Connecting GSK-3ß Inhibitory Activity with IKK-ß 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-3ß, IKK-ß, and ROCK-1 kinases are implicated in the pathomechanism of Alzheimer's disease due to their involvement in the misfolding and accumulation of amyloid ß (Aß) and tau proteins, as well as inflammatory processes. Among these kinases, GSK-3ß plays the most crucial role. In this study, we present compound 62, a novel, remarkably potent, competitive GSK-3ß 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.

Identification of a novel GSK3ß inhibitor involved in abrogating KRas dependent pancreatic tumors in Wnt/beta-catenin and NF-kB dependent manner.

Pancreatic cancer is an aggressive malignancy with a poor survival rate because it is difficult to diagnose the disease during its early stages. The currently available treatments, which include surgery, chemotherapy and radiation therapy, offer only limited survival benefit. Pharmacological interventions to inhibit Glycogen Synthase Kinase-3beta (GSK3ß) activity is an important therapeutic strategy for the treatment of pancreatic cancer because GSK3ß is one of the key factors involved in the onset, progression as well as in the acquisition of chemoresistance in pancreatic cancer. Here, we report the identification of MJ34 as a potent GSK3ß inhibitor that significantly reduced growth and survival of human mutant KRas dependent pancreatic tumors. MJ34 mediated GSK3ß inhibition was seen to induce apoptosis in a ß-catenin dependent manner and downregulate NF-kB activity in MiaPaCa-2 cells thereby impeding cell survival and anti-apoptotic processes in these cells as well as in the xenograft model of pancreatic cancer. In vivo acute toxicity and in vitro cardiotoxicity studies indicate that MJ34 is well tolerated without any adverse effects. Taken together, we report the discovery of MJ34 as a potential drug candidate for the therapeutic treatment of mutant KRas-dependent human cancers through pharmacological inhibition of GSK3ß.

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.

Tumor microenvironment-modulating oncolytic adenovirus combined with GSK-3ß inhibitor enhances antitumor immune response against bladder cancer.

Bladder cancer is a common type of cancer around the world, and the majority of patients are diagnosed with non-muscle-invasive bladder cancer (NMIBC). Although low-risk NMIBC has a good prognosis, the disease recurrence rate and development of treatment-refractory disease remain high in intermediate- to high-risk NMIBC patients. To address these challenges for the treatment of NMIBC, a novel combination therapy composed of an oncolytic adenovirus (oAd) co-expressing interleukin (IL)-12, granulocyte-macrophage colony-stimulating factor (GM-CSF), and relaxin (RLX; HY-oAd) and a clinical-stage glycogen synthase kinase (GSK)-3ß inhibitor (9-ING-41; elraglusib) was investigated in the present report. Our findings demonstrate that HY-oAd and 9-ING-41 combination therapy (HY-oAd+9-ING-41) exerted superior inhibition of tumor growth compared with respective monotherapy in a syngeneic NMIBC tumor model. HY-oAd+9-ING-41 induced high-level tumor extracellular matrix (ECM) degradation and a more potent antitumor immune response than the respective monotherapy. In detail, HY-oAd+9-ING-41 induced superior accumulation of intratumoral T cells, prevention of immune cell exhaustion, and induction of tumor-specific adaptive immune response compared to either monotherapy. Collectively, these results demonstrate that the combination of HY-oAd and 9-ING-41 may be a promising approach to elicit a potent antitumor immune response against bladder cancer.

Multistate Method to Efficiently Account for Tautomerism and Protonation in Alchemical Free-Energy Calculations.

