Design and Characterization of a Novel eEF2K Degrader with Potent Therapeutic Efficacy Against Triple-Negative Breast Cancer.

Dysregulated eEF2K expression is implicated in the pathogenesis of many human cancers, including triple-negative breast cancer (TNBC), making it a plausible therapeutic target. However, specific eEF2K inhibitors with potent anti-cancer activity have not been available so far. Targeted protein degradation has emerged as a new strategy for drug discovery. In this study, a novel small molecule chemical is designed and synthesized, named as compound C1, which shows potent activity in degrading eEF2K. C1 selectively binds to F8, L10, R144, C146, E229, and Y236 of the eEF2K protein and promotes its proteasomal degradation by increasing the interaction between eEF2K and the ubiquitin E3 ligase ßTRCP in the form of molecular glue. C1 significantly inhibits the proliferation and metastasis of TNBC cells both in vitro and in vivo and in TNBC patient-derived organoids, and these antitumor effects are attributed to the degradation of eEF2K by C1. Additionally, combination treatment of C1 with paclitaxel, a commonly used chemotherapeutic drug, exhibits synergistic anti-tumor effects against TNBC. This study not only generates a powerful research tool to investigate the therapeutic potential of targeting eEF2K, but also provides a promising lead compound for developing novel drugs for the treatment of TNBC and other cancers.

Eukaryotic elongation factor 2 kinase inhibitor, A484954 induced diuresis via nitric oxide production in spontaneously hypertensive rats.

Eukaryotic elongation factor 2 (eEF2) kinase (eEF2K) is a protein kinase that inactivates eEF2, a protein that mediates a peptidyl-tRNA translocation during an elongation step of protein synthesis. We have previously shown that eEF2K was involved in pathogenesis of essential and pulmonary hypertension. A484954 (7-amino-1-cyclopropyl-3-ethyl-2,4-dioxo-1,2,3,4-tetrahydropyrido[2,3-d] pyrimidine-6-carboxamide), a selective eEF2K inhibitor, is a membrane permeable small molecule. We have previously shown that A484954 lowered blood pressure and induced diuretic effects in spontaneously hypertensive rats (SHR) due to an increase in renal blood flow. Here we aimed to reveal mechanisms underlying the diuretic effects of A484954 in SHR. A484954-induced diuresis and increase in urinary Na+ excretion were inhibited by N (G)-nitro-L-arginine methyl ester (L-NAME), a nitric oxide (NO) synthase inhibitor. A484954 increased mRNA expression of angiotensin type 2 receptor (AT2R) and nuclear factor-erythroid 2-related factor 2 (Nrf2). In summary, we for the first time revealed that A484954 induces diuresis in SHR at least partly via the activation of NO/Nrf2/AT2R pathway.

Structure of the complex between calmodulin and a functional construct of eukaryotic elongation factor 2 kinase bound to an ATP-competitive inhibitor.

The calmodulin-activated α-kinase, eukaryotic elongation factor 2 kinase (eEF-2K), serves as a master regulator of translational elongation by specifically phosphorylating and reducing the ribosome affinity of the guanosine triphosphatase, eukaryotic elongation factor 2 (eEF-2). Given its critical role in a fundamental cellular process, dysregulation of eEF-2K has been implicated in several human diseases, including those of the cardiovascular system, chronic neuropathies, and many cancers, making it a critical pharmacological target. In the absence of high-resolution structural information, high-throughput screening efforts have yielded small-molecule candidates that show promise as eEF-2K antagonists. Principal among these is the ATP-competitive pyrido-pyrimidinedione inhibitor, A-484954, which shows high specificity toward eEF-2K relative to a panel of "typical" protein kinases. A-484954 has been shown to have some degree of efficacy in animal models of several disease states. It has also been widely deployed as a reagent in eEF-2K-specific biochemical and cell-biological studies. However, given the absence of structural information, the precise mechanism of the A-484954-mediated inhibition of eEF-2K has remained obscure. Leveraging our identification of the calmodulin-activatable catalytic core of eEF-2K, and our recent determination of its long-elusive structure, here we present the structural basis for its specific inhibition by A-484954. This structure, which represents the first for an inhibitor-bound catalytic domain of a member of the α-kinase family, enables rationalization of the existing structure-activity relationship data for A-484954 variants and lays the groundwork for further optimization of this scaffold to attain enhanced specificity/potency against eEF-2K.

