3.1.1 Anthraquinone derivatives

In 1997, Neidle and Hurley group reported the first quadruplex-interactive ligands. They developed 2,6-Diaminoalkylamido-anthraquinone, BSU-1051 (Figure 3), a telomerase inhibitor molecule with a telomerase IC₅₀ of 23 µM (Sun et al., 1997). From there on, wide range of the disubstituted amido-anthraquinone family such as 1,4-, 1,5-, 2,6-, and 2,7- isomers were developed with substituents at various positions on the chromophore by changing side chain length and size. The nature of the side chain terminal groups were further modified by systemic structure-function studies. Molecular modelling studies showed that anthraquinone compounds bind to G4 by a “threading intercalation” mode with π–π overlapping, where any flexible side chains can protrude into wider grooves of G4. But since the same binding mode is also observed for dsDNA, these compounds are not that much selective and specific for quadruplex DNA moreover their cytotoxicity can be high.

To reduce the cytotoxicity, a carbonyl group was removed from the anthraquinone moiety and a series of disubstituted 2,7-amido-fluorenones(2,7-FO) were designed and synthesized thus preventing redox cycling (Perry et al., 1999). These 2,7-FO derivatives have low telomerase IC₅₀ values of 8–12 µM with up to 10-fold reduced cytotoxicity in different human cancer cell lines, compared with that of their parent anthraquinone derivatives. This slightly less telomerase activity of 2,7-FO derivatives with that of parent molecule is due to decreased electron deficiency in the chromophore and the curved scaffold which is not favorable for π-stacking interaction with G4 structures (Perry et al., 1999). A more recent study suggests that introduction of morpholino substituent and two or more different side groups onto the fluorenone scaffold results in higher activity and selectivity to G-Quadruplex. They further deciphered that switching the side group from position 4 to 2 and establishing a positive charge increase the specificity of fluorenones to hybrid G-Quadruplex form (Alcaro et al., 2010). Tetracyclic anthraquinone analogues with another heterocycle attached to the anthraquinone core in place of side chains were reported. These G-Quadruplex interacting compounds also show anti-telomerase activity. Benzonaphtho-furan compound like tricyclic anthraquinones are modified to tetracyclic derivative. Molecular models showed that tetracyclic compounds provide more surface area suitable for stacking interaction with G-quartet.

3.1.2 Acridine derivatives

Chromophore containing positively charged central ring has been extensively used for designing G4 binding ligands as the positively charged ring is likely to be complementary to the negative electrostatic potential channel of G-Quadruplexes. Thus, to overcome the problem of the specificity and selectivity of anthraquinones for quadruplex DNA and duplex DNA, Neidle and his groups modified the acridine core and its side chains by introducing a pyridine ring in the ligand core in place of quinine which increase the positive charge of the ligand core thus enhancing the electrostatic interaction with the negatively charged G-quartet channel (Harrison et al., 1999). They synthesized a series of 3,6-disubstituted acridines including BSU 6039 (Figure 4) (Haider et al., 2003). But these compounds failed to increase telomerase inhibition probably because of the insufficient basicity of the pyridine ring nitrogen. Based on X-ray crystal structures and molecular modelling study of telomeric G4 and BSU 6039 complex, disubstituted acridines were further modified by replacing the core with acridine, changing the substitution pattern using polyamine. Using this strategy a variety of 3,6,9-trisubstituted acridines were synthesized (Harrison et al., 2003). The basicity of the pyridine ring nitrogen is increased by the incorporation of the anilino group at the 9-position, allowing the ligand core to be protonated at physiological pH. The 3,6,9-scaffold brings the 9-position substituents significantly deeper into the cavity of this groove as compared to disubstituted acridines providing an additional interaction with the third groove. Due to incorporation of the anilino group at the 9-position, the 3,6,9 acridine scaffold can interact better with the surface of this groove without creating steric clashes, especially when they carry a positive charge (Harrison et al., 2003). Two side chains at 3- and 6-positions were subjected to the two widest grooves. 3,6,9-Trisubstituted acridines analogues have much stronger binding affinity and specificity for quadruplex DNA over duplex DNA (10–70-fold greater binding constants), whereas 3,6- disubstituted acridines have similar binding constants for duplex and quadruplex DNA. BRACO-19, being a member of 3,6,9-trisubstituted acridines, showed very high binding affinity for quadruplex DNA over duplex DNA and high anti-telomerase activity in range of 10–20 nM (Moore et al., 2006a). Further study identified the antitumor activity of BRACO-19 in human xenografts (Burger et al., 2005). Stevens et al. reported that the N-methylated pentacyclic acridinium salts are another class of pentacyclic acridine derivatives. RHPS4, a pentacyclic acridine 3,11-difluoro-6,8,13-trimethyl(8H)-quino[4,3,2-kl] acridinium methylsulfate (Figure 4), is one such compound under this class which exhibits telomerase inhibitory activity (^telIC₅₀ 0.33 µM by TRAP assay) and inhibit cell proliferation within 2–3 weeks at non-cytotoxic concentrations (Bejugam et al., 2007). This high activity is due to the strong electrostatic interaction between the positively charged ligand core, brought by methylated quaternary nitrogen and negatively charged quadruplex center. However, this small-sized aromatic compound can also intercalate into duplex DNA with modest selectivity. Based on the finding that oxidized riboflavin binds to an intramolecular G-quartet with moderate affinity, trisubstituted isoalloxazines were also synthesized. These compounds lacks duplex selectivity but some trisubstituted isoalloxazines were found to be approximately 15-fold more selective for c-kit promoter than that of telomeric G4 (Bejugam et al., 2007). This result indicates that coplanar tricyclic small molecules can selectively bind to G4 over duplex DNA. Recent studies have shown that simple anthracenes with tethered side chains at appropriate positions can also act as selective G-Quadruplex ligands.

3.1.3 Bis-aryl compounds

Bis-aryl compounds are one of the most extensively used G4 stabilizing ligand. These compounds contain two symmetrical aromatic ring connected to the core, thus giving a “butterfly” like appearance. A variety of aromatic rings have been reported including, quinolinium, triazole, benzimidazole, and substituted phenyl rings. Linkers like urea, benzene, diketene, pyridine, benzene are generally attached to the core. Due to the flexible rotation of these linkers, these molecules can adapt different structural conformation so that they can fit into groove and loops of G4, showing higher binding affinity.