The majority of drug-like molecules contain at least one ionizable group, and many common drug scaffolds are subject to tautomeric equilibria. Thus, these compounds are found in a mixture of protonation and/or tautomeric states at physiological pH. Intrinsically, standard classical molecular dynamics (MD) simulations cannot describe such equilibria between states, which negatively impacts the prediction of key molecular properties in silico. Following the formalism described by de Oliveira and co-workers (J. Chem. Theory Comput. 2019, 15, 424-435) to consider the influence of all states on the binding process based on alchemical free-energy calculations, we demonstrate in this work that the multistate method replica-exchange enveloping distribution sampling (RE-EDS) is well suited to describe molecules with multiple protonation and/or tautomeric states in a single simulation. We apply our methodology to a series of eight inhibitors of factor Xa with two protonation states and a series of eight inhibitors of glycogen synthase kinase 3ß (GSK3ß) with two tautomeric states. In particular, we show that given a sufficient phase-space overlap between the states, RE-EDS is computationally more efficient than standard pairwise free-energy methods.

Combined in vivo effect of N-acylethanolamine-hydrolyzing acid amidase and glycogen synthase kinase-3ß inhibition to treat multiple sclerosis.

The current pharmacological approaches to multiple sclerosis (MS) target its inflammatory and autoimmune components, but effective treatments to foster remyelination and axonal repair are still lacking. We therefore selected two targets known to be involved in MS pathogenesis: N-acylethanolamine-hydrolyzing acid amidase (NAAA) and glycogen synthase kinase-3ß (GSK-3ß). We tested whether inhibiting these targets exerted a therapeutic effect against experimental autoimmune encephalomyelitis (EAE), an animal model of MS. The combined inhibition of NAAA and GSK-3ß by two selected small-molecule compounds, ARN16186 (an NAAA inhibitor) and AF3581 (a GSK-3ß inhibitor), effectively mitigated disease progression, rescuing the animals from paralysis and preventing a worsening of the pathology. The complementary activity of the two inhibitors reduced the infiltration of immune cells into the spinal cord and led to the formation of thin myelin sheaths around the axons post-demyelination. Specifically, the inhibition of NAAA and GSK-3ß modulated the over-activation of NF-kB and STAT3 transcription factors in the EAE-affected mice and induced the nuclear translocation of ß-catenin, reducing the inflammatory insult and promoting the remyelination process. Overall, this work demonstrates that the dual-targeting of key aspects responsible for MS progression could be an innovative pharmacological approach to tackle the pathology.

Identifying potential Alzheimer's disease therapeutics through GSK-3ß inhibition: A molecular docking and dynamics approach.

Emerging as a promising drug target for Alzheimer's disease (AD) therapy, glycogen synthase kinase 3ß (GSK-3ß) has garnered attention. This study sought to rigorously scrutinize a compendium of natural compounds retrieved from the ZINC database through pharmacodynamic experiments, employing a 1 H-indazole-3-carboxamide (INDZ) scaffold, to identify compounds capable of inhibiting the GSK-3ß protein. Utilizing a multi-step approach, the study involved pharmacophore analysis, followed by molecular docking to select five promising ligands for further investigation. Subsequently, ESMACS simulations were employed to assess the stability of the ligand-protein interactions. Evaluation of the binding modes and free energy of the ligands revealed that five compounds (2a-6a) exhibited crucial interactions with the active site residues. Furthermore, various methodologies, including hydrogen bond and clustering analyses, were utilized to ascertain their inhibitory potential and elucidate the factors contributing to ligand binding in the protein's active site. The findings from MMPBSA/GBSA analysis indicated that these five selected small molecules closely approached the IC50 value of the reference ligand (OH8), yielding energy values of -34.85, -32.58, -31.71, and -30.39 kcal/mol, respectively. Additionally, an assessment of the interactions using hydrogen bond and dynamic analyses delineated the effective binding of the ligands with the binding pockets in the protein. Through computational analysis, we obtained valuable insights into the molecular mechanisms of GSK-3ß, aiding in the development of more potent inhibitors.

Impact of GSK-3ß and CK-1δ on Wnt signaling pathway in alzheimer disease: A dual target approach.

