Molecular mechanism and biological activity of G-quadruplex ligands, a new potential class of antitumoral agents

Besides the canonical double-helix structure (B-DNA), DNA can fold into different types of secondary structures, such as left-handed Z-DNA, A-motif, hairpin, triplex and tetraplex (G-quadruplex and i-motif). Among these, G-quadruplexes (G4) have aroused a lot of interest in the scientific panorama due to their presence all over the genome. In fact, G4 forming sequences are present at telomeres, in the promoters of oncogenes, in 5’-untranslated regions (5’-UTR), in introns and in the non-coding RNA. These features assess to G4 a crucial role in important biological processes, such as DNA replication, transcriptional regulation, and genome stability. Interestingly, several bioinformatics analysis demonstrated an enrichment of G4 forming sequences in the promoters of human oncogenes, suggesting that the stabilization of these structures could play an essential role in carcinogenesis. These evidence defined G4 associated with oncogenes as a relevant class of new potential drug targets for the development of novel anticancer therapy. Consequently, many scientists have focused their research on seeking small molecular inducers and stabilizers of G-quadruplexes. RHPS4 is a pentacyclic acridine with a high selectivity for quadruplex DNA structure. During last years, many works demonstrated, both in vitro and in vivo, the antitumor efficacy of the compound and elucidated partially the molecular mechanisms of RHPS4 al telomeric level. Based on this background, the aims of the project were i) to better investigate the molecular mechanisms responsible for the biological effects of G4-ligands, in particular analyzing the ability of the compound to bind and modulate other targets besides telomeres and ii) to screen and to identify new compounds with improved biological effects. The results revealed a “two-hit” antitumor activity of RHPS4, in which the ligand acts on both cancer cells and microenvironment, blocking tumor growth and progression. In particular, the data demonstrated that, beside the effect of the G4 ligand on telomeres, RHPS4 was able to target the G4 forming sequence in the promoter of VEGFR2. The resulting stabilization of the structure induced the down regulation of both gene and protein expression of VEGFR2, thus impairing the angiogenesis in epithelial cells. Moreover, several RHPS4-derivated compounds were screened and some of them were identified as new promising G4-stabilizing telomere targeting agents. These ligands were superior to the RHPS4 in terms of both toxicological profile and biological effects.