First reported bis-aryl compounds were bis-quinolinium triazines (compound 12459) (Gomez et al., 2003). These compounds show low cytotoxicity and acts as a potent human telomerase inhibitor (^telIC₅₀ = 0.13 µM) (Riou et al., 2002). Later derivatives of bisquinolinium compounds with a pyridodicarboxamide core (e.g., Compounds 360A) were synthesized and these compounds were found to be 100 times more selective to G4 than duplex DNA (Lemarteleur et al., 2004). Internal syn-syn hydrogen bond favors these ligands to interact G4 with high selectivity. Later Phen-DC derivatives (Figure 3) were synthesized with an expanded aromatic core, by replacing pyridine with bipyridine or phenanthroline thereby showing higher selectivity and specificity toward G4 than duplex DNA (Monchaud et al., 2008). PhenDC derivatives also exhibit anti-telomerase activity with ^telIC₅₀ value of 0.3 µM.

Ligands with diarylurea skeleton are extensively studied due to their drug like characteristics. These ligands contains 1,2,3-triazole rings attached to a diphenyl urea moiety (Drewe & Neidle, 2008). These ligands can orient themselves by positioning urea oxygen atom over the ion channel and allowing deep penetration of the side chains into G4 grooves by triazole–phenyl bond rotation thus stabilizing G-quartet more efficiently by π-stacking interactions. Further modelling studies suggested that the ortho- and meta-substituted analogues are optimal for G4-DNA affinity as they can adopt a “square-planar” conformation, than that of the para-substituted ligands which are sterically too large for optimal interaction with the G-quartet. Also, by the formation of an intramolecular H-bond, ortho-substituted analogues are conformationally constrained by the urea NH and the triazole-N3 atom, which enforces the desired “square-planar” conformation (Drewe & Neidle, 2008).

Due to the presence of three rotatable bonds in the scaffold, triphenylpyridine has one of the most rigid and diverse skeletons as G4 ligand. The side chains positioned by central core help to discriminate different G4 loops. Compounds with two amine chains linked directly to the aryl rings, with piperazine(N-Ppz) substituents confers specificity and the highest affinity to human telomeric and c-KIT G4-DNA, but fails to discriminate between them. Data of different substitution reaction, suggests that the protonation of the amine is critical. According to FRET study, compounds with a para-thiomethylphenyl or para-bromophenyl substituent on the central pyridine ring and four carbon atom length side chains, confers the higher stability and specificity toward all G4-DNA sequences over dsDNA. Compounds containing thiophene, furan and piperonal units exhibit much higher specificity for G4 over dsDNA but with moderate stabilization and ligands with alkyl amine phenyl ether, bulky benzyl ether and para-dimethylaminophenyl 4-substituents on the central pyridine present the poorest G4 stabilization (Table 1).

3.1.4 Oxazole derivatives

Telomestatin (SOT-095; Figure 3), a macrocyclic natural product that was isolated from Streptomyces annulatus 3533- SV4 in 2001 by Shin-ya et al. (2001), consists of seven oxazole rings and one thiazoline ring and is currently the most effective in-vitro telomerase inhibitor, with an ^telIC₅₀ value of 5 nM obtained from TRAP assay. This compound selectively binds to intramolecular G4 rather than intermolecular quadruplex and specific to G4 than duplex DNA with more than 70-fold preference. Docking and Molecular dynamics study confirmed that both enantiomer (R and S) of telomestatin binds with four guanine residues by the help of stacking interaction as well as interaction free energy thereby stabilizing the parallel G4 at a greater extent (Stefano et al., 2012). Anti-proliferative role of telomestatin has been reported from wide range of cancer cell lines like breast cancer (Tahara et al., 2006), myeloid leukaemia (Sumi et al., 2004), human pancreatic carcinoma (Liu et al., 2005) etc. Later, telomestatin has also been used to stabilizes and to throw light on the biological roles of other G4s formed at the promoter regions of various oncogenes like, BCL-2 (Dexheimer et al., 2006), c-MYC (Seenisamy et al., 2005), VEGF (Sun et al., 2005) etc. But certain disadvantages like low solubility in water makes intracellular delivery of the drug difficult. Furthermore, difficulty in large scale synthesis of telomestatin restricts its wider use.

Recently hexaoxazole macrocyles (HXDV), composed of two symmetric trioxazoles linked by amino acids, such as, valine and bistrioxazole acetate have been synthesized by Rice et al. (Barbieri et al., 2007; Minhas et al., 2006) Due to its telomestatin-like concave shape, HXDV (Figure 3) has been reported to show high specificity toward G4 by entropically driven “terminal capping” binding mode. To increase the water solubility of hexaoxazole macrocycles, amino acid linkers are modified by changing a valine residue to lysine and HXDL has been synthesized. HXDL interacts with G4 with higher affinity because of positively charged amine groups of lysine at physiological pH (Rzuczek et al., 2008). Hexaoxazole with an arginine linker has been found to have telomestatin-like activity. On the other hand, bistrioxazole acetates, having a macrocyclic bisamide structure, were synthesized with a ^telIC₅₀ ⁼ 2 µM (Tera et al., 2007).

