Selective Binding of Distamycin A Derivative to G-Quadruplex Structure [d(TGGGGT)]4

Guanine-rich nucleic acid sequences can adopt G-quadruplex structures stabilized by layers of four Hoogsteen-paired guanine residues. Quadruplex-prone sequences are found in many regions of human genome and in the telomeres of all eukaryotic organisms. Since small molecules that target G-quadruplexes have been found to be effective telomerase inhibitors, the identification of new specific ligands for G-quadruplexes is emerging as a promising approach to develop new anticancer drugs. Distamycin A is known to bind to AT-rich sequences of duplex DNA, but it has recently been shown to interact also with G-quadruplexes. Here, isothermal titration calorimetry (ITC) and NMR techniques have been employed to characterize the interaction between a dicationic derivative of distamycin A (compound 1) and the [d(TGGGGT)]4 quadruplex. Additionally, to compare the binding behaviour of netropsin and compound 1 to the same target, a calometric study of the interaction between netropsin and [d(TGGGGT)]4 has been performed. Experiments show that netropsin and compound 1 are able to bind to [d(TGGGGT)]4 with good affinity and comparable thermodynamic profiles. In both cases the interactions are entropically driven processes with a small favourable enthalpic contribution. Interestingly, the structural modifications of compound 1 decrease the affinity of the ligand toward the duplex, enhancing the selectivity

New anti-telomerase, anti-cancer drugs: a physico-chemical approach

Human 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

Chitosan-based nanoparticles studied by isothermal titration calorimetry

In the last decade, there has been a growing interest on chitosan-based nanomaterials. Chitosan is a polymer exceptionally versatile, biodegradable, biocompatible and with good capacity of mucoadhesivity and permeation-enhancing effect. These features make chitosan a perfect material for the fabrication of polymeric nanoparticles for a variety of applications in the field of pharmaceutics, nutraceutics or cosmetics. This paper discuss on the role of isothermal titration calorimetry (ITC) in the creation of protocols for the preparation of chitosan-based nanoparticles, as well as the role of calorimetry to find chitosan-coating conditions to offer to nanoparticles the desired proprieties for the delivery of drugs, biologics and vaccines. Although several papers of the current literature show the employment of ITC in chitosan-based nanosystems, most of them lack a thermodynamic description. Here, we highlight on two types of systems: chitosan-coating nanoparticles and chitosan-containing nanoparticles. The thermodynamic properties and the energetic aspects of the overall interactions are discussed

Nuovi ligandi per G-quadruplex: un approccio chimico-fisico nella terapia anti-cancro

In tutte le cellule eucariotiche umane il DNA telomerico è costituito da una sequenza altamente ripetuta 5’TTAGGG3’. Ad ogni ciclo replicativo il DNA telomerico si riduce a partire dall’estremità 3’ costituita da singolo filamento fino ad una lunghezza critica che conduce la cellula all'apoptosi. Nell’80-85% delle cellule tumorali si osserva la sovraespressione della telomerasi, un enzima che catalizza l’allungamento del DNA telomerico determinando una proliferazione incontrollata delle cellule tumorali. Qualsiasi strategia capace di inibire la telomerasi può essere considerata una potenziale terapia anti-cancro. Le sequenze TTAGGG ricche di guanine presenti alle estremità telomeriche a singolo filamento hanno la capacità di ripiegarsi su se stesse formando delle strutture note come quadruple eliche. La formazione di tali strutture è in grado di inibire l’attività della telomerasi. E’ di estremo interesse lo studio di composti in grado di legare in maniera specifica le quadruple eliche e dei fattori che regolano gli equilibri in gioco. La conoscenza degli aspetti chimico-fisici che regolano l’interazione tra quadrupla elica e ligando rappresenta un punto chiave in vista di future applicazioni biomediche. Lo studio dell’energetica d’interazione tra nuovi ligandi e le strutture target ha permesso di chiarire la natura delle forze che guidano il processo di binding e di far luce sugli elementi strutturali utili ad ottimizzare le proprietà di legame, fornendo le basi strutturali per una futura progettazione razionale di nuove classi di farmaci ad attività anti-tumorale

G-quadruplex unfolding in higher-order DNA structures

G-quadruplex unfolding within a sequence of two quadruplex units was characterized by gel electrophoresis, calorimetry and spectroscopy. The obtained results suggest that the kinetics and thermodynamics of the individual quadruplex unfolding are affected by its interaction with other DNA secondary structural elements

Thermodynamic Signature of Secondary Nano-emulsion Formation by Isothermal Titration Calorimetry.

The stabilization of oil in water nano-emulsions by means of a polymer coating is extremely important; it prolongs the shelf life of the product and makes it suitable for a variety of applications ranging from nutraceutics to cosmetics and pharmaceutics. To date, an effective methodology to assess the best formulations in terms of thermodynamic stability has yet to be designed. Here, we perform a complete physicochemical characterization based on isothermal titration calorimetry (ITC) compared to conventional dynamic light scattering (DLS) to identify polymer concentration domains that are thermodynamically stable and to define the degree of stability through thermodynamic functions depending upon any relevant parameter affecting the stability itself, such as type of polymer coating, droplet distance, etc. For instance, the method was proven by measuring the energetics in the case of two different biopolymers, chitosan and poly-l-lysine, and for different concentrations of the emulsion coated with poly-l-lysine