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 number and distances of positive charges of polyamine side chains in a series of perylene diimides significantly influence their ability to induce G-quadruplex structures and inhibit human telomerase

We have synthesized eight polyamine perylene diimides to conjugate the efficiency of perylene derivatives in stabilizing G-quadruplex structures and the polyamines' biological activity, due to specific interactions with different DNA domains. Our study was carried out by investigating the ability of these derivatives to induce inter- and intramolecular G-quadruplex structures by polyacrylamide gel electrophoresis (PAGE) and to inhibit telomerase in a modified TRAP assay. The two properties appear to be satisfactorily correlated and they show that the number and distances of positive charges in the side chains dramatically influence both these features. Although our previous studies on perylene derivatives with mono-positively charged side chains indicated that self assembly in aqueous solution leads to a major efficiency, the result observed with the spermine derivative suggests that a too strong aggregation is unfavourable, because it determines a lower solubility of the compounds.     

Perylene side chains modulate G-quadruplex conformation in biologically relevant DNA sequences

The stabilisation of different G-quadruplex intra- and intermolecular structures by a number of perylene derivatives characterised by side chains ending with linear or cyclic amines was investigated by electrophoretic (EMSA) and spectroscopic (CD) techniques. The G-rich sequences included the biologically relevant human telomeric TTAGGG runs and the NHE region of the c-myc oncogene. The test compounds could be subdivided into two families: derivatives carrying a cyclic amine in the side chains, which show a reduced binding to the G-quadruplex form, and linear amine congeners, exhibiting enhanced affinity. The latter efficiently induce pairing of multiple DNA chains, while the former are not able to overcome the original folding of the nucleic acid sequence which is preserved in the complex. Remarkably, addition of the perylenes to G-rich sequences paired in a double helical form results in G-quadruplex induction by weak binders only. This is likely related to the ability of strong G-quadruplex binders, but not of weak G-quadruplex binders, to efficiently intercalate into the double-stranded arrangement, which becomes stabilised and is not prone to undergo denaturation and subsequent G-quadruplex folding essentially for kinetic reasons. Hence, two apparently conflicting requirements emerge from this work. In fact, linear alkylamino terminals in the perylene side chains are capable of strong and selective G-quadruplex recognition, but only cyclic amine end groups favour duplex-quadruplex transitions that are likely crucial to produce biological and pharmacological effects in living systems.     

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.     

Lysinyl macrocyclic hexaoxazoles: synthesis and selective G-quadruplex stabilizing properties

Macrocyclic hexaoxazoles having one or two lysinyl side chains in which the terminal nitrogen is either a primary amine, N,N-dimethylamine, or an acetamide have been synthesized. Sodium ion has been found to be beneficial to the macrocyclization step by acting as a template around which the linear polyoxazole can organize. Each of the targeted compounds selectivity stabilizes G-quadruplex versus duplex DNA. Compounds with one valine and one lysine residue display the best combination of G-quadruplex stabilizing ability with no detectable stabilization of duplex DNA.     

Tri-, tetra- and heptacyclic perylene analogues as new potential antineoplastic agents based on DNA telomerase inhibition

A recent approach in anticancer chemotherapy envisages telomerase as a potentially useful target. An attractive strategy deals with the development of compounds able to stabilize telomeric DNA in the G-quadruplex folded structure and, among them, a prominent position is found in the perylenes. With the aim to further investigate the role of drug structure, in view of possible pharmaceutical applications, we synthesized a series of compounds related to PIPER, a well-known perylene-based telomerase inhibitor. We modified the number of condensed aromatic rings and introduced different side chains to modulate drug protonation state and extent of self-aggregation. Effective telomerase inhibition was induced by heptacyclic analogues only, some showing a remarkably wide selectivity index with reference to inhibition of Taq polymerase. G-quadruplex stabilization was monitored by circular dichroism and melting experiments. Cell cytotoxicity measurements indicated a poor short-term cell killing ability for the best G-quartet binders. Besides the presence of a planar seven-condensed ring system, the introduction of a cyclic amine in the side chains critically affects the selectivity window.     

