Discovery of Selective, Substrate-Competitive, and Passive Membrane Permeable Glycogen Synthase Kinase-3β Inhibitors: Synthesis, Biological Evaluation, and Molecular Modeling of New <i>C</i>‑Glycosylflavones

Glycogen synthase kinase-3β (GSK-3β) is a key enzyme responsible for tau hyperphosphorylation and is a viable therapeutic target of Alzheimer’s disease (AD). We developed a new class of GSK-3β inhibitors based on the 6-<i>C</i>-glycosylflavone isoorientin (<b>1</b>). The new inhibitors are passive membrane permeable and constitutively attenuate GSK-3β mediated tau hyperphosphorylation and amyloid neurotoxicity in an AD cellular model. Enzymatic assays and kinetic studies demonstrated that compound <b>30</b> is a GSK-3β substrate-competitive inhibitor with distinct kinase selectivity, isoform-selectivity and over 310-fold increased potency as compared to <b>1</b>. Structure–activity relationship analyses and in silico modeling suggest the mechanism of actions by which the hydrophobic, π–cation, and orthogonal multipolar interactions of <b>30</b> with the substrate site are critical for the GSK-3β inhibition and selectivity. The results provide new insights into GSK-3β drug discovery. The new inhibitors are valuable chemical probes and drug leads with therapeutic potential to tackle AD and other GSK-3β relevant diseases

Effect of BEX on lipotoxic MCP-1 production.

<p>Production of MCP-1 was measured in the cell media supernatant (A–C) and cytosolic fraction (D–F) of 3T3-L1 (A, D), C2C12 (B, E), and Hepa6 (C, F) murine cells treated with palmitic acid (PA, 0.4 mM) in combination with BEX (125 µg/ml or 0.5%, v/v) or ethanol vehicle (0.5%, v/v). Concentrations were determined by cytometric bead array immunodetection against a reconstituted MCP-1 standard. Mean ± standard error over 3 trials, n ≥300 beads in any trial. Mean results were compared between treatments of the same cell type and at the same time point. Differences between means are statistically significant if columns do not share any common letters (p<0.05, one-way ANOVA with Bonferroni’s post hoc test).</p

Molecular structures of tricin and 7-O-methyl-tricin (7 MT), two compounds isolated from BEX.

<p>Molecular structures of tricin and 7-O-methyl-tricin (7 MT), two compounds isolated from BEX.</p

Effect of BEX on MCP-1 mRNA expression.

<p>Levels of MCP-1 mRNA expression in 3T3-L1 (A), C2C12 (B), and Hepa6 (C) murine cells in response to palmitic acid (PA, 0.4 mM) in combination with BEX (125 µg/ml or 0.5%, v/v) or ethanol vehicle (0.5%, v/v) at different time points and differentiation states (3T3-L1 and C2C12). Fold change calculated via comparative cycle threshold (−ΔΔCt) normalized to beta-glucuronidase (GUSβ). Other housekeeping genes such as 18 S ribosomal RNA (18 S), hypoxanthine-guanine phosphoribosyltransferase (HPRT), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were also used and yielded similar results as GUSβ. Mean ± standard deviation of at least 4 qRT-PCRs per cell type. Mean results were compared between treatments of the same cell type and at the same time point. Differences between means are statistically significant if columns do not share any common letters (p<0.05, one-way ANOVA with Tukey’s multiple comparison test).</p

Dose-dependent effects of tricin, 7-O-methyl-tricin (7 MT), Fraction K, and BEX on lipotoxic MCP-1 production and cell viability.

<p>(A) MCP-1 concentrations in cell culture media from 3T3-L1 cells cultured in 96 well plates and treated for 20 hours with PA (0.4 mM) and one of the following: tricin (black triangles), 7 MT (light grey hollow squares), Fraction K (grey diamonds), or BEX (dark grey crosses). Concentrations were determined by cytometric bead array immunodetection against a reconstituted MCP-1 standard. Mean ± standard error of 3 samples per dose, n ≥300 beads per sample. (B) Viability of 3T3-L1 cells treated with tricin (black triangles), 7 MT (light grey hollow squares), Fraction K (grey diamonds), or BEX (dark grey crosses) added to maintenance medium. All values are normalized to 3T3-L1 cells from the same plate treated with maintenance medium without flavonoids or PA (normalized average represented by a red dashed line). Mean ± standard error of three trials, n ≥3 per trial. (C) Viability of 3T3-L1 cells treated with ethanol vehicle (outlined white), Fraction K (grey), or BEX (black). Mean ± standard error of three trials, n ≥4 per trial. All values normalized to 3T3-L1 cells from the same plate treated with only maintenance medium (normalized average represented by a red dashed line). <i>p</i> values were obtained by performing a Student’s <i>t</i>-test between doses.</p

Protective effect of BEX on palmitic acid (PA)-induced lipotoxicity.

