1,721,095 research outputs found
New Anti-Telomerase Anti-Cancer Agents
No full textThe unlimited proliferative potential of cancer cells depends on telomere maintenance. Optimal telomerase activity requires in chromosomal DNA an unfolded single-stranded telomeric overhang. This overhang may adopt an unusual structure called G-quadruplex, composed of guanine G-quartet which forms in the presence of cations. Therefore, ligands that selectively bind to and stabilize telomeric G-quadruplex structures could act
as telomerase inhibitors. However, the development of a new class of anticancer drugs based on targeting the telomeric G-quadruplex structure is hampered by a lack of any structural information about the full length overhang because its size is refractory to structure determination by NMR and crystallography. To date there are no reported thermodynamic binding studies for drug interactions with the functionally relevant higher-order telomeric quadruplex structures. Current literature studies on Gquadruplexes formed by telomeric DNA and their interaction with pharmacologically interesting molecules are mostly limited to the analysis of the oligonucleotide formed by only four TTAGGG repeats (such as d(TTAGGG)4 sequences or slight variants thereof), able to fold into a single quadruplex structure. A telomeric single-stranded overhang is actually constituted of tens of TTAGGG repeats, hence it is able to form several consecutive quadruplex structures (multimers) in the overhang region.
Recently obtained, unpublished experimental data from our lab have indicated that the binding properties of longer telomeric sequence (n>4) could be different from the those
obtained with the short DNA telomeric sequence (n=4).
The need of a systematic study of the structure and binding properties of the longer telomeric DNA sequences is thus compelling. We thus first propose an innovative approach to obtain reliable structural models for the long telomeric DNA overhang that can be used as new targets for chemotherapy. Second, we propose a consistent structure-based strategy to design and test (in vitro and in vivo) ligands that specifically bind to these physiological relevant structures. In summary, the
research program consists of the following four main steps:
1) Determination of the structure and stability of the single-stranded telomeric DNA overhang.
2) Rational design of ligands using virtual screening.
3) Experimental determination of the affinity in vitro of the potential ligands (designed according to the step 2) for the target structures.
4) Biological testing of the more promising ligands derived from the analysis of the step 2 and 3
Higher-order Quadruplex Structures
No full textStructural studies have shown that four G-tracts along a DNA strand are the minimal requirement for intramolecular G-quadruplex formation. Longer DNA sequences containing multiple of four G-tracts could, in principle, form higher-order structures based on multiple G-quadruplex blocks. This later condition is abundantly verified for the telomeric single-stranded overhang (~ 200 nt) constituted of tens of TTAGGG repeats, thus opening new interesting questions about the structure of the “real” telomeric DNA. How many quadruplex units form in the human telomeric overhang ? Which type of quadruplex topologies ? Do they interact or not ? What about their binding properties ? Although, many of these questions are still unanswered, they recently began to be object of experimental and computational studies. The question about the existence and relevance of these higher-order quadruplex structures in the human genome is now an interesting and stimulating research topic in the quadruplex field. The recent results, the unsolved problems and the future prospective on the intramolecular telomeric higher-order quadruplex structures are the main topic of this review. Other studies on long telomeric RNA sequences and on other intramolecular (non telomeric) DNA higher order quadruplex structures are also presented
New anti-telomerase, anti-cancer drugs: a physico-chemical approach
