GSK-3β Inhibitor Research Articles

Decrease of KATP channel expression through D3 receptor-mediated GSK3β signaling alleviates levodopa-induced dyskinesia (LID) in Parkinson's disease mouse model.

The standard Parkinson's disease (PD) treatment is L-3,4-dihydroxyphenylalanine (L-DOPA); however, its long-term use may cause L-DOPA-induced dyskinesia (LID). Aberrant activation of medium spiny neurons (MSNs) contributes to LID, and MSN excitability is regulated by dopamine D3 receptor (D3R) and ATP-sensitive potassium (KATP) channel activity. Nevertheless, it remains unclear if D3R and KATP channels may be linked in the context of LID. Wild-type and tyrosine hydroxylase (TH)-specific Kir6.2 knockout mice were injected with 6-hydroxydopamine (6-OHDA) to generate a PD mouse model, then chronically treated with L-DOPA to induce LID. Analyses included immunohistochemical staining, biochemical endpoints, and behavior tests. The mechanisms by which D3R/KATP channels regulate LID in the PD/LID mouse model were probed by treatment with a D3R antagonist, KATP channel opener and glycogen synthase kinase 3β (GSK3β) inhibitor, followed by evaluation of abnormal involuntary movements (AIMs). The D3R antagonist FAUC365 alleviated LID, reducing AIMs and protecting against degeneration of the nigrostriatal pathway, which occurred through a direct interaction between D3Rs and KATP channels. In line with this mechanism, activation of D3R/GSK3β signaling increased KATP channel expression in the striatum of PD/LID mice. Additionally, the KATP channel opener Diz slowed LID progression and preserved nigrostriatal projections. Consistently, mice with TH-specific knockout of Kir6.2 exhibited reduced PD-like symptoms and less severe LID. D3Rs act through GSK3β signaling to regulate expression of KATP channels, which may subsequently modulate LID. Inhibition of KATP channels in TH-positive cells is sufficient to reduce AIMs in a mouse model of PD/LID.

Integrated computational approaches for identification of potent pyrazole-based glycogen synthase kinase-3β (GSK-3β) inhibitors: 3D-QSAR, virtual screening, docking, MM/GBSA, EC, MD simulation studies.

Glycogen synthase kinase-3β (GSK-3β) has emerged as a crucial target due to its substantial contribution in various cellular processes. Dysfunctional GSK-3β activity can lead to ion channel disturbances, sustain abnormal excitability, and contribute to the pathogenesis of epilepsy and other GSK-3β-related disorders. A set of 82 pyrazole analogs was utilized to study its structural features using a three-dimensional quantitative structure-activity relationship (3D-QSAR), virtual screening, molecular docking, and molecular dynamics. The QSAR model, validated using internal and external methods, demonstrated robustness with a high correlation coefficient r2training = 0.99, cross-validation coefficient q2 = 0.79, r2test = 0.69, and r2external = 0.74. The "Average of Actives" in the Activity Atlas model identified 17 molecules as active. Subsequent pharmacophore-based virtual screening of 17 actives yielded 70 compounds, which were selected as the prediction set to determine the potential GSK-3β inhibitors. Docking studies pinpointed compound P66 as the promising lead compound, with a docking score of - 10.555kcal/mol. These findings were further supported by electrostatic potential (ESP), electrostatic complementarity (EC), and Molecular Mechanics/Generalized Born Surface Area (MM/GBSA) analyses. Furthermore, a 500ns molecular dynamics (MD) simulation confirmed the structural and conformational stability of the lead complex throughout the simulation period. As a result, this study suggests that compound P66 holds the potential to be a potent lead candidate for the inhibition of GSK-3β, offering a novel therapeutic approach for GSK-3β related disorders, including epilepsy.

Palmitoyl-L-carnitine induces tau phosphorylation and mitochondrial dysfunction in neuronal cells.

