G-quadruplex Interaction Research Articles

Specific recognition and stabilization of monomeric and multimeric G-quadruplexes by cationic porphyrin TMPipEOPP under molecular crowding conditions

Ligands targeting telomeric G-quadruplexs are considered good candidates for anticancer drugs. However, current studies on G-quadruplex ligands focus exclusively on the interactions of ligands and monomeric G-quadruplexes under dilute conditions. Living cells are crowded with biomacromolecules, and the ∼200-nucleotide G-rich single-stranded overhang of human telomeric DNA has the potential to fold into multimeric G-quadruplex structures containing several G-quadruplex units. Studies on interactions between ligands and multimeric G-quadruplexes under molecular crowding conditions could provide a new route for screening specific telomeric G-quadruplex-targeting ligands. Herein, TMPipEOPP, a cationic porphyrin derivative designed by us, was demonstrated as a promising multimeric telomeric G-quadruplex ligand under molecular crowding conditions. It could highly specifically recognize G-quadruplexes. It could also promote the formation of G-quadruplexes and stabilize them. Detailed studies showed that TMPipEOPP interacted with monomeric G-quadruplexes in sandwich-like end-stacking mode of quadruplex/TMPipEOPP/quadruplex and interacted with multimeric human telomeric G-quadruplexes by intercalating into the pocket between two adjacent G-quadruplex units. The pocket size greatly affected TMPipEOPP binding. A larger pocket was advantageous for the intercalation of TMPipEOPP. This work provides new insights into the ligand-binding properties of multimeric G-quadruplexes under molecular crowding conditions and introduces a new route for screening anticancer drugs targeting telomeric G-quadruplexes.

A Light Trigger for DNA Nanotechnology

DNA minicircles with light-activated ‘caged’ interaction modules—based either on sticky ends or G-quadruplex interactions—do not form dimers until they are irradiated with near-UV light (not harmful for DNA). This technology offers a level of spatiotemporal (hybridization) control for functional nanoarchitectures.

Abstract 2517: Hybrid benzofused-biaryl polyamides with selective telomeric G-quadruplex stabilization potential

Abstract Guanine-rich nucleic acids can fold into four-stranded G-quadruplex structures which are found in telomeric DNA repeats as well as in sequences in the promoter and other regulatory regions of genes. Small molecules that can selectively bind and stabilize quadruplex structure are of increasing significance as potential anticancer agents. We recently reported a series of novel biaryl polyamides with significant selectively towards G-quadruplex compared to duplex DNA, and modest selectivity between different quadruplex types such as c-kit1, c-kit2 and HT4 (Chemical Commun., 4097, 2009). These molecules were based on a distamycin scaffold but included biaryl building blocks in place of pyrroles to switch preference from duplex to quadruplex DNA. This alteration in shape ensured that the molecules had low affinity for duplex DNA while increasing their interaction with G-quadruplex structure, since the ligands had similar dimensions. We have now synthesized a 34-member second-generation biaryl polyamide library based on the previously obtained structural information. Initial assessment of the G-quadruplex interaction of library members was carried out using a FRET-based melting assay. Two compounds (KN-88 and KN-119) containing hybrid benzofused and biaryl building blocks provided significant selective stabilization of human Telomeric G-quadruplex. At 1 µM they stabilize human Telomeric G-quadruplex by 21 and 18 °C, respectively, while showing insignificant affinity for duplex DNA. FRET competition assays with C-kit quadruplexes and CT DNA further confirmed selective stabilization of telomeric quadruplexes. CD titrations of KN-88 and KN-119 with h-telo, c-kit1 and c-kit2 quadruplex-forming DNA sequences showed concentration-dependent enhancement of major CD signals, and in some cases the ligands induced the folding of a particular quadruplex structure. Kd values for KN-88 and KN-119 for telomeric quadruplex sequences were determined by CD titration and were found to be 2.8 × 106 and 7.0 × 106, respectively. Short-term growth inhibitory experiments against a panel of tumour cell lines (MiaPaCa2, A549, MCF7, HeLa, U87MG and A431) gave low micromolar IC50 values for these two compounds, and significant selectivity for tumour cell lines compared to the non-tumour fibroblast WI38 cell line. Given their low molecular weight, good water solubility and cellular penetration properties, molecules of this type have potential as therapeutic agents and reagents that can selectively probe quadruplex structure and/or selectively down-regulate signaling pathways in cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2517. doi:10.1158/1538-7445.AM2011-2517

