title: Application of a peptide-based assay to characterize inhibitors targeting protein kinases from yeast.
abstract: Chemical molecules that inhibit protein kinase activity are important tools to assess the functions of protein kinases in living cells. To develop, test and characterize novel inhibitors, a convenient and reproducible kinase assay is of importance. Here, we applied a biotinylated peptide-based method to assess adenosine triphosphate-competitive inhibitors that target the yeast kinases Hog1, Elm1 and Elm1-as. The peptide substrates contained 13 amino acids, encompassing the consensus sequence surrounding the phosphorylation site. To test whether the lack of distal sites affects inhibitor efficacy, we compared the peptide-based assay with an assay using full-length protein as substrate. Similar inhibitor efficiencies were obtained irrespective of whether peptide or full-length protein was used as kinase substrates. Thus, we demonstrate that the peptide substrates used previously (Dinér et al. in PLoS One 6(5):e20012, 2011) give accurate results compared with protein substrates. We also show that the peptide-based method is suitable for selectivity assays and for inhibitor screening. The use of biotinylated peptide substrates provides a simple and reliable assay for protein kinase inhibitor characterization. The utility of this approach is discussed.
authors.affiliation: Department of Chemistry and Molecular Biology, University of Gothenburg, 405 30, Göteborg, Sweden.
authors.forename: Jenny
authors.initials: J
authors.lastname: Veide Vilg
authors.forename: Sita
authors.initials: S
authors.lastname: Dahal
authors.forename: Thomas
authors.initials: T
authors.lastname: Ljungdahl
authors.forename: Morten
authors.initials: M
authors.lastname: Grøtli
authors.forename: Markus J
authors.initials: MJ
authors.lastname: Tamás
mesh_terms: D004305:Dose-Response Relationship, Drug; D020128:Inhibitory Concentration 50; D020928:Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D010455:Peptides / Q000378:metabolism*; D047428:Protein Kinase Inhibitors / Q000737:chemistry / Q000494:pharmacology*; D011494:Protein Kinases / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D054852:Small Molecule Libraries; D015003:Yeasts / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism*
chemical_list: D010455:Peptides; D047428:Protein Kinase Inhibitors; D029701:Saccharomyces cerevisiae Proteins; D054852:Small Molecule Libraries; D011494:Protein Kinases; C088589:HOG1 protein, S cerevisiae; D020928:Mitogen-Activated Protein Kinases