• acridine;
  • anticancer;
  • drug;
  • drug-like;
  • in vivo;
  • medicinal chemistry;
  • pharmacology;
  • quadruplex;
  • telomerase;
  • telomere


  1. Top of page
  2. Abstract
  3. Introduction
  4. Telomeric quadruplex ligands – possible mechanisms of action
  5. G-quadruplex ligands as drugs
  6. Acknowledgement
  7. References

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.


  1. Top of page
  2. Abstract
  3. Introduction
  4. Telomeric quadruplex ligands – possible mechanisms of action
  5. G-quadruplex ligands as drugs
  6. Acknowledgement
  7. References

Human telomeres comprise tandem repeats of the DNA motif (TTAGGG) together with associated telomeric 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 quadruplexes 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 telomeric 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 significant telomerase expression appears to be a tumour suppressor mechanism [3]. Telomeres in telomerase-negative somatic cells are gradually shortened as a 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 telomerase (hTERT in humans) has reverse transcriptase enzymatic activity and synthesizes TTAGGG repeats on to the end of the 3′ single-stranded overhang. Inhibition of hTERT by siRNA, antisense or small-molecule inhibitors selectively inhibits cancer cell growth and strongly suggests that induction of telomere shortening is a viable therapeutic strategy [18].

Folding the single-stranded telomeric DNA substrate of telomerase into a four-stranded quadruplex structure 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 quadruplex-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 reliable in vitro telomerase assays [23,24]. The majority of G-quadruplex ligands contain a polycyclic heteroaromatic 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 quadruplex affinity and telomerase inhibitory potency. More recent reports have demonstrated that nonconjugated compounds that are synthetically more accessible than telomestatin can have potency against telomerase and quadruplex selectivity [26–29].


Figure 1.  Structures of quadruplex-binding ligands.

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Telomeric quadruplex ligands – possible mechanisms of action

  1. Top of page
  2. Abstract
  3. Introduction
  4. Telomeric quadruplex ligands – possible mechanisms of action
  5. G-quadruplex ligands as drugs
  6. Acknowledgement
  7. References

The classic model of telomerase inhibition and consequent telomere attrition leading to senescence and apoptosis requires that cells with a mean telomere length of 5 kb, a 24 h cell-doubling time and a subsequent loss of ∼ 100 nucleotides per round of replication would reach critical telomere shortening in ∼ 40–50 days [30,31]. This was indeed the observation in dominant-negative telomerase transfection experiments, but would be therapeutically challenging for human cancer treatment. Initial findings using G-quadruplex ligands showed very different behaviour, with senescence occurring within 7–10 days after cells were first treated, and little evidence of concomitant telomere 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 characteristic DNA damage foci using an antibody to the damage response protein γH2AX [36], or by a significant 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 protein 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 followed 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 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 telomere replication is unaffected in mouse embryonic fibroblast (i.e. untransformed) cell lines [40].


Figure 2.  Schematic of mechanism of action of the telomeric quadruplex ligand BRACO-19.

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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 quadruplex 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

  1. Top of page
  2. Abstract
  3. Introduction
  4. Telomeric quadruplex ligands – possible mechanisms of action
  5. G-quadruplex ligands as drugs
  6. Acknowledgement
  7. References

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 progressed 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 polycyclic 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 concur 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 diethylamine, 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 effective 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 features 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 penetration, (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 hydrophobic 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-availability (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.

Table 1.   Selected in vivo data on quadruplex-binding ligands. Tumour responses have been estimated from survival curves and other available data. Single-agent studies. i.p., intraperitoneal; i.v., intravenous.
G4 ligandXenograft modelMean initial tumour size Dosage (mg·kg−1)Tumour responseDays to complete responseRef
  1. 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.

TMPyP4MX-1 mammary tumor100 mga10, 20; i.p.Survival increase from 45% to 75%6057
TMPyP4PC-3 human prostate carcinoma60 mg40; i.p.60% tumour shrinkage1857
TelomestatinU937 human lymphoma1395 mm31580% tumour shrinkage2141
BRACO-19UXF1138L human uterine carcinoma68 mm3 2; i.p.96% tumour shrinkage + some complete remissions2832
BRACO-19A431 human epithelial carcinoma1080 mm3 2; i.p.Not significant54
QuarfloxinMDA-MB-231 human breast cancer> 125 mm3 6.25, 15.5; i.v.50% tumour shrinkage3758
QuarfloxinMIA PaCa-2 human pancreatic cancer> 125 mm3 5; i.v.59% tumour shrinkage3558
RHPS4UXF1138L human uterine carcinoma5 × 5 mm 5; oral30% tumour shrinkage2833
RHPS4M14, LP, LM melanoma300–350 mg10; i.p.40–51% tumour weight reduction1556
RHPS4bCG5 breast carcinoma300 mg15; i.v.75% tumour shrinkage3035

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 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 molecule.

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 activity in MX-1 mammary tumours and PC-3 human prostate 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 fluoroquinolone 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 promoter 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 derivatives [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 combination 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 oncogene that is disregulated in gastrointestinal cancer cells (inhibiting c-kit expression), and telomeric quadruplexes. The inhibition of c-kit expression and telomerase activity take place at the ligand concentrations required to halt cell growth and proliferation [60].

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 ligandXenograft modelInitial tumour sizeDosage (mg·kg−1)Drug 2Tumour responseRef
  1. a A number of other combinations, with a range of anticancer drugs, were also reported in this study.

BRACO-19A431 epidermal carcinoma1080 mm32Paclitaxel68% tumour shrinkage54
AS1410A549 lung carcinoma10 mm31Cis-platinum∼ 75% tumour shrinkage59
RHPS4UXF1138L human uterine carcinoma5 × 5 mm5TaxolComplete remissions33
RHPS4aHCT116, HT29 colorectal carcinomas300–350 mg10Irinotecan80% tumour weight reduction56


  1. Top of page
  2. Abstract
  3. Introduction
  4. Telomeric quadruplex ligands – possible mechanisms of action
  5. G-quadruplex ligands as drugs
  6. Acknowledgement
  7. References

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.


  1. Top of page
  2. Abstract
  3. Introduction
  4. Telomeric quadruplex ligands – possible mechanisms of action
  5. G-quadruplex ligands as drugs
  6. Acknowledgement
  7. References
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