Current Pharmaceutical Design - Volume 18, Issue 14, 2012

The anticancer pharmacological treatment is typically affected by a non optimized therapeutic index. In the last decade several novel molecular targets have been identified and when possible used to design innovative anticancer agents. Among them a special fold of the DNA, known as G-quadruplex conformation, has been found particularly attractive for some important reasons. Firstly, it is exclusive of guanine-rich DNA sequences that are able to create, due to the formation of Hoogsteen hydrogen bonds, a structural core different from the most common Watson and Crick double helix. Secondly, it is expressed especially in the telomeric regions of the genome undergoing undefined repeating sequence in correlation with uncontrolled cell proliferation. Thirdly, some oncogenes and promoters, such as c-kit, myc, contain the sequence prerequisites to fold into a G-quadruplex conformation. On the basis of these considerations, an emergent approach for innovative anticancer agents is based on the stabilization of this conformation via the identification of ligands that are able to selectively bind it. Moreover, in 2009 the Nobel prize for Medicine has been assigned to three scientists that discovered the role of telomers and telomerases confirming this research area as an “hot topic” in the anticancer field. In this special issue international experts with multidisciplinary competencies contribute to highlight the G-quadruplex impact in the development of novel therapeutic and diagnostic agents. A general description about occurrence and genomic location of this special DNA conformation is introduced by Paeschke and coworkers [1]. In the scientific community the DNA G-quadruplex conformational polymorphism is one of the major disputed topic, treated by Alcaro and co-workers by docking simulations using as probes both enantiomers of the most active natural compound telomestatin [2]. Finding out ligands that are able to recognize the G-quadruplex core of specific DNA sequences is a new goal for the modern medicinal chemists. The state-of-the-art in the currently available methodologies is described by the research groups of Randazzo [3] and Jaumot [4].....

DNA and RNA regions containing tracts of guanines can form very stable secondary structures called G-quadruplex (G4). Genomic sequences with the potential to form G4 (G4-motifs) are abundant across species. In all analyzed genomes G4 motifs are found near promoter regions and double strand break sites and at telomeres. Telomeres are very G-rich and prone for G4 formation. Therefore they are routinely used in in vitro and in vivo experiments to elucidate the function of G4 structures in telomere metabolism. Recently various labs demonstrated that telomere length maintenance is mediated via G4 structures. Telomere-binding proteins specifically bind to G4 structure and regulate this structure throughout the cell cycle.

Human telomeres are comprised of d(TTAGGG) repeats involved in the formation of G-quadruplex DNA structures. Ligands stabilizing these G-quadruplex DNA structures are potential inhibitors of the cancer cell-associated enzyme telomerase. In human cells, telomerase adds multiple copies of the 5'-GGTTAG-3' motif to the end of the G-strand of the telomere and in the majority of tumor cells it results over-expressed. Several structural studies have revealed a diversity of topologies for telomeric quadruplexes, which are sensitive to the nature of the cations present, to the flanking sequences, and probably also to concentration, as confirmed by the different conformations deposited in the Protein Data Bank (PDB). The existence of different polymorphisms in the DNA quadruplex and the absence of a uniquely precise binding site prompted us to carefully compare the two different docking approaches: MOLINE and Auto- Dock. As target we have selected six different experimental models of the human telomeric sequence d[AG3(T2AG3)3] based on three Gtetrads and as ligands the telomestatin isomers, whose the S enantomer is experimentally known to recognize the G-quadruplex better than the R one. In this communication we discuss the different binding modes of the well known strong telomestatin G-quadruplex binder form the thermodynamic and the geometrical points of view. With respect to this last issue we propose an easy approach to classify binding modes of G-quadruplex ligands based on a single angle descriptor as tool for the quick analysis of the binding modes.