No full textHuman telomeric DNA terminates with a 3’ single-stranded overhang containing tandem repeats of the sequence TTAGGG. These G-rich overhangs are prone to fold back to form G-quadruplex structures stabilized by consecutive G-tetrads each containing four guanines involved in Hoogsteen hydrogen bonds. G-quadruplex structures can inhibit the activity of telomerase, the enzyme that adds telomeric repeats to the ends of chromosomes and maintains the proliferation of cancer cells. Inhibition of telomerase can stop tumor growth and thus small molecules capable to interfere with telomere maintenance inducing the formation of quadruplex structures, are considered to be potential anti-cancer agents. In this perspective my thesis project concerns the characterization of the human telomeric DNA and aims to the study of new anti-telomerase agents that specifically bind telomeric DNA stabilizing the G-quadruplex structures. The first part of my thesis project concerns the study of π-π stacking ligands. The aim was to solve the nature of the binding mode and stoichiometry of the cationic porphyrin TMPyP4 to several human telomeric G-quadruplex structures. Another possible quadruplex drug studied in this thesis was the three side-chained triazatruxene derivative, termed azatrux. The binding of azatrux to the human telomeric G-quadruplex, was explored in presence of 40 % PEG 200 to simulate the crowding conditions existing inside the cell. The binding of azatrux to the tetramolecular parallel [d(TGGGGT)]4 quadruplex and to another biologically relevant G-quadruplex (oncogene promoter c-Kit87up) and to duplex DNA in the presence and absence of crowding conditions was characterized The last part of the Ph.D. study was focused on the characterisation of groove binder ligands. The binding of a dicationic derivative of distamycin A (compound 1) with the [d(TGGGGT)]4 quadruplex were explored by isothermal titration calorimetry (ITC), and compared to the binding behaviour of netropsin to the same target. Furthermore the affinity of some new ligands with the grooves of DNA-quadruplex [d(TGGGGT)]4 were studied. These ligands were derivatives of a molecule obtained from a previous virtual screening study. The interaction has been evaluated, analysing the displacement of these ligands from the grooves of DNA-quadruplex by distamycin A, the best groove binder identified until now. The derivatives have been tested over double-stranded DNA to demonstrate whether there is an enhanced selectivity for quadruplex DNA compared to the duplex structure
INTERAZIONI TRA ACIDI NUCLEICI, PROTEINE E POTENZIALI FARMACI ANTITUMORALI: UN APPROCCIO CHIMICO-FISICO
No full textStudying the effect of crowding and dehydration on DNA G-quadruplexes
No full textIntracellular environment is crowded with biomolecules that occupy a significant fraction (up to 40%) of the cellular volume, with a total concentration in the range 300-400mg/ml. Recently, the effect of crowding/dehydrating agents on the DNA G-quadruplexes has become a subject of an increasing interest. Crowding and/or dehydrating agents have been used to simulate how G-quadruplexes behave under cell-mimicking conditions characterized by a large excluded volume and a lower water activity. Indeed, the presence of both steric crowding and a lower water activity can affect G-quadruplex stability, their folding/unfolding kinetics, as well as their binding processes with proteins or small ligands. Many of these effects can be explored experimentally by measuring the dependence of the conformational stability, isomerisation kinetics and equilibria on the concentration of cosolutes which do not interact with the molecules (G-quadruplexes) under investigation. Spectroscopic methodologies, like circular dichroism, UV and fluorescence, have been widely employed to study G-quadruplexes in dilute solution. Here we focus on some aspects that need to be taken into account when employing such techniques in the presence of large amount of a cosolute. Additionally, we discuss possible problems/artifacts that arise in setting experiments in presence of these commonly employed cosolutes and in interpreting the results
Quadruplex-forming oligonucleotides as tools in anticancer therapy and aptamers design: energetic aspects
No full textRecent investigations on the G-quadruplex motif propose a new strategy for the making of antitumour drugs. Quadruplex-drug complexes have been suggested to inhibit telomerase activity; further, aptamers based on the quadruplex motif have been proved useful as tools aimed at binding and inhibiting particular proteins, thus serving as pharmaceutically active agents. However, the design of new aptamers is difficult because many factors affecting their activity and stability have not still been clarified. The knowledge of the energetics of quadruplex formation is a crucial point in view of their potential therapeutic utilization both as targets as well as therapeutic agents. In this review the energetic aspects of both quadruplex assembly and quadruplex-ligand interactions are discussed together with a summary of recent studies on physico-chemical properties in solution of quadruplex structures obtained from synthetic aptamers, including PNA-DNA chimeras
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