Marie-Paule Teulade-Fichou

Telomerase inhibitors based on quadruplex ligands selected by a fluorescence assay

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 in vitro an intramolecular quadruplex structure, which has been shown to inhibit telomerase activity. We used a fluorescence assay to identify molecules that stabilize G-quadruplexes. Intramolecular folding of an oligonucleotide with four repeats of the human telomeric sequence into a G-quadruplex structure led to fluorescence excitation energy transfer between a donor (fluorescein) and an acceptor (tetramethylrhodamine) covalently attached to the 5′ and 3′ ends of the oligonucleotide, respectively. The melting of the G-quadruplex was monitored in the presence of putative G-quadruplex-binding molecules by measuring the fluorescence emission of the donor. A series of compounds (pentacyclic crescent-shaped dibenzophenanthroline derivatives) was shown to increase the melting temperature of the G-quadruplex by 2–20°C at 1 μM dye concentration. This increase in Tm value was well correlated with an increase in the efficiency of telomerase inhibition in vitro. The best telomerase inhibitor showed an IC50 value of 28 nM in a standard telomerase repeat amplification protocol assay. Fluorescence energy transfer can thus be used to reveal the formation of four-stranded DNA structures, and its stabilization by quadruplex-binding agents, in an effort to discover new potent telomerase inhibitors.

Reevaluation of telomerase inhibition by quadruplex ligands and their mechanisms of action

Quadruplex ligands are often considered as telomerase inhibitors. Given the fact that some of these molecules are present in the clinical setting, it is important to establish the validity of this assertion. To analyze the effects of these compounds, we used a direct assay with telomerase-enriched extracts. The comparison of potent ligands from various chemical families revealed important differences in terms of effects on telomerase initiation and processivity. Although most quadruplex ligands may lock a quadruplex-prone sequence into a quadruplex structure that inhibits the initiation of elongation by telomerase, the analysis of telomerase-elongation steps revealed that only a few molecules interfered with the processivity of telomerase (i.e., inhibit elongation once one or more repeats have been incorporated). The demonstration that these molecules are actually more effective inhibitors of telomeric DNA amplification than extension by telomerase contributes to the already growing suspicion that quadruplex ligands are not simple telomerase inhibitors but, rather, constitute a different class of biologically active molecules. We also demonstrate that the popular telomeric repeat amplification protocol is completely inappropriate for the determination of telomerase inhibition by quadruplex ligands, even when PCR controls are included. As a consequence, the inhibitory effect of many quadruplex ligands has been overestimated.

Ligands playing musical chairs with G-quadruplex DNA: A rapid and simple displacement assay for identifying selective G-quadruplex binders

We report here the details of G4-FID (G-quadruplex fluorescent intercalator displacement), a simple method aiming at evaluating quadruplex-DNA binding affinity and quadruplex- over duplex-DNA selectivity of putative ligands. This assay is based on the loss of fluorescence upon displacement of thiazole orange from quadruplex- and duplex-DNA matrices. The original protocol was tested using various quadruplex- and duplex-DNA targets, and with a wide panel of G-quadruplex ligands belonging to different families (i.e. from quinacridines to metallo-organic ligands) likely to display various binding modes. The reliability of the assay is further supported by comparisons with FRET-melting and ESI-MS assays.

G-quadruplex-induced instability during leading-strand replication.

G‐quadruplexes are four‐stranded nucleic acid structures whose biological functions remain poorly understood. In the yeast S. cerevisiae, we report that G‐quadruplexes form and, if not properly processed, pose a specific challenge to replication. We show that the G‐quadruplex‐prone CEB1 tandem array is tolerated when inserted near ARS305 replication origin in wild‐type cells but is very frequently destabilized upon treatment with the potent Phen‐DC3 G‐quadruplex ligand, or in the absence of the G‐quadruplex‐unwinding Pif1 helicase, only when the G‐rich strand is the template of leading‐strand replication. The orientation‐dependent instability is associated with the formation of Rad51–Rad52‐dependent X‐shaped intermediates during replication detected by two‐dimensional (2D) gels, and relies on the presence of intact G‐quadruplex motifs in CEB1 and on the activity of ARS305. The asymmetrical behaviour of G‐quadruplex prone sequences during replication has implications for their evolutionary dynamics within genomes, including the maintenance of G‐rich telomeres.

Engineering Bisquinolinium/Thiazole Orange Conjugates for Fluorescent Sensing of G-Quadruplex DNA**

Lighting up: A G-quadruplex-specific fluorescent probe was designed combining the specificity of the pyridodicarboxamide motif for guanine quadruplexes and the fluorescence properties of thiazole orange. While the assembly of the two partners through a flexible linker leads to a nonselective probe, merging them in a single, rigid scaffold leads to a dye that elicits the properties required for G-quadruplex sensing.

