Kinase GSK3β Research Articles

Artesunate-driven autophagy: a shield against liver hypoxia/reoxygenation insult in rats via modulation of GLP1R, the chief metabolic kinase AMPK, mTOR, ULK1, P70S6K, cyclin D1, Akt, and GSK3β

BackgroundHepatic hypoxia/reoxygenation (H/R) insult is a critical issue in hepatic transplant and surgeries, profoundly influencing postoperative prognosis. One crucial pathomechanism in this condition is impaired autophagy flux, which disrupts liver homeostasis. Artesunate, an antimalarial drug, has shown potential in providing hepatoprotection against H/R injury; however, whether it can modulate disrupted autophagy to enhance hepatoprotection remains unclear.Purpose of the studyAccordingly, we delved into the potential mechanism(s) through which artesunate modulates the autophagy process in a hepatic H/R injury model.Methods and resultsRats were categorized into three groups, viz. sham operated, H/R, and artesunate-treated (50 mg/kg, i.p). Disease regression was evaluated microscopically, and molecular alternations were assessed biochemically using ELISA and western blotting techniques. Mechanistic analysis revealed that artesunate administration at reperfusion time significantly upregulated the gene expression of GLP1R protein expression of p-AMPK, accompanied by a downregulation in those of p-mTOR, and its target molecule p-ULK1, presenting the first trail to initiate autophagy. Additionally, artesunate reduced H/R-induced hepatic upregulated protein expression of p-mTOR/P70S6K cue, and cyclin D1 content, which positively correlated with the mTOR/P70S6K axis. Moreover, artesunate sharply upregulated active p-Akt, which in turn phosphorylated/inactivated GSK3β, a cascade that indirectly promotes autophagy. Consequently, artesunate increased the hepatic beclin-1 and LC3-II to further uphold its autophagic capacity. The hepato-therapeutic effectiveness of artesunate was further evidenced by reduced serum ALT and AST levels, along with diminished hepatic histopathological alterations.ConclusionArtesunate protected liver by triggering autophagy partly by modulating the GLP1R/AMPK/mTOR/ULK1, GLP1R/AMPK/mTOR/P70S6K, cyclin D1, and Akt/GSK3β trajectories providing a significant therapeutic potential in managing hepatic H/R insult.

Design, synthesis, and biological evaluation of tetrahydropyrimidine analogue as GSK-3β/Aβ aggregation inhibitor and anti-Alzheimer’s agent

The complex nature of Alzheimer’s disease (AD) etiopathology is among the principal hurdles to developing effective anti-Alzheimer agents. Tau pathology and Amyloid-β (Aβ) accumulation are hallmarks and validated therapeutic strategies of AD. GSK-3β is a serine/threonine kinase involved in tau phosphorylation. Its excessive activity also contributes to the production of Aβ plaques, making GSK-3β an attractive AD target. Taking this into account, In this article, we outline the design, synthesis, and biological validation of a focused library of 1,2,3,4-tetrahydropyrimidine based derivatives as inhibitors of GSK-3β, tau phosphorylation, and Aβ accumulation. The inhibitory activity of forty nine synthetic compounds was tested against GSK-3β and other AD-relevant kinases. The kinetic experiments revealed the mode of GSK-3β inhibition by the most potent compound 44. The in- vitro drug metabolism and pharmacokinetic studies were thereafter performed. The anti-aggregation activity of the most potent GSK-3β inhibitor was tested using AD transgenic Caenorhabditis elegans (C. elegans) strain CL2006 for quantification of Aβ plaques and BR5706 C. elegans strain for tau pathology evaluation. We then evaluated the blood–brain barrier permeability and got promising results. Therefore, we present compound 44 as a potential ATP-competitive GSK-3β inhibitor with good metabolism and pharmacokinetic profile, anti-aggregation properties for amyloid beta protein, and reduction in tau-phosphorylation levels. We recommend more investigation into compound 44-based small molecules as possible targets for AD disease-modifying treatments.

Structural and functional effects of phosphopriming and scaffolding in the kinase GSK-3β.

