Asperuloside Suppresses the Development of Depression through Wnt3α/GSK-3ß Signal Pathway in Rats.

Depressive disorder is the most common mental disorder with significant economic burden and limited treatments. Traditional Chinese medicine monomer has emerged as a promising non-pharmacological treatment for reducing depressive symptoms. The aim of this study was to investigate the antidepressant-like effects of asperuloside (ASP) and its mechanism. The depression-like behaviors of chronic unpredictable mild stress (CUMS)-exposed rats were evaluated by behavioral tests. At the same time, the behaviors of rats treated with different concentrations of ASP (10, 20, 40 mg/kg) were also evaluated. RNA sequencing was performed to screen for dysregulated genes following ASP treatment. The Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis was performed to state the enriched pathways. Protein expression was detected by Western blotting. With the increase of ASP concentration (over 20 mg/kg), the depression-like behaviors of the rats were alleviated, which was manifested as the increase of the number of entries in the central zone, decrease of immobility time, and the increase of swimming time, sucrose preference, and body weight. ASP activated the Wnt3α/glycogen synthase kinase 3ß (GSK-3ß)/ß-catenin signaling pathway in vivo. Knockdown of ß-catenin reversed the effects of ASP on regulating depression-like behaviors. ASP alleviates depression-like behaviors by activating the Wnt3α/GSK-3ß/ß-catenin signaling pathway, indicating that ASP may be a potential therapeutic drug for treatment of depression.

Association Between Type 2 Diabetes Mellitus and Alzheimer's Disease: Common Molecular Mechanism and Therapeutic Targets.

Diabetes mellitus (DM) and Alzheimer's disease (AD) rates are rising, mirroring the global trend of an aging population. Numerous epidemiological studies have shown that those with Type 2 diabetes (T2DM) have an increased risk of developing dementia. These degenerative and progressive diseases share some risk factors. To a large extent, the amyloid cascade is responsible for AD development. Neurofibrillary tangles induce neurodegeneration and brain atrophy; this chain reaction begins with hyperphosphorylation of tau proteins caused by progressive amyloid beta (Aß) accumulation. In addition to these processes, it seems that alterations in brain glucose metabolism and insulin signalling lead to cell death and reduced synaptic plasticity in AD, before the onset of symptoms, which may be years away. Due to the substantial evidence linking insulin resistance in the brain with AD, researchers have coined the name "Type 3 diabetes" to characterize the condition. We still know little about the processes involved, even though current animal models have helped illuminate the links between T2DM and AD. This brief overview discusses insulin and IGF-1 signalling disorders and the primary molecular pathways that may connect them. The presence of GSK-3ß in AD is intriguing. These proteins' association with T2DM and pancreatic ß-cell failure suggests they might be therapeutic targets for both disorders.

Neuroprotection of isoorientin against microglia activation induced by lipopolysaccharide via regulating GSK3ß, NF-κb and Nrf2/HO-1 pathways.

Background: Isoorientin (ISO), a flavone C-glycoside, is a glycogen synthase kinase 3ß (GSK3ß) substrate-competitive inhibitor. ISO has potential in treatment of Alzheimer's disease (AD). An excessive activation of GSK3ß can lead to neuroinflammation causing neuronal damage. Microglia cells, as resident immune cells of the central nervous system, mediate neuroinflammation. Here, we studied the effects of ISO on microglial activation to alleviate neuroinflammation.Methods: Effects of ISO were observed upon the stimulation of mouse microglia BV2 or SIM-A9 cells by lipopolysaccharide (LPS). Lithium chloride (LiCl) was the positive control as a GSK3ß inhibitor. The release of TNF-α and NO were analyzed by ELISA and Griess assays, while expressions of COX-2, Iba-1, BDNF, GSK3ß, NF-κB p65, IκB, Nrf2 and HO-1 were detected by Western blotting. In the co-culture model of SIM-A9 cells and differentiated SH-SY5Y human neuroblastoma cells, effects of ISO on microglia-mediated neuronal damage were evaluated with the MTS assay.Results: ISO significantly inhibited the production of TNF-α (p < 0.01), NO (p < 0.001) and the expression of COX-2 (p < 0.01) and Iba-1 (p < 0.05) induced by LPS, and increased BDNF. The cell viability of SH-SY5Y was inhibited by LPS in the co-culture, which was prevented by ISO pretreatment. ISO increased the expression of p-GSK3ß (Ser9), IκB and HO-1 in the cytoplasm, decreased NF-κB p65 and increased Nrf2 in the nucleus compared with the LPS group.Conclusion: ISO attenuated the activation of microglia through regulating the GSK3ß, NF-κB and Nrf2/HO-1 signaling pathways to exert neuroprotection.

