Author: Marina Buyanova

Publisher:

Published: 2021

Total Pages: 0

ISBN-13:

Modern drug discovery extends far beyond conventional small molecule drugs. Novel drug modalities, including proteins, peptides, and nucleic acids, hold enormous potential as therapeutic agents against currently undruggable targets. However, broad application of biomolecular modalities is limited by their inability to cross the cell membrane and engage intracellular targets. Cell-penetrating peptides (CPPs) are highly efficient delivery vehicles capable of transporting a wide range of membrane-impermeable cargos into the cell. My dissertation research is focused on the application of CPPs for the development of cell-permeable therapeutics and vectors for intracellular delivery of biological cargos. Chapter 2 aims to develop cyclic CPPs with improved cell-penetrating activity by optimizing the structure of CPP12, the most active CPP previously reported. Modification of the Nal residue yielded a significantly improved CPP12 analog, CPP12-2. The analog exhibited up to 4-fold higher cytosolic delivery efficiency than CPP12, especially at low CPP concentration. We identified that the improved cytosolic delivery efficiency is a result of a more efficient endosomal escape. CPP12-2 successfully delivered peptidyl cargo into mammalian cells and can be applied as an intracellular transporter for other biologically active molecules. Chapter 3 involves the development of novel biodegradable arginine-rich polypeptides for siRNA delivery into the cytosol of mammalian cells. Once inside the cell, siRNAs are able to specifically knockdown virtually any disease-relevant gene. We designed, synthesized, and evaluated several polypeptide vectors in intracellular siRNA delivery. The best-performing vector, CRC-CPP9 polymer, exhibited biodegradability and minimal toxicity and was moderately efficient in gene silencing. Although our efforts were not highly successful, they provided useful insights into the strategies to design other nucleic acid delivery systems. Chapter 4 focuses on the development of cell-permeable bicyclic peptide inhibitors against mutant KRas protein. Mutant KRas is a critical driver of tumorigenesis found in the deadliest cancers and is considered the “Holy Grail” of drug discovery. Starting from a previously identified ligand, we designed and screened a 2nd generation combinatorial peptide library. A medicinal chemistry optimization campaign resulted in the discovery of a lead compound 4-27 with improved binding affinity, cellular permeability, and proteolytical stability. The lead compound has demonstrated potent antitumoral activity both in vitro and in vivo. Peptide 4-27 is a promising therapeutic agent for the treatment of Ras-driven cancers.