Glycogen synthase kinase 3β (GSK-3β) targets specific signaling pathways in response to distinct upstream signals. We used structural and functional studies to dissect how an upstream phosphorylation step primes the Wnt signaling component β-catenin for phosphorylation by GSK-3β and how scaffolding interactions contribute to this reaction. Our crystal structure of GSK-3β bound to a phosphoprimed β-catenin peptide confirmed the expected binding mode of the phosphoprimed residue adjacent to the catalytic site. An aspartate phosphomimic in the priming site of β-catenin adopted an indistinguishable structure but reacted approximately 1000-fold slower than the native phosphoprimed substrate. This result suggests that substrate positioning alone is not sufficient for catalysis and that native phosphopriming interactions are necessary. We also obtained a structure of GSK-3β with an extended peptide from the scaffold protein Axin that bound with greater affinity than that of previously crystallized Axin fragments. This structure neither revealed additional contacts that produce the higher affinity nor explained how substrate interactions in the GSK-3β active site are modulated by remote Axin binding. Together, our findings suggest that phosphopriming and scaffolding produce small conformational changes or allosteric effects, not captured in the crystal structures, that activate GSK-3β and facilitate β-catenin phosphorylation. These results highlight limitations in our ability to predict catalytic activity from structure and have potential implications for the role of natural phosphomimic mutations in kinase regulation and phosphosite evolution.

Multi-signal regulation of the GSK-3β homolog Rim11 controls meiosis entry in budding yeast

Starvation in diploid budding yeast cells triggers a cell-fate program culminating in meiosis and spore formation. Transcriptional activation of early meiotic genes (EMGs) hinges on the master regulator Ime1, its DNA-binding partner Ume6, and GSK-3β kinase Rim11. Phosphorylation of Ume6 by Rim11 is required for EMG activation. We report here that Rim11 functions as the central signal integrator for controlling Ume6 phosphorylation and EMG transcription. In nutrient-rich conditions, PKA suppresses Rim11 levels, while TORC1 retains Rim11 in the cytoplasm. Inhibition of PKA and TORC1 induces Rim11 expression and nuclear localization. Remarkably, nuclear Rim11 is required, but not sufficient, for Rim11-dependent Ume6 phosphorylation. In addition, Ime1 is an anchor protein enabling Ume6 phosphorylation by Rim11. Subsequently, Ume6-Ime1 coactivator complexes form and induce EMG transcription. Our results demonstrate how various signaling inputs (PKA/TORC1/Ime1) converge through Rim11 to regulate EMG expression and meiosis initiation. We posit that the signaling-regulatory network elucidated here generates robustness in cell-fate control.

Connecting GSK-3β Inhibitory Activity with IKK-β or ROCK-1 Inhibition to Target Tau Aggregation and Neuroinflammation in Alzheimer's Disease-Discovery, In Vitro and In Cellulo Activity of Thiazole-Based Inhibitors.

GSK-3β, IKK-β, and ROCK-1 kinases are implicated in the pathomechanism of Alzheimer's disease due to their involvement in the misfolding and accumulation of amyloid β (Aβ) and tau proteins, as well as inflammatory processes. Among these kinases, GSK-3β plays the most crucial role. In this study, we present compound 62, a novel, remarkably potent, competitive GSK-3β inhibitor (IC50 = 8 nM, Ki = 2 nM) that also exhibits additional ROCK-1 inhibitory activity (IC50 = 2.3 µM) and demonstrates anti-inflammatory and neuroprotective properties. Compound 62 effectively suppresses the production of nitric oxide (NO) and pro-inflammatory cytokines in the lipopolysaccharide-induced model of inflammation in the microglial BV-2 cell line. Furthermore, it shows neuroprotective effects in an okadaic-acid-induced tau hyperphosphorylation cell model of neurodegeneration. The compound also demonstrates the potential for further development, characterized by its chemical and metabolic stability in mouse microsomes and fair solubility.

Regulation of Glycogen Synthase Kinase-3β by Phosphorylation and O-β-Linked N-Acetylglucosaminylation: Implications on Tau Protein Phosphorylation.

