Loop lengths of G-quadruplex structures affect the G-quadruplex DNA binding selectivity of the RGG motif in Ewing's sarcoma

The G-quadruplex nucleic acid structural motif is a target for designing molecules with potential anticancer properties. To achieve therapeutic selectivity by targeting the G-quadruplex, the molecules must be able to differentiate between the DNA of different G-quadruplexes. We recently reported that the Arg-Gly-Gly repeat (RGG) of the C-terminus in Ewing's sarcoma protein (EWS), which is a group of dominant oncogenes that arise due to chromosomal translocations, is capable of binding to G-quadruplex telomere DNA and RNA via arginine residues and stabilize the G-quadruplex DNA form in vitro. Here, we show that the RGG of EWS binds preferentially to G-quadruplexes with longer loops, which is not related to the topology of the G-quadruplex structure. Moreover, the G-quadruplex DNA binding of the RGG in EWS depends on the phosphate backbone of the loops in the G-quadruplex DNA. We also investigated the G-quadruplex DNA binding activity of the N- and C-terminally truncated RGG to assess the role of the regions in the RGG in G-quadruplex DNA binding. Our findings indicate that the RGG and the other arginine-rich motif of residues 617-656 of the RGG in EWS are important for the specific binding to G-quadruplex DNA. These findings will contribute to the development of molecules that selectively target different G-quadruplex DNA.     

Selective G-quadruplex ligands: The significant role of side chain charge density in a series of perylene derivatives

The human telomeric G-quadruplex structure is a promising target for the design of cancer drugs. The selectivity of G-quadruplex ligands with respect to duplex genomic DNA is of especial importance. The high selectivity of polyamine conjugated perylene derivatives appears to be regulated by side-chain charge density, as indicated by data from a FRET melting assay and induced CD spectroscopy.     

Perylene diimide G-quadruplex DNA binding selectivity is mediated by ligand aggregation

Two N,N'-disubstituted perylene diimide G-quadruplex DNA ligands, PIPER [N,N'-bis-(2-(1-piperidino)ethyl)-3,4,9,10-perylene tetracarboxylic acid diimide] and Tel01 [N,N'-bis-(3-(4-morpholino)-propyl)-3,4,9,10-perylene tetracarboxylic acid diimide] were studied. Visible absorbance, resonance light scattering, and fluorescence spectroscopy were used to characterize the pH-dependent aggregation of these ligands. The G-quadruplex DNA binding selectivity of these ligands as monitored by absorption spectroscopy is also pH-dependent. The ligands bind to both duplex and G-quadruplex DNA under low pH conditions, where the ligands are not aggregated. At higher pH, where the ligands are extensively aggregated, the apparent G-quadruplex DNA binding selectivity is high.     

The role of positive charges on G-quadruplex binding small molecules: Learning from bisaryldiketene derivatives

Background

G-quadruplexes are promising therapeutic targets for small molecules. In general, the introduction of steady positive charges through the in situ alkylation of nitrogen atoms within potential G-quadruplex ligands can significantly improve their quadruplex binding and stabilization abilities. However, our previous studies on bisaryldiketene derivatives showed that the derivative M4, whose central piperidone moiety is quaternized, exhibits a poor G-quadruplex stabilization ability.

Methods

To clarify this unusual finding, CD, ITC, UV and NMR analyses were performed to determine the binding behaviors of M4 and its non-quaternized analog M2 to G-quadruplex DNA [d(TGGGT)]₄. Molecular modeling approaches were also employed to help illustrate ligand–quadruplex DNA interactions.

Results

The CD melting and ITC analyses revealed that M2 exhibited much stronger stabilization and binding abilities to [d(TGGGT)]₄ compared to M4. Moreover, the CD and ITC analyses in combination with UV, NMR and MD simulations revealed that M2 tended to be end-stacked on the G-quartet, whereas M4 tended to be bound in the groove region. Analysis of the electrostatic potential showed that the charged surface of M4 was more positive than that of M2 and other reported ligands that bind to the G-quadruplex via end-stacking interactions.

