Characterization of the molecular interactions between resveratrol derivatives and death-associated protein kinase 1.

Death-associated protein kinase 1 (DAPK1), a Ca2+/calmodulin-regulated serine/threonine kinase, regulates cell apoptosis and autophagy and has been implicated in the pathogenesis of Alzheimer's disease (AD). Targeting DAPK1 may be a promising approach for treating AD. In our previous study, we found that a natural polyphenol, resveratrol (1), is a moderate DAPK1 inhibitor. In the present study, we investigated the interactions between natural and synthetic derivatives of 1 and DAPK1. Binding assays including intrinsic fluorescence quenching, protein thermal shift and isothermal titration calorimetry indicated that oxyresveratrol (3), a hydroxylated derivative, and pinostilbene (5), a methoxylated derivative, bind to DAPK1 with comparable affinity to 1. The enzymatic assay showed that 3 more effectively inhibits the intrinsic ATPase activity of DAPK1 compared with 1. Crystallographic analysis revealed that the binding modes of the methoxylated derivatives were different from those of 1 and 3, resulting in a unique interaction. Our results suggest that 3 may be helpful in treating AD and provide a clue for the development of promising DAPK1 inhibitors.

Targeting Death-Associated Protein Kinases for Treatment of Human Diseases: Recent Advances and Future Directions.

The death-associated protein kinase (DAPK) family is a member of the calcium/calmodulin-regulated serine/threonine protein kinase family, and studies have shown that its role, as its name suggests, is mainly to regulate cell death. The DAPK family comprises five members, including DAPK1, DAPK2, DAPK3, DRAK1 and DRAK2, which show high homology in the common N-terminal kinase domain but differ in the extra-catalytic domain. Notably, previous research has suggested that the DAPK family plays an essential role in both the development and regulation of human diseases. However, only a few small-molecule inhibitors have been reported. In this Perspective, we mainly discuss the structure, biological function, and role of DAPKs in diseases and the currently discovered small-molecule inhibitors, providing valuable information for the development of the DAPK field.

Identification of potential death-associated protein kinase-1 (DAPK1) inhibitors by an integrated ligand-based and structure-based computational drug design approach.

Death-associated protein kinase 1 (DAPK1) is a calcium/calmodulin (Ca2+/CaM)-dependent serine/threonine kinase that is abundantly expressed in the memory- and cognition-related brain areas. DAPK1 is associated with several pathological hallmarks of Alzheimer's disease (AD); it is an attractive target for designing a novel DAPK1 inhibitor as an effective therapeutic treatment for AD. In the present study, we have used an integrated ligand-based and structure-based drug design method to identify DAPK1 inhibitors. The pharmacophoric features of compound 38 G (PDB ID 4TXC) were mapped, and the models were evaluated using enrichment factor (EF) and goodness of hit (GH) score. The selected models were used to screen Zinc 15 compounds library. The identified hits were passed through drug-likeliness and PAINS filtering. The docking study was performed in three steps to yield molecules with good binding energy and ligand-target interactions. Finally, three hits were obtained, that is, ZINC000020648330, ZINC000006755051 and ZINC000020650468, which were subjected to rigorous molecular dynamics simulation. All three hits exhibited optimal stability under simulated conditions and low predicted toxicity.Communicated by Ramaswamy H. Sarma.

Computational Dissection of the Role of Trp305 in the Regulation of the Death-Associated Protein Kinase-Calmodulin Interaction.

