Up-regulation of miRNA-151-3p enhanced the neuroprotective effect of dexmedetomidine against ß-amyloid by targeting DAPK-1 and TP53.

Alzheimer's disease (AD) is the most common cause of dementia and is the leading lethal disease among the elderly. Dexmedetomidine (Dex) has been reported to have multiple neuroprotective effects, but its effect against beta-amyloid (Aß) has not been completely determined and understood. Dex can activate both α2 adrenoceptor/cAMP/PKA and imidazoline I receptors/ERK1/2 signals. To determine which signal is critical for the effect of Dex on Aß toxicity, we treated SH-SY5Y and PC12 cells with inhibitors of α2 adrenoceptor and ERK1/2. Dex suppressed the apoptosis of neuronal cells and production of reactive oxygen species induced by Aß. These suppressive effects were attenuated by both inhibitors. As indicated by western blot, Dex stimulates both pro-apoptosis (activating death-associated protein kinase 1 [DAPK-1] and p53) and anti-apoptotic (up-regulating bcl-2 and bcl-xL) signals in Aß-treated neuronal cells. This effect is likely associated with ERK1/2 signaling because ERK1/2 inhibitor disrupts the effect of Dex on these signals. To eliminate the pro-apoptotic effect of Dex while retaining its anti-apoptosis action, we screened miRNA-151-3p to target DAPK-1 and p53. Transfection with miRNA-151-3p mimics suppressed DAPK-1 and TP53 expression induced by Dex and increased Nrf-2 and SOD expression. More importantly, increasing miRNA-151-3p enhanced the anti-apoptotic and antioxidative effects of Dex in Aß-treated neuronal cells. Overall, this study revealed that Dex additionally stimulated pro-apoptosis signaling, although it suppressed Aß-induced apoptosis of neuronal cells. miRNA-151-3p enhanced the neuroprotective effect of Dex against Aß by targeting DAPK-1 and TP53.

Reprofiling of pyrimidine-based DAPK1/CSF1R dual inhibitors: identification of 2,5-diamino-4-pyrimidinol derivatives as novel potential anticancer lead compounds.

Hybridization of reported weakly active antiproliferative hit 5-amino-4-pyrimidinol derivative with 2-anilino-4-phenoxypyrimidines suggests a series of 2,5-diamino-4-pyrimidinol derivatives as potential antiproliferative agents. Few compounds belonging to the proposed series were reported as CSF1R/DAPK1 inhibitors as anti-tauopathies. However, the correlation between CSF1R/DAPK1 signalling pathways and cancer progression provides motives to reprofile them against cancer therapy. The compounds were synthesised, characterized, and evaluated against M-NFS-60 cells and a kinase panel which bolstered predictions of their antiproliferative activity and suggested the involvement of diverse molecular targets. Compound 6e, the most potent in the series, showed prominent broad-spectrum antiproliferative activity inhibiting the growth of hematological, NSCLC, colon, CNS, melanoma, ovarian, renal, prostate and breast cancers by 84.1, 52.79, 72.15, 66.34, 66.48, 51.55, 55.95, 61.85, and 60.87%, respectively. Additionally, it elicited an IC50 value of 1.97 µM against M-NFS-60 cells and good GIT absorption with Pe value of 19.0 ± 1.1 × 10-6 cm/s (PAMPA-GIT). Molecular docking study for 6e with CSF1R and DAPK1 was done to help to understand the binding mode with both kinases. Collectively, compound 6e could be a potential lead compound for further development of anticancer therapies.

Death-associated protein kinase (DAPK) family modulators: Current and future therapeutic outcomes.

Serine/threonine kinases (STKs) represent the majority of discovered kinases to date even though a few Food and Drug Administration approved STKs inhibitors are reported. The third millennium came with the discovery of an important group of STKs that reshaped our understanding of several biological signaling pathways. This family was named death-associated protein kinase family (DAPK family). DAPKs comprise five members (DAPK1, DAPK2, DAPK3, DRAK1, and DRAK2) and belong to the calcium/calmodulin-dependent kinases domain. As time goes on, the list of biological functions of this family is constantly updated. The most extensively studied member is DAPK1 (based on the publications number and Protein Data Bank reported crystal structures) that plays fundamental biological roles depending on the cellular context. DAPK1 regulates apoptosis, autophagy, contributes to the pathogenesis of Alzheimer's disease, acts as a tumor suppressor, inhibits metastasis, mediates the body responses to viral infections, and regulates the synaptic plasticity and depression. For their biological roles, several DAPKs' modulators have been reported for treatment of many diseases as well as acting as probe compounds to facilitate the understanding of the biological functions elicited by this family. Despite that, the number of reported modulators is still limited and more research needs to be conducted on the discovery of novel strategies to activate or inhibit this family. In this report, we aim at drawing more attention to this family by reviewing the recent updates regarding the structure, biological roles, and regulation of this family. In addition, the small-molecule modulators of this family are reviewed in details with their potential therapeutic outcomes evaluated to help medicinal chemists develop more potent and selective possible drug candidates.

