MINIREVIEW
Retraction:
Human telomeric G-quadruplex: targeting with small
molecules
Amit Arora, Niti Kumar, Tani Agarwal and Souvik Maiti
Proteomics and Structural Biology Unit, Institute of Genomics and Integrative Biology, Delhi, India
Retraction: The following review from FEBS Journal, ‘Human telomeric G-quadruplex: targeting with small molecules’ by
Amit Arora, Niti Kumar, Tani Agarwal and Souvik Maiti, published online on 27
th
November 2009 in Wiley InterScience
(www.interscience.wiley.com), has been retracted by agreement between the authors, the journal Editor-in-Chief Professor
Richard Perham and Blackwell Publishing Ltd. The retraction has been agreed due to overlap between this review and the
following reviews: Published in Organic & Biomolecular Chemistry, ‘A hitchhiker’s guide to G-quadruplex ligands’ by David
Monchaud and Marie-Paule Teulade-Fichou. Volume 6 Issue 4, 2008, pages 627–636. Published in BioChimie, ‘Targeting tel-
omeres and telomerase’ by Anne De Cian, Laurent Lacroix, Ce
´
line Douarre, Nassima Temime-Smaali, Chantal Trentesaux,
Jean-Franc¸ ois Riou and Jean-Louis Mergny. Volume 90 Issue 1, 2008, pages 131–155.
Introduction
Aberrant cellular proliferation is associated with the
infinite extension of telomeric ends, mediated by unusu-
ally high telomerase activity or caused by abnormal
overexpression of the proto-oncogenes generally
required for cellular growth and differentiation [1–3].
As an anticancer strategy, efforts have been invested in
targeting and lowering telomerase activity, which is
often found to be overexpressed in cancerous cells [4,5].
However, the problem associated with telomerase tar-
geting is that cells can adopt telomerase-independent
mechanisms for telomere maintenance called alternative
lengthening of telomere [6]. This leads to skepticism

discusses the future outlook for designing more effective G-quadruplex
interacting ligands.
Abbreviations
Dppz, dipyridophenazine; NCQ, neomycin capped quinacridine.
FEBS Journal 277 (2010) 1345 ª 2009 The Authors Journal compilation ª 2009 FEBS 1345
mechanisms of telomere maintenance are targeted, by
impinging on the structure and function of telomeric
ends [7]. Telomeres can organize structurally into
different conformations, for example, the G-rich
single-stranded DNA overhang can adopt an unusual
four-stranded DNA quadruplex structure, and stabiliza-
tion of this structure by ligands would render the 3¢
overhangs unavailable for hybridization with the telo-
merase template for the extension of telomeric ends [7].
Further, ligand binding and stabilization of the quadru-
plex structure affect the recruitment of telomere-associ-
ated proteins required for the capping and maintenance
of telomeric ends. In addition to telomeric ends, the pro-
moter regions of proto-oncogenes also harbor G-quad-
ruplex motifs. It has been observed that targeting the
quadruplex motif in the promoter of proto-oncogenes
with quadruplex-interacting ligands decreases the tran-
scriptional activity of these proto-oncogene and helps to
combat aberrant proliferation. These observations are
encouraging many laboratories to synthesize quadru-
plex-interacting ligands. Given the rich diversity of
G-quadruplex scaffolds and their propensity to inter-
convert, it will be a challenge to identify small molecules
that exhibit recognition selectivity for diverse scaffolds
at the cellular level. There is a general notion that bind-

structurally related corroles [21], have also been
described. An important advance in the porphyrin series
came with the design of a diselenosapphyrin Se
2
SAP
N
N
N
N
N
N
NH
N
N
HN
NH
N
N
N
N
HN
N
N
N
N
N
N
TMPyP4
3,4-TMPyPz
N

FEBS Journal 277 (2010) 1345 ª 2009 The Authors Journal compilation ª 2009 FEBS
(Fig. 1), with an expanded porphyrin core [22,23].
Se
2
SAP showed 50-fold selectivity for quadruplex DNA
over duplex DNA and was able to discriminate among
the various forms of G-quadruplex DNA.
Ligands with protonable side arms
These ligands follow the presence of protonable side
arms (e.g. amine groups) around an aromatic core
which makes the molecule water soluble, with the
charge(s) far from the hydrophobic center. Bis-
amidoanthraquinone is one example which has been
shown to be a G-quadruplex ligand, telomerase inhibi-
tor [24] and possesses IC
50
values in the low lm range
[25,26]. To address selectivity problems, Neidle and
co-workers modified the core and side arms of the
initial ligands from anthraquinone to fluorenone [27],
then acridone [28] and acridine [29,30]. A crystal struc-
ture of a complex of BSU6039 (Fig. 2), a member of
the 3,6-disubstituted acridine series with G-quadruplex
was obtained [31]. Based on the concept of p-stacking
interactions and electrostatic interactions between the
quadruplex and the ligand, an optimized prototype
BRACO-19 (Fig. 2) was designed, which was able to
interact with the G-quadruplex structure [32,33].
BRACO-19 has also been shown to inhibit cancer cell
proliferation [34]. Modification of the 9-amino substi-