Animal Models of Haploinsufficiency Revealed the Isoform-Specific Role of GSK-3 in HFD-Induced Obesity and Glucose Intolerance.

Glycogen synthase kinase 3 (GSK-3), a serine-threonine kinase with two isoforms (α and ß) is implicated in the pathogenesis of Type 2 diabetes mellitus (T2D). Recently, we reported the isoform-specific role of GSK-3 in T2D using homozygous GSK-3α/ß Knock-Out mice. While the homozygous inhibition models are idealistic in a preclinical setting, they do not mimic the inhibition seen with pharmacological agents. Hence, in this study, we sought to investigate the dose-response effect of GSK-3α/ß inhibition in the pathogenesis of obesity-induced T2D. Specifically, to gain insight into the dose-response effect of GSK-3 isoforms in T2D, we generated tamoxifen-inducible global GSK-3α/ß heterozygous mice. GSK-3α/ß heterozygous and control mice were fed a high-fat diet (HFD) for sixteen weeks. At baseline, the body weight and glucose tolerance of GSK-3α heterozygous and controls were comparable. In contrast, at baseline, a modest but significantly higher body weight (higher lean mass) was seen in GSK-3ß heterozygous compared to controls. Post-HFD, GSK-3α heterozygous and controls displayed a comparable phenotype. However, GSK-3ß heterozygous were significantly protected against obesity-induced glucose intolerance. Interestingly, the improved glucose tolerance in GSK-3ß heterozygous animals was dampened with chronic HFD-feeding, likely due to significantly higher fat mass and lower lean mass in the GSK-3ß animals. These findings suggest that GSK-3ß is the dominant isoform in glucose metabolism. However, to avail of the metabolic benefits of GSK-3ß inhibition, it is critical to maintain a healthy weight.

Recent Molecular Targets and Their Ligands for the Treatment of Alzheimer Disease.

Alzheimer's disease is a multifaceted neurodegenerative disease. Cholinergic dysfunction, amyloid ß toxicity, tauopathies, oxidative stress, neuroinflammation are among the main pathologies of the disease. Ligands targeting more than one pathology, multi-target directed ligands, attract attention in the recent years to tackle Alzheimer's disease. In this review, we aimed to cover different biochemical pathways, that are revealed in recent years for the pathology of the disease, as druggable targets such as cannabinoid receptors, matrix metalloproteinases, histone deacetylase and various kinases including, glycogen synthase kinase-3, mitogen-activated protein kinase and c-Jun N-terminal kinase, and their ligands for the treatment of Alzheimer's disease in the hope of providing more realistic insights into the field.

The therapeutic value of thiazole and thiazolidine derivatives in Alzheimer's disease: a systematic literature review.

Background and purpose: Alzheimer's disease (AD) is a common neurodegenerative disease and the fifth leading cause of death among the elderly. The development of drugs for AD treatment is based on inhibiting cholinesterase (ChE) activity and inhibiting amyloid-beta peptide and tau protein aggregations. Many in vitro findings have demonstrated that thiazole-and thiazolidine-based compounds have a good inhibitory effect on ChE and other elements involved in the AD pathogenicity cascade. Experimental approach: In the present review, we collected available documents to verify whether these synthetic compounds can be a step forward in developing new medications for AD. A systematic literature search was performed in major electronic databases in April 2021. Twenty-eight relevant in vitro and in vivo studies were found and used for data extraction. Findings/Results: Findings demonstrated that thiazole-and thiazolidine-based compounds could ameliorate AD's pathologic condition by affecting various targets, including inhibition of ChE activity, amyloid-beta, and tau aggregation in addition to cyclin-dependent kinase 5/p25, beta-secretase-1, cyclooxygenase, and glycogen synthase kinase-3ß. Conclusion and implications: Due to multitarget effects at micromolar concentration, this review demonstrated that these synthetic compounds could be considered promising candidates for developing anti-Alzheimer drugs.

