Specific binding of telomeric G-quadruplexes by hydrosoluble perylene derivatives inhibits repeat addition processivity of human telomerase

Telomerase is responsible for the immortal phenotype of cancer cells and telomerase inhibition may specifically target cancer cell proliferation. Ligands able to selectively bind to G-quadruplex telomeric DNA have been considered as telomerase inhibitors but their mechanisms of action have often been deduced from a non-quantitative telomerase activity assay (TRAP assay) that involves a PCR step and that does not provide insight on the mechanism of inhibition. Furthermore, quadruplex ligands have also been shown to exert their effects by affecting association of telomere binding proteins with telomeres. Here, we use quantitative direct telomerase activity assays to evaluate the strength and mechanism of action of hydrosoluble perylene diimides (HPDIs). HPDIs contain a perylene moiety and different numbers of positively charged side chains. Side chain features vary with regard to number and distances of the charges. IC₅₀ values of HPDIs were in the low micromolar (0.5–5 μM) range depending on the number and features of the side chains. HPDIs having four side chains emerged as the best compounds of this series. Analysis of primer elongation products demonstrated that at low HPDI concentrations, telomerase inhibition involved formation of telomeric G-quadruplex structures, which inhibited further elongation by telomerase. At high HPDI concentrations, telomerase inhibition occurred independently of G-quadruplex formation of the substrate. The mechanism of action of HPDIs and their specific binding to G-quadruplex DNA was supported by PAGE analysis, CD spectroscopy and ESI-MS. Finally, competition Telospot experiments with duplex DNA indicated specific binding of HPDIs to the single-stranded telomeric substrates over double stranded DNA, a result supported by competitive ESI-MS. Altogether, our results indicate that HPDIs act by stabilizing G-quadruplex structures in single-stranded telomeric DNA, which in turn prevents repeat addition processivity of telomerase.     

Tetrasubstituted naphthalene diimide ligands with selectivity for telomeric G-quadruplexes and cancer cells

A series of tetrasubstituted naphthalene diimide compounds with N-methylpiperazine end groups has been synthesized and evaluated as G-quadruplex ligands. They have high affinity and selectivity for telomeric G-quadruplex DNA over duplex DNA. CD studies show that they induce formation of a parallel G-quadruplex topology. They inhibit the binding of hPOT1 and topoisomerase IIIα to telomeric DNA and inhibit telomerase activity in MCF7 cells. The compounds have potent activity in a panel of cancer cell lines, with typical IC(50) values of ～0.1 μM, and up to 100-fold lower toxicity in a normal human fibroblast cell line.     

Perylene diimides with different side chains are selective in inducing different G-quadruplex DNA structures and in inhibiting telomerase

Four N,N'-disubstituted perylene diimides, having different side chains, have been studied for their ability in inducing G-quadruplex DNA structures. We found that electrostatic interactions between ligands side chains and DNA grooves play a main role not only in the amount of G-quadruplex formed, but also in selecting its topology. Moreover, such compounds show also a different ability to inhibit telomerase. The correlation of these findings suggests the intriguing possibility that different G-quadruplex structures could differently inhibit the enzyme.     

BRACO19 analog dimers with improved inhibition of telomerase and hPot 1

Human chromosomes terminate with telomeres, which contain double-stranded G-rich, repetitive DNA followed by a single-stranded overhang of the G-rich sequence. Single-stranded oligonucleotides containing G-rich telomeric repeats have been observed in vitro to fold into a variety of G-quadruplex topologies depending on the solution conditions. G-quadruplex structures are notable in part because G-quadruplex ligands inhibit both the enzyme telomerase and other telomere-binding proteins. Because telomerase is required for growth by the majority of cancers, G-quadruplex-stabilizing ligands have become an attractive platform for anticancer drug discovery. Here, we present the preparation and biochemical activities of a novel series of 3,6-disubstituted acridine dimers modeled after the known G-quadruplex ligand BRACO19. These BRACO19 Analog Dimer (BAD) ligands were shown to bind to human telomeric DNA and promote the formation of intramolecular G-quadruplexes in the absence of monovalent cations. As expected, the BAD ligands bound to telomeric DNA with a 1:1 stoichiometry, whereas the parent compound BRACO19, a monomer, bound with a 2:1 stoichiometry. The BAD ligands exhibited potent inhibition of human telomerase with IC₅₀ values similar to or lower than those of BRACO19. Furthermore, the BAD ligands displayed greater potency in the inhibition of hPot1 and increased selectivity for G-quadruplex DNA when compared to BRACO19. Collectively, these experiments support the hypothesis that there is an increased potency and selectivity to be gained in the design of G-quadruplex-stabilizing agents that incorporate multiple interactions.     

