Chemical modification and characterization of proteins and peptides for pharmaceutical and biomedical applications

Proteins and peptides have been recognized for having a wide variety of roles in human body. Thanks to their favorable features, once properly modified, they represent a good starting point for the design of therapeutics and diagnostics. The four research lines presented in this work can be inserted in this context. The first project is aimed at the development of an enzymatic treatment for hypoxanthine-guanine phosphoribosyltransferase (HPRT) deficiency-related overproduction of uric acid, as in the case of the Lesch†"Nyhan disease (LND) which is also characterized by neurological manifestations. We proposed an uricolytic treatment, exploting a triad of enzymes composed by recombinant Urate oxidase (Uox), (S)-5-hydroxyisourate (HIU) hydrolase (Urah) and (S)-2-oxo-4-hydroxy-4-carboxy-5-ureidoimidazoline (OHCU) decarboxylase (Urad), in a form suitable for prolonged administration. This enzymatic pathway is able to convert uric acid in its much more soluble metabolite (S)-allantoin in most mammals but not in hominoids, that lost these enzymes during evolution. Conjugation with the polymer polyethylene glycol (PEG), known to increase proteins circulating half-life and to decrease their immunogenicity, has been used. The three recombinant enzymes have shown to maintain an acceptable degree of activity after PEGylation and lyophilization, granting long term storage of the formulation. A combination of the three enzymes, in appropriate ratios, demonstrated to effectively convert uric acid to (S)-allantoin in vitro, posing the basis for future tests in a mouse model of disease. Conjugation with PEG was also carried out in a second project aimed at the development of haemoglobin-based oxygen carriers (HBOCs) in which the genetic manipulation of human haemoglobin (Hb) has been combined with its surface modification. Hb mutants, produced in recombinant form by collaborators at the University of Essex, have been engineered by adding cysteines on alfa and beta surface chains obtaining A12 and A13 Hb mutants, respectively. The genetically added cysteines have been used for the direct attachment of PEG molecules obtaining homogeneous products in terms of derivatization. Interestingly, the oxygen binding profile of Hb mutants remains almost the same after PEGylation. These preliminary data might demonstrate that further genetic manipulation of Hb, for example to avoid autooxidation or NO scavenging, should lead to mutants in which PEGylation will not affect the oxygen binding properties. In the last two research lines synthetic peptides, obtained by solid phase peptide synthesis, have been used for therapeutic and diagnostic purpose. The third project aimed at the development of a biosensing device consisting of three elements: an aptamer sequence, a nanostructured support, and a peptide labeled with a solvatochromic dye. The idea is to immobilize, on the nanostructure support, aptamers hybridized with labeled-peptides, that can be displaced in the presence of S. aureus inducing a colorimetric switch in the visible region. Within this multicenter project, we i) characterized the spectroscopic properties of the synthesized polar-sensitive dyes; the best candidate MediaChrom 15c was chosen and better characterized, more generally, for biological applications; ii) characterized, using circular dichroism and fluorescence spectroscopy, the interaction between the selected aptamer(s) and the peptides that were designed and synthesized. The resulting construct has been tested, with positive results, for its capability to give a naked-eyes detectable signal as a consequence of the interaction with the aptamer. In the last work, the Leucine Zipper motif of c-Maf transcription factor was obtained by solid phase peptide synthesis (University of Milan) and characterized in terms of structural features and dimerization properties. The results highlighted the tight coupling between dimerization and folding, adding new insights in the poorly characterized Maf proteins family. Potential peptide inhibitors of c-Maf dimerization were tested in vitro for their effect on dimerization of the isolated LZ fragment. Some of these compounds proved to bind c-Maf LZ, promoting a disordered-to-structured transition through an increase in helical content and inhibiting c-Maf LZ dimerization. Our results showed that solid phase peptide synthesis of protein domains and analysis of secondary and quaternary structure by CD and MALDI TOF techniques allow to build up a simple experimental platform to test LZ dimerization inhibitors. Rationally designed peptide inhibitors provide a promising lead for further development of more selective, stable and bioavailable peptidic or peptidomimetic drugs targeting diseases whose molecular basis relies on protein†"protein recognition, as is the case, e.g., of multiple myeloma.