Discovery of New Inhibitors of eEF2K from Traditional Chinese Medicine Based on In Silico Screening and In Vitro Experimental Validation.

Eukaryotic elongation factor 2 kinase (eEF2K) is a highly conserved α kinase and is increasingly considered as an attractive therapeutic target for cancer as well as other diseases. However, so far, no selective and potent inhibitors of eEF2K have been identified. In this study, pharmacophore screening, homology modeling, and molecular docking methods were adopted to screen novel inhibitor hits of eEF2K from the traditional Chinese medicine database (TCMD), and then cytotoxicity assay and western blotting were performed to verify the validity of the screen. Resultantly, after two steps of screening, a total of 1077 chemicals were obtained as inhibitor hits for eEF2K from all 23,034 compounds in TCMD. Then, to verify the validity, the top 10 purchasable chemicals were further analyzed. Afterward, Oleuropein and Rhoifolin, two reported antitumor chemicals, were found to have low cytotoxicity but potent inhibitory effects on eEF2K activity. Finally, molecular dynamics simulation, pharmacokinetic and toxicological analyses were conducted to evaluate the property and potential of Oleuropein and Rhoifolin to be drugs. Together, by integrating in silico screening and in vitro biochemical studies, Oleuropein and Rhoifolin were revealed as novel eEF2K inhibitors, which will shed new lights for eEF2K-targeting drug development and anticancer therapy.

Eukaryotic elongation factor 2 kinase inhibitor, A484954 inhibits perivascular sympathetic nerve stimulation-induced vasoconstriction in isolated renal artery.

Eukaryotic elongation factor 2 (eEF2) kinase (eEF2K) repressively regulates protein translation through phosphorylating eEF2. We previously showed that expression and activity of eEF2K are increased in isolated mesenteric arteries from spontaneously hypertensive rats (SHR) contributing to development of essential hypertension. Furthermore, we have recently shown that 7-Amino-1-cyclopropyl-3-ethyl-1,2,3,4-tetrahydro-2,4-dioxopyrido[2,3-d]pyrimidine-6-carboxamide (A484954), a selective eEF2K inhibitor, induces endothelium-dependent relaxation in isolated mesenteric arteries from SHR inducing an antihypertensive effect. In order to test the hypothesis that inhibition of eEF2K activity induces vasodilatation by suppressing sympathetic nerve activity, we examined the effects of A484954 on perivascular sympathetic nerve stimulation-induced contraction in isolated renal artery from normotensive and hypertensive rats. Electrodes were placed near the isolated renal arteries that were applied with transmural nerve stimulation (TNS). Then, contraction of the arteries was isometrically measured. A484954 inhibited TNS-induced contraction. The A484954-mediated inhibition of TNS-induced contraction was significantly prevented by NG-nitro-L-arginine methyl ester. In SHR isolated renal artery, TNS-induced contraction was enhanced compared with normotensive Wistar rats. Furthermore, A484954-mediated inhibition of TNS-induced contraction in SHR was enhanced compared with Wistar rats. In conclusion, this study demonstrates for the first time that A484954 inhibits perivascular sympathetic nerve stimulation-induced vasoconstriction at least in part perhaps through nitric oxide (NO) release from NO-operating nerve.

EEF2K silencing inhibits tumour progression through repressing SPP1 and synergises with BET inhibitors in melanoma.

