UR-144, synthetic cannabinoid receptor agonist, induced cardiomyoblast toxicity mechanism comprises cytoplasmic Ca2+ and DAPK1 related autophagy and necrosis.

UR-144, a cannabinoid receptor agonist, is widely used alone or in combination with other synthetic cannabinoids (SCs) all over the world. At overdose, cardiovascular symptoms have been reported and the underlying molecular mechanisms of these adverse effects are not known. It is highly important to clarify the toxic effects of UR-144 for the treatment of poisoning. In the present study, the molecular mechanism of cytotoxic effects of UR-144 is evaluated on a cardiomyoblastic cell line using WST-1 and LDH assays. Apoptosis/necrosis, autophagy, and ROS (reactive oxygen species) levels were determined using flow cytometry. Cytoplasmic Ca2+ levels were measured by using a fluorogenic calcium-binding dye. Released and cytoplasmic troponin T levels, a specific marker of cardiotoxicity, were examined with western blot. For the evaluation of the role of DAPK1, on UR-144-induced cell death, DAPK1 activity and DAPK1 protein level were investigated. Its cytotoxic effects increased in a dose-dependent manner for WST-1 and LDH assays, while membrane damage, one of the signs of necrotic cell death, was more remarkable than damage to mitochondria. Cytoplasmic Ca2+ levels rose after high-dose UR-144 treatment and inhibition of DAPK1 activity ameliorated UR-144-induced cytotoxicity. Released troponin T significantly increased at a dose of 200 µM. ROS and total antioxidant capacity of cells were both reduced following high dose UR-144 treatment. The results indicated that UR-144-induced autophagic and necrotic cell death might be a consequence of elevated cytoplasmic Ca2+ levels and DAPK1 activation. However, in vivo/clinical studies are needed to identify molecular mechanisms of cardiotoxic effects of UR-144.

Regulation of DAPK1 by Natural Products: An Important Target in Treatment of Stroke.

Stroke is a sudden neurological disorder that occurs due to impaired blood flow to an area of the brain. Stroke can be caused by the blockage or rupture of a blood vessel in the brain, called ischemic stroke and hemorrhagic stroke, respectively. Stroke is more common in men than women. Atrial fibrillation, hypertension, kidney disease, high cholesterol and lipids, genetic predisposition, inactivity, poor nutrition, diabetes mellitus, family history and smoking are factors that increase the risk of stroke. Restoring blood flow by repositioning blocked arteries using thrombolytic agents or endovascular therapy are the most effective treatments for stroke. However, restoring circulation after thrombolysis can cause fatal edema or intracranial hemorrhage, and worsen brain damage in a process known as ischemia-reperfusion injury. Therefore, there is a pressing need to find and develop more effective treatments for stroke. In the past, the first choice of treatment was based on natural compounds. Natural compounds are able to reduce the symptoms and reduce various diseases including stroke that attract the attention of the pharmaceutical industry. Nowadays, as a result of the numerous studies carried out in the field of herbal medicine, many useful and valuable effects of plants have been identified. The death-associated protein kinase (DAPK) family is one of the vital families of serine/threonine kinases involved in the regulation of some biological functions in human cells. DAPK1 is the most studied kinase within the DAPKs family as it is involved in neuronal and recovery processes. Dysregulation of DAPK1 in the brain is involved in the developing neurological diseases such as stroke. Natural products can function in a variety of ways, including reducing cerebral edema, reducing brain endothelial cell death, and inhibiting TNFα and interleukin-1ß (IL-1ß) through regulating the DAPK1 signal against stroke. Due to the role of DAPK1 in neurological disorders, the aim of this article was to investigate the role of DAPK1 in stroke and its modulation by natural compounds.

Death-Associated Protein Kinase 3 Inhibitors Identified by Virtual Screening for Drug Discovery in Cancer and Hypertension.

