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.

Proposed hypothesis of GSK-3 ß inhibition for stimulating Wnt/ß-catenin signaling pathway which triggers liver regeneration process.

Almost every human organ has a poor ability to regenerate, notable exceptions are liver, skin, gut, etc. Molecular and cellular underpinnings of liver regeneration might pave the way for novel treatments concerned with chronic liver disorder. Such treatments would eliminate the disadvantages of liver transplantation, such as a scarcity of donor organs, a lengthy waitlist, significant medical expenses, surgical complications, and the necessity for lifelong immunosuppressive medications. Advancement in the development of regenerative therapy is giving hope to those suffering from end-stage liver disorder. The regeneration process is unique, intricate, and well coordinated, which involve the interaction of numerous signaling pathways, cytokines, and growth factor. Various signaling pathways for liver regeneration are HO-1/BER pathway, Tweak/Fn14 signaling pathway, Hippo pathway, Wnt/beta-catenin pathway, Hedgehog signaling pathway, bile acids repairing pathway, serotonin (5HT) pathway, estrogen pathway, thyrotropin-releasing hormone (TRH) pathway, insulin repairing pathway, etc. The in vitro scientific literature revealed that numerous GSK-3 ß inhibitors (LY 2090314, AR-A014418, Tideglusib, Solasodine, CHIR99021, 9-ING-41, SB-216763) play an important role in stimulating the liver regeneration process. Similarly, from the above discussion, the direction is highlighted to emphasize the proposed molecular Wnt/ß-catenin signaling pathway which is associated with GSK-3 ß inhibition for the induction of the repairing and regeneration process.

Discovery of novel ß-carboline derivatives as selective AChE inhibitors with GSK-3ß inhibitory property for the treatment of Alzheimer's disease.

The natural product harmine, a representative ß-carboline alkaloid from the seeds of Peganum harmala L. (Zygophyllaceae), possesses a broad spectrum of biological activities. In this study, a novel series of harmine derivatives containing N-benzylpiperidine moiety were identified for the treatment of Alzheimer's disease (AD). The results showed that all the derivatives possessed significant anti-acetylcholinesterase (AChE) activity and good selectivity over butyrylcholinesterase (BChE). In particular, compound ZLWH-23 exhibited potent anti-AChE activity (IC50 = 0.27 µM) and selective BChE inhibition (IC50 = 20.82 µM), as well as acceptable glycogen synthase kinase-3 (GSK-3ß) inhibition (IC50 = 6.78 µM). Molecular docking studies and molecular dynamics simulations indicated that ZLWH-23 could form stable interaction with AChE and GSK-3ß. Gratifyingly, ZLWH-23 exhibited good selectivity for GSK-3ß over multi-kinases and very low cytotoxicity towards SH-SY5Y, HEK-293T, HL-7702, and HepG2 cell lines. Importantly, ZLWH-23 displayed efficient reduction against tau hyperphosphorylation on Ser-396 site in Tau (P301L) 293T cell model. Collectively, harmine-based derivatives could be considered as possible drug leads for the development of AD therapies.

Potential role of Drug Repositioning Strategy (DRS) for management of tauopathy.

Tauopathy is a term that has been used to represent a pathological condition in which hyperphosphorylated tau protein aggregates in neurons and glia which results in neurodegeneration, synapse loss and dysfunction and cognitive impairments. Recently, drug repositioning strategy (DRS) becomes a promising field and an alternative approach to advancing new treatments from actually developed and FDA approved drugs for an indication other than the indication it was originally intended for. This paradigm provides an advantage because the safety of the candidate compound has already been established, which abolishes the need for further preclinical safety testing and thus substantially reduces the time and cost involved in progressing of clinical trials. In the present review, we focused on correlation between tauopathy and common diseases as type 2 diabetes mellitus and the global virus COVID-19 and how tau pathology can aggravate development of these diseases in addition to how these diseases can be a risk factor for development of tauopathy. Moreover, correlation between COVID-19 and type 2 diabetes mellitus was also discussed. Therefore, repositioning of a drug in the daily clinical practice of patients to manage or prevent two or more diseases at the same time with lower side effects and drug-drug interactions is a promising idea. This review concluded the results of pre-clinical and clinical studies applied on antidiabetics, COVID-19 medications, antihypertensives, antidepressants and cholesterol lowering drugs for possible drug repositioning for management of tauopathy.

