Báo cáo khoa học: Human telomeric G-quadruplex: The current status of telomeric G-quadruplexes as therapeutic targets in human cancer pdf

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
Introduction
Human telomeres comprise tandem repeats of the
DNA motif (TTAGGG) together with associated telo-
meric proteins [1–3], as well as other more transiently
associated DNA-repair and damage-response proteins
such as Ku [4]. The terminal 150–250 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
[6–8]. 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 [9–11]. 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 clarified; 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  80–85% 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-
nificant telomerase expression appears to be a tumour
suppressor mechanism [3]. Telomeres in telomerase-
negative somatic cells are gradually shortened as a
Keywords
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:
(Received 25 June 2009, revised 5 October
2009, accepted 6 October 2009)
doi:10.1111/j.1742-4658.2009.07463.x
The 3¢-ends of human chromosomal DNA terminate in short single-
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.
1118 FEBS Journal 277 (2010) 1118–1125 ª 2009 The Author Journal compilation ª 2009 FEBS
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 enzyme’s catalytic activity [19] because
it ensures that the 3¢-end is inaccessible to hybridize
with the telomerase RNA template, the essential first
step in the catalytic cycle. The induction of quadruplex
stabilization and telomerase inhibition by a quadru-
plex-binding small molecule was first demonstrated

using a disubstituted anthraquinone derivative [20].
Many quadruplex-binding ligands have been reported
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 first such compound [25] to show both high quad-
ruplex affinity 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 [26–29].
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
Fig. 1. Structures of quadruplex-binding
ligands.
S. Neidle G-quadruplexes as cancer drug targets
FEBS Journal 277 (2010) 1118–1125 ª 2009 The Author Journal compilation ª 2009 FEBS 1119
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
 40–50 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 findings using G-quad-
ruplex ligands showed very different behaviour, with
senescence occurring within 7–10 days after cells were
first 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 significant 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, p16
INK4a
kinase and p53 pathways [32–35]
which can be visualized by the appearance of charac-
teristic DNA damage foci using an antibody to the
damage response protein cH2AX [36], or by a signifi-
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, chiefly 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
Fig. 2. Schematic of mechanism of action of the telomeric quadruplex ligand BRACO-19.
G-quadruplexes as cancer drug targets S. Neidle
1120 FEBS Journal 277 (2010) 1118–1125 ª 2009 The Author Journal compilation ª 2009 FEBS
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
fibroblast (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-affinity in vitro telomeric quad-
ruplex binding, with a K
a
value of at least 10

6
m
)1
; (b)
a low level of binding to duplex DNA, with a K
a
value
at least 10
2
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
insufficiently 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 confirmed by several crystallographic and
NMR studies of G-quadruplex–ligand complexes [42–
47]. There is no evidence from these studies of classic
intercalation between G-quartets and all analyses con-
cur in finding that ligands stack onto a terminal
G-quartet of a quadruplex. Substituents are normally
short acyclic chains, such as -(CH
2
)
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 [42–47], although these also indicate that the
flexibility of the TTA loops is ligand dependent, and
therefore structural information for a given class
of ligand would be highly desirable. Also, there are
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 affinity (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 clarified, 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 significant 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 nonspecific 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 modifications 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 findings
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
S. Neidle G-quadruplexes as cancer drug targets
FEBS Journal 277 (2010) 1118–1125 ª 2009 The Author Journal compilation ª 2009 FEBS 1121
appears able to penetrate significant 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 affinity 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 fluoroquino-
lone derivative Quarfloxin (CX-3543) is included. It is
currently in clinical trials so its pharmacological profile
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 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.
G4 ligand Xenograft model
Mean initial
tumour size
Dosage
(mgÆkg
)1
) Tumour response
Days to
complete
response Ref
TMPyP4 MX-1 mammary tumor 100 mg
a

10, 20; i.p. Survival increase from
45% to 75%
60 57
TMPyP4 PC-3 human prostate
carcinoma
60 mg 40; i.p. 60% tumour shrinkage 18 57
Telomestatin U937 human lymphoma 1395 mm
3
15 80% tumour shrinkage 21 41
BRACO-19 UXF1138L human uterine
carcinoma
68 mm
3
2; i.p. 96% tumour shrinkage
+ some complete
remissions
28 32
BRACO-19 A431 human epithelial
carcinoma
1080 mm
3
2; i.p. Not significant – 54
Quarfloxin MDA-MB-231 human
breast cancer
> 125 mm
3
6.25, 15.5; i.v. 50% tumour shrinkage 37 58
Quarfloxin MIA PaCa-2 human
pancreatic cancer
> 125 mm

3
5; i.v. 59% tumour shrinkage 35 58
RHPS4 UXF1138L human uterine
carcinoma
5 · 5 mm 5; oral 30% tumour shrinkage 28 33
RHPS4 M14, LP, LM melanoma 300–350 mg 10; i.p. 40–51% tumour weight
reduction
15 56
RHPS4
b
CG5 breast carcinoma 300 mg 15; i.v. 75% tumour shrinkage 30 35
a
Animals were initially treated with cyclophosphamide to minimize tumour burden.
b
RHPS4 was reported to have an antitumour effect in a
number of other tumour types in this study.
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.
G4 ligand Xenograft model
Initial tumour
size
Dosage
(mgÆkg
)1
) Drug 2 Tumour response Ref
BRACO-19 A431 epidermal carcinoma 1080 mm
3
2 Paclitaxel 68% tumour shrinkage 54
AS1410 A549 lung carcinoma 10 mm
3

1 Cis-platinum  75% tumour shrinkage 59
RHPS4 UXF1138L human uterine
carcinoma
5 · 5 mm 5 Taxol Complete remissions 33
RHPS4
a
HCT116, HT29 colorectal
carcinomas
300–350 mg 10 Irinotecan 80% tumour weight reduction 56
a
A number of other combinations, with a range of anticancer drugs, were also reported in this study.
G-quadruplexes as cancer drug targets S. Neidle
1122 FEBS Journal 277 (2010) 1118–1125 ª 2009 The Author Journal compilation ª 2009 FEBS
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
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-
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].
Acknowledgement
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.
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