Tuning G-quadruplex vs double-stranded DNA recognition in regioisomeric lysyl-peptidyl-anthraquinone conjugates

Anthraquinone is a versatile scaffold to provide effective DNA binders. This planar system can be easily conjugated to protonable side chains: the nature of the lateral groups and their positions around the tricyclic moiety largely affect the DNA recognition process in terms of binding affinity and mode, as well as sequence and structure of the target nucleic acid. Starting from an anthracenedione system symmetrically functionalized with N-terminal lysyl residues, we incremented the length of side chains by introducing a Gly, Ala, or Phe spacer, characterized by different flexibility, lipophilicity, and bulkiness. Moreover, 2,6, 2,7, 1,8, and 1,5 regioisomers were examined to yield a small bis(lysyl-peptidyl) anthracenedione library. By merging spectroscopic, enzymatic, and cellular results, we showed that the proper combination of a basic aminoacid (Lys) with a more hydrophobic residue (Phe) can provide selective G-quadruplex recognition, in particular when side chains are located at positions 2,6 or 2,7. In fact, while these derivatives effectively bind G-quadruplex structures, they behave at the same time as rather poor double-stranded DNA intercalators. As a result, the Lys-Phe substituted anthraquinones are poorly cytotoxic but still able to promote a senescence mechanism in cancer cells. This combination of chemical and biological properties foresees potentially valuable applications in anticancer medicinal chemistry.     

The relationship between ligand aggregation and G-quadruplex DNA selectivity in a series of 3,4,9,10-perylenetetracarboxylic acid diimides

Human telomeres are comprised of d(TTAGGG) repeats that are capable of forming G-quadruplex DNA structures. Ligands that bind to and stabilize these G-quadruplex DNA structures are potential inhibitors of the cancer cell-associated enzyme telomerase. Other potential biological uses of G-quadruplex targeting ligands have been proposed. One particularly challenging aspect of the contemplated uses of G-quadruplex targeting ligands is their selectivity for G-quadruplex DNA versus double-stranded DNA structures. We have previously reported the observation that two structurally related 3,4,9,10-perylenetetracarboxylic acid diimide-based G-quadruplex DNA ligands, PIPER [N,N'-bis(2-(1-piperidino)ethyl)-3,4,9,10-perylenetetracarboxylic acid diimide] and Tel01 [N,N'-bis(3-(4-morpholino)propyl)-3,4,9,10-perylenetetracarboxylic acid diimide], have different levels of G-quadruplex DNA binding selectivity at pH 7 as determined by absorbance changes in the presence of different DNA structures [Kerwin, S. M., Chen, G., Kern, J. T., and Thomas, P. W. (2002) Bioorg. Med. Chem. Lett. 12, 447-450]. Here we report that the less G-quadruplex DNA selective ligand PIPER can unwind double-stranded, closed circular plasmid DNA, as determined by a topoisomerase I assay. A model for the interaction of Tel01 with the G-quadruplex DNA structure formed by d(TAGGGTTA) was determined from NMR experiments. This model is similar to the previously published model for PIPER bound to the same G-quadruplex DNA and failed to provide a structural basis for the observed increased selectivity of Tel01 interaction with G-quadruplex DNA. In contrast, investigation into the aggregation state of Tel01 and PIPER as well as other 3,4,9,10-perylenetetracarboxylic acid diimide analogues bearing basic side chains demonstrates that ligand aggregation is correlated with G-quadruplex DNA binding selectivity. For all six analogues examined, those ligands that were aggregated at pH 7 in 70 mM potassium phosphate, 100 mM KCl, 1 mM EDTA buffer also demonstrated G-quadruplex DNA binding selectivity under these buffer conditions. Ligands that were not aggregated under these conditions display much lower levels of G-quadruplex DNA selectivity. The aggregation state of these ligands is extremely sensitive to the buffer pH. Tel01, which is aggregated at pH 7, is not aggregated at pH 6.4, where it demonstrates only modest G-quadruplex DNA binding selectivity, and PIPER in pH 8.5 buffer is both aggregated and highly G-quadruplex DNA-selective. To our knowledge, these studies demonstrate the first DNA structure selectivity as achieved through pH-mediated ligand aggregation. The potential impact of these findings on the selectivity of other classes of G-quadruplex DNA ligands is discussed.     

Simian virus 40 large T-antigen G-quadruplex DNA helicase inhibition by G-quadruplex DNA-interactive agents

