Characterization of G4–G4 Crosstalk in the <i>c‑KIT</i> Promoter Region

The proximal promoter of <i>c-KIT</i> contains a peculiar domain that consists of three short G-rich sequences that are close together and can fold into noncanonical DNA secondary structures called G-quadruplexes (G4). Here, we focused on a sequence containing two consecutive G4 (kit2 and kit*). By electrophoretic, surface plasmon resonance, and spectroscopic techniques, we demonstrated that they retain the ability to fold into G4 upon being inserted into the extended sequence. Here, we highlighted the occurrence of crosstalk between the two forming units. This previously unexplored G4–G4 interaction modulates both the conformation and the stability of the overall arrangement of the <i>c-KIT</i> promoter. It is not supported by stacking of single nucleotides but refers to a G4–G4 interaction surface surrounded by a two-nucleotides loop that might represent a reliable unprecedented target for anticancer therapy

The energetic contribution for (K34)₂Ni(II) binding towards tested G-quadruplexes is differently modulated.

<p>Energetic contributions describing the interaction between tested G-quadruplexes and (K34)₂Ni(II) at 37°C in 10 mM Tris, 20 mM KCl, pH 7.5. The associated enthalpic variations (ΔH) are shown in black, the entropic ones (-TΔS) in white and the Gibbs energy changes (ΔG) in grey.</p

Effect of G-Quadruplex Polymorphism on the Recognition of Telomeric DNA by a Metal Complex

<div><p>The physiological role(s) played by G-quadruplexes renders these ‘non-canonical’ DNA secondary structures interesting new targets for therapeutic intervention. In particular, the search for ligands for selective recognition and stabilization of G-quadruplex arrangements has led to a number of novel targeted agents. An interesting approach is represented by the use of metal-complexes, their binding to DNA being modulated by ligand and metal ion nature, and by complex stoichiometry. In this work we characterized thermodynamically and stereochemically the interactions of a Ni(II) bis-phenanthroline derivative with telomeric G-quadruplex sequences using calorimetric, chiroptical and NMR techniques. We employed three strictly related sequences based on the human telomeric repeat, namely Tel22, Tel26 and wtTel26, which assume distinct conformations in potassium containing solutions. We were able to monitor specific enthalpy/entropy changes according to the structural features of the target telomeric sequence and to dissect the binding process into distinct events. Interestingly, temperature effects turned out to be prominent both in terms of binding stoichiometry and ΔH/ΔS contributions, while the final G-quadruplex-metal complex architecture tended to merge for the examined sequences. These results underline the critical choice of experimental conditions and DNA sequence for practical use of thermodynamic data in the rational development of effective G-quadruplex binders.</p> </div

(K34)₂Ni(II)-G-quadruplex bound form shows conserved CD spectra features.

<p>Dichroic spectra of 4 µM Tel26 (Panel A), wtTel26 (Panel B), Tel22 (Panel C) or single strand scrambled scT22 (Panel D) in the presence of increasing concentrations of (K34)₂Ni(II) at 25°C in 10 mM Tris, 20 mM KCl, at pH 7.5. Arrows indicate changes upon addition of the metal complex, curves in bold indicate spectra of the free oligonucleotide and at 1∶1 and 2∶1 ligand:DNA ratios in panels A–C. In panel D the solid line corresponds to the CD contribution of DNA alone, the dotted line represents the dichroic spectrum at saturating concentrations of (K34)₂Ni(II).</p

Thermodynamic parameters derived from ITC titrations describing the interaction of (K34)₂Ni(II) with Tel26 and wtTel26 at 25°C in 10 mM Tris, 20 mM KCl, pH 7.5.

*<p>: the applied model of sequential binding sites considers the binding events as exactly integral numbers. Reported data are the average of three independent measurements.</p

Ligand saturation is modulated by DNA folding.

