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