| Name | Bruno |
| Surname | Pagano |
| Institution | University of Naples – Federico II |
| bruno.pagano@unina.it | |
| Address | Department of Pharmacy, University of Naples "Federico II", Via D. Montesano 49, 80131, Naples, Italy |
G-quadruplex stabilizers are an established opportunity in anticancer chemotherapy. To circumvent the antiproliferative effects of G4 ligands, cancer cells recruit PARP enzymes at telomeres. Herein, starting from the structural similarity of a potent G4 ligand previously discovered by our group and a congeneric PARP inhibitor, a library of derivatives was synthesized to discover the first dual G4/PARP ligand. We demonstrate that a properly decorated thieno[3,2-c]quinolin-4(5H)-one stabilizes the G4 fold in vitro and in cells, induces a DNA damage response localized to telomeres, inhibits PARylation in cells, and has an antiproliferative effect in BRCA2 deficient tumor cells.
In this study, we report the first atomic resolution structure of a stable G-hairpin formed by a natively occurring DNA sequence. An 11-nt long G-rich DNA oligonucleotide, 5'-d(GTGTGGGTGTG)-3', corresponding to the most abundant sequence motif in irregular telomeric DNA from Saccharomyces cerevisiae (yeast), is demonstrated to adopt a novel type of mixed parallel/antiparallel fold-back DNA structure, which is stabilized by dynamic G:G base pairs that transit between N1-carbonyl symmetric and N1-carbonyl, N7-amino base-pairing arrangements. Although the studied sequence first appears to possess a low capacity for base pairing, it forms a thermodynamically stable structure with a rather complex topology that includes a chain reversal arrangement of the backbone in the center of the continuous G-tract and 3'-to-5' stacking of the terminal residues. The structure reveals previously unknown principles of the folding of G-rich oligonucleotides that could be applied to the prediction of natural and/or the design of artificial recognition DNA elements. The structure also demonstrates that the folding landscapes of short DNA single strands is much more complex than previously assumed.
G-quadruplexes (G4s) are higher-order DNA structures typically present at promoter regions of genes and telomeres. Here, the G4 formation decreases the replicative DNA at each cell cycle, finally leading to apoptosis. The ability to control this mitotic clock, particularly in cancer cells, is fascinating and passes through a rational understanding of the ligand/G4 interaction. We demonstrate that an accurate description of the ligand/G4 binding mechanism is possible using an innovative free-energy method called funnel-metadynamics (FM), which we have recently developed to investigate ligand/protein interaction. Using FM simulations, we have elucidated the binding mechanism of the anticancer alkaloid berberine to the human telomeric G4 (d[AG3(T2AG3)3]), computing also the binding free-energy landscape. Two ligand binding modes have been identified as the lowest energy states. Furthermore, we have found prebinding sites, which are preparatory to reach the final binding mode. In our simulations, the ions and the water molecules have been explicitly represented and the energetic contribution of the solvent during ligand binding evaluated. Our theoretical results provide an accurate estimate of the absolute ligand/DNA binding free energy ([Formula: see text] = -10.3 ± 0.5 kcal/mol) that we validated through steady-state fluorescence binding assays. The good agreement between the theoretical and experimental value demonstrates that FM is a most powerful method to investigate ligand/DNA interaction and can be a useful tool for the rational design also of G4 ligands.
G-quadruplex (G4) structures are key elements in the regulation of cancer cell proliferation and their targeting is deemed to be a promising strategy in anticancer therapy.
Guanine-rich DNA motifs can form non-canonical structures known as G-quadruplexes, whose role in tumorigenic processes makes them attractive drug-target candidates for cancer therapy. Recent studies revealed that the folding and unfolding pathways of G-quadruplexes proceed through a quite stable intermediate named G-triplex.
G-Quadruplex-binding compounds are currently perceived as possible anticancer therapeutics. Here, starting from a promising lead, a small series of novel hydrazone-based compounds were synthesized and evaluated as G-quadruplex binders. The in vitro G-quadruplex-binding properties of the synthesized compounds were investigated employing both human telomeric and oncogene promoter G-quadruplexes with different folding topologies as targets. The present investigation led to the identification of potent G-quadruplex stabilizers with high selectivity over duplex DNA and preference for one G-quadruplex topology over others. Among them, selected derivatives have been shown to trap G-quadruplex structures in the nucleus of cancer cells. Interestingly, this behavior correlates with efficient cytotoxic activity in human osteosarcoma and colon carcinoma cells.
In the last decades, nano-oncologicals bearing a polyethylene glycol (PEG) coating are being emerging as biomimetic devices able to drive their drug cargo to solid tumors through passive mechanisms. To improve selectivity toward cancer cells, nanocarriers decorated with the small ligand folate have been widely investigated. Nevertheless, a great challenge remains the effective exposition of folate on nanoparticles (NPs), which is a key prerequisite to ensure the correct binding to receptor and the following endocytic uptake. On these premises, in this study we propose a novel strategy to produce core-shell folate-targeted NPs based on diblock copolymers of poly(ε-caprolactone) (PCL) and PEG through the aid of (2-hydroxypropyl)-β-cyclodextrin (HPβCD). PCL4300-PEG2000 and PCL4300-PEG2000-Fol copolymers were synthesized, characterized and employed to produce NPs without and with HPβCD by a melting/sonication procedure. Colloidal properties of targeted NPs produced with HPβCD demonstrated a highly extended conformation of PEG chains in the shell, an enhanced interaction with a specific antibody against folate and a higher uptake in cells overexpressing folate receptor. Overall, these results suggest that proper manipulation of PEG shell conformation through HPβCD can represent a novel non-covalent strategy to modify shell features.
Cancer stem cells (CSCs) have been identified in several solid malignancies and are now emerging as a plausible target for drug discovery. Beside the questionable existence of CSCs specific markers, the expression of CD133 was reported to be responsible for conferring CSC aggressiveness. Here, we identified two G-rich sequences localized within the introns 3 and 7 of the CD133 gene able to form G-quadruplex (G4) structures, bound and stabilized by small molecules. We further showed that treatment of patient-derived colon CSCs with G4-interacting agents triggers alternative splicing that dramatically impairs the expression of CD133. Interestingly, this is strongly associated with a loss of CSC properties, including self-renewing, motility, tumor initiation and metastases dissemination. Notably, the effects of G4 stabilization on some of these CSC properties are uncoupled from DNA damage response and are fully recapitulated by the selective interference of the CD133 expression.In conclusion, we provided the first proof of the existence of G4 structures within the CD133 gene that can be pharmacologically targeted to impair CSC aggressiveness. This discloses a class of potential antitumoral agents capable of targeting the CSC subpopulation within the tumoral bulk.
DNA-minor-groove-binding ligands are potent antineoplastic molecules. The antibiotic distamycin A is the prototype of one class of these DNA-interfering molecules that have been largely used in vitro. The affinity of distamycin A for DNA is well known, and the structural details of the complexes with some B-DNA and G-quadruplex-forming DNA sequences have been already elucidated. Here, we show that distamycin A binds S100β, a protein involved in the regulation of several cellular processes. The reported affinity of distamycin A for the calcium(II)-loaded S100β reinforces the idea that some biological activities of the DNA-minor-groove-binding ligands arise from the binding to cellular proteins.
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.