Structure Elucidation of Medically Important Proteins by X-ray Crystallography

Since its conception over a century ago, X-ray crystallography (XRC) has become the most successful method used to elucidate the structures and functions of biological molecules at atomic resolution. The extensive use of XRC has led to meaningful discoveries across many scientific fields, notably its contributions to rational drug design. Traditional drug discovery relies on the use of trial-and-error based approaches in cellular and animal models of disease to identify chemical probes that elicit desirable therapeutic effects based off changes in phenotype. However, this approach lacks critical information in regards to the biological target in which the compound interacts with. In contrast, the use of rational drug design presents the opportunity to identify chemical probes that target specific protein targets of known medical importance and study their interactions using three dimensional structures that can be used to suggest new drug candidates. The main focus of my research presented in this dissertation aims to utilize XRC to discover novel therapeutics. In this work, I begin by describing the use of structure-based drug discovery for the rational design of hydrocarbon-stapled peptides that block Focal Adhesion Kinase (FAK) scaffolding in cancer (Chapter 2). FAK is an intracellular tyrosine kinase that has been linked to many cancers through its interaction with Paxillin LD motifs as it relates to tumor growth, invasion, metastasis, and suppression of apoptosis. The results of this study demonstrate the effectiveness hydrocarbon-stapling has on the native Paxillin LD2 sequence with ~50 fold greater binding affinity by surface plasmon resonance (SPR) that can be explained by the unique structural interactions observed by XRC. Next, I present a series of methods which lays the foundations for the discovery of novel anti-bacterial drugs that target 3-Deoxy-D-manno-octulosonate-8-phosphate (KDO8P) Synthase, a critical enzyme in the biosynthesis of gram-negative lipopolysaccharides (Chapter 3).