Degree

Doctor of Philosophy (PhD)

Department

Cain Department of Chemical Engineering

Document Type

Dissertation

Abstract

The ubiquitin-proteasome system (UPS) is a biochemical pathway primarily responsible for the degradation of damaged proteins. The UPS is regulated by E3 ubiquitin ligases, which recognize specific degradation sequences, or degrons, on a protein leading to protein ubiquitination and subsequent degradation by the proteasome. Proteolysis targeting chimeras (PROTACs) have emerged as a novel therapeutic agent to selectively degrade ‘undruggable’ targets; however, many peptide-based PROTACs suffer low cell permeability and high protease susceptibility. The goal of this work is to utilize degron-based substrates that function as cell penetrating peptides (CPPs) in a PROTAC to selectively degrade a target. The first part of this study focuses on characterizing CPPs to further understand how their incorporation in PROTACs alters cellular uptake. D-chirality amino acids are not recognized by proteases, suggesting peptides containing non-natural amino acids will remain stable in cells. To explore this, a library of D-chirality peptides was synthesized which demonstrated enhanced stability and permeability compared to L-chirality peptides; however, it was found that their lifetime in cells was shortened due to cellular expulsions. CPPs are often used to transport various cargoes such as DNA, peptides, or proteins into cells. To study the effect of the addition of cargo, peptide sequences of varying length and charge were added to two CPPs. It was observed that net charge of the cargo, but not length, significantly affected uptake of the CPP/cargo complex. The second part of the study focuses on characterizing a library of peptide-based PROTACs. Previous work has identified long-lived, cell permeable peptides containing a β-hairpin motif which function as primary degrons. These degron/CPPs were combined with a known binding sequence for the protein Tau to create a PROTAC with increased stability compared to unstructured PROTACs. These PROTACs were capable of degrading Tau in a proteasome-dependent manner confirming the potential of incorporating a β-hairpin sequence motif into next generation PROTACs. Following this confirmation, a new class of PROTACs was designed to induce the degradation of PTP1B. Unfortunately, following the incorporation of linkers, the PROTACs appear to enhance PTP1B rather than degrade it, which merits the need for further study of this specific PROTAC.

Date

1-19-2023

Committee Chair

Melvin, Adam T.

DOI

10.31390/gradschool_dissertations.6043

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