Displacement Assay for the Detection of Stabilizers of Inactive Kinase Conformations

Targeting protein kinases with small molecules outside the highly conserved ATP pocket to stabilize inactive kinase conformati oils is becoming a more desirable approach in kinase inhibitor research, since these molecules have advanced pharmacological properties compared to compounds exclusively targeting the ATP pocket. Traditional screening approaches for kinase inhibitors arc often based on enzyme activity, but they may miss inhibitors that stabilize inactive kinase conformations by enriching the active state of the kinase. Here we present the development of a kinase binding assay employing a pyraZOIOUrea type III inhibitor and enzyme fragment complementation (EFQ technology that is suitable to screen stabilizers ofenzyniatically inactive kinases. To validate this assay system, we report tile binding characteristics of a series of kinase inhibitors to inactive p38(x and JNK2. Additionally, we present protein X-ray crystallography studies to examine the binding modes of potent quinolinc-based DFG-out binders in p38a.