Pyridostatin analogues promote telomere dysfunction and long-term growth inhibition in human cancer cells

Article abstract of DOI:10.1039/c2ob25830g

The synthesis, biophysical and biological evaluation of a series of G-quadruplex interacting small molecules based on a N,N′-bis(quinolinyl) pyridine-2,6-dicarboxamide scaffold is described. The synthetic analogues were evaluated for their ability to stabilize telomeric G-quadruplex DNA, some of which showed very high stabilization potential associated with high selectivity over double-stranded DNA. The compounds exhibited growth arrest of cancer cells with detectable selectivity over normal cells. Long-time growth arrest was accompanied by senescence, where telomeric dysfunction is a predominant mechanism together with the accumulation of restricted DNA damage sites in the genome. Our data emphasize the potential of a senescence-mediated anticancer therapy through the use of G-quadruplex targeting small molecules based on the molecular framework of pyridostatin.

Full text of DOI:10.1039/c2ob25830g

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PAPER
Pyridostatin analogues promote telomere dysfunction and long-term growth
inhibition in human cancer cells†
Sebastian Müller,a Deborah A. Sanders,a Marco Di Antonio,a Stephanos Matsis,a Jean-François Riou,b
Raphaël Rodrigueza and Shankar Balasubramaniana,c,d
Received 1st May 2012, Accepted 21st June 2012
DOI: 10.1039/c2ob25830g
The synthesis, biophysical and biological evaluation of a series of G-quadruplex interacting small
molecules based on a N,N′-bis(quinolinyl)pyridine-2,6-dicarboxamide scaffold is described. The synthetic
analogues were evaluated for their ability to stabilize telomeric G-quadruplex DNA, some of which
showed very high stabilization potential associated with high selectivity over double-stranded DNA. The
compounds exhibited growth arrest of cancer cells with detectable selectivity over normal cells. Long-
time growth arrest was accompanied by senescence, where telomeric dysfunction is a predominant
mechanism together with the accumulation of restricted DNA damage sites in the genome. Our data
emphasize the potential of a senescence-mediated anticancer therapy through the use of G-quadruplex
targeting small molecules based on the molecular framework of pyridostatin.
10
sequence (TTAGGG)n and the human telomeric G-quadruplex
Introduction
has been extensively studied in vitro.11,12 It has been postulated
that these motifs affect telomere elongation,13 capping14 and
Nucleic acids can adopt various non-Watson–Crick secondary
structures including G-quadruplexes, a supramolecular architec-
replication in vivo.15 The production of telomeric repeat-contain-
ture that can arise from certain G-rich sequences. Such structures
comprise Hoogsteen hydrogen bonded guanines leading to the
formation of G-quartets that stack via π–π interactions. The rela-
tive orientation of the strands and loop configurations can vary,
giving rise to polymorphic conformations that can depend on the
primary nucleic acid sequence, temperature, solvent and salt
composition.1–4
There has been experimental data in support of G-quadruplex
formation in the genomic DNA of several organisms.5–8 Lipps,
Rhodes and co-workers provided the first strong evidence for
their existence in the ciliate Stylonychia. In their report, the
authors demonstrated that folding into these motifs at the telo-
meric region of chromosomes is regulated by the Telomere End
Binding Proteins (TEBP) α and β in a cell cycle-dependent
manner, and might act as telomere capping structures.9 Human
telomeres comprise a highly repetitive G-rich DNA repeat
ing RNA (TERRA) may also suggest a role during transcrip-
tion.16 In a seminal paper, Zahler et al. showed that certain
G-rich telomeric sequences can form G-quadruplexes in vitro,
rendering them resistant to extension by the reverse transcriptase
telomerase.17 Successive rounds of replication leads to progress-
ive telomere shortening in normal cells, an event that is
alleviated by telomerase conferring infinite proliferative capacity
in ∼85% of cancer cells.18 These findings prompted us to design
selective G-quadruplex interacting small molecules with the
aim to develop a novel telomere based anticancer therapy.19
However, recent reports have suggested that such molecules can
induce telomere dysfunction in a telomerase independent
manner.14,20,21
Telomeres comprise a protein complex named shelterin,22
which covers and protects the ends of telomeres from the DNA-
damage response machinery that promote non-homologous
end-joining and homologous recombination. Small molecules
interacting with G-quadruplexes have been shown to induce telo-
mere dysfunctions by competing for binding with the shelterin
components Telomeric Repeat Factor 1 (TRF1), Telomeric
Repeat Factor 2 (TRF2) and Protection of Telomeres 1 (POT1),
leading to progressive telomere shortening, anaphase bridges
and aberrant chromosome segregation.14,15,23 In addition, dys-
functional telomeres have been shown to activate the DNA
damage response, resulting in cell cycle checkpoint activation
and growth arrest.24 Several putative G-quadruplex sequences,
besides the telomeric repeat, have been identified in the human
aDepartment of Chemistry, University of Cambridge, Lensfield Road,
Cambridge, CB2 1EW, UK. E-mail: rr324@cam.ac.uk,
sb10031@cam.ac.uk; Tel: +44 (0)1223 336347
bRegulation et Dynamique des Genomes, Museum National d’Histoire
Naturelle, INSERM U565, CNRS UMR 7196, Paris, France
cCancer Research UK, Cambridge Research Institute, Li Ka Shing
Center, Cambridge, CB2 0RE, UK
dSchool of Clinical Medicine, University of Cambridge, Cambridge,
CB2 0SP, UK
†Electronic supplementary information (ESI) available: Experimental
procedures and characterization of building blocks and pyridostatin
analogues and FRET-melting profiles. See DOI: 10.1039/c2ob25830g
This journal is © The Royal Society of Chemistry 2012
Org. Biomol. Chem., 2012, 10, 6537–6546 | 6537

