Structural Insight into the Interactions between Death-Associated Protein Kinase 1 and Natural Flavonoids

Abstract

Death-associated protein kinase 1 (DAPK1) is a 160 kDa serine/threonine protein kinase that belongs to the Ca(2+)/calmodulin-dependent protein kinase subfamily. DAPK1 is a possible target for the treatment of acute ischemic stroke and endometrial adenocarcinomas. In the present study, we investigated the binding characteristics of 17 natural flavonoids to DAPK1 using a 1-anilinonaphthalene-8-sulfonic acid competitive binding assay and revealed that morin was the strongest binder among the selected compounds. The crystallographic analysis of DAPK1 and 7 selected flavonoid complexes revealed the structure-binding affinity relationship in atomic-level detail. It was suggested that the high affinity of morin could be accounted for by the ionic interaction between 2'-OH and K42 and that such an interaction would not take place with either cyclin-dependent protein kinases or PIM kinases because of their broader entrance regions. Thus, morin would be a more selective inhibitor of DAPK1 than either of these other types of kinases. In addition, we found that the binding of kaempferol to DAPK1 was associated with a chloride ion. The present study provides a better understanding of the molecular properties of the ATP site of DAPK1 and may be useful for the design of specific DAPK1 inhibitors.

Structural and thermodynamic analyses of interactions between death-associated protein kinase 1 and anthraquinones

“…The catalytic domain of the DAPK1 protein (residues 1-285) was prepared using an Escherichia coli system as described previously (Yokoyama et al, 2015). The purity was assessed by SDS-PAGE with Coomassie Brilliant Blue (CBB) staining and was estimated to be greater than 90%.…”

“…Wilbek and coworkers identified an imidazopyramidazine compound (CPR005231) as a DAPK1 inhibitor with a K d of 0.24 mM (Wilbek et al, 2015). In addition, during the course of our research on the interaction of DAPK1 with natural flavonoids, we found that several natural flavonoids bind to DAPK1 with different binding affinities via various binding modes, and that the binding affinities can be drastically modulated by a slight change of the functional group (Yokoyama et al, 2015). These results led us to the idea that a DAPK1 inhibitor might be discovered among anthraquinone derivatives, including emodin and quinalizarin.…”

“…The molecular topology data for purpurin were obtained from the PRODRG server (Schü ttelkopf & van Aalten, 2004;van Aalten et al, 1996). Structure refinement could directly be started without molecular replacement using the PDB data for a DAPK-morin complex, from which the water and morin molecules were omitted (PDB entry 5auy), as the crystal of the DAPK1-purpurin complex was isomorphous to the crystal of the DAPK1-morin complex (Yokoyama et al, 2015). The crystal of the DAPK1-purpurin complex belonged to space group P2 1 2 1 2 1 , with unit-cell parameters a = 46.7, b = 62.9, c = 88.6 Å .…”

“…The 8-anilino-1-naphthalenesulfonic acid (ANS) competitive binding assay was carried out as described previously (Yokoyama et al, 2015). In brief, the fluorescence intensities of ANS were measured in a 96-well black microplate (Thermo Fisher Scientific) using a GENios plate reader (Tecan Group, Mä nnedorf, Switzerland) with excitation and emission wavelengths of 360 and 465 nm, respectively.…”

High-content screen using zebrafish (Danio rerio) embryos identifies a novel kinase activator and inhibitor

“…We also evaluated the interaction between compound 1 and DAPK1 (5AUT.pdb) 16 using molecular modeling docking studies (Figure 6C and D). Since compound 1 activates/stimulates DAPK1, we posited that ATP would be in the ATP binding pocket and allosterically modulate it.…”