Alzheimer's disease (AD) is characterized by cognitive decline and memory loss, involving mechanisms such as tau hyperphosphorylation and mitochondrial dysfunction. Increasing evidence suggests that age-related alterations in metabolite levels are crucial for the pathogenesis of AD. Here, we analyzed serum metabolites from mice of various ages (2, 4, 14, and 21 months old) using mass spectrometry. We identified palmitoyl-L-carnitine as a key metabolite with significantly increased levels in aged mice. In vitro experiments with SH-SY5Y neuronal cells demonstrated that palmitoyl-L-carnitine treatment enhanced tau phosphorylation, increased mitochondrial fission, and elevated intracellular calcium levels. Furthermore, the increased levels of tau phosphorylation were significantly reduced by the inhibition of GSK-3β, CDK5, and calpain, indicating that tau kinases activated by calcium overload are directly involved in the increase of tau phosphorylation. Considering that mitochondrial fission is related to mitochondrial dysfunction, we propose that the elevated level of serum palmitoyl-L-carnitine during aging contributes to AD pathology through these pathways. These findings highlight the significant role of lipid metabolism in neurodegeneration and offer potential therapeutic targets for age-related diseases, including AD.

In Silico and In Vitro Evaluation of Quinoline Derivatives as Potential Inhibitors of AChE, BACE1, and GSK3β for Neurodegenerative Diseases Treatment.

Neurodegenerative diseases are characterized by the structural and functional loss of neurons, affecting populations worldwide. Enzymes like acetylcholinesterase (AChE), beta-site APP cleaving enzyme-1 (BACE1), and glycogen synthase kinase 3-beta (GSK3β), contribute to their development. This study explores in silico and in vitro assays of quinoline analogues as potential inhibitors of these enzymes. In silico analyses highlighted derivative SQ6 as the most potent inhibitor for all proteins. In vitro assays confirmed that the quinoline derivatives had a modulating action on the three targets. SQ6 showed a significant AChE inhibition of 94.6%. Regarding the inhibition of GSK3β and BACE1, derivatives SQ6-SQ9, with the quinoline linked to the sulfonamide nitrogen, showed inhibition values above 40%. SQ7 and SQ9 were not considered cytotoxic for cell proliferation in human glioblastoma, and SQ6 did not show cytotoxicity at 7.8 and 3.9 µg mL-1. In murine fibroblasts, SQ6 presented a result similar to that observed for cell proliferation in human glioblastoma, while SQ7 and SQ9 were non-cytotoxic below 62.5 µg mL-1. These findings underscore compound SQ6 as the first potential multitarget enzyme inhibitor for treating neurodegenerative diseases.

9-Methylfascaplysin Prevents Neuroinflammation and Synaptic Damage via Cell-Specific Inhibition of Kinases inAPP/PS1 Transgenic Mice.

Alzheimer's disease (AD) is a leading neurodegenerative disorder without effective treatments. The nonlinear dynamic nature of AD pathophysiology suggested that multiple pharmacological actions of anti-AD drugs should be elucidated. 9-Methylfascaplysin (9-MF) was previously designed and synthesized as a novel anti-AD candidate. In this study, 9-MF at low concentrations significantly prevented cognitive impairments with similar efficacy as donepezil in APP/PS1 transgenic mice. In addition, 9-MF potently reduced β-amyloid (Aβ)-associated neuroinflammation and tau-associated synaptic damage invivo. 9-MF-regulated microglia-specific differentially phosphorylated proteins (DPPs) were mainly enriched in neuroinflammation, while 9-MF-regulated neuron-specific DPPs were enriched in synaptic regulation, as revealed by a quantitative phosphoproteomic approach. A phosphoproteome-kinome algorithm further identified that rho-associated coiled-coil kinase 2 (ROCK2) and glycogen synthase kinase 3β (GSK3β) ranked high in 9-MF-downregulated kinase perturbations. 9-MF possessed high affinities for ROCK2 and GSK3β, which was confirmed by invitro kinase activity assay. The protective effects of 9-MF were abolished by ROCK2 knockdown in Aβ-treated BV2 microglial cells, and by GSK3β knockdown in glyceraldehyde-treated SH-SY5Y neuronal cells, respectively. All these results supported that 9-MF produced anti-AD effects via cell-specific inhibition of ROCK2 and GSK3β in microglia and neurons, respectively.

