Despite the hundreds of kinase inhibitors currently in discovery and preclinical phases, the number of FDA-approved kinase inhibitors remains very low by comparison, a discrepancy which reflects the challenges which accompanies kinase inhibitor development. Targeting protein kinases with ATP-competitive inhibitors has been the classical approach to inhibit kinase activity, but the highly conserved nature of the ATP-binding site often contributes to the poor inhibitor selectivity. To address this problem, we developed a high-throughput screening technology that can discriminate for inhibitors, which stabilize inactive kinase conformations by binding within allosteric pockets in the kinase domain. Here, we describe how to use the Fluorescence Labels in Kinases approach to measure the K(d) of ligands as well as how to kinetically characterize the binding and dissociation of ligands to the kinase. We also describe how this technology can be used to rapidly screen small molecule libraries in high throughput.
Keywords: DFG-out; FLiK; allosteric inhibitor; biosensor; fluorescence; kinase.