MicroRNA-98 attenuates cardiac ischemia-reperfusion injury through inhibiting DAPK1 expression.

Cardiovascular ischemic disease is a large class of diseases that are harmful to human health. The significant role of microRNAs (miRNAs) in terms of controlling cardiac injury has been reported in latest studies. MiR-98 is very important in regulating the apoptosis, the differentiation, the growth as well as the metastasis of cells. Nevertheless, the effect of miR-98 in the cardiac ischemia reperfusion (I/R) injury has rarely been investigated. In the current research, we found that the miR-98 expression was down-regulated in the cardiomyocytes subjected to hypoxia/reoxygenation (H/R) and in the myocardium of the I/R rats. In addition, over-expression of miR-98 could significantly reduce the myocardial oxidative stress and ischemic injury as well as cell apoptosis. In agreement, similar findings were demonstrated in H9c2 cells subjected to H/R injury. Bioinformatic analysis using MiRanda and TargetScan and luciferase activity assay confirmed death-associated protein kinase 1 (DAPK1) as a direct target of miR-98. These findings suggest that miR-98 may be exploited as a novel molecular marker or therapeutic target for myocardial I/R injury. © 2018 IUBMB Life, 71(1):166-176, 2019.

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.

Inhibition of death-associated protein kinase 1 attenuates the phosphorylation and amyloidogenic processing of amyloid precursor protein.

Extracellular deposition of amyloid-beta (Aß) peptide, a metabolite of sequential cleavage of amyloid precursor protein (APP), is a critical step in the pathogenesis of Alzheimer's disease (AD). While death-associated protein kinase 1 (DAPK1) is highly expressed in AD brains and its genetic variants are linked to AD risk, little is known about the impact of DAPK1 on APP metabolism and Aß generation. In this study, we demonstrated a novel effect of DAPK1 in the regulation of APP processing using cell culture and mouse models. DAPK1, but not its kinase deficient mutant (K42A), significantly increased human Aß secretion in neuronal cell culture models. Moreover, knockdown of DAPK1 expression or inhibition of DAPK1 catalytic activity significantly decreased Aß secretion. Furthermore, DAPK1, but not K42A, triggered Thr668 phosphorylation of APP, which may initiate and facilitate amyloidogenic APP processing leading to the generation of Aß. In Tg2576 APPswe-overexpressing mice, knockout of DAPK1 shifted APP processing toward non-amyloidogenic pathway and decreased Aß generation. Finally, in AD brains, elevated DAPK1 levels showed co-relation with the increase of APP phosphorylation. Combined together, these results suggest that DAPK1 promotes the phosphorylation and amyloidogenic processing of APP, and that may serve a potential therapeutic target for AD.

Novel Functions of Death-Associated Protein Kinases through Mitogen-Activated Protein Kinase-Related Signals.

Death associated protein kinase (DAPK) is a calcium/calmodulin-regulated serine/threonine kinase; its main function is to regulate cell death. DAPK family proteins consist of DAPK1, DAPK2, DAPK3, DAPK-related apoptosis-inducing protein kinases (DRAK)-1 and DRAK-2. In this review, we discuss the roles and regulatory mechanisms of DAPK family members and their relevance to diseases. Furthermore, a special focus is given to several reports describing cross-talks between DAPKs and mitogen-activated protein kinases (MAPK) family members in various pathologies. We also discuss small molecule inhibitors of DAPKs and their potential as therapeutic targets against human diseases.

Overexpression of microRNA-133a inhibits ischemia-reperfusion-induced cardiomyocyte apoptosis by targeting DAPK2.

