Therefore, it becomes imperative that the Keywords alkaloids; anticancer agent; click chemistry; ethidium derivative; G-quadruplex; human telomere; metallo-organic complex; N-methylated
Trang 1Human telomeric G-quadruplex: targeting with small
molecules
Amit 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Agarwal and Souvik Maiti, published online on 27thNovember 2009 in Wiley InterScience (www.interscience.wiley.com), has been retracted by agreement between the authors, the journal Editor-in-Chief Professor Richard Perham and Blackwell Publishing Ltd The retraction has been agreed due to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, Ce´line Douarre, Nassima Temime-Smaali, Chantal Trentesaux, Jean-Franc¸ois 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: souvik@igib.res.in
(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.
Trang 2mechanisms 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
N
N
NH
N
N
HN
N N N HN N
N
N N
N
Se
N
Se
N
N
N N
OH HO
TQMP
Fig 1 Chemical structures of N-methylated ligands: TMPyP4, 3,4-TMPyPz, TQMP,
Se2SAP.
Trang 3(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 IC50values 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-stacking
interactions 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 duquadru-plex 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 quinacriacri-dines (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
Trang 4unique 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
N HN
Cryptolepine
N HN N
H 3 C
H 3 C
O
CH 3
y
HN
R
HN O
Triazacyclopentaphenanthrene
X HN
N
Quindoline, X= NH or O and
R= different groups
R
BRACO 19
N
N O O
PIPER
R = H, BSU6039
R = NH-p-C 6 H 4 -N(CH 3 ) 2 ,
N
N
N
N HN
N
N
N N
HN NH
R 1
NH(CH 2 ) n N(CH 3 ) 2 NH
NH
HN
HN
R 2
BOQ1 Benzoindoloquinoline
Fig 2 Chemical structures of ligands with protonable side arms: BSU6039, BRACO-19, PIPER, RHPS4, MMQ 3 , BOQ1,
benzoindoloquinoline, cryptolepine, triazacyclopentaphenanthrene, quindoline.
Trang 5found 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-containloop-contain-ing
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-quadruplex ligands
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
N +
O
CI
-N
N O
H N (CH 2 ) n
R N
O (CH 2 ) n
9-N substituted Berberine derivatives, R substituted Berberine derivatives, R represents different groups
O O
N
O
OH N
OH
O
Isaindigotone Berberine
Fig 3 Chemical structures of alkaloid-based G-quadruplex ligands:
berberine, 9-N-substituted derivatives and isaindigotone.
Trang 6peptide O
NH
N N
9-peptide acridine
O O N
X O
O X O
O
H
H
O O
O
O
HN NH
O
O 1: X = NH 3
2: X = NHCOCH CH NH +
H O 2: X = NHCOCH 2 CH 2 NH 3
Furan based ligands
O
HO
O
NH 2
O
HO
H 2 N
OH OH
O NH 2
NH 2 O
O HO
HN
OH
O 2
HN O
O HO
NH
O N
NH HN N O
O 2
NH 2
NH 2
NH 2
NH
NH 2
NH
O N
HN O
N
Tri-oxazole macrocycles
R 2
N
R 1
O
N N R 1 O
Trisubstituted isoalloxazines, R1 and R2 represents different groups
O
O
O
N N
N N
O O
N
O
R R
( ) n
( ) n
Diarylurea based Ligands
NH N
N N
peptide peptide
3,6- 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.
Trang 7core (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 as
G-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 107m)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 IC50value 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
H 2 N
NH 2
N +
+
NH 2
N
N
H 2 N
N N
N N
N
d a i L 9 4 1 e a e i z i r T s
v i
t
a
i
r
e
m
u
i
d
i
h
t
E
N
N N
NH HN
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-DC 3 ).
N O
O N
O
N N R
NH
R HN O
O O
N O N
O O
O
R = CH(CH 3 ) 2 R = CH 2 OH
Telomestatin Hexa-oxazole macrocycles
Fig 6 Chemical structures of neutral ligands: telomestatin and hexa-oxazole macrocycles.
Trang 8duplex 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 telomeric
G-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.
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
Trang 9Proteins 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⁄ oligosaccharide
binding 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-posinega-tive 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
Trang 10of 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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