Linking of Antitumor trans NHC-Pt(II) Complexes to G-Quadruplex DNA Ligand for Telomeric Targeting.

G-quadruplex structures (G4) are promising anticancerous targets. A great number of small molecules targeting these structures have already been identified through biophysical methods. In cellulo, some of them are able to target either telomeric DNA and/or some sequences involved in oncogene promotors, both resulting in cancer cell death. However, only a few of them are able to bind to these structures G4 irreversibly. Here we combine within the same molecule the G4-binding agent PDC (pyridodicarboxamide) with a N-heterocyclic carbene-platinum complex NHC-Pt already identified for its antitumor properties. The resulting conjugate platinum complex NHC-Pt-PDC stabilizes strongly G-quadruplex structures in vitro, with affinity slightly affected as compared to PDC. In addition, we show that the new conjugate binds preferentially and irreversibly the quadruplex form of the human telomeric sequence with a profile in a way different from that of NHC-Pt thereby indicating that the platination reaction is oriented by stacking of the PDC moiety onto the G4-structure. In cellulo, NHC-Pt-PDC induces a significant loss of TRF2 from telomeres that is considerably more important than the effect of its two components alone, PDC and NHC-Pt, respectively.

Dominant Driving Forces in Human Telomere Quadruplex Binding-Induced Structural Alterations.

Recently various pathways of human telomere (ht) DNA folding into G-quadruplexes and of ligand binding to these structures have been proposed. However, the key issue as to the nature of forces driving the folding and recognition processes remains unanswered. In this study, structural changes of 22-mer ht-DNA fragment (Tel22), induced by binding of ions (K(+), Na(+)) and specific bisquinolinium ligands, were monitored by calorimetric and spectroscopic methods and by gel electrophoresis. Using the global model analysis of a wide variety of experimental data, we were able to characterize the thermodynamic forces that govern the formation of stable Tel22 G-quadruplexes, folding intermediates, and ligand-quadruplex complexes, and then predict Tel22 behavior in aqueous solutions as a function of temperature, salt concentration, and ligand concentration. On the basis of the above, we believe that our work sets the framework for better understanding the heterogeneity of ht-DNA folding and binding pathways, and its structural polymorphism.

Short loop length and high thermal stability determine genomic instability induced by G-quadruplex-forming minisatellites.

G-quadruplexes (G4) are polymorphic four-stranded structures formed by certain G-rich nucleic acids, with various biological roles. However, structural features dictating their formation and/or function in vivo are unknown. In S. cerevisiae, the pathological persistency of G4 within the CEB1 minisatellite induces its rearrangement during leading-strand replication. We now show that several other G4-forming sequences remain stable. Extensive mutagenesis of the CEB25 minisatellite motif reveals that only variants with very short (≤ 4 nt) G4 loops preferentially containing pyrimidine bases trigger genomic instability. Parallel biophysical analyses demonstrate that shortening loop length does not change the monomorphic G4 structure of CEB25 variants but drastically increases its thermal stability, in correlation with the in vivo instability. Finally, bioinformatics analyses reveal that the threat for genomic stability posed by G4 bearing short pyrimidine loops is conserved in C. elegans and humans. This work provides a framework explanation for the heterogeneous instability behavior of G4-forming sequences in vivo, highlights the importance of structure thermal stability, and questions the prevailing assumption that G4 structures with short or longer loops are as likely to form in vivo.

Interactions of Pt-ttpy with G-Quadruplexes Originating from Promoter Region of the c-myc Gene Deciphered by NMR and Gel Electrophoresis Analysis.

This study provides insights into the interactions of Pt-ttpy, that is, a metallo-organic heterocycle-comprising platinum(II) complex of terpyridine, and G-quadruplexes adopted by G-rich DNA from the transcriptional regulatory element of the c-myc gene, a well-known attractive target for artificial modulation of oncogene expression. A previously noted drug-like potential of Pt-ttpy relies on its antiproliferative activity on cancer cells and its increased selectivity for G-quadruplex binding attributed to the combination of distinct interacting modes. The predominant interaction between the herein used models of a parallel G-quadruplex exhibiting short propeller-type loops and Pt-ttpy occurs through stacking to the outer G-quartets. The presence of adenine versus thymine residue at the 5'-end overhanging region allows the coordinative binding of Pt-ttpy to the G-quadruplex structure. Interestingly, Pt-ttpy triggers the formation of the G-quadruplex even in the absence of cations. Furthermore, NMR-based characterisation revealed common structural features of Pt-ttpy-G-quadruplex complexes in the presence and absence of cations, which indicate that cations may be expelled from the cores of the corresponding structures.

