Retraction: Human telomeric G-quadruplex: targeting with small molecules

MINIREVIEW

Retraction:Human telomeric G-quadruplex: targeting with smallmoleculesAmit 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 27th 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(www.interscience.wiley.com), has been retracted by agreement between the authors, the journal Editor-in-Chief Professor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, Celine Douarre, Nassima Temime-Smaali, Chantal Trentesaux,

Jean-Francois 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

about the use of telomerase inhibitors as anticancer

agents because cells can quickly switch to alternative

mechanisms and hence become resistant to telomerase

inhibitors. Therefore, it becomes imperative that the

Keywords

alkaloids; anticancer agent; click chemistry;

ethidium derivative; G-quadruplex; human

telomere; metallo-organic complex;

N-methylated ligands; proto-oncogenes;

telomerase

Correspondence

S. Maiti, Proteomics and Structural Biology

Unit, Institute of Genomics and Integrative

Biology, CSIR, Mall Road, Delhi 110 007,

India

Fax: +91 11 2766 7471

Tel: +91 11 2766 6156

E-mail: [email protected]

(Received 25 June 2009, revised 1

September 2009, accepted 28 September

2009)

doi:10.1111/j.1742-4658.2009.07461.x

Over the past few decades, numerous small molecules have been designed

to specifically and selectively target the unusual secondary structure in

DNA called the G-quadruplex. Because these ligands have been shown to

selectively inhibit the growth of cancer cells, they have become a central

focus for the development of novel anticancer agents. However, there are

many challenges which demand greater effort in order to devise strategies

for rational drug design with utmost selectivity. This minireview aims to

reflect recent developments in the design of G-quadruplex ligands and also

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-

ers that stabilize the G-quadruplex structure may pave

the way for the discovery of novel anticancer agents. A

key feature of appropriate molecules are large flat

aromatic systems involved in p-stacking with the G-tet-

rad platform and possess reasonable water solubility,

i.e. the molecule should display both hydrophobic and

hydrophilic characteristics. Various scaffolds have been

synthesized to date, and both computational and chemi-

cal–biological approaches have been used to understand

quadruplex–ligand interaction.

N-methylated ligands

N-methylated ligands, quaternized on the aromatic ring

nitrogens, have been thoroughly exploited because of

their low electron density, which in turn leads to

increased p-stacking of the aromatic part as well as

reasonable water solubility without the need for cationic

side chains. TMPyP4 is the pivotal example of this

family of ligands (Fig. 1). This tetracationic porphyrin

has been shown to have high affinity for G-quadruplex,

to efficiently inhibit telomerase and is also known to

downregulate the expression of oncogenes such as c-myc

or k-ras, along with its potency to convert antiparallel

topologies to parallel forms of quadruplexes [8–15].

Despite the nonselectivity of TMPyP4 for the quadru-

plex structure [16–18], interest in this particular mole-

cule has never declined. TQMP68 and 3,4-TMPyPz

(Fig. 1) [19] are two examples of porphyrin-based tetra-

cationic macrocycles, which have been shown to bind

efficiently to quadruplex DNA. TMPyP4-related ligands

carrying 1–3 N-methylpyridinium arms [20], as well as

structurally related corroles [21], have also been

described. An important advance in the porphyrin series

came with the design of a diselenosapphyrin Se2SAP

N

N

NN N

N

NH

N

N

HN

NH N

NN

N

HN

N

NN

N

NN

TMPyP4 3,4-TMPyPz

N HN NNHO OH

Se

N

SeN

NH HN

NNN

N N

N N

OHHO

TQMPSe2SAP

TQMP

Fig. 1. Chemical structures of N-methylated

ligands: TMPyP4, 3,4-TMPyPz, TQMP,

Se2SAP.

Quadruplex interacting molecules A. Arora et al.

FEBS Journal 277 (2010) 1345 ª 2009 The Authors Journal compilation ª 2009 FEBS

(Fig. 1), with an expanded porphyrin core [22,23].

