D'Aria, Federica (2020) Calorimetric and spectroscopic investigation of biomolecules for therapeutic applications. [Tesi di dottorato]

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Abstract

Molecular recognition is the key for all biological processes. Such phenomena can be either intermolecular, as for the biding of a ligand to a macromolecule, or intramolecular, as for the denaturation of proteins or nucleic acids. In recent decades, DNA-ligand, protein-ligand or protein-DNA interactions have been the subject of numerous physico-chemical studies. The understanding of the energetics both of biomolecules stability and of their binding with other (bio)molecules is extremely interesting in biochemistry, biotechnology, and especially in the pharmaceutical field for a targeted structure-based drug design. Calorimetric and spectroscopic methodologies, combined to computational studies and biological assays, are essential for drug discovery. Indeed, thermodynamic stability of macromolecules as well as the energetics of their interaction with potential drugs are an essential complement to structural data for the optimization of lead compounds. Specifically, in this Ph.D. thesis, studies have been addressed to investigate:  Physico-chemical factors affecting drug bioavailability (Chapter 3).  Thermodynamic stability of G-quadruplexes (G4s) in oncogene promoters and their interactions with ligands (Chapter 4).  Effects of epigenetic modifications on G4s stability (Chapter 5). Chapter 3 describes how the combination of the appropriate pH, solvent, temperature, and mixing time can improve the complexation between quercetin, a natural compound characterized by interesting pharmacological activities, and hydroxypropyl--cyclodextrin, a commonly used drug carrier. Chapter 4 focuses on the energetics of the interaction between KRAS proto-oncogene G4 and new putative anticancer drugs. This study led to the identification of a series of molecules able to selectively interact with G4s and characterized by cytotoxic activity on cancer cell lines. Finally, in Chapter 5, it was investigated, through mass spectrometry experiments, the capability of two modified sequences of KIT proto-oncogene, containing 5-methylcytosine and 5-carboxylcytosine, to fold into G4. The results proved that, despite the epigenetic modifications, these sequences were able to fold into G4, even though with a slower kinetics, as the unmodified sequence.

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