Kinase Inhibitors

Volume 795 of the series Methods in Molecular Biology pp 109-118


Kinase Inhibitor Selectivity Profiling Using Differential Scanning Fluorimetry

  • Oleg FedorovAffiliated withStructural Genomics Consortium, Oxford University Email author 
  • , Frank H. NiesenAffiliated withStructural Genomics Consortium, Oxford University
  • , Stefan KnappAffiliated withStructural Genomics Consortium, Oxford University

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Fast, robust, and inexpensive screening methods are the heart of drug discovery processes. Moreover, it is useful to have access to several established assay formats, for validation purposes. If a targeted protein is an enzyme, the logical and widely used approach is the direct measurement of the effect of the added ligands on its activity. A variety of enzymatic assay formats have been successfully applied for inhibitor screening of protein kinases. However, enzymatic assays require an active enzyme with a known substrate and often time-consuming assay optimization. Several alternative approaches have been recently developed that detect binding of ligands to proteins. This chapter overviews and provides the experimental protocol of the successful application of differential scanning fluorimetry (DSF) in our laboratory for fast and robust screening of medium-sized (<10,000) inhibitor libraries. DSF monitors the thermal stabilization of the native protein structure upon ligand binding. It allows selectivity profiling of any protein kinase without prior knowledge of either substrate or activity of the kinase under investigation. Comparative studies revealed that generated data is highly reproducible and correlates well with the results from other ligand binding methodologies, direct binding constants as well as enzymatic assays.

Key words

Kinase Protein stability Inhibitor selectivity Compound screening Differential scanning fluorimetry