Glycogen synthase kinase 3 (GSK3) plays a pivotal role in signaling pathways involved in insulin metabolism and the pathogenesis of neurodegenerative disorders. In particular, the GSK3β isoform is implicated in Alzheimer's disease (AD) as one of the key kinases involved in the hyperphosphorylation of tau protein, one of the neuropathological hallmarks of AD. As a constitutively active serine/threonine kinase, GSK3 is inactivated by Akt/PKB-mediated phosphorylation of Ser9 in the N-terminal disordered domain, and for most of its substrates, requires priming (prephosphorylation) by another kinase that targets the substrate to a phosphate-specific pocket near the active site. GSK3 has also been shown to be post-translationally modified by O-linked β-N-acetylglucosaminylation (O-GlcNAcylation), with still unknown functions. Here, we have found that binding of Akt inhibits GSK3β kinase activity on both primed and unprimed tau substrates. Akt-mediated Ser9 phosphorylation restores the GSK3β kinase activity only on primed tau, thereby selectively inactivating GSK3β toward unprimed tau protein. Additionally, we have shown that GSK3β is highly O-GlcNAcylated at multiple sites within the kinase domain and the disordered N- and C-terminal domains, including Ser9. In contrast to Akt-mediated regulation, neither the O-GlcNAc transferase nor O-GlcNAcylation significantly alters GSK3β kinase activity, but high O-GlcNAc levels reduce Ser9 phosphorylation by Akt. Reciprocally, Akt phosphorylation downregulates the overall O-GlcNAcylation of GSK3β, indicating a crosstalk between both post-translational modifications. Our results indicate that specific O-GlcNAc profiles may be involved in the phosphorylation-dependent Akt-mediated regulation of GSK3β kinase activity.

Conversion of dendritic cells into tolerogenic or inflammatory cells depends on the activation threshold and kinetics of the mTOR signaling pathway

BackgroundRestoring impaired peripheral immune tolerance is the primary challenge in treating autoimmune diseases. Our previous research demonstrated the effectiveness of small spleen peptides (SSPs), a fraction of low molecular weight proteins, in inhibiting the progression of psoriatic arthritis, even in the presence of high levels of the proinflammatory cytokine TNFα in the bloodstream. When specifically targeting dendritic cells (DCs), SSPs transform them into tolerogenic cells, which efficiently induce the development of regulatory Foxp3+ Treg cells. In this study, we provide further insights into the mechanism of action of SSPs.ResultsWe found that SSPs stimulate the activation of the mTOR signaling pathway in dendritic cells, albeit in a different manner than the classical immunogenic stimulus LPS. While LPS-induced activation is rapid, strong, and sustained, the activity induced by SSPs is delayed, less intense, yet still significant. These distinct patterns of activation, as measured by phosphorylation of key components of the pathway are also observed in response to other immunogenic and tolerogenic stimuli such as GM-CSF + IL-4 or IL-10 and TGFβ. The disparity in mTOR activation between immunogenic and tolerogenic stimuli is quantitative rather than qualitative. In both cases, mTOR activation primarily occurs through the PI3K/Akt signaling axis and involves ERK and GSK3β kinases, with minimal involvement of AMPK or NF-kB pathways. Furthermore, in the case of SSPs, mTOR activation seems to involve adenosine receptors. Additionally, we observed that DCs treated with SSPs exhibit an energy metabolism with high plasticity, which is typical of tolerogenic cells rather than immunogenic cells.ConclusionHence, the decision whether dendritic cells enter an inflammatory or tolerogenic state seems to rely on varying activation thresholds and kinetics of the mTOR signaling pathway.

Titanium dioxide nanoparticles oral exposure induce osteoblast apoptosis, inhibit osteogenic ability and increase lipogenesis in mouse

Titanium dioxide nanoparticles (TiO2-NPs) are widely used in food, paint, coating, cosmetic, and composite orthodontic material. As a common food additive, TiO2-NPs can accumulate in various organs of human body, but the effect and underlying mechanism of bone remain unclear. Here mice were exposed to TiO2-NPs by oral gavage, and histological staining of femoral sections showed that TiO2-NPs reduced bone formation and enhanced osteoclast activity and lipogenesis, contributing to decreased trabecula bone. Transmission electron microscope (TEM) as well as biochemical and flow cytometry analysis of osteoblast exhibited that TiO2-NPs accumulated in osteoblast cytoplasm and impaired mitochondria ultrastructure with increased reactive oxygen species (ROS) and lipid hyperoxide, resulting in osteoblast apoptosis. In terms of mechanism, TiO2-NPs treatment inhibited expression of AKT and then increased pro-apoptotic protein Bax expression which was failure to form heterodimers with decreased anti-apoptotic Bcl-2, activating downstream Caspase-9 and Caspase-3 and inducing apoptosis. Additionally, TiO2-NPs suppressed Wnt3a level and then activated anti-Glycogen synthesis kinase (GSK-3β) phosphorylation, and ultimately resulted in degradation of β-catenin which down-regulated Runt-related transcription factor 2 (Runx2) and Osterix, inhibiting expression of osteogenic related proteins. Together, these results revealed that exposure of TiO2-NPs induced apoptosis and inhibited osteoblast differentiation through suppressing PI3K/AKT and Wnt/β-catenin signaling pathways, resulting in reduction of trabecula bone.