Alzheimer's disease (AD) is an enigmatic neurological illness that offers few treatment options. Recent exploration has highlighted the crucial connection of the Wnt signaling pathway in AD pathogenesis, shedding light on potential therapeutic targets. The present study focuses on the dual targeting of glycogen synthase kinase-3ß (GSK-3ß) and casein kinase-1δ (CK-1δ) within the framework of the Wnt signaling pathway as a possible technique for AD intervention. GSK-3ß and CK-1δ are multifunctional kinases known for their roles in tau hyperphosphorylation, amyloid processing, and synaptic dysfunction, all of which are major hallmarks of Alzheimer's disease. They are intricately linked to Wnt signaling, which plays a pivotal part in sustaining neuronal function and synaptic plasticity. Dysregulation of the Wnt pathway in AD contributes to cognitive decline and neurodegeneration. This review delves into the molecular mechanisms by which GSK-3ß and CK-1δ impact the Wnt signaling pathway, elucidating their roles in AD pathogenesis. We discuss the potential of small-molecule inhibitors along with their SAR studies along with the multi-targetd approach targeting GSK-3ß and CK-1δ to modulate Wnt signaling and mitigate AD-related pathology. In summary, the dual targeting of GSK-3ß and CK-1δ within the framework of the Wnt signaling pathway presents an innovative and promising avenue for future AD therapies, offering new hope for patients and caregivers in the quest to combat this challenging condition.

Glycogen Synthase Kinase-3 Beta (GSK3ß) as a Potential Drug Target in Regulating Osteoclastogenesis: An Updated Review on Current Evidence.

Glycogen synthase kinase 3-beta (GSK3ß) is a highly conserved protein kinase originally involved in glucose metabolism, insulin activity, and energy homeostasis. Recent scientific evidence demonstrated the significant role of GSK3ß in regulating bone remodelling through involvement in multiple signalling networks. Specifically, the inhibition of GSK3ß enhances the conversion of osteoclast progenitors into mature osteoclasts. GSK3ß is recognised as a pivotal regulator for the receptor activator of nuclear factor-kappa B (RANK)/receptor activator of nuclear factor-kappa B ligand (RANKL)/osteoprotegerin (OPG), phosphatidylinositol-3-kinase (PI3K)/protein kinase B (AKT), nuclear factor-kappa B (NF-κB), nuclear factor-erythroid 2-related factor 2 (NRF2)/Kelch-like ECH-associated protein 1 (KEAP1), canonical Wnt/beta (ß)-catenin, and protein kinase C (PKC) signalling pathways during osteoclastogenesis. Conversely, the inhibition of GSK3ß has been shown to prevent bone loss in animal models with complex physiology, suggesting that the role of GSK3ß may be more significant in bone formation than bone resorption. Divergent findings have been reported regarding the efficacy of GSK3ß inhibitors as bone-protecting agents. Some studies demonstrated that GSK3ß inhibitors reduced osteoclast formation, while one study indicated an increase in osteoclast formation in RANKL-stimulated bone marrow macrophages (BMMs). Given the discrepancies observed in the accumulated evidence, further research is warranted, particularly regarding the use of GSK3ß silencing or overexpression models. Such efforts will provide valuable insights into the direct impact of GSK3ß on osteoclastogenesis and bone resorption.

Design, Synthesis, and Biological Evaluation of Novel Tetrahydroacridin Hybrids with Sulfur-Inserted Linkers as Potential Multitarget Agents for Alzheimer's Disease.

Alzheimer's disease (AD) is a complex neurodegenerative disease that can lead to the loss of cognitive function. The progression of AD is regulated by multiple signaling pathways and their associated targets. Therefore, multitarget strategies theoretically have greater potential for treating AD. In this work, a series of new hybrids were designed and synthesized by the hybridization of tacrine (4, AChE: IC50 = 0.223 µM) with pyrimidone compound 5 (GSK-3ß: IC50 = 3 µM) using the cysteamine or cystamine group as the connector. The biological evaluation results demonstrated that most of the compounds exhibited moderate to good inhibitory activities against acetylcholinesterase (AChE) and glycogen synthase kinase 3ß (GSK-3ß). The optimal compound 18a possessed potent dual AChE/GSK-3ß inhibition (AChE: IC50 = 0.047 ± 0.002 µM, GSK-3ß: IC50 = 0.930 ± 0.080 µM). Further molecular docking and enzymatic kinetic studies revealed that this compound could occupy both the catalytic anionic site and the peripheral anionic site of AChE. The results also showed a lack of toxicity to SH-SY5Y neuroblastoma cells at concentrations of up to 25 µM. Collectively, this work explored the structure-activity relationships of novel tetrahydroacridin hybrids with sulfur-inserted linkers, providing a reference for the further research and development of new multitarget anti-AD drugs.