BACKGROUND: Despite the remarkable breakthroughs achieved in the management of metastatic melanoma using immunotherapy and targeted therapies, long-term clinical efficacy is often compromised due to dose-limiting toxicity and innate or acquired resistance. Therefore, it is of vital importance to further explore the molecular mechanisms underlying melanoma progression and identify new targeted therapeutic approaches. METHODS: The function of eukaryotic elongation factor-2 kinase (EEF2K) in melanoma were investigated in vitro and in vivo. RNA-seq and chromatin immunoprecipitation (ChIP) assay were undertaken to explore the mechanisms. The antitumor effect of bromodomain and extra terminal domain (BET) inhibitors combined with cytarabine were assessed in melanoma both in vitro and in vivo. RESULTS: EEF2K silencing markedly attenuated the malignant phenotypes of melanoma cells, including proliferation, migration, invasion and metastasis. In contrast, EEF2K overexpression promoted melanoma cell proliferation, migration and invasion. Mechanistically, we demonstrated that EEF2K upregulates the phosphorylation of STAT3 (p-STAT3) at Tyr705, which binds to the promoter region of SPP1 and enhances its transcription, thus facilitating melanoma progression. Transfection-induced re-expression of SPP1 partly negated the inhibitory effect of EEF2K silencing on melanoma, whereas inhibition of SPP1 or STAT3 significantly abolished the efficacy of EEF2K on melanoma cells. Intriguingly, EEF2K silencing combined with BET inhibitor treatment further inhibited cell proliferation and promoted apoptosis in melanoma. We further screened the US FDA-approved antitumour drug library and identified cytarabine as a potential clinically applicable EEF2K inhibitor that could synergise with BET inhibitors in melanoma treatment. CONCLUSION: EEF2K/p-STAT3/SPP1 may be a novel oncogenic pathway in melanoma progression, which could be a target for novel combination therapy for melanoma.

Eukaryotic elongation factor 2 kinase inhibitor, A484954 induces diuretic effect via renal vasorelaxation in spontaneously hypertensive rats.

Eukaryotic elongation factor 2 (eEF2) kinase (eEF2K), alternatively known as calmodulin-dependent protein kinase III, inhibits protein translation via phosphorylating its sole substrate, eEF2. We previously demonstrated that expression and activity of eEF2K change in mesenteric artery from spontaneously hypertensive rats (SHR) with aging and that eEF2K is involved in pathogenesis of essential hypertension. In addition, we have recently revealed that acute intravenous injection with A484954, a selective eEF2K inhibitor, lowers blood pressure specifically in SHR partly via inducing vasorelaxation. In this study, we examined whether A484954 induces diuretic effect. After male SHR and normotensive Wistar Kyoto rats (WKY) were given a single intraperitoneal injection of A484954 (2.5 mg/kg, 0.5-9 h), urine was collected using metabolic cage. Contraction of isolated renal arteries form SHR was isometrically measured. While A484954 did not induce diuretic effect in WKY, it increased urine output, water intake, and urinary sodium excretion in SHR. A484954 (10 µM) induced vasorelaxation in isolated renal arteries, which was inhibited by a ß-adrenergic receptor antagonist, propranolol. It was confirmed that A484954 increased renal blood flow in SHR as measured by renal ultrasonography. In summary, it was for the first time revealed that A484954 induces diuretic effect in SHR at least partly via renal vasorelaxation through ß-adrenergic receptor.

Novel inhibitors of eukaryotic elongation factor 2 kinase: In silico, synthesis and in vitro studies.

Eukaryotic elongation factor 2 kinase (eEF2K) is an unusual alpha kinase whose expression is highly upregulated in various cancers and contributes to tumor growth, metastasis, and progression. More importantly, eEF2K expression is associated with poor clinical outcome and shorter patient survival in breast, lung and ovarian cancers. Therefore, eEF2K is an emerging molecular target for development of novel targeted therapeutics and precision medicine in solid cancers. Currently, there are not any available potent and specific eEF2K inhibitors for clinical translation. In this study, we designed and synthesized a series of novel compounds with coumarin scaffold with various substitutions and investigated their effects in inhibiting eEF2K activity using in silico approaches and in vitro studies in breast cancer cells. We utilized an amide substitution at position 3 on the coumarin ring with their pharmacologically active groups containing pyrrolidine, piperidine, morpholine and piperazine groups with (CH2)2 bridged for aliphatic amides. Due to their ability to form covalent binding to the target enzyme, we also investigated the effects of boron containing groups on functionalized coumarin ring (3 compounds) and designed novel aliphatic and aromatic derivatives of coumarin scaffolds (10 compounds) and phenyl ring with boron groups (4 compounds). The Glide/SP module of the Maestro molecular modeling package was used to perform in silico analysis and molecular docking studies. According to our combined results, structure activity relationship (SAR) was performed in detail. Among the newly designed, synthesized, and tested compounds, our in vitro findings revealed that several compounds displayed a highly effective eEF2K inhibition at submicromolar concentrations in in vitro breast cancer cells. In conclusion, we identified novel compounds that can be used as eEF2K inhibitors and that they should be further evaluated by in vivo preclinical tumor models studies for antitumor efficacy and clinical translation.

