Summary Protein kinases are key regulators of cellular signal transduction with crucial roles for healthy cell biology and pathogenesis. Accordingly many drugs have been developed to address the dysregulation of these enzymes, notably in oncology. Around 50 small molecule kinase inhibitors have already been approved, and hundreds of such drugs are the object of clinical investigation to combat these disease drivers with targeted therapeutic approaches. Due to the conserved structure of the kinase domain across the kinome, compounds may not only act on their intended targets but may also bind and inhibit other proteins. Thus, many kinase inhibitors display a promiscuous target profile that makes selectivity profiling indispensable in order to understand the positive and negative actions of the drugs regardless of their use as chemical probes or therapeutic agents. Chemical proteomics has developed into a powerful technique for investigating drug–protein interactions. One successful example is the Kinobeads technology that features broadly selective small molecule kinase inhibitors immobilized on sepharose beads for affinity enrichment of native kinases out of any tissue or cell lysates. Target deconvolution of kinase inhibitors is achieved through a competitive set-up in which the lysate is treated with increasing concentrations of an inhibitor of interest before affinity enrichment with the Kinobeads matrix. This results in a competition between the Kinobeads and the compound for the active sites of all targets present in the lysate. For each engaged target, reduced binding to Kinobeads ensues, leading to dose-dependent intensity reduction of the target peptides in the mass spectrometric read-out. Analysis of the many dose–response curves obtained helps to establish the target space of a molecule. In this chapter we describe the principle, experimental details, and applications of the Kinobeads technology. We first draw the reader's attention to the theoretical background and important points to consider when developing a chemical proteomics profiling assay. We then disclose our latest standard operating procedure to achieve a successful Kinobeads profiling assay. Next, we summarize representative application examples of the Kinobeads in different stages of the drug discovery pipeline for human and non-human proteomes. Finally we propose a perspective of the future opportunities of the Kinobeads approach in drug discovery.     «

Summary Protein kinases are key regulators of cellular signal transduction with crucial roles for healthy cell biology and pathogenesis. Accordingly many drugs have been developed to address the dysregulation of these enzymes, notably in oncology. Around 50 small molecule kinase inhibitors have already been approved, and hundreds of such drugs are the object of clinical investigation to combat these disease drivers with targeted therapeutic approaches. Due to the conserved structure of the kinas...     »