MINIREVIEW

Human telomeric G-quadruplex: The current status

of telomeric G-quadruplexes as therapeutic targets
in human cancer
Stephen Neidle
Cancer Research UK Biomolecular Structure Group, University of London, UK

Keywords The 3-ends of human chromosomal DNA terminate in short single-

acridine; anticancer; drug; drug-like; in vivo;
medicinal chemistry; pharmacology;
quadruplex; telomerase; telomere
Correspondence
Stephen Neidle, Cancer Research UK
Biomolecular Structure Group, The School
of Pharmacy, University of London,
29-39 Brunswick Square, London
WC1N 1AX, UK
Fax: +44 207 753 5970
Tel: +44 207 753 5969
E-mail: stephen.neidle@pharmacy.ac.uk
stranded guanine-rich tandem-repeat sequences. In cancer cells, these are
associated with the telomere-maintenance enzyme telomerase together with
the end-binding protein hPOT1. Small molecules that can compete with
these proteins and induce the single-stranded DNA to form quadruplex
ligand complexes are, in effect, able to expose these 3-ends, which results
in the activation of a DNA damage response and selective inhibition of cell
growth. Several of these G-quadruplex binding molecules have shown
promising anticancer activity in tumour xenograft models, which indicate
that the approach may be applicable to the treatment of a wide range of
human cancers. This minireview summarizes the available data on these
compounds and the challenges posed for drug discovery.

(Received 25 June 2009, revised 5 October

2009, accepted 6 October 2009)

doi:10.1111/j.1742-4658.2009.07463.x

Introduction
Human telomeres comprise tandem repeats of the
DNA motif (TTAGGG) together with associated telo-
meric proteins [13], as well as other more transiently
associated DNA-repair and damage-response proteins
such as Ku [4]. The terminal 150250 nucleotides at
the extreme 3-ends of telomeres are single-stranded
[5], but are protected from higher order aggregation by
binding to multiple repeats of a single-stranded DNA
binding protein (hPOT1 in humans), which in turn
interacts with other proteins in the core telomere
complex, notably TPP1, to regulate telomerase action
in cancer cells, and thereby maintain telomere length
[68]. Loss of hPOT1 deprotects telomeres and initiates
DNA damage-response mediated cell death. Small
molecules that stabilize the single strand into higher
order (G-quadruplex) structures compete with hPOT1
and also initiate this response [911]. Thus, quadruplex
formation at the single-strand overhang may itself be a
DNA damage signal, producing responses analogous
to those of other mediators of telomere damage [12].
The biological function of induced telomeric quadru-
plexes remains to be fully claried; an end-protective
role has been suggested, there is evidence of functional
interactions involving poly(ADP-ribose) polymerase-1
[13] and in ciliates at least, quadruplex structures are
involved in telomerase recruitment [14,15]. However,
to date, there is no direct evidence of a role for telo-
meric G-quadruplexes in the functioning of telomeres
in normal human cells.
Telomerase is overexpressed in  8085% of cancer
cells and primary tumours [16,17] and maintains
telomere length homeostatis (acting as a tumour
promoter). Telomere shortening in the absence of sig-
nicant telomerase expression appears to be a tumour
suppressor mechanism [3]. Telomeres in telomerase-
negative somatic cells are gradually shortened as a