A distinct protein posttranslational modifications-linked OsATL32-OsPPKL2-OsGSK2 loop modulates rice immunity against blast disease.

Protein posttranslational modifications play crucial roles in plant immunity through modulating a complicated signaling network mediated by different hormones. We previously demonstrated that OsATL32, an ATL-type E3 ligase, negatively contributes to rice immunity against Magnaporthe oryzae. Here, we show that OsATL32 forms a loop with OsPPKL2 and OsGSK2 through distinct protein posttranslational modifications to modulate rice immunity. OsATL32 ubiquitinates OsPPKL2, a protein phosphatase with Kelch-like repeat domains that exerts positive roles in regulating rice immunity against M. oryzae and chitin-triggered immune responses, for degradation. The glycogen synthase kinase 2 (OsGSK2), which acts as a negative regulator of rice immunity against M. oryzae and chitin-triggered immune responses, phosphorylates OsATL32 to elevate its protein stability and E3 ligase activity on OsPPKL2. Moreover, OsPPKL2 directly dephosphorylates OsGSK2, affecting its kinase activity on substrates including OsATL32 for phosphorylation. Like OsGSK2 as a BR signaling repressor, OsATL32 negatively regulates BR signaling; conversely, OsPPKL2 plays a positive role in BR signaling. These findings provide a molecular mechanism in which OsATL32 serves as a node connecting BR signaling and immunity by associating with OsPPKL2 and OsGSK2, assembling into a distinct protein posttranslational modifications-linked loop that functions in rice BR signaling and immunity.

GSK3 regulation Wnt/ß-catenin signaling affects adipogenesis in bovine skeletal muscle fibro/adipogenic progenitors.

Clarifying the cellular origin and regulatory mechanisms of intramuscular fat (IMF) deposition is crucial for improving beef quality. Here, we used single-nucleus RNA sequencing to analyze the structure and heterogeneity of skeletal muscle cell populations in different developmental stages of Yanbian cattle and identified eight cell types in two developmental stages of calves and adults. Among them, fibro/adipogenic progenitors (FAPs) expressing CD29 (ITGA7)pos and CD56 (NCAM1)neg surface markers were committed to IMF deposition in beef cattle and expressed major Wnt ligands and receptors. LY2090314/XAV-939 was used to activate/inhibit Wnt/ß-catenin signal. The results showed that the blockade of Glycogen Synthase Kinase 3 (GSK3) by LY2090314 promoted the stabilization of ß-catenin and reduced the expression of genes related adipogenic differentiation (e.g., PPARγ and C/EBPα) in bovine FAPs, confirming the anti-adipogenic effect of GSK3. XAV-939 inhibition of the Wnt/ß-catenin pathway promoted the lipid accumulation capacity of FAPs. Furthermore, we found that blocking GSK3 enhanced the paracrine effects of FAPs-MuSCs and increased myotube formation in muscle satellite cells (MuSCs). Overall, our results outline a single-cell atlas of skeletal muscle development in Yanbian cattle, revealed the role of Wnt/GSK3/ß-catenin signaling in FAPs adipogenesis, and provide a theoretical basis for further regulation of bovine IMF deposition.

GSK3: A potential target and pending issues for treatment of Alzheimer's disease.

