
19.43; found: C, 71.07; H, 7.39; N, 19.4.
% cell viability = [(FI(570) of treated cells - FI(570) of plain DMSO)/
(FI(570) of untreated cells - FI(570) of plain DMSO)] × 100.
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Long-term Cell Viability Assay. In a typical long-term viability
assay, ~5.0 × 104 cells per well were grown in 6 well tissue
culture plates and treated with a sub-cytotoxic concentration
of (5 μM) or an equivalent of 0.1 volume % DMSO. The cells in
untreated and ligand-treated wells were counted and re-
seeded with half population of the cells. This process was
continued for total 15 days. Finally, cell population vs time
(days) plots were generated.
duplex DNA. The ligand’s G4 DNA-specific fluorescence “turn-
on” phenomenon helped in discriminating G4 DNA with that of
duplex-DNA. Each of the ligands induced topological
transformation of the K+-stabilized mixed-hybrid G4 DNA to a
thermodynamically more stable parallel G4 DNA structure. The
monomeric ligand, M2 could induce the formation of dimeric
G4 DNA even at low K+ ion concentration. However, the
dimeric ligand, D4 generated
intermolecular G4 DNA structures from a 9-mer human
telomeric DNA sequence. The ligands M2 D2 D3, and D4
a dimeric and tetrameric
Annexin V-FITC and PI Staining Assay. An Annexin V-FITC and
PI Apoptosis Detection kit (Sigma) was used to detect
apoptosis in the ligands treated HeLa cells. In a typical
experiment, cells were seeded at 0.25 million cells population
in 6 well cell culture plates. After 24 h, cells were incubated
with each ligand for 48 h at a final concentration of 20 µM. The
cells were then washed properly with DPBS buffer followed by
trypsinization and resuspended in 1X binding buffer (HEPES
buffer containing 0.14 M NaCl and 2.5 mM CaCl2). Thereafter,
Annexin V-FITC conjugate was added to each of the cell
suspensions and incubated for ~10 min at room temperature
keeping the same protected from light. Then, each cell
,
,
showed complete telomerase inhibition with IC50 values in the
sub-micromolar concentration range. Among all the molecules,
the dimeric ligand D4 showed highest telomerase inhibition
activity (IC50, 0.25 µM) which was higher by more than four
times to that of monomer. Interestingly, the activity shown by
the dimeric bis-benzimidazole ligands present them as the
most potential telomerase inhibitors among all the bis-
benzimidazole ligands reported till date.18,28,29 The absorption
titration, CD spectroscopy, fluorescence titration and
electrophoretic mobility analysis and the telomerase inhibition
activity unambiguously demonstrated the superiority of the
dimeric ligands over the monomeric ones. Among the dimeric
molecules, the ligands with piperazine moiety in the linker
showed greater efficiency than the ligands with a neutral,
hexaethylene glycol based spacer. The ligands demonstrated
significant antiproliferative activity toward cancer cell lines
while unaffecting the telomerase negative primary HFF cells.
The ligand induced cytotoxicity was due to apoptosis as
confirmed by Annexin V-PI dual staining (FACS) and PI nuclear
counterstaining (fluorescence microscopy). The cellular
suspension was incubated with PI solution at
concentration of 1µg/ml for ~5 min. Samples were then
analyzed using FACS Calibur flow cytometer (Becton-
a final
a
Dickinson). Data obtained from the flow cytometry were
analyzed using WinMDI 2.9 software by considering gated cell
population for the detection of % apoptotic cells.
Fluorescence Microscopy. To visualize the cellular
internalization of the ligands and apoptotic nuclei,
fluorescence microscopic studies were performed. In a typical
experiment, cells were cultured on a glass cover slip placed in
12 well cell culture plates. Cells were then treated with each
ligand (24 h, internalization and 48 h, apoptosis) followed by
washing properly with DPBS. The cells were then fixed in 4%
paraformaldehyde solution for ~10 min. Subsequently, cells
were rinsed with DPBS and treated with 0.1% Triton-X-100 for
~5 min to permeabilize the cell membrane. Then the cells were
washed with DPBS, and the glass cover slips were taken out
and nuclei were counterstained with PI. Finally, the
overstaining was removed by repeated washing with DPBS and
then autoclaved milli-Q, the cover slips were mounted on glass
internalization studies revealed
a
prominent nuclear
localization of ligands in cancer cells in comparison with that of
normal cells. The co-cultures experiments revealed a distinct
cellular distribution pattern of ligands toward cancer and
normal cells. Concisely, we present a series of potent dimeric
carbazole based bis-benzimidazole derivatives depicting the
importance of linker design for improved G4 DNA binding and
their prospects as telomere targeting putative anticancer
drugs.
slides and observed under
(Olympus IX81).
a fluorescence microscope
Acknowledgements
This work was supported by J. C. Bose Fellowship, DST, to S.B.
B.M. acknowledges IISc for a Research Associate fellowship.
The human foreskin fibroblast (HFF) cells were a kind gift from
the Manipal University, Manipal.
For the co-culture experiments, HT1080 and HFF cells were
mixed in an equal concentration of about 1.5 × 106 cells/ml
and plated in 12 well cell culture plates. Next day, the cells
were incubated with ligands for 24 h and the samples were
processed as discussed above.
Notes and references
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G-quadruplex DNA binding dimeric ligands and their telomerase inhibition activity
137x60mm (150 x 150 DPI)