Death-associated protein kinase 1 (DAPK1), as a calcium/calmodulin (CaM) regulated serine/threonine kinase, functions in apoptotic and autophagy pathways and represents an interesting drug target for inflammatory bowel disease and Alzheimer's disease. The crystal structure of the DAPK1 catalytic domain and the autoregulatory domain (ARD) in complex with CaM provides an understanding of CaM-dependent regulation of DAPK1 activity. However, the molecular basis of how distinct Trp305 (W305Y and W305D) mutations in the ARD modulate different DAPK1 activities remains unknown. Here, we performed multiple, µs-length molecular dynamics (MD) simulations of the DAPK1-CaM complex in three different (wild-type, W305Y, and W305D) states. MD simulations showed that the overall structural complex did not change significantly in the wild-type and W305Y systems, but underwent obvious conformational alteration in the W305D system. Dynamical cross-correlation and principal component analyses revealed that the W305D mutation enhanced the anti-correlated motions between the DAPK1 and CaM and sampled a broader distribution of conformational space relative to the wild-type and W305Y systems. Structural and energetical analyses further exhibited that CaM binding was unfavored in response to the W305D mutation, resulting in the decreased binding of CaM to the W305D mutant. Furthermore, the hydrogen bonds and salt bridges responsible for the loss of CaM binding on the interface of the DAPK1-CaM complex were identified in the W305D mutant. This result may provide insights into the key role of Trp305 in the regulation of CaM-mediated DAPK1 activity.

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

Death-associated protein kinase 1 (DAPK1) is a serine/threonine protein kinase that regulates apoptosis and autophagy. DAPK1 is considered to be a therapeutic target for amyloid-ß deposition, endometrial adenocarcinomas and acute ischemic stroke. Here, the potent inhibitory activity of the natural anthraquinone purpurin against DAPK1 phosphorylation is shown. Thermodynamic analysis revealed that while the binding affinity of purpurin is similar to that of CPR005231, which is a DAPK1 inhibitor with an imidazopyridazine moiety, the binding of purpurin was more enthalpically favorable. In addition, the inhibition potencies were correlated with the enthalpic changes but not with the binding affinities. Crystallographic analysis of the DAPK1-purpurin complex revealed that the formation of a hydrogen-bond network is likely to contribute to the favorable enthalpic changes and that stabilization of the glycine-rich loop may cause less favorable entropic changes. The present findings indicate that purpurin may be a good lead compound for the discovery of inhibitors of DAPK1, and the observation of enthalpic changes could provide important clues for drug development.

Structural Basis for Recruitment of DAPK1 to the KLHL20 E3 Ligase.

BTB-Kelch proteins form the largest subfamily of Cullin-RING E3 ligases, yet their substrate complexes are mapped and structurally characterized only for KEAP1 and KLHL3. KLHL20 is a related CUL3-dependent ubiquitin ligase linked to autophagy, cancer, and Alzheimer's disease that promotes the ubiquitination and degradation of substrates including DAPK1, PML, and ULK1. We identified an "LPDLV"-containing motif in the DAPK1 death domain that determines its recruitment and degradation by KLHL20. A 1.1-Å crystal structure of a KLHL20 Kelch domain-DAPK1 peptide complex reveals DAPK1 binding as a loose helical turn that inserts deeply into the central pocket of the Kelch domain to contact all six blades of the ß propeller. Here, KLHL20 forms salt-bridge and hydrophobic interactions including tryptophan and cysteine residues ideally positioned for covalent inhibitor development. The structure highlights the diverse binding modes of ß-propeller domains versus linear grooves and suggests a new target for structure-based drug design.

Death-Associated Protein Kinase 1 Phosphorylation in Neuronal Cell Death and Neurodegenerative Disease.

Regulated neuronal cell death plays an essential role in biological processes in normal physiology, including the development of the nervous system. However, the deregulation of neuronal apoptosis by various factors leads to neurodegenerative diseases such as ischemic stroke and Alzheimer's disease (AD). Death-associated protein kinase 1 (DAPK1) is a calcium/calmodulin (Ca2+/CaM)-dependent serine/threonine (Ser/Thr) protein kinase that activates death signaling and regulates apoptotic neuronal cell death. Although DAPK1 is tightly regulated under physiological conditions, DAPK1 deregulation in the brain contributes to the development of neurological disorders. In this review, we describe the molecular mechanisms of DAPK1 regulation in neurons under various stresses. We also discuss the role of DAPK1 signaling in the phosphorylation-dependent and phosphorylation-independent regulation of its downstream targets in neuronal cell death. Moreover, we focus on the major impact of DAPK1 deregulation on the progression of neurodegenerative diseases and the development of drugs targeting DAPK1 for the treatment of diseases. Therefore, this review summarizes the DAPK1 phosphorylation signaling pathways in various neurodegenerative diseases.

