Grasso, Nicola (2024) Design, synthesis, and characterization of peptide-based ligands targeting G-quadruplex DNA structures. [Tesi di dottorato]

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Abstract

Noncanonical nucleic acid structures, particularly G-quadruplexes (G4s), emerged as promising anticancer target due to their potential for selective interactions. The unique stacking of planar G-tetrads in G4 creates distinct electrostatic and hydrophobic interaction surfaces that can be specifically targeted by proteins and small molecules. Additionally, the structural flexibility of G4s, including their diverse folding patterns and topologies, offers multiple specific binding sites for proteins and ligands. Their dynamic nature allows for specific recognition by proteins that can differentiate between folded and unfolded states. G4s are now recognized as key players in several critical biological processes, such as the regulation of gene expression, the maintenance of genomic stability, and telomere function. However, these biological roles are tightly regulated by proteins that bind and modulate G4 formation and stability. Dysregulation of these G4-interacting proteins may significantly contribute to the onset of various diseases, particularly cancer and neurodegenerative disorders. Thus, these unique nucleic acids structures have become attractive targets for therapeutic drug design. Over the past decades, several classes of compounds have been developed to stabilize and target G4s. However, despite the growing number of known G4-targeting ligands, there remains a need for highly selective and specific molecules. Peptides may represent promising candidates for this role, offering the potential to act as precise G4-targeting agents. This PhD thesis aims to demonstrate the potential of protein-derived peptides as targeting agents for G4 DNA and their anticancer activity. A dual approach was employed to identify these peptides. In the first approach, structural data from existing G4-protein complexes were used as a starting point. In the second, the amino acid composition of known G4-binding proteins was analysed to identify common motifs responsible for G4 recognition. The interactions between these peptides and G4 structures were thoroughly characterized using various biophysical techniques, providing key insights for the design of peptide-based G4 ligands. The most promising peptides were further investigated for their anticancer activity against specific cell lines. Additionally, this thesis explores the simultaneous targeting of both G4 structures, and the proteins involved in their recognition, offering the potential for more effective and targeted therapeutic strategies. To this end, an Artificial Intelligence (AI)-powered virtual screening (VS) method was initially used to identify potential inhibitors of the PARP1 protein. This was followed by a classical docking-based VS approach using the available G4 experimental structure, leading to the identification of potential dual-acting candidates. These compounds were experimentally validated for their ability to inhibit PARP1 and stabilize G4 structures, and the most promising compounds were further tested to assess their antiproliferative effects against specific cancer cell lines. The thesis is organized into four main chapters: CHAPTER 1 provides an overview on non-canonical nucleic acid secondary structures, in particular G-quadruplexes and their biological roles in genomic regions. CHAPTER 2 discusses the main methodologies employed in the studies presented in the following chapters. CHAPTER 3 reports on the identification of G4-binding peptides and the characterisation of their interaction with G4s by employing several biophysical techniques. The most promising peptides were further investigated for their anticancer activity against specific cell lines. CHAPTER 4 describes the potential of combining Artificial Intelligence and structure-based methods to discover multitarget compounds that simultaneously inhibit the poly(ADP-ribose) polymerase (PARP)1 enzyme and stabilize G4 structures. These compounds were experimentally validated using biophysical techniques and evaluated for their biological activity, demonstrating their promise as effective therapeutic agents. The last section of this PhD thesis includes the GENERAL CONCLUSIONS and REFERENCES. Finally, the APPENDIX presents the scientific articles published and cited throughout the thesis. PAPER III is not included in the APPENDIX as it is still in preparation

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