Divergent modulation of Src-family kinase regulatory interactions with ATP-competitive inhibitors.

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.

Active site profiling reveals coupling between domains in SRC-family kinases.

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 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 have a distinct preference for the autoinhibited forms of these kinases. Furthermore, we show that ligands that have 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.

Affinity-based probes based on type II kinase inhibitors.

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.

Protein kinase affinity reagents based on a 5-aminoindazole scaffold.

Affinity reagents that target protein kinases are powerful tools for signal transduction research. Here, we describe a general set of kinase ligands based on a 5-aminoindazole scaffold. This scaffold can readily be derivatized with diverse binding elements and immobilized analogs allow selective enrichment of protein kinases from complex mixtures.

Biochemical mechanisms of resistance to small-molecule protein kinase inhibitors.

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 past 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.

Chemical genomic and proteomic methods for determining kinase inhibitor selectivity.

The clinical success of the Bcr-Abl tyrosine kinase inhibitor Gleevec((R)) and the recent clinical approval of a number of small molecule drugs that target protein kinases have intensified the search for novel protein kinase inhibitors. Since most small molecule kinase inhibitors target the highly conserved ATP-binding pocket of this enzyme family, the target selectivity of these molecules is a major concern. Due to the large size of the human kinome, it is a formidable challenge to determine the absolute specificity of a given protein kinase inhibitor, but recent technological developments have made substantial progress in achieving this goal. This review summarizes some of the most recent experimental techniques that have been developed for the determination of protein kinase inhibitor selectivity. Special emphasis is placed on the results of these screens and the general insights that they provide into kinase inhibitor target selectivity.