EEF1A2 promotes PTEN-GSK3β-SCF complex-dependent degradation of Aurora kinase A and is inactivated in breast cancer.

The translation elongation factor eEF1A promotes protein synthesis. Its methylation by METTL13 increases its activity, supporting tumor growth. However, in some cancers, a high abundance of eEF1A isoforms is associated with a good prognosis. Here, we found that eEF1A2 exhibited oncogenic or tumor-suppressor functions depending on its interaction with METTL13 or the phosphatase PTEN, respectively. METTL13 and PTEN competed for interaction with eEF1A2 in the same structural domain. PTEN-bound eEF1A2 promoted the ubiquitination and degradation of the mitosis-promoting Aurora kinase A in the S and G2 phases of the cell cycle. eEF1A2 bridged the interactions between the SKP1-CUL1-FBXW7 (SCF) ubiquitin ligase complex, the kinase GSK3β, and Aurora-A, thereby facilitating the phosphorylation of Aurora-A in a degron site that was recognized by FBXW7. Genetic ablation of Eef1a2 or Pten in mice resulted in a greater abundance of Aurora-A and increased cell cycling in mammary tumors, which was corroborated in breast cancer tissues from patients. Reactivating this pathway using fimepinostat, which relieves inhibitory signaling directed at PTEN and increases FBXW7 expression, combined with inhibiting Aurora-A with alisertib, suppressed breast cancer cell proliferation in culture and tumor growth in vivo. The findings demonstrate a therapeutically exploitable, tumor-suppressive role for eEF1A2 in breast cancer.

FBW7/GSK3β mediated degradation of IGF2BP2 inhibits IGF2BP2-SLC7A5 positive feedback loop and radioresistance in lung cancer

BackgroundThe development of radioresistance seriously hinders the efficacy of radiotherapy in lung cancer. However, the underlying mechanisms by which radioresistance occurs are still incompletely understood. The N6-Methyladenosine (m6A) modification of RNA is involved in cancer progression, but its role in lung cancer radioresistance remains elusive. This study aimed to identify m6A regulators involved in lung cancer radiosensitivity and further explore the underlying mechanisms to identify therapeutic targets to overcome lung cancer radioresistance.MethodsBioinformatic mining was used to identify the m6A regulator IGF2BP2 involved in lung cancer radiosensitivity. Transcriptome sequencing was used to explore the downstream factors. Clonogenic survival assays, neutral comet assays, Rad51 foci formation assays, and Annexin V/propidium iodide assays were used to determine the significance of FBW7/IGF2BP2/SLC7A5 axis in lung cancer radioresistance. Chromatin immunoprecipitation (ChIP)-qPCR analyses, RNA immunoprecipitation (RIP) and methylated RNA immunoprecipitation (MeRIP)-qPCR analyses, RNA pull-down analyses, co-immunoprecipitation analyses, and ubiquitination assays were used to determine the feedback loop between IGF2BP2 and SLC7A5 and the regulatory effect of FBW7/GSK3β on IGF2BP2. Mice models and tissue microarrays were used to verify the effects in vivo.ResultsWe identified IGF2BP2, an m6A “reader”, that is overexpressed in lung cancer and facilitates radioresistance. We showed that inhibition of IGF2BP2 impairs radioresistance in lung cancer both in vitro and in vivo. Furthermore, we found that IGF2BP2 enhances the stability and translation of SLC7A5 mRNA through m6A modification, resulting in enhanced SLC7A5-mediated transport of methionine to produce S-adenosylmethionine. This feeds back upon the IGF2BP2 promoter region by further increasing the trimethyl modification at lysine 4 of histone H3 (H3K4me3) level to upregulate IGF2BP2 expression. We demonstrated that this positive feedback loop between IGF2BP2 and SLC7A5 promotes lung cancer radioresistance through the AKT/mTOR pathway. Moreover, we found that the ubiquitin ligase FBW7 functions with GSK3β kinase to recognize and degrade IGF2BP2.ConclusionsCollectively, our study revealed that the m6A “reader” IGF2BP2 promotes lung cancer radioresistance by forming a positive feedback loop with SLC7A5, suggesting that IGF2BP2 may be a potential therapeutic target to control radioresistance in lung cancer.

