G-quadruplex (G4) and i-motif (iM) are four-stranded non-canonical nucleic acid structural arrangements. Recent evidences suggest that these DNA structures exist in living cells and could be involved in several cancer-related processes, thus representing an attractive target for anticancer drug discovery. Efforts toward the development of G4 targeting compounds have led to a number of effective bioactive ligands. Herein, employing several biophysical methodologies, we studied the ability of some well-known G4 ligands to interact with iM-forming DNA. The data showed that the investigated compounds are actually able to interact with both DNA in vitro, thus acting de facto as multi-target-directed agents. Interestingly, while all the compounds stabilize the G4, some of them significantly reduce the stability of the iM. The present study highlights the importance, when studying G4-targeting compounds, of evaluating also their behavior toward the i-motif counterpart.
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.
This communication reports on a possible distinct role of HMGB1 protein. Biophysical studies revealed that HMGB1 binds and stabilizes the G-quadruplex of the KRAS promoter element that is responsible for most of the transcriptional activity. Biological data showed that inhibition of HMGB1 increases KRAS expression. These results suggest that HMGB1 could play a role in the gene transcriptional regulation via the functional recognition of the G-quadruplex.
HMGB1 is a ubiquitous non-histone protein, which biological effects depend on its expression and subcellular location. Inside the nucleus, HMGB1 is engaged in many DNA events such as DNA repair, transcription and telomere maintenance. HMGB1 has been reported to bind preferentially to bent DNA as well as to noncanonical DNA structures like 4-way junctions and, more recently, to G-quadruplexes. These are four-stranded conformations of nucleic acids involved in important cellular processes, including telomere maintenance. In this frame, G-quadruplex recognition by specific proteins represents a key event to modulate physiological or pathological pathways. Herein, to get insights into the telomeric G-quadruplex DNA recognition by HMGB1, we performed detailed biophysical studies complemented with biological analyses. The obtained results provided information about the molecular determinants for the interaction and showed that the structural variability of human telomeric G-quadruplex DNA may have significant implications in HMGB1 recognition. The biological data identified HMGB1 as a telomere-associated protein in both telomerase-positive and -negative tumor cells and showed that HMGB1 gene silencing in such cells induces telomere DNA damage foci. Altogether, these findings provide a deeper understanding of telomeric G-quadruplex recognition by HMGB1 and suggest that this protein could actually represent a new target for cancer therapy.
The i-motif DNA, also knowna si -DNA, is an oncanonical DNAs econdary structure formed by cytosine-rich sequences,c onsisting of two intercalated parallel-stranded duplexes held together by hemi-protonated cytosine-cytosine + (C:C + )base pairs.The growing interest in the i-DNAstructure as atarget in anticancer therapyincreases the need for tools for arapid and meaningful interpretation of the spectroscopic data of i-DNAsamples.Herein, we analyzed the circular dichroism (CD) and thermal difference UV-absorbance spectra (TDS) of 255 DNAs equences by means of multivariate data analysis, aiming at unveiling peculiar spectral regions that could be used as diagnostic features during the analysis of i-DNA-forming sequences.
Targeting
G-quadruplex structures is currently viewed as a promising
anticancer strategy. Searching for potent and selective G-quadruplex
binders, here we describe a small series of new monohydrazone derivatives
designed as analogues of a lead which was proved to stabilize G-quadruplex
structures and increase R loop levels in human cancer cells. To investigate
the G-quadruplex binding properties of the new molecules,
in vitro
biophysical studies were performed employing both
telomeric and oncogene promoter G-quadruplex-forming sequences. The
obtained results allowed the identification of a highly selective
G-quadruplex ligand that, when studied in human cancer cells, proved
to be able to stabilize both G-quadruplexes and R loops and showed
a potent cell killing activity associated with the formation of micronuclei,
a clear sign of genome instability.
A chemoproteomic-driven approach was used to investigate the interaction network between human telomeric G-quadruplex DNA and nuclear proteins. We identified novel G-quadruplex binding partners, able to recognize these DNA structures at chromosome ends, suggesting a possible, and so far unknown, role of these proteins in telomere functions.
Background: DNA G-quadruplex (G4) structures represent potential anti-cancer targets. In this study, we compared the effect of two G4-targeting compounds, C066-3108 and the gold standard BRACO-19. Methods: In breast and prostate cancer cells, cytotoxicity induced by both molecules was measured by a sulforhodamine B assay. In breast cancer cells, cycle, apoptosis, the formation of G4 structures, calreticulin and high mobility group box 1 (HMGB1), as well as T cell activation, were analyzed by flow cytometry and adenosine triphosphate (ATP) by luminescence. Results: Both ligands inhibited cell survival and induced DNA damage. In MCF-7 cells, G4 ligands increased the subG0/G1 phase of the cell cycle inducing apoptosis and reduced intracellular ATP. In untreated MCF-7 cells, we observed a slight presence of G4 structures associated with the G2/M phase. In MDA-MB231 cells, G4 ligands decreased the G1 and enhanced the G2/M phase. We observed a decrease of intracellular ATP, calreticulin cell surface exposure and an increase of HMGB1, accompanied by T cell activation. Both compounds induced G4 structure formation in the subG0/G1 phase. Conclusions: Our data report similar effects for both compounds and the first evidence that G4 ligands induce the release of danger signals associated with immunogenic cell death and induction of T cell activation.
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