Interactions of the human telomeric DNA with terbium-amino acid complexes

The human telomeric DNA can form four-stranded structures: the G-rich strand adopts a G-quadruplex conformation stabilized by G-quartets and the C-rich strand may fold into an I-motif based on intercalated C.C(+) base pairs. There is intense interests in the design and synthesis of compounds which can target telomeric DNA and inhibit the telomerase activity. Here we report the thermodynamic studies of the two newly synthesized terbium-amino acid complexes bound to the human telomeric G-quadruplex and I-motif DNA which were studied by means of UV-Visible, DNA meltings, fluorescence and circular dichroism. These two complexes can bind to the human telomeric DNA and have shown different features on DNA stability, binding stoichiometry, and sequence-dependent fluorescence enhancement. To our knowledge, this is the first report to show terbium-amino acid complexes can interact with the human telomeric DNA.     

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.     

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.     

Duplex and quadruplex DNA binding and photocleavage by trioxatriangulenium ion

The stable trioxatriangulenium ion (TOTA) has previously been shown to bind to and photooxidize duplex DNA, leading to cleavage at G residues, particularly 5'-GG-3' repeats. Telomeric DNA consists of G-rich sequences that may exist in either duplex or G-quadruplex forms. We have employed electrospray ionization mass spectrometry (ESI-MS) to investigate the interactions between TOTA and duplex DNA or G-quadruplex DNA. A variety of duplex decamer oligodeoxynucleotides form complexes with TOTA that can be detected by ESI-MS, and the stoichiometry and fragmentation patterns observed are commensurate with an intercalative binding mode. TOTA also forms complexes with four-stranded and hairpin-dimer G-quadruplex oligodeoxynucleotides that can be detected by ESI-MS. Both the stoichiometry and the fragmentation patterns observed by ESI-MS are different than those observed for G-tetrad end-stacking binding ligands. We have carried out (1)H NMR titrations of a four-stranded G-quadruplex in the presence of TOTA. Addition of up to 1 equiv of TOTA is accompanied by pronounced upfield shifts of the G-tetrad imino proton resonances in the NMR, which is similar to the effect observed for G-tetrad end-stacking ligands. At higher ratios of added TOTA, there is evidence for additional binding modes. Duplex DNA containing either human telomeric repeats (T(2)AG(3))(4) or the Tetrahymena telomeric repeats (T(2)G(4))(4) are readily photooxidized by TOTA, the major sites of oxidation being the central guanine residues in each telomeric repeat. These telomeric repeats were incorporated into duplex/quadruplex chimeras in which the repeats adopt a G-quadruplex structure. Analysis by denaturing polyacrylamide gel electrophoresis reveals significantly less TOTA photocleavage of these quadruplex telomeric repeats when compared to the duplex repeats.     

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.     

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.     

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.     

Selective interaction of ethidium derivatives with quadruplexes: an equilibrium dialysis and electrospray ionization mass spectrometry analysis

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 telomerase is a relevant target in oncology, and telomerase inhibitors have been proposed as new potential anticancer agents. In this paper, we have analyzed the selectivity of four ethidium derivatives and ethidium itself toward different G-quadruplex species, with electrospray mass spectrometry and competitive equilibrium dialysis and evaluated their inhibitory properties against telomerase. A selectivity profile may be obtained through electrospray ionization mass spectrometry (ESI-MS), which is in fair agreement with competitive equilibrium dialysis data. It also provides unambiguous data on the number of binding sites per nucleic acid (maximal number of two ethidium derivatives per quadruplex, in agreement with external stacking). Our experiments also demonstrate that one compound (4) is the most active and selective G-quadruplex ligand within this series and the most selective telomerase inhibitor in a modified TRAP-G4 assay.     

