Synthesis of cyclic peptides as molecular probes inhibiting the formation of PEX5-PTS1 complex

The aim of the project is to obtain a cyclic peptide, ligand of the peroxisomal protein PEX5, as a molecular probe for the study of peroxisome biogenesis and function. Peroxisomes are unique organelles whose functions vary according to the type of cell and the stage of development of the organism. They play a very important role in cellular metabolism. The most important biochemical processes taking place in peroxisomes are β-oxidation of fatty acids, detoxification and protection against oxidative stress. In Trypanosoma parasites, the function of the peroxisome is played by the glycosome in which glycolysis takes place, the process of supplying the cell with ATP. For the proper functioning of peroxisomes, cytoplasmic enzymes are necessary, which have special signaling segments (PTS), directing them to the interior of the peroxisome through the receptors circulating in the cytoplasm (PEX5 and PEX7). Most peroxisomal enzymes contain either the first or the second type of targeting signal (PTS1 and PTS2). Our project aims to obtain a cyclic peptide that will competitively bind to PEX5, blocking the formation of the PTS1/PEX5 protein complex, and thus prevent the targeting of enzymes to the target organelle. The PTS1 stretch is a C-terminal peptide with the amino acid sequence Ser-Lys-Leu. The starting point for the design of the cyclic peptide was the analysis of the PTS1 motif binding pocket structure and the method of sequence binding by the PEX5 protein. The designed peptide should have high affinity for PEX5 and inhibit the formation of the PEX5/PTS1 complex. At the same time, this peptide has a cyclic structure (ring closure by a hydrophobic, aliphatic bridge), which should increase its metabolic stability and the ability to penetrate biological membranes.

Peptide synthesis was planned following standard Fmoc peptide synthesis procedures on a solid support (resin). The first stage of the project will be the synthesis of indispensable non-natural, commercially available amino acids: (S) -2 - ((tert-butoxycarbonyl) amino) -4,4-dimethylhex-5-enoate and N-5- (but-3-en-1- yl) -N-2- (tert-butoxycarbonyl) -L-glutamine.

The next stage of the research task consists in attaching the C-terminal amino acid to the 2-chlorotrityl resin and then extending the peptide chain.

The next step in the synthesis is the selective cleavage of the peptide from the resin while maintaining orthogonal groups protecting the side chains of amino acids. The last, key stage of the synthesis is cyclization by means of metathesis, reduction of the double bond and deprotection of peptide functional groups in the environment of trifluoroacetic acid.

The obtained cyclic peptide will be tested for the dissociation capacity of the PEX5/PTS1 complex (e.g. by the fluorescence polarization (FP) method). The results of the described scientific activity will allow to determine the validity of planning another project aimed at optimization of cyclic peptides, inhibitors of PEX5/PTS1 complex formation.

We assume that such compounds could be valuable molecular probes that will help better understand the PEX pathway and the mechanism of protein transport to peroxisomes. They may also become potential drugs against diseases caused by trypanosomes (Chagas disease, African sleeping sickness).