A collection of 26 polyammonium cyclophane-type macrocycles with a large structural diversity has been screened for G-quadruplex recognition. A two-step selection procedure based on the FRET-melting ...assay was carried out enabling identification of macrocycles of high affinity (ΔT1/2 up to 30°C) and high selectivity for the human telomeric G-quadruplex. The four selected hits possess sophisticated architectures, more particularly the presence of a pendant side-arm as well as the existence of a particular topological arrangement appear to be strong determinants of quadruplex binding. These compounds are thus likely to create multiple contacts with the target that may be at the origin of their high selectivity, thereby suggesting that this class of macrocycles offers unique advantages for targeting G-quadruplex-DNA.
Glycosyl conjugation to drugs is a strategy being used to take advantage of glucose transporters (GLUT) overexpression in cancer cells in comparison with non-cancerous cells. Its extension to the ...conjugation of drugs to thiosugars tries to exploit their higher biostability when compared to O-glycosides. Here, we have synthesized a series of thiosugar naphthalene diimide conjugates as G-quadruplex ligands and have explored modifications of the amino sidechain comparing dimethyl amino and morpholino groups. Then, we studied their antiproliferative activity in colon cancer cells, and their antiparasitic activity in T. brucei and L. major parasites, together with their ability to bind quadruplexes and their cellular uptake and location. We observed higher toxicity for the sugar-NDI-NMe2 derivatives than for the sugar-NDI-morph compounds, both in mammalian cells and in parasites. Our experiments indicate that a less efficient binding to quadruplexes and a worse cellular uptake of the carb-NDI-morph derivatives could be the reasons for these differences. We found small variations in cytotoxicity between O-carb-NDIs and S-carb-NDIs, except against non-cancerous human fibroblasts MRC-5, where thiosugar-NDIs tend to be less toxic. This leads to a notable selectivity for β-thiomaltosyl-NDI-NMe212 (9.8 fold), with an IC50 of 0.3 μM against HT-29 cells. Finally, the antiparasitic activity observed for the carb-NDI-NMe2 derivatives against T. brucei was in the nanomolar range with a good selectivity index in the range of 30- to 69- fold.
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•A series of thiosugar naphthalene diimide (NDI) conjugates as G-quadruplex ligands has been synthesized.•Anti-proliferative and antiparasitic activity of the new compounds were measured.•Sugar-NDI with dimethyl amino groups showed higher toxicity than those with morpholino groups.•S-carb-NDIs were less toxic on healthy cells than their O-carb-NDI analogues.•β-thiomaltosyl-NDI-NMe212 showed an IC50 of 0.3 μM against HT-29 cells with 9.8 fold selectivity.
Two of the title compounds (
14a,
19) bearing cationic side chains present high selectivity for telomeric G-quadruplex over duplex DNA.
The design and synthesis of 2,6-diphenylthiazolo3,2-
...b1,2,4triazoles characterized by a large aromatic building block bearing cationic side chains are reported. These molecules are evaluated as telomeric G-quadruplex stabilizers and for their selectivity towards duplex DNA by competition experiments. Two compounds (
14a,
19) were found active with high selectivity for telomeric G-quadruplex over duplex DNA.
It has been shown that the use of conformationally pH-switchable lipids can drastically enhance the cytosolic drug delivery of lipid vesicles. Understanding the process by which the pH-switchable ...lipids disturb the lipid assembly of nanoparticles and trigger the cargo release is crucial to optimize the rational design of pH-switchable lipids. Here, we gather morphological observations (FF-SEM, Cryo-TEM, AFM, confocal microscopy), physicochemical characterization (DLS, ELS), as well as phase behavior studies (DSC,
H NMR, Langmuir isotherm, and MAS NMR) to propose a mechanism of pH-triggered membrane destabilization. We demonstrate that the switchable lipids are homogeneously incorporated with other co-lipids (DSPC, cholesterol, and DSPE-PEG
) and promote a liquid-ordered phase insensitive to temperature variation. Upon acidification, the protonation of the switchable lipids triggers a conformational switch altering the self-assembly properties of lipid nanoparticles. These modifications do not lead to a phase separation of the lipid membrane; however, they cause fluctuations and local defects, which result in morphological changes of the lipid vesicles. These changes are proposed to affect the permeability of vesicle membrane, triggering the release of the cargo encapsulated in the lipid vesicles (LVs). Our results confirm that pH-triggered release does not require major morphological changes, but can result from small defects affecting the lipid membrane permeability.
