Systematic Evaluation of Benchmark G4 Probes and G4 Clinical Drugs using three Biophysical Methods: A Guideline to Evaluate Rapidly G4-Binding Affinity.

G-quadruplex DNA structures (G4) are proven to interfere with most genetic and epigenetic processes. Small molecules binding these structures (G4 ligands) are invaluable tools to probe G4-biology and address G4-druggability in various diseases (cancer, viral infections). However, the large number of reported G4 ligands (>1000) could lead to confusion while selecting one for a given application. Herein we conducted a systematic affinity ranking of 11 popular G4 ligands vs 5 classical G4 sequences using FRET-melting, G4-FID assays and SPR. Interestingly SPR data globally align with the rankings obtained from the two semi-quantitative assays despite discrepancies due to limits and characteristics of each assay. In the whole, PhenDC3 emerges as the most potent binder irrespective of the G4 sequence. Immediately below PDS, PDC-360A, BRACO19, TMPyP4 and RHPS4 feature strong to medium binding again with poor G4 topology discrimination. More strikingly, the G4 drugs Quarfloxin, CX5461 and c-PDS exhibit weak affinity with all G4s studied. Finally, NMM and Cu-ttpy showed heterogeneous behaviors due, in part, to their physicochemical particularities poorly compatible with screening conditions. The remarkable properties of PhenDC3 led us to propose its use for benchmarking FRET-melting and G4-FID assays for rapid G4-affinity evaluation of newly developed ligands.

In vitro effects of Trastuzumab Emtansine (T-DM1) and concurrent irradiation on HER2-positive breast cancer cells.

BACKGROUND: To determine the effects of concurrent irradiation and T-DM1 on HER2-positive breast cancer cell lines. METHODS: Five human breast cancer cell lines (in vitro study) presenting various levels of HER2 expression were used to determine the potential therapeutic effect of T-DM1 combined with radiation. The toxicity of T-DM1 was assessed using viability assay and cell cycle analysis was performed by flow cytometry after BrdU incorporation. HER2 cells were irradiated at different dose levels after exposure to T-DM1. Survival curves were determined by cell survival assays (after 5 population doubling times). RESULTS: The results revealed that T-DM1 induced significant lethality due to the intracellular action of DM1 on the cell cycle with significant G2/M phase blocking. Even after a short time incubation, the potency of T-DM1 was maintained and even enhanced over time, with a higher rate of cell death. After irradiation alone, the D10 (dose required to achieve 10% cell survival) was significantly higher for high HER2-expressing cell lines than for low HER2-expressing cells, with a linearly increasing relationship. In combination with irradiation, using conditions that allow cell survival, T-DM1 does not induce a radiosensitivity. CONCLUSIONS: Although there is a linear correlation between intrinsic HER2 expression and radioresistance, the results indicated that T-DM1 is not a radiation-sensitizer under the experimental conditions of this study that allowed cell survival. However, further investigations are needed, in particular in vivo studies before reaching a final conclusion.

Differential behaviour of cationic triphenylamine derivatives in fixed and living cells: triggering and imaging cell death.

Triphenylamines are on/off fluorescent DNA minor groove binders, allowing nuclear staining of fixed cells. By contrast, they accumulate in the cytoplasm of living cells and efficiently trigger cell apoptosis upon prolonged visible light irradiation. This process occurs concomitantly with their subcellular re-localization to the nucleus, enabling fluorescence imaging of apoptosis.

N-phenyl-carbazole-based two-photon fluorescent probes: strong sequence dependence of the duplex vs quadruplex selectivity.

Herein we report on the synthesis and DNA recognition properties of a series of three N-phenyl carbazole-based light-up probes initially designed for two-photon absorption. The vinylic derivatives (Cbz-2Py, Cbz-3Py) display strong fluorescence enhancement when bound to various duplex- and quadruplex-forming oligonucleotides whereas the oxazole derivative is not fluorescent in DNA. Determination of affinity constants by fluorimetric titrations evidenced that Cbz-2Py has a clear preference for AT-rich duplex structures. Circular Dichroism (CD) measurements confirmed the sequence-dependent binding of this compound and suggest insertion in the minor groove as shown by a strong induced CD (ICD) signal and further supported by molecular modeling. Altogether the data indicate that duplex vs quadruplex selectivity of the dyes is strongly dependent on the sequence of the duplex. Finally, the dyes exhibit high two-photon absorption cross-sections (up to 540GM in glycerol) and allow a fine and bright staining of nuclear DNA with low background fluorescence as shown by one and two-photon confocal microscopy imaging of fixed cells.