To examine microRNA-133a (miR-133a) endogenous expression in cardiomyocytes after ischemia-reperfusion (I/R) injury and study the effects of miR-133a overexpression on I/R injury-induced cardiomyocyte apoptosis. Dual-Luciferase Reporter Assay detected dynamic expression of miR-133a. In an in vitro hypoxia-reoxygenation (HR) injury model and an in vivo rat model of I/R injury, rat cardiomyocytes were transfected with miR-133a mimic to test the effects of miR-133a overexpression on apoptosis. MiR-133a and Death Associated Protein Kinase 2 (DAPK2) mRNA expression was measured using real-time-PCR, and DAPK2 protein expression was detected by western blotting. Annexin V-fluorescein isothiocyanate/propidium iodide (PI) double-staining measured the apoptosis rate in H9C2 cells and transferase dUTP nick end labeling assay quantified the cardiomyocyte apoptosis rate in tissues obtained from in vivo the rat model. DAPK2 is a target of miR-133a. Both in vitro and in vivo results confirmed that after expression of miR-133a mimics, miR-133a levels increased, which was accompanied by decrease in DAPK2 mRNA and protein expression. In H9C2 cells, HR injury caused a sharp decrease in miR-133a expression and a significant upregualtion of DAPK2 mRNA and protein levels. However, exogenous miR-133a expression led to a significant reduction in DAPK2 mRNA and protein levels despite HR injury. Similar results were obtained from in vivo I/R injury model. After HR injury or I/R injury the apoptosis rate of myocardial cells was highly elevated and decreased significantly only after transfection of miR-133a into cardiomyocytes. MiR-133a overexpression may inhibit I/R injury-mediated cardiomyocyte apoptosis by targeting DAPK2, leading to reduced DAPK2 protein, thus miR-133a may potentially have a high therapeutic value in I/R injury.

Synthesis of 6,8,9 poly-substituted purine analogue libraries as pro-apoptotic inducers of human leukemic lymphocytes and DAPK-1 inhibitors.

A 18-member library of 6,8,9-poly-substituted purines was prepared from pyrimidines, primary alcohols, and N,N-dimethylamides under basic conditions via a novel one-pot synthetic pathway controlled by amide sizes and the novel analogues were tested against two leukemia cell lines: Jurkat (acute T cell leukemia) and K562 (chronic erythroleukemia) cells. Compounds having a benzoxy group at C6 position of the aromatic ring exhibited antiproliferative activity in Jurkat cells whereas all compounds induced a lower effect on K562 cells. Analysis of cell cycle, Annexin-V staining, and cleavage of initiator caspases assays showed that the active purine analogues induce cell death by apoptosis. Based on these results, a new purine derivative was synthesized, 6-benzyloxy-9-tert-butyl-8-phenyl-9H-purine (6d), which displayed the highest activity of the series against Jurkat cell lines. Finally, (33)P-radiolabeled kinase assays using 96 recombinant human kinases known to be involved in apoptotic events were performed. Just one of the kinases tested, DAPK-1, was inhibited 50% or more by the phenotypic hits at 10 µM, suggesting that the inhibition of this target could be responsible for the induction of cell death by apoptosis. In agreement with the phenotypic results, the most active antiproliferative agent, 6d, displayed also the lowest IC50 value against recombinant DAPK1 (2.5 µM), further supporting the potential role of this protein on the observed functional response. DAPK-1 inhibition led by 6d together with its pro-apoptotic properties against the Jurkat line makes it an interesting candidate to further investigate the role of DAPK1 kinase in triggering apoptosis in cancer cells, a role which is attracting recent interest.

De Novo Fragment Design for Drug Discovery and Chemical Biology.

Automated molecular de novo design led to the discovery of an innovative inhibitor of death-associated protein kinase 3 (DAPK3). An unprecedented crystal structure of the inactive DAPK3 homodimer shows the fragment-like hit bound to the ATP pocket. Target prediction software based on machine learning models correctly identified additional macromolecular targets of the computationally designed compound and the structurally related marketed drug azosemide. The study validates computational de novo design as a prime method for generating chemical probes and starting points for drug discovery.

Intervention of death-associated protein kinase 1-p53 interaction exerts the therapeutic effects against stroke.

BACKGROUND AND PURPOSE: Death-associated protein kinase 1 (DAPK1) interacts with the tumor suppressor gene p53 via a direct binding of a death domain of DAPK1 to a DNA-binding motif (DM) of p53 (p53DM) and converges multiple cell death pathways in stroke. The goals of this study are to determine whether disruption of DAPK1-p53 interaction is therapeutically effective against stroke. METHODS: We synthesized a membrane-permeable p53DM peptide (Tat-p53DM) and tested the therapeutic effects of Tat-p53DM in a mouse model with stroke. RESULTS: We showed that Tat-p53DM blocked DAPK1-p53 interaction in brain cells in vivo. When administered 6 hours after stroke onset in adult male mice, Tat-p53DM was still therapeutically effective against brain damages and improved neurological functions. CONCLUSIONS: DAPK1-p53 interaction is a preferred target for therapeutic intervention of stroke.

Death-associated protein kinase 3 mediates vascular structural remodelling via stimulating smooth muscle cell proliferation and migration.