Se2SAP 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 IC50 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-stackinginteractions 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-

tuent of BRACO-19 from an aniline to a difluoroben-

zylamine group was also carried out to circumvent the

problem of cellular uptake of BRACO-19 [35]. There

are reports of interaction studies between G-quadru-

plex DNA and the perylene diimide PIPER (Fig. 2)

[36]. This molecule consists of a broader hydrophobic

core, with two external amine appendages. This family

of compounds displayed moderate telomerase inhibi-

tion activity but showed 42-fold selectivity for quadru-

plex DNA over duplex DNA at pH 7.0 and pH 8.5

[37–39]. Biological experiments showed the cellular

uptake of such ligands, thereby suggesting them as

potential G-quadruplex binders [40,41]. Well-known

duplex-binding ligands like daunomycin [42], dista-

mycin and netropsin [43] have also been tested as

G-quadruplex-binding ligands. Several flavonoid

[44,45] or steroid derivatives [46] have also been shown

to bind quadruplexes with variable efficiency.

Another class of ligands known as pentacyclic acri-

dines and quinacridines (which possess a crescent

shape) likely to maximize overlap with the guanines of

the accessible G-quartet, has been developed. RHPS4

(Fig. 2) is one example of a pentacyclic acridine-based

G-quadruplex interacting ligand. A number of in vitro

and in vivo reports have shown that RHPS4 is a poten-

tial molecule for cancer therapy because it targets

G-quadruplex structures at the telomeric ends [47].

This molecule has recently also been used in preclinical

trials for solid tumors [48]. MMQ3 (Fig. 2), a com-

pound of the quinacridine family, showed remarkable

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-

nal amino group at the 9-position of berberine resulted

in increased binding with G-quadruplex, thus leading

to higher inhibitory effects on the amplification of

21-mer telomeric DNA and on telomerase activity.

Recently, the interaction of 9-N-substituted berberine

derivatives (Fig. 3) with c-myc quadruplex has also

been studied and the results indicated that these deriv-

atives may selectively induce and stabilize the forma-

tion of intramolecular parallel G-quadruplex in c-myc,

thus leading to downregulation of the transcription of

c-myc in the HL60 lymphomas cell line [65]. In addi-

tion to berberine, other alkaloids such as palmatine

and sanguinarine also demonstrate moderate quadru-

plex-binding abilities; however, the introduction of

protonable functional groups might further enhance

their recognition and stabilization abilities. Isoindigo-

tone (Fig. 3), a naturally occurring alkaloid with a

A. Arora et al. Quadruplex interacting molecules


unique asymmetric chromophore comprising an ali-

phatic five-member ring in the middle core has been

shown to serve as a new scaffold for unfused aromatic

quadruplex ligands. The introduction of at least two

cationic side chains to the chromophore resulted in

enhanced selectivity and solubility [66]. Interestingly,

in a pharmacophore-based virtual screening, two non-

planar alkaloid-based G-quadruplex ligands were

N O2N

N

N

HN

CryptolepineN

HN

NH3C

H3C

O

CH3y

HNR

HNO

Triazacyclopentaphenanthrene

X

HN

N

Quindoline, X= NH or O and R= different groups

O O

R

N NH

N NH

N

O O

H H

BRACO 19

N NN

O O

N NN

OO

PIPER

R = H, BSU6039

R = NH-p-C6H4-N(CH3)2,

N

F N

N HN

N

N

N

HN

N

N

RHPS4 MMQ3

NNHNNH

R1

NH(CH2)nN(CH3)2

NH

NHN N

HN

HN HNN

R2

N N

BOQ1 Benzoindoloquinoline

Fig. 2. Chemical structures of ligands with

protonable side arms: BSU6039, BRACO-19,

PIPER, RHPS4, MMQ3, BOQ1,

benzoindoloquinoline, cryptolepine,

triazacyclopentaphenanthrene, quindoline.



found. These two ligands exhibit good capability for

G-quadruplex stabilization and prefer binding to para-

lleled G-quadruplex rather than to duplex DNA. These

results have shown that planar structures are not

essential for G-quadruplex stabilizers, which may rep-

resent a new class of G-quadruplex-targeted agents as

potential antitumor drugs [67].