3.1.5 Porphyrin derivatives

Porphyrins are one of the most well studied binding agents for duplex DNA. They show intercalating interaction while binding to dsDNA. The aromatic rings of porphyrin provide a planar arrangement which allows them to bind to the G4s by stacking interaction with the Guanine tetrads. Porphyrins have four N-methylated pyridines. Water solubility and π–π stacking interactions are increased by the polycationic moiety due to ionic configuration and decreased electron density of the core aromatic system. Hurley et al. reported TMPyP4 as the type molecule of this class (Figure 3). CD melting analysis and NMR experiments, divulge that TMPyP4 (tetra-(N-methyl-4-pyridyl)porphyrin) binds both dsDNA and G-Quadruplex structures (parallel and antiparallel) with only two fold greater affinity for G4 than duplex DNA (Dexheimer et al., 2006; Liu et al., 2005; Wheelhouse et al., 1998). Thus, TMPyP4 showed poor selectivity for G4 over duplex DNA. Later, it was found that TMPyP4 binds and stabilizes different G4 in oncogenic promoters like K-RAS, c-MYC, BCl-2, etc. CD melting and ITC experiments suggested that, TMPyP4 binds to c-MYC Pu27 G4 at a stoichiometry of 4:1 (TMPyP4:Pu27). But, its isomer TMPyP2 (tetra-(N-methyl-2-pyridyl) porphyrin showed very weak affinity to G4s. From the NMR Chemical shift, it was clearly observed that TMPyP4 binds to G4 by external stacking interaction. Recently, from the crystal structure of TMPyP4 and human bimolecular telomeric G4 complex, it was found that TMPyP4 stacks upon TTA nucleotides either at the external loop or at the 5′ region of the G4, involving hydrogen bonding between the base pairs which are not taking part in the G4 formation (Phan et al., 2005). As TMPyP4 is a standardized molecular skeleton to probe G4, this porphyrin skeleton is modified to increase G-Quadruplex selectivity and specificity. By replacing the cationic pyridinium ring of TMPyP4 with a phenol ring which possess a quaternary ammonium moiety, TQMP, a non-pyridinium cationic porphyrin has been synthesized, showing 30-fold increase in G4 selectivity than TMPyP4 through specific groove and loop binding (Wang et al., 2006).

Later to increase specificity, expanded porphyrin derivative, 5,10,15,20-[tetra(N-methyl-3-pyridyl)]-26,28-diselenasapphyrin chloride (Se2SAP) was synthesized and reported to bind selectively to G4-DNA with a 50-fold increase over duplex DNA (Seenisamy et al., 2005). Se2SAP was found to bind selectively to c-MYC G-Quadruplex even in the presence of other quadruplexes and duplex DNA and convert parallel c-MYC quadruplex into mixed parallel and anti-parallel hybrid conformation (Freyer et al., 2007). Se2SAP can even bind and stabilize bcl-2 promoter quadruplex into mixed anti-parallel and parallel hybrid structures (Dexheimer et al., 2006). But due to the low synthetic yields this ligand failed to draw further attention. At present, pentacationic manganese (III) porphyrin, containing four flexible cationic arms attached to the central aromatic core, is the most specific ligand that can discriminate between G4 and dsDNA, increasing the selectivity by 10,000 folds (Dixon et al., 2007). Other TMPyP4 variants, such as, cationic N-confused porphyrin (Zorzan et al., 2016), pyridyl-substituted corrole (Ma et al., 2009), porphyrazines (Gonçalves et al., 2006) and phthalocyanines have been recently reported. Among them, Tetramethylpyridinium porphyrazines (TMPyPz), nonsymmetrical phthalocyanine azo derivatives, was found to show higher binding affinity (by 100 fold) to human telomeric G4 than duplex DNA by 100-fold compared to TMPyP4 (Gonçalves et al., 2006). Zinc complex (Zn-TMPyPz) derivatives was found to show similar affinity and selectivity (Ren et al., 2007). Later water soluble octa-cationic zinc phthalocyanine (ZnPc) was also found to be an effective G-Quadruplex binding ligand with anti-telomerase activity (^telIC₅₀ = 0.23 µM) (Ren et al., 2007).

3.1.6 Quinacridine analogues

In 2001, Mergny et al. (2001b) reported a new class of crescent shaped dibenzophenanthroline (quinacridine) pentacyclic ligands containing five fused aromatic rings in a linear arrangement and extended amino side chains that increase the propensity of overlapping with the G-quartet and electrostatic interaction with grooves. Many quinacridine derivatives, including mono ortho quinacridine (MOQ) and mono meta quinacridine (MMQ) were already synthesized and CD melting studies suggest that these ligands bind to the human intramolecular quadruplex and have anti-telomerase activity (Hounsou et al., 2007). The two active compounds like disubstituted quinacridines (Figure 4) and cyclic-bis-quinacridine BOQ have ΔT_m values of 19.7 and 12.5°C, and IC₅₀ values of 0.028 and 0.5 µm, respectively (Mergny et al., 2001). Cyclic bis-quinacridine (BOQ1), scaffold is mainly composed of two quinacridines connected by polyamine linkers, having greater quadruplex selectivity than mono-quinacridine compounds. BOQ1 binds to G4 preferentially (ΔT_m = +28°C) over duplex DNA, due to the steric hindrance effect of the macro-cyclic scaffold which makes it more favorable for groove/loop interactions. Thus, BOQ1 appears as one of the most potential scaffold targeting G4. Recently, a novel platinum–quinacridine hybrid (PtMPQ) composed of single functional Pt moiety and mono-para-quinacridine, was reported to interact with quadruplex DNA by a dual covalent- noncovalent binding mode (Bertrand et al., 2007).

3.1.7 Berberine derivatives

Berberine, a plant alkaloid (Figure 3), was first isolated from Chinese herbs and reported to have anti-microbial activity but later it was established as a G4 stabilizing agent. In 1999, berberine was reported to have anti-telomerase activity (Naasani et al., 1999) and many 1,3-substituted berberine derivatives are synthesized by Neidle et al. These derivatives bind to G4 with very high affinity (Binding constants K_i = 2.05 × 10⁻⁶ M⁻¹ to 3.06 × 10⁻⁶ M⁻¹) (Franceschin et al., 2006; Zhang et al., 2007). They also found that piperidino-berberine has better stabilizing effect on G-Quadruplex and increased anti-telomerase activity. Investigation into the mechanism of binding of berberine to human telomeric G4-DNA using different DNA topologies by Moraca et al. revealed that in case of antiparallel folding, berberine seems to preferentially bind to G4-DNA loops and grooves, whereas in the hybrid-type conformations the binding probably occurs at the terminal G-tetrads. Using funnel-metadynamics simulations they also found that in case of biologically relevant parallel G4-DNA [PDB ID: 3r6r] two berberine molecules stacked at the 5′-end assuming an antiparallel orientation, whereas the other two are bound to the 3′-end in a parallel orientation (Moraca et al., 2017). This marked an important step in finding out the mode of interaction of molecules bearing similar chemical structure. Huang et al. synthesized 9-subsituted berberines having an alkyl side chain with a terminal amino group, which can efficiently interact G4 with greater binding affinity than berberine. 9-substituted berberines were again modified by the incorporation of one extra pyridine ring on berberine scaffold and thus, quinolino-benzo dihydroisoquindolium (QBDI) compounds (Figure 3) were synthesized, which have enhanced G4 interacting potential due to the increased aromaticity of the QBDI backbone. Later, it was reported that these 9-substituted berberine and QBDI derivatives possess higher selectivity for G-Quadruplex DNA in c-MYC oncogene than the others.