Glycogen synthase kinase-3 (GSK3), consisting of GSK3α and GSK3ß subtypes, is a complex protein kinase that regulates numerous substrates. Research has observed increased GSK3 expression in the brains of Alzheimer's disease (AD) patients and models. AD is a neurodegenerative disorder with diverse pathogenesis and notable cognitive impairments, characterized by Aß aggregation and excessive tau phosphorylation. This article provides an overview of GSK3's structure and regulation, extensively analyzing its relationship with AD factors. GSK3 overactivation disrupts neural growth, development, and function. It directly promotes tau phosphorylation, regulates amyloid precursor protein (APP) cleavage, leading to Aß formation, and directly or indirectly triggers neuroinflammation and oxidative damage. We also summarize preclinical research highlighting the inhibition of GSK3 activity as a primary therapeutic approach for AD. Finally, pending issues like the lack of highly specific and affinity-driven GSK3 inhibitors, are raised and expected to be addressed in future research. In conclusion, GSK3 represents a target in AD treatment, filled with hope, challenges, opportunities, and obstacles.

Molecular mechanisms and therapeutic potential of lithium in Alzheimer's disease: repurposing an old class of drugs.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder characterized by cognitive decline and memory loss. Despite advances in understanding the pathophysiological mechanisms of AD, effective treatments remain scarce. Lithium salts, recognized as mood stabilizers in bipolar disorder, have been extensively studied for their neuroprotective effects. Several studies indicate that lithium may be a disease-modifying agent in the treatment of AD. Lithium's neuroprotective properties in AD by acting on multiple neuropathological targets, such as reducing amyloid deposition and tau phosphorylation, enhancing autophagy, neurogenesis, and synaptic plasticity, regulating cholinergic and glucose metabolism, inhibiting neuroinflammation, oxidative stress, and apoptosis, while preserving mitochondrial function. Clinical trials have demonstrated that lithium therapy can improve cognitive function in patients with AD. In particular, meta-analyses have shown that lithium may be a more effective and safer treatment than the recently FDA-approved aducanumab for improving cognitive function in patients with AD. The affordability and therapeutic efficacy of lithium have prompted a reassessment of its use. However, the use of lithium may lead to potential side effects and safety issues, which may limit its clinical application. Currently, several new lithium formulations are undergoing clinical trials to improve safety and efficacy. This review focuses on lithium's mechanism of action in treating AD, highlighting the latest advances in preclinical studies and clinical trials. It also explores the side effects of lithium therapy and coping strategies, offering a potential therapeutic strategy for patients with AD.

Naringin Prevents Diabetic-Induced Dysmetabolism in Male Wistar Rats by Modulating GSK-3 Activities and Oxidative Stress-Dependent Pathways.

Type 2 diabetes mellitus (T2DM), characterized by insulin resistance and glucose dysmetabolism, is a major metabolic disorder accompanied with health and financial burden. Recently, research findings showed that orange peel extract (OPE) has health benefits such as improved insulin sensitivity and glucose metabolism. The present study aimed at establishing the role of naringin from OPE on T2DM-induced glucose and lipid dysmetabolism. Thirty male (30) Wistar rats were randomized into five groups: control, diabetes, diabetes + naringin, diabetes + orange peel, and diabetes + metformin. Oral administration was once per day for 28 days. After 28 days of treatment, naringin ameliorated the diabetes-induced increase in blood sugar, homeostatic model assessment (HOMA) IR, triglyceride, total cholesterol, triglyceride/high density lipoprotein, total cholesterol/high density lipoprotein, triglyceride glucose index, glucose synthase kinase-3, lactate, lactate dehydrogenase, malondialdehyde, c-reactive protein, and tumor necrosis factor α compared with the diabetic untreated animals. Furthermore, naringin reversed diabetes-induced decrease in serum insulin, HOMA B, HOMA S, quantitative insulin-sensitivity check index, high-density lipoprotein, total antioxidant capacity, superoxide dismutase, catalase, glucose transporter-4, and hepatic glycogen. This study showed that naringin prevented diabetes-induced dysglycemia and dyslipidemia via glucose synthase kinase-3 and oxidative stress-dependent pathways.

Transcriptional Regulation Analysis Provides Insight into the Function of GSK3ß Gene in Diannan Small-Ear Pig Spermatogenesis.