Death-associated protein kinase 3 (DAPK3) is a serine/threonine protein kinase that regulates apoptosis, autophagy, transcription, and actin cytoskeleton reorganization. DAPK3 induces morphological alterations in apoptosis when overexpressed, and it is considered a potential drug target in antihypertensive and anticancer drug development. In this article, we report new findings from a structure-guided virtual screening for discovery of phytochemicals that could modulate the elevated expression of DAPK3, and with an eye to anticancer drug discovery. We used the Indian Medicinal Plants, Phytochemistry and Therapeutics (IMPPAT), a curated database, as part of the methodology. The potential initial hits were identified based on their physicochemical properties and binding affinity toward DAPK3. Subsequently, various filters for drug likeness followed by interaction analysis and molecular dynamics (MD) simulations for 100 nsec were performed to explore the conformational sampling and stability of DAPK3 with the candidate molecules. Notably, the data from all-atom MD simulations and principal component analysis suggested that DAPK3 forms stable complexes with ketanserin and rotenone. In conclusion, this study supports the idea that ketanserin and rotenone bind to DAPK3, and show stability, which can be further explored as promising scaffolds in drug development and therapeutics innovation in clinical contexts such as hypertension and various types of cancer.

Estrogen-dependent hypersensitivity to diabetes-evoked cardiac autonomic dysregulation: Role of hypothalamic neuroinflammation.

AIMS: To investigate if autonomic dysregulation is exacerbated in female rats, subjected to diabetes mellitus (DM), via a paradoxical estrogen (E2)-evoked provocation of neuroinflammation/injury of the hypothalamic paraventricular nucleus (PVN). MAIN METHODS: We measured cardiac autonomic function and conducted subsequent PVN neurochemical studies, in DM rats, and their respective controls, divided as follows: male, sham operated (SO), ovariectomized (OVX), and OVX with E2 supplementation (OVX/E2). KEY FINDINGS: Autonomic dysregulation, expressed as sympathetic dominance (higher low frequency, LF, band), only occurred in DM E2-replete (SO and OVX/E2) rats, and was associated with higher neuronal activity (c-Fos) and higher levels of TNFα and phosphorylated death associated protein kinase-3 (p-DAPK3) in the PVN. These proinflammatory molecules likely contributed to the heightened PVN oxidative stress, injury and apoptosis. The PVN of these E2-replete DM rats also exhibited upregulations of estrogen receptors, ERα and ERß, and proinflammatory adenosine A1 and A2a receptors. SIGNIFICANCE: The E2-dependent autonomic dysregulation likely predisposes DM female rats and women to hypersensitivity to cardiac dysfunction. Further, upregulations of proinflammatory mediators including adenosine A1 and A2 receptors, TNFα and DAPK3, conceivably explain the paradoxical hypersensitivity of DM females to PVN inflammation/injury and the subsequent autonomic dysregulation in the presence of E2.

Dihydroartemisinin induces apoptosis and autophagy-dependent cell death in cholangiocarcinoma through a DAPK1-BECLIN1 pathway.

Cholangiocarcinoma (CCA) is a very aggressive cancer arising from the malignant transformation of cholangiocytes. Intrahepatic CCA is associated with reactive inflammation and intense fibrosis of the hepatobiliary tract. Dihydroartemisinin (DHA), the active compound found in Artemisia annua, has been shown to possess anti-tumor activity in a variety of human cancers, including hepatoma. Here, we tested the ability of DHA to specifically kill CCA cells and have investigated the underlying mechanisms. DHA induced both apoptosis and autophagy-dependent caspase-independent cell death in many CCA cell lines, while being slightly toxic to immortalized cholangiocytes. DHA induced the expression of many apoptosis- and autophagy-related genes in CCA cells. In particular, it greatly induced the expression of DAPK1, and reduced the interaction of BECLIN1 with BCL-2 while promoting its interaction with PI3KC3. Genetic silencing of DAPK1 prevented DHA-induced autophagy. Pharmacologic and genetic inhibition of BECLIN1 function prevented autophagy and cell death induced by DHA in CCA cells. These data unravel a novel pathway of DHA cancer toxicity and open the possibility to introduce DHA in the therapeutic regimen for the treatment of CCA.

DAPK1-ERK signal mediates oxygen glucose deprivation reperfusion induced apoptosis in mouse N2a cells.

