Noncanonical DNA-binding proteins: from identification to next generation peptide-based anticancer therapeutics

Background: G-quadruplexes and i-motifs are biologically relevant noncanonical DNA secondary structures, whose existence in vivo has been unambiguously demonstrated by their visualization in human cells. The compelling evidence that such noncanonical structures act as active regulators of cancer-related genomic processes made them an emerging research topic and an attractive target for gene regulation and anticancer drug design. Hypothesis: Formation, stabilization, and resolution of such noncanonical structures are regulated by nucleic acid-directed proteins that can activate/deactivate specific pathways. Despite their importance, the identity of most of these proteins remain unknown, as well as how they work and what is their biological impact on cancer cells. The identification of the proteins that specifically recognize noncanonical structures is crucial to understand the elusive molecular mechanisms in which such DNA motifs are involved, with important implications also in the field of drug discovery. Aims: This project aims to identify and investigate the nuclear proteins able to selectively recognize G-quadruplex and i-motif structures in the same transcriptional regulatory element of selected cancer-related human gene promoters. Starting from the identified proteins, this project also aims to develop peptides/peptidomimetics able to target such DNA structures with high affinity and selectivity, as well as lower toxicity and increased functionality compared to the traditional small molecule drugs. Experimental Design: The identification of the interactors of the selected gene promoter i-motifs and G-quadruplexes will be achieved in a direct fashion by a chemical proteomics approach that we have already proven to be a successful strategy. A combined use of biophysical and biological experiments will be employed to investigate the selected proteins and understand their role, as well as to study the properties of the developed peptides. Expected Results: We expect to identify the proteins that specifically recognize the G-quadruplex and i-motif structures in the same location of the genome and to characterize their interaction from a structural and functional point of view. Then, by exploiting the noncanonical DNA-recognition motifs of the discovered proteins, we expect to obtain peptide-based anticancer agents combining the advantages of therapeutic proteins and small-molecule drugs. Impact On Cancer: The discovery of the nuclear proteins that specifically recognize noncanonical DNA structures and the elucidation of their functions will contribute to expand the knowledge on these structures and their roles in cancer, and to shed light on the complex regulatory mechanisms of gene expression. Moreover, our findings will have a significant impact in the field of drug discovery. Indeed, we will exploit the molecular determinants of these protein-DNA interactions to develop in 5 years' time a new generation of molecularly targeted anticancer therapeutics.