Fluorescence-based melting assays for studying quadruplex ligands

The telomeric G-rich single-stranded DNA can adopt in vitro an intramolecular quadruplex structure, which has been shown to directly inhibit telomerase activity. The reactivation of this enzyme in immortalized and most cancer cells suggests that telomeres and telomerase are relevant targets in oncology, and telomere ligands and telomerase inhibitors have been proposed as new potential anticancer agents. In this paper, we have analysed the FRET method used to measure the stabilization and selectivity of quadruplex ligands towards the human telomeric G-quadruplex. The stabilization value depends on the nature of the fluorescent tags, the incubation buffer, and the method chosen for T(m) calculation, complicating a direct comparison of the results obtained by different laboratories.     

G-quadruplex preferentially forms at the very 3' end of vertebrate telomeric DNA

Human chromosome ends are protected with kilobases repeats of TTAGGG. Telomere DNA shortens at replication. This shortening in most tumor cells is compensated by telomerase that adds telomere repeats to the 3′ end of the G-rich telomere strand. Four TTAGGG repeats can fold into G-quadruplex that is a poor substrate for telomerase. This property has been suggested to regulate telomerase activity in vivo and telomerase inhibition via G-quadruplex stabilization is considered a therapeutic strategy against cancer. Theoretically G-quadruplex can form anywhere along the long G-rich strand. Where G-quadruplex forms determines whether the 3′ telomere end is accessible to telomerase and may have implications in other functions telomere plays. We investigated G-quadruplex formation at different positions by DMS footprinting and exonuclease hydrolysis. We show that G-quadruplex preferentially forms at the very 3′ end than at internal positions. This property provides a molecular basis for telomerase inhibition by G-quadruplex formation. Moreover, it may also regulate those processes that depend on the structure of the very 3′ telomere end, for instance, the alternative lengthening of telomere mechanism, telomere T-loop formation, telomere end protection and the replication of bulky telomere DNA. Therefore, targeting telomere G-quadruplex may influence more telomere functions than simply inhibiting telomerase.     

Telomerase recognizes G-quadruplex and linear DNA as distinct substrates

Telomeric DNA can assemble into a nonlinear, higher-order conformation known as a G-quadruplex. Here, we demonstrate by electrospray ionization mass spectrometry that the two repeat telomeric sequence d(TGGGGTTGGGGT) from Tetrahymena thermophila gives rise to a novel parallel four-stranded G-quadruplex in the presence of sodium. The G-quadruplex directly interacts with the catalytic subunit of Tetrahymena telomerase (TERT) with micromolar affinity, and the presence of telomerase RNA is not obligatory for this interaction. Both N- and C-terminal halves of TERT bind the G-quadruplex independently. This G-quadruplex is a robust substrate for both recombinant and cell extract-derived telomerase in vitro. Furthermore, the G-quadruplex weakens the affinity of wild-type telomerase for the incoming nucleotide (dTTP) and likely perturbs the nucleotide binding pocket of the enzyme. In agreement with this, a lysine to alanine substitution at amino acid 538 (K538A) within motif 1 of TERT dramatically reduces the ability of telomerase to extend G-quadruplex but not linear DNA. The K538A mutant retains binding affinity for the quadruplex. This suggests that telomerase undergoes changes in conformation in its active site to specifically accommodate binding and subsequent extension of G-quadruplex DNA. We propose that telomerase recognizes G-quadruplex DNA as a substrate that is distinct from linear DNA.     

Design, synthesis, biophysical and biological studies of trisubstituted naphthalimides as G-quadruplex ligands

A series of trisubstituted naphthalimides have been synthesized and evaluated as telomeric G-quadruplex ligands by biophysical methods. Affinity for telomeric G-quadruplex AGGG(TTAGGG)(3) binding was first screened by fluorescence titrations. Subsequently, the interaction of the telomeric G-quadruplex with compounds showing the best affinity has been studied by isothermal titration calorimetry and UV-melting experiments. The two best compounds of the series tightly bind the telomeric quadruplex with a 2:1 drug/DNA stoichiometry. These derivatives have been further evaluated for their ability to inhibit telomerase by a TRAP assay and their pharmacological properties by treating melanoma (M14) and human lung cancer (A549) cell lines with increasing drug concentrations. A dose-dependent inhibition of cell proliferation was observed for all cellular lines during short-term treatment.     

