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.     

Structural Investigations on the Anti‐HIV G‐Quadruplex‐Forming Oligonucleotide TGGGAG and Its Analogues: Evidence for the Presence of an A‐Tetrad

Several anti‐HIV aptamers adopt DNA quadruplex structures. Among these, “Hotoda's aptamer” (base sequence TGGGAG) was one of the first to be discovered. Although it has been the topic of some recent research, no detailed structural investigations have been reported. Here we report structural investigations on this aptamer and analogues with related sequences, by using UV, CD, and NMR spectroscopy as well as electrophoretic techniques. The addition of a 3′‐end thymine has allowed us to obtain a single, investigable quadruplex structure. Data clearly point to the presence of an A‐tetrad. Furthermore, the effects of the incorporation of an 8‐methyl‐2′‐deoxyguanosine at the 5′‐end of the G‐run were investigated.     

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.     

Multivalent Interactions between an Aromatic Helical Foldamer and a DNA G‐Quadruplex in the Solid State

Quinoline‐based oligoamide foldamers have been identified as a potent class of ligands for G‐quadruplex DNA. Their helical structure is thought to target G‐quadruplex loops or grooves and not G‐tetrads. We report a co‐crystal structure of the antiparallel hairpin dimeric DNA G‐quadruplex (G₄T₄G₄)₂ with tetramer 1 —a helically folded oligo‐quinolinecarboxamide bearing cationic side chains—that is consistent with this hypothesis. Multivalent foldamer–DNA interactions that modify the packing of (G₄T₄G₄)₂ in the solid state are observed.     

The 5′‐AG5CC‐3′ Fragment from the Human CPEB3 Ribozyme Forms an Ultrastable Parallel RNA G‐Quadruplex

An unusually thermostable G‐quadruplex is formed by a sequence fragment of a naturally occurring ribozyme, the human CPEB3 ribozyme. Strong evidence is provided for the formation of a uniquely stable intermolecular G‐quadruplex structure consisting of five tetrad layers, by using CD spectroscopy, UV melting curves, 2D NMR spectroscopy, and gel shift analysis. The cationic porphyrin TMPyP4 destabilizes the complex.     

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.     

Fluorine‐Mediated Editing of a G‐Quadruplex Folding Pathway

A (3+1)‐hybrid‐type G‐quadruplex was substituted within its central tetrad by a single 2′‐fluoro‐modified guanosine. Driven by the anti‐favoring nucleoside analogue, a novel quadruplex fold with inversion of a single G‐tract and conversion of a propeller loop into a lateral loop emerges. In addition, scalar couplings across hydrogen bonds demonstrate the formation of intra‐ and inter‐residual F ??? H8?C8 pseudo‐hydrogen bonds within the modified quadruplexes. Alternative folding can be rationalized by the impact of fluorine on intermediate species on the basis of a kinetic partitioning mechanism. Apparently, chemical or other environmental perturbations are able to redirect folding of a quadruplex, possibly modulating its regulatory role in physiological processes.     

Ligand‐Mediated G‐Quadruplex Induction in a Double‐Stranded DNA Context by Cyclic Imidazole/Lysine Polyamide

G‐quadruplex (G4) DNA is often observed as a DNA secondary structure in guanine‐rich sequences, and is thought to be relevant to pharmacological and biological events. Therefore, G4 ligands have attracted great attention as potential anticancer therapies or in molecular probe applications. Here, we designed cyclic imidazole/lysine polyamide (cIKP) as a new class of G4 ligand. It was readily synthesized without time‐consuming column chromatography. cIKP selectively recognized particular G4 structures with low nanomolar affinity. Moreover, cIKP exhibited the ability to induce G4 formation of the promoter of G4‐containing DNA in the context of stable double‐stranded DNA (dsDNA) under molecular crowding conditions. This cIKP might be applicable as a molecular probe for the detection of potential G4‐forming sequences in dsDNA.     

Structure‐Based Design of a Monosaccharide Ligand Targeting Galectin‐8

Galectin‐8 is a β‐galactoside‐recognising protein that has a role in the regulation of bone remodelling and is an emerging new target for tackling diseases with associated bone loss. We have designed and synthesised methyl 3‐O‐[1‐carboxyethyl]‐β‐d ‐galactopyranoside (compound 6 ) as a ligand to target the N‐terminal domain of galectin‐8 (galectin‐8N). Our design involved molecular dynamics (MD) simulations that predicted 6 to mimic the interactions made by the galactose ring as well as the carboxylic acid group of 3′‐O‐sialylated lactose (3′‐SiaLac), with galectin‐8N. Isothermal titration calorimetry (ITC) determined that the binding affinity of galectin‐8N for 6 was 32.8 μm , whereas no significant affinity was detected for the C‐terminal domain of galectin‐8 (galectin‐8C). The crystal structure of the galectin‐8N– 6 complex validated the predicted binding conformation and revealed the exact protein–ligand interactions that involve evolutionarily conserved amino acids of galectin and also those unique to galectin‐8N for recognition. Overall, we have initiated and demonstrated a rational ligand design campaign to develop a monosaccharide‐based scaffold as a binder of galectin‐8.     