Interaction of telomestatin with the telomeric single-strand overhang

The extremities of chromosomes end in a G-rich single-stranded overhang that has been implicated in the onset of the replicative senescence. The repeated sequence forming a G-overhang is able to adopt a peculiar four-stranded DNA structure in vitro called a G-quadruplex, which is a poor substrate for telomerase. Small molecule ligands that selectively stabilize the telomeric G-quadruplex induce telomere shortening and a delayed growth arrest. Here we show that the G-quadruplex ligand telomestatin has a dramatic effect on the conformation of intracellular G-overhangs. Competition experiments indicate that telomestatin strongly binds in vitro and in vivo to the telomeric overhang and impairs its single-stranded conformation. Long-term treatment of cells with telomestatin greatly reduces the G-overhang size, as evidenced by specific hybridization or telomeric oligonucleotide ligation assay experiments, with a concomitant delayed loss of cell viability. In vivo protection experiments using dimethyl sulfate also indicate that telomestatin treatment alters the dimethyl sulfate effect on G-overhangs, a result compatible with the formation of a local quadruplex structure at telomeric overhang. Altogether these experiments strongly support the hypothesis that the telomeric G-overhang is an intracellular target for the action of telomestatin.     

Spectroscopic study and G-quadruplex DNA binding affinity of two bioactive papaverine-derived ligands

The interactions of G-quadruplex DNA with two oxidation products of papaverine, 6a,12a-diazadibenzo-[a,g]fluorenylium derivative (1) and 2,3,9,10-tetramethoxy-12-oxo-12H-indolo[2,1-a]isoquinolinium cation (2) were investigated. Their activity against telomerase was assessed using the conventional telomeric repeat amplification protocol (TRAP) assay. Effect of TRAP buffer and oligonucleotide length on the DNA-binding affinity of 1 and 2 were also studied. Three quadruplex-forming oligonucleotides with human telomeric sequence: dG₃(T₂AG₃)₃ (htel21), dAG₃(T₂AG₃)₃ (htel22), and d(T₂AG₃)₄ (htel24) were used in these investigations. Both ligands were capable of interacting with G4 DNA with binding stoichiometry indicating that two ligand molecules bind to G-quadruplex, which agrees with the binding model of end-stacking on terminal G-tetrads. Circular dichroism spectra revealed that preferences of quadruplex-forming oligonucleotide to adopt a particular topological structure may be also affected by the external ligand that binds to quadruplex. Telomerase activity was suppressed at very low ligand 1 and ligand 2 concentrations with an appreciable selectivity comparing with inhibition of Taq polymerase.     

Synthesis and evaluation of 9-O-substituted berberine derivatives containing aza-aromatic terminal group as highly selective telomeric G-quadruplex stabilizing ligands

A series of new 9-O-substituted berberine derivatives (4a–j) as telomeric quadruplex ligands was synthesized and evaluated. The results from biophysical and biochemical assay indicated that introducing of positive charged aza-aromatic terminal group into the side chain of 9-position of berberine significantly improved the binding ability with G-quadruplex, and exhibited the inhibitory effect on the hybridization and on telomerase activity. These derivatives showed excellent selectivity for telomeric G-quadruplex DNA over duplex.     

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.     

Benzoindoloquinolines interact with DNA tetraplexes and inhibit telomerase

Telomeric G-rich single-stranded DNA can adopt a G-tetraplex structure which has been shown to inhibit telomerase activity. We have examined benzoindoloquinolines derivatives for their ability to stabilize an intramolecular G-quadruplex. The increase in T(m) value of the G-quadruplex was associated with telomerase inhibition in vitro.     

A DNA polymerase stop assay for G-quadruplex-interactive compounds.

We have developed and characterized an assay for G-quadruplex-interactive compounds that makes use of the fact that G-rich DNA templates present obstacles to DNA synthesis by DNA polymerases. Using Taq DNA polymerase and the G-quadruplex binding 2, 6-diamidoanthraquinone BSU-1051, we find that BSU-1051 leads to enhanced arrest of DNA synthesis in the presence of K+by stabilizing an intramolecular G-quadruplex structure formed by four repeats of either TTGGGG or TTAGGG in the template strand. The data provide additional evidence that BSU-1051 modulates telomerase activity by stabilization of telomeric G-quadruplex DNA and point to a polymerase arrest assay as a sensitive method for screening for G-quadruplex-interactive agents with potential clinical utility.     