GSK-3β inhibitor amplifies autophagy-lysosomal pathways by regulating TFEB in Parkinson's disease models

Parkinson's disease (PD), a common neurodegenerative disorder characterized by degeneration of the substantia nigra and a marked increase in Lewy bodies in the brain, primarily manifests as motor dysfunction. Glycogen synthase kinase-3 beta (GSK-3β) is known to play a critical role in various pathological processes of neurodegenerative diseases. However, the impact of GSK-3β inhibitors on PD progression and the underlying molecular mechanisms responsible for the effects have not been fully elucidated. Using in vitro and mouse models of 1-methyl-4-phenylpyridine (MPP+)-or methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced PD, we found that inhibition of GSK-3β activity alleviated mitochondrial damage, cell apoptosis, and neuronal cell loss by promoting the nuclear translocation of transcription factor EB (TFEB), thereby amplifying the autophagy-lysosomal pathway (ALP). Importantly, siRNA silencing of the TFEB gene impaired the GSK-3β inhibitor-mediated activation of the ALP pathway, thus negating the metabolic support required for neuronal functional improvement. Short-term treatment with the GSK-3β inhibitor significantly ameliorated motor dysfunction and improved motor coordination in model mice with MPTP-induced PD. GSK-3β inhibition increased the ALP and TFEB activities in the mice, thereby reducing α-synuclein aggregation and neuronal damage. In conclusion, our study demonstrates that inhibition of GSK-3β activity can delay the pathological processes of PD via promotion of the TFEB–ALP pathway, potentially providing a novel therapeutic target for this neurodegenerative disorder.

Molecular Docking, Bioinformatic Analysis, and Experimental Verification for the Effect of Naringin on ADHD: Possible Inhibition of GSK-3β and HSP90

Background/Objectives: One of the most abundant and growing neurodevelopmental disorders in recent decades is attention deficit hyperactivity disorder (ADHD). Many trials have been performed on using drugs for the improvement of ADHD signs. This study aimed to detect the possible interaction of naringin with Wnt/β-catenin signaling and its putative anti-inflammatory and protective effects in the mouse ADHD model based on bioinformatic, behavioral, and molecular investigations. Furthermore, molecular docking was applied to investigate possible interactions with the GSK-3β and HSP90 proteins. Methods: Male Swiss albino mice were divided into four groups, a normal control group, monosodium glutamate (SGL) control, SGL + naringin 50 mg/kg, and SGL + naringin 100 mg/kg. The psychomotor activity of the mice was assessed using the self-grooming test, rope crawling test, and attentional set-shifting task (ASST). In addition, biochemical analyses were performed using brain samples. Results: The results of the SGL group showed prolonged grooming time (2.47-folds), a lower percentage of mice with successful crawling on the rope (only 16.6%), and a higher number of trials for compound discrimination testing in the ASST (12.83 ± 2.04 trials versus 5.5 ± 1.88 trials in the normal group). Treatment with naringin (50 or 100 mg per kg) produced significant shortening in the grooming time (31% and 27% reductions), as well as a higher percentage of mice succeeding in crawling with the rope (50% and 83%, respectively). Moreover, the ELISA assays indicated decreased dopamine levels (0.36-fold) and increased TNF-α (2.85-fold) in the SGL control group compared to the normal mice, but an improvement in dopamine level was observed in the naringin (50 or 100 mg per kg)-treated groups (1.58-fold and 1.97-fold). Similarly, the PCR test showed significant declines in the expression of the Wnt (0.36), and β-catenin (0.33) genes, but increased caspase-3 (3.54-fold) and BAX (5.36-fold) genes in the SGL group; all these parameters were improved in the naringin 50 or 100 mg/kg groups. Furthermore, molecular docking indicated possible inhibition for HSP90 and GSK-3β. Conclusions: Overall, we can conclude that naringin is a promising agent for alleviating ADHD symptoms, and further investigations are required to elucidate its mechanism of action.

Discovery of Two GSK3β Inhibitors from Sophora flavescens Ait. using Structure-based Virtual Screening and Bioactivity Evaluation.