G-quadruplex stabilizer 3,6-bis(1-methyl-4-vinylpyridinium)carbazole diiodide induces accelerated senescence and inhibits tumorigenic properties in cancer cells

Carbazole derivatives that stabilized G-quadruplex DNA structure formed by human telomeric sequence have been designed and synthesized. Among them, 3,6-bis(1-methyl-4-vinylpyridinium)carbazole diiodide (BMVC) showed an increase in G-quadruplex melting temperature by 13 degrees C and has a potent inhibitory effect on telomerase activity. Treatment of H1299 cancer cells with 0.5 mumol/L BMVC did not cause acute toxicity and affect DNA replication; however, the BMVC-treated cells ceased to divide after a lag period. Hallmarks of senescence, including morphologic changes, detection of senescence-associated beta-galactosidase activity, and decreased bromodeoxyuridine incorporation, were detected in BMVC-treated cancer cells. The BMVC-induced senescence phenotype is accompanied by progressive telomere shortening and detection of the DNA damage foci, indicating that BMVC caused telomere uncapping after long-term treatments. Unlike other telomerase inhibitors, the BMVC-treated cancer cells showed a fast telomere shortening rate and a lag period of growth before entering senescence. Interestingly, BMVC also suppressed the tumor-related properties of cancer cells, including cell migration, colony-forming ability, and anchorage-independent growth, indicating that the cellular effects of BMVC were not limited to telomeres. Consistent with the observations from cellular experiments, the tumorigenic potential of cancer cells was also reduced in mouse xenografts after BMVC treatments. Thus, BMVC repressed tumor progression through both telomere-dependent and telomere-independent pathways.     

Label-free fluorescence method for screening G-quadruplex ligands

G-quadruplex ligands can interfere with the telomere structure, telomere elongation/replication, and proliferation of cancer cells. A key element in the development of potent G-quadruplex ligands is the screening of large chemical libraries of potential candidates. Here, we describe a simple fluorescence method for screening of G-quadruplex ligands. The method is based on the ability of G-quadruplex ligands to displace hemin from G-quadruplex-based DNAzyme, resulting in a decrease of its catalytic activity on the fluorescence-developing reaction between p-hydroxyphenylacetic acid and H(2)O(2). The method eliminates the requirement for expensive and time-consuming preparation of labeled DNA. Our method provides a simple, cheap, and sensitive approach to screen G-quadruplex ligands (potential antitumor drugs).     

The relationship between ligand aggregation and G-quadruplex DNA selectivity in a series of 3,4,9,10-perylenetetracarboxylic acid diimides

Human telomeres are comprised of d(TTAGGG) repeats that are capable of forming G-quadruplex DNA structures. Ligands that bind to and stabilize these G-quadruplex DNA structures are potential inhibitors of the cancer cell-associated enzyme telomerase. Other potential biological uses of G-quadruplex targeting ligands have been proposed. One particularly challenging aspect of the contemplated uses of G-quadruplex targeting ligands is their selectivity for G-quadruplex DNA versus double-stranded DNA structures. We have previously reported the observation that two structurally related 3,4,9,10-perylenetetracarboxylic acid diimide-based G-quadruplex DNA ligands, PIPER [N,N'-bis(2-(1-piperidino)ethyl)-3,4,9,10-perylenetetracarboxylic acid diimide] and Tel01 [N,N'-bis(3-(4-morpholino)propyl)-3,4,9,10-perylenetetracarboxylic acid diimide], have different levels of G-quadruplex DNA binding selectivity at pH 7 as determined by absorbance changes in the presence of different DNA structures [Kerwin, S. M., Chen, G., Kern, J. T., and Thomas, P. W. (2002) Bioorg. Med. Chem. Lett. 12, 447-450]. Here we report that the less G-quadruplex DNA selective ligand PIPER can unwind double-stranded, closed circular plasmid DNA, as determined by a topoisomerase I assay. A model for the interaction of Tel01 with the G-quadruplex DNA structure formed by d(TAGGGTTA) was determined from NMR experiments. This model is similar to the previously published model for PIPER bound to the same G-quadruplex DNA and failed to provide a structural basis for the observed increased selectivity of Tel01 interaction with G-quadruplex DNA. In contrast, investigation into the aggregation state of Tel01 and PIPER as well as other 3,4,9,10-perylenetetracarboxylic acid diimide analogues bearing basic side chains demonstrates that ligand aggregation is correlated with G-quadruplex DNA binding selectivity. For all six analogues examined, those ligands that were aggregated at pH 7 in 70 mM potassium phosphate, 100 mM KCl, 1 mM EDTA buffer also demonstrated G-quadruplex DNA binding selectivity under these buffer conditions. Ligands that were not aggregated under these conditions display much lower levels of G-quadruplex DNA selectivity. The aggregation state of these ligands is extremely sensitive to the buffer pH. Tel01, which is aggregated at pH 7, is not aggregated at pH 6.4, where it demonstrates only modest G-quadruplex DNA binding selectivity, and PIPER in pH 8.5 buffer is both aggregated and highly G-quadruplex DNA-selective. To our knowledge, these studies demonstrate the first DNA structure selectivity as achieved through pH-mediated ligand aggregation. The potential impact of these findings on the selectivity of other classes of G-quadruplex DNA ligands is discussed.     

