The fine architecture of guanine-rich regions within oncogene promoters

Suppression of oncogenes transcription represents an ideal tool to integrate the currently available therapeutics to treat several cancer types and to overcome the potential occurrence of resistance. An experimentally validated mechanism of intervention is represented by the induction/stabilization of G-quadruplex structures in genes promoter by small molecules. G-quadruplexes are DNA non-canonical secondary structures consisting of stacked G-quartets, cyclic arrangements of four guanine residues held together by Hoogsteen hydrogen bonds and stabilized by a central cation. At the moment, none of the identified G-quadruplex ligands reached the clinic. Several reasons can contribute to this poor outcome comprising both the plastic structural features of nucleic acids and the multiple metabolic pathways which might be affected when a small molecule interacts with G-quadruplex structures. Thus, one of the major issues lies on the proper structural analysis of targeted G-quadruplex. To overcome this bias, in this Ph.D.’s thesis kinetic and thermodynamic behaviours of G-quadruplexes have been characterized to obtain an improved description of these structures as potential pharmaceutical targets. The study has been focused on G-rich sequences within c-KIT and EGFR oncogene promoters. By applying a set of complementary structural and biophysical approaches, the folding pathways of these G-quadruplexes and influence of flanking regions in terms of structural stability and folding rearrangement have been described. The obtained information indicates that the promoter architecture might be not properly derived by analysis of minimal G-quadruplex forming sequences at the thermodynamic equilibrium, commonly used for screening assayes. Indeed, reported data suggest the existence of different unique mechanisms/pathways involved in the regulation of these oncogenes transcription which comprise kinetically favored folding intermediates or unprecedented structural arrangements. The final outcome of Ph.D.‘s project is a deeper understanding of nucleic acid tridimensional arrangement of EGFR and c-KIT promoters which might help in setting up new drug-design programs based on models of G-quadruplex target more closely related to the physiological ones.