Ser289 phosphorylation activates both DAPK1 and DAPK2 but in response to different intracellular signaling pathways.

DAPK1 and DAPK2 are calmodulin (CaM)-regulated protein kinases that share a high degree of homology in their catalytic and CaM regulatory domains. Both kinases function as tumor suppressors, and both have been implicated in autophagy regulation. Over the years, common regulatory mechanisms for the two kinases as well as kinase-specific ones have been identified. In a recent work, we revealed that DAPK2 is phosphorylated on Ser289 by the metabolic sensor AMPK, and that this phosphorylation enhances DAPK2 catalytic activity. Notably, Ser289 is conserved between DAPK1 and DAPK2, and was previously found to be phosphorylated in DAPK1 by RSK. Intriguingly, Ser289 phosphorylation was conversely reported to inhibit the pro-apoptotic activity of DAPK1 in cells. However, as the direct effect of this phosphorylation on DAPK1 catalytic activity was not tested, indirect effects were not excluded. Here, we compared Ser289 phosphorylation of the two kinases in the same cells and found that the intracellular signaling pathways that lead to Ser289 phosphorylation are mutually-exclusive and different for each kinase. In addition, we found that Ser289 phosphorylation in fact enhances DAPK1 catalytic activity, similar to the effect on DAPK2. Thus, Ser289 phosphorylation activates both DAPK1 and DAPK2, but in response to different intracellular signaling pathways.

Regulation of the Expression of DAPK1 by SUMO Pathway.

Death Associated Protein Kinase 1 (DAPK1) is an important signaling kinase mediating the biological effect of multiple natural biomolecules such as IFN-γ, TNF-α, curcumin, etc. DAPK1 is degraded through both ubiquitin-proteasomal and lysosomal degradation pathways. To investigate the crosstalk between these two DAPK1 degradation pathways, we carried out a screen using a set of ubiquitin E2 siRNAs at the presence of Tuberous Sclerous 2 (TSC2) and identified that the small ubiquitin-like molecule (SUMO) pathway is able to regulate the protein levels of DAPK1. Inhibition of the SUMO pathway enhanced DAPK1 protein levels and the minimum domain of DAPK1 protein required for this regulation is the kinase domain, suggesting that the SUMO pathway regulates DAPK1 protein levels independent of TSC2. Suppression of the SUMO pathway did not enhance DAPK1 protein stability. In addition, mutation of the potential SUMO conjugation sites on DAPK1 kinase domain did not alter its protein stability or response to SUMO pathway inhibition. These data suggested that the SUMO pathway does not regulate DAPK1 protein degradation. The exact molecular mechanism underlying this regulation is yet to be discovered.

DAPK1: a Novel Pathology and Treatment Target for Alzheimer's Disease.

Alzheimer's disease (AD) is the most common neurodegenerative disease and seriously damages the health of elderly population. Clinical drug research targeting at classic pathology hallmarks, such as amyloid-ß (Aß) and tau protein, failed to achieve effective cognitive improvement, suggesting that the pathogenesis of AD is much complicated, and there are still other unknown and undetermined important factors. Death-associated protein kinase 1 (DAPK1) is a calcium/calmodulin-dependent serine/threonine kinase that plays an important role in various neuronal injury models. Mounting evidence has demonstrated that DAPK1 variants are associated with AD risk. The activation of DAPK1 is also involved in AD-related neurodegeneration in the brain. Exploring the roles of DAPK1 in AD might help us understand the pathogenic mechanisms and find a novel promising therapeutic target in AD. Therefore, in this review, we comprehensively summary the main progress of DAPK1 in the AD studies from genetic risk, neuropathological process, and clinical potential implications.

Validation of chemical genetics for the study of zipper-interacting protein kinase signaling.

