Synthesis of L-alanyl-pyrrolidine based prolyl oligopeptidase ligands with five-membered heteroaromatics at the 2-position on the pyrrolidine ring

Prolyl oligopeptidase (PREP) is a serine protease that is widely found throughout the human body and especially in the brain. The primary function of PREP is thought to be the hydrolysis of the carboxyl side bond of proline residues in oligopeptides. PREP is also shown to increase the dimerization and aggregation of α-synuclein and downregulate the protein phosphatase 2A mediated autophagy in the cell via direct protein-protein interactions (PPI). The accumulation of α-synuclein aggregates in cell is known to cause α-synucleinopathies such as Parkinson’s disease. This makes the PPIs of PREP an attractive target for drug research. The mechanisms of the PPIs of PREP are still poorly understood. Recent studies have shown that these PPIs can be modulated with ligands lacking high inhibitory activity for the proteolytic activity. These studies show that the IC50-value of the ligand does not correlate with ligands ability to affect the PPIs of PREP. Ligands that could selectively modulate the PPIs of PREP without inhibiting the proteolytic activity of PREP could give valuable information on the mechanisms of the PPIs and on how to modulate them. It is hypothesized that the ligands could bind to PREP at a site that does not interfere with its proteolytic activity, and ligand binding is assumed to restrict the dynamic structure of PREP and thereby also modulating the PPIs of PREP. The aim of this study was to synthetize novel peptidic PREP ligands and study their effects on the proteolytic activity of PREP and the PPIs of PREP. The aim was to find and identify ligands and structures that would modulate the PPIs of PREP and observe how the IC50-values of the ligands would correlate. L-Alanyl-pyrrolidine was selected as the scaffold for the compound series and the five-membered heteroaromatics, imidazole, triazole and tetrazole, were added to the 2-position of the pyrrolidine ring. In this position there is an electrophilic group in many PREP inhibitors, although these heteroaromatics are not electrophiles. The scaffold was also expanded by adding phenyalkyl groups with different linker lengths were added to the N-terminal side of the alanine. The ligands were synthesized using four synthesis routes which were based on synthesis methods found in literature. The IC50-values and the effects on α-synuclein dimerization and autophagic flux were determined for five synthetized compounds. The tested compounds were all weak PREP inhibitors and showed no strong activity in the α-synuclein dimerization and autophagy assays. Despite the weak activities in the assays, the importance of the linker length in the phenyalkyl group was shown. Changing the linker by one methylene group had noticeable effect on the overall activity. The results also demonstrate a lack of correlation between the IC50-values and the effects on α-synuclein dimerization and autophagic flux, which further confirms the lack of correlation between the proteolytic function and the PPIs of PREP which was observed also in previous studies.