Inhibiting Eukaryotic Elongation Factor 2 Kinase: An Update on Pharmacological Small-Molecule Compounds in Cancer.

Eukaryotic elongation factor 2 kinase (eEF2K), a member of the atypical protein kinase family of alpha-kinases, is well-known as a negative regulator of protein synthesis by phosphorylating eEF2. Notably, eEF2K functions as a key regulator of several cellular processes, leading to tumorigenesis. To date, some small-molecule compounds have been reported as potential eEF2K inhibitors in cancer drug discovery. However, an ideal targeted drug design still faces huge challenges. Alternatively, other design strategies, such as repurposed drugs, dual-target drugs, and drug combination strategies, provide insights into the improvement of cancer treatment. Here, we summarize the crucial eEF2K-modulating pathways in cancer, including AMPK, REDD1, and Src. Moreover, we discuss the inhibition of eEF2K with single-target inhibitors, repurposed drugs, dual-target inhibitors, drug combination strategies, and other emerging technologies for therapeutic purposes. Together, these inspiring findings provide insights into a promising strategy for inhibiting eEF2K with small-molecule compounds to improve potential cancer therapy.

Inhibition of eEF2K synergizes with glutaminase inhibitors or 4EBP1 depletion to suppress growth of triple-negative breast cancer cells.

The eukaryotic elongation factor-2 kinase, eEF2K, which restricts protein translation elongation, has been identified as a potential therapeutic target for diverse types of malignancies including triple negative breast cancer (TNBC). However, the contexts in which eEF2K inhibition is essential in TNBC and its consequences on the proteome are largely unknown. Here we show that genetic or pharmacological inhibition of eEF2K cooperated with glutamine (Gln) starvation, and synergized with glutaminase (GLS1) inhibitors to suppress growth of diverse TNBC cell lines. eEF2K inhibition also synergized with depletion of eukaryotic translation initiation factor 4E-binding protein 1 (eIF4EBP1; 4EBP1), a suppressor of eukaryotic protein translation initiation factor 4E (eIF4E), to induce c-MYC and Cyclin D1 expression, yet attenuate growth of TNBC cells. Proteomic analysis revealed that whereas eEF2K depletion alone uniquely induced Cyclin Dependent Kinase 1 (CDK1) and 6 (CDK6), combined depletion of eEF2K and 4EBP1 resulted in overlapping effects on the proteome, with the highest impact on the 'Collagen containing extracellular matrix' pathway (e.g. COL1A1), as well as the amino-acid transporter, SLC7A5/LAT1, suggesting a regulatory loop via mTORC1. In addition, combined depletion of eEF2K and 4EBP1 indirectly reduced the levels of IFN-dependent innate immune response-related factors. Thus, eEF2K inhibition triggers cell cycle arrest/death under unfavourable metabolic conditions such as Gln-starvation/GLS1 inhibition or 4EBP1 depletion, uncovering new therapeutic avenues for TNBC and underscoring a pressing need for clinically relevant eEF2K inhibitors.

Neuroprotective Effect of Kinase Inhibition in Ischemic Factor Modeling In Vitro.

The contribution of many neuronal kinases to the adaptation of nerve cells to ischemic damage and their effect on functional neural network activity has not yet been studied. The aim of this work is to study the role of the four kinases belonging to different metabolic cascades (SRC, Ikkb, eEF2K, and FLT4) in the adaptive potential of the neuron-glial network for modeling the key factors of ischemic damage. We carried out a comprehensive study on the effects of kinases blockade on the viability and network functional calcium activity of nerve cells under ischemic factor modeling in vitro. Ischemic factor modelling was performed on day 14 of culturing primary hippocampal cells obtained from mouse embryos (E18). The most significant neuroprotective effect was shown in the blockade of FLT4 kinase in the simulation of hypoxia. The studies performed revealed the role of FLT4 in the development of functional dysfunction in cerebrovascular accidents and created new opportunities for the study of this enzyme and its blockers in the formation of new therapeutic strategies.