<p>3T3-L1 (A), and Hepa6 cells (B) were grown to confluency in 96-well plates and treated with 0.4 mM PA for 24 hours in combination with BEX (125 µg/ml or 0.5%, v/v) or ethanol (0.5%, v/v) as a solvent control. Cell viability was measured using a MTS assay. Mean ± standard deviation over 5 trials, n ≥3 wells per trial. Differences between means are statistically significant if the columns do not share any common letters (p<0.001, one-way ANOVA, Tukey’s multiple comparison test).</p

<i>C</i>‑Glycosylflavones Alleviate Tau Phosphorylation and Amyloid Neurotoxicity through GSK3β Inhibition

Alzheimer’s disease (AD) is the most common brain disorder worldwide. Aberrant tau hyperphosphorylation and accumulation play critical roles in the formation of neurofibrillary tangles highly associated with neuronal dysfunction and cognitive impairment in AD pathogenesis. Glycogen synthase kinase-3β (GSK3β) is a key kinase responsible for tau hyperphosphorylation. Selective inhibition of GSK3β is a promising strategy in AD therapy. Corn silks (CS, <i>Zea mays</i> L.) have been traditionally used as a medicinal herb and recently noted for their potentially cognitive benefits. However, the neuroprotective components of CS and their molecular mechanism have received little attention to date. As part of our effort screening phytochemicals against a broad panel of kinases targeting AD tauopathy, we found inhibition of GSK3β by CS extracts. Subsequent bioassay-guided fractionation led to the isolation and identification of two 6-<i>C</i>-glycosylflavones, isoorientin (<b>1</b>) and 3′-methoxymaysin (<b>2</b>), with selective inhibition against GSK3β in vitro. Enzyme kinetics and molecular docking studies demonstrated that <b>1</b> specifically inhibited GSK3β via an ATP noncompetitive mechanism, acting as a substrate competitive inhibitor of GSK3β. Further in vitro cellular studies demonstrated that <b>1</b> effectively attenuated tau phosphorylation mediated by GSK3β and was neuroprotective against β-amyloid-induced tau hyperphosphorylation and neurotoxicity in SH-SY5Y cells. The <i>C</i>-glycosylflavones represent new lead candidates with a novel mechanism of action for the development of AD phytopharmaceuticals

Cytotoxic Effects of 24-Methylenecyloartanyl Ferulate on A549 Nonsmall Cell Lung Cancer Cells through MYBBP1A Up-Regulation and AKT and Aurora B Kinase Inhibition

Lung cancer is the second most prevalent cancer. Nonsmall cell lung cancer (NSCLC) is the most common type of lung cancer. The low efficacy in current chemotherapies impels us to find new alternatives to prevent or treat NSCLC. Rice bran oil is cytotoxic to A549 cells, a NSCLC cell line. Here, we identified 24-methylenecyloartanyl ferulate (24-mCAF) as the main component responsible for the cytotoxicity in A549 cells. An iTRAQ-based quantitative proteomics analysis revealed that 24-mCAF inhibits cell proliferation and activates cell death and apoptosis. 24-mCAF induces up-regulation of Myb binding protein 1A (MYBBP1A), a tumor suppressor that halts cancer progression. 24-mCAF inhibits the activity of AKT and Aurora B kinase, two Ser/Thr kinases involved in MYBBP1A regulation and that represent important targets in NSCLC. This study provides the first insight of the effect of 24-mCAF, the main component of rice bran oil, on A459 cells at the cellular and molecular levels

Neopetrocyclamines A and B, Polycyclic Diamine Alkaloids from the Sponge <i>Neopetrosia</i> cf <i>exigua</i>

Two new polycyclic alkaloids, neopetrocyclamines A and B (<b>1</b> and <b>2</b>), along with the known metabolites papuamine (<b>3</b>) and haliclonadiamine (<b>4</b>), were isolated from the Indonesian sponge <i>Neopetrosia</i> cf <i>exigua</i>. Neopetrocyclamine A contains a formamidinium moiety, a rare functional group. While these compounds share the same basic biosynthetic building blocks, the size of the ring system differs in <b>1</b> and <b>2</b> because of the formamidinium moiety. Biological evaluations of <b>1</b>–<b>4</b> revealed that papuamine is cytotoxic against glioblastoma SF-295 cells (GI₅₀ = 0.8 μM)