title: Detection of allosteric kinase inhibitors by displacement of active site probes.
abstract: Non-adenosine triphosphate (ATP) competitive, allosteric inhibitors provide a promising avenue to develop highly selective small-molecule kinase inhibitors. Although this class of compounds is growing, detection of such inhibitors can be challenging as standard kinase activity assays preferentially detect compounds that bind to active kinases in an ATP competitive manner. We have previously described a time-resolved fluorescence resonance energy transfer (TR-FRET)-based kinase binding assay using the competitive displacement of ATP competitive active site fluorescent probes ("tracers"). Although this format has gained acceptance, published data with this and related formats are almost entirely without examples of non-ATP competitive compounds. Thus, this study addresses whether this format is useful for non-ATP competitive inhibitors. To this end, 15 commercially available non-ATP competitive inhibitors were tested for their ability to displace ATP competitive probes. Despite the diversity of both compound structures and their respective targets, 14 of the 15 compounds displaced the tracers with IC(50) values comparable to literature values. We conclude that such binding assays are well suited for the study of non-ATP competitive inhibitors. In addition, we demonstrate that allosteric inhibitors of BCR-Abl and MEK bind preferentially to the nonphosphorylated (i.e., inactive) form of the kinase, indicating that binding assays may be a preferred format in some cases.
authors.affiliation: Life Technologies Corporation, Madison, WI 53719, USA.
authors.forename: Connie S
authors.initials: CS
authors.lastname: Lebakken
authors.forename: Laurie J
authors.initials: LJ
authors.lastname: Reichling
authors.forename: Jason M
authors.initials: JM
authors.lastname: Ellefson
authors.forename: Steven M
authors.initials: SM
authors.lastname: Riddle
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000494:Allosteric Regulation / Q000187:drug effects; D001667:Binding, Competitive; D020134:Catalytic Domain / Q000187:drug effects; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D031541:Fluorescence Resonance Energy Transfer; D016044:Fusion Proteins, bcr-abl / Q000037:antagonists & inhibitors / Q000378:metabolism; D006801:Humans; D020128:Inhibitory Concentration 50; D048369:MAP Kinase Kinase 1 / Q000037:antagonists & inhibitors / Q000378:metabolism; D011485:Protein Binding; D047428:Protein Kinase Inhibitors / Q000737:chemistry / Q000494:pharmacology*; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism
chemical_list: D047428:Protein Kinase Inhibitors; D000255:Adenosine Triphosphate; D011505:Protein-Tyrosine Kinases; D016044:Fusion Proteins, bcr-abl; D048369:MAP Kinase Kinase 1; C482125:MAP2K1 protein, human