Targeting pancreatic cancer with a G-quadruplex ligand

The integrity of telomeres in most cancer cells is maintained by the action of the telomerase enzyme complex, which catalyzes the synthesis of telomeric DNA repeats in order to replace those lost during replication. Telomerase is especially up-regulated in metastatic cancer and is thus emerging as a major therapeutic target. One approach to telomerase inhibition involves the sequestration of the single-stranded 3' ends of telomeric DNA into higher-order quadruplex structures. We have recently shown that tetra-substituted naphthalene diimide compounds are potent quadruplex-stabilizing molecules with telomerase inhibitory activity in cells. We show here that one such compound, BMSG-SH-3, which has been optimized by computer modeling, has significant in vivo antitumor activity against a model for pancreatic cancer, a cancer that is especially resistant to current therapies. A large reduction in telomerase activity in treated tumors was observed and the naphthalene diimide compound was found to be selectively localized in the treated tumors. We find that the expression of the therapeutically important chaperone protein HSP90, a regulator of telomerase is also reduced in vivo by BMSG-SH-3 treatment. The compound is a potent stabilizer of two G-quadruplex sequences found in the promoter region of the HSP90 gene, as well as a G-quadruplex from human telomeric DNA. It is proposed that the simultaneous targeting of these quadruplexes may be an effective anti-tumor strategy.     

Binding of gemini bisbenzimidazole drugs with human telomeric G-quadruplex dimers: effect of the spacer in the design of potent telomerase inhibitors

The study of anticancer agents that act via stabilization of telomeric G-quadruplex DNA (G4DNA) is important because such agents often inhibit telomerase activity. Several types of G4DNA binding ligands are known. In these studies, the target structures often involve a single G4 DNA unit formed by short DNA telomeric sequences. However, the 3'-terminal single-stranded human telomeric DNA can form higher-order structures by clustering consecutive quadruplex units (dimers or n-mers). Herein, we present new synthetic gemini (twin) bisbenzimidazole ligands, in which the oligo-oxyethylene spacers join the two bisbenzimidazole units for the recognition of both monomeric and dimeric G4DNA, derived from d(T2AG3)4 and d(T2AG3)8 human telomeric DNA, respectively. The spacer between the two bisbenzimidazoles in the geminis plays a critical role in the G4DNA stability. We report here (i) synthesis of new effective gemini anticancer agents that are selectively more toxic towards the cancer cells than the corresponding normal cells; (ii) formation and characterization of G4DNA dimers in solution as well as computational construction of the dimeric G4DNA structures. The gemini ligands direct the folding of the single-stranded DNA into an unusually stable parallel-stranded G4DNA when it was formed in presence of the ligands in KCl solution and the gemini ligands show spacer length dependent potent telomerase inhibition properties.     

Stabilizing parallel G-quadruplex DNA by a new class of ligands: Two non-planar alkaloids through interaction in lateral grooves

Human DNA sequences consisting of tandem guanine (G) nucleotides can fold into a four-stranded structure named G-quadruplex via Hoogsteen hydrogen bonding. As the sequences forming G-quadruplex exist in essential regions of eukaryotic chromosomes and are involved in many important biological processes, the study of their biological functions has currently become a hotspot. Compounds selectively binding and stabilizing G-quadruplex structures have the potential to inhibit telomerase activity or alter oncogene expression levels and thus may act as antitumor agents. Most of reported G-quadruplex ligands generally have planar structures which stabilize G-quadruplex by π–π stacking. However, based on a pharmacophore-based virtual screening two non-planar G-quadruplex ligands were found. These two ligands exhibit good capability for G-quadruplex stabilization and prefer binding to paralleled G-quadruplex rather than to duplex DNA. The binding of these ligands to G-quadruplex may result from groove binding at a 2:1 stoichiometry. These results have shown that planar structures are not essential for G-quadruplex stabilizers, which may represent a new class of G-quadruplex-targeted agents as potential antitumor drugs.     

Interaction of an acridine dimer with DNA quadruplex structures.

The reactivation of telomerase activity in most cancer cells supports the concept that telomerase is a relevant target in oncology, and telomerase inhibitors have been proposed as new potential anticancer agents. The telomeric G-rich single-stranded DNA can adopt an intramolecular G-quadruplex structure in vitro, which has been shown to inhibit telomerase activity. The C-rich sequence can also adopt a quadruplex (intercalated) structure (i-DNA). Two acridine derivatives were shown to increase the melting temperature of the G- quadruplex and the C-quadruplex at 1 microM dye concentration. The increase in Tm value of the G-quadruplex was associated with telomerase inhibition in vitro. The most active compound, "BisA", showed an IC(50) value of 0.75 microM in a standard TRAP assay.