Synthesis and Binding Studies of Novel Diethynyl‐Pyridine Amides with Genomic Promoter DNA G‐Quadruplexes

Herein, we report the design, synthesis and biophysical evaluation of novel 1,2,3-triazole-linked diethynyl-pyridine amides and trisubstituted diethynyl-pyridine amides as promising G-quadruplex binding ligands. We have used a Cu(I)-catalysed azide-alkyne cycloaddition click reaction to prepare the 1,2,3-triazole-linked diethynyl-pyridine amides. The G-quadruplex DNA binding properties of the ligands have been examined by using a Förster resonance energy transfer (FRET) melting assay and surface plasmon resonance (SPR) experiments. The investigated compounds are conformationally flexible, having free rotation around the triple bond, and exhibit enhanced G-quadruplex binding stabilisation and specificity between intramolecular promoter G-quadruplex DNA motifs compared to the first generation of diaryl-ethynyl amides (J. Am. Chem. Soc. 2008, 130, 15950-15956). The ligands show versatility in molecular recognition and promising G-quadruplex discrimination with 2-50-fold selectivity exhibited between different intramolecular promoter G-quadruplexes. Circular dichroism (CD) spectroscopic analysis suggested that at higher concentration these ligands disrupt the c-kit2 G-quadruplex structure. The studies validate the design concept of the 1,3-diethynyl-pyridine-based scaffold and demonstrate that these ligands exhibit not only significant selectivity over duplex DNA but also variation in G-quadruplex interaction properties based on small chemical changes in the scaffold, leading to unprecedented differential recognition of different DNA G-quadruplex sequences.

Structure, location and interactions of G‐quadruplexes

Four-stranded G-rich DNA structures called G-quadruplexes have been the subject of increasing interest recently. Experimental and computational techniques have been used to implicate them in important biological processes such as transcription and translation. In this minireview, I discuss how they form, what structures they adopt and with what stability. I then discuss the computational approaches used to predict them on a genomic scale and how the information derived can be combined with experiments to understand their biological functions. Other minireviews in this series deal with G-quadruplex nucleic acids and human disease [Wu Y & Brosh RM Jr (2010) FEBS J] and making sense of G-quadruplex and i-motif function in oncogene promoters [Brooks TA et al. (2010) FEBS J].

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.

Specific DNA G‐quadruplexes bind to ethanolamines

A significant number of G-quadruplex-forming sequences have been revealed in human genome by bioinformatic searches, implying that G-quadruplexes may be involved in important biological processes and may be new chemotherapeutic targets. Therefore, it is important to discover the potential interactions of G-quadruplexes with other molecules or groups. Here we describe a class of G-quadruplexes, which can bind to ethanolamine groups that widely exist in biomolecules and drug molecules. The specific interaction of these G-quadruplexes with ethanolamine groups was identified by high performance affinity chromatography (HPAC) using immobilized ethanolamine and diethanolamine as stationary phase reagents. The circular dichroism (CD) spectra and native polyacrylamide gel electrophoresis (PAGE) show that these ethanolamine binding quadruplexes adopt an intramolecularly parallel structure. The relationship of ethanolamine binding and G-quadruplexe structure provides new clues for the G-quadruplex-related studies as well as for the molecular designs of therapeutic reagents.

Applications of isothermal titration calorimetry in biophysical studies of G-quadruplexes.

G-quadruplexes are higher-order nucleic acids structures formed by G-rich sequences that are stabilized by tetrads of hydrogen-bonded guanine bases. Recently, there has been growing interest in the study of G-quadruplexes because of their possible involvement in many biological processes. Isothermal titration calorimetry (ITC) has been proven to be a useful tool to study the energetic aspects of G-quadruplex interactions. Particularly, ITC has been applied many times to determine the thermodynamic properties of drug-quadruplex interactions to screening among various drugs and to address drug design. In the present review, we will focus on the ITC studies of G-quadruplex structures and their interaction with proteins and drugs and the most significant results will be discussed.

Single Molecule Force Spectroscopy of Guanine Quadruplex DNA

A single molecule force spectroscopy study was performed to examine the formation and stability of guanine quadruplexes using various G-rich oligomers. The study examined the differences in interactions of four-stranded and two-stranded G-quadruplexes. The two-stranded G-quadruplex system was used for dynamic force spectroscopy measurements. Rupture force dependency on the log loading rate was found to be non-linear. Using recently developed microscopic models, the apparent kinetic and thermodynamic parameters for the G-quadruplex system were estimated. The microscopic models predict barrier widths that are consistent with the bond length of the quadruplex strand.