Autophagy acts as a safeguard mechanism against G-quadruplex ligand-mediated DNA damage

G-quadruplex ligands have attracted considerable interest as novel anticancer therapeutics due to their capability to interfere with guanosine-rich DNA/RNA sequences, such as telomeres. Elucidation of the structures of telomeric G-quadruplexes has led, in the past few years, to the rational development of effective G-quadruplex-stabilizing small molecules. In the present study, we showed that short-term exposure of melanoma cells to Ant1,5—an anthracene-based ligand able to stabilize telomeric G-quadruplexes—impaired cell growth without inducing cell senescence or apoptosis. Conversely, drug-treated cells were characterized by the occurrence of typical biochemical and morphological features associated with autophagy, such as an increase in the lipidated form of the autophagic marker LC3B and the accumulation of autophagosomes. Such drug-induced autophagy occurred as a consequence of DNA damage induction, at least in part dependent on drug-mediated telomere uncapping, through a pathway converging on the cyclin-dependent kinase inhibitor 1A (CDKN1A/p21). Indeed, melanoma cells depleted for CDKN1A did not show evidence of autophagic markers upon exposure to Ant1,5. The inhibition of autophagy by a pharmacologic inhibitor or through RNAi-mediated depletion of the ATG5 gene enhanced the cytotoxic activity of Ant1,5, as revealed by the marked increase in drug-induced apoptosis. Our data outline a molecular scenario in which G-quadruplex ligand-induced telomeric dysfunctions and DNA damage are translated into an autophagic response and provide the first evidence of autophagy as a safeguard mechanism activated by melanoma cells to counteract G-quadruplex ligand-mediated cellular stress.     

Structure of a G-quadruplex-ligand complex

Stabilisation of G-quadruplex structures formed from telomeric DNA, by means of quadruplex-selective ligands, is a means of inhibiting the telomerase enzyme from catalysing the synthesis of telomeric DNA repeats. In order to understand the molecular basis of ligand-quadruplex recognition, the crystal structure has been determined of such a complex, at 1.75A resolution. This complex is between a dimeric antiparallel G-quadruplex formed from the Oxytricha nova telomeric DNA sequence d(GGGGTTTTGGGG), and a di-substituted aminoalkylamido acridine compound. The structure shows that the acridine moiety is bound at one end of the stack of G-quartets, within one of the thymine loops. It is held in place by a combination of stacking interactions and specific hydrogen bonds with thymine bases. The stability of the ligand in this binding site has been confirmed by a 2ns molecular dynamics simulation.     

Telomeric repeat mutagenicity in human somatic cells is modulated by repeat orientation and G-quadruplex stability

Telomeres consisting of tandem guanine-rich repeats can form secondary DNA structures called G-quadruplexes that represent potential targets for DNA repair enzymes. While G-quadruplexes interfere with DNA synthesis in vitro, the impact of G-quadruplex formation on telomeric repeat replication in human cells is not clear. We investigated the mutagenicity of telomeric repeats as a function of G-quadruplex folding opportunity and thermal stability using a shuttle vector mutagenesis assay. Since single-stranded DNA during lagging strand replication increases the opportunity for G-quadruplex folding, we tested vectors with G-rich sequences on the lagging versus the leading strand. Contrary to our prediction, vectors containing human [TTAGGG]₁₀ repeats with a G-rich lagging strand were significantly less mutagenic than vectors with a G-rich leading strand, after replication in normal human cells. We show by UV melting experiments that G-quadruplexes from ciliates [TTGGGG]₄ and [TTTTGGGG]₄ are thermally more stable compared to human [TTAGGG]₄. Consistent with this, replication of vectors with ciliate [TTGGGG]₁₀ repeats yielded a 3-fold higher mutant rate compared to the human [TTAGGG]₁₀ vectors. Furthermore, we observed significantly more mutagenic events in the ciliate repeats compared to the human repeats. Our data demonstrate that increased G-quadruplex opportunity (repeat orientation) in human telomeric repeats decreased mutagenicity, while increased thermal stability of telomeric G-quadruplexes was associated with increased mutagenicity.