Development of an Efficient G‐Quadruplex‐Stabilised Thrombin‐Binding Aptamer Containing a Three‐Carbon Spacer Molecule

The thrombin‐binding aptamer (TBA), which shows anticoagulant properties, is one of the most studied G‐quadruplex‐forming aptamers. In this study, we investigated the impact of different chemical modifications such as a three‐carbon spacer (spacer‐C₃), unlocked nucleic acid (UNA) and 3′‐amino‐modified UNA (amino‐UNA) on the structural dynamics and stability of TBA. All three modifications were incorporated at three different loop positions (T3, T7, T12) of the TBA G‐quadruplex structure to result in a series of TBA variants and their stability was studied by thermal denaturation; folding was studied by circular dichroism spectroscopy and thrombin clotting time. The results showed that spacer‐C₃ introduction at the T7 loop position (TBA‐SP7) significantly improved stability and thrombin clotting time while maintaining a similar binding affinity as TBA to thrombin. Detailed molecular modelling experiments provided novel insights into the experimental observations, further supporting the efficacy of TBA‐SP7. The results of this study could provide valuable information for future designs of TBA analogues with superior thrombin inhibition properties.     

Indolo[3,2‐c]quinoline G‐Quadruplex Stabilizers: a Structural Analysis of Binding to the Human Telomeric G‐Quadruplex

A library of 5‐methylindolo[3,2‐c]quinolones (IQc) with various substitution patterns of alkyldiamine side chains were evaluated for G‐quadruplex (G4) binding mode and efficiency. Fluorescence resonance energy transfer melting assays showed that IQcs with a positive charge in the heteroaromatic nucleus and two weakly basic side chains are potent and selective human telomeric (HT) and gene promoter G4 stabilizers. Spectroscopic studies with HT G4 as a model showed that an IQc stabilizing complex involves the binding of two IQc molecules (2,9‐bis{[3‐(diethylamino)propyl]amino}‐5‐methyl‐11H‐indolo[3,2‐c]quinolin‐5‐ium chloride, 3 d ) per G4 unit, in two non‐independent but equivalent binding sites. Molecular dynamics studies suggest that end‐stacking of 3 d induces a conformational rearrangement in the G4 structure, driving the binding of a second 3 d ligand to a G4 groove. Modeling studies also suggest that 3 d , with two three‐carbon side chains, has the appropriate geometry to participate in direct or water‐mediated hydrogen bonding to the phosphate backbone and/or G4 loops, assisted by the terminal nitrogen atoms of the side chains. Additionally, antiproliferative studies showed that IQc compounds 2 d (2‐{[3‐(diethylamino)propyl]amino}‐5‐methyl‐11H‐indolo[3,2‐c]quinolin‐5‐ium chloride) and 3 d are 7‐ to 12‐fold more selective for human malignant cell lines than for nonmalignant fibroblasts.     

Aromatic Core Extension in the Series of N‐Cyclic Bay‐Substituted Perylene G‐Quadruplex Ligands: Increased Telomere Damage,Antitumor Activity,and Strong Selectivity for Neoplastic over Healthy Cells

Based on previous work on both perylene and coronene derivatives as G‐quadruplex binders, a novel chimeric compound was designed: N,N′‐bis[2‐(1‐piperidino)‐ethyl]‐1‐(1‐piperidinyl)‐6‐[2‐(1‐piperidino)‐ethyl]‐benzo[ghi]perylene‐3,4:9,10‐tetracarboxylic diimide (EMICORON), having one piperidinyl group bound to the perylene bay area (positions 1, 12 and 6, 7 of the aromatic core), sufficient to guarantee good selectivity, and an extended aromatic core able to increase the stacking interactions with the terminal tetrad of the G‐quadruplex. The obtained “chimera” molecule, EMICORON, rapidly triggers extensive DNA damage of telomeres, associated with the delocalization of telomeric protein protection of telomeres 1 (POT1), and efficiently limits the growth of both telomerase‐positive and ‐negative tumor cells. Notably, the biological effects of EMICORON are more potent than those of the previously described perylene derivative (PPL3C), and more interestingly, EMICORON appears to be detrimental to transformed and tumor cells, while normal fibroblasts expressing telomerase remain unaffected. These results identify a new promising G‐quadruplex ligand, structurally and biologically similar on one side to coronene and on the other side to a bay‐monosubstituted perylene, that warrants further studies.     

3‐Hydroxy‐2‐[1′‐phenyl‐3′‐(p‐chlorophenyl)‐4′‐pyrazolyl]‐4‐oxo‐4H‐1‐benzopyran as a spectrophotometric reagent for trace determination of tungsten(vi)

3‐Hydroxy‐2‐[1′‐phenyl‐3′‐(p‐chlorophenyl)‐4′‐pyrazolyl]‐4‐oxo‐4H‐1‐benzopyran solution in acetone forms a yellow‐coloured complex with tungsten(vi ) in perchloric acid medium which is extractable into chloroform after equilibration. In this study, tests were carried out on this compound and it was found to be quite stable and to obey Beer’s law. Interactions with various ions were studied and the molar absorptivity, Sandell’s sensitivity and relative standard deviation were also investigated. Job’s method, the mole ratio method and the equilibrium‐shift method were also used. The ratio of metal to ligand was measured using a method that was simpler, more accurate, sensitive, rapid and selective as compared with existing methods. This method is applicable to the analysis of various samples of varied composition.     

Expanding the Potential of G‐Quadruplex Structures: Formation of a Heterochiral TBA Analogue

In order to expand the potential applications of G‐quadruplex structures, we explored the ability of heterochiral oligodeoxynucleotides based on the thrombin‐binding aptamer (TBA) sequence to fold into similar complexes, with particular focus on their resistance in biological environments. A combination of CD and NMR techniques was used. Similarly to TBA, the ODN ggTTggtgtggTTgg (lower case letters indicate L residues) is able to fold into a chair‐like antiparallel G‐quadruplex structure, but has a slightly higher thermal stability. The discovery that heterochiral ODNs are able to form stable G‐quadruplex structures opens up new possibilities for their development in several fields, as aptamers, sensors and, as recently shown, as catalysts for enantioselective reactions.     

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.