Glycogen synthase kinase-3ß (GSK3ß) not only plays a crucial role in regulating sperm maturation but also is pivotal in orchestrating the acrosome reaction. Here, we integrated single-molecule long-read and short-read sequencing to comprehensively examine GSK3ß expression patterns in adult Diannan small-ear pig (DSE) testes. We identified the most important transcript ENSSSCT00000039364 of GSK3ß, obtaining its full-length coding sequence (CDS) spanning 1263 bp. Gene structure analysis located GSK3ß on pig chromosome 13 with 12 exons. Protein structure analysis reflected that GSK3ß consisted of 420 amino acids containing PKc-like conserved domains. Phylogenetic analysis underscored the evolutionary conservation and homology of GSK3ß across different mammalian species. The evaluation of the protein interaction network, KEGG, and GO pathways implied that GSK3ß interacted with 50 proteins, predominantly involved in the Wnt signaling pathway, papillomavirus infection, hippo signaling pathway, hepatocellular carcinoma, gastric cancer, colorectal cancer, breast cancer, endometrial cancer, basal cell carcinoma, and Alzheimer's disease. Functional annotation identified that GSK3ß was involved in thirteen GOs, including six molecular functions and seven biological processes. ceRNA network analysis suggested that DSE GSK3ß was regulated by 11 miRNA targets. Furthermore, qPCR expression analysis across 15 tissues highlighted that GSK3ß was highly expressed in the testis. Subcellular localization analysis indicated that the majority of the GSK3ß protein was located in the cytoplasm of ST (swine testis) cells, with a small amount detected in the nucleus. Overall, our findings shed new light on GSK3ß's role in DSE reproduction, providing a foundation for further functional studies of GSK3ß function.

Toll-like receptor 4-dependent innate immune responses are mediated by intracrine corticosteroids and activation of glycogen synthase kinase-3ß in astrocytes.

Reactive astrocytes are important pathophysiologically and synthesize neurosteroids. We observed that LPS increased immunoreactive TLR4 and key steroidogenic enzymes in cortical astrocytes of rats and investigated whether corticosteroids are produced and mediate astrocytic TLR4-dependent innate immune responses. We found that LPS increased steroidogenic acute regulatory protein (StAR) and StAR-dependent aldosterone production in purified astrocytes. Both increases were blocked by the TLR4 antagonist TAK242. LPS also increased 11ß-hydroxysteroid dehydrogenase type 1 (11ß-HSD1) and corticosterone production, and both were prevented by TAK242 and by siRNAs against 11ß-HSD1, StAR, or aldosterone synthase (CYP11B2). Knockdown of 11ß-HSD1, StAR, or CYP11B2 or blocking either mineralocorticoid receptors (MR) or glucocorticoid receptors (GR) prevented dephosphorylation of p-Ser9GSK-3ß, activation of NF-κB, and the GSK-3ß-dependent increases of C3, IL-1ß, and TNF-α caused by LPS. Exogenous aldosterone mimicked the MR- and GSK-3ß-dependent pro-inflammatory effects of LPS in astrocytes, but corticosterone did not. Supernatants from astrocytes treated with LPS reduced MAP2 and viability of cultured neurons except when astrocytic StAR or MR was inhibited. In adrenalectomized rats, intracerebroventricular injection of LPS increased astrocytic TLR4, StAR, CYP11B2, and 11ß-HSD1, NF-κB, C3 and IL-1ß, decreased astrocytic p-Ser9GSK-3ß in the cortex and was neurotoxic, except when spironolactone was co-injected, consistent with the in vitro results. LPS also activated NF-κB in some NeuN+ and CD11b+ cells in the cortex, and these effects were prevented by spironolactone. We conclude that intracrine aldosterone may be involved in the TLR4-dependent innate immune responses of astrocytes and can trigger paracrine effects by activating astrocytic MR/GSK-3ß/NF-κB signaling.

Glycogen synthase kinase 3 activity enhances liver inflammation in MASH.