Impact of G-quadruplex structures and intronic polymorphisms rs17878362 and rs1642785 on basal and ionizing radiation-induced expression of alternative p53 transcripts.

G-quadruplex (G4) structures in intron 3 of the p53 pre-mRNA modulate intron 2 splicing, altering the balance between the fully spliced p53 transcript (FSp53, encoding full-length p53) and an incompletely spliced transcript retaining intron 2 (p53I2 encoding the N-terminally truncated Δ40p53 isoform). The nucleotides forming G4s overlap the polymorphism rs17878362 (A1 wild-type allele, A2 16-base pair insertion) which is in linkage disequilibrium with rs1642785 in intron 2 (c.74+38 G>C). Biophysical and biochemical analyses show rs17878362 A2 alleles form similar G4 structures as A1 alleles although their position is shifted with respect to the intron 2 splice acceptor site. In addition basal FSp53 and p53I2 levels showed allele specific differences in both p53-null cells transfected with reporter constructs or lymphoblastoid cell lines. The highest FSp53 and p53I2 levels were associated with combined rs1642785-GG/rs17878362-A1A1 alleles, whereas the presence of rs1642785-C with either rs17878362 allele was associated with lower p53 pre-mRNA, total TP53, FSp53 and p53I2 levels, due to the lower stability of transcripts containing rs1642785-C. Treatment of lymphoblastoid cell with the G4 binding ligands 360A or PhenDC3 or with ionizing radiation increased FSp53 levels only in cells with rs17878362 A1 alleles, suggesting that under this G4 configuration full splicing is favoured. These results demonstrate the complex effects of intronic TP53 polymorphisms on G4 formation and identify a new role for rs1642785 on mRNA splicing and stability, and thus on the differential expression of isoform-specific transcripts of the TP53 gene.

Photo-cross-linking probes for trapping G-quadruplex DNA.

We have developed a straightforward synthetic pathway to a set of six photoactivatable G-quadruplex ligands with a validated G4-binding motif (the bisquinolinium pyridodicarboxamide PDC-360A) tethered through various spacers to two different photo-cross-linking groups: benzophenone and an aryl azide. The high quadruplex-versus-duplex selectivity of the PDC core was retained in the new derivatives and resulted in selective alkylation of two well-known G-quadruplexes (human telomeric G4 and oncogene promoter c-myc G4) under conditions of harsh competition. The presence of two structurally different photoactivatable functions allowed the selective alkylation of G-quadruplex structures at specific nucleobases and irreversible G4 binding. The topology and sequence of the quadruplex matrix appear to influence strongly the alkylation profile, which differs for the telomeric and c-myc quadruplexes. The new compounds are photoactive in cells and thus provide new tools for studying G4 biology.

Synthesis and characterization of a new photoinduced switchable ß-cyclodextrin dimer.

This paper reports an efficient preparation of bridged bis-ß-CD AZO-CDim 1 bearing azobenzene as a linker and exhibiting high solubility in water. The photoisomerization properties were studied by UV-vis and HPLC and supported by ab initio calculations. The cis/trans ratio of AZO-CDim 1 is 7:93 without irradiation and 37:63 after 120 min of irradiation at 365 nm; the reaction is reversible after irradiation at 254 nm. The photoinduced, switchable binding behavior of AZO-CDim 1 was evaluated by ITC, NMR and molecular modeling in the presence of a ditopic adamantyl guest. The results indicate that AZO-CDim 1 can form two different inclusion complexes with an adamantyl dimer depending on its photoinduced isomers. Both cavities of cis-AZO-CDim 1 are complexed simultaneously by two adamantyl units of the guest forming a 1:1 complex while trans-AZO-CDim 1 seems to lead to the formation of supramolecular polymers with an n:n stoichiometry.

Solution structure of a G-quadruplex bound to the bisquinolinium compound Phen-DC(3).

Phen-DC3 is a highly promising compound that specifically targets G-quadruplexes, with potent biological effects observed in vivo. We used NMR spectroscopy to solve the structure of the complex formed between Phen-DC3 and an intramolecular G-quadruplex derived from the c-myc promoter. Structural information revealed that Phen-DC3 interacts with the quadruplex through extensive π-stacking with guanine bases of the top G-tetrad. On the basis of our structure, modifications are proposed for the development of this compound for selective targeting of a specific G-quadruplex conformation.