Kushen (Sophora flavescens Ait.) has a long history of medicinal use in China due to its medicinal values, such as antibacterial, antiviral, and anti-inflammatory. Rapid discovery of the components and the medicinal effects exerted by Kushen will help elucidate the science of Kushen in curing diseases. GSK3β (glycogen synthase kinase-3 beta) is a protein kinase with a wide range of physiological functions, such as antibacterial, antiviral, and anti-inflammatory. The discovery of inhibitors targeting GSK3β from Kushen was not only helpful for the rapid discovery of the components responsible for the efficacy of Kushen but also important for the development of novel drugs. In this study, the chemical composition of Kushen was extracted from the TMSCP database. Molecular docking, GSK3β enzyme assay, and molecular dynamics simulations were used to discover the GSK3β inhibitors from the chemical composition of Kushen. A total of 113 chemical compositions of Kushen were extracted from the TMSCP database. Molecular docking indicated that 15 chemical compositions of Kushen scored better than -8 kcal/mol against GSK3β. GSK3β enzyme assay demonstrated several inhibitory activities of kushenol I and kushenol F with IC50 values of 7.53 ± 2.55 μM and 4.96 ± 1.29 μM, respectively. Molecular dynamics simulations were used to reveal the interactions of kushenol I and kushenol F with GSK3β from structural and energetic perspectives. Kushenol I and kushenol F could be the material basis for the antibacterial, antiviral, and anti-inflammatory properties of Kushen.

Mechanisms of Differential Sensitivity to Ethanol-Induced Apoptosis in Mouse Spinal Cord at Different Developmental Stages-Akt/GSK Signaling and BAX.

The current study investigated differences in ethanol-induced apoptosis of spinal cord dorsal horn neurons at different developmental stages and the molecular mechanisms involved. A mouse ethanol intervention model was established on postnatal days 4, 7, and 12. Primary cells were derived from the spinal cord at postnatal day 4. Western blotting, immunofluorescence, and flow cytometry were used to detect apoptosis-related proteins in the spinal cord and primary cells. Kyoto Encyclopedia of Genes and Genomes enrichment analysis of differentially expressed genes originating from the Gene Expression Omnibus dataset GSE184615 was conducted. Effects on Akt/GSK3β pathway proteins were investigated using the GSK3β inhibitor AR-A014418, and the Akt inhibitor DHA. Lentiviral knockdown and overexpression of intervening GSK3β were used in HT22 cell lines to investigate the effects of alcohol on GSK 3β and caspase proteins. J-aggregates, reactive oxygen species assays, and calcein-AM assays were used to investigate mitochondrial function and cell viability. Ethanol caused downregulation of Akt activity and upregulation of GSK3β activity and apoptosis. DHA, AR-A014418, and knockdown of GSK3β effectively counteracted ethanol-induced apoptosis, whereas overexpression of GSK3β enhanced the injury process. PI3K activity was unchanged during these processes. Fluorescence colocalization analysis indicated that BAX was translocated to mitochondria during the apoptotic process. BAX was downregulated as the spinal cord developed, consistent with a reduced susceptibility to ethanol-induced apoptosis. Akt/GSK3β signaling and BAX together determine the direction of alcohol-induced apoptosis and its susceptibility to change during developmental stages in the spinal cord.

The contributory role of GSK3β in hypertension exacerbating atherosclerosis by regulating the OMA1/PGC1α pathway.

Atherosclerosis is closely related to endothelial dysfunction and hypertension. GSK3β is a critical regulator in atherosclerosis. This study was carried out to investigate the effects of GSK3β on hypertension exacerbating atherosclerosis in vitro and in vivo. L-NAME + HFD-ApoE-/- mice were used for this study for 12 weeks, and their endothelial dysfunction and inflammation were analyzed. Oil red O and H&E staining revealed that treatment with LiCl, an inhibitor of GSK3β, reduced atherosclerotic lesions and lipid accumulation. The levels of lipid homeostasis and oxidation stress were attenuated following LiCl administration. LiCl-treated ApoE-/- mice showed lowered blood pressure. LiCl also suppressed the expressions of Drp1, Bax, ICAM1, VCAM1 and TNF-α compared to HFD + L-NAME induced mice and oxLDL + L-NAME-treated Human aorta endothelial cell line(HAECs). LiCl treatment increased the expressions of MFN2 and Bcl2. Mitotracker-red, MitoSOX and JC-1 staining indicated that LiCl treatment reduced mitochondrial division and ROS production, increased mitochondrial ΔΨm compared to oxLDL + L-NAME-treated HAECs. The expression of OMA1 was decreased by LiCl treatment, while PGC1α expression was increased. In HAECs, we found that OMA1 knockdown increased mitochondrial function and the expression of PGC1α. We also demonstrated LiCl increased OMA1 ubiquitination compared with the Control group, thus decreased OMA1 expression. Furthermore, siOMA1 antagonized the increased protein expressions of ICAM1, VCAM1, TNF-α, Bax and Drp1, decreased the protein expressions of Bcl2 and MFN2 by siPGC1α. Taken together, we demonstrated that GSK3β could play a contributory role in hypertension exacerbating atherosclerosis by regulating the OMA1/PGC1α pathway and inhibiting mitochondrial function.