A new method for the study of G-quadruplex ligands

A new method for the study of G-quadruplex ligands was developed, in which the interaction of G-quadruplexes with ligands can be judged by the naked eye, eliminating the need for any expensive machines.

Interrogation of G-quadruplex–protein interactions

The ability to accurately examine the interaction of G-quadruplex DNA with proteins is essential for revealing the biological roles of these unusual DNA structures. In this regard, there are four primary G-quadruplex-related activities of proteins that have been studied including simple equilibrium binding, promotion or catalysis of G-quadruplex formation, dissociation of G-quadruplex structures, and covalent modification of G-quadruplexes, which includes both nucleolytic cleavage and nucleotide addition. Here, assays used to examine the interactions of G-quadruplexes with proteins will be reviewed and specific methods to study the interactions of G-quadruplexes from telomeric DNA sequences with a variety of proteins will be described. Importantly, this review emphasizes the importance of evaluating the integrity of the G-quadruplex being studied as single sequences can often form a variety of folded structures.

Design, synthesis, and biological evaluation of a series of fluoroquinoanthroxazines with contrasting dual mechanisms of action against topoisomerase II and G-quadruplexes.

Topoisomerase inhibitors are important and clinically effective drugs, while G-quadruplex-interactive compounds that disrupt telomere maintenance mechanisms have yet to be proven useful in the clinic. If G-quadruplex-interactive compounds are to be clinically useful, it will most likely be in combination with more established cytotoxic agents. We have previously reported on a family of topoisomerase II inhibitors that also interact with G-quadruplexes. On the basis of previously established structure-activity relationships (SARs) for compounds that are able to inhibit topoisomerase II or interact with G-quadruplex to varying degrees, we have now designed and synthesized four new fluoroquinoanthroxazines (FQAs) that have different profiles of mixed topoisomerase II poisoning effects and G-quadruplex interactions. The biological profiles of the four new compounds were determined with respect to G-quadruplex interaction (polymerase stop and photocleavage assays) and topoisomerase II interaction (DNA cleavage and kDNA decatenation assays), alongside cytotoxicity tests with matched pairs of topoisomerase II-resistant and topoisomerase II-sensitive cells and with telomerase (+) and ALT (+) cell lines (ALT = alternative lengthening of telomeres). From this study, we have identified two FQAs with sharply contrasting profiles of potent G-quadruplex interaction with a weak topoisomerase II poisoning effect, and vice versa, for further evaluation to determine the optimum combination of these activities in subsequent in vivo studies.

G-quadruplex interaction of 3,6,9-trisubstituted acridines leads to specific telomerase inhibition, induction of senescence and telomere shortening

G-quadruplex interaction of 3,6,9-trisubstituted acridines leads to specific telomerase inhibition, induction of senescence and telomere shortening

Telomestatin, a potent telomerase inhibitor that interacts quite specifically with the human telomeric intramolecular g-quadruplex.

Telomestatin is a natural product isolated from Streptomyces anulatus 3533-SV4 and has been shown to be a very potent telomerase inhibitor. The structural similarity between telomestatin and a G-tetrad suggested to us that the telomerase inhibition might be due to its ability either to facilitate the formation of or trap out preformed G-quadruplex structures, and thereby sequester single-stranded d[T(2)AG(3)](n) primer molecules required for telomerase activity. Significantly, telomestatin appears to be a more potent inhibitor of telomerase (5 nM) than any of the previously described G-quadruplex-interactive molecules. In this communication we provide the first experimental evidence that telomestatin selectively facilitates the formation of or stabilizes intramolecular G-quadruplexes, in particular, that produced from the human telomeric sequence d[T(2)AG(3)](4). A simulated annealing (SA) docking approach was used to study the binding interactions of telomestatin with the intramolecular antiparallel G-quadruplex structure. Each intramolecular G-quadruplex molecule was found to bind two telomestatin molecules (unpublished results). A 2:1 model for the telomestatin bound in the external stacking mode in an energy minimized complex with the human telomeric basket-type G-quadruplex was constructed. Our observation that a G-quadruplex-interactive molecule without significant groove interactions is able to reorient in a G-quadruplex structure proints to the importance of core interaction with an asymmetric G-quadruplex structure in producing selective binding. Furthermore, the G-quadruplex interactions of telomestatin are more selective for the intramolecular structure in contrast to other G-quadruplex-interactive agents, such as TMPyP4.