Anti-Inflammatory Effect of IKK-Activated GSK-3ß Inhibitory Peptide Prevented Nigrostriatal Neurodegeneration in the Rodent Model of Parkinson's Disease.

Parkinson's disease (PD) is a progressive movement disorder caused by nigrostriatal neurodegeneration. Since chronically activated neuroinflammation accelerates neurodegeneration in PD, we considered that modulating chronic neuroinflammatory response might provide a novel therapeutic approach. Glycogen synthase kinase 3 (GSK-3) is a multifunctional serine/threonine protein kinase with two isoforms, GSK-3α and GSK-3ß, and GSK-3ß plays crucial roles in inflammatory response, which include microglial migration and peripheral immune cell activation. GSK-3ß inhibitory peptide (IAGIP) is specifically activated by activated inhibitory kappa B kinase (IKK), and its therapeutic effects have been demonstrated in a mouse model of colitis. Here, we investigated whether the anti-inflammatory effects of IAGIP prevent neurodegeneration in the rodent model of PD. IAGIP significantly reduced MPP+-induced astrocyte activation and inflammatory response in primary astrocytes without affecting the phosphorylations of ERK or JNK. In addition, IAGIP inhibited LPS-induced cell migration and p65 activation in BV-2 microglial cells. In vivo study using an MPTP-induced mouse model of PD revealed that intravenous IAGIP effectively prevented motor dysfunction and nigrostriatal neurodegeneration. Our findings suggest that IAGIP has a curative potential in PD models and could offer new therapeutic possibilities for targeting PD.

Addressing a Trapped High-Energy Water: Design and Synthesis of Highly Potent Pyrimidoindole-Based Glycogen Synthase Kinase-3ß Inhibitors.

In small molecule binding, water is not a passive bystander but rather takes an active role in the binding site, which may be decisive for the potency of the inhibitor. Here, by addressing a high-energy water, we improved the IC50 value of our co-crystallized glycogen synthase kinase-3ß (GSK-3ß) inhibitor by nearly two orders of magnitude. Surprisingly, our results demonstrate that this high-energy water was not displaced by our potent inhibitor (S)-3-(3-((7-ethynyl-9H-pyrimido[4,5-b]indol-4-yl)(methyl)amino)piperidin-1-yl)propanenitrile ((S)-15, IC50 value of 6 nM). Instead, only a subtle shift in the location of this water molecule resulted in a dramatic decrease in the energy of this high-energy hydration site, as shown by the WaterMap analysis combined with microsecond timescale molecular dynamics simulations. (S)-15 demonstrated both a favorable kinome selectivity profile and target engagement in a cellular environment and reduced GSK-3 autophosphorylation in neuronal SH-SY5Y cells. Overall, our findings highlight that even a slight adjustment in the location of a high-energy water can be decisive for ligand binding.

Anti-fibrotic effect of 6-bromo-indirubin-3'-oxime (6-BIO) via regulation of activator protein-1 (AP-1) and specificity protein-1 (SP-1) transcription factors in kidney cells.

PAI-1 and CTGF are overexpressed in kidney diseases and cause fibrosis of the lungs, liver, and kidneys. We used a rat model of unilateral ureteral obstruction (UUO) to investigate whether 6-BIO, a glycogen synthase kinase-3ß inhibitor, attenuated fibrosis by inhibiting PAI-1 and CTGF in vivo. Additionally, TGFß-induced cellular fibrosis was observed in vitro using the human kidney proximal tubular epithelial cells (HK-2), and rat interstitial fibroblasts (NRK49F). Expression of fibrosis-related proteins and signaling molecules such as PAI-1, CTGF, TGFß, αSMA, SMAD, and MAPK were determined in HK-2 and NRK49F cells using immunoblotting. To identify the transcription factors that regulate the expression of PAI-1 and CTGF the promoter activities of AP-1 and SP-1 were analyzed using luciferase assays. Confocal microscopy was used to observe the co-localization of AP-1 and SP-1 to PAI-1 and CTGF. Expression of PAI-1, CTGF, TGFß, and α-SMA increased in UUO model as well as in TGFß-treated HK-2 and NRK49F cells. Furthermore, UUO and TGFß treatment induced the activation of P-SMAD2/3, SMAD4, P-ERK 1/2, P-P38, and P-JNK MAPK signaling pathways. PAI-1, CTGF, AP-1 and SP-1 promoter activity increased in response to TGFß treatment. However, treatment with 6-BIO decreased the expression of proteins and signaling pathways associated with fibrosis in UUO model as well as in TGFß-treated HK-2 and NRK49F cells. Moreover, 6-BIO treatment attenuated the expression of PAI-1 and CTGF as well as the promoter activities of AP-1 and SP-1, thereby regulating the SMAD and MAPK signaling pathways, and subsequently exerting anti-fibrotic effects on kidney cells.