genome25 with an enrichment in promoter regions of several
proto-oncogenes26 such as c-kit,27,28 c-myc,29,30 bcl-231 and
K-ras.32 Small molecules have been proposed to interact with
promoter quadruplexes and to alter the expression of those genes
in cellulo.29,30,33,34 These motifs have also been identified in
RNA35–37 where they might have a functional role by modula-
ting translation21,38 and alternative splicing.39 Further evidence
for the existence of G-quadruplex motifs in human cells has
been provided by recent work from Gibbons and co-workers,
demonstrating that the alpha thalassemia/mental retardation syn-
drome X-linked (ATRX) helicase binds to clusters of G-quadru-
plex motifs, hence modulating the expression of these genes.40
Numerous studies on synthetic molecules that interact with
G-quadruplexes have helped demonstrate the existence and eluci-
date putative biological roles of these nucleic acid struc-
tures.41–50 Previously reported quinoline-based molecules
selectively recognize G-quadruplex structures over double-
stranded DNA with high potency, making this chemical moiety
an attractive feature to exploit for the design of G-quadruplex
ligands.51–54 We previously reported a G-quadruplex stabilizing
synthetic small molecule, based on a N,N′-bis(quinolinyl)pyri-
dine-2,6-dicarboxamide scaffold (Fig. 1), which we named pyri-
dostatin (1). We demonstrated the suitability of the molecule to
target telomeres in cells.23 The design of these molecules was
initially based on structural features shared by a potent G-quad-
ruplex binding molecule54 with particular emphasis on (1) the
ability to adopt a flat but flexible conformation, facilitated by an
internal hydrogen bonding network, prone to adapt to the
dynamic and polymorphic nature of diverse G-quadruplex struc-
tures; (2) an optimal electronic density of the aromatic surface to
enable π–π interactions with the G-tetrad tuned by substituents
(for instance alkoxy or halogens capable of altering the electron
density) and (3) the presence of free nitrogen lone pairs able to
coordinate with a molecule of water55 or alternatively to seques-
ter a monovalent potassium cation in the centre, thus locking the
flat surface of the molecule and facilitating the interaction with
G-quartets (Fig. 1). These key features distinguish pyridostatin
from other structurally related G-quadruplex interacting
molecules.51–54 Prior to this work, we showed that pyridostatin,
the lead compound of this family, induces telomere dysfunction
by competing for binding with telomere associated proteins such
as human POT1.23 Furthermore, we have illustrated the ability of
a biotinylated analogue to mediate the selective pull-down of
telomeric fragments from genomic DNA by means of affinity
matrix isolation. During the course of this study, we have
demonstrated the high selectivity of pyridostatin analogues
towards G-quadruplex nucleic acids, regardless of sequence
variability and structure polymorphism, compared to double-
stranded DNA.56 Recent work in our laboratory has proven that
pyridostatin alters transcription and replication of particular
human genomic loci containing high G-quadruplex clustering
within the coding region, which encompasses telomeres57 and
selected genes such as the proto-oncogene SRC.58 These results
emphasize that selective G-quadruplex ligands represent a new
class of DNA damaging agents with limited sites of action
throughout the genome, in contrast to well-known therapeutic
agents previously reported to inducing genome-wide DNA
damage in a stochastic manner associated with high cytotoxicity.
These results prompted us to synthesize structural variants
based on the N,N′-bis(quinolinyl)pyridine-2,6-dicarboxamide
scaffold to further explore the scope of their use as anti-cancer
agents. We have used this family of compounds by keeping the
central scaffold of pyridostatin intact and varying the substitu-
ents, including different cationic, neutral and glycosidic side-
chains. These groups were systematically altered to gain further
insights into structure–activity relationships. Herein, we describe
an assessment of their potential to stabilize the telomeric
G-quadruplex (H-Telo) and a selection of genomic promoter
quadruplexes using a Förster Resonance Energy Transfer
(FRET)-melting assay initially introduced by Mergny and
Maurizot.59 Furthermore, we have measured their growth inhibi-
tory properties against a panel of human cancer cell lines as a
first potency readout, and finally have explored the phenotype
induced at telomeres.
Results
Chemical synthesis of pyridostatin analogues
In this study, 26 small molecules based on the N,N′-bis(quino-
linyl)pyridine-2,6-dicarboxamide scaffold were synthesized in
two to seven synthetic steps as shown in Schemes 1 and 2. We
chose to systematically vary side chain lengths and their chemi-
cal functionalities, keeping the central scaffold of pyridostatin
unaltered. Synthetic analogues were obtained by coupling one
mole equivalent of pyridine-based building blocks with two
mole equivalents of quinoline-based building blocks as depicted
in Scheme 1. The central core was synthesized either from cheli-
damic acid (2) or the commercially available pyridine-2,6-dicar-
bonyl dichloride (3). Compounds 4a and 4c were prepared by
reacting 2 with thionyl chloride followed by quenching the reac-
tion mixture with cold methanol (MeOH). The products were
separated by aqueous work-up yielding 29% of 4a and 28% of
4c. Compound 4b was prepared in 90% yield from 2 in toluene–
MeOH using (trimethylsilyl)diazomethane (Me3SiCHN2). Inter-
mediate 4a was further functionalized via a Mitsunobu reaction,
mixing diisopropyl azodicarboxylate (DIAD) and triphenylphos-
phine in tetrahydrofuran (THF) at 0 °C then room temperature.
The resulting 4-substituted 2,6-pyridine-dimethylesters were
then deprotected in the presence of sodium hydroxide (NaOH) in
Fig. 1 Molecular structure of pyridostatin (1). The design rationale is
indicated on the structure.
