Interaction of G-Quadruplexes with Nonintercalating Duplex-DNA

Review pubs.acs.org/bc

Interaction of G-Quadruplexes with Nonintercalating Duplex-DNA Minor Groove Binding Ligands Akash K. Jain‡ and Santanu Bhattacharya*,‡,† ‡

Department of Organic Chemistry, Indian Institute of Science, Bangalore 560 012, India Chemical Biology Unit, Jawaharlal Nehru Centre for Advanced Scientific Research, Bangalore 560 012, India

†

ABSTRACT: The enzyme telomerase synthesizes the G-rich DNA strands of the telomere and its activity is often associated with cancer. The telomerase may be therefore responsible for the ability of a cancer cell to escape apoptosis. The G-rich DNA sequences often adopt tetra-stranded structure, known as the G-quadruplex DNA (G4-DNA). The stabilization of the telomeric DNA into the G4-DNA structures by small molecules has been the focus of many researchers for the design and development of new anticancer agents. The compounds which stabilize the G-quadruplex in the telomere inhibit the telomerase activity. Besides telomeres, the G4-DNA forming sequences are present in the genomic regions of biological significance including the transcriptional regulatory and promoter regions of several oncogenes. Inducing a G-quadruplex structure within the G-rich promoter sequences is a potential way of achieving selective gene regulation. Several G-quadruplex stabilizing ligands are known. Minor groove binding ligands (MGBLs) interact with the double-helical DNA through the minor grooves sequence-specifically and interfere with several DNA associated processes. These MGBLs when suitably modified switch their preference sometimes from the duplex DNA to G4-DNA and stabilize the G4-DNA as well. Herein, we focus on the recent advances in understanding the Gquadruplex structures, particularly made by the human telomeric ends, and review the results of various investigations of the interaction of designed organic ligands with the G-quadruplex DNA while highlighting the importance of MGBL-G-quadruplex interactions.

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enzyme, called helicase, facilitates the replication of each strand. Complete replication of the overhanging end of the chromosome cannot be accomplished by conventional DNA polymerases.4 In almost all eukaryotes, the chromosomal endreplication problem is solved by the enzyme telomerase, a ribonucleoprotein.5 A portion of the RNA subunit of telomerase provides the nucleic acid template that the DNA cannot provide for itself.6,7 The enzyme telomerase is upregulated in nearly 80−90% of the cancer cells, leading to an abnormal growth of cells.7 Telomerase inhibition therefore comprises a key strategy for the development of anticancer agents. This is because studies so far have shown that the telomerase inhibitors can stop the proliferation of the cancer cells or cause apoptosis of the cancer cells, while they have no effect on most of the normal cells.8 The telomere cap is composed of telomerase (having the components hTERT, hTERC, Hsp90, Tp1, and so forth), telomere-associated proteins (like TRF1, TRF2, Tankyrase, TIN2, POT1, Dykserin, and so forth), and telomeric DNA repair proteins (for instance, MRE11, RAD50, KU70, XRCC5/ KU80, and H2AX, etc.). This machinery maintains the length of

INTRODUCTION

At the ends of the chromosomes, i.e., the telomeres, DNA does not consist of a complex protein-coding sequence. Instead, these are made of a simple sequence such as TTGGGG in the ciliate Tetrahymena1 or TTAGGG in humans,2 which are repeated a few hundred times in humans and fewer times in ciliates. The bulk of telomeric DNA adopts a double-helical conformation keeping the GT-rich sequence paired with its CA-rich complement. However, the 3′-end of such DNA protrudes as a single-stranded overhang in all the eukaryotes studied.2−5 This DNA sequence binds to specific proteins, which cap the chromosome ends either directly or by inducing a particular DNA structure.5 DNA in the cell nucleus is copied and transcribed by enzymes such as DNA and RNA polymerases. DNA synthesis is catalyzed by DNA polymerase, an enzyme which has crucial role in the DNA replication. A DNA polymerase elongates the new DNA strand from 5′ to 3′ direction, by adding a free nucleotide onto the growing 3′-end of the new strand. It never begins the synthesis of a new chain, and therefore, it needs a primer to add the first nucleotide at the 3′-OH group of the primer. The first two bases of the primer are always the RNA bases, while other bases may be either DNA or RNA. Primers are synthesized by an enzyme, primase. Conversion of the duplex DNA into a single-stranded DNA, with the help of an © 2011 American Chemical Society

Received: May 30, 2011 Revised: September 14, 2011 Published: November 10, 2011 2355

dx.doi.org/10.1021/bc200268a | Bioconjugate Chem. 2011, 22, 2355−2368

Bioconjugate Chemistry

Review

the telomere.9 A G-quadruplex selective ligand triggers the folding of the G-rich repeats of DNA into a G4-DNA structure. In turn, this inhibits the binding of telomerase and its associated proteins with telomere, thereby stopping the telomere elongation process.9 Since G4-DNAs do not serve as substrates for the telomerase, these structures have been the focus of much research in medicinal and biological chemistry as attractive targets for the anticancer drug design.7,9,10 All G4DNAs contain a basic repeating and stacking motif, the Gquartet structure, which is formed by four guanine bases and held in a plane by the Hoogsteen mode of hydrogen bonds (Figure 1A).7−11 DNA is a potential target for several

dynamic mixture of four intramolecular quadruplexes which differ in their loop arrangements.15,19 Similarly, a 22-mer G4DNA forming sequence, c-kit87up, in the promoter region of ckit forms a unique intramolecular quadruplex in K+ solution.21 Presently, much of the research effort involving G-quadruplex DNA is directed toward the study of G4-DNA formed by human telomeric repeats d[T2AG3]n, particularly a 21-mer sequence d[T2AG3]3G3 and a 22-mer d[AG3(T2AG3)3]. Altering the TTAGGG to TTAGAG in any one of the repeats of T2AG3 converts an intramolecular to an intermolecular Gquadruplex with varying degrees of parallel and antiparallelstranded character, depending on the length of the incubation time, the concentration of the ion present in solution (Li+, Na+, or K+), and the exact DNA sequence.22 A human telomeric DNA repeat sequence can form two related types of the G4DNA structures, known as hybrid-1 or hybrid-2 structures (Figure 1B), depending on the flanking segments, out of which the hybrid-2 type is predicted to be the major structural component in the human telomeric DNA.23−26 These solution structures are different from the K+-stabilized crystal structure of the G4-DNA derived from 22-mer sequence d(AG3[T2AG3]3).26 Other possible G4-DNA conformations adopted by short human telomeric sequences and quadruplex-forming sequences in other genes were already reviewed.27 Recently, a dimeric structure for the eight-repeat human telomeric sequence containing 2-folded quadruplex units was reported on the basis of high-resolution G4-DNA structure obtained for the four-repeat sequence (Figure 1C). 28 Different topologies of the G4-DNA (in telomeric and genomic regions) having some unusual intermediates as shown by NMR experiments were reviewed recently.29 Drug−DNA complex formation may be divided into two broad areas via (i) covalent and (ii) noncovalent interactions. 30 Covalent interactions are generally irreversible. DNA alkylating agents like nitrogen mustards fall under this category. Also cisplatin cross-links to DNA, specifically through the N-7 atom of purines of DNA base pairs impeding their hydrogen bond forming capacity with cDNA bases. Such compounds inhibit transcription, translation, and replication of DNA and have long been used as anticancer drugs.30−32 Intercalative and nonintercalative drugs represent the major classes of noncovalently interacting compounds.33 Intercalators generally insert between the layers of nucleic acid base pairs and disrupt the shape of the double-helix significantly, thereby preventing replication and transcription often by untwisting the DNA.30,31,33 Nonintercalative molecules interact with the duplex DNA through its major and minor grooves. These grooves differ in their electrostatic potential, hydrogen bonding characteristics, steric requirements, hydrophobicity, hydration, and microenvironmental polarity.30,31,33−39 Most of the DNA binding proteins associate through the major groove of the duplex DNA, while small molecular mass ligands bind via both the major and minor grooves of DNA.33 DNA minor groove binding ligands (MGBLs) are generally “crescent-shaped” molecules which recognize the minor grooves in a sequencespecific manner. Oligopeptides (netropsin, distamycin-A),34,40 benzimidazoles (Hoechst 33258, Hoechst 33342),37 DAPI,33 berenil,33 bis-quaternary cations (SN 6999, SN 7167, SN 18071),31 cyanine dyes (DTDC, DODC),31 and so forth are some of the well-known MGBLs of low molecular mass. These molecules typically recognize 3−5 base pairs, and some of their analogues including dimers recognize even longer stretches of DNA.34−42