3.1.8 Quindoline derivatives

Molecular modelling study of anthraquinones and acridines derivatives revealed that a tricyclic chromophore is not specific for stacking interaction to G-quartet terminal. The disubstituted derivatives (Figure 4) of the natural product quindoline with two alkyl amino groups at the 2,7-positions or at the 2,10-positions, showed ^telIC₅₀ values in the range of 6–16 µM (Caprio et al., 2000) and another class of tetracyclic chromophore ligands containing four aromatic rings, benzo[b]naphtho [2,3-d]furan have been reported to have ^telIC₅₀ value of 7.0 µM. Another report suggests that 11-subsitituted quindolines show stronger anti-telomerase activity than the disubstituted quindolines, with ^telIC₅₀ values of 0.4–12 µM (Zhou et al., 2006) due to the presence of the nitrogen atom in the pyridine ring of quindoline. Substituted amino groups at the 11-position act as electron-donating groups which can enhance the basicity of the pyridine nitrogen atom (Zhou et al., 2006). This nitrogen atom in the pyridine ring can be protonated at physiological pH, thus increasing the electrostatic interaction between the positively charged quindoline scaffold and the negatively charged center of the G4. This 11-substituted quindoline also triggers G-rich sequence in c-MYC promoter to fold into a G4 and downregulate their cellular expressions (Ou et al., 2007).

3.1.9 Polyamides

Distamycin A (Dist-A; Figure 4) is a highly studied antibiotic which binds to the narrow, deep A/T sequence of the minor groove of B-DNA because of its curved helical polyamide structure. It binds to duplex DNA in a drug/DNA stoichiometry of 2:1 (Asagi et al., 2010). Later, it has been reported that Dist-A interacts with diverse array of parallel G-Quadruplex sequences. In 2007, Randazzo et al. reported the NMR structure of Dist-A-[d(TGGGGT)]₄ complex. They found that two dimers formed by four Dist-A molecules binds to two opposite grooves of the quadruplex through the interaction with G-quartets and sugar-phosphate backbone by the help of π–π stacking interaction. It also achieves a dipole-dipole interaction between G4 and the pyrrole ring and the carbonyl group of Dist-A (Martino et al., 2007). Different polyamides have been synthesized by using distamycin scaffold. Amide-linked oligopyrroles, 2,4 and 2,5-pyrrole polyamides show selectivity toward duplex DNA than G4 but selectivity toward G4 can be increased in these molecules by changing the heterocyclic skeleton (Moore et al., 2006b). These ligands show anti cell proliferative activity in human lung carcinoma (A549), human colorectal carcinoma (HT-29) and against breast cancer with IC₅₀ values in micromolar range (Rahman et al., 2009).

3.1.10 Perylene, coronene and napthalene diimide

Perylene is one of the most suitable G4 binding ligands due to its electron-rich polyaromacity and flat geometry. By the help of molecular docking studies, Fedoroff et al. synthesized PIPER (Figure 4) based on the perylene scaffold with ^telIC₅₀ values in the low-µM range (Fedoroff et al., 1998). NMR study indicates that PIPER binds to many G4 structures in 1:1, 1:2, or 2:1 stoichiometric ratios (Han et al., 1999). Later, PIPER derivatives were synthesized by the modification of the aromatic core and side chain lengths. Protonated or alkylated amino groups of the side chains increases the binding affinity with G4. Further incorporation of cationic side chains [DAPER4C(1,6)] increased the water solubility of hydrophobic perylene system (Michelia et al., 2009).

Coronene is another polyaromatic system used as a ligand. Many coronene derivatives were synthesized, which can stabilize G4-DNA. They also show anti-telomerase activity (Franceschin et al., 2007). Later, many naphthalene diimides substitutes were synthesized by incorporating DNA alkylating side chains varying groups by flexible spacers and/or spermidine- and spermine-like side chains with altering length between inner nitrogen atoms to increase G4 selectively (Hampel et al., 2010; Marchetti et al., 2015). These compounds have been shown to increase stabilization of telomeric and cKIT2 quadruplex sequence. The hydrophobic planar core of these molecules stacks on to G4 and the additional ionic interactions with the loops are increased due to the alkylated amino side chains.

3.2.1 Telomere

Telomere in general protects the chromosome end from shortening and regulates senescence and apoptosis. Telomerase, a telomeric G4 binding protein, plays a key role in tumorigenesis (Calabrese et al., 2018; Chen & Yang, 2012). This, together with the fact that telomerase is over-expressed in >85% of all tumors and across all tumor types, suggest that embarrassment of telomerase would be an operative anticancer therapeutic tactic (Burger et al., 2005). Since telomerase binds to telomeric G4, the later becomes a fine druggable target for cancer treatment. The most well studied ligand that was first developed against telomere G4 to inhibit telomerase was the N,N′-(9-((4-(dimethylamino)phenyl)amino)acridine-3,6-diyl)bis(3-(pyrrolidine-1-yl)propanamide), or BRACO-19 (B19; Figure 5) in short. The specification of B19 to G4 is due to an acridine core and a nitrogen atom in the heterocyclic frame that could be protonated at physiological environments (Ruggiero & Richter, 2018). As a result, the electron deficiency in the chromophore was amplified, with subsequent boost of the G4 interaction. These classes of compounds muddle to G4 via π–π interaction and additionally requests tertiary amine groups, since it is protonated at physiological pH, in their side chain to interact with the grooves. Telomestatin is another naturally occurring compound that binds most tightly to the telomeric G4 (Table 2). Though it has an EC₅₀ value of 5 nM, research by De Cian et al. (2007) show that it might not be that potent as it was claimed. Based on the idea of alkylating hybrid ligands that could combine the aptophore properties of flat aromatic moieties with reactive chemical groups Doria et al. developed the model of a new ligand where the naphthalendiimide core (NDI) was trussed to (NDI–QMPs) quinone methides. They synthesized a new class of tri- and tetra-substituted NDIs in order to conquer poor water solubility of the ensuing alkylated oligomers and investigated the G4 reversible recognition and triggerable covalent damage–stabilization of telomeric G4 structures (Doria et al., 2012).

