ABSTRACT
Glycogen synthase kinase-3ß (GSK-3ß) is a potential target in the field of Alzheimer's disease drug discovery. We recently reported a new class of 9H-pyrimido[4,5-b]indole-based GSK-3ß inhibitors, of which 3-(3-((7-chloro-9H-pyrimido[4,5-b]indol-4-yl)(methyl)amino)piperidin-1-yl)propanenitrile (1) demonstrated promising inhibitory potency. However, this compound underwent rapid degradation by human liver microsomes. Starting from 1, we prepared a series of amide-based derivatives and studied their structure-activity relationships against GSK-3ß supported by 1 µs molecular dynamics simulations. The biological potency of this series was substantially enhanced by identifying the eutomer configuration at the stereocenter. Moreover, the introduction of an amide bond proved to be an effective strategy to eliminate the metabolic hotspot. The most potent compounds, (R)-3-(3-((7-chloro-9H-pyrimido[4,5-b]indol-4-yl)(methyl)amino)piperidin-1-yl)-3-oxopropanenitrile ((R)-2) and (R)-1-(3-((7-bromo-9Hpyrimido[4,5-b]indol-4-yl)(methyl)amino)piperidin-1-yl)propan-1-one ((R)-28), exhibited IC50 values of 480 nM and 360 nM, respectively, and displayed improved metabolic stability. Their favorable biological profile is complemented by minimal cytotoxicity and neuroprotective properties.
Subject(s)
Glycogen Synthase Kinase 3 beta/antagonists & inhibitors , Protein Kinase Inhibitors/chemistry , Protein Kinase Inhibitors/pharmacology , Animals , CHO Cells , Cell Line , Cricetulus , Drug Discovery , Drug Evaluation, Preclinical/methods , Drug Stability , Female , Glycogen Synthase Kinase 3 beta/chemistry , Glycogen Synthase Kinase 3 beta/metabolism , Humans , Indoles/chemistry , Male , Microsomes, Liver/drug effects , Microsomes, Liver/metabolism , Molecular Dynamics Simulation , Protein Kinase Inhibitors/pharmacokinetics , Structure-Activity RelationshipABSTRACT
Bearing in mind that pain and major depressive disorder (MDD) often share biological pathways, this condition is classified as depression-pain syndrome. Mounting evidence suggests that oxidative stress is implicated in the pathophysiology of this syndrome. The development of effective pharmacological interventions for the depression-pain syndrome is of particular importance as clinical treatments for this comorbidity have shown limited efficacy. Therefore, the present study aimed to evaluate whether the 3,5-dimethyl-1-phenyl-4-(phenylselanyl)-1H-pyrazole (SePy) was able to reverse the depression-pain syndrome induced by intracerebroventricular (i.c.v) streptozotocin (STZ) in mice and the possible modulation of oxidative and nitrergic pathways in its effect. The treatment with SePy (1 and 10 mg/kg) administered intragastrically (i.g.) reversed the increased immobility time in the tail suspension test, decreased grooming time in the splash test, latency time to nociceptive response in the hot plate test, and the response frequency of Von Frey hair (VFH) stimulation induced by STZ (0.2 mg/4 µl/per mouse). Additionally, SePy (10 mg/kg, i.g.) reversed STZ-induced alterations in the levels of reactive oxygen species, nitric oxide, and lipid peroxidation and the superoxide dismutase and catalase activities in the prefrontal cortices (PFC) and hippocampi (HC) of mice. Treatment with SePy (10 mg/kg, i.g.) also reversed the STZ-induced increased expression of inducible nitric oxide synthase (iNOS) and glycogen synthase kinase 3 beta (GSK3ß) in the PFC and HC. An additional molecular docking investigation found that SePy binds to the active site of iNOS and GSK3ß. Altogether, these results indicate that the antidepressant-like effect of SePy is accompanied by decreased hyperalgesia and mechanical allodynia, which were associated with its antioxidant effect.