Mature neurons from iPSCs unveil neurodegeneration-related pathways in mucopolysaccharidosis type II: GSK-3ß inhibition for therapeutic potential.

Mucopolysaccharidosis (MPS) type II is caused by a deficiency of iduronate-2-sulfatase and is characterized by the accumulation of glycosaminoglycans (GAGs). Without effective therapy, the severe form of MPS II causes progressive neurodegeneration and death. This study generated multiple clones of induced pluripotent stem cells (iPSCs) and their isogenic controls (ISO) from four patients with MPS II neurodegeneration. MPS II-iPSCs were successfully differentiated into cortical neurons with characteristic biochemical and cellular phenotypes, including axonal beadings positive for phosphorylated tau, and unique electrophysiological abnormalities, which were mostly rescued in ISO-iPSC-derived neurons. RNA sequencing analysis uncovered dysregulation in three major signaling pathways, including Wnt/ß-catenin, p38 MAP kinase, and calcium pathways, in mature MPS II neurons. Further mechanistic characterization indicated that the dysregulation in calcium signaling led to an elevated intracellular calcium level, which might be linked to compromised survival of neurons. Based on these dysregulated pathways, several related chemicals and drugs were tested using this mature MPS II neuron-based platform and a small-molecule glycogen synthase kinase-3ß inhibitor was found to significantly rescue neuronal survival, neurite morphology, and electrophysiological abnormalities in MPS II neurons. Our results underscore that the MPS II-iPSC-based platform significantly contributes to unraveling the mechanisms underlying the degeneration and death of MPS II neurons and assessing potential drug candidates. Furthermore, the study revealed that targeting the specific dysregulation of signaling pathways downstream of GAG accumulation in MPS II neurons with a well-characterized drug could potentially ameliorate neuronal degeneration.

APE1 inhibition enhances ferroptotic cell death and contributes to hepatocellular carcinoma therapy.

Ferroptosis, a regulated form of cell death triggered by iron-dependent lipid peroxidation, has emerged as a promising therapeutic strategy for cancer treatment, particularly in hepatocellular carcinoma (HCC). However, the mechanisms underlying the regulation of ferroptosis in HCC remain to be unclear. In this study, we have identified a novel regulatory pathway of ferroptosis involving the inhibition of Apurinic/apyrimidinic endonuclease 1 (APE1), a key enzyme with dual functions in DNA repair and redox regulation. Our findings demonstrate that inhibition of APE1 leads to the accumulation of lipid peroxidation and enhances ferroptosis in HCC. At the molecular level, the inhibition of APE1 enhances ferroptosis which relies on the redox activity of APE1 through the regulation of the NRF2/SLC7A11/GPX4 axis. We have identified that both genetic and chemical inhibition of APE1 increases AKT oxidation, resulting in an impairment of AKT phosphorylation and activation, which leads to the dephosphorylation and activation of GSK3ß, facilitating the subsequent ubiquitin-proteasome-dependent degradation of NRF2. Consequently, the downregulation of NRF2 suppresses SLC7A11 and GPX4 expression, triggering ferroptosis in HCC cells and providing a potential therapeutic approach for ferroptosis-based therapy in HCC. Overall, our study uncovers a novel role and mechanism of APE1 in the regulation of ferroptosis and highlights the potential of targeting APE1 as a promising therapeutic strategy for HCC and other cancers.

GSK3ß Inhibition Ameliorates Atherosclerotic Calcification.

