Common G-Quadruplex Binding Agents Found to Interact With i-Motif-Forming DNA: Unexpected Multi-Target-Directed Compounds (original) (raw)
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Analytical Chemistry, 2012
The study of ligand interaction with G-quadruplex DNA is an active research area, because many ligands are shown to bind G-quadruplex structures, showing anticancer effects. Here, we show, for the first time, how fluorescence correlation spectroscopy (FCS) can be used to study binding kinetics of ligands with G-quadruplex DNA at the single molecule level. As an example, we study interaction of a benzo-phenoxazine ligand (Cresyl Violet, CV) with antiparallel and (3 + 1) hybrid G-quadruplex structures formed by human telomeric sequence. By using simple modifications in FCS setup, we describe how one can extract the reaction kinetics from diffusion-coupled correlation curves. It is found that the ligand (CV) binds stronger, by an order of magnitude, to a (3 + 1) hybrid structure, compared to an antiparallel one. Ensemble-averaged time-resolved fluorescence experiments are also carried out to obtain the binding equilibrium constants (K) of ligand-quadruplex interactions in bulk solution for the first time, which are found to match very well with FCS results. Global analysis of FCS data provides association (k +) and dissociation (k −) rates of the ligand in the two structures. Results indicate that stronger ligand binding to the (3 + 1) hybrid structure is controlled by the dissociation rate, rather than the association rate of ligand in the quadruplexes. Circular dichroism (CD) and induced-CD spectra show that the ligand not only binds at different conformations in the quadruplexes, but also induces antiparallel structure to form a mixed-type hybrid structure in Na + solution. However, in K + solution, the ligand stabilizes the (3 + 1) hybrid structure. Molecular docking studies predict the possible differences in binding sites of the ligand inside two quadruplexes, which strongly support the experimental observations. Results suggest that different binding modes of the ligand to the quadruplex structures actually assist the alteration of structures differently.
Interaction of G-Quadruplexes with Nonintercalating Duplex-DNA Minor Groove Binding Ligands
Bioconjugate Chemistry, 2011
The enzyme telomerase synthesizes the G-rich DNA strands of the telomere and its activity is often associated with cancer. The telomerase may be therefore responsible for the ability of a cancer cell to escape apoptosis. The G-rich DNA sequences often adopt tetra-stranded structure, known as the G-quadruplex DNA (G4-DNA). The stabilization of the telomeric DNA into the G4-DNA structures by small molecules has been the focus of many researchers for the design and development of new anticancer agents. The compounds which stabilize the G-quadruplex in the telomere inhibit the telomerase activity. Besides telomeres, the G4-DNA forming sequences are present in the genomic regions of biological significance including the transcriptional regulatory and promoter regions of several oncogenes. Inducing a G-quadruplex structure within the G-rich promoter sequences is a potential way of achieving selective gene regulation. Several G-quadruplex stabilizing ligands are known. Minor groove binding ligands (MGBLs) interact with the double-helical DNA through the minor grooves sequence-specifically and interfere with several DNA associated processes. These MGBLs when suitably modified switch their preference sometimes from the duplex DNA to G4-DNA and stabilize the G4-DNA as well. Herein, we focus on the recent advances in understanding the Gquadruplex structures, particularly made by the human telomeric ends, and review the results of various investigations of the interaction of designed organic ligands with the G-quadruplex DNA while highlighting the importance of MGBL-G-quadruplex interactions.
State-of-the-Art Methodologies for the Discovery and Characterization of DNA G-Quadruplex Binders
Current Pharmaceutical Design, 2012
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.
Journal of Medicinal Chemistry, 2013
Nowadays, it has been demonstrated that DNA G-quadruplex arrangements are involved in cellular aging and cancer, thus boosting the discovery of selective binders for these DNA secondary structures. By taking advantage of available structural and biological information on these structures, we performed a high throughput in silico screening of commercially available molecules databases by merging ligand-and structure-based approaches by means of docking experiments. Compounds selected by the virtual screening procedure were then tested for their ability to interact with the human telomeric G-quadruplex folding by circular dichroism, fluorescence spectroscopy, and photodynamic techniques. Interestingly, our screening succeeded in retrieving a new promising scaffold for G-quadruplex binders characterized by a psoralen moiety.