A diarylheptanoid derivative mediates glycogen synthase kinase 3β to promote the porcine muscle satellite cell proliferation: Implications for cultured meat production

Skeletal muscle stem cells, or satellite cells, are vital for cultured meat production, driving proliferation and differentiation to form muscle fibers in vitro. However, these abilities are often compromised after long-term in vitro culturing due to a loss of their stemness characteristics. Therefore, effective pharmacological agents that enhance satellite cell proliferation and maintain stemness ability are needed for optimal cell growth for cultured meat production. In this study, the effects of the identified glycogen synthase kinase 3β (GSK3β) inhibitors, ASPP 049, a diarylheptanoid isolated from Curcuma comosa rhizomes, and CHIR 99021 on porcine muscle satellite cell (PMSC) proliferation and Wnt/β-catenin signaling pathway were investigated. We found that both compounds enhanced cell viability and proliferation while preserving the stemness marker, as evidenced by increased expression of the skeletal muscle stem cell marker, Pax7 protein. Molecular dynamics simulations showed that ASPP 049 and CHIR 99021 exhibited differing binding affinities, primarily through hydrophobic interactions, suggesting potential for the design of more potent inhibitors in the future. Despite its weaker binding, ASPP 049 still showed significant effects on the regulation of the Wnt/β-catenin signaling pathway via increased phosphorylation of GSK3β at Ser9 and decreased the phosphorylation of β-catenin at Ser33, Ser37, and Thr41, thereby subsequently activating Wnt transcriptional activity. This study highlights the potential of ASPP 049 and CHIR 99021 to enhance PMSC proliferation and maintain stemness ability, offering a promising avenue for improving cultured meat production.

Aβ -induced excessive mitochondrial fission drives type H blood vessels injury to aggravate bone loss in APP/PS1 mice with Alzheimer's diseases.

Alzheimer's diseases (AD) patients suffer from more serious bone loss than cognitively normal subjects at the same age. Type H blood vessels were tightly associated with bone homeostasis. However, few studies have concentrated on bone vascular alteration and its role in AD-related bone loss. In this study, APP/PS1 mice (4- and 8-month-old) and age-matched wild-type mice were used to assess the bone vascular alteration and its role in AD-related bone loss. Transmission electron microscopy, immunofluorescence staining and iGPS 1.0 software database were utilized to investigate the molecular mechanism. Mitochondrial division inhibitor (Mdivi-1) and GSK-3β inhibitor (LiCl) were used to rescue type H blood vessels injury and verify the molecular mechanism. Our results revealed that APP/PS1 mice exhibited more serious bone blood vessels injury and bone loss during ageing. The bone blood vessel injury, especially in type H blood vessels, was accompanied by impaired vascularized osteogenesis in APP/PS1 mice. Further exploration indicated that beta-amyloid (Aβ) promoted the apoptosis of vascular endothelial cells (ECs) and resulted in type H blood vessels injury. Mechanistically, Aβ-induced excessive mitochondrial fission was found to be essential for the apoptosis of ECs. GSK-3β was identified as a key regulatory target of Aβ-induced excessive mitochondrial fission and bone loss. The findings delineated that Aβ-induced excessive mitochondrial fission drives type H blood vessels injury, leading to aggravate bone loss in APP/PS1 mice and GSK-3β inhibitor emerges as a potential therapeutic strategy.

VP3.15 reduces acute cerebellum damage after germinal matrix-intraventricular hemorrhage of the preterm newborn