On the basis of growing evidence for G-quadruplex DNA structures in genomic DNA and the presumed need to resolve these structures for DNA replication, the G-quadruplex DNA unwinding ability of a prototypical replicative helicase, SV40 large T-antigen (T-ag), was investigated. Here, we demonstrate that this G-quadruplex helicase activity is robust and comparable to the duplex helicase activity of T-ag. Analysis of the SV40 genome demonstrates the presence of sequences that may form intramolecular G-quadruplexes, which are the presumed natural substrates for the G-quadruplex helicase activity of T-ag. A number of G-quadruplex-interactive agents as well as new perylene diimide (PDI) derivatives have been investigated as inhibitors of both the G-quadruplex and the duplex DNA helicase activities of T-ag. A unique subset of these G-quadruplex-interactive agents inhibits the G-quadruplex DNA unwinding activity of T-ag, relative to those reported to inhibit G-quadruplex DNA unwinding by RecQ-family helicases. We also find that certain PDIs are both potent and selective inhibitors of the G-quadruplex DNA helicase activity of T-ag. Surface plasmon resonance and fluorescence spectroscopic G-quadruplex DNA binding studies of these T-ag G-quadruplex helicase inhibitors have been carried out, demonstrating the importance of attributes in addition to binding affinity for G-quadruplex DNA that may be important for inhibition. The identification of potent and selective inhibitors of the G-quadruplex helicase activity of T-ag provides tools for probing the specific role of this activity in SV40 replication.     

Carbazole-based fluorescent probes for G-quadruplex DNA targeting with superior selectivity and low cytotoxicity

G-quadruplex DNA plays a very important role in clinical diagnosis and fluorescence analysis has attracted extensive attention. A class of carbazole-based fluorescent probes for the detection of G-quadruplex DNA was established in this work. In this system, the installation of an oligo(ethylene glycol) chain on the scaffold will improve the water-solubility and biocompatibility. The presence of styrene-like different side groups could tune the selectivity toward G-quadruplex DNA binding. Results revealed that the substitution pattern and position gave a great influence on the ability for the discrimination of the G-quadruplex from other DNA structures. Especially, probe E1 bound to G-quadruplex DNA with superior selectivity, which exhibiting almost no fluorescence response in the presence of non-G-quadruplex DNA structures. Comprehensive analyses revealed that E1 could bind both ends of the G-quadruplex, resulting in a significant increase of fluorescence emission intensity. Cellular uptake assay suggested that E1 could pass through membrane and enter living cells with low cytotoxicity.     

Design,synthesis and evaluation of 4,7-diamino-1,10-phenanthroline G-quadruplex ligands

A series of 4,7-diamino-1,10-phenanthroline derivatives carrying positively charged side chains has been synthesized, and their G-quadruplex interaction evaluated by circular dichroism (CD) and surface plasmon resonance (SPR). In absence of side chains, 4,7-diamino-1,10-phenanthroline exhibits a weak but significant G-quadruplex stabilizing effect, compared to no stabilization by 1,10-phenanthroline. We hypothesize that this effect is due to increased basicity of the phenanthroline nitrogens and protonation or ion chelation to form a central positive charge which stack on the G-tetrad above the central ionic column. Introduction of positively charged side chains results in compounds with appreciable G-quadruplex stabilizing properties and high aqueous solubility, with the longer side chains giving more potent compounds. Ligands carrying guanidine side chains in general show higher quadruplex stabilizing activity and distinctly slower kinetic properties than their amino and dimethylamino analogues, possibly due to specific hydrogen bond interactions with the G-quadruplex loops.     

Natural and synthetic G-quadruplex interactive berberine derivatives

The interaction of the natural alkaloid berberine with various G-quadruplex DNA structures and its ability to inhibit telomerase have been examined and compared with those of a synthetic piperidino derivative and the related compound coralyne. The results show that these molecules have selectivity for G-quadruplex compared to duplex DNA, and that their aromatic moieties play a dominant role in quadruplex binding.     

Perylene ligand wrapping G-quadruplex DNA for label-free fluorescence potassium recognition

A perylene ligand, N,N-bis-(1-aminopropyl-3-propylimidazol salt)-3,4,9,10-perylene tetracarboxylic acid diimide ligand (PDI), which consisted of π-conjugated perylene moiety and hydrophilic side chains with positively charged imidazole rings, was used to wrap G-quadruplex for fluorescence turn-on K(+) recognition. Electrostatic attraction between PDI's positively charged imidazole rings and DNA's negatively charged phosphate backbones enabled PDI to accumulate on DNA. Upon trapping K(+), these G-rich DNA sequences transitioned to G-quadruplex. Subsequently, PDI ligands wrapped G-quadruplex, in which the flat aromatic core of PDI ligand interacted with G-quartet through π-π stacking and the side chains were positioned in grooves through electrostatic interactions. Consequently, the interaction mode change and conformational transition from PDI stacked G-sequence to PDI wrapped G-quadruplex led to PDI fluorescence enhancement, which was readily monitored as the detection signal. This strategy excluded the sequence tagging step and exhibited high selectivity and sensitivity towards K(+) ion with the linear detection range of 10-150nM. Besides, PDI ligands may hold diagnostic and therapeutic application potentials to human telomere and cancer cells.     