Nowadays, the molecular basis of interaction between low molecular weight compounds and biological macromolecules is the subject of numerous investigations aimed at the rational design of molecules with specific therapeutic applications. In the last decades, it has been demonstrated that DNA quadruplexes play a critical role in several biological processes both at telomeric and gene promoting levels thus providing a great stride in the discovery of ligands able to interact with such a biologically relevant DNA conformation. So far, a number of experimental and computational approaches have been successfully employed in order to identify new ligands and to characterize their binding to the DNA. The main focus of this review is the description of these methodologies, placing a particular emphasis on computational methods, isothermal titration calorimetry (ITC), mass spectrometry (MS), nuclear magnetic resonance (NMR), circular dichroism (CD) and fluorescence spectroscopies.

The present paper reviews the recent advances in and applications of experimental techniques used to study interactions between G-quadruplex structures and ligands that are potentially of pharmaceutical interest. Several instrumental techniques are used to study such interactions. The application of spectroscopic techniques such as molecular absorption, circular dichroism, molecular fluorescence, mass spectrometry and nuclear magnetic resonance are reviewed and we discuss the type of information (qualitative or quantitative) that can be obtained from the use of each technique. Additionally, the application of complementary techniques such as surface plasmon resonance, isothermal titration calorimetry and different methods based on biochemistry is considered. For each technique, the main applications are presented and they are classified according to the family of the ligand and the type of G-quadruplex forming sequence (human telomeric or promoter region of oncogenes) considered.

DNA is the chemotherapeutic target for treating diseases of genetic origin. Besides well-known double-helical structures (A, B, Z, parallel stranded-DNA etc.), DNA is capable of forming several multi-stranded structures (triplex, tetraplex, i-motif etc.) which have unique biological significance. The G-rich 3'-ends of chromosomes, called telomeres, are synthesized by telomerase, a ribonucleoprotein, and over-expression of telomerase is associated with cancer. The activity of telomerase is suppressed if the G-rich region is folded into the four stranded structures, called G-quadruplexes (G4-DNAs) using small synthetic ligands. Thus design and synthesis of new G4- DNA ligands is an attractive strategy to combat cancer. G4-DNA forming sequences are also prevalent in other genomic regions of biological significance including promoter regions of several oncogenes. Effective gene regulation may be achieved by inducing a G4-DNA structure within the G-rich promoter sequences. To date, several G4-DNA stabilizing ligands are known. DNA groove binders interact with the duplex B-DNA through the grooves (major and minor groove) in a sequence-specific manner. Some of the groove binders are known to stabilize the G4-DNA. However, this is a relatively under explored field of research. In this review, we focus on the recent advances in the understanding of the G4-DNA structures, particularly made from the human telomeric DNA stretches. We summarize the results of various investigations of the interaction of various organic ligands with the G4-DNA while highlighting the importance of groove binder-G4-DNA interactions.

Small molecules that can induce and stabilize G-quadruplex DNA structures represent a novel approach for anti-cancer and anti-parasitic therapy and extensive efforts have been directed towards discovering lead compounds that are capable of stabilizing quadruplexes. The purpose of this study is to explore conformational modifications in a series of heterocyclic dications to discover structural motifs that can selectively bind and stabilize specific G-quadruplexes, such as those present in the human telomere. The G-quadruplex has various potential recognition sites for small molecules; however, the primary interaction site of most of these ligands is the terminal tetrads. Similar to duplex-DNA groove recognition, quadruplex groove recognition by small molecules offers the potential for enhanced selectivity that can be developed into a viable therapeutic strategy. The compounds investigated were selected based on preliminary studies with DB832, a bifuryl-phenyl diamidine with a unique telomere interaction. This compound provides a paradigm that can help in understanding the optimum compound-DNA interactions that lead to quadruplex groove recognition. DNA recognition by the DB832 derivatives was investigated by biophysical experiments such as thermal melting, circular dichroism, mass spectrometry and NMR. Biological studies were also performed to complement the biophysical data. The results suggest a complex binding mechanism which involves the recognition of grooves for some ligands as well as stacking at the terminal tetrads of the human telomeric G-quadruplex for most of the ligands. These molecules represent an excellent starting point for further SAR analysis for diverse modes of quadruplex recognition and subsequent structure optimization for drug development.