Background & Aims: Metabolic dysfunction-associated steatohepatitis (MASH) is characterized by excessive circulating toxic lipids, hepatic steatosis, and liver inflammation. Monocyte adhesion to liver sinusoidal endothelial cells (LSECs) and transendothelial migration (TEM) are crucial in the inflammatory process. Under lipotoxic stress, LSECs develop a proinflammatory phenotype known as endotheliopathy. However, mediators of endotheliopathy remain unclear. Methods: Primary mouse LSECs isolated from C57BL/6J mice fed chow or MASH-inducing diets rich in fat, fructose, and cholesterol (FFC) were subjected to multi-omics profiling. Mice with established MASH resulting from a choline-deficient high-fat diet (CDHFD) or FFC diet were also treated with two structurally distinct GSK3 inhibitors (LY2090314 and elraglusib [9-ING-41]). Results: Integrated pathway analysis of the mouse LSEC proteome and transcriptome indicated that leukocyte TEM and focal adhesion were the major pathways altered in MASH. Kinome profiling of the LSEC phosphoproteome identified glycogen synthase kinase (GSK)-3ß as the major kinase hub in MASH. GSK3ß-activating phosphorylation was increased in primary human LSECs treated with the toxic lipid palmitate and in human MASH. Palmitate upregulated the expression of C-X-C motif chemokine ligand 2, intracellular adhesion molecule 1, and phosphorylated focal adhesion kinase, via a GSK3-dependent mechanism. Congruently, the adhesive and transendothelial migratory capacities of primary human neutrophils and THP-1 monocytes through the LSEC monolayer under lipotoxic stress were reduced by GSK3 inhibition. Treatment with the GSK3 inhibitors LY2090314 and elraglusib ameliorated liver inflammation, injury, and fibrosis in FFC- and CDHFD-fed mice, respectively. Immunophenotyping using cytometry by mass cytometry by time of flight of intrahepatic leukocytes from CDHFD-fed mice treated with elraglusib showed reduced infiltration of proinflammatory monocyte-derived macrophages and monocyte-derived dendritic cells. Conclusion: GSK3 inhibition attenuates lipotoxicity-induced LSEC endotheliopathy and could serve as a potential therapeutic strategy for treating human MASH. Impact and Implications: LSECs under lipotoxic stress in MASH develop a proinflammatory phenotype known as endotheliopathy, with obscure mediators and functional outcomes. The current study identified GSK3 as the major driver of LSEC endotheliopathy, examined its pathogenic role in myeloid cell-associated liver inflammation, and defined the therapeutic efficacy of pharmacological GSK3 inhibitors in murine MASH. This study provides preclinical data for the future investigation of GSK3 pharmacological inhibitors in human MASH. The results of this study are important to hepatologists, vascular biologists, and investigators studying the mechanisms of inflammatory liver disease and MASH, as well as those interested in drug development.

Dietary sucrose determines the regulatory activity of lithium on gene expression and lifespan in Drosophila melanogaster.

The amount of dietary sugars and the administration of lithium both impact the lifespan of the fruit fly Drosophila melanogaster. It is noteworthy that lithium is attributed with insulin-like activity as it stimulates protein kinase B/Akt and suppresses the activity of glycogen synthase kinase-3 (GSK-3). However, its interaction with dietary sugar has largely remained unexplored. Therefore, we investigated the effects of lithium supplementation on known lithium-sensitive parameters in fruit flies, such as lifespan, body composition, GSK-3 phosphorylation, and the transcriptome, while varying the dietary sugar concentration. For all these parameters, we observed that the efficacy of lithium was significantly influenced by the sucrose content in the diet. Overall, we found that lithium was most effective in enhancing longevity and altering body composition when added to a low-sucrose diet. Whole-body RNA sequencing revealed a remarkably similar transcriptional response when either increasing dietary sucrose from 1% to 10% or adding 1 mM LiCl to a 1% sucrose diet, characterized by a substantial overlap of nearly 500 differentially expressed genes. Hence, dietary sugar supply is suggested as a key factor in understanding lithium bioactivity, which could hold relevance for its therapeutic applications.

Mechanistic insights into SARS-CoV-2 spike protein induction of the chemokine CXCL10.