AIMS: Death-associated protein kinase 1 (DAPK1) is a kinase found to promote neuronal apoptosis induced by ischemia. Extracellular signal-regulated kinase (ERK) was identified as a key molecule in DAPK1 signaling. However, the mechanisms of neuronal ischemia reperfusion injury remain unknown. Here, we investigate the influence of DAPK1-ERK signal on neuronal apoptosis following ischemia reperfusion. METHODS: Mouse N2a cells were used in this study and primary cultured neurons along with mice were adopted as supplements. Oxygen glucose deprivation (OGD) or administration of N-methyl-d-aspartate (NMDA) and glycine was performed on cells while middle cerebral artery occlusion (MCAO) model on mice. DAPK1 knocking down was achieved by lentiviral-delivered shRNA. Protein expressions were evaluated by western blots. Protein-protein binding was confirmed by co-immunoprecipitation and immunofluorescent assay. Apoptosis of cells was measured by flow cytometry and lacate dehydrogenase (LDH) leakage assay. RESULTS: Ischemia reperfusion resulted in increased DAPK1 and ERK activation as well as aggravated apoptosis in a time-dependent manner. DAPK1 was proved to bind to ERK during reperfusion following OGD, MCAO and excitotoxicity model. Interception of this binding by knocking down DAPK1 led to nuclear translocation of ERK and reduced apoptosis. CONCLUSION: Our study revealed the DAPK1-ERK signal as a potential mechanism contributing to neuronal apoptosis in response to ischemia reperfusion. Disruption of this signal pathway could be a promising therapeutic target against stroke.

Molecular mechanisms of protein kinase regulation by calcium/calmodulin.

Many human protein kinases are regulated by the calcium-sensor protein calmodulin, which binds to a short flexible segment C-terminal to the enzyme's catalytic kinase domain. Our understanding of the molecular mechanism of kinase activity regulation by calcium/calmodulin has been advanced by the structures of two protein kinases-calmodulin kinase II and death-associated protein kinase 1-bound to calcium/calmodulin. Comparison of these two structures reveals a surprising level of diversity in the overall kinase-calcium/calmodulin arrangement and functional readout of activity, as well as complementary mechanisms of kinase regulation such as phosphorylation.

Ethanol dose-dependently elicits opposing regulatory effects on hippocampal AMPA receptor GluA2 subunits through a zeta inhibitory peptide-sensitive kinase in adolescent and adult Sprague-Dawley rats.

AMPA receptor GluA2 subunits are strongly implicated in cognition, and prior work suggests that these subunits may be regulated by atypical protein kinase C (aPKC) isoforms. The present study assessed whether hippocampal and cortical AMPA receptor GluA2 subunit regulation may be an underlying factor in known age-related differences to cognitive-impairing doses of ethanol, and if aPKC isoforms modulate such responses. Hippocampal AMPA receptor GluA2 subunit, protein kinase Mζ (PKMζ), and PKCι/λ expression were elevated during adolescence compared to adults. 1 h following a low-dose (1.0-g/kg) ethanol exposure, hippocampal AMPA receptor GluA2 subunit serine 880 phosphorylation was decreased in adolescents, but was increased in adults. Age-dependent changes in GluA2 subunit phosphorylation were paralleled by alterations in aPKC isoforms, and zeta inhibitory peptide (ZIP) administration prevented ethanol-induced increases in both in adults. Ethanol-induced changes in GluA2 subunit phosphorylation were associated with delayed regulation in synaptosomal GluA2 subunit expression 24 h later. A higher ethanol dose (3.5-g/kg) failed to elicit changes in most measures in the hippocampus at either age. Similar to the hippocampus, analysis of cerebral cortical tissue also revealed age-related declines. However, no demonstrable effects were found following a low-dose ethanol exposure at either age. High-dose ethanol exposure reduced adolescent GluA2 subunit phosphorylation and aPKC isoform expression that were again accompanied by delayed reductions in synaptosomal GluA2 subunit expression. Together, these results suggest that GluA2-containing AMPA receptor modulation by aPKC isoforms is age-, region- and dose-dependently regulated, and may potentially be involved in developmentally regulated ethanol-induced cognitive impairment and other ethanol behaviors.

A novel multi-target drug screening strategy directed against key proteins of DAPk family.

Screening and identifying multi-target ligands becomes a daunting task when there are very few matching pharmacophoric features among the proteins. Herein, we describe a novel screening strategy to identify multi-target ligands for proteins having varying pharmacophoric features with their ligands. This strategy was adopted to identify multi-target ligands for death-associated protein kinase (DAPk) family. The role of the kinase activity of DAPk in eukaryotic cell apoptosis and the ability of bioavailable DAPk inhibitors to rescue neuronal death after brain injury have made it a drug-discovery target for neurodegenerative disorders. In this work, we employed a novel strategy using the existing computational approaches to design multi-target inhibitors, which can potentially inhibit one or any combination of the three DAPk family members. The strategy employs a combination of merged pharmacophore matching, database screening and molecular docking to reliably identify potential multi-target inhibitors targeted against DAPk protein family.