Abstract
G-quadruplexes (G4) are non-canonical DNA and/or RNA secondary structures formed in guanine-rich regions. Given their over-representation in specific regions in the genome such as promoters ...and telomeres, they are likely to play important roles in key processes such as transcription, replication or RNA maturation. Putative G4-forming sequences (G4FS) have been reported in humans, yeast, bacteria, viruses and many organisms. Here we present the first mapping of G-quadruplex sequences in Dictyostelium discoideum, the social amoeba. 'Dicty' is an ameboid protozoan with a small (34 Mb) and extremely AT rich genome (78%). As a consequence, very few G4-prone motifs are expected. An in silico analysis of the Dictyostelium genome with the G4Hunter software detected 249-1055 G4-prone motifs, depending on G4Hunter chosen threshold. Interestingly, despite an even lower GC content (as compared to the whole Dicty genome), the density of G4 motifs in Dictyostelium promoters and introns is significantly higher than in the rest of the genome. Fourteen selected sequences located in important genes were characterized by a combination of biophysical and biochemical techniques. Our data show that these sequences form highly stable G4 structures under physiological conditions. Five Dictyostelium genes containing G4-prone motifs in their promoters were studied for the effect of a new G4-binding porphyrin derivative on their expression. Our results demonstrated that the new ligand significantly decreased their expression. Overall, our results constitute the first step to adopt Dictyostelium discoideum as a 'G4-poor' model for studies on G-quadruplexes.
Paraspeckles are RNA–protein structures within the nucleus of mammalian cells, capable of orchestrating various biochemical processes. An overexpression of the architectural component of ...paraspeckles, a long non-coding RNA called NEAT1 (Nuclear Enriched Abundant Transcript 1), has been linked to a variety of cancers and is often associated with poor patient prognosis. Thus, there is an accumulating interest in the role of paraspeckles in carcinogenesis, however there is a limited understanding of how NEAT1 expression is regulated. Here, we demonstrate that both nuclear G-quadruplex (G4) and paraspeckle formation are significantly increased in a human breast cancer cell line compared to non-tumorigenic breast cells. Moreover, we identified and characterized G4-forming sequences within the NEAT1 promoter and demonstrate stabilization of G4 DNA with a G4-stabilizing small molecule results in a significant alteration in both paraspeckle formation and NEAT1 expression. This G4-mediated alteration of NEAT1 at both the transcriptional and post-transcriptional levels was evident in U2OS osteosarcoma cells, MCF-7 breast adenocarcinoma and MDA-MB-231 triple negative breast cancer cells.
•NEAT1_2 lncRNA and DNA G-quadruplexes are altered in multiple cancers .•We identify and characterise potential G-quadruplexes in the NEAT1 gene promoter.•Stabilization of G-quadruplexes alters NEAT1_2 expression and paraspeckle formation.
G-quadruplexes (G4) are found at important genome regions such as telomere ends and oncogene promoters. One prominent strategy to explore the therapeutic potential of G4 is stabilized it with ...specific ligands.
We report the synthesis of new phenanthroline, phenyl and quinoline acyclic bisoxazole compounds in order to explore and evaluate the targeting to c-myc and human telomeric repeat 22AG G4 using FRET-melting, CD-melting, NMR, fluorescence titrations and FID assays.
The design strategy has led to potent compounds (Phen-1 and Phen-2) that discriminate different G4 structures (human telomeric sequences and c-myc promoter) and selectively stabilize G4 over duplex DNA. CD studies show that Phen-2 binds and induces antiparallel topologies in 22AG quadruplex and also binds c-myc promotor, increasing their Tm in about 12°C and 30°C respectively. In contrast, Phen-1 induces parallel topologies in 22AG and c-myc, with a moderate stabilization of 4°C for both sequences. Consistent with a CD melting study, Phen-2 binds strongly (K=106 to 107M−1) to c-myc and 22AG quadruplexes.
