The Potential of Peptide Chemistry: from Small Molecules up to Innovative Protein Engineering

This thesis is composed of two sections. PART I Design, Synthesis and Functionalization of Cyclic Peptides Targeting Chemokine Receptor 4 In the chemokine receptor subfamily, CXCR4 dictates directional cell migration during routine immune surveillance and activates leukocytes for the inflammatory response, towards gradients of the CXCL12 ligand. Although CXCL12/CXCR4 axis evolved to benefit the host, inappropriate regulation or expression of these proteins is involved in pathological conditions, including infection, chronic inflammatory diseases, atherosclerosis, and more than 20 different human cancers. Notably, overexpression of CXCR4 has been associated with increased metastatic potential and poor prognosis in many solid tumors. CXCR4/CXCL12 pairing plays a central role in Paget’s hypothesis of “seed-and-soil”, and thus targeting the pathway is not only a logic therapeutic approach but also a strategy in blocking cancer progression or malignant transformation. There is, therefore, an impetus for non-invasive means to sensitively identify tumours prone to progression. As such, the aim of this work was to develop a number of novel CXCR4 antagonists to assess their suitability as imaging radiotracers for positron emission tomography (PET). As part of our ongoing efforts in identifying new CXCR4 antagonists, a CXCL12-derived small cyclic peptide, which selectively interacted with CXCR4, was identified. Although the sequence possessed an interesting in vitro and in vivo pharmacological profile, it suffered from degradation in biological fluids and its IC50 was in the low micromolar range. Herein, to enhance both the CXCR4 affinity and metabolic stability, I embarked on a lead optimization campaign. Efforts to elucidate the necessary residues for the interaction with the receptor and the critical stereocenters of mine sequences systematically included tools such as alanine scanning and D-aminoacid scanning. Next, the overall, dynamic and topographical requirements and electronic properties of the cyclic region of the obtained compounds were investigated. Inspired by the promising results, the newly generated potent, selective and functional antagonist has been tailored as a target-specific CXCR4 imaging probe. Exploring the flexibility of the receptor binding pocket, a study of the optimal site of attachment and an optimization of suitable linker units was carried on. To meet the clinical need for a highly specific and sensitive tool for the chemokine receptor 4 assessment and quantification in vivo, a CXCR4 targeted nuclear probe has been developed. Despite the additional moieties, the final compound showed high affinity to the chemokine CXCR4 receptor and the collected results suggest that the natGa is a promising agent for preclinical non-invasive PET imaging. PART II Rescue of C-terminal Destabilized Proteins by Use of D-Amino Acids In eukaryotic cells, the ubiquitin–proteasome pathway acutely regulates the protein turnover. The covalent attachment of ubiquitin to lysine residues of targeted proteins is a signal for the recognition by the proteasome. However, it’s been recently discovered that efficient proteasomal proteolysis requires an additional intrinsically disordered region at which the enzyme complex engages the substrate and initiates degradation. On the other hand, although biomolecules composed of mirror image amino acids are of particular interest for their non immunogenic properties, the reason of their unique biological stability remains relatively unexplored. The main challenge with probing the stability of proteins containing non-natural functionalities is the delivery of such proteins into the cytosol. Inspired by nature, researchers exploited the ability of toxins to spread through the body and started to investigate the sophisticated bacterial A-B toxin-mediated delivery nanomachines. Working as a macromolecular syringe, the pathogen injects his toxins into the cytoplasm of host cells in a directly and selectively way. Thus, I recruited the disarmed version of Bacillus anthracis toxin, the versatile LFN/PA platform, for efficiently deliver chemical entities such as mirror image polypeptides across the bilayer phospholipid membrane. I used the catalytic domain of sortase A from Staphylococcus aureus (SrtA) as a “molecular stapler”: this enzyme-mediated tailoring has allowed for the facile and specific fixing of protein-peptide fusions, safeguarding their functionality. Further, a standardized and rigorous western blot workflow has been drawn up to provide highly sensitive, consistent, reproducible and as quantitative as possible data of the cytosolic delivered fraction of chimera proteins. Thus, I related the rate of proteasomal degradation of proteins containing mixed chirality peptides. It has been shown that L-sequences, acting as an unstructured region, were able to achieve lower steady state concentrations when compared to D-cargos, which rescued the protein from the proteasomal-mediated degradation, suggesting that stereospecific interactions are required for the accessibility to the narrow catalytic pore. It was also found that the C-terminus group plays a role in the degradation pathway: peptides presenting a –CONH2 as a C-termini are less stable compared to those with the – COOH. Protein quantification via Western blotting allowed us to detect even this feature, while using an SRM-based quantification we wouldn’t be able to ascertain such a small difference in the molecular weight of the targeted proteins. With the goal of understanding complex cell biological processes in the detailed quantitative manner, Anthrax toxin system has been successfully leveraged to deliver mixed chirality peptides into the cytosol. The resulted information could be used to discover novel regulatory principles and to develop strategies to interfere with cellular processes therapeutically or to engineer cellular metabolis