3.2.2 hTERT

As mentioned earlier, the transcriptional instigation of this telomerase reverse transcriptase (hTERT) gene is crucial during tumorigenesis, its expression remains repressed in adult somatic cells (Saha et al., 2017). Thus, the expression of hTERT gene is highly regulated by several transcription factors as well as epigenetic state. It also harbors a G4 structure in its core promoter which regulates the expression of the catalytic subunit of the telomerase. Mutations in the hTERT promoter has been reported in >75% of glioblastomas, melanomas and in aggressive thyroid cancer and most importantly all these mutations are found to be in the G-rich region which forms quadruplex (Chaires et al., 2014; Huang et al., 2013; Vogelstein et al., 2013; Xing et al., 2014). Metastasis suppressor non-metastatic 2 (NME2) and RE1-silencing transcription factor (REST)–lysine-specific histone demethylase 1 (LSD1) co-repressor complex associates with the G4 in hTERT promoter to repress the expression of telomerase (Saha et al., 2017). In most of the cancer the hTERT G4 gets resolved to duplex DNA and in absence of G4 structure the NME2 and REST co-repressor complex fails to bind to the promoter resulting in the high level of telomerase expression leading to cancer and tumorogenesis. So technically stabilizing the hTERT G4 in cancer cell seemed to be novel therapeutic avenue. On treating with eleven G4 specific ligands ∼50%–85% downregulation of hTERT is seen in HT1080 cells (Figure 2) (Saha et al., 2017). But the problem is these ligands are not hTERT G4 specific ligand rather they bind to any G4 structures. Gomez et al. (2004) developed a 2,4,6-triamino-1,3,5-triazine derivative, ligand 12459, a potent G4 stabilizer present in hTERT intron 6 (Figure 5). It downregulates the expression of telomerase by altering the hTERT splicing pattern, thereby reducing the expression of active (+α+β) transcript and over-express inactive (−β) transcript. 12459 induces the guanine-rich region present in the 5′ end of intron 6 of hTERT to form quadruplex (Gomez et al., 2004). It is also more specific to hTERT G4 than telomeric quadruplex. Compared to telomestatin and B19, 12459 was found to be more potent in achieving 50% inhibition in annealing the guanine-rich strand with its complementary strand. The IC₅₀ value of the reaction was found to be 1.75 ± 0.37 µM for G4TERT1 sequence, which is way less than telomestatin and B19 which had IC₅₀ value of 5.0 ± 1.7 and 5.7 ± 1.1, respectively (Gomez et al., 2004). It also induces massive telomeric dysfunction along with DNA damage at 0.5–1 µM concentration (Douarre et al., 2013). 12459 reforms the hTERT alternative RNA splicing in A549 cells, thus suggesting that 12459 affects hTERT mRNA processing rather than hTERT DNA transcription leading to the downregulation of telomerase activity previously observed in A549 cells. The IC₅₀ values of 12459 were found to be 1.75, 2 and 6.25 mM for G4TERT1, G4TERT2 and VNTR6-1, respectively. Two other potent G-Quadruplex ligands, telomestatin and BRACO19, did not amend splicing and transcript levels of hTERT, suggesting that the change in the RNA splicing is specific for triazine derivative (Gomez et al., 2004). Hence to conclude we can assume that 12459 induces the formation of G4 structure causing alternative splicing to reduce active telomerase formation. Stabilizing the G4 also allows the NME2 and REST co-repressor complex to bind to it and repress telomerase expression. Many structurally related Ru-polypyridyl complexes has already been reported to have higher affinity toward G-Quadruplex. Using Time-Resolved Infrared Spectroscopy, Devereux et al. (2020) showed the binding of the light-switch compound, [Ru(phen)₂(dppz)]^{2 +}, to quadruplex DNA in solution. Recently the first X-ray crystal structure of a quadruplex, d(TAGGGTT) bound Ru^IIdppz derivative has been reported. Besides organic G4-ligands, metal complexes based G4-ligands have recently attracted a lot of interest because of their selectivity. A series of TMPyP4 complexes with various metal centers, including main-group metals and transition metals (Ni(II), Mn(III), Mg(II), Cu(II), Zn(II), Pd(II), Pt(II), Fe(III), Co(II) etc.) have been synthesized. All these TMPyP4-metal complexes efficiently found to stabilize the hTelomere quadruplex and inhibit telomerase activity in vitro, due to the π-stacking abilities and cationic charge. Gold(III)–TMPyP4 complex has also been reported as an hTelomere G4 stabilizer and a potent telomerase inhibitor (Cao et al., 2017). Another novel platinum-quinacridine hybrid, composed of a monofunctional Pt moiety and a G-Quadruplex ligand (mono-para-quinacridine or MPQ), has been synthesized and reported to interact with quadruplex DNA (22AG, oligonucleotide that mimics the human telomeric repeat) via a dual noncovalent/covalent binding mode (Bertrand et al., 2007).

3.2.3 BCL-2

Certo et al. (2006) found that being the central protein to maintain a balance between apoptosis and survival, tumor cells depend upon the BCL-2 for their survival. Aberrant expression of BCL-2 is also responsible for neurodegenerative disease like Parkinson, Alzheimer's and multiple sclerosis (Kuhlmann et al., 1999; Marshall et al., 1997; Satou et al., 1995). There are G-Quadruplex located upstream of major promoter P1 of BCl-2 (Dexheimer et al., 2006). Several organic molecules are reported to bind to BCL-2 G4. But they fail to discriminate between BCL-2 G4 and other G4 in cell, thereby hampering their advancement as clinical drugs. A derivatives of furo[2,3-d]pyridazin-4(5H)-one (FP3; Figure 5) could selectively bind to the BCL-2 quadruplex and stabilize it; thereby down-regulating the expression and transcription of BCL-2 gene (Amato et al., 2018). The pyridazinone moiety was selected because of its anti-cancerous, anti-inflammatory and anti-thrombotic property. FP3 also was able to discriminate and selectively binds to BCL-2 G4. It showed substantial cytotoxicity in Jurkat cells with 30% inhibition at 10 µM concentration and 40% reduction in Bcl-2 protein on treatment with 25 µM for 24 hr (Table 2). Jana et al. recently confirmed that another quindoline derivative, chelerythrine, interacts with BCL-2 G4. Even though it shows higher quadruplex-to-duplex selectivity and induce selective cytotoxicity to varied cancer cells, the molecule is found to interact with telomeric and other oncogenic quadruplexes (Jana et al., 2017).