Zipper-interacting protein kinase (ZIPK) is a Ser/Thr kinase that mediates a variety of cellular functions. Analogue-sensitive kinase technology was applied to the study of ZIPK signaling in coronary artery smooth muscle cells. ZIPK was engineered in the ATP-binding pocket by substitution of a bulky gatekeeper amino acid (Leu93) with glycine. Cell-permeable derivatives of pyrazolo[3,4-d]pyrimidine provided effective inhibition of L93G-ZIPK (1NM-PP1, IC50 , 1.0 µM; 3MB-PP1, IC50 , 2.0 µM; and 1NA-PP1, IC50 , 8.6 µM) but only 3MB-PP1 had inhibitory potential (IC50 > 10 µM) toward wild-type ZIPK. Each of the compounds also attenuated Rho-associated coiled-coil containing protein kinase (ROCK) activity under experimental conditions found to be optimal for inhibition of L93G-ZIPK. In silico molecular simulations showed effective docking of 1NM-PP1 into ZIPK following mutational enlargement of the ATP-binding pocket. Molecular simulation of 1NM-PP1 docking in the ATP-binding pocket of ROCK was also completed. The 1NM-PP1 inhibitor was selected as the optimal compound for selective chemical genetics in smooth muscle cells since it displayed the highest potency for L93G-ZIPK relative to WT-ZIPK and the weakest off-target effects against other relevant kinases. Finally, the 1NM-PP1 and L93G-ZIPK pairing was effectively applied in vascular smooth muscle cells to manipulate the phosphorylation level of LC20, a previously defined target of ZIPK.

Targeting Pim Kinases and DAPK3 to Control Hypertension.

Sustained vascular smooth muscle hypercontractility promotes hypertension and cardiovascular disease. The etiology of hypercontractility is not completely understood. New therapeutic targets remain vitally important for drug discovery. Here we report that Pim kinases, in combination with DAPK3, regulate contractility and control hypertension. Using a co-crystal structure of lead molecule (HS38) in complex with DAPK3, a dual Pim/DAPK3 inhibitor (HS56) and selective DAPK3 inhibitors (HS94 and HS148) were developed to provide mechanistic insight into the polypharmacology of hypertension. In vitro and ex vivo studies indicated that Pim kinases directly phosphorylate smooth muscle targets and that Pim/DAPK3 inhibition, unlike selective DAPK3 inhibition, significantly reduces contractility. In vivo, HS56 decreased blood pressure in spontaneously hypertensive mice in a dose-dependent manner without affecting heart rate. These findings suggest including Pim kinase inhibition within a multi-target engagement strategy for hypertension management. HS56 represents a significant step in the development of molecularly targeted antihypertensive medications.

Structural basis of mitochondrial receptor binding and constriction by DRP1.

Mitochondrial inheritance, genome maintenance and metabolic adaptation depend on organelle fission by dynamin-related protein 1 (DRP1) and its mitochondrial receptors. DRP1 receptors include the paralogues mitochondrial dynamics proteins of 49 and 51 kDa (MID49 and MID51) and mitochondrial fission factor (MFF); however, the mechanisms by which these proteins recruit and regulate DRP1 are unknown. Here we present a cryo-electron microscopy structure of full-length human DRP1 co-assembled with MID49 and an analysis of structure- and disease-based mutations. We report that GTP induces a marked elongation and rotation of the GTPase domain, bundle-signalling element and connecting hinge loops of DRP1. In this conformation, a network of multivalent interactions promotes the polymerization of a linear DRP1 filament with MID49 or MID51. After co-assembly, GTP hydrolysis and exchange lead to MID receptor dissociation, filament shortening and curling of DRP1 oligomers into constricted and closed rings. Together, these views of full-length, receptor- and nucleotide-bound conformations reveal how DRP1 performs mechanical work through nucleotide-driven allostery.

Non-canonical activation of DAPK2 by AMPK constitutes a new pathway linking metabolic stress to autophagy.

