Structural studies on human and parasite enzymes related to disease

Macromolecular X-ray crystallography plays a key role in the drug discovery process, providing essential clues to explain the mechanism of action of protein targets and to elucidate the inhibitor binding mode. The detailed analysis of crystal structures is precious to conceive powerful inhibitors, through a rational structure-based drug discovery approach, that may lead to the development of new drugs. During my PhD, I had the opportunity to face two relevant topics related to human diseases, namely human and parasite Heat shock protein 90 (Hsp90) and human 14-3-3σ, and to study them through X-ray crystallography. In the first chapter, X-ray crystallography was exploited to characterize the role on nucleotide binding of both Arg97 in the N-terminal domain (NTD) of Leishmania braziliensis Hsp90 and of Lys112 in the human counterpart to find new determinants to selectively target the parasite enzyme. In details, we investigated the contribution of parasite Arg97 through the introduction of this residue, by site-directed mutagenesis, in the human protein generating the “leishmanized” variant of human Hsp90-NTD. The alanine variant was also generated to deeply understand the role of this non-conserved residue in human and parasite proteins. A variety of ADP and ATP analogues and cAMP were used to probe the role of these residues. According to our structural results residue Arg97 of Leishmania braziliensis Hsp90-NTD and Lys112 in the human protein are not crucial for substrate binding, making them not exploitable for the development of selective inhibitors targeting parasite Hsp90 over the human counterpart. In the second chapter, an X-ray crystallographic screening was performed on human 14-3- 3σ using small molecular weight compounds to identify new modulators of proteinprotein interactions (PPIs) involving this target. As a matter of fact, human 14-3-3σ protein is crucial for various cellular cascades, making it a target for the development of new anticancer and antileukemia drugs. The crystal structure of h14-3-3σ was solved in complex with pyridoxal phosphate (PLP) and inosine monophosphate (IMP). Notably, our structures revealed the fundamental contribution of the phosphate moiety present in both compounds for h14-3-3σ recognition and binding. The structural informationachieved will guide the rational design of new h14-3-3σ PPIs inhibitors based on these innovative scaffolds.