G-quadruplex structures in TP53 intron 3: role in alternative splicing and in production of p53 mRNA isoforms

The tumor suppressor gene TP53, encoding p53, is expressed as several transcripts. The fully spliced p53 (FSp53) transcript encodes the canonical p53 protein. The alternatively spliced p53I2 transcript retains intron 2 and encodes Δ40p53 (or ΔNp53), an isoform lacking first 39 N-terminal residues corresponding to the main transactivation domain. We demonstrate the formation of G-quadruplex structures (G4) in a GC-rich region of intron 3 that modulates the splicing of intron 2. First, we show the formation of G4 in synthetic RNAs encompassing intron 3 sequences by ultraviolet melting, thermal difference spectra and circular dichroism spectroscopy. These observations are confirmed by detection of G4-induced reverse transcriptase elongation stops in synthetic RNA of intron 3. In this region, p53 pre-messenger RNA (mRNA) contains a succession of short exons (exons 2 and 3) and introns (introns 2 and 4) covering a total of 333 bp. Site-directed mutagenesis of G-tracts putatively involved in G4 formation decreased by ~30% the excision of intron 2 in a green fluorescent protein-reporter splicing assay. Moreover, treatment of lymphoblastoid cells with 360A, a synthetic ligand that binds to single-strand G4 structures, increases the formation of FSp53 mRNA and decreases p53I2 mRNA expression. These results indicate that G4 structures in intron 3 regulate the splicing of intron 2, leading to differential expression of transcripts encoding distinct p53 isoforms.

The importance of metal geometry in the recognition of G-quadruplex-DNA by metal-terpyridine complexes

A family of terpyridine metallo-organic complexes has been designed and its recognition properties of G-quadruplex-DNA investigated. The series combines easy synthetic access and good affinity-selectivity ratio for quadruplex-DNA. Our study also highlights that the geometry of the metal center strongly governs the ability of the compounds to discriminate quadruplex from duplex-DNA.

Synthetic Strategies to Derivatizable Triphenylamines Displaying High Two-Photon Absorption

A versatile synthetic strategy to access a set of highly fluorescent pi-conjugated triphenylamines bearing a functional linker at various positions on one phenyl ring is described. These compounds were designed for large two-photon absorption (2PA) and in particular for labeling of biomolecules. The monoderivatized trisformylated or trisiodinated intermediates described herein allow introduction of a large variety of electron-withdrawing groups required for large 2PA as well as a panel of chemical functions suitable for coupling to biomolecules. The monoderivatized three-branched compounds and in particular the benzothiazole (TP-3Bz) series show remarkable linear (high extinction coefficients and high quantum yield) and nonlinear (high 2-photon cross sections) optical properties. Interestingly the presence of functional side chains does not disturb the two-photon absorption. Finally, monoderivatized two-branched derivatives also appear to be valuable candidates. Altogether the good optical properties of the new derivatizable pi-conjugated TPA combined with their small size and their compatibility with bioconjugation protocols suggest that they represent a new chemical class of labels potentially applicable for the tracking of biomolecules using two-photon scanning microscopy.

G-quadruplex recognition by quinacridines: a SAR, NMR, and biological study.

The synthesis of a novel group of quinacridine‐based ligands (MMQs) is described along with an evaluation of their G‐quadruplex binding properties. A set of biophysical assays was applied to characterize their interaction with DNA quadruplexes: FRET–melting experiments and equilibrium microdialysis were used to evaluate their quadruplex affinity and their ability to discriminate quadruplexes across a broad panel of DNA structures. All data collected support the proposed model of interaction of these compounds with G‐quadruplexes, which is furthermore confirmed by a solution structure determined by 2D NMR experiments. Finally, the activity of the MMQ series against tumor cell growth is reported, and the data support the potential of quadruplex‐interactive compounds for use in anticancer approaches.

Vinyl-pyridinium triphenylamines : Novel far-red emitters with high photostability and two-photon absorption properties for staining DNA

A series of mono‐, bis‐ and trisvinyl‐pyridinium triphenylamines (TP‐py) has been synthesised and evaluated for its one‐ and two‐photon absorption (2PA) induced‐fluorescence properties under biological conditions. Interestingly, these compounds are only weakly fluorescent in water, whereas their fluorescence emissions are strongly restored (exaltation factors of 20–100) upon binding to double‐stranded DNA. Additional measurements in glycerol indicate that the fluorescence increases are the result of immobilisation of the dyes in the DNA matrix, which inhibits rotational de‐excitation modes. This particular feature is especially remarkable in the case of the bis and tris derivatives (TP‐2 py, TP‐3 py), which each display a high affinity (Kd ∼μM) for dsDNA. TPIF measurements have shown that TP‐2 py and TP‐3 py each have a large 2PA cross section (δ up to 700 GM) both in glycerol and in the presence of DNA, which ranks them amongst the best 2PA biological fluorophores. Finally, one‐ and two‐photon confocal imaging in cells revealed that these compounds perform red staining (λem=660–680 nm) of nuclear DNA with excellent contrast. The remarkable optical properties of the TP‐py series, combined with their high photostability and their easy synthetic access, make these compounds extremely attractive for use in confocal and 2PA microscopy.