Autophagy is an intracellular degradation process essential for adaptation to metabolic stress. DAPK2 is a calmodulin-regulated protein kinase, which has been implicated in autophagy regulation, though the mechanism is unclear. Here, we show that the central metabolic sensor, AMPK, phosphorylates DAPK2 at a critical site in the protein structure, between the catalytic and the calmodulin-binding domains. This phosphorylation activates DAPK2 by functionally mimicking calmodulin binding and mitigating an inhibitory autophosphorylation, providing a novel, alternative mechanism for DAPK2 activation during metabolic stress. In addition, we show that DAPK2 phosphorylates the core autophagic machinery protein, Beclin-1, leading to dissociation of its inhibitor, Bcl-XL. Importantly, phosphorylation of DAPK2 by AMPK enhances DAPK2's ability to phosphorylate Beclin-1, and depletion of DAPK2 reduces autophagy in response to AMPK activation. Our study reveals a unique calmodulin-independent mechanism for DAPK2 activation, critical to its function as a novel downstream effector of AMPK in autophagy.

Exploring putative inhibitors of Death Associated Protein Kinase 1 (DAPK1) via targeting Gly-Glu-Leu (GEL) and Pro-Glu-Asn (PEN) substrate recognition motifs.

Recently, a new signaling complex Death Associated Protein Kinase 1 (DAPK1) ̶ N-methyl-D-aspartate receptor subtype 2B (NMDAR2B or NR2B) engaged in the neuronal death cascade was identified and it was found that after stroke injury, N-methyl-D-aspartate glutamate (NMDA) receptors interact with DAPK1 through NR2B subunit and lead to excitotoxicity via over-activation of NMDA receptors. An acute brain injury, such as stroke, is a serious life-threatening medical condition which occurs due to poor blood supply to the brain and further leads to neuronal cell death. During a stroke, activated DAPK1 migrates towards the extra-synaptic site and binds to NR2B subunit of NMDA receptor. It is this DAPK1-NR2B interaction that arbitrates the pathological processes like apoptosis, necrosis, and autophagy of neuronal cells observed in stroke injury, hence we aimed to inhibit this vital interaction to prevent neuronal damage. In the present study, using PubChem database, we applied an integrative approach of virtual screening and molecular dynamic simulations and identified a potential lead compound 11 that interrupts DAPK1-NR2B interaction by competing with both ATP and substrate for their binding sites on DAPK1. This inhibitor was found potent and considerably selective to DAPK1 as it made direct contact with the ATP binding sites as well as substrate recognition motifs: Gly-Glu-Leu (GEL) and Pro-Glu-Asn (PEN). Further in vitro and in vivo experiments are demanded to validate the efficacy of compound 11 nevertheless, it can be considered as suitable starting point for designing DAPK1 inhibitors.

Contextualization of drug-mediator relations using evidence networks.

BACKGROUND: Genomic analysis of drug response can provide unique insights into therapies that can be used to match the "right drug to the right patient." However, the process of discovering such therapeutic insights using genomic data is not straightforward and represents an area of active investigation. EDDY (Evaluation of Differential DependencY), a statistical test to detect differential statistical dependencies, is one method that leverages genomic data to identify differential genetic dependencies. EDDY has been used in conjunction with the Cancer Therapeutics Response Portal (CTRP), a dataset with drug-response measurements for more than 400 small molecules, and RNAseq data of cell lines in the Cancer Cell Line Encyclopedia (CCLE) to find potential drug-mediator pairs. Mediators were identified as genes that showed significant change in genetic statistical dependencies within annotated pathways between drug sensitive and drug non-sensitive cell lines, and the results are presented as a public web-portal (EDDY-CTRP). However, the interpretability of drug-mediator pairs currently hinders further exploration of these potentially valuable results. METHODS: In this study, we address this challenge by constructing evidence networks built with protein and drug interactions from the STITCH and STRING interaction databases. STITCH and STRING are sister databases that catalog known and predicted drug-protein interactions and protein-protein interactions, respectively. Using these two databases, we have developed a method to construct evidence networks to "explain" the relation between a drug and a mediator.  RESULTS: We applied this approach to drug-mediator relations discovered in EDDY-CTRP analysis and identified evidence networks for ~70% of drug-mediator pairs where most mediators were not known direct targets for the drug. Constructed evidence networks enable researchers to contextualize the drug-mediator pair with current research and knowledge. Using evidence networks, we were able to improve the interpretability of the EDDY-CTRP results by linking the drugs and mediators with genes associated with both the drug and the mediator. CONCLUSION: We anticipate that these evidence networks will help inform EDDY-CTRP results and enhance the generation of important insights to drug sensitivity that will lead to improved precision medicine applications.