G-quadruplex stabilization and high telomerase inhibi-
tory activity [49]. An NMR structure for a complex of
MMQ1 (dipropylamino analogue of MMQ3) with tet-
ramolecular quadruplex is also available [50]. Another
compound BOQ1 (Fig. 2), a dimeric macrocyclic quin-
acridine, showed improved quadruplex stabilization,
better overall selectivity than the monomeric series and
efficient telomerase inhibitory activity [51–53]. The
crescent-shape particularity of quinacridine has also
been found in several other ligands (Fig. 2), such as
indoloquinolines [54–56], cryptolepine and its
analogues [57], quindolines [58–61] and triazacyclo-
pentaphenantrene [62].
Alkaloid-based ligands
Alkaloid-based ligands like berberine (Fig. 3) and its
synthetic derivative have been examined for G-quadru-
plex binding and their ability to inhibit telomerase.
Results show that these molecules have selectivity for
G-quadruplex compared with duplex DNA, and that
their aromatic moieties play a dominant role in quad-
ruplex binding. Our group has also investigated the
complete thermodynamic profile of the berberine–telo-
meric quadruplex interaction using spectroscopic,
calorimetric and molecular modeling studies [63]. Fur-
thermore, interaction of 9-substituted derivatives with
human telomeric DNA indicated that these
compounds can induce and stabilize the formation of
antiparallel telomeric G-quadruplex in the presence or
absence of metal cations [64]. Introduction of a side
chain with the proper length of methylene and a termi-

N
N
N
HN
Cryptolepine
N
HN
N
H
3
C
H
3
C
O
CH
3
y
HN
R
HN
O
Triazacyclopentaphenanthrene
X
HN
N
Quindoline, X= NH or O and
R= different groups
OO
R

H
N
N
N
N
HN
N
N
RHPS4 MMQ3
N
N
HN
NH
R
1
NH(CH
2
)
n
N(CH
3
)
2
NH
NH
NN
HN
HN
HN
N

degree of selectivity, although they appeared to be
moderate telomerase inhibitors. A compound named
neomycin-capped quinacridine (Fig. 4) was developed
in which neomycin and a quinacridine moiety were
conjugated to target the loop and G-quartet of the
quadruplex, respectively [69]. Neomycin-capped quin-
acridine showed preferential binding to loop-contain-
ing quadruplexes compared with nonloop-containing
quadruplexes, along with efficient quadruplex stabiliza-
tion and strong telomerase inhibitory activity, thus
fully validating the design. The presence of three
amino appendages on the same face of the tri-oxazole
macrocycles (Fig. 4) resulted in selective stabilization
of one form of quadruplex over another, as shown by
the preferential binding of tri-oxazole macrocycles to
c-kit quadruplex rather than the human telomeric one
[70]. Furthermore, isoalloxazines (Fig. 4) have also
been shown to bind to c-kit quadruplex with 14-fold
selectively over the telomeric quadruplex [71], thereby
opening up a possibility for the design of a second
generation of ligands capable of selectively altering the
expression of a given gene. Recently, copper(I)-cata-
lyzed ‘click’ chemistry was used to design a series of
diarylurea ligands (Fig. 4). These ligands demonstrated
a high degree of selective telomeric G-quadruplex sta-
bilization and were not cytotoxic in several cancer cell
lines [72]. Moreover, urea-based nonpolycyclic
aromatic ligands with alkylaminoanilino side chains as
G-quadruplex DNA interacting agents have been
developed (Fig. 4) [73]. Using spectroscopic experi-

and mutagenic properties of ethidium bromide,
researchers developed a novel and safer series of
G-quadruplex ligands, derived from triazine [77–79].
One of the member of the series known as 12459
(Fig. 5) displayed selective stabilization of G-quadru-
plex and also strongly inhibited telomerase activity.
Triazines were followed by a structurally related bis-
quinolinium series containing a pyridodicarboxamide
X
H
N
R
N
+
O
CI
-
N
N
O
H
N
(CH
2
)
n
R
N
O
(CH