Endothelial-mesenchymal transition (EndMT) drives endothelium to contribute to atherosclerotic calcification. In a previous study, we showed that glycogen synthase kinase-3ß (GSK3ß) inhibition induced ß-catenin and reduced mothers against DPP homolog 1 (SMAD1) in order to redirect osteoblast-like cells towards endothelial lineage, thereby reducing vascular calcification in Matrix Gla Protein (Mgp) deficiency and diabetic Ins2Akita/wt mice. Here, we report that GSK3ß inhibition or endothelial-specific deletion of GSK3ß reduces atherosclerotic calcification. We also find that alterations in ß-catenin and SMAD1 induced by GSK3ß inhibition in the aortas of Apoe-/- mice are similar to Mgp-/- mice. Together, our results suggest that GSK3ß inhibition reduces vascular calcification in atherosclerotic lesions through a similar mechanism to that in Mgp-/- mice.

Synthesis of Unsymmetrical Squaramides as Allosteric GSK-3ß Inhibitors Promoting ß-Catenin-Mediated Transcription of TCF/LEF in Retinal Pigment Epithelial Cells.

The glycogen synthase kinase 3ß (GSK-3ß) is a ubiquitous enzyme that is a validated target for the development of potential therapeutics useful in several diseases including retinal degeneration. Aiming at developing an innovative class of allosteric inhibitors of GSK-3ß potentially useful for retinal degeneration, we explored the class of squaramides. The developed compounds (6 a-l) were obtained through a nontoxic one-pot synthetic protocol, which employs low-cost goods and avoids any purification step. Ethanol was used as the reaction solvent, simultaneously allowing the pure reaction products' recovery (by precipitation). Out of this set of squaramides, 6 j stood out, from computational and enzymatic converging data, as an ATP non-competitive inhibitor of GSK-3ß of micromolar potency. When engaged in cellular studies using retinal pigment epithelial cells (ARPE-19) transfected with a luciferase reporter gene under the control of T-cell factor/lymphoid enhancer factor (TCF/LEF) binding sites, 6 j was able to dose-dependently induce ß-catenin nuclear accumulation, as shown by the increased luciferase activity at a concentration of 2.5 µM.

Synthesis and biological evaluation of thieno[3,2-c]pyrazol-3-amine derivatives as potent glycogen synthase kinase 3ß inhibitors for Alzheimer's disease.

Glycogen synthase kinase 3ß (GSK-3ß) catalyses the hyperphosphorylation of tau protein in the Alzheimer's disease (AD) pathology. A series of novel thieno[3,2-c]pyrazol-3-amine derivatives were designed and synthesised and evaluated as potential GSK-3ß inhibitors by structure-guided drug rational design approach. The thieno[3,2-c]pyrazol-3-amine derivative 16b was identified as a potent GSK-3ß inhibitor with an IC50 of 3.1 nM in vitro and showed accepted kinase selectivity. In cell levels, 16b showed no toxicity on the viability of SH-SY5Y cells at the concentration up to 50 µM and targeted GSK-3ß with the increased phosphorylated GSK-3ß at Ser9. Western blot analysis indicated that 16b decreased the phosphorylated tau at Ser396 in a dose-dependent way. Moreover, 16b effectively increased expressions of ß-catenin as well as the GAP43, N-myc, and MAP-2, and promoted the differentiated neuronal neurite outgrowth. Therefore, the thieno[3,2-c]pyrazol-3-amine derivative 16b could serve as a promising GSK-3ß inhibitor for the treatment of AD.

Development of inhibitors targeting glycogen synthase kinase-3ß for human diseases: Strategies to improve selectivity.

Glycogen synthase kinase-3ß (GSK-3ß) is a conserved serine/threonine kinase that participates in the transmission of multiple signaling pathways and plays an important role in the occurrence and development of human diseases, such as metabolic diseases, neurological diseases and cancer, making it to be a potential and promising drug target. To date, copious GSK-3ß inhibitors have been synthesized, but only few have entered clinical trials. Most of them exerts poor selectivity, concomitant off-target effects and side effects. This review summarizes the structural characteristics, biological functions and relationship with diseases of GSK-3ß, as well as the selectivity profile and therapeutic potential of different categories of GSK-3ß inhibitors. Strategies for increasing selectivity and reducing adverse effects are proposed for the future design of GSK-3ß inhibitors.

