Molecular Recognition of Parallel DNA Quadruplex d(TTAGGGT)4 by Mitoxantrone: Binding with 1:2 Stoichiometry Leading to Thermal Stabilization and Telomerase Inhibition

The interaction of the anthraquinone derivative mitoxantrone, a semisynthetic anti‐cancer drug with two non‐planar side chains, with heptamer G‐quadruplex d(TTAGGGT)₄, which contains the human telomere DNA sequence, was evaluated by differential scanning calorimetry, fluorescence Job plotting, absorption, and NMR and CD spectroscopy. Binding led to thermal stabilization of DNA (ΔT_m=13–20 °C). The spectra revealed that two mitoxantrone molecules bind externally at two sites of the DNA quadruplex as monomers, by partial insertion of the chromophore and side‐chain interaction at the grooves. The inhibition of telomerase (IC₅₀=2 μm), as determined by a TRAP assay, can be attributed to thermal stabilization of the DNA quadruplex because of the interactions with mitoxantrone. The studies revealed highly specific molecular recognition between a ligand and a parallel‐stranded G‐quadruplex; this might serve as a platform for the rational design of new drugs.     

Exploring the Interaction of Curaxin CBL0137 with G-Quadruplex DNA Oligomers

Curaxins and especially the second-generation derivative curaxin CBL0137 have important antitumor activities in multiple cancers such as glioblastoma, melanoma and others. Although most of the authors suggest that their mechanism of action comes from the activation of p53 and inactivation of NF-kB by targeting FACT, there is evidence supporting the involvement of DNA binding in their antitumor activity. In this work, the DNA binding properties of curaxin CBL0137 with model quadruplex DNA oligomers were studied by ¹H NMR, CD, fluorescence and molecular modeling. We provided molecular details of the interaction of curaxin with two G-quadruplex structures, the single repeat of human telomere d(TTAGGGT)₄ and the c-myc promoter Pu22 sequence. We also performed ¹H and ³¹P NMR experiments were also performed in order to investigate the interaction with duplex DNA models. Our data support the hypothesis that the interaction of curaxin with G-quadruplex may provide a novel insight into the DNA-binding properties of CBL0137, and it will be helpful for the design of novel selective DNA-targeting curaxin analogues.     

Synthesis of small molecules targeting multiple DNA structures using click chemistry

The ability of small molecules to target DNA forms the basis of many clinically used antitumour agents. This study examines the effects of novel 9-aminoacridine carboxamides, synthesised by click chemistry based upon the reactions of either 9-(2-azidoethyl)amino or 9-propargylaminoacridine compounds, on various types of DNA tertiary structures. This gave either monomeric or dimeric compounds, the dimeric derivatives being the first unsymmetrical acridine dimers to be described. The compounds were assayed for duplex DNA, quadruplex DNA and four-way junction DNA binding. Their antiproliferative activity in the Human promyelocytic leukaemia cell line, HL60, was also assessed. Although for some of the compounds, notably the acridine 4-carboxamides, activity correlated with DNA binding affinity, for others it did not, with the rigidly linked dimers in particular showing a complicated relationship between 3- and 4-carboxamide structure and biological activity. The monomeric 3-carboxamides were more effective at stabilising G-quadruplex structures and also gave more hits in the four-way junction stabilisation assay. There is clear evidence from the binding of the 3-carboxamides that these compounds destabilise the open X form of the junction at lower concentrations and stabilise the X-stacked at higher concentrations. This might have implications for the biological activity of these compounds against proteins that bind to the Holliday junction (HJ).     

Binding Properties of RNA Quadruplex of SARS-CoV-2 to Berberine Compared to Telomeric DNA Quadruplex

Previous studies suggest that berberine, an isoquinoline alkaloid, has antiviral potential and is a possible therapeutic candidate against SARS-CoV-2. The molecular underpinnings of its action are still unknown. Potential targets include quadruplexes (G4Q) in the viral genome as they play a key role in modulating the biological activity of viruses. While several DNA-G4Q structures and their binding properties have been elucidated, RNA-G4Qs such as RG-1 of the N-gene of SARS-CoV-2 are less explored. Using biophysical techniques, the berberine binding thermodynamics and the associated conformational and hydration changes of RG-1 could be characterized and compared with human telomeric DNA-G4Q 22AG. Berberine can interact with both quadruplexes. Substantial changes were observed in the interaction of berberine with 22AG and RG-1, which adopt different topologies that can also change upon ligand binding. The strength of interaction and the thermodynamic signatures were found to dependent not only on the initial conformation of the quadruplex, but also on the type of salt present in solution. Since berberine has shown promise as a G-quadruplex stabilizer that can modulate viral gene expression, this study may also contribute to the development of optimized ligands that can discriminate between binding to DNA and RNA G-quadruplexes.     