Click chemistry-based ligands

Click chemistry is a chemical approach which synthe-

sizes new drug-like molecules by joining readily avail-

able smaller units together using simple chemical

reactions aided by catalysts to reduce large enthalpy

hurdles. Neidle and co-workers [68] introduced the

concept of click chemistry in designing G-quadruplex

binding ligands. The resulting bistriazole derivatives

showed good quadruplex stabilization with a high

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-

ments, it was demonstrated that they have significant

selectivity over duplex DNA, and also for particular

G-quadruplexes. Preliminary biological studies using

short-term cell growth inhibition assays showed that

some of the ligands have cancer cell selectivity,

although they appear to have low potency for intracel-

lular telomeric G-quadruplex structures, suggesting

that their cellular targets may be other, possibly

oncogene-related, quadruplexes. Balasubramanian and

co-workers [74] developed a series of trisubstituted acri-

dine–peptide conjugates (Fig. 4) and explored the abil-

ity of these ligands to recognize and discriminate

between different quadruplexes derived from the human

telomere, and c-kit and N-ras proto-oncogenes. Our

group reported the binding properties of 18- and

24-membered cyclic oligopeptides (Fig. 4) developed

from a novel furan amino acid, 5-(aminomethyl)-2-furan-

carboxylic acid, to G-quadruplex. Comparative analysis

of the binding data of these ligands with G-quadruplex

and double-strand DNA shows that 24-membered cyclic

peptides are highly selective for telomeric G-quadruplex

structures and thus can be used as a scaffold to

target quadruplex structures at the genomic level [75].

Ethidium derivatives as G-quadruplexligands

Mergny and co-workers [76] reported the use of ethidi-

um derivatives as G-quadruplex ligands (Fig. 5). The

results showed G-quadruplex stability and telomerase

inhibition activity as well as quadruplex over duplex

selectivity. However, because of the well-known toxic

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 HN

R

N+

OCI-

N

NOHN

(CH2)n

R

N

O

(CH2)n

9-N substituted Berberine derivatives, Rsubstituted Berberine derivatives, Rrepresents different groups

OO

NO

OHN

OH

OIsaindigotone

Berberine

Fig. 3. Chemical structures of alkaloid-based G-quadruplex ligands:

berberine, 9-N-substituted derivatives and isaindigotone.



peptide

O

O O

NH

N NH

NNH

N

9-peptide acridine

OONH

XO

O

X

OO

O O

H

H

NH HN

O

O

O

O

HNNHO

O OHN

O1: X = NH3

+

2: X = NHCOCH CH NH +

O O

HN

O2: X = NHCOCH2CH2NH3

Furan based ligands

O

HO

O

NH2

OHOH2N

OHOHO NH2

NH2O

OHO

HNOH

O

2

HNO

O

HO

NH

ONO

NH

NH HN

NO

O

2

NH2 NH2

NH2

NH

NH2NH

O

NHN O

O NH2

HNNH

N

Tri-oxazole macrocycles

R2

N

NCQ N NHN

R1

O

NN R1

OTrisubstituted isoalloxazines, R1 and R2 represents different groups

HN

HN

ONH

NH

O

HN NH

O

O ON N

N N

N NO

O

HN NHO O

NH

R NH

RO

( )n( )n

RR

( )n

O O( )n

Diarylurea based Ligands

NH

N

N NH

NH

NH

NH

O Opeptidepeptide

O O3,6- bis acridine-peptide conjugates

Fig. 4 Chemical structures of click chemis-

try-based G-quadruplex ligands: neomycin

capped quinacridine (NCQ), tri-oxazole

macrocycles, trisubstituted isoalloxazines,

diarylurea ligands, 3, 6-bis acridine–peptide

conjugates, 9-peptide acridine and 5-(amino-

methyl)-2-furancarboxylic acid (furan)-based

G-quadruplex ligands.



core (Fig. 5) [80,81]. Two compounds of the pyridodi-

carboxamide series, namely 307A and 360A, exhibited

a high degree of quadruplex stabilization, exquisite

quadruplex over duplex selectivity, and were able to

induce efficient inhibition of telomerase. These com-

pounds have also been shown to induce delayed

growth arrest and apoptosis in immortalized cell lines.

These results are particularly impressive with regard to

the structural simplicity of the series and its two-step

synthesis [81]. Remarkably, tritiated 360A has also

been shown to localize preferentially at the telomeric

regions of chromosomes, thus providing new evidence

of the existence of quadruplex in a cellular context

[82]. Pyridodicarboxamide derivatives have also been

shown to induce the formation of tetramolecular

quadruplexes and act as molecular chaperones, thereby

proving their efficiency as G-quadruplex binders [83].