Progress in the Development of Eukaryotic Elongation Factor 2 Kinase (eEF2K) Natural Product and Synthetic Small Molecule Inhibitors for Cancer Chemotherapy.

Eukaryotic elongation factor 2 kinase (eEF2K or Ca2+/calmodulin-dependent protein kinase, CAMKIII) is a new member of an atypical α-kinase family different from conventional protein kinases that is now considered as a potential target for the treatment of cancer. This protein regulates the phosphorylation of eukaryotic elongation factor 2 (eEF2) to restrain activity and inhibit the elongation stage of protein synthesis. Mounting evidence shows that eEF2K regulates the cell cycle, autophagy, apoptosis, angiogenesis, invasion, and metastasis in several types of cancers. The expression of eEF2K promotes survival of cancer cells, and the level of this protein is increased in many cancer cells to adapt them to the microenvironment conditions including hypoxia, nutrient depletion, and acidosis. The physiological function of eEF2K and its role in the development and progression of cancer are here reviewed in detail. In addition, a summary of progress for in vitro eEF2K inhibitors from anti-cancer drug discovery research in recent years, along with their structure-activity relationships (SARs) and synthetic routes or natural sources, is also described. Special attention is given to those inhibitors that have been already validated in vivo, with the overall aim to provide reference context for the further development of new first-in-class anti-cancer drugs that target eEF2K.

Fluoxetine regulates eEF2 activity (phosphorylation) via HDAC1 inhibitory mechanism in an LPS-induced mouse model of depression.

BACKGROUND: Selective serotonin reuptaker inhibitors, including fluoxetine, are widely studied and prescribed antidepressants, while their exact molecular and cellular mechanism are yet to be defined. We investigated the involvement of HDAC1 and eEF2 in the antidepressant mechanisms of fluoxetine using a lipopolysaccharide (LPS)-induced depression-like behavior model. METHODS: For in vivo analysis, mice were treated with LPS (2 mg/kg BW), fluoxetine (20 mg/kg BW), HDAC1 activator (Exifone: 54 mg/kg BW) and NH125 (1 mg/kg BW). Depressive-like behaviors were confirmed via behavior tests including OFT, FST, SPT, and TST. Cytokines were measured by ELISA while Iba-1 and GFAP expression were determined by immunofluorescence. Further, the desired gene expression was measured by immunoblotting. For in vitro analysis, BV2 cell lines were cultured; treated with LPS, exifone, and fluoxetine; collected; and analyzed. RESULTS: Mice treated with LPS displayed depression-like behaviors, pronounced neuroinflammation, increased HDAC1 expression, and reduced eEF2 activity, as accompanied by altered synaptogenic factors including BDNF, SNAP25, and PSD95. Fluoxetine treatment exhibited antidepressant effects and ameliorated the molecular changes induced by LPS. Exifone, a selective HDAC1 activator, reversed the antidepressant and anti-inflammatory effects of fluoxetine both in vivo and in vitro, supporting a causing role of HDAC1 in neuroinflammation allied depression. Further molecular mechanisms underlying HDAC1 were explored with NH125, an eEF2K inhibitor, whose treatment reduced immobility time, altered pro-inflammatory cytokines, and NLRP3 expression. Moreover, NH125 treatment enhanced eEF2 and GSK3ß activities, BDNF, SNAP25, and PSD95 expression, but had no effects on HDAC1. CONCLUSIONS: Our results showed that the antidepressant effects of fluoxetine may involve HDAC1-eEF2 related neuroinflammation and synaptogenesis.

Eukaryotic elongation factor 2 kinase inhibitor, A484954 lowered blood pressure in spontaneously hypertensive rats via inducing vasorelaxation.