Death-associated protein kinase 3 (DAPK3) also known as zipper-interacting kinase is a serine/threonine kinase that mainly regulates cell death and smooth muscle contraction. We have previously found that protein expression of DAPK3 increases in the mesenteric artery from spontaneously hypertensive rats (SHRs) and that DAPK3 mediates the development of hypertension in SHRs partly through promoting reactive oxygen species-dependent vascular inflammation. However, it remains to be clarified how DAPK3 controls smooth muscle cell (SMC) proliferation and migration, which are also important processes for hypertension development. We, therefore, sought to investigate whether DAPK3 affects SMC proliferation and migration. siRNA against DAPK3 significantly inhibited platelet-derived growth factor (PDGF)-BB-induced SMC proliferation and migration as determined by bromodeoxyuridine (BrdU) incorporation and a cell counting assay as well as a Boyden chamber assay respectively. DAPK3 siRNA or a pharmacological inhibitor of DAPK3 inhibited PDGF-BB-induced lamellipodia formation as determined by rhodamine-phalloidin staining. DAPK3 siRNA or the DAPK inhibitor significantly reduced PDGF-BB-induced activation of p38 and heat-shock protein 27 (HSP27) as determined by Western blotting. In ex vivo studies, PDGF-BB-induced SMC out-growth was significantly inhibited by the DAPK inhibitor. In vivo, the DAPK inhibitor significantly prevented carotid neointimal hyperplasia in a mouse ligation model. The present results, for the first time, revealed that DAPK3 mediates PDGF-BB-induced SMC proliferation and migration through activation of p38/HSP27 signals, which may lead to vascular structural remodelling including neointimal hyperplasia. The present study suggests DAPK3 as a novel pharmaceutical target for the prevention of hypertensive cardiovascular diseases.

Death associated protein kinases: molecular structure and brain injury.

Perinatal brain damage underlies an important share of motor and neurodevelopmental disabilities, such as cerebral palsy, cognitive impairment, visual dysfunction and epilepsy. Clinical, epidemiological, and experimental studies have revealed that factors such as inflammation, excitotoxicity and oxidative stress contribute considerably to both white and grey matter injury in the immature brain. A member of the death associated protein kinase (DAPk) family, DAPk1, has been implicated in cerebral ischemic damage, whereby DAPk1 potentiates NMDA receptor-mediated excitotoxicity through interaction with the NR2BR subunit. DAPk1 also mediate a range of activities from autophagy, membrane blebbing and DNA fragmentation ultimately leading to cell death. DAPk mRNA levels are particularly highly expressed in the developing brain and thus, we hypothesize that DAPk1 may play a role in perinatal brain injury. In addition to reviewing current knowledge, we present new aspects of the molecular structure of DAPk domains, and relate these findings to interacting partners of DAPk1, DAPk-regulation in NMDA-induced cerebral injury and novel approaches to blocking the injurious effects of DAPk1.

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.

MiR-141 attenuates sepsis-induced cardiomyopathy by targeting DAPK1.

OBJECTIVE: To discuss the possible effects of microRNA-141 (miR-141) in sepsis-induced cardiomyopathy (SIC) via targeting death-associated protein kinase 1 (DAPK1). METHODS: An SIC mouse model was constructed by abdominal injection of lipopolysaccharide (LPS) and divided into control, LPS, LPS + pre-miR-141, and LPS + anti-miR-141 groups. Hemodynamic indicators and heart function indexes of mice were detected. ELISA was used to determine the serum levels of inflammatory cytokines, while TUNEL staining to observe the apoptosis of myocardial cells of mice, as well as qRT-PCR and Western blotting to clarify the expression of miR-141 and DAPK1. Lastly, in vitro experiment was also conducted on the primary neonatal rat ventricular cardiomyocytes (NRVCMs) to validate the results. RESULTS: Mice in the LPS group, as compared to the control group, had lower left ventricular ejection fraction, left ventricular fractional shortening, left ventricular systolic pressure, and ±dp/dt, but a higher left ventricular end-diastolic pressure, while the serum expression of IL-1ß, IL-6, TNF-α, and cTn-T was up-regulated evidently with the increased apoptotic index of myocardial tissues. However, miR-141 and Bcl-2/Bax were down-regulated with elevated DAPK1 and cleaved caspase-3. The above changes were ameliorated in mice from the LPS + pre-miR-141 group relative to the LPS group, while those in the LPS + anti-miR-141 group were further deteriorated. In vitro experiment showed that miR-141 overexpression could reduce the apoptosis of LPS-induced NRVCMs and the levels of inflammatory cytokines with the increased cell viability. CONCLUSION: MiR-141 could decrease inflammatory response and reduce myocardial cell apoptosis by targeting DAPK1, thereby playing the promising protective role in SIC.