Conclusions

The results indicated that the different positively charged surfaces of M2 and M4 might be the key reason for their different binding modes. These different binding modes also lead to different binding affinities and stabilization abilities for [d(TGGGT)]₄.

General significance

These results provide new clues for the rational design of G-quadruplex-binding small molecules with steady positive charges.     

Benzo(h)quinoline derivatives as G-quadruplex binding agents

G-quadruplexes are unusual structures formed from guanine-rich sequences of nucleic acids. G-quadruplexes have been postulated to play important roles in a number of biological systems including gene regulation and the inhibition of enzyme function. Recently, our laboratory reported on the synthesis and evaluation of a triaza-cyclopentaphenanthrene compound which bound to G-quadruplexes with good affinity and selectivity. This compound contains a 4-pyridone group which has not been previously utilized in other quadruplex binding agents. In this Letter, we describe the synthesis and evaluation of 4-pyridone containing 2- and 3-carboxy-benzoquinolines as G-quadruplex binding agents. We find that these compounds are capable of binding G-quadruplexes with a K_a in the range of 3 × 10⁵ M^?1 and with a 10-fold selectivity for quadruplex over duplex DNA.     

The influence of positional isomerism on G-quadruplex binding and anti-proliferative activity of tetra-substituted naphthalene diimide compounds

The synthesis together with biophysical and biological evaluation of a series of tetra-substituted naphthalene diimide (ND) compounds, are presented. These compounds are positional isomers of a recently-described series of quadruplex-binding ND derivatives, in which the two N-methyl-piperidine-alkyl side-chains have now been interchanged with the positions of side-chains bearing a range of end-groups. Molecular dynamics simulations of a pair of positional isomers are in accord with the quadruplex stabilization and biological data for these compounds. Analysis of structure–activity data indicates that for compounds where the side-chains are not of equivalent length then the positional isomers described here tend to have improved cell proliferation potency and in some instances, superior quadruplex stabilization ability.     

Sequences in the HSP90 promoter form G-quadruplex structures with selectivity for disubstituted phenyl bis-oxazole derivatives

The HSP90 protein is an important target in cancer. We report here that stable quadruplex DNAs can be formed from a promoter sequence in the HSP90 gene, on the basis of melting, circular and NMR studies, and show that these can be selectively targeted by non-macrocyclic quadruplex-stabilizing phenyl bis-oxazole derivatives. These do not bind significantly to duplex DNA and show low stabilization of the human telomeric quadruplex. These results suggest an approach to targeting HSP90 at the DNA level.     

Disubstituted 1,8-dipyrazolcarbazole derivatives as a new type of c-myc G-quadruplex binding ligands

A series of 1,8-dipyrazolcarbazole (DPC) derivatives (6a-6d, 7a-7d) designed as G-quadruplex ligands have been synthesized and characterized. The FRET-melting and SPR results showed that the DPC derivatives could well recognize G-quadruplex with strong discrimination against the duplex DNA. In addition, the DPC derivatives showed much stronger stabilization activities and binding affinities for c-myc G-quadruplex rather than telomeric G-quadruplex. Therefore, their interactions with c-myc G-quadruplex were further explored by means of CD spectroscopy, PCR-stop assay, and molecular modeling. In cellular studies, all compounds showed strong cytotoxicity against cancer cells, while weak cytotoxicity towards normal cells. RT-PCR assay showed that compound 7b could down-regulate c-myc gene expression in Ramos cell line, while had no effect on c-myc expression in CA46 cell line with NHE III(1) element removed, indicating its effective binding with G-quadruplex on c-myc oncogene in vivo.     