Guanine-rich nucleic acid sequences are known to form G-quadruplex - four-stranded DNA or RNA structures stabilized by an array of Hoogsteen hydrogen bonds. G-quadruplex structures are involved in the modulation of gene expression at the transcription and translation levels. Accordingly, G-quadruplexes are considered as novel therapeutic targets for anticancer drug development. In this review, the authors provide a brief, up-to-date summary of G-quadruplex binding ligands, including naturally occurring molecules, synthetic compounds, and molecules identified by computational database screening. The key structural motifs of G-quadruplex binding ligands, that is, an aromatic core and basic side chains, are addressed in the context of how these molecules interact with G-quadruplex. A better understanding of these interactions would facilitate the rational design of ligands selective for DNA or RNA G-quadruplex.

G-quadruplex structure is a four-stranded form of DNA, which is associated with cancer cell proliferation. G-quadruplexstabilized ligands have the potential to interfere with telomere replication by blocking the elongation procedure catalyzed by telomerase, and therefore have the potential to be anti-cancer drugs. A considerable number of novel compounds capable of targeting G-quadruplex at high affinity and specificity have been reported. Among them, several G-quadruplex ligands have shown promising anti-cancer activity in tumor xenograft models, and entered phase II clinical trials on cancer patients. This review summarized recent developments of Gquadruplex ligands as anti-cancer drugs and several powerful strategies to discover novel G-quadruplex ligands as anti-cancer drug candidates by screening natural product extracts and structural optimization of previously identified typical compounds.

Regulation of genetic functions based on targeting DNA or RNA sequences with complementary oligonucleotides is especially attractive in the post-genome era. Oligonucleotides can be rationally designed to bind their targets based on simple nucleic acid base pairing rules. However, the use of natural DNA and RNA oligonucleotides as targeting probes can cause numerous off-target effects. In addition, natural nucleic acids are prone to degradation in vivo by various nucleases. To address these problems, nucleic acid mimics such as peptide nucleic acids (PNA) have been developed. They are more stable, show less off-target effects, and, in general, have better binding affinity to their targets. However, their high affinity to DNA can reduce their sequence-specificity. The formation of alternative DNA secondary structures, such as the G-quadruplex, provides an extra level of specificity as targets for PNA oligomers. PNA probes can target the loops of G-quadruplex, invade the core by forming PNA-DNA guanine-tetrads, or bind to the open bases on the complementary cytosine-rich strand. Not only could the development of such G-quadruplex-specific probes allow regulation of gene expression, but it will also provide a means to clarify the biological roles G-quadruplex structures may possess.

Due to the lack of structural guidelines about G-quadruplex ligands, rational design cannot be the only approach to discover potent G4-ligands. As a complementary approach, screening of chemical library may provide interesting scaffolds known as hits provided that specific tools are available. In this work, the Institut Curie-CNRS chemical library was firstly screened by chemoinformatics methods. Similarity estimations by comparison with reference compounds (Phen-DC3, 360A, MMQ12) provided a set of molecules, which were then evaluated by high-throughput G4-FID (HT-G4-FID) against various G-quadruplex DNA. A full investigation of the most interesting molecules, using the HT-G4-FID assay and molecular modeling, supplied an interesting structure-activity relationship confirming the efficiency of this general approach. Overall, we demonstrated that HT-G4-FID coupled with screening of chemical libraries is a powerful tool to identify new G4-DNA binding scaffolds.