Figure 1. (A) Structure of a G-quartet is shown, in which the hydrogen bonding patterns are also indicated. (B) Schematic model of the DNA secondary structure in human telomeres. The hybrid-type telomeric G-quadruplex structures can be readily folded and stacked end to end to form compact-stacking structures for multimers in the elongated telomeric DNA. Figure adapted from ref 23, with permission of the Oxford University Press. (C) Average structure from molecular dynamics simulation of the Hybrid-12 model; dT and dA residues, in the junction region between the quadruplex units, are shown in red and blue, respectively. Figure adapted from the ref 28, with permission of the American Chemical Society.

anticancer drugs and there are many possible mechanisms of action.12 G-quadruplex interactive compounds inhibit telomerase by stabilizing the single-stranded 3′-telomere ends as G4DNA.7,11 Besides telomeres, the G4-DNA forming sequences occur in biologically relevant regions of the genome including the immunoglobulin switch regions,13 the transcriptional regulatory regions of a number of genes such as the insulin gene,14 and also the promoter regions of several oncogenes such as c-kit, cmyc, c-myb, K-RAS, bcl-2, and many others.15−18 Formation of G-quadruplex structures from these G-rich promoter sequences induced by a small molecular ligand provides a means of achieving selective gene regulation and thereby target a protein, the product of gene expression. This has been demonstrated for the c-myc oncogene at the nuclease hypersensitivity element (NHE) III1 which is responsible for up to 90% of c-myc transcription.15,19 Topology of these G-quadruplex structures depends on the DNA sequence, number of telomeric repeats, oligodeoxynucleotide (ODN) concentration, and an appropriate stabilizing cation.20 A 27-mer G4-DNA forming sequence in c-myc forms a 2356



Review

MGBLs bind with the non B-DNA and multistranded DNA structures too. There are reports on the studies of the complexes of MGBLs involving ps-DNA31 and triplex31,43 and G-quadruplex DNA.44 Herein, we provide an overview of the nature of interaction of G-quadruplex DNA with low molecular mass organic ligands with a special emphasis on MGBLs. Since G-quadruplexes are involved in several important cellular functions, the studies of these complexes should be useful in the design and discovery of some effective chemotherapeutic agents.

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G-QUADRUPLEXES AND ORGANIC LIGANDS

G-quadruplex DNA structures have affinity for small, positively charged organic ligands. G4-DNAs have several sites of interaction with an organic ligand, mainly through the Gtetrads and the grooves. G-quadruplex stabilizing molecules (particularly the end-stacking ones) often have the following features, e.g., a central symmetrical and a planar core, an extended delocalized π-electron system, tertiary amines which become positively charged due to the protonation under physiological conditions of pH, and the side chains that are able to form hydrogen bonds with the DNA bases and with the phosphate backbone of the loops.45 Many end-stacking ligands fit this description, although this is not always a general rule. Thus, the compounds that bind with the G4-DNA grooves may have entirely different molecular features. In 1997, a crystal structure of G4-DNA formed by hexanucleotide TG4T in Na+ solution was elucidated. This showed several distinct structural features different from that of the duplex DNA.46 This includes notably the possession of four quasi-equivalent grooves along with a pronounced channel of negative electrostatic potential running through the center of the planes of the G-quartets, allowing the metal ions to be coordinated between the planes in a bipyramidal antiprismatic manner. Structural properties of the G4-DNA grooves vary according to molecularity and type (strand orientation) of the G-quadruplex.47 Four-stranded intermolecular G4-DNA has four identical grooves that are of about the same size as the minor groove of B-DNA.48 In contrast to the parallel-stranded G-quadruplexes, the four grooves in an antiparallel quadruplex are not identical, e.g., foldover monomeric G4-DNAs with diagonal loops have one wide groove, two medium-sized grooves, and one narrow groove. 49 Taking these features into account, many compounds were synthesized to target the G4-DNA formed by the human telomeric DNA sequence.50 Structures of some small organic cation-G4-DNA complexes have been recently reviewed27 and are supposed to be helpful in finding out new structure-based leads for the design of anticancer drugs. The intramolecular G4DNA made by the sequence d[AG3(T2AG3)3] has seven major binding sites available for a drug, made up of two G-tetrads for end-stacking (parallel-loop and diagonal-loop), one wide groove, two medium-sized grooves, one narrow groove, and top parallel loop binding sites (Figure 2). Thus, the geometries and functional groups of the G-quadruplexes are different from that of the duplex DNA. A drug can bind to multiple sites suggested by its skeleton and functional groups present in it. Thus, although 3,6-bis(1-methyl-4-vinylpyridinium iodine) carbazole (BMVC, 1) exerts a strong interaction with all the sites, its binding with the end-stacked G-quartets is the most favorable.51

Figure 2. Molecular structure of 3,6-Bis(1-methyl-4-vinylpyridinium iodine) carbazole (BMVC, top) and a sketch of the G4-DNA structure made by human 22-mer telomeric DNA showing its binding sites for a drug (bottom). Figure adapted from ref 51, with permission of the American Chemical Society.