During a SARS-CoV-2 infection, macrophages recognize viral components resulting in cytokine production. While this response fuels virus elimination, overexpression of cytokines can lead to severe COVID-19. Previous studies suggest that the spike protein (S) of SARS-CoV-2 can elicit cytokine production via the transcription factor NF-κB and the toll-like receptors (TLRs). In this study, we found that: (i) S and the S2 subunit induce CXCL10, a chemokine implicated in severe COVID-19, gene expression by human macrophage cells (THP-1); (ii) a glycogen synthase kinase-3 inhibitor attenuates this induction; (iii) S and S2 do not activate NF-κB but do activate the transcription factor IRF; (iv) S and S2 do not require TLR2 to elicit CXCL10 production or activate IRF; and (v) S and S2 elicit CXCL10 production by peripheral blood mononuclear cells (PBMCs). We also discovered that the cellular response, or lack thereof, to S and S2 is a function of the recombinant S and S2 used. While such a finding raises the possibility of confounding LPS contamination, we offer evidence that potential contaminating LPS does not underly induced increases in CXCL10. Combined, these results provide insights into the complex immune response to SARS-CoV-2 and suggest possible therapeutic targets for severe COVID-19.

Delivery of AKT1 phospho-forms to human cells reveals differential substrate selectivity.

Protein kinase B (AKT1) is a serine/threonine kinase that regulates fundamental cellular processes, including cell survival, proliferation, and metabolism. AKT1 activity is controlled by two regulatory phosphorylation sites (Thr308, Ser473) that stimulate a downstream signaling cascade through phosphorylation of many target proteins. At either or both regulatory sites, hyperphosphorylation is associated with poor survival outcomes in many human cancers. Our previous biochemical and chemoproteomic studies showed that the phosphorylated forms of AKT1 have differential selectivity toward peptide substrates. Here, we investigated AKT1-dependent activity in human cells, using a cell-penetrating peptide (transactivator of transcription, TAT) to deliver inactive AKT1 or active phospho-variants to cells. We used enzyme engineering and genetic code expansion relying on a phosphoseryl-transfer RNA (tRNA) synthetase (SepRS) and tRNASep pair to produce TAT-tagged AKT1 with programmed phosphorylation at one or both key regulatory sites. We found that all TAT-tagged AKT1 variants were efficiently delivered into human embryonic kidney (HEK 293T) cells and that only the phosphorylated AKT1 (pAKT1) variants stimulated downstream signaling. All TAT-pAKT1 variants induced glycogen synthase kinase (GSK)-3α phosphorylation, as well as phosphorylation of ribosomal protein S6 at Ser240/244, demonstrating stimulation of downstream AKT1 signaling. Fascinatingly, only the AKT1 variants phosphorylated at S473 (TAT-pAKT1S473 or TAT-pAKT1T308,S473) were able to increase phospho-GSK-3ß levels. Although each TAT-pAKT1 variant significantly stimulated cell proliferation, cells transduced with TAT-pAKT1T308 grew significantly faster than with the other pAKT1 variants. The data demonstrate differential activity of the AKT1 phospho-forms in modulating downstream signaling and proliferation in human cells.

Synthesis and Evaluation of 5-(Heteroarylmethylene)hydantoins as Glycogen Synthase Kinase-3ß Inhibitors.

Glycogen synthase kinase-3 (GSK-3) is a serine/threonine kinase which plays a center role in the phosphorylation of a wide variety of proteins, generally leading to their inactivation. As such, GSK-3 is viewed as a therapeutic target. An ever-increasing number of small organic molecule inhibitors of GSK-3 have been reported. Phenylmethylene hydantoins are known to exhibit a wide range of inhibitory activities including for GSK-3ß. A family of fourteen 2-heterocycle substituted methylene hydantoins (14, 17-29) were prepared and evaluated for the inhibition of GSK-3ß at 25 µM. The IC50 values of five of these compounds was determined; the two best inhibitors are 5-[(4'-chloro-2-pyridinyl)methylene]hydantoin (IC50 = 2.14 ± 0.18 µM) and 5-[(6'-bromo-2-pyridinyl)methylene]hydantoin (IC50 = 3.39 ± 0.16 µM). The computational docking of the compounds with GSK-3ß (pdb 1q41) revealed poses with hydrogen bonding to the backbone at Val135. The 5-[(heteroaryl)methylene]hydantoins did not strongly inhibit other metalloenzymes, demonstrating poor inhibitory activity against matrix metalloproteinase-12 at 25 µM and against human carbonic anhydrase at 200 µM, and were not inhibitors for Staphylococcus aureus pyruvate carboxylase at concentrations >1000 µM.