6538 | Org. Biomol. Chem., 2012, 10, 6537–6546
This journal is © The Royal Society of Chemistry 2012

Scheme 2 Synthetic route to compounds 27–33. (i) 27, 29–31: 11,
CuSO4·5H2O, sodium L-ascorbate, RN3, H2O–tBuOH, rt, overnight; 28,
32, 33: 12, CuSO4·5H20, Sodium L-ascorbate, RN3, H2O–tBuOH, rt,
overnight.
Scheme 1 Synthetic route to compounds 1 and 8–26. (i) 4a, 4c:
SOCl2, MeOH, 0 °C then rt, 2 h; 4b: toluene–MeOH, Me3SiCHN2, rt,
30 min; (ii) 5a–c, 5f: ROH, triphenylphosphine, DIAD, THF, 0 °C to rt,
3 d; then for 5a–f: NaOH (aq.), MeOH, rt, 1 h; (iii) 7a–e: ROH, triphenyl-
phosphine, DIAD, THF,
0 °C to rt, 3 d; 7f: toluene–MeOH,
tert-butyl dicarbonate)-protected derivative, avoiding chromato-
graphic purification, which then afforded the target molecule
using standard TFA deprotection in DCM. Final products
1, 8–33 were further purified by high performance liquid chromato-
graphy (HPLC) to ensure high purity for the biophysical, in vitro
and in vivo assays used in our subsequent studies. Molecules
22–26 all lack a side chain at position 4 of the pyridine ring,
thus enabling us to investigate the relevance of that substitution
for G-quadruplex stabilization and ensuing associated biological
effects. Compounds 1, 8–14 carry side chains with varying
chemical functionalities on the pyridine core as well as the qui-
noline moiety, which may influence the recognition towards
different quadruplexes and relative conformations. We also intro-
duced a chlorine onto the pyridine core (15–19) as well as a side
chain containing a fluorinated benzene ring (20–21), which have
the potential to participate in interactions with G-quadruplex
loops or change the electron density of the pyridine core, and may
thus provide additional selectivity over duplex DNA (ds-DNA).61
Compounds 27–33 were synthesized using the copper modified
Huisgen reaction (Scheme 2), which enabled the introduction of
complex functionalities such as sugar-containing asymmetric
centers, capable of multiple hydrogen bond networking.
Me3SiCHN2, rt, 30 min; (iv) 1, 8–21: 5a–f, 1-chloro-N,N,2-trimethyl-
propenyl-amine, DCM, 2 h, then triethylamine, 0 °C, 1 h then rt, then
7a–f, overnight; then for 1, 8, 9, 11, 12, 15, 16, 20: TFA–DCM, rt, 1 h;
(v) 22–26: 2, triethylamine, DCM, 0 °C, 1 h, then 7a–f overnight; then
for 22, 23: TFA–DCM, rt, 1 h.
H2O–MeOH to afford the pyridine-based building blocks 5a–f
in 57–77% yield. The quinoline-based building blocks were
obtained in 53–71% yield from commercially available starting
material 2-amino-quinolinone (6) either via a similar Mitsunobu
reaction to provide 7a–e, or through a direct methylation in the
presence of toluene–MeOH containing Me3SiCHN2. Building
blocks 5a–f were reacted with Ghosez’s reagent (1-chloro-
N,N,2-trimethylpropenyl-amine) and then with triethylamine,
followed by the addition of 7a–f to obtain products 1, 8–21 in
61–93% yield. Compounds 8–9, 11–12, 15–16 and 20 were then
deprotected using trifluoroacetic acid (TFA) in dichloromethane
(DCM). Compound 3 was reacted with 7a–g in the presence of
triethylamine to provide compounds 22–26 in 62–82% yield.
Finally, compounds 27–33 were synthesized in 22–67% yield
from 11 and 12 using the copper catalyzed alkyne–azide 1,3-
dipolar cycloaddition as shown in Scheme 2.
Compounds 31–33 were conveniently obtained with retention
of configuration, in mild conditions and without the use of pro-
tecting groups, highlighting the flexibility of this protocol.60 It is
noteworthy that the condensed products of building blocks 5b,
5d and 5f with 7a–b, 7f could be precipitated from hot aceto-
nitrile (MeCN) yielding either the final molecule, or a Boc (di-
Biophysical evaluation of G-quadruplex stabilization by
pyridostatin analogues
In order to study the interaction of the molecules with telomeric
G-quadruplex DNA, we performed a Förster resonance energy
This journal is © The Royal Society of Chemistry 2012
Org. Biomol. Chem., 2012, 10, 6537–6546 | 6539

transfer (FRET)-melting assay59 using the human telomeric
G-quadruplex-forming sequence (H-Telo) and a ds-DNA as
targets. The data are described in Table S1.† A large number of
molecules of this family showed up to 35 K stabilization of
H-Telo at 1 μM in melting experiments. None of the molecules
studied showed any detectable stabilization of ds-DNA at this
concentration, demonstrating the suitability of the scaffold for
cell-based assays. Molecules 1, 8–10, which contain multiple
amine functionalities, exhibited maximal stabilization for H-Telo
at 1 μM ligand (Fig. 2, Table S1†).
fluorine and amine substituents. However, this is not the case for
compounds 31–33, since the sugar moieties might be involved
in stabilizing non-covalent interactions, raising the ΔTm-values
recorded for these compounds to above 30 K. The nature of the
amine on the side chain at position R2 was found to have a small
influence on the stabilization potential of the analogues follow-
ing the general trend: –NMe2 ≥ –NH2 ≥ –pyrrolidine. Compar-
ing ΔTm-values for 1 and 8, 15–16, 18–19, 22–23, 25–28 and
31–32, respectively, showed that a three-carbon side chain gives
slightly higher stabilization potentials than two-carbon side
chains. Finally, we have investigated the selectivity of each
molecule for G-quadruplex over ds-DNA by measuring the
melting of H-Telo in the presence of increasing amounts of each
analogue and 50 mole equivalents of unlabelled ds-DNA compe-
titor. Negligible changes in melting temperatures were observed,
further demonstrating a high specificity of these compounds for
quadruplex DNA over ds-DNA, as was shown before for this
type of scaffold23,56 (see Fig. 2). Encouraged by these results,
we next evaluated the stabilization properties of these com-
pounds over various G4 structures before studying their aptitude
to induce a phenotype in cell-based assays (see Fig. 3).
