Resumen de Synthesis of biaryl bicyclic peptides for recognition of protein surfaces
The present thesis is based on the development and optimization of a solid-phase strategy to prepare biaryl bicyclic pentapeptides. Suzuki-Miyaura cross-coupling reactions were used to obtain the biaryl bridge. The metholodogy was compatible with the introduction of amino acids with functional groups in their side-chains (lysine, arginine and serine).
The bicyclic pentapeptides were considered privileged structures regarding the evaluated properties. The introduction of the biaryl stapled enhanced passive diffusion permeability across the blood-brain barrier (BBB). Furthermore, protease resistance in human serum was also improved due to the presence of the biaryl. Moreover, no cytotoxicity was observed in HeLa cells at 500µM concentration.
The developed methodology was expanded to a new kind of compounds. While the compounds synthesized in chapter 1 displayed a phe-phe stapled, the peptides prepared in chapter 3, presented the stapling between two tryptophans (trp-trp). Permeability studies demonstrated that the nature of the stapled was critical to improve the passive transport across the BBB. Cyclic peptides with bromine at different positions (5, 6 or 7) of the indole group of tryptophan were also synthesized. These compounds showed high passive permeability across the BBB. High cell survival rates in HeLa cells were observed for these peptides, except for the analog with bromine at position 5 of the indole.
We aimed to evaluate the recovery of the self-assembly of a mutated p53 protein with some of the previously described peptides.
Protein p53 is known as the “genome guardian”. The alteration of the balance of p53 pathway is mainly related with cancer. We were interested in the region that controls the proper folding of the protein, named the tetramerization domain. This domain is a dimer of dimer that contains 37 residues. The most relevant point mutation in this region is R337H. This mutant is mainly associated with the atypically frequent cases of pediatric adrenocortical carcinoma (ACC) in southern Brazil. Moreover, it is also associated with high prevalence of breast cancer in women from this region.
The native p53TD protein, as well, as the R337H mutant were synthesized and characterized by circular dichroism (CD). The tetramer formed by R337H mutant was unstable, especially at higher pH. In order to stabilize the folding of this mutant, we used two different peptides prepared during the synthesis as ligands for this protein. Previous studies in our group demonstrated that calixarenes displaying guanidinium groups were able to recover the self-assembly of R337H mutant. Therefore, the selected peptides were from the arginine family. The bicyclic peptide, cyclo(Arg-(4&)Phe-Arg-(4&)Phe-D-Pro), was used as ligand of the protein R337H mutant. Unfolding experiments by CD did not show a relevant stabilization of the tetramer formed by the mutant in the presence of this ligand. Nevertheless, a more promising result was obtained for the linear stapled peptide, H-Arg-(4&)Phe-Arg-(4&)Phe-D-Pro-OH. The thermal stability of the protein R337H mutant in the presence of the linear stapled ligand was increased, transition temperature was 7ºC higher. Therefore, we could find an interesting application of one of these peptides synthesized by our previously optimized methodology.
