Targeting DNA i-motif structures: a new frontier in anticancer therapy

Background Non-canonical DNA secondary structures can be formed by sequences that are widely distributed, but not randomly located, throughout the human genome and play crucial roles in a number of biological functions. Guanine-rich stretches of DNA can form unusual structures, called G-quadruplexes, known to play key roles in cancer. Wherever there is a G-quadruplex forming sequence in one strand, the complementary strand contains a cytosine-rich sequence that may be capable of forming another non-canonical structure, known as i-motif. Hypothesis G-quadruplex/i-motif forming sequences are frequently located in or near the regulatory regions of numerous oncogenes and in the terminal regions of chromosomes, the telomeres, thus implying their involvement in a variety of cellular functions. There are evidences that small molecules able to bind i-motif structures can modulate gene expression and enhance telomerase inhibition. Despite the i-motif structures are now well-validated biological targets, very limited efforts have been carried out to date to develop ligands able to target them. A cutting-edge frontier in biomedicine is thus bringing i-motifs into focus as drug targets for the treatment of cancer. Aims DNA i-motifs have distinctive structural features that make them unique compared to other DNA secondary structures. This can be strategic to design selective and thus low toxic ligands to be used in anticancer therapy. Therefore, this project aims at the discovery of new drug-like molecules specifically binding and promoting i-motif structures in human telomere and oncogene promoter regions. These molecules can act as antitumor agents by themselves, but they could also be used in synergy with G- quadruplex ligands. Experimental Design The experimental design is divided in six tasks. Task 1 is dedicated to the search of i-motif forming sequences. Task 2 is focused on their biophysical study, both from the structural and energetic point of view. Task 3 is focused on the determination of their three-dimensional structure. These structures will be used as targets in virtual screening calculations (Task 4), which will identify potential ligands whose binding properties will be evaluated by biophysical methodologies (Task 5). Finally, the best molecules will be biologically tested on a panel of human cancer cell lines (Task 6). Expected Results We expect to find ligands able to selectively bind i-motif DNA structures and to exert anticancer activity. The discovered compounds will have drug-like scaffolds that could represent the staring point for the development of a new generation of molecules to be used in anticancer therapy. The chemical features of these ligands will be analyzed to determine the structural requisites that are essential for the selective binding of i-motif structures. Impact On Cancer This research project aims at the discovery of anti-cancer candidate drugs specifically binding and stabilizing telomeric and oncogene promoter i-motif DNA under physiological conditions. The discovery of such molecules would be invaluable to the field of cancer biology. In addition, our studies will contribute to expand the current knowledge on the roles of i-motifs in cancer, with enormous potential repercussions.