New triarylamine-based far-red DNA stainers with high two-photon absorption properties.

A series of red emitting vinyl-triphenyamines (TP) have been synthesized and evaluated for their two photon absorption (2PA) properties. These compounds are virtually non fluorescent in the free state but exhibit a bright red fluorescence upon binding to double-stranded DNA, both in one- and two-photon absorption. This feature allows one- and two-photon confocal imaging in cells of nuclear DNA with an excellent contrast. Derivatizable analogues for covalent bioconjugation to oligonucleotides are described and variation on the structure is discussed.

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.

Thiazole orange: a useful probe for fluorescence sensing of G-quadruplex-ligand interactions.

Fluorimetric titrations were performed to gain insight into parameters that govern the association of thiazole orange (TO) and G-quadruplex-DNA (G4-DNA). Use of loop-containing and loop-lacking quadruplexes evidenced the critical influence of the loops on the stoichiometry of the association and on the fluorescence exaltation of TO. We subsequently tried to benefit from this sensitivity to evaluate the influence of G4-DNA cationic environment on ligand binding via a recently reported G4-FID assay.

Molecular recognition of quadruplex DNA by quinacridine derivatives.

The interaction of monomeric and dimeric quinacridines with quadruplex DNA has been investigated using a variety of biophysical methods. Both series of compounds were shown to exhibit a high affinity for the G4 conformation with two equivalent binding sites. As shown from the SPR and dialysis experiments the macrocyclic dimer appears more selective than its monomeric counterpart.

Direct photocleavage of HIV-DNA by quinacridine derivatives triggered by triplex formation.

Amino-p-quinacridine compounds (PQs) have been shown to stabilize strongly and specifically triple-helical DNA. Moreover, these derivatives display photoactive properties that make them efficient DNA cleavage agents. We exploited these two properties (triplex-specific binding and photoactivity) to selectively cleave a double-stranded (ds)DNA sequence present in the HIV-1 genome. Cleavage was first carried out on a linearized plasmid (3300 bp) containing the HIV polypurine tract (PPT) that allowed targeting by a triplex-forming oligonucleotide (TFO). PQ(3)(), the most active compound of the series, efficiently cleaved double-stranded DNA in the vicinity of the PPT when this sequence had formed a triplex with a 16-mer TFO. Investigation of the cleavage at the molecular level was addressed on a short DNA fragment (56 bp); the photoinduced cleavage by PQ(3)() occurred only in the presence of the triple helix. Nevertheless, unusual cleavage patterns were observed: damage was observed at guanines located 6-9 bp away from the end of the triple helical site. This cleavage is very efficient (up to 60%), does not require alkaline treatment, and is observed on both strands. A quinacridine-TFO conjugate produced the same cleavage pattern. This observation, along with others, excludes the hypothesis of a triplex-induced allosteric binding site of PQ(3 )()adjacent to the damaged sequence and indicates that PQ(3 )()preferentially binds in the vicinity of the 5'-triplex junction. Irradiation in the presence of TFO-conjugates with acridine (an intercalative agent) and with the tripeptide lys-tryp-lys led to a complete inhibition of the photocleavage reaction. These results are interpreted in terms of competitive binding and of electron-transfer quenching. Together with the findings of simple mechanistic investigations, they led to the conclusion that the photoinduced damage proceeds through a direct electron transfer between the quinacridine and the guanines. This study addresses the chemical mechanism leading to strand breakage and characterizes the particular photosensitivity of the HIV-DNA target sequence which could be an oxidative hot spot for addressed photoinduced strand scission by photosensitizers.