3.2.4 c-MYC

A triaryl-substituted imidazole/carbazole conjugate, with two 1-methylpiperazine side chains and a phenyl moiety in the imidazole ring, IZCZ-3 (Figure 5), can selectively bind c-MYC G-Quadruplex. It was seen that on removing the 1-methylpiperazine side chain the selectivity to c-MYC quadruplex was reduced. The 1-methylpiperazine helps to increase electrostatic interaction and the phenyl moiety reduce its binding affinities with antiparallel or hybrid G-Quadruplexes thus focusing it to bind to parallel c-MYC G-Quadruplexes (Hu et al., 2018). This ligand has the four-clover leaf like structure which as shown in Figure 5, stacked perfectly on the terminal G-quartet plane of c-MYC via π–π interaction, and the positively charged central imidazole moiety was located in the ion channel of the c-MYC G-Quadruplex, leading to a relatively low binding energy of −8.7 kcal/mol (Hu et al., 2018), thereby showing significant anti-cancer activity by arresting cancer cells at G0/G1 cell cycle. Another molecule 6-methyl-3-(naphthalen-2-yl)-8a-(4-methylpyridin-2-yl)-indolizinone(indolizinone) also selectively recognize c-MYC G-Quadruplex in 1:1 binding model (Yao et al., 2017). Thus, it was seen that molecule with aromatic group can stack onto the quadruplex and interact strongly (Yao et al., 2017) (Table 2). A series of Zn(II) complexes with amido-armed phthalocyanines has also been reported with excellent affinities and specificities for hTelomere G4. These molecules also showed the strongest binding (K_D = 2 nM) when interacting with c-MYC G-Quadruplex DNA (Alzeer et al., 2009). A penta-coordinated Au(III) pyrazolylpyridine complex has been reported to interact strongly and selectively with c-MYC quadruplex DNA, through π–π stacking (Suntharalingam et al., 2010).

3.2.5 KRAS

KRAS is another most frequently mutated yet undruggable oncogene in human cancer. To target KRAS at gene level and develop new therapeutic strategy, one regioisomers of 5-methyl-indolo[3,2-c]quinoline derivatives (IQc:3e; Figure 5) with a range of alkyl diamine side chains was designed to target DNA promoter of the KRAS gene. IQc:3e has flat extended heteroaromatic surface that can stack onto G-quartets through π–π stacking and the basic side chains can hydrogen bond and establish electrostatic interactions with the phosphate backbone and loops/grooves of G4-DNA, either directly or mediated via water molecules. It was seen that molecules lacking alkyldiamines as their side chain produced lower affinity toward KRAS gene. The IQc:3e has IC₅₀ value of 1.8 and 1.98 µM against KRAS dependent colon cancer cell line HCT-116 and pancreatic cancer cell line MiaPaCa2, respectively (Table 2). Whereas it has higher IC₅₀ value for KRAS independent breast cancer cell line (IC50 = 11.40 µM) and lung fibroblasts WI-38 cell line (IC₅₀ = 10.08 µM), respectively (Lavrado et al., 2015). Later, a series of novel multicarbazole derivatives, tricarbazolebenzene (TCB), has been synthesized to enhance G4 selectivity and stabilization. Further it was shown that C3-symmetry and a crescent shaped architecture incorporated by heteropolyaromatic multicarbazole scaffold impart both G4 interaction and selectivity. Ou et al. reported that multicarbazoles can bind selectively to G-Quadruplex over duplex DNA and show a higher stabilization for KRAS G4 compared to telomeric and other promoter G-Quadruplexes. They also reported the first high-resolution (1.6 Å) crystal structure of KRAS G-Quadruplex as a 5′-head-to-head dimer with extensive poly-A π-stacking interactions (Ou et al., 2020). Zinc complex of an isopropylguanidinium- modified phthalocyanine has also been reported as potent stabilizer KRAS promoter G-Quadruplex DNA (Membrino et al., 2010).

3.2.6 cKIT

Another gene c-KIT is generally involved in diverse biological function namely, maintenance of hematopoietic stem cell, melanogenesis, erythropoiesis, spermatogenesis, etc including cancer (Ray et al., 2008). Its dysregulation gives rise to plethora of diseases including cancer. Its gain-of-function mutation is linked to gastrointestinal stromal tumors (>70%), mast cell tumors (>70%) and some acute myeloid leukemia (>68%) (Zorzan et al., 2016). At first tyrosine kinase inhibitor (TKI) was used to treat cancer overexpressing c-kit. But soon cancer cells developed drug resistant against TKI leading toward the failure of those drugs. Just upstream c-KIT transcription start site, there are two G4 forming element, KIT1 and KIT2, regulating the c-KIT expression and hence they offer an excellent target for drug. Two anthraquinone derivative with (AQ1) and without (AQ7) phenylalanine moiety in its side chain was found to selectively bind to the c-KIT G4. The binding affinity drastically reduced on removing the phenylalanine moiety. Based on Alamar Blue cytotoxicity test, AQ1 (Figure 5) was found to have an IC₅₀ value of 1.65 and 3.00 µM for HCG27 and MCF-7 cells, respectively (Zorzan et al., 2016). Whereas AQ7, being a better c-KIT quadruplex binder, showed meagre to no cytotoxicity. This is could be due to the inability of AQ7 in reaching intracellular target due to its large side chain. This proves that affinity does not corresponds to cytotoxicity every time. Another derivative of indenoisoquinolines (ISb) also selectively targeted c-KIT G4. Previously indenoisoquinoline derivatives have revealed antiproliferative properties in several human cancer cell lines (Morrell et al., 2007a, 2007b). ISb particularly stabilized c-KIT1 G4 having IC₅₀ value of 4.1 ± 0.43 µM and 0.3 ± 0.034 in c-KIT overexpressed Gastrointestinal stromal tumor GIST882 cell line and colorectal cancer cell line HT-29, respectively (Table 2) (Bejugam et al., 2010). Recently an indolo-naphthyridine derivative discovered through structure-based pharmacophore models has shown strong affinity for c-KIT1 G4 as the result of the π–π stacking and two hydrogen bond interactions betrothed by morpholinopropyl side chain with the backbone residues of c-KIT1 G4. But being a non-optimized compound, this molecule could only impart very low cytotoxicity on HT-29 and A2780 cell lines (Catalano et al., 2019).