Similarly, introducing other functions at position 4 of the pyri-
dine moiety such as a chlorine (compounds 15–19) or sugars
(compounds 31–33) improved the stabilization properties of the
named compounds towards H-Telo compared to the unsubsti-
tuted analogues. This data set suggests that cation–dipole inter-
actions as well as hydrogen and halogen-bonding interactions
enable the improvement of the binding properties of the main
scaffold towards G-quadruplex motifs. Conversely, removal of
amine functionalities at positions R1 and R2, as for compounds
13–14, was found to reduce the stabilizing potential of the corre-
sponding scaffold, thus further demonstrating the need for polar
functionalities to promote G-quadruplex stabilization. Ligands
carrying an alkyne functionality (11–12) induced moderate ΔTm-
values, most likely due to the lack of the above mentioned stabi-
lizing interactions. Ligands 27–29 also showed moderate stabil-
ization of H-Telo, which was rather surprising for compound 29,
since this analogue contains three positively charged functional-
ities that have been shown to be stability-enhancers for all the
other members of this family. Additionally, the introduction of a
negatively charged functionality to afford 30 hampered the
ability of this analogue to interact with H-Telo, presumably due
to electrostatic repulsion with the negatively charged phosphate
backbone of the DNA. The 4-fluorobenzyloxy substitution at
position R1 of ligands 20 and 21 resulted in lower ΔTm values of
8.8 K and 4.3 K, respectively. These results are consistent with
those observed for alkyne substituted analogues and suggest that
the steric hindrance imposed by the benzene and triazole rings at
the central position may lead to a weaker contribution of the
Evaluation of cell viability upon exposure to analogues of
pyridostatin
The molecules were assessed for their ability to inhibit cell
growth using a luminescent cell viability assay. We investigated
growth inhibition after 3 days of exposure to compounds 1, 8–33
on a panel of four human cell lines: HeLa (adenocarcinoma),
HT1080 (fibrosarcoma), U2OS (osteosarcoma), and WI-38
(normal lung fibroblasts), the latter being non-cancerous. The
data are summarized in Fig. 3 and Table S2.† Molecules
1, 9–10, 15–19 and 22–26 showed growth inhibition at high
nanomolar to low micromolar concentrations against the panel of
Fig. 2 FRET-melting competition results at 1 μM for 1, 8–33 in the
presence of 50 mol. equiv. of unlabeled ds-DNA against H-Telo. Values
are expressed as ΔTm. Black: ΔTm in the absence of ds-DNA. Grey: ΔTm
in the presence of ds-DNA. Errors denote the standard deviation of at
least three independent experiments.
Fig. 3 Representation of FRET melting values for various G-quadru-
plex structures (top) and IC50-values of growth inhibition after 72 h
(bottom) treatment with compounds 1, 8–33. Errors denote the standard
deviation of at least three independent experiments.
6540 | Org. Biomol. Chem., 2012, 10, 6537–6546
This journal is © The Royal Society of Chemistry 2012

cell lines. Compound 9 displayed the lowest values of the series
with IC50-values ranging from 0.2 to 0.5 μM. This suggests that
positively charged side chains may be required for improving the
water solubility and cellular uptake of the apolar central skeleton,
in addition to participating in favorable non-covalent interactions
with the DNA targets. It is noteworthy that most of the com-
pounds showed generally lower IC50-values for the cancer cell
lines than for the normal cell line WI-38. For example, pyri-
dostatin (1) exhibited an 18.5-fold selectivity for HT1080 cells
over WI-38 cells. Analogues 17 and 27 were found to be the
most selective molecules for HeLa and U2OS with a 6.0- and
5.2-fold difference in growth inhibition over WI-38 cells,
respectively. Notably, compounds 27–33, which contain a tri-
azole at position R1, showed relatively higher IC50-values as
compared to other analogues, which might reflect differences in
cell permeability and uptake of these analogues, since the sugar
functionalized compound retained remarkable stabilization pro-
perties. Ligand 14, which contains no functionalities, did not
exhibit growth inhibitory properties at the concentrations used,
thus reflecting on the poor solubility properties of this analogue
and weak G-quadruplex stabilization potential. These results are
in agreement with our previous reports23,58 showing that this
family of small molecules promotes DNA double strand break
formation, which results in cell cycle arrest and growth
inhibition.
compounds (Fig. 4) at their respective IC50-values. Some of the
molecules caused a strong reduction of population doublings
indicating a long-term growth arrest of HT1080 cells, notably
1, 15 and 17 after 6 days, 9 after 12 days and 10 and 21 after 21
days of exposure. Compounds 20 and 33 also caused a decrease
in population doublings over time, with a flattening of the curve
starting after 19 and 25 days of exposure, respectively.