N
H
N
9-peptide acridine
O
O
N
H
X
O
O
X
O
O
O
O
H
H
NH
HN
O
O
O
O
HN
NH
O
O
O
H

H
2
N
H
3
Furan based ligands
O
HO
O
NH
2
O
HO
H
2
N
OH
OH
O
NH
2
NH
2
O
O
HO
HN
OH
O
2

HN
O
O
NH
2
HN
NH
N
Tri-oxazole macrocycles
R
2
N
NCQ
N N
H
N
R
1
O
N
N
R
1
O
Trisubstituted isoalloxazines, R1 and
R2 represents different groups
H
N
H
N

( )
n
( )
n
R
R
( )
n
O
O
( )
n
Diarylurea based Ligands
NH
N
N
N
H
N
H
N
H
N
H
O
O
peptide
peptide
O O
3,6- bis acridine-peptide conju

has been extended with the synthesis of phenanthroline
analogues. Phenanthroline-DC (Fig. 5) showed a
perfect geometrical match with a G-quartet and was
found to be remarkably more selective than telomesta-
tin, thus confirming the great potential of bisquino-
linium compounds [84,85].
Metallo-organic complex as
G-quadruplex ligand
A class of metallo-organic complexes has emerged as
highly interesting molecules because of their easy syn-
thetic access and their promising G-quadruplex bind-
ing properties. This approach is based on the
hypothesis that the central metal core could be posi-
tioned over the cation channel of the quadruplex,
thereby optimizing stacking interactions between the
surrounding chelating agent and the accessible G-quar-
tet [30]. The presence of a cationic or highly polarized
nature is also a further advantage in promoting an
association with the negatively charged G-quadruplex
DNA. The first reported examples described the inser-
tion of a metal in the central cavity of TMPyP4 and
their use as Cu(II)– [86,87], Mn(III)– [88], etc. A spec-
tacular 10 000-fold selectivity for quadruplex over
duplex has been measured by SPR for the highly cat-
ionic Mn(III)–porphyrin complex [88]. Moreover,
Cu(II)– and Pt(II)–terpyridine complexes can also be
obtained in one-step or two-step processes and these
ligands possess high affinity and high selectivity for the
G-quadruplex [89]. Recently, a series of platinum(II)
complexes containing dipyridophenazine (dppz) and

N
NH
2
N
+
NN
N
+
NH
2
N
N
H
2
N
N
H
N
N
N
N
H
dnagiL95421desabenizairTsevitav
iredmuidihtE
N
N
H
HN
O
O

N
N
R
ON
N
O
N
NH
N
N
R
HN
O
O
O
N
O
N
O
O
N
O
O
R = CH(CH
3
)
2
R = CH
2
OH

are also summarised in Table 1.
Mode of action of telomeric
G-quadruplex binding ligands
The unlimited proliferative potential of cancer cells
depends on telomere maintenance, which in turn
makes telomeres and telomerase an attractive target
for cancer therapy [104]. Most telomere-targeted
antitumour strategies address the telomerase-depen-
dent mechanism of telomere maintenance. It is well
reported that formation of intramolecular G-quadru-
plexes by the telomeric G-rich strand inhibits telomerase
activity [105]. Therefore, ligand-induced stabilization
of intramolecular telomeric G-quadruplexes provides
an attractive strategy for the development of antican-
cer agents. Molecules that target telomeric DNA were
initially considered to be telomerase inhibitors
[24,106,107]. However, this strategy cannot be
considered feasible as a cancer-specific approach
because normal cells also have telomeres and bear
quadruplex potential. Nevertheless, it is possible that
telomeres from normal and cancer cells exhibit differ-
ences in structure or accessibility and that a telomere
ligand could exhibit selective toxicity. In this mini-
review, we also discuss the role of G-quadruplex
binding ligand on telomerase enzyme, as well as the
direct effect on telomeres, thus altering telomere
maintenance.
Proteins of the telomerase complex
More than 30 proteins have been proposed to be asso-
ciated with the telomerase enzyme complex (Table S1