The challenge of G-quadruplexes is that the G-rich sequences can adopt various G4 structures and possibly interconvert among them, particularly under the change of environmental conditions. Both NMR and circular dichroism (CD) show the spectral conversion of d[AG3(T2AG3)3] (HT22) from Na-form to K-form after Na+/K+ ion exchange. No appreciable change on the induced CD spectra of BMVC molecule and the single molecule tethered particle motion of HT22 in Na+ solution upon K+ titration suggests that the spectral conversion is unlikely due to the structural conversion via fully unfolded intermediate. Although a number of mechanisms were proposed for the spectral change induced by the Na+/K+ ion exchange, determining the precise structures of HT22 in K+ solution may be essential to unravel the mechanism of the structural conversion. Thus, development of a new method for separating different structures is of critical importance for further individual verification. In the second part of this review, we describe a new approach based on “micelleenhanced ultrafiltration” method for DNA structural separation. The BMVC, a G-quadruplex ligand, is first modified and then forms a large size of emulsion after ultrasonic emulsification, together with its different binding affinities to various DNA structures; for the first time, we are able to separate different DNA structures after membrane filtration. Verification of the possible structural conversion and investigation of structural diversity among various G4 structures are essential for exploring their potential biological roles and for developing new anticancer drugs.

Highly specific and tight-binding nucleic acid aptamers have been selected against a variety of molecular targets for over 20 years. A significant proportion of these oligonucleotides display G-quadruplex structures, particularly for DNA aptamers, that enable molecular recognition of their ligands. G-quadruplex structures couple a common scaffold to varying loop motifs that act in target recognition. Here, we review DNA G-quadruplex aptamers and their ligands from a structural and functional perspective. We compare the diversity of DNA G-quadruplex aptamers selected against multiple ligand targets, and consider structure with a particular focus on dissecting the thrombin binding aptamer - thrombin interaction. Therapeutic and analytical applications of DNA G-quadruplex aptamers are also discussed. Understanding DNA G-quadruplex aptamers carries implications not only for therapeutics and diagnostics, but also in the natural biochemistry of guanine-rich nucleic acids.

The use of nucleic acids as drugs represents a consistently growing approach. Different therapeutical strategies take advantage of the biological and biophysical properties of DNA and RNA to properly modulate activity of selected targets. A peculiar characteristic of these molecules is their structural flexibility which allows them to assume distinct foldings depending upon their sequence and/or environment. During the last twenty years this has led to the theoretical and experimental development of oligonucleotide aptamers, short sequences which can recognize a target with specificity and affinity comparable to antibodies. A leading example is represented by the Thrombin aptamer (15fTBA), a 15-mer DNA selected by its high affinity for the exosite I (fibrinogen binding site) of the coagulation factor. The very stable protein-DNA complex formation is the result of complementarities between the two macromolecules promoted by the aptamer sequence and folding as well as of electrostatic interactions generated by the charge balance at the binding site/s. Here, we investigated the relative role of these contributions and their involvement in defining the biological properties of the resulting complex. Thus we compared the Thrombin binding and inhibition properties of TBA to those of unrelated single stranded oligonucleotides. Additionally, the differences between the two protein exosites were assessed by using 29hTBA, a longer (29-mer) aptamer known to bind exosite II (heparin binding site). A subtle balance of aptamer folding and sequence is shown to cooperate with charge density for effective and selective recognition of exosite I or exosite II by TBAs.

The thrombin binding aptamer (TBA) is a well characterized chair-like, antiparallel quadruplex structure that binds specifically to thrombin at nanomolar concentrations and therefore it has interesting anticoagulant properties. In this article we review the research involved in the development of new TBA derivatives with improved anticoagulant properties as well as the use of the TBA as a model compound for the study of quadruplex structures. Specifically, we describe the impact of modified nucleosides and non-natural backbones in the guanine tetrads or in the loops and the introduction of pendant groups at the 3' or 5'-ends. The modified oligonucleotides are shown to be excellent tools for the understanding of the molecular structure of the TBA and its folding properties. Finally, we review the use of the TBA-Thrombin recognition system for the development of analytical tools based on the TBA folding.