Moreover, the pyridodicarboxamide family of ligands

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 asG-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

C-deprotonated 2-phenylpyridine (N-CH) ligands have

been developed and their G-quadruplex DNA-binding

potential assayed. [PtII(dppz-COOH)(N-C)]CF3SO3

(1; dppz-COOH = 11-carbotxydipyrido [3, 2-a: 2¢,3¢-c] phenazine) binds G-quadruplex DNA through an

external end-stacking mode with a binding affinity in

the order of 107 m)1. Using a biotinylated-primer

extension telomerase assay, the same molecule was also

shown to be an effective inhibitor of human telomerase

in vitro, with an IC50 value of 760 nm [90].

Neutral ligands

The category of neutral ligands is not the largest but it

includes the paradigm for G-quadruplex recognition,

namely telomestatin (Fig. 6). This is isolated from

Streptomyces annulatus [91] and has been extensively

studied because it appears to be one of the most inter-

esting G-quadruplex ligands [10,91–100]. Indeed, this

molecule greatly stabilizes the G-quadruplex and

appears to be one of the most selective G-quadruplex

ligands: > 70-fold. The total absence of an affinity for

N

HN

H2N

NH2

N+

N NN+

NH2

NN

H2N

NH

N

N

N

NH

dnagiL95421desabenizairTsevitaviredmuidihtE

NNH HN

O O

NNNH HN

N NNH HN

N N

O O

PDC core

PhenDC3

Fig. 5. Chemical structures of ethidium derivatives as G-quadruplex

ligands: ethidium derivatives, 2, 4, 6-triamino-1, 3, 5-triazine deriva-

tive (12459), pyridodicarboxamide (PDC) core and phenanthroline

analogues (Phen-DC3).

SO O

ON N

N

O

O N

ON

O

NN

RO N

N ONNH

N NR

HN

O

O

O

NO

NO

O

N O

O R = CH(CH3)2 R = CH2OH

Telomestatin Hexa-oxazole macrocycles

Fig. 6. Chemical structures of neutral ligands: telomestatin and

hexa-oxazole macrocycles.



duplex DNA because of its neutral character and cyc-

lic shape justifies the initial strong bend towards this

molecule. It has also been reported to inhibit the pro-

liferation of telomerase-positive cells, by modifying the

conformation and length of the telomeres, and the dis-

sociation of telomere-related proteins from telomeres.

Nevertheless, two major drawbacks of telomestatin are

that it is difficult to obtain and it has poor water solu-

bility. The complete synthesis of telomestatin seems to

be highly complex and is not compatible with large-

scale preparation [101]. There are also a few reports in

literature on macrocyclic hexaoxazole ligands and their

interaction with telomeric G-quadruplex [102].

HXDV and HXLV-AC (Fig. 6) are two synthetic

hexaoxazole-containing macrocyclic compounds which

have been characterized for their cytotoxic activities

against human cancer cells. Their detailed binding and

thermodynamics of interaction with the intramolecular

(3+1) G-quadruplex structural motif formed in the

presence of K+ ions by human telomeric DNA has

also been reported [103].

Examples of ligands belonging to different classes

are also summarised in Table 1.

Mode of action of telomericG-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

in [108]). It has been shown that the active complex is

composed of three different components, hTERT, hTR

and dyskerin, with two copies of each. However, regu-

lation of the relative expression levels of hTERT, hTR

and dyskerin is poorly understood. It is known that

methylation of the promoter and 50 exons may lead to

repression of hTERT expression in normal cells [109].

Recently, evidence for a molecular link between choles-

terol-activated receptor Ck and hTERT transcription

has been reported [110]. Moreover, it has also been

shown that the core hTR promoter is activated by Sp1

and is repressed by Sp3 [111,112].

Table 1. Examples of ligands belonging to different classes.

Class Compound

N-methylated ligands TMPyP4, TQMP68, 3,4-TMPyPz, tetramethylpyridiniumporphyrazines, corroles, Se2SAP

Ligands with protonable

side arms

Bisamidoanthraquinone, fluorenone, acridone, acridine, BSU6039, BRACO-19, benzylamino-acridine,

perylene diimide PIPER

Daunomycin, distamycin and netropsin, flavonoids, steroids

RHPS4, MMQ3, MMQ1, BOQ1, indoloquinolines, cryptolepine analogues, quindolines and

triazacyclopentaphenantrene

Alkaloid-based ligands Berberine derivatives, palmatine sanguinarine, isoindigotone