1118 FEBS Journal 277 (2010) 11181125 2009 The Author Journal compilation 2009 FEBS
S. Neidle
consequence of the end-replication effect, and once
telomeric DNA is at a critically short length, cells
enter p53 and Rb-dependent replicative senescence,
and ultimately apoptosis. The catalytic subunit of telo-
merase (hTERT in humans) has reverse transcriptase
enzymatic activity and synthesizes TTAGGG repeats
on to the end of the 3 single-stranded overhang. Inhi-
bition of hTERT by siRNA, antisense or small-mole-
cule inhibitors selectively inhibits cancer cell growth
and strongly suggests that induction of telomere short-
ening is a viable therapeutic strategy [18].
Folding the single-stranded telomeric DNA substrate
of telomerase into a four-stranded quadruplex struc-
ture inhibits the enzymes catalytic activity [19] because
it ensures that the 3-end is inaccessible to hybridize
with the telomerase RNA template, the essential rst
step in the catalytic cycle. The induction of quadruplex
stabilization and telomerase inhibition by a quadru-
plex-binding small molecule was rst demonstrated
using a disubstituted anthraquinone derivative [20].
Many quadruplex-binding ligands have been reported
Fig. 1. Structures of quadruplex-binding
ligands.
FEBS Journal 277 (2010) 11181125 2009 The Author Journal compilation 2009 FEBS
G-quadruplexes as cancer drug targets
subsequently [18,21,22], although relatively few have
been evaluated in cell-based assays, or even with reli-
able in vitro telomerase assays [23,24]. The majority of
G-quadruplex ligands contain a polycyclic heteroaro-
matic core, although it is clear that this is not an
essential requirement for quadruplex binding. Several
effective quadruplex-binding ligands do not have this
feature. The cyclic polyamine telomestatin (Fig. 1) was
the rst such compound [25] to show both high quad-
ruplex afnity and telomerase inhibitory potency.
More recent reports have demonstrated that nonconju-
gated compounds that are synthetically more accessible
than telomestatin can have potency against telomerase
and quadruplex selectivity [2629].
Telomeric quadruplex ligands possible
mechanisms of action
The classic model of telomerase inhibition and conse-
quent telomere attrition leading to senescence and
apoptosis requires that cells with a mean telomere
1119
G-quadruplexes as cancer drug targets
length of 5 kb, a 24 h cell-doubling time and a sub-
sequent loss of  100 nucleotides per round of repli-
cation would reach critical telomere shortening in
 4050 days [30,31]. This was indeed the observation
in dominant-negative telomerase transfection experi-
ments, but would be therapeutically challenging for
human cancer treatment. Initial ndings using G-quad-
ruplex ligands showed very different behaviour, with
senescence occurring within 710 days after cells were
rst treated, and little evidence of concomitant telo-
mere shortening [11,18,32]. This has subsequently been
shown to be characteristic of the G-quadruplex ligand
class as a whole, and the observations of on-target
in vivo activity within clinically useful timescales are
encouraging signs that signicant single-agent clinical
utility may be eventually achievable with appropriate
compounds.
The quadruplex-binding acridine ligands BRACO-19
and RHPS4 (Fig. 1), in common with telomestatin,
induce rapid replicative senescence in cancer cells and
activate the same DNA damage response that follows
DNA double-strand breaks. This involves in particular
ATM, p16INK4a kinase and p53 pathways [3235]
which can be visualized by the appearance of charac-
Fig. 2. Schematic of mechanism of action of the telomeric quadruplex ligand BRACO-19.
1120
S. Neidle
teristic DNA damage foci using an antibody to the
damage response protein cH2AX [36], or by a signi-
cant population of cells undergoing end-to-end fusions
in metaphase [37]. Such changes are analogous to
those produced when the telomeric protein TRF2 is
knocked out. This response is a consequence of the
displacement of bound proteins from the single-
stranded overhang, chiey hPOT1, as well as possible
uncapping of telomerase from the ends. There are
likely to be multiple mechanisms involved, some of
which at least have cross-talk between them (Fig. 2).
For example, hPOT1 interacts with the telomeric pro-
tein Tpp1 and facilitates telomere length regulation
by telomerase, and hPOT1 displacement disregulates
telomerase function [7,8]. Also, although the classic
telomerase inhibition model does not appear to be fol-
lowed by G-quadruplex-binding agents, cancer cells
generally have marked telomere length heterogeneity,
with some having extremely short (< 1 kb) telomeres.
It has been suggested that these cells are not only
sensitive to senescence, but also that their viability is
critical to the cell population overall [38,39], although
it is not clear to what extent telomere shortening,
initially considered to be an essential marker of
FEBS Journal 277 (2010) 11181125 2009 The Author Journal compilation 2009 FEBS
S. Neidle
telomerase inhibition, is relevant to the short-term
effects of telomeric G-quadruplex ligands. Q-FISH
studies have shown that telomestatin is localized at
telomeres during replication and importantly, that telo-