Eukaryotic elongation factor 2 (eEF2) kinase (eEF2K) suppresses protein translation. We previously reported eEF2K expression was upregulated in mesenteric arteries (MA) from spontaneously hypertensive rats (SHR). We have recently revealed A484954, an eEF2K inhibitor, acutely suppressed vasopressor agonists-induced increase of blood pressure (BP) in normal Wistar rats. In this study, we examined the acute effects of A484954 on BP in SHR and explored underlying mechanisms. BP was measured by a carotid cannulation method in SHR. Isometric contraction in MA from SHR was measured. Endothelial nitric oxide synthase (eNOS) dimerization was measured by low-temperature sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting. A484954 lowered BP in 15-week-old SHR. A484954 induced relaxation in MA from both 4- and 7-9-week-old SHR. In MA from 4-week-old SHR, A484954-induced relaxation was inhibited almost completely by a NOS inhibitor, NG-nitro-l-arginine methyl ester (l-NAME) and significantly by a ß blocker, propranolol. In MA from 7-9-week-old SHR, on the other hand, A484954-induced relaxation was inhibited partly either by l-NAME, indomethacin, a cyclooxygenase inhibitor, or l-NAME + indomethacin. A484954 promoted the dimerization of eNOS in human endothelial cells. In summary, we have revealed A484954 lowers BP in SHR perhaps through the vasorelaxation via the production of endothelium-derived relaxing factors.

NMDA-induced nitric oxide generation and CREB activation in central nervous system is dependent on eukaryotic elongation factor 2 kinase.

The NMDA receptor is crucial to several functions in CNS physiology and some of its effects are mediated by promoting nitric oxide production from L-arginine and activation of signaling pathways and the transcription factor CREB. Our previous work demonstrated in retinal cells that increasing intracellular free L-arginine levels directly correlates to nitric oxide (NO) generation and can be promoted by protein synthesis inhibition and increase of free L-arginine concentration. Eukaryotic elongation factor 2 kinase (eEF2K), a calcium/calmodulin-dependent kinase, is also known to be activated by NMDA receptors leading to protein synthesis inhibition. Here we explored how does eEF2K participate in NMDA-induced NO signaling. We found that when this enzyme is inhibited, NMDA loses its ability to promote NO synthesis. On the other hand, when NO synthesis is increased by protein synthesis inhibition with cycloheximide or addition of exogenous L-arginine, eEF2K has no participation, showcasing a specific link between this enzyme and NMDA-induced NO signaling. We have previously shown that inhibition of the canonical NO signaling pathway (guanylyl cyclase/cGMP/cGK) blocks CREB activation by glutamate in retinal cells. Interestingly, pharmacological inhibition of eEF2K fully prevents CREB activation by NMDA, once again demonstrating the importance of eEF2K in NMDA receptor signaling. In summary, we demonstrated here a new role for eEF2K, directly controlling NMDA-dependent nitrergic signaling and modulating L-arginine availability in neurons, which can potentially be a new target for the study of physiological and pathological processes involving NMDA receptors in the central nervous system.

Eukaryotic elongation factor-2 kinase (eEF2K) signaling in tumor and microenvironment as a novel molecular target.

Eukaryotic elongation factor-2 kinase (eEF2K), an atypical member of alpha-kinase family, is highly overexpressed in breast, pancreatic, brain, and lung cancers, and associated with poor survival in patients. eEF2K promotes cell proliferation, survival, and aggressive tumor characteristics, leading to tumor growth and progression. While initial studies indicated that eEF2K acts as a negative regulator of protein synthesis by suppressing peptide elongation phase, later studies demonstrated that it has multiple functions and promotes cell cycle, angiogenesis, migration, and invasion as well as induction of epithelial-mesenchymal transition through induction of integrin ß1, SRC/FAK, PI3K/AKT, cyclin D1, VEGF, ZEB1, Snail, and MMP-2. Under stress conditions such as hypoxia and metabolic distress, eEF2K is activated by several signaling pathways and slows down protein synthesis and helping cells to save energy and survive. In vivo therapeutic targeting of eEF2K by genetic methods inhibits tumor growth in various tumor models, validating it as a potential molecular target. Recent studies suggest that eEF2K plays a role in tumor microenvironment cells by monocyte chemoattractant protein-1 (MCP-1) and accumulation of tumor-associated macrophages. Due to its clinical significance and the pivotal role in tumorigenesis and progression, eEF2K is considered as an important therapeutic target in solid tumors. However, currently, there is no specific and potent inhibitor for translation into clinical studies. Here, we aim to systematically review current knowledge regarding eEF2K in tumor biology, microenvironment, and development of eEF2K targeted inhibitors and therapeutics.