Compounds 16, 19, 22–24, 26 and 27 did not show any
additional decrease in population doublings over the time-course
measured, as compared to what would be expected for incu-
bation at the IC50. No change of population doublings was
observed over the time course measured for compounds that did
not have a substitution at position 4 of the pyridine ring (com-
pounds 22–26). It is worth noting that 97–99% of the control
and the cells treated at the respective IC50-value of each com-
pound (1, 9–10, 15–17, 19–24, 26–27 and 33) were viable
during the long-term treatment as observed by trypan blue
nuclear exclusion, indicative of growth arrest as opposed to cyto-
toxicity induced by this family of compounds. In addition, the
total cell number reached a plateau after long-term exposure
without noticeable decrease, which also supports the absence of
major cell killing. Our results indicate that complex mechanisms
of action for these compounds are taking place. One of these
mechanisms may involve the induction of dysfunctional
telomeres, as previously shown for 1, due to a competition for
binding of these analogues with telomere binding proteins and
ensuing telomere shortening. To verify this hypothesis we inves-
tigated the influence of these analogues on telomere G-overhang
integrity in HT1080 cells.
Long-term growth inhibitory effects of pyridostatin analogues
towards HT1080 cells
We investigated the long-term growth effects on HT1080 cells of
a selection of molecules, namely 1, 9–10, 15–17, 19–24, 26–27
and 33, by incubating cells over a period of 30 days with the
G-overhang shortening and cellular senescence induced by
analogues of pyridostatin
Small molecules that can compete for binding with shelterin
components have been shown to induce telomere shortening,
G-overhang degradation and replicative senescence in cultured
cells.23,47,56,62 We chose compounds 9–10, 15, 17 and 33, since
these analogues displayed moderate to high stabilization of the
telomeric G-quadruplex H-Telo as judged by FRET-melting
assays and long-term growth arrest occurring between 6 to 25
days of treatment. A β-galactosidase assay performed on
HT1080 cells after 8 days of incubation with compounds 9–10,
15, 17 and 33 at their respective IC50 revealed the presence of
senescent cells (Fig. S4†). No senescent cells were observed in
mock treated cells.
We decided to measure telomere G-overhang lengths after
treatment by employing a non-denaturing hybridization assay.62
The experiments were performed using a 21C oligonucleotide
(5′-(CCCTAA)3CCC-3′) complementary to the 3′-G-overhang.
G-overhang signals were assessed over a time course of 12 days,
at five time-points (0 d, 3 d, 6 d, 9 d and 12 d). We found that all
five analogues induced a decrease in the hybridization signal as
compared to total genomic DNA stained with ethidium bromide
(EtBr, Fig. 5). Consistent with a selective effect induced by the
compounds, mock treated cells did not show any noticeable
change in the hybridization signal. This showed that the
telomeric G-overhang decreased in length over the time-course
upon exposure to the small molecules. Interestingly, the kinetics
Fig. 4 Studies of long-term growth effect of compounds 1, 9, 10, 15,
17, 20, 21 and 33 on HT1080 cells. CON denotes control cells, which
were not treated with compound. Measurements were taken at the
respective IC50 values of the compounds.
This journal is © The Royal Society of Chemistry 2012
Org. Biomol. Chem., 2012, 10, 6537–6546 | 6541

We demonstrated that diverse simple and complex functional fea-
tures could be introduced before or after the assembly of the
main scaffold, which confers additional flexibility to our syn-
thetic scheme. Functionalization via click chemistry, to yield
molecules 27–33, was attractive for chain derivatization at a late
stage of the synthesis.60 Molecules of this family have the ability
to stabilize G-quadruplexes formed by the H-Telo sequence in
the presence of an excess of ds-DNA (Fig. 2) and also a selec-
tion of promoter quadruplexes (Fig. S2 and Table S1†) with very
high ΔTm values, demonstrating the versatility of this type of
scaffold to bind G-quadruplex motifs with varying loop
sequence. This unique property as a potent generic G-quadruplex
binder rationalizes in part the ability of these compounds to
target other genomic locations containing putative G-quadruplex
forming motifs and to promote genomic instability at well
defined genomic locations.23,58
The nature of the substitution at the pyridine moiety had a
great influence on the stabilization potential of the molecules for
H-Telo following the general trend of: amine > Cl > sugar > H >
alkoxy. Positively charged functionalities and sugars conferred
higher stabilization, most likely due to their potential for electro-
static and hydrogen bonding interactions with the nucleic acid
target. Accordingly, substitution with uncharged, aromatic or
aliphatic side chains generally had an adverse effect on the
stabilization potential of the molecule, which could be due to
electronic repulsion or steric clash. Modifications in the length
of the side chain linked to the quinoline moiety had a less strik-
ing effect, but generally stabilization was higher for chain
lengths of three carbons over two carbons. Notably, the stabili-
zation potential for G-quadruplexes was almost eliminated for 14
with methoxy-substitutions on both R1 and R2. This shows that
the side chains play a crucial role in the potential of the
molecules to stabilize G-quadruplex nucleic acids. Clearly, the
nature of side chains affected the water solubility properties of
each analogue and may reflect differences observed in biophysi-
cal and biological assays.
Most of the molecules showed high nanomolar to low micro-
molar IC50-values for short-term (72 h) growth inhibition against
the four cell lines investigated. Some of the compounds, in par-
ticular pyridostatin (1), 17, 24, and 27, showed a considerable
difference in their growth-inhibitory properties between the
different cell-lines investigated. Molecules 17, 24 and 27 were
found to be more potent for cancer cell lines over WI-38. Com-
pounds 17 and 24 are more selective towards the cancer cell
lines than other analogues that carry a hydrogen or chlorine at
position R1. This shows how the variation of the substitution at
R2 can be used to modulate differential growth inhibitory proper-
ties from one cell line to another, which may be exploited
further to develop small molecules empowered with selective
anti-proliferative properties.