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G-QUADRUPLEXES IN NON-TELOMERIC REGIONS As mentioned earlier, G4-DNA forming motifs have been identified and characterized in functional regions (e.g., promoters and enhancers) of many oncogenes such as c-kit, c-myc, c-myb, K-RAS, bcl-2, and other cell growth-related genes.13−21,29,52 Most of these G4-DNA motifs in the gene promoter regions have physical chemical properties and structural characteristics that might make them interact strongly with putative drugs.18 Further, they have such diverse structures that a high degree of selectivity might be possible.18,21,29 The initial report on genome-wide analyses highlighting multiple regulatory roles of G4-DNA structure appeared in 2006, 53 followed by a number of recent reports.17,54−56 G4-DNA formation in genes may affect the cellular function in different possible ways. G4-DNA formation in the insulin-linked polymorphic region upstream of the insulin gene enhanced its transcription, where single/double mutations that disrupted the G4-DNA structure afforded reduced promoter activity. 14 On the other hand, G4-DNA also acts as a “silencer element” for certain oncogenes.15,18,57 Several proteins from different organisms that interact with the G4-DNA have been reported.58−60 They play an important role in transcription of certain genes and can be classified by function into five major groups. These are those that (i) increase the stability of G4DNA, (ii) destabilize G4-DNA in a noncatalytic way, (iii) unwind catalytically G4-DNA in an ATP-dependent fashion, (iv) promote the formation of G4-DNA, or (v) specifically cleave DNA at or adjacent to a quadruplex forming domain. G4-DNA structures in the promoter regions have generally been targeted by end-stacking ligands, which have been already reviewed.18

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GROOVE BINDING LIGANDS FOR G-QUADRUPLEX DNA As discussed above, the bulk of the research concerning the G4DNA binding ligands has focused heavily on the ligands having aromatic rings that could potentially stack on the terminal G4planes on both ends of the G4-DNA. Other potential binding 2357



Review

Figure 3. Molecular structures of benzimidazole-based ligands: Hoechst 33258, terbenzimidazoles, ‘V’-shaped bisbenzimidazoles, 67 benzobisimidazoles,68 symmetrical isomeric bisbenzimidazoles used to study tunable binding with the duplex DNA in presence of metal ion, 63 symmetrical ‘V’-shaped bisbenzimidazoles based on 1,3-phenylene-bis(piperazinyl benzimidazole) [R 1 = H, R2 = CH2CH2OH;69 R1 = OH or OEt, R2 = Me or CH2CH2OH],70 benzobisimidazoles,68 symmetrical isomeric bisbenzimidazoles used to study tunable binding with the duplex DNA in presence of metal ion.63

A(G.G.G.G) pentad.61 The structure-based design of ligands and their consequent synthesis to achieve significant binding with individual grooves of defined G4-DNAs is still an ongoing challenge in the field. Since the dimensions of the G4-DNA grooves differ with the type of the G4-DNA, donor−acceptor patterns of the H-bonding sites also vary.62 Thus, successful implementation of the groove binding is an important strategy to selectively target a particular G4-DNA structure.

sites include the four grooves of the G4-DNA. The width, depth, and accessibility of these four grooves depend on the strand alignment and the topologies of the connecting loops. The structural differences between the duplex and the G4-DNA grooves offer an alternative yet important strategy for the design and synthesis of new ligands for differentiation between the two nucleic acid structures. The grooves are protected by the double-chain-reversal loops, but edgewise and diagonal loops do not have such restrictions. The G4-plane is made by the Watson−Crick base pairing scheme, and the major groove edges of guanines and minor groove edges are still available for further recognition. It was shown that an adenine can align with the minor groove edge of a G4-plane guanine, to form a sheared G.A noncanonical pair, leading to the creation of a

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FEATURES OF THE GROOVE BINDING LIGANDS Benzimidazole Based Scaffolds. The benzimidazole moiety is structurally related to the purine bases and is found in several biologically relevant natural compounds. Bisbenzimidazole-based Hoechst 33258 generally binds with 4−5

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Figure 4. (A) Chemical structures of symmetrical ‘V’-shaped bisbenzimidazole (8a) and its linear isomer (9). 69 (B) Optimized structures at the B3LYP/6-31G* level of theory of 8a and 9 (adapted from the ref 69, with permission of the American Chemical Society). (C) Structures of the Gtetrad along with interatomic distances.

contiguous AT base pairs of the duplex DNA (2, Figure 3). In contrast, the terbenzimidazoles bind with extended sequences of DNA (3, Figure 3).37−39 Other benzimidazole derivatives were also studied to modulate their binding affinity toward duplex and other higher-order DNA structures. 30,31,37,43 Interestingly, three symmetrical isomeric bisbenzimidazole compounds show very different binding affinity toward ATrich duplex DNA (Figure 3).63 Hoechst 33258 binds with the G4-DNA formed by the DNA sequence derived from human cmyc promoter region, d(G4AG3TG4AG3TG4A2G2TG4). Using UV, fluorescence spectroscopic, and SPR techniques, a binding constant of the order of 106 M−1 was estimated.64 Anisotropy measurements and higher lifetime obtained from time-resolved decay experiments reveal that Hoechst 33258 bound to G4DNA is rotationally restricted in a less polar environment than in the bulk buffered medium. As Hoechst 33258 is known to bind specifically to A−T-rich regions of the duplex DNA, the likelihood of Hoechst 33258 interacting with the AAGGT loop of the G4-DNA was proposed.64 Later on, the binding was examined by NMR data too.65 Upon addition of Hoechst 33258 to the solution of a G4-DNA made from a 24-mer sequence d[TGAG3TG4 AG3TG4A2G2] (pu24), the imino protons of the top G4-plane’s residues are found to be most perturbed (shifted toward upfield and broadened).65 This is an

observation which is consistent with the stacking of these ligands on the top G-tetrad. Further, imino proton of G13, which is involved in the terminal G-quartet formation, is greatly broadened, suggesting multiple stacking configurations of Hoechst 33258 over G13. Recently, Hoechst 33258 was shown to stabilize the G4-DNA, a sequence derived from the human telomeric repeats in solution having Na+ ions, but not K+.66 However, in the molecular crowding conditions mimicked by the addition of polyethylene glycol (PEG), it lost its Gquadruplex binding affinity and consequent telomerase inhibition activity.66 The negative effect of the molecular crowding could be attributed to the reduced affinity between the ligands and the G4-DNA, possibly as a consequence of decreased water activity and enhanced viscosity of the medium. Two symmetrical V-shaped bisbenzimidazole derivatives were prepared and one of them was shown to induce the G4-DNA formation and stabilize it.67 The benzimidazole constructs that are more effective have pyridine as the central aromatic core and substitutions on benzimidazoles at 3- and 6positions (4a), making a planar and relatively nonflexible conformation because of the intramolecular H-bonding between the imidazole hydrogen and the nitrogen of pyridine. This compound (4a) also showed effective nuclease resistance activity.67 Other molecules having benzene as the central ring 2359