Targeting DNA G‐Quadruplexes with Helical Small Molecules

We previously identified quinoline‐based oligoamide helical foldamers and a trimeric macrocycle as selective ligands of DNA quadruplexes. Their helical structures might permit targeting of the backbone loops and grooves of G‐quadruplexes instead of the G‐tetrads. Given the vast array of morphologies G‐quadruplex structures can adopt, this might be a way to achieve sequence selective binding. Here, we describe the design and synthesis of molecules based on macrocyclic and helically folded oligoamides. We tested their ability to interact with the human telomeric G‐quadruplex and an array of promoter G‐quadruplexes by using FRET melting assay and single‐molecule FRET. Our results show that they constitute very potent ligands—comparable to the best so far reported. Their modes of interaction differ from those of traditional tetrad binders, thus opening avenues for the development of molecules specific for certain G‐quadruplex conformations.     

Fluorescent Probes for G‐Quadruplex Structures

Mounting evidence supports the presence of biologically relevant G‐quadruplexes in single‐cell organisms, but the existence of endogenous G‐quadruplex structures in mammalian cells remains highly controversial. This is due, in part, to the common misconception that DNA and RNA molecules are passive information carriers with relatively little structural or functional complexity. For those working in the field, however, the lack of available tools for characterizing DNA structures in vivo remains a major limitation to addressing fundamental questions about structure–function relationships of nucleic acids. In this review, we present progress towards the direct detection of G‐quadruplex structures by using small molecules and modified oligonucleotides as fluorescent probes. While most development has focused on cell‐permeable probes that selectively bind to G‐quadruplex structures with high affinity, these same probes can induce G‐quadruplex folding, thereby making the native conformation of the DNA or RNA molecule (i.e., in the absence of probe) uncertain. For this reason, modified oligonucleotides and fluorescent base analogues that serve as “internal” fluorescent probes are presented as an orthogonal means for detecting conformational changes, without necessarily perturbing the equilibria between G‐quadruplex, single‐stranded, and duplex DNA. The major challenges and motivation for the development of fluorescent probes for G‐quadruplex structures are presented, along with a summary of the key photophysical, biophysical, and biological properties of reported examples.     

Effect of ATRX and G‐Quadruplex Formation by the VNTR Sequence on α‐Globin Gene Expression

ATR‐X (α‐thalassemia/mental retardation X‐linked) syndrome is caused by mutations in chromatin remodeler ATRX. ATRX can bind the variable number of tandem repeats (VNTR) sequence in the promoter region of the α‐globin gene cluster. The VNTR sequence, which contains the potential G‐quadruplex‐forming sequence CGC(GGGGCGGGG)_n, is involved in the downregulation of α‐globin expression. We investigated G‐quadruplex and i‐motif formation in single‐stranded DNA and long double‐stranded DNA. The promoter region without the VNTR sequence showed approximately twofold higher luciferase activity than the promoter region harboring the VNTR sequence. G‐quadruplex stabilizers hemin and TMPyP4 reduced the luciferase activity, whereas expression of ATRX led to a recovery in reporter activity. Our results demonstrate that stable G‐quadruplex formation by the VNTR sequence downregulates the expression of α‐globin genes and that ATRX might bind to and resolve the G‐quadruplex.     

Mixed‐Ligand Mononuclear Copper(II) Complex: Crystal Structure and Anticancer Activity

A novel copper(II) complex with mixed ligands including β‐[(3‐formyl‐5‐methyl‐2‐hydroxy‐benzylidene)amino]propionic acid anion and 1,10′‐phenanthroline was synthesized, and its crystal structure was thoroughly characterized. It exerted excellent inducing apoptosis, anti‐angiogenesis and antiproliferative properties in vitro. The complex can bind human serum albumin (HSA) at physiological pH conditions. Remarkably, it can induce formation of the mixed parallel/antiparallel G‐quadruplex structures in the G‐rich sequence of the proximal vascular endothelial growth factor (VEGF) promoter, and stabilize these G‐quadruplex structures, which provide an opportunity for anti‐angiogenesis chemotherapeutics. Furthermore, the complex showed a strong uptake, and exhibited multiple anticancer functions by inhibiting the expression of p‐Akt and p‐Erk1/2 proteins and by upregulating the levels of reactive oxygen species (ROS). Because of the reported results, this new copper(II) complex qualifies itself as a potential anticancer drug candidate.     