Evaluation of by disubstituted acridone derivatives as telomerase inhibitors: the importance of G-quadruplex binding

The synthesis and evaluation of a group of 2,6-, 2,7- and 3,6-bis-aminoalkylamido acridones are reported, which show a similar level of activity against telomerase in vitro compared to their acridine counterparts. Computer modelling and calculations of relative binding energies suggest an equivalent binding mode to human intramolecular G-quadruplex DNA, but with significantly reduced affinity, as a result of the limited delocalisation of the acridone chromophore compared to the acridine system. Thermal melting studies on acridone and acridine quadruplex complexes using a FRET approach support these predictions. Long-term cell proliferation studies at sub-cytotoxic doses with two representative acridones using the SKOV3 cell line, show that neither compound produces growth arrest, in contrast with the effects produced by the tri-substituted acridine compound BRACO-19. It is concluded that telomerase inhibitory activity is a necessary though by itself insufficient property in order for cellular growth arrest to occur at sub-toxic concentrations, and that tight quadruplex binding is also required.     

G-quadruplex DNA cleavage preference and identification of a perylene diimide G-quadruplex photocleavage agent using a rapid fluorescent assay

A rapid fluorescence assay for G-quadruplex DNA cleavage was used to investigate the preference of TMPyP4 photochemical and Mn·TMPyP4 oxidative cleavage. Both agents most efficiently cleave the c-Myc promoter G-quadruplex. Direct PAGE analysis of selected assay samples showed that for a given cleavage agent, different cleavage products are formed from different G-quadruplex structures. Cleavage assays carried out in the presence of excess competitor nucleic acid structures revealed the binding selectivity of cleavage agents, while comparisons with duplex cleavage efficiency employing a dual-labeled hairpin oligonucleotide revealed neither agent prefers G-quadruplex over duplex substrates. Finally, this assay was used to identify the perylene diimide Tel11 as a photocleavage agent for the c-Myc G-quadruplex.     

The aggregation and G-quadruplex DNA selectivity of charged 3,4,9,10-perylenetetracarboxylic acid diimides

Two N,N'-disubstituted perylene diimide G-quadruplex DNA ligands, Tel11 (N,N'-bis-[3-(4-methyl-morpholin-4-yl)-propyl]-3,4,9,10-perylenetetracarboxylic acid diimide diiodide) and Tel12 (N,N'-bis-[(3-phosphono)-propyl]-3,4,9,10-perylenetetracarboxylic acid diimide tetrapotassium salt) were synthesized and studied. Visible absorbance spectroscopy, resonance light scattering, and fluorescence spectroscopy were utilized to explore the aggregation state, affinity for various DNA structures, and G-quadruplex selectivity of these ligands. The water-soluble ligands exist in a monomer-dimer equilibrium with the cationic Tel11 exhibiting a greater affinity for various DNA structures than the anionic Tel12. Tel12 has greater selectivity for G-quadruplex DNA over double-stranded DNA than Tel11.     

Selective interactions of perylene derivatives having different side chains with inter- and intramolecular G-quadruplex DNA structures. A correlation with telomerase inhibition

While the importance of the aromatic core in small organic molecules, studied as G-quadruplex mediated telomerase inhibitors, appears well studied by a number of researches, the role of side chains has been less well characterized. In this paper, we have studied the ability of six perylene derivatives with different side chains to induce both inter- and intramolecular G-quadruplex structures. The distance between the aromatic core and the positive charges in the side chains emerges as a significant molecular feature in G-quadruplex formation. Furthermore, the G-quadruplex formation appears also related to drugs 'self-association', influenced by the side chains basicity. The different efficiencies of the six perylene derivatives in interacting both with inter- and intramolecular G-quadruplex structures satisfactorily correlate with telomerase inhibition in cell-free systems.     