3.2.7 rDNA

In genome perhaps, the densest population of G4 after telomere is present in rDNA having 12% more G4 than rest of the genome. There are approximately 1,500 G4 motifs in rDNA non-transcribed strand that act as transcription activator (Sabouri et al., 2014). RNA Polymerase I complex (Pol I) is responsible for transcription of rRNA genes and as a result control ribosome biogenesis. Studies by Derenzini et al. have found that in cancer the Pol I activity and rRNA synthesis is remarkably high (Derenzini et al., 2000). Nucleolin stabilizes these G4 structure and enhances the activity of Pol I, to synthesize more rRNA (Hanakahi et al., 1999; Rickards et al., 2007). Thus, this nucleolin/G4 complex offers a potential therapeutic target to treat cancer. A fluoroquinolone derivative called quarfloxin (Figure 5) have been developed which targets and disrupts nucleolin/G4 complex inducing apoptosis in cancer cells. This is the first molecule to go to Phase II clinical trial (Drygin et al., 2009). It is selective in disrupting rRNA synthesis and does not inhibit DNA or protein synthesis. It has an IC₅₀ value of 3.3 µmol/L (Table 2) (Hanakahi et al., 1999). This molecule is highly selective and kills cancer cells associated with over-expressive ribosome biogenesis. It does not hamper cellular effects associated with other G4 structures of other oncogenes. Quorfloxin is also resistant to multi drug resistant and hence has been developed as a key therapeutic molecule.

3.2.8 VEGF

Vascular endothelial growth factor (VEGF) plays a pivotal role for tumor angiogenesis by stirring the growth, migration, survival and permeability of endothelial cells thereby causing metastases. There are several VEGF targeting drugs available in market such as bevacizumab (Hurwitz et al., 2004; Sandler et al., 2006) sorafenib (Escudier et al., 2007) etc. The VEGF promoter contains a guanine-rich region which can fold to G4 structure and provides a platform for binding of multiple transcription factor such as sp1, Ap2 and Egr1 to regulate VEGF expression at transcriptional level (Sun et al., 2008). This calls for a novel methodology for rational drug design targeting VEGF quadruplex. Wu et al. (1840) synthesized quindoline derivatives and introduced 5-N-methyl and 7-fluoro groups to augment its interaction with the VEGF G-Quadruplex DNA. The IC₅₀ of SYUIQ-FM05 (Figure 5) was found to be 1.0 ± 0.4 μM which is much lower compared to its parent molecule SYUIQ-05 (IC₅₀ = 16.6 ± 0.7 μM; Table 2). Electron donating alkyl group, Fluorine and N,N-dimethylpropane-1,3-diamine side chain thus made it more potent and VEGF specific drug. The positive charge in the 5-N-position contributes to electrostatic interaction and improves the π–π stacking to quadruplexes. The alkyl amino group at the 11-position increases basicity of the quinolone nitrogen, which allows in situ protonation of the 5-N-atom at physiological pH and the cationic ligand core to contact the negatively polarized quartet center. Further VEGF transcription was suppressed dramatically at 0.5 µM concentration. But it also targets c-KIT and BCL-2 G4, thus lacks selectivity (Cammas et al., 2015; Wu et al., 1840).

3.2.9 c-Myb

c-Myb gene was first associated with leukemia, later it was found to be linked with lymphomas, colonic tumors and several other solid tumors. The role of c-Myb gene in regulating cellular proliferation, differentiation and apoptosis is well established thus is an important switch in cancer therapy. The mammalian c-myb promoter contains three highly conserved GGA motif (Q3, Q2, Q1). Matsugami et al. (2001) solved the structure of one GGA G-Quadruplex motif and found that it forms a intramolecular or intermolecular quadruplex with a tetrad:heptad:heptad:tetrad (T:H:H:T) structure (Palumbo et al., 2008). Cui and Yuan (2011) demonstrated using ESI-MS that dehydrocorydaline, a natural compound can distinguish duplex from the cmyb quadruplex and bind to it specifically. This compound binds to the quadruplex with 1:2 binding stoichiometry to the cmyb quadruplex and increases the melting temperature by 18°C which is high enough for the molecule to be treated as a c-myb stabilizing agent (Cui & Yuan, 2011). Furthermore, it prefers the end-stacking mode with one molecule at each end site through π–π interaction with the G-Quadruplex. Another ligand dehydroevodiamine isolated from Evodia rutaecarpa is found to bind selectively with this promoter (Li et al., 2017). This ligand having hypotensive (Yang et al., 1990), antiamnesic and antiarrythmic (Loh et al., 2014) properties, binds to all three quadruplex structures. One interesting fact is that both compounds are natural alkaloid extracted from a Chinese herbal medicine thus the ligands have far fewer side effects. Li et al. after screening several molecules found topotecan (Figure 6), a small molecule with aromatic system having more affinities toward c-myb quadruplex. The binding is further strengthened by its heteroatoms which augments the electrostatic interaction with G-Quadruplex (Li et al., 2018). But this molecule stabilizes the quadruplex structure and enhances the transcriptional as well as translational activity of the c-myb gene. Hence topotecan acts as an enhancer of mRNA and protein expression (Li et al., 2018).