Germinal matrix-intraventricular hemorrhage (GM-IVH) is one of the most common complications of the preterm newborn. The pathology of the GM-IVH is not completely understood and even regions distant from the lesion area are severely affected. It has been suggested that cerebellar diaschisis may underlie the neurodevelopmental problems that many of these kids show, including cerebral palsy, attention deficit disorders or hyperactivity. Additionally, GM-IVH has no successful treatment. VP3.15 is a dual action phosphodiesterase 7 (PDE7) and glycogen synthase kinase-3β (GSK-3β) inhibitor that limits neuroinflammation and neuronal loss. Therefore, it might also provide a relevant tool to reduce complications associated with GM-IVH. We have used a murine model of GM-IVH to analyze the short and long-term effects of VP3.15 in brain pathology and behavioral complications. In our hands, the induction of unilateral GM-IVH to P7 CD1 mice results in a short-term (P14) compromise of the cerebellar neuronal population and Purkinje cells arborization, an increase of microglia burden in the nuclei and an overall increase of punctuate cerebellar hemorrhages. Whereas brain alterations are no longer observed in the long term (P110), these animals present overt hyperactivity when analyzed in the adulthood, supporting the long-term behavioral impairment. Also, hyperactivity significantly correlates with ipsi and contralateral cerebellar sizes, neuronal densities and myelin basic protein levels. Importantly, treatment with VP3.15 significantly reduces neuronal loss, Purkinje cells simplification, the presence of cerebellar hemorrhages, as well as hyperactivity. Altogether, our data support the neuroprotective effects of VP3.15 in GM-IVH of the PT.

Targeting Canonical Wnt-signaling Through GSK-3β in Arrhythmogenic Cardiomyopathy: Conservative or Progressive?

Arrhythmogenic cardiomyopathy is a primary myocardial disease and a major cause of sudden death in all populations of the world. Canonical Wnt signalling is a critical pathway controlling numerous processes including cellular differentiation, hypertrophy and development. GSK3β is a ubiquitous serine/threonine kinase, which acts downstream of Wnt to promote protein ubiquitination and proteasomal degradation. Several studies now suggest that inhibiting GSK3β can prevent and reverse key pathognomonic features of ACM in a range of experimental models. However, varying concerns are reported throughout the literature including the risk of paradoxical arrhythmias, cancer and off-target effects in upstream or downstream pathways. CLINICAL RELEVANCE: In light of the start of the phase 2 TaRGET clinical trial, designed to evaluate the potential therapeutic efficacy of GSK3β inhibition in patients with arrhythmogenic cardiomyopathy, this report aims to review the advantages and disadvantages of this strategy.

Morinda officinalis iridoid glycosides, as an inhibitor of GSK-3β, alleviates rheumatoid arthritis through inhibition of NF-κB and JAK2/STAT3 pathway.

Morinda officinalis iridoid glycosides (MOIG) showed potential benefits in the treatment of rheumatoid arthritis (RA), but their exact mechanism has yet to be explored. To evaluate the effects of MOIG on RA, and explore the potential targets and molecular mechanism of MOIG in RA. The collagen-induced arthritis (CIA) rats were used to evaluate the effects of MOIG on RA. The proliferation, migration and invasion of fibroblast-like synoviocytes (FLSs) stimulated with or without tumor necrosis factor (TNF)-α were examined by CCK-8, wound healing and transwell assays, respectively. IF and WB were applied to investigate related mechanism in FLSs. The molecular docking, molecular dynamics simulation, CETSA and siRNA were used to analyze the interaction of MOIG with target. Finally, the adjuvant-induced arthritis (AA) mice model with gene knockdown was used to confirm the effect of MOIG on glycogen synthase kinase-3β (GSK-3β). MOIG significantly alleviated the paw swelling and synovial hyperplasia in CIA rats. Moreover, MOIG suppressed proliferation, migration and invasion, the secretion of inflammatory factors, and the expression of adhesion related proteins in TNF-α-stimulated FLSs. MOIG also inhibited the activation of Janus activating kinase 2 (JAK2)/signal transducer and activator of transcription 3 (STAT3) and nuclear factor kappa-B (NF-κB) signaling pathway in FLSs. Interestingly, the plant metabolites in MOIG had a good affinity with GSK-3β, and inhibition of GSK-3β attenuated the effects of MOIG on FLSs. Knockdown GSK-3β gene could inhibit the paw swelling and inflammatory indicators, decrease the arthritis score and synovial hyperplasia, reduce the phosphorylation of p65 and STAT3 in AA mice, thereby suppressing the NF-κB and STAT3 signaling activation, and MOIG treatment had no significant effects on AA mice with si-GSK-3β. MOIG alleviates joint inflammation in RA through inhibition NF-κB and JAK2/STAT3 pathway via suppression of GSK-3β in FLSs, which provides supports for MOIG as a promising therapeutic agent of RA.