Delivery of Small Molecule EF2 Kinase Inhibitor for Breast and Pancreatic Cancer Cells Using Hyaluronic Acid Based Nanogels.

PURPOSE: To evalauted natural polymeric biomaterials including hyaluronic acid (HA) and its copolymeric form HA:Suc nanoparticles (NPs) as drug carrier systems for delivery of hydrophobic small molecule kinase EF2-kinase inhibitor in breast and pancreatic cancer cells. METHODS: In vitro cellular uptake studies of Rhodamine 6G labaled HA:Suc nanoparticles were evaluated by using flow cytometry analysis and fluorescent microscopy in breast (MDA-MB-231 and MDA-MB-436) and pancreatic cancer cells (PANC-1 and MiaPaca-2). Besides, in vitro release study of compound A (an EF2-kinase inhibitor) as a model hydrophobic drug was performed in the cancer cells. RESULTS: These biological evaluation studies indicated that HA and HA:Suc NPs provided a highly effective delivery of compound A were into breast and pancreatic cancer cells, leading to significant inhibition of cell proliferation and colony formation of breast and pancreatic cancer cells. CONCLUSION: HA-sucrose NPs incorporating an EF2-Kinase inhibitor demonstrate significant biologic activity in breast and pancreatic cancer cells. This is the first study that shows natural polymeric drug carriers succesfully deliver a hydrofobic cancer drug into cancer cells. Graphical Abstract Nanoparticles based on HA:Suc are effective in delivering hydrofobic cancer drugs in breast and pancreatic cancers.

SIRT1 Protects the Heart from ER Stress-Induced Injury by Promoting eEF2K/eEF2-Dependent Autophagy.

Many recent studies have demonstrated the involvement of endoplasmic reticulum (ER) stress in the development of cardiac diseases and have suggested that modulation of ER stress response could be cardioprotective. Previously, we demonstrated that the deacetylase Sirtuin 1 (SIRT1) attenuates ER stress response and promotes cardiomyocyte survival. Here, we investigated whether and how autophagy plays a role in SIRT1-afforded cardioprotection against ER stress. The results revealed that protective autophagy was initiated before cell death in response to tunicamycin (TN)-induced ER stress in cardiac cells. SIRT1 inhibition decreased ER stress-induced autophagy, whereas its activation enhanced autophagy. In response to TN- or isoproterenol-induced ER stress, mice deficient for SIRT1 exhibited suppressed autophagy along with exacerbated cardiac dysfunction. At the molecular level, we found that in response to ER stress (i) the extinction of eEF2 or its kinase eEF2K not only reduced autophagy but further activated cell death, (ii) inhibition of SIRT1 inhibited the phosphorylation of eEF2, (iii) eIF2α co-immunoprecipitated with eEF2K, and (iv) knockdown of eIF2α reduced the phosphorylation of eEF2. Our results indicate that in response to ER stress, SIRT1 activation promotes cardiomyocyte survival by enhancing autophagy at least through activation of the eEF2K/eEF2 pathway.

Intranasal Insulin Increases Synaptic Protein Expression and Prevents Anesthesia-Induced Cognitive Deficits Through mTOR-eEF2 Pathway.

General anesthesia increases the risk for cognitive impairment and Alzheimer's disease (AD) in vulnerable individuals such as the elderly. We previously reported that prior administration of insulin through intranasal delivery can prevent the anesthesia-induced cognitive impairment and biochemical changes in the brain. However, little is known about the underlying molecular mechanisms. Here, we report that general anesthesia resulted in downregulation of mammalian/mechanistic target of rapamycin (mTOR) and eukaryotic elongation factor 2 (eEF2) in the brain along with reduction of presynaptic proteins and brain-derived neurotrophic factor and cognitive impairment in aged mice. Prior administration of intranasal insulin prevented these anesthesia-induced changes. These results suggest the involvement of the mTOR-eEF2 signaling pathway in the anesthesia-induced brain changes and cognitive impairment and in the prevention of these changes with insulin. Correlation analyses and the use of eEF2 kinase inhibitor further support our conclusions. These studies shed light on the molecular mechanism by which anesthesia and insulin could act on synaptic proteins and cognitive function.