Specialization among iron-sulfur cluster helicases to resolve G-quadruplex DNA structures that threaten genomic stability.

G-quadruplex (G4) DNA, an alternate structure formed by Hoogsteen hydrogen bonds between guanines in G-rich sequences, threatens genomic stability by perturbing normal DNA transactions including replication, repair, and transcription. A variety of G4 topologies (intra- and intermolecular) can form in vitro, but the molecular architecture and cellular factors influencing G4 landscape in vivo are not clear. Helicases that unwind structured DNA molecules are emerging as an important class of G4-resolving enzymes. The BRCA1-associated FANCJ helicase is among those helicases able to unwind G4 DNA in vitro, and FANCJ mutations are associated with breast cancer and linked to Fanconi anemia. FANCJ belongs to a conserved iron-sulfur (Fe S) cluster family of helicases important for genomic stability including XPD (nucleotide excision repair), DDX11 (sister chromatid cohesion), and RTEL (telomere metabolism), genetically linked to xeroderma pigmentosum/Cockayne syndrome, Warsaw breakage syndrome, and dyskeratosis congenita, respectively. To elucidate the role of FANCJ in genomic stability, its molecular functions in G4 metabolism were examined. FANCJ efficiently unwound in a kinetic and ATPase-dependent manner entropically favored unimolecular G4 DNA, whereas other Fe-S helicases tested did not. The G4-specific ligands Phen-DC3 or Phen-DC6 inhibited FANCJ helicase on unimolecular G4 ∼1000-fold better than bi- or tetramolecular G4 DNA. The G4 ligand telomestatin induced DNA damage in human cells deficient in FANCJ but not DDX11 or XPD. These findings suggest FANCJ is a specialized Fe-S cluster helicase that preserves chromosomal stability by unwinding unimolecular G4 DNA likely to form in transiently unwound single-stranded genomic regions.

Visualizing the quadruplex: from fluorescent ligands to light-up probes.

Detection of quadruplex structures by visual methods is a major challenge of the quadruplex nucleic acid research area. Consequently, considerable efforts are under way for the discovery of quadruplex specific agents endowed with fluorescence properties. In this review chapter we propose a comprehensive and critical overview of the diverse molecular design and strategies that have been described to identify quadruplex-selective fluorescent probes. Innovative compounds as well as classical DNA dyes are reviewed. The compounds have been divided into three classes: (1) "light-up" probes that display a strong enhancement upon G4 binding, (2) "light-off" probes that display a decreased fluorescence upon binding, and (3) permanent probes ("tagged" G4-binders) that exhibit no variation of fluorescence but display quadruplex binding specificity. The labeling performances of probes in various analytical contexts (in solution, in gel, at the level of chromosomes, and in fixed cells) are also reported and commented on when available. Finally we address the strengths and weaknesses of each probe class and highlight the critical features that must be addressed in developing a practicable quadruplex-specific labeling agent.

Cationic pentaheteroaryls as selective G-quadruplex ligands by solvent-free microwave-assisted synthesis.

We report herein a solvent-free and microwaved-assisted synthesis of several water soluble acyclic pentaheteroaryls containing 1,2,4-oxadiazole moieties (1-7). Their binding interactions with DNA quadruplex structures were thoroughly investigated by FRET melting, fluorescent intercalator displacement assay (G4-FID) and CD spectroscopy. Among the G-quadruplexes considered, attention was focused on telomeric repeats together with the proto-oncogenic c-kit sequences and the c-myc oncogene promoter. Compound 1, and to a lesser extent 2 and 5, preferentially stabilise an antiparallel structure of the telomeric DNA motif, and exhibit an opposite binding behaviour to structurally related polyoxazole (TOxaPy), and do not bind duplex DNA. The efficiency and selectivity of the binding process was remarkably controlled by the structure of the solubilising moieties.

Effects of a halogenated G-quadruplex ligand from the pyridine dicarboxamide series on the terminal sequence of XpYp telomere in HT1080 cells.