S-SCAM inhibits Axin-dependent synaptic function of GSK3ß in a sex-dependent manner.

S-SCAM/MAGI-2 gene duplication is associated with schizophrenia (SCZ). S-SCAM overexpression in the forebrain induces SCZ-like phenotypes in a transgenic (Tg) mouse model. Interestingly, S-SCAM Tg mice show male-specific impairments in synaptic plasticity and working memory. However, mechanisms underlying the sex-specific deficits remain unknown. Here we report that S-SCAM Tg mice have male-specific deficits in synaptic GSK3ß functions, as shown by reduced synaptic protein levels and increased inhibitory phosphorylation of GSK3ß. This GSK3ß hyper-phosphorylation was associated with increased CaMKII activities. Notably, synaptic levels of Axin1, to which GSK3ß binds in competition with S-SCAM, were also reduced in male S-SCAM Tg mice. We demonstrated that Axin-binding is required for the S-SCAM overexpression-induced synaptic GSK3ß reduction. Axin stabilization using XAV939 rescued the GSK3ß deficits and restored the temporal activation of GSK3ß during long-term depression in S-SCAM overexpressing neurons. Interestingly, synaptic Axin2 levels were increased in female S-SCAM Tg mice. Female sex hormone 17ß-estradiol increased Axin2 expression and increased synaptic GSK3ß levels in S-SCAM overexpressing neurons. These results reveal the role of S-SCAM in controlling Axin-dependent synaptic localization of GSK3ß. Moreover, our studies point out the pathological relevance of GSK3ß hypofunction found in humans and contribute to understanding the molecular underpinnings of sex differences in SCZ.

GSK-3ß inhibition protects human nucleus pulposus cell against oxidative stress-inducing apoptosis through mitochondrial pathway.

BACKGROUND: Oxidative stress in the intervertebral disc leads to nucleus pulposus (NP) degeneration by inducing cell apoptosis. However, the molecular mechanisms underlying this process remain unclear. Increasing evidence indicates that GSK-3ß is related to cell apoptosis induced by oxidative stress. In this study, we explored whether GSK-3ß inhibition protects human NP cell against apoptosis under oxidative stress. METHODS AND RESULTS: Immunofluorescence staining was used to show the expression of GSK-3ß in human NP cells (NPCs). Flow cytometry, mitochondrial staining and western blot (WB) were used to detect apoptosis of treated NPCs, changes of mitochondrial membrane potential and the expression of mitochondrial apoptosis-related proteins using GSK-3ß specific inhibitor SB216763. Co-Immunoprecipitation (Co-IP) was used to demonstrate the interaction between GSK-3ß and Bcl-2. We delineated the protective effect of GSK-3ß specific inhibitor SB216763 on human NPCs apoptosis induced by oxidative stress in vitro. Further, we showed SB216763 exert the protective effect by preservation of the mitochondrial membrane potential and inhibition of caspase 3/7 activity during oxidative injury. The detailed mechanism underlying the antiapoptotic effect of GSK-3ß inhibition was also studied by analyzing mitochondrial apoptosis pathway in vitro. CONCLUSIONS: We concluded that the GSK-3ß inhibitor SB216763 protected mitochondrial membrane potential to delay nucleus pulposus cell apoptosis by inhibiting the interaction between GSK-3ß and Bcl-2 and subsequently reducing cytochrome c(Cyto-C) release and caspase-3 activation. Together, inhibition of GSK-3ß using SB216763 in NPCs may be a favorable therapeutic strategy to slow intervertebral disc degeneration.

A non-catalytic scaffolding activity of hexokinase 2 contributes to EMT and metastasis.