Death-Associated Protein Kinase Activity Is Regulated by Coupled Calcium/Calmodulin Binding to Two Distinct Sites.

The regulation of many protein kinases by binding to calcium/calmodulin connects two principal mechanisms in signaling processes: protein phosphorylation and responses to dose- and time-dependent calcium signals. We used the calcium/calmodulin-dependent members of the death-associated protein kinase (DAPK) family to investigate the role of a basic DAPK signature loop near the kinase active site. In DAPK2, this loop comprises a novel dimerization-regulated calcium/calmodulin-binding site, in addition to a well-established calcium/calmodulin site in the C-terminal autoregulatory domain. Unexpectedly, impairment of the basic loop interaction site completely abolishes calcium/calmodulin binding and DAPK2 activity is reduced to a residual level, indicative of coupled binding to the two sites. This contrasts with the generally accepted view that kinase calcium/calmodulin interactions are autonomous of the kinase catalytic domain. Our data establish an intricate model of multi-step kinase activation and expand our understanding of how calcium binding connects with other mechanisms involved in kinase activity regulation.

A Novel Recombinant Anti-CD22 Immunokinase Delivers Proapoptotic Activity of Death-Associated Protein Kinase (DAPK) and Mediates Cytotoxicity in Neoplastic B Cells.

The serine/threonine death-associated protein kinases (DAPK) provide pro-death signals in response to (oncogenic) cellular stresses. Lost DAPK expression due to (epi)genetic silencing is found in a broad spectrum of cancers. Within B-cell lymphomas, deficiency of the prototypic family member DAPK1 represents a predisposing or early tumorigenic lesion and high-frequency promoter methylation marks more aggressive diseases. On the basis of protein studies and meta-analyzed gene expression profiling data, we show here that within the low-level context of B-lymphocytic DAPK, particularly CLL cells have lost DAPK1 expression. To target this potential vulnerability, we conceptualized B-cell-specific cytotoxic reconstitution of the DAPK1 tumor suppressor in the format of an immunokinase. After rounds of selections for its most potent cytolytic moiety and optimal ligand part, a DK1KD-SGIII fusion protein containing a constitutive DAPK1 mutant, DK1KD, linked to the scFv SGIII against the B-cell-exclusive endocytic glyco-receptor CD22 was created. Its high purity and large-scale recombinant production provided a stable, selectively binding, and efficiently internalizing construct with preserved robust catalytic activity. DK1KD-SGIII specifically and efficiently killed CD22-positive cells of lymphoma lines and primary CLL samples, sparing healthy donor- or CLL patient-derived non-B cells. The mode of cell death was predominantly PARP-mediated and caspase-dependent conventional apoptosis as well as triggering of an autophagic program. The notoriously high apoptotic threshold of CLL could be overcome by DK1KD-SGIII in vitro also in cases with poor prognostic features, such as therapy resistance. The manufacturing feasibility of the novel CD22-targeting DAPK immunokinase and its selective antileukemic efficiency encourage intensified studies towards specific clinical application. Mol Cancer Ther; 15(5); 971-84. ©2016 AACR.

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

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.

Death-associated protein kinase: A molecule with functional antagonistic duality and a potential role in inflammatory bowel disease (Review).

The cytoskeleton-associated serine/threonine kinase death-associated protein kinase (DAPK) has been described as a cancer gene chameleon with functional antagonistic duality in a cell type and context specific manner. The broad range of interaction partners and substrates link DAPK to inflammatory processes especially in the gut. Herein we summarize our knowledge on the role of DAPK in different cell types that play a role under inflammatory conditions in the gut. Besides some promising experimental data suggesting DAPK as an interesting drug target in inflammatory bowel disease there are many open questions regarding direct evidence for a role of DAPK in intestinal inflammation.