Development of a Small Molecule Regulated Cre Recombinase and A Chemical-Genetic Strategy for the Investigation of Kinase Non-Catalytic Function using Covalent Conformation-Selective Inhibitors

Abstract

Chapter 1: Development of a Small Molecule Regulated Cre Recombinase Our lab has previously developed a chemical genetic method for controlling signaling enzymes with a small molecule. In this method, the endogenous regulatory domains of an enzyme of interest are replaced with a protein-protein interaction that acts as an artificial regulatory domain. This synthetic enzyme can be controlled by a potent, selective and cell permeable small molecule that disrupts the protein-protein interaction that serves as the artificial regulatory domain. Cre (Causes Recombination) Recombinase is a site-specific tyrosine recombinase from bacteriophage P1. Cre is commonly used to facilitate conditional knock-in, knock-out and inversion studies is mammals8. This chapter describes the development of a Cre Recombinase whose activity can be controlled in cells with the small molecule-regulated switch that we previously reported. Our small molecule-regulated Cre Recombinase allows rapid, reversible, and dose-dependent activation of Cre recombinase and has the potential to be used to conditionally activate or inactivate gene expression in vivo. Applications of our small molecule-regulated Cre Recombinase and efforts to determine the mechanism of small molecule regulation are described. Chapter 2: A Chemical-Genetic Strategy for the Investigation of Kinase Non-Catalytic Function using Covalent Conformation-Selective Inhibitors Protein kinases are a large family of >530 signaling proteins that allow a cell to respond appropriately to a variety of external stimuli (Manning et. al. 2002). Most current research on protein kinases focuses on studying their catalytic activity, but recent evidence has shown that the non-catalytic role of kinases–including scaffolding and DNA binding–is essential to cell survival (Rauch et. al 2011). A major challenge in studying the non-catalytic roles of protein kinases is the lack of selective molecular tools for studying this aspect of kinase function. As a result, the non-catalytic functions of most proteins kinases are not well understood. A major focus of research in the field has been development of inhibitors that selectively bind distinct ATP-binding site conformations of kinases. Specifically, Type I inhibitors (Taylor et al, 2011), which bind catalytically active kinases, and type II inhibitors, which bind to catalytically inactive forms of the ATP-binding site (Ranjitkar et al, 2010; Ranjitkar et al, 2014; Seeliger et al, 2009; Okram et al, 2006). In many kinases, the conformation of the ATP-binding site and regulatory domains are allosterically coupled. Stabilizing different ATP-binding site forms with conformation-selective inhibitors has the potential to divergently modulate the non-catalytic roles of protein kinases in the cell (Rauch et al, 2011). The second half of this thesis describes work analyzing the divergent impacts of conformationally-selective inhibitors on the non-catalytic functions of Src Family Kinases (SFKs) in cells.