Protein kinases, key regulators of intracellular signal transduction, have emerged as an important class of drug targets. Chemical proteomic tools that facilitate the functional interrogation of protein kinase active sites are powerful reagents for studying the regulation of this large enzyme family and for performing inhibitor selectivity screens. Here we describe a new crosslinking strategy that enables rapid and quantitative profiling of protein kinase active sites in lysates and live cells. Applying this methodology to the SRC-family kinases (SFKs) SRC and HCK led to the identification of a series of conformation-specific, ATP-competitive inhibitors that display a distinct preference for autoinhibited forms of these kinases. Furthermore, we show that ligands that demonstrate this selectivity are able to modulate the ability of the regulatory domains of SRC and HCK to engage in intermolecular binding interactions. These studies provide insight into the regulation of this important family of tyrosine kinases.

Multidomain protein kinases, central controllers of signal transduction, use regulatory domains to modulate catalytic activity in a complex cellular environment. Additionally, these domains regulate noncatalytic functions, including cellular localization and protein–protein interactions. Src-family kinases (SFKs) are promising therapeutic targets for a number of diseases and are an excellent model for studying the regulation of multidomain kinases. Here, we demonstrate that the regulatory domains of the SFKs Src and Hck are divergently affected by ligands that stabilize two distinct inactive ATP-binding site conformations. Conformation-selective, ATP-competitive inhibitors differentially modulate the ability of the SH3 and SH2 domains of Src and Hck to engage in intermolecular interactions and the ability of the kinase–inhibitor complex to undergo post-translational modification by effector enzymes. This surprising divergence in regulatory domain behavior by two classes of inhibitors that each stabilize inactive ATP-binding site conformations is found to occur through perturbation or stabilization of the αC helix. These studies provide insight into how conformation-selective, ATP-competitive inhibitors can be designed to modulate domain interactions and post-translational modifications distal to the ATP-binding site of kinases.

Protein kinases have emerged as one of the most frequently targeted families of proteins in drug discovery. While the development of small-molecule inhibitors that have the potency and selectivity necessary to be effective cancer drugs is still a formidable challenge, there have been several notable successes in this area over the last decade. However, in the course of the clinical use of these inhibitors, it has become apparent that drug resistance is a recurring problem. Because kinase inhibitors act by targeting a specific kinase or set of kinases, there is a strong selective pressure for the development of mutations that hinder drug binding but preserve the catalytic activity of these enzymes. To date, resistance mutations to clinically-approved kinase inhibitors have been identified in a number of kinases. This review will highlight recent work that has been performed to understand how mutations in the kinase catalytic domain confer drug resistance. In addition, recent experimental efforts to predict potential sites of clinical drug resistance will be discussed. KeywordsDFG motif; Gatekeeper residue; Hydrogen bond; IC 50 ; Imatinib; K d ; P-loop; Protein kinase Reversible protein phosphorylation cascades represent a central theme in cellular signal transduction. Protein kinases are the single family of enzymes that catalyze the transfer of the γ-phosphate group from adenosine 5'-triphosphate (ATP) to a target protein, and thus are key regulators of these phosphorylation pathways (1). Due to the central role that these enzymes play in cellular behavior, it is not surprising that misregulated protein kinase activity contributes to a number of diseases including cancer, inflammation and diabetes (2). Currently, there are dozens of small-molecule protein kinase inhibitors undergoing clinical evaluation, with eleven approved for clinical use (3,4).The catalytic domains of protein kinases are bi-lobal with a smaller N-terminal lobe comprised mainly of β-strands and a larger α-helical C-terminal lobe (Figure 1, top left panel) (5,6). These lobes are joined by a segment known as the hinge region, which outlines a narrow hydrophobic cleft where ATP binds. The adenine ring of ATP makes key hydrogen-bonding contacts with the amide backbone of the hinge region. The α-and β-phosphate groups of ATP are aligned for catalysis via an interaction with a divalent magnesium ion and a conserved catalytic lysine (7,8). Protein substrates bind in an extended conformation along a shallow groove on the Clobe, which allows the residue that will be phosphorylated to accept the γ-phosphate of ATP. Adjacent to the ATP-binding cleft is a 20-30 residue long activation loop that increases the catalytic activity of most kinases when phosphorylated (9). The activation loop contains the highly conserved Asp-Phe-Gly (DFG) motif, the conformation of which is directly coupled to * Corresponding author. Department of Chemistry, University of Washington, Seattle, WA, 98195, Phone: 206-543-1653, maly@chem.washington.edu. NIH Public Access...

Protein kinases are key components of most mammalian signal transduction networks and are therapeutically relevant drug targets. Efforts to study protein kinase function would benefit from new technologies that are able to profile kinases in complex proteomes. Here, we describe active site-directed probes for profiling kinases in whole cell extracts and live cells. These probes contain general ligands that stabilize a specific inactive conformation of the ATP-binding sites of protein kinases, as well as trifluoromethylphenyl diazirine and alkyne moieties that allow covalent modification and enrichment of kinases, respectively. A diverse group of serine/threonine and tyrosine kinases were identified as specific targets of these probes in whole cell extracts. In addition, a number of kinase targets were selectively labeled in live cells. Our chemical proteomics approach should be valuable for interrogating protein kinase active sites in physiologically relevant environments.