Synthesis and interactions of sulfated C5a-receptor and CHIPS mimics

Abstract

Post-translational modifications are modifications of peptides or proteins after they have been synthesized in the ribosomes. These post-translational modifications (PTM’s) are intended to augment functional diversity of the 20 natural L-amino acids of which proteins are constructed. PTM’s have important functions and most are crucial for the tasks of the modified proteins. Sulfation was recognized as a post-translational modification of proteins in 1954 and it was slowly realized that tyrosine sulfation is a widespread modification of excreted and membrane bound peptides and proteins. Sulfation is catalyzed by two membrane bound tyrosylprotein sulfotransferases (TPST) located in the trans Golgi, which use PAPS as universal sulfate donor. Although sulfation is believed to be a widespread and biologically very important PTM, it has not been studied in great detail. This is mainly because of the instability of the sulfate group, especially under acidic conditions. In this thesis a new sequence independent synthesis method for the site selective introduction of multiple sulfated tyrosine residues into bioactive peptides is described. This method is applied on the synthesis of sulfated Leu-enkephalin and a collection of site selective sulfated N-terminal fragments of the Complement factor 5b Receptor (C5aR). This N-terminal part of the C5aR forms the binding site of the Chemotaxis Inhibitory Proteins of Staphylococcus aureus (CHIPS), an immunomodulatory protein excreted by S. aureus to prevent the C5aR from being activated by its natural ligand C5a. By preventing the activation of the C5aR, CHIPS can prevent the recruitment of specific white blood cells to the site of infection. The (over)activation of the C5a-receptor has been linked to several serious inflammatory diseases and therefore CHIPS and mimics of CHIPS could form a very interesting new class of anti-inflammatory compounds. The synthesized sulfated fragments of the C5aR are used to study the binding of CHIPS to the C5a-Receptor by ITC and NMR spectroscopy. The structure of CHIPS in complex with the N-terminus of the C5aR is elucidated, which reveals crucial structural elements within CHIPS for tight binding to the C5aR. These structural elements are selected and combined to design and synthesize new classes of C5aR inhibitors. One class based on the versatile TAC-scaffold were successfully synthesized but appeared to show no detectable affinity towards the sulfated C5aR fragments. A promising class of CHIPS mimics, based on a shortened CHIPS fragment including the fragments which interact with the C5aR, showed a low micromolar affinity towards the sulfated N-terminal part of the C5aR. The designed mimic CHOPS is a promising lead structure for development of new anti-inflammatory drug candidates.