Non-canonical four-stranded structures called G-quadruplexes can form among telomere repeats during its replication. Small molecule ligands able to interact and to stabilize G-quadruplexes were shown to disrupt the binding of essential telomeric components, such as POT1 and to trigger a telomeric dysfunction associated with a delayed growth arrest in tumor cells. We describe here the chemical synthesis and the G-quadruplex binding properties of three halogenated analogs of the 360A ligand that belongs to the 2,6 pyridine dicarboxamide series. 360A is now commonly used as a benchmark both for biophysical and cellular assays as this compound was shown to display a potent affinity and selectivity for telomeric G-quadruplex DNA over duplex DNA and to induce delayed growth inhibition in HT1080 tumor cell line. Two biophysical assays indicate that, in most cases, the presence of the halogen atom seems to slightly improve the interaction with the telomeric quadruplex. For stability reasons, the bromo derivative (360A-Br) was selected for the cellular assays. Since POT1 participates to the fine tuning of the C-strand end resection during telomere replication, we investigated the effect of 360A-Br to alter the terminal nucleotide composition of XpYp telomere in HT1080 cells using C-STELA. HT1080 cells treated for up to 24 days with 360A-Br presented some minor but significant variations of C-strand terminal nucleotide composition, also observed with a partial siRNA depletion of POT1. The relevance of these minor modifications of the telomeric C-strand resection induced by 360A-Br in HT1080 cells are discussed.

A streptavidin paramagnetic-particle based competition assay for the evaluation of the optical selectivity of quadruplex nucleic acid fluorescent probes.

Although quadruplex nucleic acids are thought to be involved in many biological processes, they are massively overwhelmed by duplex DNA in the cell. Small molecules, able to probe quadruplex nucleic acids with high optical selectivity, could possibly achieve the visualization of these processes. The aim of the method described herein is to evaluate quickly the optical selectivity of quadruplex nucleic acid probes, in isothermal conditions, using widely available materials, small quantities of oligonucleotides and virtually any kind and quantity of biological competitor. The assay relies on the use of streptavidin-coated paramagnetic particles and biotinylated quadruplex forming oligonucleotides, allowing a quick and easy separation of the quadruplex target from the competitor. In the present study, two quadruplex nucleic acids (the DNA and RNA human telomeric repeats) have been used as targets while a duplex DNA oligonucleotide, total DNA, total RNA, another quadruplex nucleic acid and a protein have been used as competitors. The optical selectivity of various probes, displaying different photophysical properties and binding selectivities, has been successfully examined, allowing the identification of a best candidate for further cell microscopy experiments. This assay allows a quick and reliable assessment of the labeling properties of a quadruplex binder in cellular environment conditions. It is an interesting alternative to gel electrophoresis experiments since it is performed in solution, has a well-resolved separation system and allows easy quantifications.

Screening of a chemical library by HT-G4-FID for discovery of selective G-quadruplex binders.

Due to the lack of structural guidelines about G-quadruplex ligands, rational design cannot be the only approach to discover potent G4-ligands. As a complementary approach, screening of chemical library may provide interesting scaffolds known as hits provided that specific tools are available. In this work, the Institut Curie-CNRS chemical library was firstly screened by chemoinformatics methods. Similarity estimations by comparison with reference compounds (Phen-DC3, 360A, MMQ12) provided a set of molecules, which were then evaluated by high-throughput G4-FID (HT-G4-FID) against various G-quadruplex DNA. A full investigation of the most interesting molecules, using the HT-G4-FID assay and molecular modeling, supplied an interesting structure-activity relationship confirming the efficiency of this general approach. Overall, we demonstrated that HT-G4-FID coupled with screening of chemical libraries is a powerful tool to identify new G4-DNA binding scaffolds.

Tridentate N-donor palladium(II) complexes as efficient coordinating quadruplex DNA binders.

Fifteen complexes of palladium, platinum, and copper, featuring five different N-donor tridentate (terpyridine-like) ligands, were prepared with the aim of testing their G-quadruplex-DNA binding properties. The fluorescence resonance energy transfer melting assay indicated a striking positive effect of palladium on G-quadruplex DNA stabilization compared with platinum and copper, as well as an influence of the structure of the organic ligand. Putative binding modes (noncoordinative π stacking and base coordination) of palladium and platinum complexes were investigated by ESI-MS and UV/Vis spectroscopy experiments, which all revealed a greater ability of palladium complexes to coordinate DNA bases. In contrast, platinum compounds tend to predominantly bind to quadruplex DNA in their aqua form by noncoordinative interactions. Remarkably, complexes of [Pd(ttpy)] and [Pd(tMebip)] (ttpy = tolylterpyridine, tMebip = 2,2'-(4-p-tolylpyridine-2,6-diyl)bis(1-methyl-1H-benzo[d]imidazole)) coordinate efficiently G-quadruplex structures at room temperature in less than 1 h, and are more efficient than their platinum counterparts for inhibiting the growth of cancer cells. Altogether, these results demonstrate that both the affinity for G-quadruplex DNA and the binding mode of metal complexes can be modulated by modifying either the metal or the organic ligand.