mere replication is unaffected in mouse embryonic
broblast (i.e. untransformed) cell lines [40].
Validation of a telomeric quadruplex mode of action
involves evidence from a number of assays. The most
important are: (a) high-afnity in vitro telomeric quad-
ruplex binding, with a Ka value of at least 106 m)1; (b)
a low level of binding to duplex DNA, with a Ka value
at least 102 less than for telomeric quadruplexes; (c)
selective inhibition of cell growth, with normal human
cell lines being relatively unaffected; (d) senescence; (e)
inhibition of telomerase activity in cells; (f) competitive
inhibition of hPOT1 binding in cells; and (g) evidence
of telomere uncapping in cells from hTERT.
G-quadruplex ligands as drugs
In vivo activity in xenograft cancer models has been
reported to date for few telomeric quadruplex ligands,
notably the trisubstituted acridine compound BRACO-
19 [32], the polycyclic compound RHSP4 [34,35] and
telomestatin [41] (Fig. 1). The telomeric DNA single-
strand overhang is a target for all these compounds, as
judged by the observations of hPOT1 and hTERT
uncapping. To date, none of these molecules has pro-
gressed beyond the experimental stage into clinical
trial, probably in part because these compounds are
insufciently drug-like. Little data is publicly available
on their ADME pharmacokinetic properties.
To date, the development of small molecules as
G-quadruplex binders has been largely based on poly-
cyclic planar aromatic compounds with at least one
substituent terminating in a cationic group [20,21].
Normally two such substituents are required. The
rationale for the planar moiety has been that this
would stack effectively onto planar G-quartets, which
has been conrmed by several crystallographic and
NMR studies of G-quadruplexligand complexes [42
47]. There is no evidence from these studies of classic
intercalation between G-quartets and all analyses con-
cur in nding that ligands stack onto a terminal
G-quartet of a quadruplex. Substituents are normally
short acyclic chains, such as -(CH2)3- with a terminal
cationic nitrogen-containing group such as diethyl-
amine, pyrrolidine or piperidine. Structure-based drug
discovery does have these few structures as starting
points [4247], although these also indicate that the
exibility of the TTA loops is ligand dependent, and
therefore structural information for a given class
of ligand would be highly desirable. Also, there are
FEBS Journal 277 (2010) 11181125 2009 The Author Journal compilation 2009 FEBS
G-quadruplexes as cancer drug targets
no experimental structural data as yet on folded
telomeric DNA sequences containing eight or twelve
TTAGGG repeats (i.e. with two or three consecutive
quadruplexes), which may be more representative of
the totality of the single-stranded overhang, and
which may be important for these ligands being able
to differentiate telomeric quadruplexes from others in
the genome.
It has long been realized that therapeutically effec-
tive quadruplex-binding ligands should have minimal
duplex DNA afnity (and therefore more generalized
toxicity), and assays for duplex:quadruplex selectivity
are routinely performed in many laboratories. The
structural requirements for selectivity have not yet
been fully claried, but mostly involve those steric fea-
tures that are incompatible with the dimensions of a
double helix. A large number of genomic DNA and
RNA G-quadruplexes may also be drug targets [48
53], many of which are involved in proliferation. It is
plausible that G-quadruplex-binding molecules even
with relatively modest selectivity between various
G-quadruplexes, may still have utility in cancer
therapeutics, provided they have low toxicity to
normal cells. Of greater practical importance is that
future G-quadruplex ligands are developed with regard
to their ability to be used as drugs, so that they have:
(a) effective and selective tumour uptake and penetra-
tion, (b) acceptable pharmacokinetic characteristics
and metabolism, and (c) a signicant therapeutic
window.
The features common to most current quadruplex
ligands, of several cationic charges and large hydro-
phobic surface area, do aid cellular uptake (probably
by active transport mechanisms), but may also enable
a high background of nonspecic binding to cellular
components, and are not consistent with oral bio-avail-
ability (although this in itself may not be an important
goal). The three positive charges on the BRACO-19
molecule are probably a factor in the inability of this
compound to penetrate larger tumours in both the
UXF1138L and A431 xenograft models [32,54]
(Table 1). Compound AS1410 was devised [55] to have
increased hydrophobicity compared with its parent
compound BRACO-19 as a result of modications to
the substituents at the 9-position. This resulted in an
increase in plasma half-life from 1 to 2 h.
The limited in vivo data available (Table 1) suggest
that telomeric quadruplex ligands may be useful for
the treatment of solid tumours; to date there is very
little data on haematological cancers. Notable ndings
include that of single-agent activity for RHSP4 in a
metastatic melanoma model, as well as in a melanoma
line resistant to the platinum drug DDP [56]. RHPS4
1121
G-quadruplexes as cancer drug targets S. Neidle