Subject(s)
Depression/drug therapy , Nitrosative Stress/drug effects , Oxidative Stress/drug effects , Pain/drug therapy , Pyrazoles/administration & dosage , Selenium/administration & dosage , Animals , Depression/chemically induced , Depression/metabolism , Glycogen Synthase Kinase 3 beta/chemistry , Glycogen Synthase Kinase 3 beta/metabolism , Injections, Intraventricular , Male , Mice , Nitric Oxide Synthase Type II/chemistry , Nitric Oxide Synthase Type II/metabolism , Nitrosative Stress/physiology , Oxidative Stress/physiology , Pain/chemically induced , Pain/metabolism , Pain Measurement/drug effects , Pain Measurement/methods , Protein Structure, Secondary , Streptozocin/administration & dosage , Streptozocin/toxicityABSTRACT
Dietary supplementation with curcumin has been previously reported to have beneficial effects in people with insulin resistance, type 2 diabetes (T2D) and Alzheimer's disease (AD). This study investigated the effects of dietary supplementation with curcumin on key peptides implicated in insulin resistance in individuals with high risk of developing T2D. Plasma samples from participants recruited for a randomised controlled trial with curcumin (180 mg/day) for 12 weeks were analysed for circulating glycogen synthase kinase-3 ß (GSK-3ß) and islet amyloid polypeptide (IAPP). Outcome measures were determined using ELISA kits. The homeostasis model for assessment of insulin resistance (HOMA-IR) was measured as parameters of glycaemic control. Curcumin supplementation significantly reduced circulating GSK-3ß (-2.4 ± 0.4 ng/mL vs. -0.3 ± 0.6, p = 0.0068) and IAPP (-2.0 ± 0.7 ng/mL vs. 0.4 ± 0.6, p = 0.0163) levels compared with the placebo group. Curcumin supplementation significantly reduced insulin resistance (-0.3 ± 0.1 vs. 0.01 ± 0.05, p = 0.0142) compared with placebo group. Dietary supplementation with curcumin reduced circulating levels of IAPP and GSK-3ß, thus suggesting a novel mechanism through which curcumin could potentially be used for alleviating insulin resistance related markers for reducing the risk of T2D and AD.
Subject(s)
Alzheimer Disease/prevention & control , Curcumin/administration & dosage , Curcumin/pharmacology , Diabetes Mellitus, Type 2/prevention & control , Dietary Supplements , Glycogen Synthase Kinase 3 beta/chemistry , Insulin Resistance , Islet Amyloid Polypeptide/blood , Alzheimer Disease/etiology , Diabetes Mellitus, Type 2/etiology , Enzyme-Linked Immunosorbent Assay , Female , Humans , Male , Middle Aged , Risk , Time FactorsABSTRACT
Oxidative stress-mediated endothelial injury is considered to be involved in the pathogenesis of various cardiovascular diseases. Farrerol, a typical natural flavanone from the medicinal plant Rhododendron dauricum L., has been reported to show protective effects against oxidative stress-induced endothelial injuries in our previous study. However, its action molecular mechanisms and targets are still unclear. In the present study, we determined whether farrerol can interact with glycogen synthase kinase 3ß- (GSK-3ß-) nuclear factor erythroid 2-related factor 2- (Nrf2-) antioxidant response element (ARE) signaling, which is critical in defense against oxidative stress. Our results demonstrated that farrerol could specifically target Nrf2 negative regulator GSK-3ß and inhibit its kinase activity. Mechanistic studies proved that farrerol could induce an inhibitory phosphorylation of GSK-3ß at Ser9 without affecting the expression level of total GSK-3ß protein and promote the nuclear translocation of Nrf2 as well as the mRNA and protein expression of its downstream target genes heme oxygenase-1 (HO-1) and NAD(P)H: quinone oxidoreductase 1 (NQO1) in EA.hy926 cells. Further studies performed with GSK-3ß siRNA and specific inhibitor lithium chloride (LiCl) confirmed that GSK-3ß inhibition was involved in farrerol-mediated endothelial protection and Nrf2 signaling activation. Moreover, molecular docking and molecular dynamics studies revealed that farrerol could bind to the ATP pocket of GSK-3ß, which is consistent with the ATP-competitive kinetic behavior. Collectively, our results firstly demonstrate that farrerol could attenuate endothelial oxidative stress by specifically targeting GSK-3ß and further activating the Nrf2-ARE signaling pathway.
