FLiK: a direct-binding assay for the identification and kinetic characterization of stabilizers of inactive kinase conformations.
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.
Chemical Genomics Centre of the Max Planck Society, Dortmund, Germany. Electronic address: simard@amgen.com.
Jeffrey R
JR
Simard
Chemical Genomics Centre of the Max Planck Society, Dortmund, Germany; Fakultät Chemie, Chemische Biologie, Technische Universität Dortmund, Dortmund, Germany. Electronic address: daniel.rauh@tu-dortmund.de.
Daniel
D
Rauh
D015081:2-Naphthylamine / Q000031:analogs & derivatives / Q000737:chemistry; D020134:Catalytic Domain; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry; D057166:High-Throughput Screening Assays; D006801:Humans; D007700:Kinetics; D008024:Ligands; D009154:Mutation; D011487:Protein Conformation; D047428:Protein Kinase Inhibitors / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D055550:Protein Stability / Q000187:drug effects; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D054852:Small Molecule Libraries; D048051:p38 Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D005456:Fluorescent Dyes; D008024:Ligands; D047428:Protein Kinase Inhibitors; D011994:Recombinant Proteins; D054852:Small Molecule Libraries; C038518:acrylodan; D015081:2-Naphthylamine; D048051:p38 Mitogen-Activated Protein Kinases
DFG-out; FLiK; allosteric inhibitor; biosensor; fluorescence; kinase