Characterization and Exploitation of Bidirectional Allosteric Coupling in Multi-Domain Tyrosine Kinases using Conformation-Selective ATP-Competitive Inhibitors

Abstract

Protein kinases are a large family of enzymes that play integral roles in cell signaling networks and are thus critical for effecting appropriate cellular responses to environmental stimuli. Much of kinase biological function has been studied in terms of catalytic activity: phosphorylation of substrate proteins as part of signaling cascades. However, recent evidence has shown that kinases play many important non-catalytic functions such as DNA-binding, scaffolding, and participating in a variety of physiologically relevant protein-protein interactions. While critical, these roles have not been thoroughly explored, in large part due to limited availability of selective ATP-competitive inhibitors. Selectivity for a specific kinase is difficult to achieve due to high structural homology between the ATP-binding sites of the 518 human kinases. Additionally, it has been shown over the past two decades that it is possible to stabilize structurally distinct ATP-binding site conformations using conformation-selective inhibitors, termed Type I and Type II inhibitors, in many kinases. In several cases, inhibition of kinase ATP-binding sites with Type I or Type II inhibitors has been shown to divergently affect cell signaling events as a result of allosteric coupling between important structural features in the ATP-binding site and distal protein-protein interaction sites on the inhibited kinase. Thus, it is important not only to build selective ATP-competitive inhibitors but to understand how their binding affects global kinase conformation through allosteric coupling. This thesis describes my work characterizing allosteric networks in multi-domain tyrosine kinases (Src-Family Kinases (SFKs) and Abl) using conformation-selective inhibitors as well as developing a method for using conformation-selective inhibitors in cells to better understand how non-catalytic function of a kinase of interest determines its role in cell signaling networks.