Ethidium derivatives Triazine 12459, pyridodicarboxamide core containing 307A and 360A, tritiated 360A phenanthroline analogues
Metallo-organic complex Cu(II)–TMpyP4 complex,Mn(III)–TMPyP4 complex, Cu(II) and Pt(II)–terpyridine complexes,
PtII(dppz-COOH)(N-C)]CF
3
SO
3
Neutral ligands Telomestatin, hexaoxazole-containing macrocyclic HXDV and HXLV-AC
Quadruplex interacting molecules A. Arora et al.
FEBS Journal 277 (2010) 1345 ª 2009 The Authors Journal compilation ª 2009 FEBS
Proteins involved in the protection of telomere
extremities (shelterin/telosomes)
During the last decade, proteins that protect the telo-
mere extremities have been identified and ascertained
to make up a complex called the telosome [113] or
shelterin [114]. This complex is principally composed
of six proteins. Three of these bind directly to the telo-
meric repeats: TRF1, TRF2 and POT1. TRF1 and
TRF2 have Myb-type DNA-binding domains [115],
whereas POT1 has two oligonucleotide ⁄ oligosaccharide
binding domains and displays a strong preference for
the single-stranded telomeric sequence relative to dou-
ble-stranded DNA [116]. The three other proteins are
TIN2, which binds TRF1 and TRF2, TPP1, which
binds TIN2 and POT1, and Rap1, which binds TRF2
[117]. TRF2 is shown to be involved in strand invasion
and T-loop formation [118,119] and in combination
with telomerase deficiency, has been most strongly
implicated in carcinogenesis [120]. Overexpression of
TRF2 also reduces telomeric, but not genomic, single-
strand break repair [121]. Recently, it has been shown

inhibition and ⁄ or telomere dysfunction. Some of these
ligands were able to downregulate telomerase expres-
sion in treated cells [79,131,134,135].
Direct effects of G-quadruplex ligands on
telomeres: induction of telomere dysfunction
Earlier studies have demonstrated a short-term
response (apoptosis) induced by G-quadruplex ligands
that could not be explained solely by telomerase inhi-
bition [79,81,131]. After just 15 days of exposure, sub-
toxic concentrations of the G-quadruplex ligands
RHPS4 or BRACO-19 can trigger growth arrest in
tumor cells, before any detectable telomere shortening
occurs [134,135]. Modifications of hTERT or hTR can
interference with the telomere capping function, which
in turn leads to short-term and massive apoptosis.
Overexpression of either hTERT or a dominant nega-
tive of hTERT in a telomerase-positive cell line
evidently does not modify the antiproliferative effect of
the triazine derivative 12459 (formula shown in Fig. 5)
[77]. The observation that BRACO-19 causes chromo-
some end-to-end fusion marked by the appearance of
p16-associated senescence led to the idea that G-quad-
ruplex ligands act primarily to disrupt the telomere
structure [136]. Such telomeric dysfunction was also
observed in cell lines treated with other quadruplex
ligands and in cell lines resistant to a triazine deriva-
tive, as evidenced by the typical images of telophase
bridges [81,137,138]. These studies suggest that the
direct target of these ligands is the telomere rather
than telomerase.

ligands display are: (a) direct stacking with quartets,
(b) loop interactions or external stacking, and (c) inter-
actions between ligand substituents ⁄ side chains and the
phosphate backbone of quadruplexes. These properties
can be optimized for different quadruplexes, which dis-
play variations in loop length, composition and topo-
logies, so as to achieve discriminative quadruplex
targeting. A recent study addressed the issue of ligand
selectivity by examining the distinct loop geometry in a
bimolecular quadruplex of Oxytrichia [142]. However,
the limited structures available for quadruplex–ligand
complexes retard this exploratory strategy of drug
design for biologically relevant quadruplex structures.
To circumvent this limitation there is the need to
adopt an integrative approach involving molecular
dynamics and biophysical techniques to obtain rapid
and accurate screening of quadruplex-interacting
ligands. Virtual screening of chemical libraries by
molecular docking is one attractive approach adopted
to identify potential scaffolds. The hits obtained from
the in silico search can be validated further through
biophysical methods involving spectroscopic and calo-
rimetric measurements, giving a quantitative idea of
the thermodynamic stability of the complex. Quadru-
plex-forming sequences have an inherent ability to
adopt diverse structures, which are influenced by their
loop length and composition. Therefore, a systematic
study of quadruplex–ligand interaction involving quad-
ruplexes of varying loop length and composition is
required. Such an attempt has been made for telomeric,

modeling studies for best candidate ligands to under-
stand their therapeutic efficacy. Because this quadru-
plex–ligand field is booming, both chemists and
biologists in conjunction could provide new molecular
principles that may contribute to the emergence of
effective anticancer therapies.
Acknowledgement
Financial support for this work from the Department
of Science and Technology (Swarnajayanti project),
Government of India, New Delhi to SM is gratefully
acknowledged.
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