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 degrees C at 1 microM dye concentration. This increase in T(m) value was well correlated with an increase in the efficiency of telomerase inhibition in vitro. The best telomerase inhibitor showed an IC(50) 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.

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.

DNA double helix destabilizing properties of cyclobisintercaland compounds and competition with a single strand binding protein.

The DNA helix destabilizing activity of a series of cyclobisintercaland compounds (CBIs) has been evaluated by measuring their ability to displace a 32P-labelled oligonucleotide primer (17-mer) hybridized to the single stranded DNA of M13. This destabilizing activity appears to be strongly dependent on the cyclic structure (the linear acyclic references are inactive) and the size of the macrocycle; both features being known to determine the preferential binding of the compound to ssDNA. Interestingly, CBIs induced the dissociation of the duplex template in a concentration range (0.5-1 microM) close to that required for the destabilizing activity of single stranded DNA binding proteins (SSBs). Therefore competition experiments between CBIs and an SSB protein (Eco SSB) for binding to a single stranded oligonucleotide target (36-mer) have been performed through gel electrophoresis and nitrocellulose binding assays and strong inhibitory effects on the formation of the SSB:36-mer complex have been observed.

Threading bis-intercalation of a macrocyclic bisacridine at abasic sites in DNA: nuclear magnetic resonance and molecular modeling study.

The macrocyclic bisacridine (CBA) has been reported previously to specifically recognize single-stranded nucleic acid structures, especially DNA hairpins. The binding of the drug with an abasic site-containing oligonucleotide, was investigated by (1)H NMR and molecular modeling. We have used a DNA undecamer, the d(C(1)G(2)C(3)A(4)C(5)X(6)C(7)A(8)C(9)G(10)C(11)) x d(G(12)C(13)G(14)T(15)G(16)T(17)G(18)T(19)G(2)(0)C(21)G(22)) duplex in which the X residue is a stable analogue of the abasic site [3-hydroxy-2-(hydroxymethyl) tetrahydrofuran]. Analysis of the NMR data reveals that the bisacridine molecule forms two different intercalation complexes in a 80/20 (+/- 10) ratio. For the major complex, a molecular modeling study was performed guided by nineteen intermolecular drug-DNA restraints, determined from NOESY spectra. In this model, the ligand interacts in the threading binding mode with an acridine ring intercalated between the C(7)-A(8) and T(15)-G(16) base pairs, while the other acridine ring resides in the abasic pocket. The two linker chains are positioned in the minor and in the major groove, respectively. A comparable study was performed to evaluate the interaction of CBA with the parent unmodified duplex in which X(6) was replaced by an adenine residue. No complex formation was observed when operating in identical conditions. This shows the selective binding of CBA to the abasic site and its potential interest to target the abasic site lesion.

Selective photocleavage of single-stranded nucleic acids by cyclobisintercaland molecules.

Irradiation of mixtures of a single-stranded circular plasmid and of a double-stranded supercoiled DNA in presence of the cyclobisintercaland compounds 2 or 3 shows that these reagents effect the selective photocleavage of the single-stranded entity. Furthermore, 2 also cleaves tRNAasp preferentially at single-stranded domains.

A macrocyclic bis-acridine shifts the equilibrium from duplexes towards DNA hairpins.

Nucleic acids can undergo dynamic conformational changes associated with the regulation of biological processes. A molecule presenting larger affinities for alternative structures relative to a duplex is expected to modify such conformational equilibria. We have previously reported that macrocyclic bis-acridine binds preferentially to single-stranded regions, especially DNA hairpins, due to steric effects. Here, we show, using gel electrophoresis, fluorescence and melting temperature experiments, that the macrocycle bis-acridine shifts an equilibrium from a duplex towards the corresponding hairpins. Competition experiments enlighten the higher affinity of the macrocycle for hairpins compared with double-stranded DNA. The macrocycle bis-acridine destabilizes a synthetic polynucleotide, by the formation of premelted areas. By extrapolation, the macrocycle bis-acridine should be able to disrupt, at least locally, genomic DNA duplexes and to stabilize unpaired areas, especially palindromic ones forming hairpins. Such macrocyclic compounds may have potential applications in the therapy of diseases involving hairpins.