ß-Sitosterol alleviates the malignant phenotype of hepatocellular carcinoma cells via inhibiting GSK3B expression.

To explore the effects of ß-Sitosterol upon hepatocellular carcinoma cell proliferation, apoptosis, migration, invasion, and epithelial-mesenchymal transition (EMT), and to investigate the underlying mechanism using network pharmacology. Human hepatocellular carcinoma cell lines (Huh-7 and HCCLM3) were expose to gradient concentrations of ß-Sitosterol (5 µg/mL, 10 µg/mL, and 20 µg/mL). Cell viability and proliferation were assessed using MTT, CCK-8, colony formation, and EdU assays.Flow cytometry was employed to evaluate cell cycle and apoptosis. Scratch and Transwell assays were performed, respectively, to detect cell migration and invasion. The levels of apoptosis-associated proteins (BAX, BCL2, and cleaved caspase3) as well as EMT-associated proteins (E-cadherin, N-cadherin, Snail, and Vimentin) were detected in Huh-7 and HCCLM3 cell lines using Western blot analysis. The drug target gene for ß-Sitosterol was screened via PubChem and subsequently evaluated for expression in the GSE112790 dataset. In addition, the expression level of glycogen synthase kinase 3 beta (GSK3B) within the Cancer Genome Atlas-Liver Hepatocellular Carcinoma (TCGA-LIHC) database was analyzed, along with its correlation to the survival outcomes of patients with hepatocellular carcinoma. The diagnostic efficiency of GSK3B was assessed by analyzing the ROC curve. Subsequently, Huh-7 and HCCLM3 cell lines were transfected with the overexpression vector of GSK3B and then treated with ß-Sitosterol to further validate the association between GSK3B and ß-Sitosterol. GSK3B demonstrated a significantly elevated expression in patients with hepatocellular carcinoma, which could predict hepatocellular carcinoma patients' impaired prognosis based on GEO dataset and TCGA database. GSK3B inhibitor (CHIR-98014) notably inhibited cell proliferation and invasion, promoted cell apoptosis and cell cycle arrest at G0/G1 phase in hepatocellular carcinoma cells. ß-Sitosterol treatment further promoted the efffects of GSK3B inhibitor on hepatocellular carcinoma cells. GSK3B overexpression has been found to enhance the proliferative and invasive capabilities of hepatocellular carcinoma cells. Furthermore it has been observed that GSK3B overexpression, it has been obsear can partially reverse the inhibitory effect of ß-Sitosterol upon hepatocellular. ß-Sitosterol suppressed hepatocellular carcinoma cell proliferation and invasion, and enhanced apoptosis via inhibiting GSK3B expression.

First in Class Dual Non-ATP-Competitive Glycogen Synthase Kinase 3ß/Histone Deacetylase Inhibitors as a Potential Therapeutic to Treat Alzheimer's Disease.

Despite recent FDA approvals, Alzheimer's disease (AD) still represents an unmet medical need. Among the different available therapeutic approaches, the development of multitarget molecules represents one of the most widely pursued. In this work, we present a second generation of dual ligands directed toward highly networked targets that are deeply involved in the development of the disease, namely, Histone Deacetylases (HDACs) and Glycogen Synthase Kinase 3ß (GSK-3ß). The synthesized compounds are highly potent GSK-3ß, HDAC2, and HDAC6 inhibitors with IC50 values in the nanomolar range of concentrations. Among them, compound 4 inhibits histone H3 and tubulin acetylation at 0.1 µM concentration, blocks hyperphosphorylation of tau protein, and shows interesting immunomodulatory and neuroprotective properties. These features, together with its ability to cross the blood-brain barrier and its favorable physical-chemical properties, make compound 4 a promising hit for the development of innovative disease-modifying agents.