Fig. 5 Telomeric G-overhang shortening assessed for 9, 10, 15, 17 and
33 in HT1080 cells by non-denaturing hybridization assay. CON rep-
resents DNA from cells grown in the absence of compound. Cells were
incubated at the respective IC50 concentrations. (a) Hybridization gel
radiograph and EtBr fluorescence picture. (b) Graph showing the %
hybridization signal against days of treatment. The radiograph signal
was normalized against the fluorescence signal. Errors denote the stan-
dard deviation from three different experiments.
of the G-overhang degradation appeared to vary, with com-
pounds 10 and 17 being the most potent to induce an early
effect, a result that is in apparent contrast with their kinetics of
cell growth arrest (Fig. 4).
These data suggest that these compounds trigger a telomeric
dysfunction associated with the induction of a phenotype remi-
niscent of cellular senescence. However, the correlation between
telomere dysfunction and long-term growth arrest for compounds
9–10, 15, 17 and 33 is complex and suggests that additional
events contribute to the growth inhibition and the induction of
senescence.
Discussion
We have shown that N,N′-bis(quinolinyl)pyridine-2,6-dicarbox-
amides in general stabilize G-quadruplex nucleic acids selec-
tively as compared to ds-DNA. Systematic changes of the side
chains can help modulate their in vitro stabilization potential for
G-quadruplexes and differentially affect short- and long-term
cell growth. Moreover, their low molecular weight and global
structural features confer drug-like properties. This constitutes a
promising starting point for the development of compounds with
differential growth inhibitory properties on different cell types
combined with low toxicity.
The synthesis of the library of compounds was efficient and
gave moderate to high yields with high purity in 2–7 synthetic
steps. In particular, the coupling of quinoline-based building
blocks with the pyridine core using Ghosez’ reagent was facile
and high yielding compared to other coupling strategies.
Long-term growth studies upon incubation with the com-
pounds showed that some compounds caused a decrease in
population doublings between 6 and 30 days treatment (Fig. 4).
In this set of experiments each compound was screened at its
IC50 value in order to detect and quantify a phenotype, thus
enabling a comparative analysis of the analogues. The six com-
pounds with the greatest differential effect on long-term growth
were either substituted with an amine side chain (1, 9–10), a
chlorine (15 and 17) or a sugar at position R1 (compound 33).
6542 | Org. Biomol. Chem., 2012, 10, 6537–6546
This journal is © The Royal Society of Chemistry 2012

Consistent with this, these compounds also exhibited very good
stabilization of the telomeric quadruplex. None of the molecules
substituted with a hydrogen at position R1 showed a considerable
decrease in population doublings upon long-term exposure.
Interestingly, all 6 analogues have either a primary amine or a
pyrrolidine on the side chain at R2. The majority of the com-
pounds that induced long-term growth arrest had a side-chain
linked by two carbons (with the exception of compound 33).
The cellular growth data suggests an important sensitivity to the
R2 substitution. Compounds 16, 19 and 22–24 exhibit good
short-term growth inhibition, rather than long-term growth
effects, which suggest a distinction in biological mechanism(s).
Potent stabilization of non-telomeric G-quadruplex structures by
FRET-melting (Fig. S2 and Table S1†) may hint that non-
telomeric G-quadruplex targeting is the predominant contribut-
ing factor to the observed short-term growth arrests. Indeed, our
previous reports23,58 have shown that pyridostatin (1) induces
DNA damage at non-telomeric loci of genomic DNA, resulting
in the activation of a cell cycle checkpoint-dependent growth
arrest.
oncogenes.67 In general, most of the N,N′-bis(quinolinyl)pyri-
dine-2,6-dicarboxamides that featured in our study showed excel-
lent stabilization of the telomeric G-quadruplex combined with
high selectivity over double-stranded DNA, making them suit-
able chemical agents to target telomeres. The compounds show
some striking growth-inhibitory effects on cancer cell-lines after
few days of exposure, some of them exhibiting a complete arrest
after long-term exposure to the drug. The differential short and
long-term inhibitory properties of the different analogues high-
lights the fact that subtle structural and functional chemical vari-
ations of the ligands can be exploited to fine-tune the biological
activity of these analogues. Our data are in agreement with a
model where both telomeric dysfunction and the accumulation
of DNA damage sites at genomic loci enriched in G-quadruplex
structures trigger a reduced cell growth associated with sene-
scence induction. The selective targeting of G-quadruplexes by
N,N′-bis(quinolinyl)pyridine-2,6-dicarboxamides constitute
a
promising angle for the development of novel anticancer drugs
that could act by impairing cancer mechanisms such as telomere
maintenance.
Other G-quadruplex ligands have been shown to stabilize
telomeric quadruplexes in human cells and cause telomere
dysfunction and shortening leading to replicative senescence in
telomerase-positive47 and alternative lengthening of telomeres
(ALT)63,64 cells. Our investigation of the effect of some
analogues (9–10, 15, 17 and 33) on the telomeric G-overhang
revealed that these derivatives also trigger a telomere dysfunction
in HT1080 cells, in agreement with our previous finding
that pyridostatin competes for binding at telomeres with the
G-overhang binding protein POT1 in HT1080 cells.23 However,
the kinetics for long-term growth inhibition and G-overhang
degradation largely differ between these compounds and suggest
additional cellular mechanisms. Interestingly, all these deriva-
tives induced senescence during long-term treatments (Fig. S4†)
but did not induce an elevated level of cell death, suggesting a
mechanism of action different from agents that cause a massive
and global DNA damage response throughout the genome. This
is in agreement with the targeting properties of N,N′-bis(quino-
linyl)pyridine-2,6-dicarboxamides towards G-quadruplexes.