Phen-1 and Phen-2 discriminated among various quadruplex topologies and exhibited high selectivity for quadruplexes over duplexes. Phen-2 retains antiparallel topologies for quadruplex 22AG and does not induce conformational changes on the parallel c-myc quadruplex although Phen-1 favors the parallel topology. NMR studies also showed that the Phen-2 binds to the c-myc quadruplex via end stacking.
Overall, the results suggest the importance of Phen-2 as a scaffold for the fine-tuning with substituents in order to enhance binding and stabilization to G4 structures. This article is part of a Special Issue entitled "G-quadruplex" Guest Editor: Dr. Concetta Giancola and Dr. Daniela Montesarchio.
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•Phen-1 and Phen-2 G-quadruplex ligands have been synthesized and characterized.•Circular dichroism, FRET, fluorescence and NMR have been employed.•The compounds bind and stabilize specifically G-quadruplex structures.•Phen-2 is the most promising ligand for binding and stabilization G4.
Scar formation after wound healing is a major medical problem. A better understanding of the dynamic nuclear architecture of the genome during wound healing could provide insights into the underlying ...pathophysiology and enable novel therapeutic strategies. Here, we demonstrate that TGF‐β‐induced fibrotic stress increases formation of the dynamic secondary DNA structures called G‐quadruplexes in skin fibroblasts, which is coincident with increased expression of collagen 1. This G‐quadruplex formation is attenuated by a small molecule inhibitor of intracellular Ca2+ influx and an anti‐fibrotic compound. In addition, we identify G‐quadruplex‐forming sequences in the promoter region of COL1A1, which encodes collagen 1, and confirm their ability to form G‐quadruplex structures under physiologically relevant conditions. Our findings reveal a link between G‐quadruplexes and scar formation that may lead to novel therapeutic interventions.
The syntheses of novel 2,4-bis(substituted-aminomethyl)phenylphenylquinazolines
and 2,4-bis(substituted-aminomethyl)phenylphenylquinolines
are reported here in six steps starting from various ...halogeno-quinazoline-2,4-(1
,3
)-diones or substituted anilines. The antiproliferative activities of the products were determined in vitro against a panel of breast (MCF-7 and MDA-MB-231), human adherent cervical (HeLa and SiHa), and ovarian (A2780) cell lines. Disubstituted 6- and 7-phenyl-bis(3-dimethylaminopropyl)aminomethylphenyl-quinazolines
,
, and
displayed the most interesting antiproliferative activities against six human cancer cell lines. In the series of quinoline derivatives, 6-phenyl-bis(3-dimethylaminopropyl)aminomethylphenylquinoline
proved to be the most active. G-quadruplexes (G4) stacked non-canonical nucleic acid structures found in specific G-rich DNA, or RNA sequences in the human genome are considered as potential targets for the development of anticancer agents. Then, as small aza-organic heterocyclic derivatives are well known to target and stabilize G4 structures, their ability to bind G4 structures have been determined through FRET melting, circular dichroism, and native mass spectrometry assays. Finally, telomerase inhibition ability has been also assessed using the MCF-7 cell line.
A new fluorescent, non‐cytotoxic perylene diimide derivative with two biotins at the peri position, PDI2B, has been synthesized. This molecule is able to interact selectively with G‐quadruplexes with ...scarce or no affinity towards single‐ or double‐stranded DNA. These features have made it possible to design a simple, effective, safe, cheap, and selective method for fishing G‐quadruplex structures in solution by use of PDI2B and streptavidin coated magnetic beads. The new cyclic method reported leads to the recovery of more than 80 % of G‐quadruplex structures from solution, even in the presence of an excess of single‐stranded or duplex DNA as competitors. Moreover, PDI2B is a G4 ligand that can display higher thermal stabilization and greater affinity for 2‐ over 3‐tetrad quadruplexes, which constitutes a novel type of behavior.
Gone fishin′: A new fluorescent, non‐cytotoxic perylene diimide derivative with two biotins at the peri position, PDI2B, has been synthesized. This molecule is able to interact selectively with G‐quadruplexes with scarce or no affinity towards single‐ or double‐stranded DNA. These features have made it possible to design a simple, effective, safe, cheap, and selective method to fish for G‐quadruplex structures in solution by use of PDI2B and streptavidin‐coated magnetic beads.
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