Subject(s)
Antioxidant Response Elements/genetics , Chromones/pharmacology , Endothelial Cells/drug effects , Glycogen Synthase Kinase 3 beta/antagonists & inhibitors , NF-E2 Transcription Factor/metabolism , Oxidative Stress/drug effects , Signal Transduction/drug effects , Antioxidants/pharmacology , Cell Line , Cell Nucleus/metabolism , Cell Survival/drug effects , Cell Survival/genetics , Chromones/chemistry , Endothelial Cells/enzymology , Endothelial Cells/metabolism , Endothelium/drug effects , Endothelium/enzymology , Endothelium/metabolism , Glycogen Synthase Kinase 3 beta/chemistry , Glycogen Synthase Kinase 3 beta/genetics , Glycogen Synthase Kinase 3 beta/metabolism , Heme Oxygenase-1/metabolism , Humans , Kinetics , Lithium Chloride/pharmacology , Molecular Docking Simulation , NAD(P)H Dehydrogenase (Quinone)/metabolism , NF-E2 Transcription Factor/genetics , Oxidative Stress/genetics , Phosphorylation , RNA, Small Interfering , Signal Transduction/geneticsABSTRACT
BACKGROUND: Wogonin has been reported to exhibit various biological activities such as anti-inflammation, anti-microbial, and anti-tumor. Previous studies have demonstrated that wogonin could down-regulate Cyclin D1 activity on multiple cancers. However, the related mechanisms have not been fully elucidated so far. PURPOSE: The aim of the current study was to explore whether wogonin can suppress hepatocellular carcinoma (HCC) progression and the mechanism of wogonin in inhibiting Cyclin D1 expression. METHODS: Herein, we assessed the anti-tumor activity of wogonin against hepatocellular carcinoma (HCC) by MTT assay, clonogenic assay, cell cycle analysis and orthotopic xenograft mouse models. Western blot, immunofluoscence assay, co-immunoprecipitation assay, docking program, surface plasmon resonance, site-directed mutagenesis assay and immunohistochemical assay were performed for exploring the underlying mechanisms of wogonin-induced growth inhibition in HCC. RESULTS: Our results showed that non-toxic dosage of wogonin (10, 20 µM) could inhibit cells proliferation and suppress cells cycle progression in MHCC97L and HepG2 cell. Moreover, the findings from the western blot and immunofluoscence assay confirmed the inhibition action of wogonin (10, 20 µM) on Cyclin D1 expression in MHCC97L cells, and wogonin (10, 20 µM) pre-treatment was capable of promoting Cyclin D1 ubiquitination and degradation in MHCC97L cell. In addition, wogonin promoted phosphorylation of Cyclin D1 on threonine-286 site, the mutation of threonine-286 to alanine-286A blocked Cyclin D1 proteolysis induced by wogonin. Wogonin-promoted Cyclin D1 phosphorylation and subsequent proteolysis may associate with the activation of GSK3beta in cancer cells. The phosphorylated form of GSK3beta (active form) expression was significantly increased after wogonin (20 µM) exposure. Molecular docking study and Biacore SPR analysis of GSK3beta mutant further validated the high-affinity wogonin binding site on GSK3beta. Moreover, in vivo studies further confirmed that phospho-GSK3beta Tyr216 was over-expressed in HCC specimens after wogonin treatment while the amount of Cyclin D1 was significantly decreased. CONCLUSION: In summary, our data reveal a novel molecular mechanism by which wogonin induces HCC cells cycle arrest and suppresses tumor proliferation.
Subject(s)
Carcinoma, Hepatocellular/drug therapy , Cyclin D1/metabolism , Flavanones/pharmacology , Glycogen Synthase Kinase 3 beta/metabolism , Liver Neoplasms/drug therapy , Animals , Antineoplastic Agents/pharmacology , Carcinoma, Hepatocellular/metabolism , Carcinoma, Hepatocellular/pathology , Cell Cycle Checkpoints/drug effects , Cell Proliferation/drug effects , Cyclin D1/genetics , Down-Regulation , Enzyme Activation/drug effects , Glycogen Synthase Kinase 3 beta/chemistry , Glycogen Synthase Kinase 3 beta/genetics , Hep G2 Cells , Humans , Liver Neoplasms/metabolism , Liver Neoplasms/pathology , Male , Mice, Inbred BALB C , Molecular Docking Simulation , Phosphorylation , Xenograft Model Antitumor AssaysABSTRACT
Alzheimer's disease is a rapidly increasing neurodegenerative disease. It is a multifactorial disease and also a global threat. Several enzymes are implicated in the disease in which Glycogen Synthase Kinase 3 beta is a key enzyme to increase the disease progression by the hyperphosphorylation of the tau protein. We have used an integrative chemoinformatics and pharmacokinetics approach for the identification of novel small molecules. We have retrieved a subset from the ZINC database (nâ¯=â¯5,36,709) and screened against GSK3ß in four steps. From here top 298 potent compounds were selected and employed for their pharmacokinetics analysis. We had seen that 29 compounds showed the key characteristics to be a novel drug candidate therefore, all these compounds were employed for redocking studies using Autodock Vina and Autodock. This analysis revealed that four compounds were showing good binding affinity. All these four compounds were employed for MDS analysis of 100â¯ns From here using a bunch of MD analyses we have found that out of four compounds GSK3ß-ZINC21011059 and GSK3ß-ZINC21011066 act as a stable protein-ligand complex. Therefore we proposed ZINC21011059 and ZINC21011066 can serve as a novel compounds against GSK3ß and predicted scaffold can be used for further optimization towards the improvement of isoform selectivity, and warranting further investigations towards their in vitro and in vivo validation of the bioactivity.
