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.

Structure-based discovery of the first allosteric inhibitors of cyclin-dependent kinase 2

“…As a matter of fact, detection of allosteric ligands using standard kinase activity assays is known to be challenging, as these methods preferentially detect compounds that bind to active kinases in an ATP-competitive manner. 22,23 Nevertheless, a 30% inhibition of kinase activity was achieved when a 100 μM concentration of compound 4 was pre-incubated with CDK2 before the addition of Cyclin A and the substrate Rb, confirming the ability of this compound to bind and partially inhibit CDK2 activity when pre-formed CDK2/Cyclin A complexes are not used (data not shown). Considering the tight binding of Cyclin A for CDK2, it is conceivable that more potent analogs of compounds 1-7 may inhibit complex formation with cyclins more effectively.…”

The glycoglycerolipid 1,2-dipalmitoyl-3-(N-palmitoyl-6′-amino-6′-deoxy-α-d-glucosyl)-sn-glycerol is no inhibitor of the human Myt1 kinase

“…Even though most type III inhibitors do not directly bind to the ATP site, the majority must either alter the active site in a way that displaces the tracer or bind close to the active site. The TR-FRET-based assay used in our previous report was, meanwhile, shown to have excellent performance concerning the identification of allosteric kinase inhibitors 14 . Based on the assay data presented, an interaction of GGL1 with Myt1 at a site close to the kinase domain appears to be unlikely.…”

Biasing Receptor Tyrosine Kinase Signaling Pathways

“…190 We fully expect that as kinase screening strategies evolve to enable the detection of inhibitors acting at allosteric sites, 191 the identification of RTK inhibitors displaying the ability to selectively perturb some, but not all, of the signaling events activated upon ligand binding and dimerization will become more common.…”