Glucocorticoid stress hormones stimulate vesicle-free Tau secretion and spreading in the brain

Chronic stress and elevated levels of glucocorticoids (GCs), the main stress hormones, accelerate Alzheimer’s disease (AD) onset and progression. A major driver of AD progression is the spreading of pathogenic Tau protein between brain regions, precipitated by neuronal Tau secretion. While stress and high GC levels are known to induce intraneuronal Tau pathology (i.e. hyperphosphorylation, oligomerization) in animal models, their role in trans-neuronal Tau spreading is unexplored. Here, we find that GCs promote secretion of full-length, primarily vesicle-free, phosphorylated Tau from murine hippocampal neurons and ex vivo brain slices. This process requires neuronal activity and the kinase GSK3β. GCs also dramatically enhance trans-neuronal Tau spreading in vivo, and this effect is blocked by an inhibitor of Tau oligomerization and type 1 unconventional protein secretion. These findings uncover a potential mechanism by which stress/GCs stimulate Tau propagation in AD.

Wnt3a/GSK3β/β-catenin Signalling Modulates Doxorubicin-associated Memory Deficits in Breast Cancer.

Chemobrain is widespread in breast cancer patients receiving chemotherapy. However, the exact mechanism, especially the associated signalling pathway, is not currently clear. This study was to evaluate the behavioural changes in breast cancer mice after chemotherapy and to further explore the role of Wnt3a/glycogen synthase kinase (GSK3β)/β-catenin signalling in chemobrain. MMTV-PyMT(+) breast cancer mice were injected intraperitoneally with doxorubicin (4mg/kg) once a week for three weeks to establish a chemobrain model. The Morris water maze (MWM) and novel object recognition (NOR) tests were performed to assess the learning and memory ability. Electron microscopy was used to observe the structural changes in the hippocampal CA1 region. The brain tissue of breast cancer mice after chemotherapy was taken out for mRNA-seq detection. Then, the expression levels and phosphorylation of key proteins in the Wnt3a/GSK3 β/β-catenin signalling pathway were evaluated through Western blotting (WB) and immunofluorescence. Doxorubicin-induced spatial and short-term memory impairment was observed in breast cancer mice, and obvious neuronal damage could be seen in the hippocampal CA1 region. Immunofluorescence staining for GSK3β was increased. Wnt signalling pathway is highly enriched from mRNA-seq analysis, with GSK3β genes at important nodes. The relative protein levels of p-PI3K, p-AKT, p-GSK3 β, Wnt3a and TCF-1 were decreased significantly, while the p-β-catenin level was increased. After injection of the GSK3β inhibitor sb216763 (1 ng/0.5µl/side), hippocampal neuronal injury was alleviated to some extent, and the changes in the expression of proteins upstream and downstream of this signalling pathway were reversed. Wnt3a/GSK3 β/β-catenin signalling is likely involved in doxorubicin-induced memory impairment. This result provides basic evidence for the further study of chemobrain in breast cancer.

Abstract PR011: Novel role of glycogen synthase kinase-3β in determining cancer cell response to PARPi through regulation of 53BP1 function

Abstract Glycogen synthase kinase-3β (GSK3β) is a multifunctional serine/threonine kinase involved in various cellular processes and signaling pathways. Accumulating evidence suggests GSK3β plays a role in cancer treatment with effects on tumorigenesis and treatment response. We have previously shown that inhibition of GSK3β protects neurons from ionizing radiation-induced apoptosis through regulation of the repair of double strand breaks (DSBs). Here, we investigate the molecular mechanism underlying GSK3β regulation of DSB repair. We illustrate that GSK3β directly interacts with 53BP1 and phosphorylates 53BP1 at threonine 334 amino acid (T334) within a region heavily phosphorylated by several stress kinases. Phosphorylation at T334 inhibits 53BP1’s function in recruitment to and retention at DSB sites as well as canonical NHEJ. In contrast, phosphorylation at Thr334 of 53BP1 enhances single strand DNA resection and promotes homologous recombination (HR) repair. Most importantly, genetic and pharmacologic inhibition of GSK3β-53BP1 signaling axis dramatically enhances the PARPi cytotoxic response of BRCA1 proficient cancer cells. The assessment of the effect of GSK3β-53BP1 axis on tumor cell response to radiation treatment is underway. In conclusion, this study establishes the connection between GSK3β kinase and DSB repair through its phosphorylation and regulation of 53BP1 property. Moreover, it demonstrates that GSK3β kinase activity uniquely results in inhibition of 53BP1 as opposed to other kinases that enhance 53BP1 function. Importantly, this novel signaling axis provides a strategy for targeting BRCA1-proficinet cancer cell resistance to PARPi. Citation Format: Fen Xia, Heba Allam, Scarlett Acklin, Mousumi Patra, Kirk West, Justin Leung. Novel role of glycogen synthase kinase-3β in determining cancer cell response to PARPi through regulation of 53BP1 function [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: DNA Damage Repair: From Basic Science to Future Clinical Application; 2024 Jan 9-11; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2024;84(1 Suppl):Abstract nr PR011.