Subject(s)
Alzheimer Disease/drug therapy , Alzheimer Disease/enzymology , Cheminformatics , Glycogen Synthase Kinase 3 beta/antagonists & inhibitors , Small Molecule Libraries/analysis , Small Molecule Libraries/therapeutic use , Binding Sites , Drug Evaluation, Preclinical , Enzyme Stability , Glycogen Synthase Kinase 3 beta/chemistry , Humans , Hydrogen Bonding , Ligands , Molecular Docking Simulation , Principal Component Analysis , Protein Conformation , Small Molecule Libraries/pharmacokinetics , Solvents , ThermodynamicsABSTRACT
Inhibition of glycogen synthase kinase 3ß (GSK-3ß) is considered to be the central therapeutic approach against Alzheimer's disease (AD). In the present study, boiled water extracts of the Kangen-karyu (KK) herbal mixture and its constituents were screened for GSK-3ß inhibitory activity. KK is used in traditional Kampo and Chinese medicines for improving cognitive function. The GSK-3ß inhibition potential was evaluated by using the Kinase-Glo luminescent kinase assay platform. Furthermore, enzyme kinetics and in silico modeling were performed by using AutoDockTools to demonstrate the mechanism of enzyme inhibition. KK extract significantly inhibited GSK-3ß in a concentration-dependent manner (IC50: 17.05 ± 1.14 µg/mL) when compared with the reference drug luteolin (IC50: 2.18 ± 0.13 µM). Among the six components of KK, extracts of Cyperi Rhizoma and Salviae Miltiorrhizae Radix significantly inhibited GSK-3ß with IC50 values of 20.68 ± 2.50 and 7.77 ± 1.38 µg/mL, respectively. Among the constituents of the roots of S. miltiorrhiza water extract, rosmarinic acid, magnesium lithospermate B, salvianolic acid A, salvianolic acid B, and salvianolic acid C inhibited GSK-3ß with IC50 values ranging from 6.97 to 135.5 µM. Salvianolic acid B was found to be an ATP-competitive inhibitor of GSK-3ß and showed the lowest IC50 value (6.97 ± 0.96 µM). In silico modeling suggested a mechanism of action by which the hydrophobic, πâ»cation, and hydrophilic interactions of salvianolic acid B at ATP and substrate sites are critical for the observed GSK-3ß inhibition. Therefore, one of the mechanisms of action of KK against AD may be the inhibition of GSK-3ß and one of the active components of KK is the root of S. miltiorrhiza and its constituents: rosmarinic acid, magnesium lithospermate B, and salvianolic acids A, B, and C. Our results demonstrate the pharmacological basis for the use of KK against AD.
Subject(s)
Alzheimer Disease/enzymology , Drugs, Chinese Herbal/pharmacology , Glycogen Synthase Kinase 3 beta/antagonists & inhibitors , Alkenes/chemistry , Alkenes/pharmacology , Alzheimer Disease/drug therapy , Benzofurans/chemistry , Benzofurans/pharmacology , Caffeic Acids/chemistry , Caffeic Acids/pharmacology , Cinnamates/chemistry , Cinnamates/pharmacology , Computer Simulation , Depsides/chemistry , Depsides/pharmacology , Dose-Response Relationship, Drug , Drugs, Chinese Herbal/chemistry , Glycogen Synthase Kinase 3 beta/chemistry , Humans , Lactates/chemistry , Lactates/pharmacology , Molecular Docking Simulation , Molecular Structure , Plant Roots/chemistry , Polyphenols/chemistry , Polyphenols/pharmacology , Rosmarinic AcidABSTRACT
The present study was performed to evaluate the insulin-like effects of zinc in normal L6 myotubes as well as its ability to alleviate insulin resistance. Glucose consumption was measured in both normal and insulin-resistant L6 myotubes. Western blotting and immunofluorescence revealed that zinc exhibited insulin-like glucose transporting effects by activating key markers that are involved in the insulin signaling cascade (including Akt, GLUT4 and GSK3ß), and downregulating members of the insulin signaling feedback cascade such as mammalian target of rapamycin (mTOR) and ribosomal protein S6 kinase (S6K1). In normal L6 myotubes, zinc enhanced glucose consumption via a mechanism that might involve the activation of Akt phosphorylation, glucose transporter 4 (GLUT4) translocation and GSK3ß phosphorylation. In contrast, zinc exerted insulin-mimetic effects in insulin-resistant L6 myotubes by upregulating Akt phosphorylation, GLUT4 translocation and GSK3ß phosphorylation, and downregulating the expression of mTOR and S6K1. In conclusion, zinc might enhance glucose consumption by modulating insulin signaling pathways including Akt-GLUT4, GSK3ß, mTOR and S6K1.