Identification of novel brain penetrant GSK-3β inhibitors toward Alzheimer’s disease therapy by virtual screening, molecular docking, dynamic simulation, and MMPBSA analysis

One of the significant therapeutic targets for Alzheimer’s disease (AD) is Glycogen Synthase Kinase-3β (GSK-3β). Inhibition of GSK-3β can prevent hyperphosphorylation of tau, and thus prevent formation and accumulation of neurofibrillary tangles and neuropil threads that block intracellular transport, trigger unfolded protein response, and increase oxidative stress, cumulatively leading to neurodegeneration. In this study, we have performed structure-based virtual screening of two small-molecule libraries from ChemDiv CNS databases using AutoDock Vina to identify hit molecules based on their binding affinities compared to that of an established GSK-3β inhibitor, indirubin-3’-monoxime (IMO). Pharmacoinformatic screening on SwissADME and pkCSM servers enabled identification of lead molecules with favorable pharmacoinformatic properties for drug likeliness, including blood brain barrier (BBB) permeability. Further, molecular dynamic simulations identified six candidate lead molecules that show stable complex formation with GSK-3β in dynamic state under physiological conditions. Principal component analysis of the dynamic state was used to plot Free Energy Landscapes (FELs) of GSK-3β-ligand complexes. STRIDE secondary structure analysis of the lowest energy conformations identified from FEL plots, and binding free energy calculations by Molecular Mechanics Poisson-Boltzmann Surface Area ((ΔGbind )MM-PBSA) of the simulation trajectories led to the identification of two ligands as potential lead molecules having favorable free energy landscape profiles as well as significantly lower (ΔGbind )MM-PBSA in dynamic state compared to that of reference inhibitor IMO. Hence, this study identifies two novel brain penetrant GSK-3β inhibitors that are likely to have therapeutic potential against Alzheimer’s disease.

Disrupted mitochondrial response to nutrients is a presymptomatic event in the cortex of the APPSAA knock-in mouse model of Alzheimer's disease.

Reduced brain energy metabolism, mammalian target of rapamycin (mTOR) dysregulation, and extracellular amyloid beta (Aβ) oligomer (xcAβO) buildup are some well-known Alzheimer's disease (AD) features; how they promote neurodegeneration is poorly understood. We previously reported that xcAβOs inhibit nutrient-induced mitochondrial activity (NiMA) in cultured neurons. We now report NiMA disruption in vivo. Brain energy metabolism and oxygen consumption were recorded in heterozygous amyloid precursor protein knock-in (APPSAA) mice using two-photon fluorescence lifetime imaging and multiparametric photoacoustic microscopy. NiMA is inhibited in APPSAA mice before other defects are detected in these Aβ-producing animals that do not overexpress APP or contain foreign DNA inserts into genomic DNA. Glycogen synthase kinase 3 (GSK3β) signals through mTORC1 to regulate NiMA independently of mitochondrial biogenesis. Inhibition of GSK3β with TWS119 stimulates NiMA in cultured human neurons, and mitochondrial activity and oxygen consumption in APPSAA mice. NiMA disruption in vivo occurs before plaques, neuroinflammation, and cognitive decline in APPSAA mice, and may represent an early stage in human AD. Amyloid beta blocks communication between lysosomes and mitochondria in vivo. Nutrient-induced mitochondrial activity (NiMA) is disrupted long before the appearance of Alzheimer's disease (AD) histopathology in heterozygous amyloid precursor protein knock-in (APPSAA/+) mice. NiMA is disrupted long before learning and memory deficits in APPSAA/+ mice. Pharmacological interventions can rescue AD-related NiMA disruption in vivo.

Danggui Shaoyao San Alleviates Early Cognitive Impairment in Alzheimer's Disease Mice Through IRS1/GSK3β/Wnt3a-β-Catenin Pathway.