Black phosphorus nanosheets inhibit glioblastoma cell migration and invasion through modulation of WNT/β-catenin and NOTCH signaling pathways

Black phosphorus nanosheet (BPNS) has recently demonstrated intrinsic anti-tumor bioactivity, but its underlying molecular mechanism remains unclear, limiting its potential applications in biomedicine. In this study, we investigated the impact of BPNS on glioblastoma cells and observed a significant dose-dependent inhibition of invasion and migration. RNA sequencing analysis revealed downregulation of genes associated with the WNT/β-catenin and NOTCH signaling pathways, both linked to invasion and migration. Mechanistically, BPNS directly binds to CSNK2A2, reducing its kinase activity, which indirectly enhances GSK-3β kinase activity. As a result, GSK-3β increases the phosphorylation level of β-catenin, leading to its degradation and subsequent inhibition of downstream molecules in the WNT/β-catenin signaling pathway. Our study uncovers the inherent biological activity of BPNS in hindering glioblastoma invasion and migration and sheds light on the molecular mechanisms, offering new directions for the nanomaterial’s biomedical applications.

Palmitic Acid Induces Posttranslational Modifications of Tau Protein in Alzheimer’s Disease–Related Epitopes and Increases Intraneuronal Tau Levels

Metabolic diseases derived from an unhealthy lifestyle have been linked with an increased risk for developing cognitive impairment and even Alzheimer’s disease (AD). Although high consumption of saturated fatty acids such as palmitic acid (PA) has been associated with the development of obesity and type II diabetes, the mechanisms connecting elevated neuronal PA levels and increased AD marker expression remain unclear. Among other effects, PA induces insulin resistance, increases intracellular calcium and reactive oxygen species (ROS) production, and reduces the NAD+/NADH ratio, resulting in decreased activity of the deacetylase Sirtuin1 (SIRT1) in neurons. These mechanisms may affect signaling pathways that impact the posttranslational modifications (PTMs) of the tau protein. To analyze the role played by PA in inducing the phosphorylation and acetylation of tau, we examined PTM changes in human tau in differentiated neurons from human neuroblastoma cells. We found changes in the phosphorylation state of several AD-related sites, namely, S199/202 and S214, that were mediated by a mechanism associated with the dysregulated activity of the kinases GSK3β and mTOR. PA also increased the acetylation of residue K280 and elevated total tau level after long exposure time. These findings provide information about the mechanisms by which saturated fatty acids cause tau PTMs that are similar to those observed in association with AD biochemical changes.Graphical

Design, synthesis and in vitro anti-proliferative evaluation of new pyridine-2,3-dihydrothiazole/thiazolidin-4-one hybrids as dual CDK2/GSK3β kinase inhibitors.

Herein, the molecular hybridization drug discovery approach was used in the design and synthesis of twelve novel pyridine-2,3-dihydrothiazole hybrids (2a,b-5a,b and 13a,b-14a,b) and fourteen pyridine-thiazolidin-4-one hybrids (6a,b-12a,b) as anti-proliferative analogues targeting CDK2 and GSK3β kinase inhibition. Almost all of the newly synthesized hybrids, including their precursors (1a,b), were evaluated for their anti-proliferative activity against three human cancer cell lines-MCF-7, HepG2 and HEp-2-as well as normal Vero cell lines. Both compounds 1a (pyridine-thiourea precursor) and 8a (pyridine-5-acetyl-thiazolidin-4-one hybrid) exhibited excellent anti-proliferative activity against HEp-2 (IC50 = 7.5 μg mL-1, 5.9 μg mL-1, respectively). Additionally, 13a (pyridine-5-(p-tolyldiazenyl-2,3-dihydrothiazole)) hybrid demonstrated excellent anti-proliferative activity against HepG2 (IC50 = 9.5 μg mL-1), with an acceptable safety profile against Vero (<45% inhibition at 100 μg mL-1) in the cases of 8a and 13a alone. The three promising anti-proliferative hybrids (1a, 8a, 13a) were selected for the assessment of their in vitro inhibitory kinase activity against CDK2/GSK3β using roscovitine (IC50 = 0.88 μg mL-1) and CHIR-99021 (IC50 = 0.07 μg mL-1) as references, respectively. Compound 13a was the most potent dual CDK2/GSK3β inhibitor (IC50 = 0.396 μg mL-1, 0.118 μg mL-1, respectively) followed by 8a (IC50 = 0.675 μg mL-1, 0.134 μg mL-1, respectively), and the weakest was 1a. To elucidate the mechanism of the most potent anti-proliferative 13a hybrid, further cell cycle analysis was performed revealing that it caused G1 cell cycle arrest and induced apoptosis. Moreover, it resulted in an increase in Bax and caspase-3 with a decrease in Bcl-2 levels in HepG2 cells compared with untreated cells. Finally, in silico drug likeness/ADME prediction for the three potent compounds as well as a molecular docking simulation study were conducted in order to explore the binding affinity and interactions in the binding site of each enzyme, which inspired their usage as anti-proliferative leads for further modification.