Porphyrin derivatives for telomere binding and telomerase inhibition

The capacity of G-quadruplex ligands to stabilize four-stranded DNA makes them able to inhibit telomerase, which is involved in tumour cell proliferation. A series of cationic metalloporphyrin derivatives was prepared by making variations on a meso-tetrakis(4-N-methyl-pyridiniumyl)porphyrin skeleton (TMPyP). The DNA binding properties of nickel(II) and manganese(III) porphyrins were studied by surface plasmon resonance, and the capacity of the nickel porphyrins to inhibit telomerase was tested in a TRAP assay. The nature of the metal influences the kinetics (the process is faster for Ni than for Mn) and the mode of interaction (stacking or external binding). The chemical alterations did not lead to increased telomerase inhibition. The best selectivity for G-quadruplex DNA was observed for Mn-TMPyP, which has a tenfold preference for quadruplex over duplex.     

Looking for Efficient G‐Quadruplex Ligands: Evidence for Selective Stabilizing Properties and Telomere Damage by Drug‐Like Molecules

There is currently significant interest in the development of G‐quadruplex‐interactive compounds, given the relationship between the ability to stabilize these non‐canonical DNA structures and anticancer activity. In this study, a set of biophysical assays was applied to evaluate the binding of six drug‐like ligands to DNA G‐quadruplexes with different folding topologies. Interestingly, two of the investigated ligands showed selective G‐quadruplex‐stabilizing properties and biological activity. These compounds may represent useful leads for the development of more potent and selective ligands.     

Rational Design,Synthesis, Biophysical and Antiproliferative Evaluation of Fluorenone Derivatives with DNA G‐Quadruplex Binding Properties

Molecular modeling studies carried out with experimental DNA models with the sequence d[AG₃(T₂AG₃)₃] suggest that the introduction of a net positive charge onto the side chain of a series of fluorenone carboxamides can improve G‐quadruplex binding. The terminal morpholino moiety was replaced with a novel N‐methylmorpholinium cation starting from two 4‐carboxamide compounds. A different substitution on the fluorenone ring was also investigated and submitted to the same quaternarization process. All compounds were analyzed for their DNA binding properties by competition dialysis methods. In vitro antiproliferative tests were carried out against two different tumor cell lines. Docking experiments were conducted by including all four known human repeat unit G‐quadruplex DNA sequences (27 experimentally determined conformations) against the most active fluorenone derivatives. The results of theoretical, biophysical, and in vitro experiments indicate two novel derivatives as lead compounds for the development of a new generation of G‐quadruplex ligands with greater potency and selectivity.     

Modeling and biological investigations of an unusual behavior of novel synthesized acridine-based polyamine ligands in the binding of double helix and G-quadruplex DNA

Three novel 2,7-substituted acridine derivatives were designed and synthesized to investigate the effect of this functionalization on their interaction with double-stranded and G-quadruplex DNA. Detailed investigations of their ability to bind both forms of DNA were carried out by using spectrophotometric, electrophoretic, and computational approaches. The ligands in this study are characterized by an open-chain (L1) or a macrocyclic (L2, L3) framework. The aliphatic amine groups in the macrocycles are joined by ethylene (L2) or propylene chains (L3). L1 behaved similarly to the lead compound m-AMSA, efficiently intercalating into dsDNA, but stabilizing G-quadruplex structures poorly, probably due to the modest stabilization effect exerted by its protonated polyamine chains. L2 and L3, containing small polyamine macrocyclic frameworks, are known to adopt a rather bent and rigid conformation; thus they are generally expected to be sterically impeded from recognizing dsDNA according to an intercalative binding mode. This was confirmed to be true for L3. Nevertheless, we show that L2 can give rise to efficient π-π and H-bonding interactions with dsDNA. Additionally, stacking interactions allowed L2 to stabilize the G-quadruplex structure: using the human telomeric sequence, we observed the preferential induction of tetrameric G-quadruplex forms. Thus, the presence of short ethylene spacers seems to be essential for obtaining a correct match between the binding sites of L2 and the nucleobases on both DNA forms investigated. Furthermore, current modeling methodologies, including docking and MD simulations and free energy calculations, provide structural evidence of an interaction mode for L2 that is different from that of L3; this could explain the unusual stabilizing ability of the ligands (L2>L3>L1) toward G-quadruplex that was observed in this study.     

Ligand‐Induced Dimerization of a Truncated Parallel MYC G‐Quadruplex

Binding of an indoloquinoline derivative with an aminoalkyl side chain to a truncated sequence from the MYC promoter region was studied through isothermal titration calorimetry (ITC). The targeted MYC3 sequence lacks 3′‐flanking nucleotides and forms a monomeric parallel quadruplex (G4) with a blunt‐ended 3′‐outer tetrad under the solution conditions employed. Analysis of ITC isotherms reveals multiple binding equilibria with the initial formation of a 1:2 ligand/quadruplex complex. Evaluation of electrophoretic mobilities as well as NMR spectral data confirm ligand‐induced dimerization of MYC3 quadruplexes with the ligand sandwiched between the two 3′‐outer tetrads. Additional ligand molecules in excess bind to the 5′‐outer tetrads of the sandwich complex. Such a ligand‐promoted G4 dimerization may be exploited for the controlled assembly or disassembly of G4 aggregates to expand on present quadruplex‐based technologies.     