Review

Figure 5. Chemical structures of netropsin, distamycin-A, and carbamoyl analogues of distamycin having 4 and 5 N-methyl-pyrrole rings and dicationic derivative76 of distamycin-A (distamycin-A analogue having 5 rings is called MEN 10716), 82 uncharged distamycin-A analogue devoid of amidinium group,83 anthracenylioxazole lexitropsin conjugates,84 and planar U-shaped byaryl polyamides.85

and substitutions at 1- and 2-positions, however, show much less affinity toward the G4-DNA. Recently, a symmetrical, linear bisbenzimidazole (5) and some benzobisimidazole compounds (6a−6c, Figure 3) were tested against the duplex and the G4-DNA.68 Linear bisbenzimidazole (5) was found to bind with the double-helical DNA, while the benzobisimidazole compounds were found to stabilize the G4-DNA preferentially over the duplex DNA. Side chains have an important role in the G-quadruplex binding and telomerase inhibition. These benzobisimidazole derivatives have a conjugated aromatic surface, which probably aids their binding with a G-quadruplex DNA via π−π stacking.68 The shape of a G-quadruplex ligand has an important role toward the affinity and stabilization of the G4-DNA. G4-DNA interactions with two isomeric bisbenzimidazole-based compounds with different shapes (‘V’-shaped, angular vs linear; Figure 3) were investigated.69 While the linear isomer (9)

shows little affinity for the monomolecular G4-DNA structure generated in the presence of Na+, the ‘V’-shaped angular isomer (8a, R1 = H, R2 = CH2CH2OH), expected to cover three guanines of the G-quartet, induces a significant structural change of the intramolecular G4-DNA, above a threshold concentration. The structural change was evident from the pronounced changes observed both in the CD spectra and from the electrophoretic experiments using the G4-DNA. Notably, this V-shaped isomer also induces the G4-DNA formation without any added cation. The ligand-G4-DNA complexes were also investigated by computational methods, which provide additional information on the structure−activity relationships (Figure 4). Dimensions of the angular isomer match with those of the G-quartet, and it should cover three guanines of the Gquartet. The linear isomer, which should cover two guanines of the G-quartet, caused less structural change and provided little stabilization to the G4-DNA. In agreement with these 2360



Review

observations, TRAP assay results with the telomerase extracted from A549 cells show that the angular isomer is a significantly more effective inhibitor of the telomerase activity.69 A series of symmetrical ‘V’-shaped bisbenzimidazoles based on 1,3-phenylene-bis (piperazinyl benzimidazole) compounds was synthesized (8b−8d, Figure 3). These compounds induce G4-DNA formation even without any added cation with the ODN sequence d(T2G4)4 relevant to Tetrahymena.70 Binding affinity of these molecules toward the G-quadruplex increases upon introduction of an electron-donating ethoxy group on the central phenyl ring. The dimensions of these compounds match with those of the G-tetrad plane as per calculations, suggesting that end-stacking as the possible mode of the G-quadruplex stabilization. Appearance of an induced circular dichoric (ICD) signal at a higher drug: DNA ratio, however, indicates the groove binding as well.70 Oligopeptides and Related Lexitropsins. Some reports are available regarding the interaction of netropsin (Nt, 10), distamycin-A (Dist-A, 11), and their other oligopeptide analogues with the G4-DNA (Figure 5). This class of molecules acts as the minor-groove binders (MGB) for the duplex DNA.34,35,42 Binding properties of these N-methyl pyrrole containing cresent-shaped oligopeptides with the duplex DNA depend on the number of pyrrole rings and the presence or absence of a leading amide (in Dist-A).34 Longer analogues (beyond four/five consecutive rings) tend to lose their selectivity toward certain AT-rich DNA sequences. This is because the curvature of such long ligands often fails to match with the pitch of the DNA double-helix, disrupting the Hbonding and van der Waals interactions necessary for the specific sequence recognition.31,34,36 Dist-A and Nt were also investigated with the three G-quadruplexes characterized by different groove widths: [d(TG4T)]4 (Q1), [d(G4T4G4)]2 (Q2), and d(G4T2G4TGTG4T2G4) (Q3) using homonuclear NMR.71 Below 2:1 ligand/G-quadruplex stoichiometry, Dist-A, in a dimeric form, binds with each groove of the tetraplex to form short-lived complexes on an NMR time scale. At higher drug/DNA ratios, a second Dist-A dimer specifically binds with the tetraplex, to give a 4:1 complex, in slow exchange with the 2:1 complex. Nt was found to be in a fast chemical exchange with all three kinds of G-quadruplexes, whereas Dist-A interacted tightly with Q1 and, to a less extent, with Q2. Both molecules possess higher affinity toward the duplex DNA than the G4-DNA. Two opposite models were proposed for the Dist-A complexed, parallel G4-DNA containing ODNs of different sequences in K+ buffers. The first one suggests that Dist-A molecules bind as dimers in the two opposite grooves of the G4-DNA [d(TG4T)]4;72,73 while the second one suggests that two Dist-A molecules stack on the terminal G-tetrad planes of the G4-DNAs made up of each of [d(TAG3T2A)]4, [d(TAG4T2)]4, and [d(TAG5T)]4 on the basis of NMR studies.74 Below 2:1 ligand−tetraplex stoichiometry, Dist-A, in a dimeric form, binds to each groove of the tetraplex to form short-lived complexes on the NMR time scale. Therefore, only one set of signals for the four strands was observed. The fast exchange behavior of the lower complex (2:1 Dist-A-G4-DNA) could be changed only to an intermediate regime by decreasing the temperature of the system. As the temperature was reduced from 300 to 280 K, there was a general broadening of the resonances, with peaks belonging to Dist-A broadening more than the other peaks, probably due to the slower reorientation of the Dist-A dimer. At higher drug/DNA ratios, a second DistA dimer specifically binds with the G4-DNA, to give a 4:1

