Identification and Characterization of a Small-Molecule Inhibitor of Death-Associated Protein Kinase 1

Death-associated protein kinase 1 (DAPK1) has been associated with N-methyl-d-aspartate (NMDA) receptor-mediated excitotoxicity, and has recently been suggested as a target for the treatment of stroke. Here, we examined DAPK1-mediated in vitro phosphorylation of the GluN2B subunit of NMDA receptors. We established a high-throughput screening assay for the protein kinase DAPK1 by using a Caliper microfluidics capillary electrophoresis system (Caliper Life Sciences/PerkinElmer) and identified a novel small-molecule imidazo-pyramidazine inhibitor (6) targeting the catalytic domain of DAPK1. The inhibitor was characterized by enzyme kinetic assays and isothermal titration calorimetry (ITC), and a high resolution crystal structure provided detailed insight into the binding mode of the inhibitor.