3.5.1 Cyanines

Cyanines are a class of dye that have the tendency to bind to the G-quartet structures. The pentamethine analogs of cyanine dye unveiled an entropically driven (Nanjunda et al., 2012) initial 1:1 binding stoichiometry with an end-stacking binding mode with a preferential binding for parallel over hybrid G-Quadruplexes followed by a weaker binding. The pentathemine linker furnished the desired elasticity for efficient stacking at the terminal tetrads of the quadruplex. Further dimethyl substitutions on both indolenine rings substantially diminish their affinity for DNA duplex (Nanjunda et al., 2013) as the bulky methyl group causes steric hindrance and fails to bind to the duplex groove. The quadruplex affinity and solubility are further enhanced by introducing ammonium cations to the heterocyclic nitrogen. Halogenation at the 5-position and the meso-carbon of the polymethine-bridge helps increasing the pharmacological features (Marcelo Zaldini et al., 2010). The dimethyl indolenine cyanine scaffold was selected based on the concepts that it could not fit into the minor groove, as with planar cyanines, but could stack on the terminal tetrads of quadruplex structure with cationic chains in the quadruplex grooves. On the other hand, another DNA sensitive cyanine dye- meso-substituted trimethine “Cyan 2” interacts strongly with both triplex (through groove binding) and G-Quadruplex DNA (through DNA intercalation) (Kovalska et al., 2011). But since cyan 2 can bind to G4-DNA only in absence of K⁺ and other cations its use as in-vivo probe is limited (Kovalska et al., 2011). Research by Tang's group shows that the size of aromatic group also plays a very important role in the quadruplex selectivity. He showed that large aromatic substituents are required for aggregation and higher selectivity toward G-Quadruplex structures whereas smaller aromatic substituents lack quadruplex selectivity suggesting that large surface area are essential for proper π–π stacking of the dye with quadruplex (Yu et al., 2015). Cyanines are thus a major group which can bind to quadruplex using end-stacking mode and act as a probe for G-Quadruplex visualization.

3.5.2 Template-assembled synthetic G-Quartets (TASQs)

G-quartets are classically templated and stabilized by cations, whereas in the absence of cations guanines aggregates to form ribbon like structures (Sessler et al., 2000). Cation-free G-quartets are rare due to the repulsion of the coplanar carbonyl groups and the high stability of less-ordered polymeric ribbons. Nikan and Sherman (2008) developed a lipophilic guanine linked cavitands named template-assembled synthetic G-Quartets (TASQ) by click chemistry which are stable at low concentration in a cation independent environment. The chemical shift value of ¹H NMR of this compound suggests that the guanine bases of TASQs are not assembled as the guanines of native quadruplex. They also found a strong intra-residue NOE among H8 and H1′ signifying a syn conformation along with glycosidic bond which thwarts the unstructured G-ribbon formation. A negligible shift (Δδ = 0.2 ppm) in the imino signal even at 50°C indicates a stable H-bond and hence a stable G-quartet structure at high temperature. These TASQs have high application as G-quartet aptamers or as G-quartet recognizing protein screens. Another water soluble TASQ named ^PNADOTASQ (DOTA-templated synthetic G-quartet), acts as both DNA and RNA G-Quadruplex Ligand (Haudecoeur et al., 2013). The DOTASQ also can catalyse pseudo-enzymatic oxidation reactions in a DNAzyme-inspired manner. Previous reports show that quadruplex/hemin complex in presence of H₂O₂ can perform peroxidase like oxidation reaction (Josephy et al., 1982). This TASQ can in fact replace quadruplex and mimic peroxidase and perform the same function albeit with a decreased affinity (Haudecoeur et al., 2013). They also have several other bionanotechnological applications that are yet to be fathomed. It is of no doubt that though research with synthetic Quadruplex (SQ) has just begun but the application and rich diversity that the field present is astounding, and its true potential is yet to be realized in nanobiotechnology.

3.5.3 Distyryl dyes

The distyryl skeletons are synthesized by condensation between pyridinium salts and aldehydes. Distyryl dyes has been used in designing of sensors for different types of analytes (Li et al., 2004). Spectroscopic studies showed that cationic dyes like 2,4-bis(4-dimethylaminostyryl)-1-methylpyridinium and its quaternary aminoalkyl derivative not only selectively binds to G-Quadruplex in micromolar concentration with far greater affinity compared to dsDNA but also can discriminate between different G-Quadruplex (Xie et al., 2013). Recent report suggested that these dyes interact with human telomeric G-Quadruplex with a very high affinity than that of oncogenic promoter G-Quadruplex. One sees an increased fluorescence intensity and melting temperature by the binding of c-MYC and c-KIT₁ G-Quadruplex with these dyes. They even can selectively detect G-Quadruplex structure from dsDNA crowded condition. For this specificity, they can be used for selective staining of G-Quadruplex DNA in polyacrylamide gel. Due to its high fluorescence enhancement and luminescence responses, this distyryl dye can be used as a promising G-Quadruplex probe.

3.5.4 BODIPY dyes

Due to its high molar absorption coefficients, sharp absorption bands, high fluorescence quantum yields and photochemical stability, BODIPY dyes has been used as probe (Ulrich et al., 2008). Recently few BODIPY based molecules has been synthesized by screening a library of molecules, which can specifically bind to parallel G-Quadruplex DNA with exposed ends and four medium grooves with higher affinity than that of anti-parallel G-Quadruplexes, dsDNA, or ssDNA. Molecular modeling study showed that these compounds prefers groove binding upon end stacking (Zhang et al., 2014). Hence, BODIPY dyes can be used as a potential groove targeted, structure sensitive G-Quadruplex probe. The importance of disaggregation and carboxymide group in these sensors for fluorescent probing has also been demonstrated. The hydrogen binding capacity of the carboxymide group with guanine and adenine is essential for high affinity docking to the quadruplex (Zhang et al., 2014).

3.5.5 Tetraphenylethene dyes

There are some non-emissive dyes whose fluorescence intensity can be enhanced by aggregate formation. Tetraphenylethene (TPE) dyes has this unique aggregation-induced emission properties. Two tetraphenylethene derivative with positively charged side arms, that is, DATPE (1,2-Bis-1,2-tetraphenylethene dibromide; (Zhang et al., 2015)) and QATPE (1,1,2,2-Tetrakistetraphenylethene tetrabromide), two and four 4- (N,N,N,-trimethylammonium)butoxy substituents, respectively, has been synthesized (Zhang et al., 2015). DATPE binds only with multimeric G-Quadruplexes, having a low in-vivo cytotoxicity and little effect on the stability of multimeric G-Quadruplexes. On the other hand, QATPE not only has a high binding affinity toward multimeric G-Quadruplex but also has a higher stabilizing activity on them. But the lower G-Quadruplex recognizing activity compared with that of DATPE, makes QATPE a promising multimeric G-Quadruplex targeting drug candidate. Due to the low cytoxicity, tetraphenylethene dye emerges as a promising probe for detection of multimeric G-Quadruplex in cells.