Evidence indicates that telomere dysfunction is one of the causes
underpinning cellular senescence.65 Therefore, the protracted
accumulation of DNA damage at telomeres using low concen-
trations of chemotherapeutic agents to induce accelerated senes-
cence is an increasingly known and investigated mechanism to
inhibit tumor cell growth renewal.66 Thus, long-term growth
arrests and senescence induced by these derivatives are note-
worthy results of a complex interplay between telomeric effects
and the accumulation of restricted DNA damage sites being gen-
erated throughout the genome via non-telomeric G-quadruplex
targeting.
Experimental
FRET-melting studies
100 μM stock solutions of oligonucleotides were prepared in
molecular biology grade DNase-free water. Further dilutions
were carried out in 60 mM potassium cacodylate buffer, pH 7.4.
FRET experiments were carried out with a 200 nM oligonucleo-
tide concentration. All labeled DNA oligonucleotides were sup-
plied by Eurogentec® Ltd and all unlabeled oligonucleotides
were supplied by IBA® GmbH. Seven dual fluorescently labeled
DNA oligonucleotides were used in these experiments: H-Telo
(5′-FAM-GGG TTA GGG TTA GGG TTA GGG-TAMRA-3′),
C-kit1 (5′-FAM-GGG AGG GCG CTG GGA GGA GGG-
TAMRA-3′), C-kit2 (5′-FAM-GGG CGG GCG CGA GGG
AGG GG-TAMRA-3′), C-myc (5′-FAM-TGA GGG TGG GTA
GGG TGG GTA A-TAMRA-3′), Bcl-2 (5′-FAM GGG CGC
GGG ACG AGG GGG GCG GG-TAMRA-3′), KRAS (5′-FAM
AGG GCG GTG TGG GAA GAG GGA AGA GGG GGA
GG-TAMRA-3′), ds-DNA (5′-FAM-TAT AGC TAT A-HEG-T
ATA GCT ATA-TAMRA-3′) which is a dual-labeled 20-mer
oligonucleotide comprising a self-complementary sequence with
a central polyethylene glycol linker able to fold into a hairpin.
The competitor was an unlabeled 26-mer oligonucleotide
(5′-CAA TCG GAT CGA ATT CGA TCC GAT TG-3′). The
donor fluorophore was 6-carboxyfluorescein (FAM) and the
acceptor fluorophore was 6-carboxytetramethylrhodamine
(TAMRA). The dual-labeled oligonucleotides were annealed at a
concentration of 400 nM by heating at 94 °C for 10 min fol-
lowed by slow cooling to rt at a controlled rate of 0.1 °C min−1
.
96-well plates were prepared by addition of 50 μl of the annealed
DNA solution to each well, followed by 50 μl solution of mo-
lecules 1, 8–33 at the appropriate concentration. For the compe-
tition experiments, competitor was added to the fluorescently
labeled quadruplex oligonucleotides sequences at an excess of
50 mol equiv. and annealed in the same solution. Measurements
were made in triplicate with an excitation wavelength of 483 nm
and a detection wavelength of 533 nm. Final analysis of the data
Conclusions
Small molecules with the ability to target telomeres are highly
desirable for the treatment of malignant neoplasia, since one of
the hallmark of cancer has been defined by the infinite proli-
ferative capacity of cells permitted by mechanisms that sustain
telomere length in addition to the ectopic expression of
This journal is © The Royal Society of Chemistry 2012
Org. Biomol. Chem., 2012, 10, 6537–6546 | 6543

was carried out using OriginPro 7.5 data analysis and graphing
software (OriginLab®).
with 2% formaldehyde and 0.2% glutaraldehyde in 1 × PBS for
15 min. This solution was then removed and the wells washed
twice with 1 × PBS. Each well was incubated with 800 μl stain-
ing solution (40 mM citric acid/sodium phosphate (pH 6.0),
0.15 M NaCl, 2 mM MgCl2, 5 mM potassium ferrocyanide,
5 mM potassium ferricyanide, 1 mg ml−1 X-gal, 5% dimethyl-
formamide (DMF)) at 37 °C for 16 h. The staining solution was
removed and the cells stored in 70% glycerol.
Cell culture
HeLa, HT1080, U2OS and WI-38 cells were cultured in T-75
flasks in Dulbecco’s Modified Eagle’s Medium (DMEM)
supplemented with 10% fetal calf serum and split at 70–80%
confluency using trypsin EDTA. The HT1080, WI-38 and U2OS
cells were obtained from the European Collection of Cell
Cultures (ECACC) and the HeLa cells were a generous gift of
Prof. Ashok Venkitaraman/Cambridge.
Non-denaturing hybridization assay
We performed a non-denaturing hybridization assay to detect the
3′ G-overhang. 5 μg of undigested genomic DNA, extracted
from cells treated with 9, 10, 15, 17, 33 or no compound at the
respective time points, were hybridized for 16 h at 50 °C with
0.5 pmol of [γ-32P] adenosine triphosphate (ATP)-labeled 21C
oligonucleotide (5′-(CCCTAA)3CCC-3′) in 30 μl hybridization
buffer (20 mM tris(hydroxymethyl)aminomethane (TRIS)·HCl
(pH 8.0), 0.5 mM EDTA, 50 mM NaCl, 10 mM MgCl2). Reac-
tions were stopped by the addition of 9 μl loading buffer (20%
glycerol, 1 mM ethylenediaminetetraacetate (EDTA), 0.2%
bromophenol blue) and size-fractioned on a 0.7% agarose gel for
3 h at 45 V. The gels were subsequently stained with ethidium
bromide (EtBr). The bottom of the gel was cut to remove
unbound probe and thus increase resolution. Gels were dried on
Whatman® filter paper at 60 °C and exposed overnight on an
autoradiography film. Ethidium bromide fluorescence and the
autoradiography film were scanned with a phosphorimager
(Typhoon™ 9210, Amersham Biosciences®). Results were
expressed as the relative hybridization signal normalized to the
fluorescent signal of EtBr.
