סמינר בכימיה אורגנית: Chemical protein synthesis as a tool for therapeutic applications: The development of mirror-image RaPID technology & New insulin analogues

Abstract:

Random nonstandard Peptides Integrated Discovery (RaPID) system is one of the most powerful methods for the selection of de novo macrocyclic peptide binders for proteins of interest by using a combination of flexible in vitro translation system (FIT) and mRNA display technology. Here, we show the development of mirror-image RaPID technology (Fig. 1) for the discovery of innate protease-resistant macrocyclic peptides that specifically bind to and inhibit Matrilysin (MMP7).

MMP7 plays a crucial role in cancer metastasis and progression, making it an attractive target for therapeutic development. 

However, the development of potent and selective MMP7 inhibitors is challenging due to the conservation of active site across various MMPs. We started by developing an approach for the chemical synthesis of the catalytic domain of MMP7, and upon optimization, we were able to synthesize both biotinylated L- and D-MMP7 in milligram quantities, which where both used in the RaPID system. One of the identified macrocyclic peptides against biotinylated D-MMP7, termed D20, was synthesized in its mirror-image form, D’20, consisting of twelve D-amino acids, one cyclic b-amino acid and a thioether bond. Notably, D’20 potently inhibited the human MMP7 with IC50 of 90 nM, and showed selectivity over other MMPs tested in this study. Moreover, D’20 inhibited the migration of pancreatic cell line CFPAC-1 while having no effect on the cell proliferation and viability. Additionally, D’20 exhibited excellent stability in human serum,  as well as in the simulated gastric and intestinal fluids. This study highlights that the mirrorimage RaPID technology can be a powerful tool to develop in vivo stable macrocyclic peptides for therapeutic applications.

Furthermore, I will discuss our latest results for the preparation of new analogues of insulin, in which a disulfide 6-11 in chain A of insulin was replaced with diselenide, allowing us to prepare Se-insulin analogues in high yield. Further, we found that these insulins were more stable at room temperature than wt-insulin, and more resistant to fibrillation. These studies bring us closer to make more stable insulin analogues for diabetes melilotus.