Amelioration of Amyloid-β Induced Alzheimer's Disease by Bacopa monnieri through Modulation of Mitochondrial Dysfunction and GSK-3β/Wnt/β-Catenin Signaling.

Alzheimer's disease (AD) is the most prevalent dementia, affecting a large number of populations. Despite being under scrutiny for decades, an effective therapeutic option is still not available. This study explores the therapeutic role of a nootropic herb Bacopa monnieri (BM) in AD-like pathological conditions produced by injecting preformed amyloid-β42 (Aβ42) fibril bilaterally into hippocampus of Wistar rats, and ethanolic extract of BM is orally administered for 4 weeks. Assessment of behavioral changes reveals that BM treatment ameliorates Aβ42-induced cognitive impairment and compromised explorative behavior. Supplementation of BM also reduces oxidative stress biomarkers, proinflammatory cytokines, and cholinesterase activity in the AD rats. Additionally, BM treatment restores Bcl-2-associated X protein (Bax)/ B-cell lymphoma 2 (Bcl-2) imbalance, increases neurotrophic factors expression, and prevents neurodegeneration validated by quantifying Nissl-positive hippocampal neurons. Interestingly, BM administration eliminates amyloid plaques in the hippocampal region and normalizes the Aβ42-induced increase in phospho-tau and total tau expression. Mechanistic investigations reveal that BM interacts with glycogen synthase kinase (GSK-3β) and restores Wnt/β-catenin signaling. BM has been used in diet as a nootropic herb for several centuries. This study highlights the anti-Alzheimer activity of BM from the behavioral to the molecular level by modulating mitochondrial dysfunction, and GSK-3β mediates the Wnt/β-catenin signaling pathway.

Glycogen Synthase Kinase 3 beta confers refractoriness to the microRNA-210-elicited exacerbation of hypoxia-reoxygenation stress-induced apoptotic cell death

Abstract Introduction Apoptotic cell death is a detrimental signatory molecular hallmark of Ischemia / Reperfusion (I/R) injury. Despite the exhaustive findings and significant advancements in the realm of I/R research, there is a paucity of viable molecular strategies that could be targeted to combat the underlying deleterious biochemical pathways that precipitate Hypoxia-Reoxygenation (H-R) stress, the cellular manifestation of I/R injury. The master hypoxamiR, microRNA-210 (miR-210), is an indispensable molecular component of the protective cellular adaptive response to hypoxia stress that paradoxically exacerbates apoptotic cell death during the cellular reoxygenation phase. While the entire repertoire of molecular entities mediating this paradoxical deleterious impact of miR-210 on H-R stress induced apoptotic cell death are largely unknown, recent studies have unveiled and posited, the evolutionary conserved serine/threonine kinase, Glycogen Synthase Kinase 3 beta (GSK3β) as a pivotal molecular focal point of the convergent and divergent signaling cascades that modulate the miR-210-regulated apoptotic cell death during the cellular reoxygenation phase. Purpose In this study, we elucidated and delineated the role of GSK3β (in)activation in the miR-210-induced exacerbation of the H-R stress-induced apoptotic cell death in AC-16 cardiomyocytes. Methods Using transient overexpression vectors and decoy / inhibition vectors to modulate miR-210 expression concomitant with the ectopic expression of the constitutive active GSK3β S9A (ca-GSK3β S9A) and the catalytically-dead dominant negative GSK3β K85A (dn-GSK3β K85A) mutants, we performed exhaustive biochemical and molecular assays determining apoptotic cell death in AC-16 cardiomyocytes subjected H-R stress. Results Caspase-3 activity analysis coupled with DNA fragmentation assays demonstrated that the H-R driven augmented GSK3β kinase activity significantly attenuates the miR-210-induced exacerbation of H-R evoked apoptotic cell death during the cellular reoxygenation phase. Resolution of the multimodal components of the apoptotic signaling pathway, unveiled that the H-R driven augmented GSK3β kinase activity modestly increases the miR-210-driven exacerbation of the intrinsic apoptotic pathway during the cellular reoxygenation phase, as molecularly inferred by the levels of the release of Cytochrome C from the mitochondria into the cytosol. However, in a stark contrast, H-R driven augmented GSK3β kinase activity concomitantly overwhelmingly attenuated the flux through the extrinsic apoptotic pathway during the cellular reoxygenation phase, as molecularly inferred by caspase-8 activity analysis. Conclusion Our study is the first to unveil that the H-R driven augmented GSK3β kinase activity during the cellular reoxygenation phase, is an inherent protective cellular adaptive response that mitigates the miR-210-orchestrated exacerbation of the cellular reoxygenation-elicited apoptotic cell death.