2,6-Diphenylthiazolo[3,2-b][1,2,4]triazoles as telomeric G-quadruplex stabilizers

The design and synthesis of 2,6-diphenylthiazolo[3,2-b][1,2,4]triazoles characterized by a large aromatic building block bearing cationic side chains are reported. These molecules are evaluated as telomeric G-quadruplex stabilizers and for their selectivity towards duplex DNA by competition experiments. Two compounds (14a, 19) were found active with high selectivity for telomeric G-quadruplex over duplex DNA.     

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.     

The influence of pH on the G-quadruplex binding selectivity of perylene derivatives

Three new perylene derivatives with branched ionizable side chains were synthesized, and their G-quadruplex binding specificities were compared by spectroscopic and electrophoretic analysis with two well-studied G-quadruplex ligands: PIPER and TmPyP4. The value of pH and consequent charge formation and self-aggregation of these perylene derivatives influences not only the type of G-quadruplex formation, but also the G-quadruplex binding selectivity.     

A new cationic porphyrin derivative (TMPipEOPP) with large side arm substituents: a highly selective G-quadruplex optical probe

The discovery of uncommon DNA structures and speculation about their potential functions in genes has brought attention to specific DNA structure recognition. G-quadruplexes are four-stranded nucleic acid structures formed by G-rich DNA (or RNA) sequences. G-rich sequences with a high potential to form G-quadruplexes have been found in many important genomic regions. Porphyrin derivatives with cationic side arm substituents are important G-quadruplex-binding ligands. For example, 5,10,15,20-Tetrakis(N-methylpyridinium-4-yl)-21H,23H-porphyrin (TMPyP4), interacts strongly with G-quadruplexes, but has poor selectivity for G-quadruplex versus duplex DNA. To increase the G-quadruplex recognition specificity, a new cationic porphyrin derivative, 5,10,15,20-tetra-{4-[2-(1-methyl-1-piperidinyl)ethoxy]phenyl} porphyrin (TMPipEOPP), with large side arm substituents was synthesized, and the interactions between TMPipEOPP and different DNA structures were compared. The results show that G-quadruplexes cause large changes in the UV-Vis absorption and fluorescence spectra of TMPipEOPP, but duplex and single-stranded DNAs do not, indicating that TMPipEOPP can be developed as a highly specific optical probe for discriminating G-quadruplex from duplex and single-stranded DNA. Visual discrimination is also possible. Job plot and Scatchard analysis suggest that a complicated binding interaction occurs between TMPipEOPP and G-quadruplexes. At a low [G-quadruplex]/[TMPipEOPP] ratio, one G-quadruplex binds two TMPipEOPP molecules by end-stacking and outside binding modes. At a high [G-quadruplex]/[TMPipEOPP] ratio, two G-quadruplexes bind to one TMPipEOPP molecule in a sandwich-like end-stacking mode.     

Two cationic porphyrin isomers showing different multimeric G-quadruplex recognition specificity against monomeric G-quadruplexes

Ligands that can interact specifically with telomeric multimeric G-quadruplexes could be developed as promising anticancer drugs with few side effects related to other G-quadruplex-forming regions. In this paper, a new cationic porphyrin derivative, m-TMPipEOPP, was synthesized and characterized. Its multimeric G-quadruplex recognition specificity under molecular crowding conditions was compared to its isomer p-TMPipEOPP. The slight structural difference accounts for different multimeric G-quadruplex recognition specificity for the two isomers. p-TMPipEOPP can barely discriminate between multimeric and monomeric G-quadruplexes. By contrast, m-TMPipEOPP can bind with multimeric but not with monomeric G-quadruplexes. p-TMPipEOPP might bind to multimeric G-quadruplexes by two modes: sandwich-like end-stacking mode and pocket-dependent intercalative mode. Increasing the pocket size between adjacent two G-quadruplex uints is beneficial for the latter mode. m-TMPipEOPP might bind to multimeric G-quadruplexes by a side binding mode, which confers m-TMPipEOPP with higher multimeric G-quadruplex recognition specificity compared to p-TMPipEOPP. m-TMPipEOPP increases the stability of multimeric G-quadruplex under both dilute and molecular crowding conditions but its G-quadruplex-stabilizing ability is a little weaker than p-TMPipEOPP. These results provide important information for the design of highly specific multimeric G-quadruplex ligands. Another interesting finding is that pocket size is an important factor in determining the stability of multimeric G-quadruplexes.     

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.