Recent advances in colorimetric biosensing have led to rapid methods for target detection, potentially leading to applications on-site, at the point-of-need. This review focuses on one such platform, the G-quadruplex-hemin based DNAzymes, which exhibit peroxidase- like activity. Since their discovery in the late 1990s, various approaches have been adopted in applying the unique catalytic properties of these DNAzymes to detecting nucleic acids, proteins, metal ions and other ligands, through the oxidation of substrate pre-cursors into colored products. G-quadruplex based DNAzymes act as modular units of G-rich DNA sequences, and hence can be synthesized cheaply and conveniently using routine oligonucleotide synthesis. Herein, we discuss the various strategies that have been developed to exploit this class of DNAzymes as candidate probes for optical detection and sensing, for a variety of chemical and biological targets.

G-quadruplexes have found increasing potential in applications such as molecular therapeutics, diagnostics, and sensing. As a consequence, small molecules capable of selectively detecting G-quadruplexes have received significant attention in recent literature. Our review here addresses representative advances in the development of luminescent G-quadruplex probes and highlights their potential applications in sensing and imaging.

G-rich nucleic acid oligomers can form G-quadruplexes built by G-tetrads stacked upon each other. The basic building block of the G-quadruplexes is similar, but the formation of different quadruplex structures is highly responsive to the strand stoichiometry, strand orientation, guanine glycosidic torsion angle, connecting loops, and the metal coordination. Because of its structural variations and different functions, G-quadruplex applied in biorecognition events can function as a versatile signaling component. A variety of strategies that incorporate G-quadruplex have also been reported. In this review, we mainly discuss G-quadruplex as signal transducer from the following aspects for biorecognition events: analyte-induced G-quadruplex reconfiguration and fluorescence enhancement of small ligand; analyte-induced G-quadruplex reconstruction and formation of DNAzyme; Stimulus-responsive G-quadruplex refolding and manipulation of electron transfer; Stimulus-responsive G-quadruplex and its combination with nanopore systems; Small ligand-responsive Gquadruplex stabilization for drug screening; Nanomaterial-reponsive G-quadruplex reformation; Target-triggered continuous formation of G-quadruplex by DNA nanomachine. We have comprehensively described the recent progress in our labs and others. Undoubtedly, bioanalytical technology and nanotechnology based on G-quadruplex will continue to grow, leading to develop new diagnostics, therapeutics and drug development.

A recent finding demonstrated that telomere DNA is transcribed into telomeric repeat-containing RNA (referred to as TERRA) in mammalian cells. The existence of TERRA RNA may reveal a new level of regulation and protection of chromosome ends that could promote valuable insight into fundamental biological processes such as cancer and aging. Revealing the structure and function of telomere RNA will be essential for understanding telomere biology and telomere-related diseases. In fact, others and we have shown by NMR and x-ray crystallography that human telomere RNA forms G-quadruplex structures. More recently, we found that human telomere RNA forms a G-quadruplex dimer in the living cells by employing a light-switching probe. Recently, researches concerning the telomere RNA G-quadruplexes have made much progress. This review highlights the structures and topologies for telomere RNA G-quadruplex and recent efforts in the design of telomere RNA G-quadruplex ligands; outlines the future challenges in the field.

G-quadruplexes are non canonical secondary structures held together by Hoogsteen bonded planar guanine quartets formed in G-rich sequences in DNA and RNA. Considerable research over the past three decades has contributed to a great deal of understanding of these unusual structures in DNA. Various factors governing the stability of DNA quadruplexes coupled with their in vivo existence have been well documented. RNA has emerged as a key regulatory player in the functioning of the cell shifting the focus to RNA Gquadruplexes which were discovered recently. RNA G-quadruplexes demonstrate immense potential for in vivo existence and function due to their inherent chemistry. We have highlighted the major findings of the field and compared them to structural aspects of DNA quadruplexes. Further, the plausible functions of RNA G-quadruplexes such as translational suppression, splicing etc. are discussed in brief, suggesting scope for an extensive role of these structures in biological systems. As the field is growing, we endeavor to review the current knowledge and evaluate the various attributes of RNA G- quadruplex structure, stability, function and applications. We have also attempted to evaluate the physical and physiological role and relevance of these motifs.