Fluorescence intercalator displacement assay for screening G4 ligands towards a variety of G-quadruplex structures.

The potential formation of G-quadruplexes in many regions of the genome makes them an attractive target for drug design. A large number of small molecules synthesized in recent years display an ability to selectively target and stabilize G-quadruplexes. To screen for G4 ligands, we modified a G4-FID (G-quadruplex Fluorescent Intercalator Displacement) assay. This test is based on the displacement of an "on/off" fluorescence probe, Thiazole Orange (TO), from quadruplex or duplex DNA matrices by increasing amounts of a putative ligand. Selectivity measurements can easily be achieved by comparing the ability of the ligand to displace TO from various quadruplex and duplex structures. G4-FID requires neither modified oligonucleotides nor specific equipment and is an isothermal experiment. This test was adapted for high throughput screening onto 96-well plates allowing the comparison of more than twenty different structures. Fifteen different known G4 ligands belonging to different families were tested. Most compounds showed a good G4 vs duplex selectivity but exhibited little, if any, specificity for one quadruplex sequence over the others. The quest for the "perfect" specific G4 ligand is not over yet!

Nucleic acids targeted to drugs: SELEX against a quadruplex ligand.

A number of small molecules demonstrate selective recognition of G-quadruplexes and are able to stabilize their formation. In this work, we performed the synthesis of two biotin-tagged G4 ligands and analyzed their interactions with DNA by two complementary techniques, FRET and FID. The compound that exhibited the best characteristics (a biotin pyridocarboxamide derivative with high stabilization of an intramolecular quadruplex and excellent duplex-quadruplex specificity) was used as bait for in vitro selection (SELEX). Among 80 DNA aptamer sequences selected, only a small minority (5/80) exhibited G4-prone motifs. Binding of consensus candidates was confirmed by SPR. These results indicate that G4 ligands that appear highly specific when comparing affinities or stabilization for one quadruplex against one duplex, do not only bind quadruplex sequences but may also recognize other nucleic motifs as well. This observation may be relevant when whole genome or transcriptome analysis of binding sites is seeked for, as unexpected binding sites may also be present.

Development of a high-throughput G4-FID assay for screening and evaluation of small molecules binding quadruplex nucleic acid structures.

G4-FID (G-quadruplex fluorescent intercalator displacement) is a simple and fast method that allows to evaluate the affinity of a compound for G-quadruplex DNA and its selectivity towards duplex DNA. This assay is based on the loss of fluorescence of thiazole orange (TO) upon competitive displacement from DNA by a putative ligand. We describe here the development of a high-throughput version of this assay performed in 96-well microplates, and fully transposable to 384-well microplates. The test was calibrated with a set of G-quadruplex ligands characterized for their ability to bind quadruplex within a large range of affinity. The comparison of the results obtained in microplates and in cuvettes was conducted indicating a full agreement. Additionally, the spectral range of the test was enlarged using two other fluorescent on/off probes whose absorption are red-shifted (TO-PRO-3) and blue-shifted (Hoechst 33258) as compared to that of TO. These labels enable to screen a large diversity of compounds with various optical properties, which was exemplified by evaluation of affinity and selectivity of the porphyrin TMPyP4 that could not be evaluated previously. Altogether, our study demonstrates that the HT-G4-FID assay offers the possibility to label a large variety of G-quadruplexes of biological interest and should enable screening of collections of putative G4-ligands of high structural diversity. It thus represents a powerful tool to bring into light new ligands able to discriminate between quadruplexes of different structures.

Study of a cyclopamine glucuronide prodrug for the selective chemotherapy of glioblastoma.

The first glucuronide prodrug of the hedgehog signaling inhibitor cyclopamine was synthesized. The carbamoyl derivatisation of cyclopamine significantly decreased its toxicity towards the U87 human glioblastoma cell line. However, when the prodrug was incubated with beta-glucuronidase in the culture media, the active drug was efficiently released thereby restoring its anti-proliferative activity.