complex, in slow exchange with the 2:1 complex, as evidenced from the appearance of separate proton resonances for the two species. This behavior may be explained assuming that the binding of the second drug pair is more favorable than the binding of the first one.72 Nt is involved in fast exchange on the NMR time scale with its binding sites on the tetraplex.72 A new modified oligonucleotide, namely, d(TGGMeGGT), having GMe (8-methyl-2′-deoxyguanosine), able to fold into a quadruplex, was used to study its interactions with Dist-A. 73 Thus, the 8-methyl group of the four dGMe of the quadruplex [d(TGGMeGGT)]4 faces right into the very central region of the grooves, pointing outward from the G4-DNAs. As a result, if Dist-A interacts with the groove of the G-quadruplex, the presence of these bulky groups in the very central region of the four grooves should prevent (or at least should limit) the insertion of Dist-A molecules. Consequently, the formation of a stable complex would be impeded. In the case of [d(TG4T)]4, Dist-A displayed a high affinity toward the G4-DNA as evidenced from the appearance of a new set of signals during the NMR titration. Furthermore, Dist-A caused a loss of the original fourfold symmetry of the free G4-DNA. During the entire titration process of the G-quadruplex [d(TGGMeGGT)]4, the four strands remain magnetically equivalent and only a general change in the chemical shift resonances are observed. Thus, the presence of the methyl groups in the central region of the grooves does affect the binding of Dist-A to the quadruplex, and hence, Dist-A interacts with the grooves of the quadruplex. The affinity between Dist-A and [d(TG4T)]4 is enhanced (∼10-fold) when the ratio of Dist-A and the quadruplex is increased.73 Dist-A was reported to stack on the terminal G4-planes having the flanking bases of the G4-DNA.74 This allowed detection of 2:1, 4:1, and 8:1 Dist-A/G4-DNA complexes. DistA also inhibits the interaction of BLM helicase (an enzyme which unwinds the G4-DNA) with the G4-DNA. Dist-A inhibited binding of the G-quadruplex by nucleoilin (Nuc)RGG9. However, Dist-A exerts no effect on the interaction of G4-DNA with either a full-length murine nucleolin (Nucleolin) or a recombinant nucleolin possessing amino acid residues 281−709 that is devoid of the acidic N-terminus (Nuc-1,2,3,4RGG9) or recombinant nucleolin having only four RBD domains (RNA binding domains, Nuc-1,2,3,4).74 These results show that Dist-A may be used to probe the interactions of G4DNAs with certain proteins. These also indicate that at least two independent modes of protein−G4-DNA interactions may be studied by the sensitivity to Dist-A. In another report, interactions of Dist-A, tel01, and DTC with the G4-DNA were studied using ESI-MS.75 When two different ligand−G4-DNA complexes were subjected to a mass spectral analysis, distinct fragmentation patterns were observed. Tel01-G4-DNA complexes underwent a facile loss of the drug and produced single-strand ODN, while Dist-A-G4-DNA complexes produced single-strand ODN ions bound with the Dist-A. DTC-G4-DNA complexes had similar fragmentation patterns.75 Thus, it may be concluded that DTC and Dist-A bind with [d(T2G5T)]4 through the grooves. Different binding behavior observed with such drug molecules suggests that a sequence-dependent interaction prevails in the G4-DNA recognition events. Recently, Nt and a dicationic derivative of Dist-A (12, Figure 5) were studied with [d (TG4T)]4 G4-DNA using NMR titrations and isothermal titration calorimetry (ITC). 76 Dicationic derivatives form 2:1 complex with the G4-DNA. 2361



Review

Interactions in both cases (Nt and the dicationic Dist-A) are entropically driven with a small favorable enthalpic contribution. The stoichiometry and thermodynamic properties of interactions of Dist-A/its analogues with G4-DNA are affected by the presence of different cations in solution. Dist-A and its two carbamoyl derivatives (13, 14; having 4 and 5 N-methylpyrrole rings, respectively, Figure 5) were reported to behave differently from the target [d(TG4T)]4 and d[AG3(T2AG3)3] G4-DNAs in K+ and Na+ solutions.77 Out of these carbamoyl derivatives, one having 5 N-methyl-pyrrole rings (14) is also called MEN 10716. Dist-A and its 4 N-methyl-pyrrole derivative bind with the parallel quadruplex structure [d(TG4T)]4 in 1:1 stoichiometry in solution containing Na+.78 Dist-A and the 4 N-methyl-pyrrole derivative bind with the investigated G4-DNA in both solution conditions; conversely, the 5 N -methyl-pyrrole derivative seems to have a lower G4DNA affinity in any case as evidenced from the ITC and 1H NMR studies.77 The 4 N-methyl-pyrrole derivative, however, showed a 10-fold higher G4-DNA affinity than the duplex DNA.78 A number of amide-linked oligo N-methyl-pyrroles based on Dist-A molecules, having 2 to 6 pyrrole rings and substitutions at different positions (2,4-substitution vs 2,5-substitution), were also synthesized. These interact with a human intramolecular G4-DNA d[G3(T2AG3)3].79 Several of these molecules show an enhanced ratio of binding constants toward the G4-DNA vs the duplex DNA compared to that of Dist-A itself or its analogue with a 2,5-disubstituted pyrrole system. The G-quadruplex binding affinity increases with the number of N-methyl-pyrrole units of this class of ligands, suggesting that such an interaction is consistent with a mixed groove/G-quartet stacking mode of binding. A qualitative molecular model of a six-repeat pyrrole polyamide molecule docked onto the crystal structure of a human 22-mer G-quadruplex structure is shown (Figure 6),

Oligoamides having up to 3 N-methyl-pyrrole (Py) or -imidazole (Im) units were shown to have less effect on the G-quadruplex stability than other known end-stacking G4-DNA ligands like Tel 01, porphyrin, DAPER, PIPER, and so forth.80,81 In contrast, oligoamides having 4 or more rings induce stabilization of the G-quadruplexes. Dist-A derivative MEN 10716 (14, Figure 5) inhibits the human telomerase enzyme in a dose-dependent manner.82 Exposure of human melanoma JR8 cell extracts and U2-OS human steogenic sarcoma cell line extracts to MEN 10716 induced a dose-dependent inhibition of the telomerase activity, with an IC50 value of 24 ± 3 mM. A chronic exposure of the H460 nonsmall cell lung cancer cells to the above drug (100 mM every other day for 50 days) induced a consistent inhibition (>85%) of the telomerase activity.82 Inhibition of the telomerase by Dist-A and its 2,5-disubstituted derivative was also determined using a modified PCR based TRAP assay. 79 These two compounds were examined in this experiment up to a concentration of 100 μM, and concomitant controls were carried out to ensure that there was no interference with the correct functioning of Taq polymerase at these concentrations. Dist-A exhibited a telEC50 value of ∼25 μM, whereas its 2,5disubstituted derivative showed no telomerase inhibitory activity even at 100 μM. However, both compounds show significant PCR inhibition, suggesting that the positive result obtained in the TRAP assay is not necessarily due to the telomerase inhibition and other mechanisms are possible.79 Dist-A also inhibits protein interactions with the G4-DNA. The utility of Dist-A as a probe of the G4-DNA−protein interactions was thus demonstrated and two separate modes of the protein−G4-DNA interactions, which may be analyzed by the sensitivity to Dist-A, were proposed.74 An uncharged Dist-A analogue, where the major change in its structure involved the replacement of the amidinium group by an uncharged N-methyl amide moiety (15, Figure 5), was used to probe the importance of the unique Coulombic interactions in the Dist-A/[d(TG4T)]4 complex formation.83 Removal of the positively charged terminal group results in an exceptional ligand−G4-DNA complex formation in which the grooves as well as the 3′-end of the telomeric DNA are occupied. Furthermore, a lack of charge in the ligand molecule does not affect the relative orientation of two molecules of the analogue forming a dimer. The positively charged amidinium moiety of the Dist-A interacts with the phosphate groups of the Gquadruplex, providing a favorable (although small) enthalpic contribution, while the analogue, where the amidinium group is replaced by the uncharged N-methyl amide moiety, cannot interact with electrostatic complementarity.83 The binding affinity of the uncharged analogue with the G4-DNA as

