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-s
Trang 1Human 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: stephen.neidle@pharmacy.ac.uk
(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
Trang 2consequence 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.
Trang 3length 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, p16INK4a 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.
Trang 4telomerase 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 Ka value of at least 106m)1; (b)
a low level of binding to duplex DNA, with a Kavalue
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
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 -(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 [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
Trang 5appears 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%
TMPyP4 PC-3 human prostate
carcinoma
BRACO-19 UXF1138L human uterine
carcinoma
+ some complete remissions
BRACO-19 A431 human epithelial
carcinoma
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
RHPS4 UXF1138L human uterine
carcinoma
reduction
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
RHPS4 UXF1138L human uterine
carcinoma
RHPS4a 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.
Trang 6tives [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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