Luminescent cell viability assay
IC50 values of growth inhibition were determined using the cell
viability assay CellTiter-Glo™ (Promega®). Cells were plated in
96 well plates at a density of 4000 cells per well in 100 μl of
media and incubated for 24 h. Compounds 1, 8–33 were added
in serial dilutions (in a range between 0–40 μM) at a volume of
100 μl per well at the respective concentrations. Cell viability
was measured after 72 h using the manufacturer’s protocol.
Measurements were taken after 20 min incubation at rt.
All measurements were made in triplicate. Final analysis of the
data was carried out using OriginPro 7.5 data analysis and graph-
ing software (OriginLab®).
Long-term growth assay and cytotoxicity
HT1080 cells were cultured in T-25 flasks as stated above and
seeded at 300 000 cells per flask initially and incubated with
1, 9–10, 15–17, 19–24, 26–27 and 33 at their respective IC50
values or no compound as a control. The cells were split after
3 or 4 days, counted with a hemicytometer and reseeded at their
initial density of 300 000 cells per flask. This procedure was
repeated every third day. All experiments were performed in
triplicate. Population doubling (PD) was calculated using the
following formula: PD = (ln(n1/n2)/ln2), n1 = initial number of
cells, n2 = final number of cells. Cytotoxicity was assessed at
each time point using a Trypan blue exclusion assay. Cells were
counted (as stated above) in a 1 : 1 mixture of media : Trypan
blue (0.4% in DMSO). Dead cells were assessed by blue nuclear
staining.
Alphabetical list of non-standard abbreviations
ALT
ATP
alternative lengthening of telomeres
adenosine triphosphate
ATRX
alpha thalassemia/mental retardation syndrome
X-linked
Boc
Di-tert-butyl dicarbonate
DIAD
DCM
diisopropyl azodicarboxylate
dichloromethane
DMEM Dulbecco’s modified Eagle medium
DMF N,N-dimethylformamide
ds-DNA double stranded DNA
EDTA
ESI
EtBr
FAM
FC
FRET
HPLC
IC50
MeOH
NaOH
NMR
PD
PBS
POT1
ethylenediaminetetraacetic acid
electrospray ionization
ethidium bromide
6-carboxyfluorescein
flash chromatography
Förster resonance energy transfer
high performance liquid chromatography
half maximal inhibitory concentration
methanol
sodium hydroxide
nuclear magnetic resonance
population doubling
β-Galactosidase assay
Senescence was assessed using a senescence β-galactosidase
staining kit (Cell Signalling Technology®) according to the
manufacturer’s protocol. HT1080 cells were cultured in T-75
flasks for 1 d and incubated with compounds 9, 10, 15, 17 and
33 at their respective IC50 values for 4 d. As a control, cells
were incubated without compound under the same conditions.
Cells were split and seeded at 4000 cells per well on a Cover
Well™ microscopy slide chamber (Invitrogen®) and incubated
with compounds 9, 10, 15, 17 and 33 at the aforementioned con-
centrations or in the absence of compound for another
4 d. Subsequently, the media was removed and the wells washed
twice with 1 × phosphate buffered saline (PBS). Cells were fixed
phosphate buffered saline
protection of telomeres 1
6544 | Org. Biomol. Chem., 2012, 10, 6537–6546
This journal is © The Royal Society of Chemistry 2012

27 H. Fernando, A. P. Reszka, J. Huppert, S. Ladame, S. Rankin,
A. R. Venkitaraman, S. Neidle and S. Balasubramanian, Biochemistry,
2006, 45, 7854.
28 S. Rankin, A. P. Reszka, J. Huppert, M. Zloh, G. N. Parkinson,
A. K. Todd, S. Ladame, S. Balasubramanian and S. Neidle, J. Am. Chem.
Soc., 2005, 127, 10584.
29 A. T. Phan, Y. S. Modi and D. J. Patel, J. Am. Chem. Soc., 2004, 126,
8710.
30 A. Siddiqui-Jain, C. Grand, D. J. Bearss and L. H. Hurley, Proc. Natl.
Acad. Sci. U. S. A., 2002, 99, 11593.
TAMRA 6-carboxytetramethylrhodamine
TERRA telomeric repeat containing RNA
TFA
THF
TEBP
TLC
Tm
trifluoroacetic acid
tetrahydrofuran
telomeric end-binding protein
thin layer chromatography
melting temperature
TRF1/2 telometic repeat factor 1/2
TRIS tris(hydroxymethyl)aminomethane
31 J. Dai, D. Chen, R. A. Jones, L. H. Hurley and D. Yang, Nucleic Acids
Res., 2006, 34, 5133.
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34 M. Bejugam, S. Sewitz, P. S. Shirude, R. Rodriguez, R. Shahid and
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Acknowledgements
We thank Cancer Research UK for programme funding and for a
studentship (S.M.), MUIR FIRB-Ideas RBID082ATK for pro-
gramme funding (M.D.A.), Ligue Nationale Contre le Cancer for
programme funding (J.F.R.), Helen Lightfoot for technical assist-
ance, and Chris Lowe for carefully proofreading this manuscript.
36 D. J. Patel, A. T. Phan and V. Kuryavyi, Nucleic Acids Res., 2007, 35,
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38 A. Bugaut, R. Rodriguez, S. Kumari, S.-T. Hsu and S. Balasubramanian,
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