Click chemistry-based

ligands

Bistriazole derivatives, neomycin-capped quinacridine, tri-oxazole macrocycles, isoalloxazines,

diarylurea-based ligands and substituted derivatives, trisubstituted acridine–peptide conjugates,

furan-based macrocycles

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)]CF3SO3

Neutral ligands Telomestatin, hexaoxazole-containing macrocyclic HXDV and HXLV-AC



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 ⁄oligosaccharidebinding 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

that TPP1 ⁄POT1–telomeric complex increases the

activity and processivity of the human telomerase core

enzyme [122,123]. Shelterin partners also associate with

Apollo, a novel component of the human telomeric

complex that, along with TRF2, appears to protect

chromosome termini from being recognized and pro-

cessed for DNA damage [124,125]. Inactivation of

either TRF2 or POT1 [126,127] leads to variation in

the overall length of the single-stranded G overhang,

aberrant homologous recombination [128] and also

induces a specific DNA damage response at most telo-

mere ends [129]. These studies are consistent with the

view that telomere ends are engaged in a peculiar

structure in order to protect their integrity.

Possible role of G-quadruplex binding ligands as

telomerase inhibitors

G-quadruplex ligands were first evaluated as telomer-

ase inhibitors and could conceivably induce telomere

shortening and replicative senescence [130]. Long-term

treatment of human cancer cells with subtoxic doses of

disubstituted triazines or telomestatin induces progres-

sive telomere shortening that correlates with the induc-

tion of senescence [77,79,131,132]. This telomere

shortening may be the result of inhibition of telomer-

ase and ⁄or telomere replication. A similar pheno-

menon was noticed in human cells treated with

telomestatin, a new steroid derivative, and BRACO-19

[46,95,133]. This may intuitively be the result of telo-

merase inhibition, but as we discuss below, such short-

term loss may also be the result of telomere replication

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.

DNA damage pathways induced by G-quadruplex

ligands

Telomeres effectively prevent the recognition of natural

chromosome ends as double-stranded breaks. It has

previously been shown that telomere shortening or the

loss of protective factors such as TRF2, TIN2 and

POT1 activates a DNA damage response pathway

[139]. In addition, initiation of a DNA damage path-

way was demonstrated in BCR-ABL-positive human

leukemia cells after telomestatin treatment character-

ized by the phosphorylation of ATM and Chk2 [131].

A similar DNA damage response ensues subsequent to

telomestatin treatment in HT1080-treated cells as

evidenced by the formation of gH2AX foci that par-

tially co-localize at the telomere, thus suggesting the

induction of telomeric dysfunction [95]. A similar

gH2AX response is elicited in the nucleus of

UXF1138L uterus carcinoma cells upon the interaction



of RHPS4 [140]. The triazine derivative 12459 also

induces either senescence or apoptosis in the human

A549 pulmonary carcinoma cell line, in a concentra-

tion- and exposure time-dependent fashion [141].

Future perspectives

Although synthesis of small molecules to target the

quadruplex is attracting attention, there are many chal-

lenges which demand greater efforts if we are to devise

strategies for rational drug design with high selectivity.

The common features that most quadruplex-interacting

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,

c-myc and c-kit quadruplex–porphyrin interactions,

thereby establishing the influence of loop length and

composition in perturbing molecular recognition of the

quadruplex and its interaction with ligand. However,

this dataset needs to be extended for other biologically

relevant quadruplexes. Another notable observation is

that ligands which demonstrate a promising perfor-

mance in vitro usually display poor biological efficacy.

This inconsistency between in vitro and in vivo results

can be attributed to molecular crowding conditions in

the cell. Most in vitro experiments neglect the role of

molecular crowding agents, which have a major influ-

ence on the structure of nucleic acids and the interac-

tion with their partners. Molecular crowding agents

perturb the quadruplex–ligand interaction by influenc-

ing the participation of water molecules. Therefore, an

important parameter that should be taken into account

during molecular dynamics and biophysical studies is

hydration and the associated changes in heat capacity

upon complex formation.

Lastly, the in vitro knowledge generated should be

extrapolated to the cellular level to evaluate the thera-

peutic potential of the ligands inhibiting telomerase

and ⁄or perturbing the molecular recognition of quad-

ruplexes and competing with transcription factor bind-

ing. Efficient molecular assays should be developed for

the accurate estimation of inhibitory effects and related

toxicity with appropriate control experiments [84].

These molecular assays should be combined with glo-

bal transcriptomic, proteomic profiling and tumor

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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Retraction: Human telomeric G-quadruplex: targeting with small molecules