Alzheimer's disease (AD) is a neurodegenerative disease characterized by Amyloid plaques and neurofibrillary tangles. We explored the potential mechanism by which Danggui Shaoyao San (DSS) modulates central glucose metabolism via the insulin receptor substrate 1 (IRS1)/glycogen synthase kinase-3β (GSK3β)/Wnt3a-β-catenin pathway, thereby exerting protective effects on cognitive functions. In vitro, HT22 cells were induced with streptozotocin (STZ) to investigate the impact of GSK3β on pathway transduction. The active components in the DSS stock solution were validated using mass spectrometry. Subsequently, an AD model in C57BL/6J mice was established through STZ injection into both ventricles. The success of the model was validated behaviorally and pathologically. The Morris Water Maze (MWM) test, immunohistochemistry, Western blotting, quantitative reverse transcription-PCR, and 18F-fluorodeoxyglucose-positron emission tomography (FDG-PET) were employed to evaluate the influence of DSS on memory and pathological changes in AD. The DSS stock solution, rich in active components, ameliorated the memory deficits in AD mice in the MWM. In vitro, GSK3β exhibited regulatory control over Wnt and β-catenin, with GSK3β inhibition mitigating β-amyloid and tau redundancies at protein and gene levels, facilitating signal transduction. In vivo, DSS impacted key targets in the IRS1/GSK3β/Wnt3a-β-catenin pathway, mitigated senile plaques resulting from amyloid β (Aβ) deposition and neurofiber tangles induced by tau hyperphosphorylation, and alleviated the decline in central glucose metabolism observed in FDG-PET. Our findings suggest that DSS potentially confers cognitive protection by alleviating central hypoglycemia through the IRS1/GSK3β/Wnt3a-β-catenin pathway. This may serve as a promising therapeutic avenue for AD.

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 β) 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/β knockout mice. Although 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/β 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/β heterozygous mice. GSK-3α/β heterozygous and control mice were fed a high-fat diet (HFD) for 16 wk. 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-3β heterozygous compared with controls. Post-HFD, GSK-3α heterozygous and controls displayed a comparable phenotype. However, GSK-3β heterozygous were significantly protected against obesity-induced glucose intolerance. Interestingly, the improved glucose tolerance in GSK-3β heterozygous animals was dampened with chronic HFD-feeding, likely due to significantly higher fat mass and lower lean mass in the GSK-3β animals. These findings suggest that GSK-3β is the dominant isoform in glucose metabolism. However, to avail the metabolic benefits of GSK-3β inhibition, it is critical to maintain a healthy weight.NEW & NOTEWORTHY The precise isoform-specific role of GSK-3 in obesity-induced glucose intolerance is unclear. To overcome the limitations of pharmacological GSK-3 inhibitors (not isoform-specific) and tissue-specific genetic models, in the present study, we created novel inducible heterozygous mouse models of GSK-3 inhibition that allowed us to delete the gene globally in an isoform-specific and temporal manner to determine the isoform-specific role of GSK-3 in obesity-induced glucose intolerance.

Determination of Potential Lead Compound from Magnolia officinalis for Alzheimer’s Disease through Pharmacokinetic Prediction, Molecular Docking, Dynamic Simulation, and Experimental Validation

Amyloid β protein (Aβ) deposition has been implicated as the molecular driver of Alzheimer’s disease (AD) progression. The modulation of the formation of abnormal aggregates and their post-translational modification is strongly suggested as the most effective approach to anti-AD. Beta-site APP-cleaving enzyme 1 (BACE1) acts upstream in amyloidogenic processing to generate Aβ, which rapidly aggregates alone or in combination with acetylcholinesterase (AChE) to form fibrils. Accumulated Aβ promotes BACE1 activation via glycogen synthase kinase-3β (GSK-3β) and is post-translationally modified by glutaminyl cyclase (QC), resulting in increased neurotoxicity. A novel multi-target inhibitor as a potential AD agent was identified using an in silico approach and experimental validation. Magnolia officinalis, which showed the best anti-AD activity in our preliminary study, was subjected to analysis, and 82 compounds were studied. Among 23 compounds with drug-likeness, blood–brain barrier penetration, and safety, honokiol emerged as a lead structure for the inhibition of BACE1, AChE, QC, and GSK-3β in docking and molecular dynamics (MD) simulations. Furthermore, honokiol was found to be an excellent multi-target inhibitor of these enzymes with an IC50 of 6–90 μM, even when compared to other natural single-target inhibitors. Taken together, the present study is the first to demonstrate that honokiol acts as a multiple enzyme inhibitor with an excellent pharmacokinetic and safety profile which may provide inhibitory effects in broad-range areas including the overproduction, aggregation, and post-translational modification of Aβ. It also provides insight into novel structural features for the design and discovery of multi-target inhibitors for anti-AD.