Figure 6. Qualitative molecular model of a six-repeat pyrrole polyamide molecule docked onto the crystal structure of the human 22-mer G-quadruplex structure. Figure adapted from the ref 79, with permission of the Royal Society of Chemistry.

where the two repeats of the polyamides located in a G4-DNA groove and the others stack on a terminal G-quartet surface. 79

Figure 7. Chemical structures of common carbocyanine dyes (19−23), dyes ETC (24), and MKT-077 (25) synthesized based on cyanine dyes. 2362



Review

measured from ITC is 9 × 105 M−1 at 25 °C. However, no comparison of binding affinity with Dist-A was given. Some anthracenyl isoxazole lexitropsin conjugates (16, 17; Figure 5) were developed recently. Plausible binding modes of these compounds after energy minimization have suggested that one of them binds with the G4-DNA via intercalation, by displacing a guanine. Other compounds bind via a stacking mode with the G-tetrad.84 G4-DNA formed by the sequence d[5′-CATGGTGGT3(G3TTA)4CCAC-3′] quench the fluorescence of ligands suggesting a π−π stacking interaction of the ligand with the G-tetrad. Some planar U-shaped biaryl polyamides (17, 18) possessing high selectivity toward the Gquadruplex over the duplex DNA were also developed. 85 Compound 17 shows high selectivity for the human telomeric G4-DNA, while compound 18 is selective for two c-kit G4DNAs. Cyanine Based Ligands. Carbocyanine dyes (19−23, Figure 7) represent another class of MGBLs which are often studied with the G4-DNA. These compounds form complexes with the minor grooves of the duplex DNA, and show pronounced exciton splitting in their ICD spectra.30,86 Stacked species are important for the recognition of G4-DNA. Studies with the duplex DNAs demonstrate that ligands that bind DNA as stacked dimers have enhanced binding affinity/selectivity over similar ligands which bind as monomers.86 Similar stacking in the G4-DNA grooves seems to be a favorable way to selectively recognize different G4-DNA structures. Such a mode of interaction probably optimizes through an induced fit component, between the stacked ligands and the G-bases of the G-quadruplexes. On the basis of different spectroscopic studies, 3,3′-diethyloxa-dicarbocyanine (20, DODC) was shown to bind with the grooves of a dimeric hairpin G-DNA.87 DODC−G4DNA interaction resulted in some unique spectral signatures, e.g., a new absorbance peak (534 nm), an ICD signal (534−626 nm), a quenching of the fluorescence intensity of the ligand on excitation with the visible light, and a strong energy transfer from DNA.87 In the electrophoresis experiments, only the G4DNA bound to DODC were detectable above the background. However, neither the duplex DNA bound to DODC nor the free G4-DNA was observable. G4-DNA was detectable even in the presence of a high concentration of the duplex DNA. This demonstrates the ability to detect a small amount of dimeric hairpin quadruplex in the presence of a large amount of the duplex DNA. The concentration ratio was limited by the capacity of the gel.87 Satellite hole spectroscopy studies also supported the groove-binding model for DODC with the G4DNA.88 A different study using competition dialysis assay suggests that DODC has a preference for the triplex DNA compared to the G4-DNA. Although no structural information is available on this molecule in the DNA-bound form, it is possible that due to the geometric features, DODC could be binding to a Gquadruplex groove rather than stacking on a G-quartet. If this is indeed true, then its affinity should be highly sensitive to the groove dimensions, which vary along with the type of the G4DNA.62 Electrospray ionization mass spectrometric patterns also support the diethylthiocarbocyanine iodide (21, DTC) binding with the G4-DNA grooves.75,89,90 Dissociation patterns of the DTC/G4-DNA complexes resemble those of Dist-A. Therefore, it is predicted that DTC interacts with the parallel G4-DNA [d(T2G5T)4] through groove-binding.75 Tel01 was found to interact via end-stacking with the G4-planes of the G4-DNA.75 However, binding to G4-DNA and telomerase

inhibition by only DTC among all the carbocyanines tested appears to be a result which is ambiguous.90 SPR studies indicate that Dist-A binds very weakly to the telomeric DNA sequence attached on a support.72 The binding of Dist-A is likely to be seen in the experimental methods such as NMR, which employs high concentrations of the ligand. 72 Screening experiments with other MGB polyamides also did not reveal any significant G4-DNA interactions. These results are contrary to other reports, where Dist-A and its analogues have been studied with the G4-DNA using NMR and ESIMS.71−73,75 DODC was shown to have higher affinity toward the G-quadruplex grooves via stacking, as suggested by the negative ICD spectra, and is presented as a more promising G4DNA groove-binding model system.91 Dist-A did not show any induced signal. A new fluorescence-based study of the G4-DNA ligands was reported using DODC and DTDC as reporter molecules along with other G-quadruplex ligands.92 N-Methyl mesoporphyrin IX (NMM), DODC, and DTDC (22) allow the monitoring of several structural features of G4-DNA, as these molecules detect G4-DNA structure specifically. These three molecules were used as reporter molecules to screen six G4-DNA ligands. The fluorescence intensity of the reporter increases when it binds to the G4-DNA. Addition of ligands can either enhance or diminish the fluorescence due to the reporter molecule. DODC and DTDC also exhibit ICD upon binding to the most of the G4-DNAs indicating groove binding. NMM competes with DODC and DTDC in binding with d(G2T2G2TGTG2T2G2). NMM also competes with DODC in binding with d(T2AG3)4.92 Recently, binding of a novel cyanine dye 3,3′-di(3-sulfopropyl)-4,5,4′,5′-dibenzo-9-ethylthiacarbocyanine triethylammonium salt (ETC, 24, Figure 7) was studied with unimolecular human telomeric G-quadruplex, owing to its common properties with the G4-DNA ligands.93 ETC also has specific interactions with an intramolecular telomeric G4-DNA via end stacking.94 ETC stacks on one specific end of the hybrid, some selective parallel and antiparallel G-quadruplexes, or on both ends of the normal parallel G4-DNA. Nearby loop structures also play an important role in the binding. Some specific lateral or unusual propeller loops “snatch” part of ETC molecule and facilitate stacking on the end G-quartet, while the diagonal or special lateral snap-back loops block the access of ETC molecule to the G4-DNA frame. Several in vitro G4-DNA related biological activities of MGBLs were also reported. G-quadruplex ligands, e.g., TMPyP4, NMM, coraylene, Dist-A, DODC, and DTC, along with perylene diimides (PDI) were investigated as inhibitors of the G-quadruplex and the duplex DNA helicase activities of the T-antigen (T-ag).95 G-quadruplex helicase activity was found to be strong and similar to that of duplex DNA helicase activity of the T-ag. Analysis of the SV40 genome demonstrates the presence of sequences that may form intramolecular Gquadruplexes, which are presumed to be natural substrates for the G4-DNA helicase activity involving the T-ag. Dist-A, which does not inhibit the G-quadruplex unwinding activity of bloom syndrome homologue (BLM) gene, was found to be a weak inhibitor of the G4-DNA unwinding activity of the T-ag. DODC weakly inhibits the G′2 (bimolecular G-quadruplex) unwinding activity of the T-ag. A number of other G4-DNA ligands including Hoechst 33258 and DTC were found to be, however, not as effective inhibitors of the G4-DNA unwinding activity of the T-ag. PDIs were found to be potent and selective inhibitors of the T-ag associated G4-DNA helicase activity. 95 The effect of DODC on telomerase activity was also explored. 2363



