Cationic calixarenes for GCase targeting, G-quadruplex recognition and DNA delivery

The design of molecular systems capable of interacting with biomacromolecules and modulating their function represents a major goal at the interface between supramolecular chemistry and chemical biology. Within this framework, calixarenes represent versatile molecular platforms in which the conformational properties of the scaffold and the spatial arrangement of the binding units can be finely tuned to suit different molecular targets and to investigate factors driving recognition phenomena. Their structural adaptability, together with the possibility of introducing a wide variety of functional groups at defined positions make calixarenes particularly suitable for the design of ligands targeting complex biomolecular systems. The present thesis work explores the development of cationic calixarenes as supramolecular derivatives for the interaction with biomedically relevant macromolecules, such as nucleic acids and enzymes, with the aim of designing and setting up non-covalent strategies for molecular recognition with possible therapeutic applications. The first experimental chapter (Chapter 2) of this thesis presents the results obtained from a study focused on calix[4]arene-based multivalent iminosugars for the interaction with the lysosomal enzyme Glucocerebrosidase (GCase). Our goal was to synthesize multivalent ligands capable of binding to mutant GCase and restoring its activity, acting as pharmacological chaperones. By exploring different ligand valency and disposition of the functional units on the scaffold, this work led to the identification of ligands that, thanks to their multivalent nature, display increased potency compared to monovalent analogues, and that are capable of restoring GCase activity as demonstrated by in vitro tests on cells. Chapter 3 describes the evaluation of p-aminomethylcalix[n]arenes as a novel class of ligands for biomedically relevant G-quadruplex structures. With the aim of developing ligands capable of G-quadruplex (G4) recognition, a library of calix[n]arene derivatives functionalized with different cationic or protonatable groups was prepared. Calix[n]arene scaffolds of different size (n = 4, 6, and 8) and with different conformational properties were considered, to investigate how varying the valency and the conformational flexibility of the scaffold influenced the ligands ability to bind to the target G4, access suitable binding sites and induce a stabilization. Exploiting different complementary techniques, the interaction of the synthesized ligands with telomeric G4s and KRAS G4 was investigated, leading us to identify ligands functionalized with cationic ammonium groups and based on mobile macrocyclic scaffolds as promising candidates to stabilize the secondary structure of the target G4s. Finally, in chapter 4, it is presented the work carried out during a six-months stay at the University of Seville, in collaboration with professors Carmen Ortiz Mellet and José Manuel García Fernández. During this period, an array of polycationic amphiphilic calix[4]arene and β-cyclodextrin derivatives was synthesized with the aim of evaluating their efficiency in nucleic acid complexation for gene delivery applications. All synthesized compounds were co-formulated with a nucleic acid substrate and the properties of the resulting nanoparticles were investigated. Most derivatives proved effective for nucleic acid complexation and, particularly the calix[4]arene-based ones, even for its protection and release. Thanks to the collaboration with Prof. Alan Lai and Ya-Jeng Chang at Academia Sinica in Taipei, Taiwan, preliminary investigation on cytokine expression revealed that some of these vectors also possess immunomodulatory properties, which might play an important role in the development of gene delivery systems.