Targeting Pathological Protein Interactions in Drug Discovery

Abstract

The interaction of proteins with themselves or other molecules is essential to biological function. The dysregulation of normal protein structure, folding, and interactions forms the basis of many human diseases. Elucidating the protein interactions which underlie various disease states is crucial to understanding disease progression and identifying protein targets for therapeutic development. This dissertation focuses on targeting pathological protein interactions in cancer, non-alcoholic fatty liver disease (NAFLD), and neurodegenerative disorders. The tools developed in this dissertation are not only relevant to these specific disease states, but can be modified to pursue novel protein targets for drug discovery in other diseases as well. Chapter 1 of this dissertation provides an explanation of the scientific background and previous research that has motivated the current work. Chapter 2 presents the first high-throughput screening (HTS) platform that detects structural changes in death receptor 5 (DR5), a membrane protein which regulates apoptosis. In this chapter we have identified small-molecules which modulate DR5 signaling and sensitize TRAIL-resistant cancer cells to TRAIL-induced apoptosis. In chapter 3, the same HTS platform is used to discover novel inhibitors of DR5-mediated TRAIL-induced apoptosis for therapeutic intervention of NAFLD and Alzheimer’s Disease. Chapter 4 provides a summary of various projects which involved research on alpha-synuclein (α-syn), the primary protein involved in Parkinson’s Disease. In chapter 4.2, yeast display is implemented as a screening platform to identify inhibitors of α-syn uptake via targeting neurexin-1β. Chapter 4.3 provides an in-depth protocol on the purification of α-syn for laboratory studies. Chapter 4.4 summarizes an attempt to create a cell-free FRET-based biosensor to target the pathological aggregation of α-syn. Chapter 4.5 details a protocol for producing dopamine-modified α-syn oligomers for laboratory studies on dopamine-induced α-syn toxicity. Finally, chapter 4.6 identifies threonine 75 in α-syn as a critical amino acid for fibrillization. This work has overall contributed to the field of drug discovery by providing novel high-throughput screening tools to study DR5 and other protein-protein interactions, and has further elucidated important aspects of α-syn aggregation to be targeted in Parkinson’s Disease.