Affinity Reagents that Target a Specific Inactive Form of Protein Kinases

SummaryA number of small-molecule inhibitors have been developed that target the catalytic domains of protein kinases that are not in an active conformation. An inactive form that has been observed in several kinases is the DFG-out conformation. This conformation is characterized by an almost 180° rotation of the conserved Asp-Phe-Gly (DFG) motif in the ATP-binding cleft relative to the active form. However, the sequence and structural determinants that allow a kinase to stably adopt the DFG-out conformation are not known. Here, we characterize a series of inhibitors based on a general pharmacophore for this inactive form. We demonstrate that modified versions of these inhibitors can be used to study the thermodynamics and kinetics of ligand binding to DFG-out-adopting kinases and for enriching these kinases from complex protein mixtures.

Graphical AbstractFigure optionsDownload full-size imageDownload high-quality image (251 K)Download as PowerPoint slideHighlights► The determinants that allow kinases to adopt different inactive forms are not known ► A pharmacophore for a specific inactive form (DFG-out) of kinases was identified ► Immobilized type II inhibitors effectively enrich DFG-out-adopting kinases ► A STE20 kinase, LOK, was identified as a DFG-out-adopting protein kinase