Inhibition of Death-associated Protein Kinase 1 protects against Epileptic Seizures in mice.

Epilepsy is a chronic encephalopathy and one of the most common neurological disorders. Death-associated protein kinase 1 (DAPK1) expression has been shown to be upregulated in the brains of human epilepsy patients compared with those of normal subjects. However, little is known about the impact of DAPK1 on epileptic seizure conditions. In this study, we aim to clarify whether and how DAPK1 is regulated in epilepsy and whether targeting DAPK1 expression or activity has a protective effect against epilepsy using seizure animal models. Here, we found that cortical and hippocampal DAPK1 activity but not DAPK1 expression was increased immediately after convulsive pentylenetetrazol (PTZ) exposure in mice. However, DAPK1 overexpression was found after chronic low-dose PTZ insults during the kindling paradigm. The suppression of DAPK1 expression by genetic knockout significantly reduced PTZ-induced seizure phenotypes and the development of kindled seizures. Moreover, pharmacological inhibition of DAPK1 activity exerted rapid antiepileptic effects in both acute and chronic epilepsy mouse models. Mechanistically, PTZ stimulated the phosphorylation of NR2B through DAPK1 activation. Combined together, these results suggest that DAPK1 regulation is a novel mechanism for the control of both acute and chronic epilepsy and provide new therapeutic strategies for the treatment of human epilepsy.

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.

Presynaptic Caytaxin prevents apoptosis via deactivating DAPK1 in the acute phase of cerebral ischemic stroke.

Death-associated protein kinase 1 (DAPK1) is a key protein that mediates neuronal death in ischemic stroke. Although the substrates of DAPK1 and molecular signal in stroke have been gradually discovered, the modulation of DAPK1 itself is still unclear. Here we first reveal that Caytaxin, a brain-specific member of BCL2/adenovirus E1B -interacting protein (BNIP-2), increases and interacts with DAPK1 as early as 2 h after middle cerebral artery occlusion (MCAO) in the penumbra area of mouse brain. Furthermore, Caytaxin binds to DAPK1 at the presynaptic site and inhibits DAPK1 catalytic activity. Silencing Caytaxin by Caytaxin shRNA (Sh-Caytaxin) enhances DAPK1 activity, deteriorates neuronal apoptosis and brain injuries both in vivo and in vitro. Thus, elevating presynaptic Caytaxin could prevent neuronal apoptosis by inhibiting DAPK1 activation in the acute stage of ischemic stroke. Caytaxin may physiologically protect neuronal cells and represent a potential prevention and therapeutic target in the early phase of cerebral ischemic stroke.

Protopanaxadiol ginsenoside Rd protects against NMDA receptor-mediated excitotoxicity by attenuating calcineurin-regulated DAPK1 activity.

Neuroprotective strategies in the treatment of stroke have been attracting a great deal of attentions. Our previous clinical and basic studies have demonstrated that protopanaxadiol ginsenoside-Rd (Rd), a monomer compound extracted from Panax ginseng or Panax notoginseng, has neuroprotective effects against ischemic stroke, probably due to its ability to block Ca2+ overload, an usual consequence of the overactivation of NMDA receptor (NMDAR). As an extending study, we explored here whether Rd exerted its neuroprotection as a novel NMDAR blocker. Our whole-cell patch-clamp results showed that Rd reduced NMDAR currents of cultured rat cortical neurons (EC50 = 7.7 µM) dose-dependently by acting on extrasynaptic NMDAR NR2b subunit. However, unexpectedly, cell transfection and radioligand binding assays revealed that Rd did not bind to the NMDAR channel directly. Alternatively, it inhibited the phosphorylation of NR2b at Ser-1303, a target of death associated protein kinase 1 (DAPK1). Moreover, cell-based and cell-free enzymatic assays showed that Rd did not inhibit the activity of DAPK1 directly, but blocked the activity of calcineurin, a key phosphatase for activating DAPK1. Importantly, other protopanaxadiol ginsenosides were also found to have potential inhibitory effects on calcineurin activity. Furthermore, as expected, calcineurin inhibition by cyclosporin A could mimic Rd's effects and protect against NMDA-, oxygen glucose deprivation- or transient ischemic stroke-induced neuronal injury. Therefore, our present study provided the first evidence that Rd could exert an inhibitive effect on NMDAR-triggered currents and sequential excitotoxicity through mitigation of DAPK1-mediated NR2b phosphorylation by attenuating calcineurin activity.