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It reduced telomerase activity in pheochromocytoma PC-12 cells in a dose-dependent manner (10−100 mM) after 12 h of treatment.96 In another study, DODC inhibited telomerase in nasopharyngeal carcinoma NPC-Tax (a control NPC cell line), but not in NPC-TW01 cells, when studied with the extracts from cells, which were treated with 0.7 mM of DODC for 1−3 days. On direct addition of DODC to the study media, inhibition was observed at 10 mM for the NPC-Tax and 500 mM for the NPC-TW01 nasopharyngeal carcinoma cells, respectively.97 DODC itself was able to induce apoptotic cell death, although it did not reduce the telomerase activity in the NPC-TW01 cells. Instead, DODC induced the cell apoptosis via a mitochondrion-mediated mechanism. DODC inhibited the uptake of another mitochondrial probe 3,3′-dihexyloxacarbocyanine iodide. By proteomic comparative analysis, it was apparent that DODC induced the increase of prohibition level in the mitochondria, indicating an occurrence of the mitochondrial perturbation. Moreover, DODC increases the levels of p53 and an 18 kDa truncated Bax on mitochondria, which in turn potentiates the release of cytochrome c for the activation of caspases.97 In a different study, at the tested concentrations, DODC had little effects on the hydrolysis of the substrates under question in presence of either K+ or Li+ ions, or on the stability of the d[T24(G3TTA)3G3] G4-DNA and on the electrophoretic mobilities of either d[T 2 4 (G 3 T 2 A) 3 G 3 ] or d[T 2 4 GTGTGAGTG 2 AG 2 TGT GAG2T].98 DODC did neither inhibit the exonuclease hydrolysis nor the telomerase activity.97 It also destabilized the G4-DNA under experimental conditions. The synergistic effects between low-dose arsenic trioxide and DODC on the apoptosis of HT1080 cells were also investigated.99 Results reveal that low-dose arsenic could block cell cycle arrest at the G2/M phase and induce apoptosis, whereas DODC could block cell cycle arrest at the G0/G1 phase but does not induce apoptosis. However, cells pretreated with DODC showed greater sensitivity to arsenic than untreated cells. DODC alone could induce the hairpin G-quadruplex formation and inhibit the telomerase activity in a dose-dependent manner. It has been predicted that DODC can synergistically enhance the apoptosis induced by arsenic, suggesting that increased cell senescence in response to arsenic is induced by an altered telomere state rather than by a loss of telomerase.99 Rhodacyanine dye MKT077 (25, Figure 7) has also been reported to have telomerase inhibition activity.100 Due to its structural similarity with DODC, an interaction with the G4-DNA may be possible for the activity.62 The binding mode of DODC with the G4-DNA is a matter of considerable interest. One can imagine four possible modes of noncovalent association: (a) intercalation between the Gquartets, (b) binding through the grooves, (c) interaction with the thymine loops, and (d) outside binding. The possibility of outside binding can be easily eliminated on the basis of the fluorescence collisional quenching data and the ICD spectra. On the basis of the ICD spectra obtained for DODC complexed with two different G-quadruplexes [d(G4T4G4)]2 and [d(G3T4G3)]2, which have similar loops, binding only to the loops is overruled. Furthermore, the dye does not bind specifically to the unimolecular hairpin Watson−Crick duplex containing a T4 loop. Thus, groove binding and intercalation remain as the two plausible modes of specific binding.87 On the basis of preliminary NMR data, DODC is suggested to bind within the loops of the G4-DNA.92 CD and fluorescence

competition studies with these ligands also suggest binding with the loops of the G4-DNA. Atomic resolution data are, however, needed to unambiguously determine the structures of the G4DNA-cyanine dye complexes.92 However, for the cyanine dye− duplex DNA complexes, the appearance of ICD spectra is due to the groove binding. Other classical MGBLs (berenil, DAPI, SN-18071, etc.) have still not been examined for their interaction with the G4-DNAs.

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CONCLUDING REMARKS AND PERSPECTIVES Telomeres, rDNA, and promoter regions have been identified as potential targets, and there are over 350 000 predicted sequences in the genome that can fold into G-quadruplex structures, raising the daunting task of determining selective targets for these drugs.17,101 G-quadruplex ligands acting on both telomeric and nontelomeric quadruplexes have significant biological importance and quite a few of them are in clinical trials.18,29,102,103 Few examples include G4-DNA binding ligands, RHPS4,104 telomestatin,105 derivative of telomestatin HXDV,106 BRACO-19,107 porphyrin TMPyP4,15 quarfloxin (CX-3543),108 307A,109 triazines,110 and so forth. Quarfloxacin was originally developed to bind to the c-myc promoter. Instead, it appears to bind ribosomal quadruplexes, interfering with the binding of the protein nucleolin, which normally binds to the G-quadruplex structures, leading to cell death in cancerous cells. Quarfloxin is in the phase II clinical trials for the treatment of carcinoid/neuroendocrine tumors. 108,111 However, it is still a long way off to get G4-DNA binding ligands as successful anticancer agents. As discussed earlier, the ligands which bind to the Gquadruplex grooves also exert stabilizing effects. The grooves of the G4-DNA structures have different characteristics than the grooves of the duplex DNA, and thus, the ligands that bind only with the grooves of the G4-quadruplex selectively should be able to recognize DNA in a structure-specific manner with high affinity. DNA minor groove binding ligands are known to be able to distinguish the variable groove widths engendered by a specific sequence.112 G4-DNA structures give many variations within the generic theme of a G-quadruplex forming motif. Therefore, ligand design and development in the future would necessitate dealing with more complex issues of structural variations. Thus, this should not be limited to duplex versus G4-DNA distinction only. Availability of alternating groove widths in the G-quadruplexes in this regard may provide a platform toward selective targeting of specific topological and conformational forms of the G4-DNA. Ligands capable of spanning two or more grooves in the G-quadruplex may be able to distinguish one form from another based upon the appropriate groove dimensions and their specific orientations. Furthermore, “designed” MGBLs should be capable of selective targeting as well. To find new quadruplex groove binding agents, the molecular nature of the G-quadruplex grooves must therefore be considered judiciously. Interestingly, all known grooves of the G-quadruplex structures that have been characterized are chemically and conformationally quite different from the minor grooves of the duplex DNA. Thus, the recognition of the grooves of these nucleic acid structures is supposed to give a higher degree of selectivity over other types of DNA structural motifs, which may not be possible in the case of most of the existing end-stacking G-quadruplex ligands. Therefore, to understand the structural and conformational requirements for the recognition of the grooves of G4-DNA, 2364