Biochimie, 2009
Human DNA sequences consisting of tandem guanine (G) nucleotides can fold into a four-stranded structure named G-quadruplex via Hoogsteen hydrogen bonding. As the sequences forming G-quadruplex exist in essential regions of eukaryotic chromosomes and are involved in many important biological processes, the study of their biological functions has currently become a hotspot. Compounds selectively binding and stabilizing G-quadruplex structures have the potential to inhibit telomerase activity or alter oncogene expression levels and thus may act as antitumor agents. Most of reported G-quadruplex ligands generally have planar structures which stabilize G-quadruplex by p-p stacking.
The role of thermodynamics and kinetics in ligand binding to G-quadruplex DNA
Chemical Communications, 2012
Molecular dynamics simulations were used to investigate the binding of four different 2,4,6-triarylpyridines to G-quadruplex DNA. Both the binding free energies, and the kinetics of binding are required to explain the measured degree of ligand induced stabilisation of the compounds, with bulky substituents having the potential to prevent the ligand from reaching the lowest energy binding site.
Biochimica et Biophysica Acta (BBA) - General Subjects, 2017
Background: Guanine-rich DNA motifs can form non-canonical structures known as Gquadruplexes, whose role in tumorigenic processes makes them attractive drug-target candidates for cancer therapy. Recent studies revealed that the folding and unfolding pathways of G-quadruplexes proceed through a quite stable intermediate named G-triplex. Methods: Virtual screening was employed to identify a small set of putative G-triplex ligands. The G-triplex stabilizing properties of these compounds were analyzed by CD melting assay. DSC, nondenaturing gel electrophoresis, NMR and molecular modeling studies were performed to investigate the interaction between the selected compound 1 and G-rich DNA structures. Cytotoxic activity of 1 was evaluated by MTT cell proliferation assay. Results: The experiments led to the identification of a promising hit that was shown to bind preferentially to G-triplex and parallel-stranded G-quadruplexes over duplex and antiparallel Gquadruplexes. Molecular modeling results suggested a partial end-stacking of 1 to the external Gtriad/G-tetrads as a binding mode. Biological assays showed that 1 is endowed with cytotoxic effect on human osteosarcoma cells. Conclusions: A tandem application of virtual screening along with the experimental investigation was employed to discover a G-triplex-targeting ligand. Experiments revealed that the selected compound actually acts as a dual G-triplex/G-quadruplex stabilizer, thus stimulating further studies aimed at its optimization. General significance: The discovery of molecules able to bind and stabilize G-triplex structures is highly appealing, but their transient state makes challenging their recognition. These findings suggest that the identification of ligands with dual G-triplex/G-quadruplex stabilizing properties may represent a new route for the design of anticancer agents targeting the G-rich DNA structures.
Biochimie, 2008
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 quadruplexand 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. that is not subjected to structural interconversion, the TBA (thrombin binding aptamer) . Finally, because quadruplex-vs. duplex-selectivity is a critical issue and because binding of both probe and ligand to duplex-DNA may be length-and sequence-dependent, we compare the results obtained with the 17-base pair duplex-DNA (dsl7) used in our initial work with a 26-bp duplex (ds26). G4-FID experiments reported herein were carried out with a set of 16 quadru-plex-ligands (from bisquinolinium to metallo-organic ligands) with two different QFOs (22AG , TBA ), in two different salt conditions (Na + , K + ) and with two different DNA control duplex-DNAs (dsl7 [31] and ds26 ).
Chemistry – A European Journal, 2019
Naphthalene diimide dyads exhibiting a different substitution pattern and linker length have been synthesized and evaluated as G-quadruplex (G4) ligands, investigating their cytotoxicity in selected cell lines. The dyads with the long C7-linker exhibit extremely low IC50 values, below 10 nM, on different cancer cell lines. Contrary, the dyads with shorter C4-linker were much less effective, with IC values increasing up to 1 µM. Among the three dyads with the longest linker, small differences in IC50 emerge, suggesting that its length plays a more important role rather than the substitution pattern. We have further shown that the dyads are able to induce cellular DNA damage response, which is not limited to the telomeric regions and is likely the origin of their cytotoxicity. Both absorption titrations and dynamic light scattering of the most cytotoxic dyads in the presence of hTel22 highlight their ability to induce effective G4 aggregation, acting as non-covalent crosslinking agents.