Efficient Synthesis of Peptide and Protein Functionalized Pyrrole-Imidazole Polyamides Using Native Chemical Ligation

The advancement of DNA-based bionanotechnology requires efficient strategies to functionalize DNA nanostructures in a specific manner with other biomolecules, most importantly peptides and proteins. Common DNA-functionalization methods rely on laborious and covalent conjugation between DNA and proteins or peptides. Pyrrole-imidazole (Py–Im) polyamides, based on natural minor groove DNA-binding small molecules, can bind to DNA in a sequence specific fashion. In this study, we explore the use of Py–Im polyamides for addressing proteins and peptides to DNA in a sequence specific and non-covalent manner. A generic synthetic approach based on native chemical ligation was established that allows efficient conjugation of both peptides and recombinant proteins to Py–Im polyamides. The effect of Py–Im polyamide conjugation on DNA binding was investigated by Surface Plasmon Resonance (SPR). Although the synthesis of different protein-Py–Im-polyamide conjugates was successful, attenuation of DNA affinity was observed, in particular for the protein-Py–Im-polyamide conjugates. The practical use of protein-Py–Im-polyamide conjugates for addressing DNA structures in an orthogonal but non-covalent manner, therefore, remains to be established.     

Phototoxic Activity and DNA Interactions of Water‐Soluble Porphyrins and Their Rhenium(I) Conjugates

In the search for alternative photosensitizers for use in photodynamic therapy (PDT), herein we describe two new water‐soluble porphyrins, a neutral fourfold‐symmetric compound and a +3‐charged tris‐methylpyridinium derivative, in which either four or one [1,4,7]‐triazacyclononane (TACN) units are connected to the porphyrin macrocycle through a hydrophilic linker; we also report their corresponding tetracationic Re^I conjugates. The in vitro (photo)toxic effects of the compounds toward the human cell lines HeLa (cervical cancer), H460M2 (non‐small‐cell lung carcinoma), and HBL‐100 (non‐tumorigenic epithelial cells) are reported. Three of the compounds are not cytotoxic in the dark up to 100 μm , and the fourfold‐symmetric couple revealed very good phototoxic indexes (PIs). The intracellular localization of all derivatives was studied in HeLa cells by confocal fluorescence microscopy. Although low nuclear localization was observed for some of them, it still prompted us to investigate their capacity to bind both quadruplex and duplex DNA; we observed significant selectivity in the tris‐methylpyridinium derivatives for G‐quadruplex interactions.     

G-quadruplex recognition by quinacridines: a SAR, NMR, and biological study

The synthesis of a novel group of quinacridine-based ligands (MMQs) is described along with an evaluation of their G-quadruplex binding properties. A set of biophysical assays was applied to characterize their interaction with DNA quadruplexes: FRET-melting experiments and equilibrium microdialysis were used to evaluate their quadruplex affinity and their ability to discriminate quadruplexes across a broad panel of DNA structures. All data collected support the proposed model of interaction of these compounds with G-quadruplexes, which is furthermore confirmed by a solution structure determined by 2D NMR experiments. Finally, the activity of the MMQ series against tumor cell growth is reported, and the data support the potential of quadruplex-interactive compounds for use in anticancer approaches.     

DNA and RNA Quadruplex-Binding Proteins

Four-stranded DNA structures were structurally characterized in vitro by NMR, X-ray and Circular Dichroism spectroscopy in detail. Among the different types of quadruplexes (i-Motifs, minor groove quadruplexes, G-quadruplexes, etc.), the best described are G-quadruplexes which are featured by Hoogsteen base-paring. Sequences with the potential to form quadruplexes are widely present in genome of all organisms. They are found often in repetitive sequences such as telomeric ones, and also in promoter regions and 5'' non-coding sequences. Recently, many proteins with binding affinity to G-quadruplexes have been identified. One of the initially portrayed G-rich regions, the human telomeric sequence (TTAGGG)_n, is recognized by many proteins which can modulate telomerase activity. Sequences with the potential to form G-quadruplexes are often located in promoter regions of various oncogenes. The NHE III₁ region of the c-MYC promoter has been shown to interact with nucleolin protein as well as other G-quadruplex-binding proteins. A number of G-rich sequences are also present in promoter region of estrogen receptor alpha. In addition to DNA quadruplexes, RNA quadruplexes, which are critical in translational regulation, have also been predicted and observed. For example, the RNA quadruplex formation in telomere-repeat-containing RNA is involved in interaction with TRF2 (telomere repeat binding factor 2) and plays key role in telomere regulation. All these fundamental examples suggest the importance of quadruplex structures in cell processes and their understanding may provide better insight into aging and disease development.