Table 1. Selected in vivo data on quadruplex-binding ligands. Tumour responses have been estimated from survival curves and other avail-
able data. Single-agent studies. i.p., intraperitoneal; i.v., intravenous.

Days to
Mean initial Dosage complete
G4 ligand Xenograft model tumour size (mgkg)1) Tumour response response Ref

TMPyP4 MX-1 mammary tumor 100 mga 10, 20; i.p. Survival increase from 60 57
45% to 75%
TMPyP4 PC-3 human prostate 60 mg 40; i.p. 60% tumour shrinkage 18 57
carcinoma
Telomestatin U937 human lymphoma 1395 mm3 15 80% tumour shrinkage 21 41
BRACO-19 UXF1138L human uterine 68 mm3 2; i.p. 96% tumour shrinkage 28 32
carcinoma + some complete
remissions
BRACO-19 A431 human epithelial 1080 mm3 2; i.p. Not significant 54
carcinoma
Quarfloxin MDA-MB-231 human > 125 mm3 6.25, 15.5; i.v. 50% tumour shrinkage 37 58
breast cancer
Quarfloxin MIA PaCa-2 human > 125 mm3 5; i.v. 59% tumour shrinkage 35 58
pancreatic cancer
RHPS4 UXF1138L human uterine 5 5 mm 5; oral 30% tumour shrinkage 28 33
carcinoma
RHPS4 M14, LP, LM melanoma 300350 mg 10; i.p. 4051% tumour weight 15 56
reduction
RHPS4b CG5 breast carcinoma 300 mg 15; i.v. 75% tumour shrinkage 30 35
a b
Animals were initially treated with cyclophosphamide to minimize tumour burden. RHPS4 was reported to have an antitumour effect in a
number of other tumour types in this study.

appears able to penetrate signicant tumour masses
(Table 1), in accord with its single net positive charge
combined with the relatively small size of this mole-
cule.
Data on two other quadruplex-binding ligands have
also been included. The porphyrin compound
TMPyP4, which does bind with high afnity to a wide
range of quadruplex nucleic acids, albeit with low
selectivity, has been reported to show anticancer activ-
ity in MX-1 mammary tumours and PC-3 human pros-
tate carcinomas [57]. Although quadruplexes in the
promoter region of the c-myc oncogene have been
suggested as a target for this compound, it is also an
established telomerase inhibitor, so action at the
telomere level should not be ruled out. In vivo data on
the recently described quadruplex-binding uoroquino-
lone derivative Quaroxin (CX-3543) is included. It is
currently in clinical trials so its pharmacological prole
has relevance to other quadruplex ligands. This agent
was initially suggested to be targeting a c-myc pro-
moter quadruplex, but is now believed to function by
selectively disrupting nucleolin rDNA quadruplex
complexes [58]. It does not show the cellular behaviour
characteristic of a telomere targeting agent.
It is encouraging for future clinical applications that
several G-quadruplex ligands show in vivo synergistic
activity (Table 2) with conventional cytotoxic agents,
such as cis-platinum, taxol and camptothecin deriva-

Table 2. In vivo studies of quadruplex-binding ligands in combination with established anticancer drugs. Tumour responses have been
estimated from survival curves and other available data. Studies in combination with established anticancer drugs.

Initial tumour Dosage

G4 ligand Xenograft model size (mgkg)1) Drug 2 Tumour response Ref

BRACO-19 A431 epidermal carcinoma 1080 mm3 2 Paclitaxel 68% tumour shrinkage 54
AS1410 A549 lung carcinoma 10 mm3 1 Cis-platinum  75% tumour shrinkage 59
RHPS4 UXF1138L human uterine 5 5 mm 5 Taxol Complete remissions 33
carcinoma
RHPS4a HCT116, HT29 colorectal 300350 mg 10 Irinotecan 80% tumour weight reduction 56
carcinomas
a
A number of other combinations, with a range of anticancer drugs, were also reported in this study.

1122 FEBS Journal 277 (2010) 11181125 2009 The Author Journal compilation 2009 FEBS
S. Neidle
tives [33,54,56,59], although the detailed mechanism of
this effect remains to be established. The order in
which the drugs are administered appears to be an
important determinant of whether a particular combi-
nation is synergistic or antagonistic. It is also possible 10
that quadruplex-binding ligands can have multiple
quadruplex targets, which could confer therapeutic
advantage. Dual targeting has been reported for a
substituted naphthalene diimide, which interacts with
quadruplexes in the promoter region of the c-kit onco-
11
gene that is disregulated in gastrointestinal cancer cells
(inhibiting c-kit expression), and telomeric quadruplexes.
The inhibition of c-kit expression and telomerase activ-
ity take place at the ligand concentrations required to
halt cell growth and proliferation [60]. 12
Acknowledgement
13
I am grateful to Cancer Research UK for Programme
Grant support and a Professorial Fellowship, and to
my colleagues for their input to the work described in
the references. 14
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