Novel Role of Glycogen Synthase Kinase-3β in Determining Cancer Cell Response to Genotoxic Stress through Regulation of 53BP1 Function

Glycogen synthase kinase-3β (GSK3β) is a multifunctional serine/threonine kinase involved in various cellular processes and signaling pathways. Accumulating evidence suggests GSK3β plays a role in cancer treatment with effects on tumorigenesis and treatment response. We have previously shown that inhibition of GSK3β protects neurons from ionizing radiation-induced apoptosis through upregulation of non-homologous end joining (NHEJ) mediated repair of double strand breaks (DSBs). Here, we investigate the molecular mechanism underlying GSK3β regulation of NHEJ. Using biochemical, molecular, and genetic approaches, we investigated the physical and functional interaction between GSK3β and 53BP1. Cultured human tumor cells were utilized as model system to further characterize how GSK3β controls 53BP1 function in DNA DSB recognition and repair. Finally, we employed a cell biology approach to genetically and pharmacologically manipulate GSK3β activity and test how the GSK3β-53BP1 axis impacts tumor cytotoxic response to PARP inhibitor (PARPi) and radiation therapy. We illustrate that GSK3β directly interacts with 53BP1 and phosphorylates 53BP1 at threonine 334 amino acid (T334) within a region heavily phosphorylated by several stress kinases. Phosphorylation at T334 inhibits 53BP1's function in recruitment to DNA DSB sites as well as canonical NHEJ. Furthermore, our results identify GSK3β regulation of 53BP1 function in NHEJ is achieved through suppression of downstream mediators, RIF1 and PTIP, and their function. In contrast, GSK3β enhances single strand DNA resection and promotes homologous recombination (HR) repair. Most importantly, genetic and pharmacologic inhibition of GSK3β-53BP1 signaling axis dramatically enhances the cytotoxic response of BRCA1-deficit cancer cells to PARPi. The assessment of the effect of GSK3β-53BP1 axis on tumor cell response to radiation treatment is underway. This study establishes the connection between GSK3β and DSB repair through its phosphorylation and regulation of 53BP1 pathways. Moreover, it demonstrates that GSK3β kinase activity uniquely results in inhibition of 53BP1 as opposed to other kinases that enhance 53BP1 function. Importantly, this novel signaling axis provides a strategy for targeting cancer cell resistance to PARPi.

The Axin scaffold protects the kinase GSK3β from cross-pathway inhibition.

Multiple signaling pathways regulate the kinase GSK3β by inhibitory phosphorylation at Ser9, which then occupies the GSK3β priming pocket and blocks substrate binding. Since this mechanism should affect GSK3β activity towards all primed substrates, it is unclear why Ser9 phosphorylation does not affect other GSK3β-dependent pathways, such as Wnt signaling. We used biochemical reconstitution and cell culture assays to evaluate how Wnt-associated GSK3β is insulated from cross-activation by other signals. We found that the Wnt-specific scaffold protein Axin allosterically protects GSK3β from phosphorylation at Ser9 by upstream kinases, which prevents accumulation of pS9-GSK3β in the Axin-GSK3β complex. Scaffold proteins that protect bound proteins from alternative pathway reactions could provide a general mechanism to insulate signaling pathways from improper crosstalk.