<p>Fraction of rearranged G-quadruplex (f) induced by increasing concentrations of (K34)₂Ni(II) at 25°C in 10 mM Tris, 20 mM KCl, pH 7.5. Oligonucleotides concentration: 4 µM.</p

(K34)₂Ni(II) shows different binding profiles towards wtTel26 and Tel26 at 25°C.

<p>ITC profiles corresponding to the titration of 25 µM wtTel26 (Panel A) or Tel26 (Panel B) with (K34)₂Ni(II) at 25°C in 10 mM Tris, 20 mM KCl, pH 7.5. Raw ITC data (top panel) and binding isotherms (bottom panel).</p

DataSheet1_G-quadruplexes formation within the promoter of TEAD4 oncogene and their interaction with Vimentin.docx

G-quadruplexes (G4s) are nucleic acid secondary structures detected within human chromosomes, that cluster at gene promoters and enhancers. This suggests that G4s may play specific roles in the regulation of gene expression. Within a distinct subgroup of G-rich domains, the formation of two or more adjacent G4 units (G4-repeats) is feasible. Recently it was shown that Vimentin, a protein highly expressed within mesenchymal cells, selectively recognizes these arrangements. Putative G4-repeats have been searched within the human gene proximal promoters by the bioinformatics tool QPARSE and they resulted to be enriched at genes related to epithelial-to-mesenchymal transition (EMT). This suggested that Vimentin binding at these sites might be relevant for the maintenance of the mesenchymal phenotype. Among all the identified sequences, in the present study we selected the one located within the promoter of the TEAD4 oncogene. TEAD4 codifies for a transcriptional enhancer factor, TEAD4, that actively promotes EMT, supporting, cell proliferation and migration. Moreover, in colorectal cancer cells TEAD4 directly enhances the expression of Vimentin. Thus, the possible interaction of Vimentin with TEAD4 promoter could highlight a positive feedback loop between these two factors, associated to important tumor metastasis related events. Here, we exploited spectroscopic and electrophoretic measurements under different conditions to address the folding behavior of the selected sequence. This allowed us to validate the folding of TEAD4 promoter into a G4-repeat able to interact with Vimentin.</p

Optimized Virtual Screening Workflow for the Identification of Novel G‑Quadruplex Ligands

G-quadruplexes, alternative DNA secondary structures present in telomeres, emerge as promising targets for the treatment of cancer, because they prevent telomere elongation and accordingly cell proliferation. Within this study, theoretically validated pharmacophore- and shape-based models as well as a theoretically validated docking protocol were generated and applied in parallel for virtual screening and the identification of novel G-quadruplex ligands. Top-ranked hits retrieved with all methods independently and in addition in a consensus approach were selected for biological testing. Of the 32 tested virtual hits seven selectively stabilized G-quadruplexes over duplex DNA in the fluorescence melting assay. For the five most active compounds, chemically closely related analogues were collected and subjected to in vitro analysis. Thereby, seven further novel G-quadruplex ligands could be identified. These molecules do not only represent novel scaffolds, but some of them are in addition even more potent G-quadruplex stabilizers than the established reference compound berberine. This study proposes an optimized in silico workflow for the identification of novel G-quadruplex stabilizers, which can also be applied in future studies. In addition, structurally novel and promising lead candidates with strong and selective G-quadruplex stabilizing properties are reported

Novel Polyamine–Naphthalene Diimide Conjugates Targeting Histone Deacetylases and DNA for Cancer Phenotype Reprogramming

A series of hybrid compounds was designed to target histone deacetylases and ds-/G-quadruplex DNAs by merging structural features deriving from Scriptaid and compound <b>1</b>. Compound <b>6</b> binds different DNA arrangements, inhibits HDACs both <i>in vitro</i> and in cells, and is able to induce a reduction of cell proliferation. Moreover, compound <b>6</b> displays cell phenotype-reprogramming properties since it prevents the epithelial to mesenchymal transition in cancer cells, inducing a less aggressive and migratory phenotype, which is one of the goals of present innovative strategies in cancer therapies