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(7) Cech, T. R. (2000) Life at the end of the chromosome: Telomeres and telomerase. Angew. Chem., Int. Ed. 39, 34−43. (8) Chen, H., Li, Y., and Tollefsbol, T. O. (2009) Strategies targeting telomerase inhibition. Mol. Biotechnol. 41, 194−199. (9) Phatak, P., and Burger, A. M. (2007) Telomerase and its potential for therapeutic intervention. Br. J. Pharmacol. 152, 1003− 1011. (10) Balasubramanian., S., and Neidle, S. (2009) G-quadruplex nucleic acids as therapeutic targets. Curr. Opin. Chem. Biol. 13, 345− 353. (11) McEachern, M. J., Krauskopf, A., and Blackburn, E. H. (2000) Telomeres and their control. Annu. Rev. Genet. 34, 331−358. (12) Hurley, L. H. (2002) DNA and its associated processes as targets for cancer therapy. Nat. Rev. Cancer 2, 188−200. (13) Sen, D., and Gilbert, W. (1988) Formation of parallel fourstranded complexes by guanine-rich motifs in DNA and its implications for meiosis. Nature 334, 364−366. (14) Catasti, P., Chen, X., Moyzis, R. K., Bradbury, E. M., and Gupta, G. (1996) Structure−function correlations of the insulin-linked polymorphic region. J. Mol. Biol. 264, 534−545. (15) Siddiqui-Jain, A., Grand, C. L., Bearss, D. J., and Hurley, L. H. (2002) Direct evidence for a G-quadruplex in a promoter region and its targeting with a small molecule to repress c-myc transcription. Proc. Natl. Acad. Sci. U.S.A. 99, 11593−11598. (16) Todd, A. K., Haider, S. M., Parkinson, G. N., and Neidle, S. (2007) Sequence occurrence and structural uniqueness of a Gquadruplex in the human c-kit promoter. Nucleic Acids Res. 35, 5799− 5808. (17) Huppert, J. L., and Balasubramanian, S. (2007) G-quadruplexes in promoters throughout the human genome. Nucleic Acids Res. 35, 406−413. (18) Balasubramanian, S., Hurley, L. H., and Neidle, S. (2011) Targrtting G-quadruplexes in gene promoters: a novel anticancer strategy. Nat. Rev. Drug Discovery 10, 261−275. (19) Ou, T.-M., Lu, Y.-J., Zhang, C., Huang, Z.-S., Wang, X.-D., Tan, J.-H., Chen, Y., Ma, D.-L., Wong, K.-Y., Tang, J. C-O., Chan, A. S-C., and Gu, L.-Q. (2007) Stabilization of G-quadruplex DNA and downregulation of oncogene c-myc by quindoline derivatives. J. Med. Chem. 50, 1465−1474. (20) Davis, J. T. (2004) G-Quartets 40 Years Later: From 5′-GMP to molecular biology and supramolecular chemistry. Angew. Chem., Int. Ed. 43, 668−669. (21) Phan, A. T., Kuryavyi, V., Burge, S., Neidle, S., and Patel, D. J. (2007) Structure of an unprecedented G-quadruplex scaffold in the human c-kit promoter. J. Am. Chem. Soc. 129, 4386−4392. (22) Pedroso, I. M., Duarte, L. F., Yanez, G., Burkewitz, K., and Fletcher., T. M. (2007) Sequence specificity of inter- and intramolecular G-quadruplex formation by human telomeric DNA. Biopolymers 87, 74−84. (23) Jixun, D., Megan, C., Chandanamali, P., Roger, A. J., and Danzhou, Y. (2007) Structure of the hybrid-2 type intramolecular human telomeric G-quadruplex in K+ solution: insights into structure polymorphism of the human telomeric sequence. Nucleic Acids Res. 35, 4927−4940. (24) Phan, A. T., Luu, K. N., and Patel, D. J. (2006) Different loop arrangements of intramolecular human telomeric (3 + 1) Gquadruplexes in K+ solution. Nucleic Acids Res. 34, 5715−5719. (25) Dai, J., Carver, M., and Yang, D. (2008) Polymorphism of human telomeric quadruplex structures. Biochimie 90, 1172−1183. (26) Parkinson, G. N., Lee, M. P., and Neidle, S. (2002) Crystal structure of parallel quadruplex from human telomeric DNA. Nature 417, 876−880. (27) Phan, A. T., Kuryavyi, V., and Patel, D. J. (2006) DNA architecture: from G to Z. Curr. Opin. Struct. Biol. 16, 288−298. (28) Petraccone, L., Trent, J. O., and Chaires, J. B. (2008) The tail of the telomere. J. Am. Chem. Soc. 130, 16530−16532. (29) Yang, D., and Okamoto, K. (2010) Structural insights into Gquadruplexes: towards new anticancer drugs. Future Med. Chem. 2, 619−646.

many of the existing MGBLs should be chemically modified logically. Toward the success of the G-quadruplex binding ligands at the genetic level, the hypothesis proposed by Huppert is relevant,111 in that the fusion of the G4-DNA recognition molecules with systems that bind to specific sequences of the duplex DNA are feasible. Tethering MGBLs that are able to recognize six or more contiguous DNA base pairs (e.g., terbenzimidazoles)37−39,43 or longer “designed” Py/Im polyamides33−36,39 might provide an increase in the specificity and binding affinity depending on the nature, length, and/or flexibility of the linkers used. Some of the MGBLs discussed so far bind with the nonnatural or model G-quadruplexes like [d(TG4T)]4 and also those made from the modified nucleotides.71−78 These MGBLs may not be of much use in developing a drug. However, MGBLs that bind with the natural G-quadruplexes may be of significant medicinal importance. These include cyanine dyes,81−88 oligopeptide-based on distamycin analogues, in particular, the longer oligopeptides,69−73 and certain benzimidazoles.64−70 Longer oligopeptides lose helical phase matching with the grooves of the duplex-DNA and thus prefer to bind with the non B-DNA structures. Carbon skeleton of benzimidazoles,69,70 oligopeptides,85 and cyanine dyes may be suitably crafted to make them G-quadruplex DNA-specific. Thus, MKT-077, a compound structurally similar to DODC, is already being used in clinical trials as an anticancer agent. 113 It shows selective cytotoxity toward cancer cells,114 which may be due to its ability to localize specifically on the mitochondria of the cancer cells.113 Examples of G-quadruplex binding MGBLs discussed in this review should therefore serve as a useful source for the design of a whole new class of highly selective groove-binding ligands with attractive biological properties.

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AUTHOR INFORMATION

Corresponding Author *E-mail: [email protected]. Phone: (91)-80-22932664. Fax: (91)-80-23600529.

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ACKNOWLEDGMENTS This work was supported by a grant from the Department of Science and Technology (DST), New Delhi, India, as J. C. Bose Fellowship to S.B. A.K.J. is thankful to DST, New Delhi, for a Fast Track Grant for young scientists.

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REFERENCES

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