GSK-3ß inhibition elicits a neuroprotection by restoring lysosomal dysfunction in neurons via facilitation of TFEB nuclear translocation after ischemic stroke.

Lysosomal dysfunction is an essential pathogenesis of autophagic neuronal injury after ischemic stroke. As a result of cerebral ischemia, transcription factor EB (TFEB) is greatly phosphorylated by prominently activated glycogen synthase kinase-3ß (GSK-3ß). This increased TFEB phosphorylation decreases its nuclear translocation and subsequently leads to reduced lysosomal biosynthesis, which ultimately results in lysosomal dysfunction. The present study is to investigate whether the lysosomal dysfunction in neurons can be restored to alleviate post-stroke damage by GSK-3ß inhibition. The GSK-3ß activity was inhibited by pre-treatment with CHIR-99021 (CHIR) for 3 days before middle cerebral artery occlusion (MCAO) surgery in rats. Besides, the lysosomal capacity was altered by pre-administration with Bafilomycin A1 (Baf-A1) and EN6, respectively. Twenty-four hours after MCAO/reperfusion, the penumbral tissues were obtained to detect the GSK-3ß, cytoplasmic and nuclear TFEB, and proteins in autophagic/lysosomal pathway by western blot and immunofluorescence, respectively. Meanwhile, the infarct volume, neurological deficits and neuron survival were assessed to evaluate the neurological outcomes elicited by GSK-3ß inhibition. The results demonstrated that the neurological injury could be significantly mitigated by GSK-3ß inhibition in MCAO + CHIR group, compared with that in MCAO group. Moreover, CHIR-facilitated TFEB nuclear translocation in neurons was coupled with reinforced lysosomal activities and attenuated autophagic substrates. However, GSK-3ß inhibition-induced neuroprotection was greatly counteracted by Baf-A1-weakened lysosomal capacity. Conversely, EN6-reinforced lysosomal activities further ameliorated the autophagic/lysosomal signaling, and synergistically alleviated the neurological damage upon GSK-3ß inhibition after MCAO/reperfusion. Our data suggests that GSK-3ß inhibition-augmented neuroprotection against ischemic stroke is elicited by restoring the lysosomal dysfunction in neurons.

Design, synthesis, in vitro antiproliferative evaluation and GSK-3ß kinase inhibition of a new series of pyrimidin-4-one based amide conjugates.

A new series of novel amide conjugates of pyrimidin-4-one and aromatic/heteroaromatic /secondary cyclic amines has been synthesized and their in vitro antiproliferative activities against a panel of 60 human cancer cell lines of nine different cancer types were tested at NCI. Among the synthesized compounds, compound (4i) showed significant anti-proliferative activity. Compound (4i) displayed most potent activity against the breast tumor cell line T-47D and CNS tumor cell line SNB-75 exhibiting a growth of 1.93 % and 14.63 %, respectively. ADMET studies of the synthesized compounds were also performed and they were found to exhibit good drug like properties. Compound (4i) was found to exhibit potential inhibitory effect over GSK-3ß with IC50 value of 71 nM. The molecular docking studies revealed that (4i) showed good binding affinity to GSK-3ß and revealed multiple H-bonding and p-cation interactions with important amino acid residues on the receptor site. Compound (4i) may thus serve as a potential candidate for further development of novel anticancer therapeutics.