The effect of pyridyl substituents on the thermodynamics of porphyrin binding to G-quadruplex DNA

Most of the G-quadruplex interactive molecules reported to date contain extended aromatic flat ring systems and are believed to bind principally by π–π stacking on the end G-tetrads of the quadruplex structure. One such molecule, TMPyP4, (5,10,15,20-tetra(N-methyl-4-pyridyl)porphyrin), exhibits high affinity and some selectivity for G-quadruplex DNA over duplex DNA. Although not a realistic drug candidate, TMPyP4 is used in many nucleic acid research laboratories as a model ligand for the study of small molecule G-quadruplex interactions. Here we report on the synthesis and G-quadruplex interactions of four new cationic porphyrin ligands having only 1, 2, or 3 (N-methyl-4-pyridyl) substituents. The four new ligands are: P(5) (5-(N-methyl-4-pyridyl)porphyrin), P(5,10) (5,10-di(N-methyl-4-pyridyl)porphyrin), P(5,15) (5,15-di(N-methyl-4-pyridyl)porphyrin), and P(5,10,15) (5,10,15-tri(N-methyl-4-pyridyl)porphyrin). Even though these compounds have been previously synthesized, we report alternative synthetic routes that are more efficient and that result in higher yields. We have used ITC, CD, and ESI-MS to explore the effects of the number of N-methyl-4-pyridyl substituents and the substituent position on the porphyrin on the G-quadruplex binding energetics. The relative affinities for binding these ligands to the WT Bcl-2 promoter sequence G-quadruplex are: K_TMPyP4 ≈ K_P(5,15) > K_P(5,10,15) >>> K_P(5,10), K_P(5). The saturation stoichiometry is 2:1 for both P(5,15) and P(5,10,15), while neither P(5) nor P(5,10) exhibit significant complex formation with the WT Bcl-2 promoter sequence G-quadruplex. Additionally, binding of P(5,15) appears to interact by an ‘intercalation mode’ while P(5,10,15) appears to interact by an ‘end-stacking mode’.     

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.     

New highly hydrosoluble and not self-aggregated perylene derivatives with three and four polar side-chains as G-quadruplex telomere targeting agents and telomerase inhibitors

Four new perylene derivatives with three and four basic side-chains are reported here as G-quadruplex interactive compounds. The new perylene derivatives are readily soluble in water and not self-aggregated, in contrast to what happens with the previously reported two side-chain perylene derivatives. All four compounds are able to induce the G-quadruplex and to inhibit 50% of telomerase activity at about 5 microM concentration, showing a similar efficiency with respect to each other. Molecular modelling studies are presented to try to explain these findings.     

Interaction of water with the G-quadruplex loop contributes to the binding energy of G-quadruplex to protein

Unlike DNA duplexes that release water upon interaction with protein, the binding of DNA G-quadruplex of the thrombin-binding aptamer (TBA) to thrombin takes up water. Here, to reveal the mechanism of water uptake, we designed four mutants of TBA (ΔT3, ΔT7, ΔT9, ΔT12), in which thymine residues (T3, T7, T9 and T12) were deleted from the loop regions of TBA G-quadruplex. For the mutants the thermodynamics and the osmolyte effects on the interactions with thrombin were investigated. The mutants ΔT3, ΔT9 and ΔT12 decreased the binding constants of the G-quadruplex to thrombin. Furthermore, an osmotic stress analysis indicated that the number of water molecules binding to the complex decreased in the mutants ΔT3 and ΔT9. The decrease in the binding affinity was related to loss of binding of the loop nucleotides to water molecules. Therefore, the interaction between loops of the G-quadruplex and water molecules contributed to the binding energy of G-quadruplex to protein. Our study suggests that water binding is essential for the binding of G-quadruplex to protein.     

Improved gelatinase a selectivity by novel zinc binding groups containing galardin derivatives

The synthesis of several analogues of galardin, a MMP inhibitor, are presented with their in vitro inhibitory activity against MMP-1 and MMP-2. These compounds contain a distinct Zinc Binding Group (ZBG). Those having a 2-acylated-heterocycle as well as a 2-arylamide function do not exhibit a good inhibition/selectivity against the enzymes tested. On the contrary, those that are based on a hydrazide scaffold present potent selectivity for MMP-2 versus MMP-1.