Chemical Proteomic Tools for Studying Protein Kinase Active Sites

Abstract

Protein kinases constitute one of the largest protein families in humans. These enzymes catalyze phosphorylation of serine, threonine or tyrosine residues in their protein substrates. As protein kinases regulate most signal transduction pathways in cells and play important roles in many cellular functions, deregulation of their activity can lead to a number of diseases including cancer, diabetes and inflammation. Targeted inhibition of protein kinases has therefore become an attractive therapeutic strategy for the treatment of a number of diseases. Small molecule inhibitors that target the active site of protein kinases can be used to study their catalytic function and regulation. We have designed and generated a set of small molecule ligands that bind to protein kinase active sites in a conformation–specific manner. These ligands have been used as proteomic tools to study the active sites of a wide range of protein kinases. These efforts have been particularly focused on a class of ligands, type II inhibitors, which stabilize an inactive conformation of the ATP–binding site, called the DFG–out. We have shown that type II inhibitors can be extensively modified and that these reagents can be used in a range of proteomic applications. These proteomic efforts have provided insight into the roles of specific kinases during signaling events. Furthermore, these chemical tools have significantly contributed towards our understanding of the structure and regulation of protein kinases.