Hexokinase 2 (HK2), which catalyzes the first committed step in glucose metabolism, is induced in cancer cells. HK2's role in tumorigenesis has been attributed to its glucose kinase activity. Here, we describe a kinase independent HK2 activity, which contributes to metastasis. HK2 binds and sequesters glycogen synthase kinase 3 (GSK3) and acts as a scaffold forming a ternary complex with the regulatory subunit of protein kinase A (PRKAR1a) and GSK3ß to facilitate GSK3ß phosphorylation and inhibition by PKA. Thus, HK2 functions as an A-kinase anchoring protein (AKAP). Phosphorylation by GSK3ß targets proteins for degradation. Consistently, HK2 increases the level and stability of GSK3 targets, MCL1, NRF2, and particularly SNAIL. In addition to GSK3 inhibition, HK2 kinase activity mediates SNAIL glycosylation, which prohibits its phosphorylation by GSK3. Finally, in mouse models of breast cancer metastasis, HK2 deficiency decreases SNAIL protein levels and inhibits SNAIL-mediated epithelial mesenchymal transition and metastasis.

Suppression of nitrosative stress and inflammation of the knee joint synovium in collagen type ii-induced rheumatoid arthritis by the inhibition of glycogen synthase kinase-3ß / Supresión del estrés nitrosativo e inflamación de la sinovial de la articulación de la rodilla en la artritis reumatoide inducida por colágeno tipo ii mediante la inhibición de la glucógeno sintasa quinasa-3 ß

SUMMARY: Rheumatoid arthritis (RA), an inflammatory autoimmune disease that causes cartilage degradation and tissue destruction, can affect synovial joints such as the knee joint. The link between the nitrosative stress enzyme inducible nitric oxide synthase (iNOS) and the cytokine interleukin-1 (IL-1β) in RA-induced knee joint synovial membrane damage with and without the incorporation of the GSK3β inhibitor TDZD-8 has never been studied. As a result, we used active immunization method with collagen type II (COII) for twenty one days to induce RA in rats. TDZD-8 (1 mg/kg; i.p.) was given daily into matched immunized rats for three weeks after day 21 (COII+TDZD-8). Blood and tissue samples were taken 42 days after immunization. A dramatic increase in rheumatoid factor (RF) blood levels, as well as considerable synovial tissue damage and inflammatory cell infiltration of the synovial membrane, were used to validate the onset of RA following COII immunization. COII immunization increased tissue levels of iNOS protein and IL- 1β mRNA and protein expression, which TDZD-8 suppressed considerably (p<0.0001). Furthermore, there was a significantly (p<0.001) positive correlation between iNOS, inflammatory biomarkers, and RF. We concluded that TDZD-8 reduced RA-induced IL-1β -iNOS axis-mediated arthritis in the rat knee joint synovium.

RESUMEN: La artritis reumatoide (AR), es una enfermedad autoinmune inflamatoria que causa la degradación del cartílago y la destrucción del tejido, pudiendo afectar las articulaciones sinoviales, como la articulación de la rodilla. No se ha estudiado el vínculo entre la óxido nítrico sintasa inducible por la enzima del estrés nitrosativo (iNOS) y la citocina interleucina-1 (IL-1β) en el daño de la membrana sinovial de la articulación de la rodilla provocado por AR con y sin la incorporación del inhibidor de GSK3β TDZD-8. Utilizamos el método de inmunización activa con colágeno tipo II (COII) durante veintiún días para inducir AR en ratas. Se administró TDZD-8 (1 mg/kg; i.p.) diariamente a ratas inmunizadas emparejadas durante tres semanas después del día 21 (COII+TDZD- 8). Se tomaron muestras de sangre y tejido 42 días después de la inmunización. Se observó un gran aumento de los niveles sanguíneos del factor reumatoideo (FR), así como un daño considerable del tejido sinovial e infiltración de células inflamatorias en la membrana sinovial, para validar la aparición de la AR después de la inmunización con COII. La inmunización con COII aumentó los niveles tisulares de la proteína iNOS y la expresión de proteína y ARNm de IL-1β, que TDZD-8 suprimió considerablemente (p<0,0001). Además, hubo una correlación positiva significativa (p<0,001) entre iNOS, biomarcadores inflamatorios y FR. Concluimos que TDZD- 8 redujo la artritis mediada por el eje IL-1β-iNOS inducida por la AR en la sinovial de la articulación de la rodilla de rata.