
Alzheimer’s disease (AD) is the most common neurodegenerative disorder accompanied with progressive memory loss and cognitive impairment. The neuropathological diagnosis of AD relies on the presence of amyloid plaques and
There are two isoforms of GSK-3, GSK-3α (51 kDa) and GSK-3β (47 kDa), encoded by separate genes, which share extensive similarities in their catalytic domains, but differ in their N- and C-terminal regions (Woodgett, 1990). Unlike other protein kinases, GSK-3 is constitutively active and is inactivated by upstream regulators in response to stimuli. Several signaling pathways regulate GSK-3 activity (Hur and Zhou, 2010). Activation of the phosphatidylinositol 3-kinase (PI3K)-AKT serine/threonine kinase 1 (AKT) pathway results in inactivation of GSK-3 through phosphorylation at N-terminal Ser9 (GSK-3β) or Ser21 (GSK-3α) (Cross
Over the past decade, various classes of small molecule GSK-3β inhibitors with diverse mechanisms have been reported for the treatment of AD. Lithium, a natural, inorganic and water-soluble GSK-3β inhibitor, reduces Tau phosphorylation and insoluble Tau levels in transgenic mice overexpressing human P301L mutant Tau (Noble
In this study, virtual screening using the docking computation combined with a compound structure similarity search was first utilized to screen compounds in a database in order to identify novel GSK-3β inhibitors. Among the top-ranked compounds, seven compounds were selected and underwent
The GOLD docking program (Verdonk
Molecular weight, H-bond donor, H-bond acceptor, octanol-water partition coefficient, and polar surface area of VB compounds were calculated using Internet software ChemDraw (http://www.perkinelmer.com/tw/category/chemdraw/). In addition, BBB permeation scores were computed using an online BBB prediction server (https://www.cbligand.org/BBB/).
The ability of the seven selected VB compounds to inhibit GSK-3β kinase was evaluated, wherein a known GSK-3β inhibitor SB-216763 (Sigma-Aldrich, St Louis, MO, USA) served as a positive control. GSK-3β kinase activity was measured in the presence of test compounds using GSK-3β Kinase Enzyme System (Promega, Madison, WI, USA). Reactions were performed at 30°C for 30 minutes in the 25 µL mixture containing 25 µM ATP, 0.2 mg/mL GSK-3β substrate, 1 ng GSK-3β, and 0.018 µM SB-216763 (44) or VB compounds. Kinase activity data were measured as relative light units (RLU) directly correlated with the amount of ADP produced by using SpectraMax L microplate reader (Molecular Devices, Sunnyvale, CA, USA).
Human SH-SY5Y cells expressing DsRed tagged pro-aggregated mutant (∆K280) of C-terminal repeat domain of Tau (TauRD, Gln244-Glu372 of the longest Tau441 isoform) (Lin
To evaluate compound cytotoxicity, 2×104 ∆K280 TauRD-DsRed SH-SY5Y cells were plated on 96-well dishes with retinoic acid (10 µM) on day 1. On day 2, the cells were treated with the test compounds (1-100 μM) for 8 h, followed by inducing ∆K280 TauRD-DsRed expression with doxycycline (2 μg/mL). On day 8, 10 μL of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (5 mg/mL) was added to the cells at 37°C for 3 h. 100 μL of lysis buffer (10% Triton X-100, 0.1 N HCl, 18% isopropanol) was then added to dishes and the absorbance of the insoluble purple formazan product at OD 570 nm was read by a FLx800 fluorescence microplate spectrophotometer (Bio-Tek, Winooski, VT, USA). IC50 (half maximal inhibitory concentration) was estimated by a method of interpolation.
∆K280 TauRD-DsRed SH-SY5Y cells were seeded in a 6-well plate (5×105/well), differentiated with retinoic acid, and treated with congo red, SB-415286, VB-030 or VB-037 (10 µM) and doxycycline as described. On day 8, cells were collected and total RNA was extracted using Trizol reagent (Invitrogen). The RNA was reverse-transcribed using high-capacity cDNA reverse transcriptase (Thermo Fisher Scientific, Waltham, MA, USA). Real-time quantitative PCR experiments were performed using 100 ng cDNA and customized Assays-by-Design probe for DsRed and HPRT1 (4326321E) using StepOnePlus Real-time PCR system (Applied Biosystems, Foster City, CA, USA). Fold change was calculated using the formula 2∆Ct, ∆CT=CT (HPRT1)–CT (DsRed), in which CT indicates cycle threshold.
As described, ∆K280 TauRD-DsRed SH-SY5Y cells were seeded in a 6-well plate and treated with retinoic acid, test compounds (10 µM) and doxycycline. On day 8, cells were collected and total proteins were prepared using lysis buffer containing 10 mM Tris-HCl pH 7.5, 150 mM NaCl, 5 mM EDTA, pH 8.0, 0.1% SDS, 1% sodium deoxycholate, 1% NP-40, as well as protease (Sigma-Aldrich) and phosphatase (Abcam, Cambridge, MA, USA) inhibitor cocktails. Proteins (25 μg) were separated on 10% SDS-polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride membranes (Sigma-Aldrich) by reverse electrophoresis. After blocking, the membrane was probed with DsRed (1:500; Santa Cruz Biotechnology, Dallas, TX, USA), GSK-3α and GSK-3β (1:500; Santa Cruz Biotechnology), p-GSK-3α (S21) (1:1000; Cell Signaling Technology, Beverly, MA, USA), p-GSK-3β (S9) (1:1000; Cell Signaling Technology), ERK and p-ERK (T202/Y204) (1:1000; Cell Signaling Technology), AKT and p-AKT (S473) (1:2000; Cell Signaling Technology), P38 and p-P38 (T180/Y182) (1:1000; Cell Signaling Technology), JNK and p-JNK (T183/Y185) (1:2000; Cell Signaling Technology), Tau (1:500; Dako, Glostrup, Denmark), p-Tau (S202) (1:2000; AnaSpec Inc., Fremont, CA, USA), p-Tau (T231 and S404) (1:2000; Sigma-Aldrich), p-Tau (S396) (1:1000; Sigma-Aldrich), and GAPDH (1:2000; MDBio Inc., Taipei, Taiwan) at 4°C overnight. After extensive washing, the immune complexes were detected by horseradish peroxidase-conjugated goat anti-mouse or goat anti-rabbit IgG antibody (1:5000; GeneTex, Irvine, CA, USA) and chemiluminescence substrate (Millipore, Billerica, MA, USA).
All experiments were performed according to the guidelines of the Institutional Animal Care and Use Committee of National Taiwan Normal University, Taipei, Taiwan. Female pregnant mice (C57BL/6J) were purchased from the National Breeding Centre for Laboratory Animals (Taipei, Taiwan). Mouse hippocampi were isolated from embryonic brains on days 16°C18 and trypsinized (0.05%) for 15 min in 37°C and cells were cultured in neurobasal plating media as described). Briefly, the hippocampal cells were plated into poly-L-lysine (100 μg/mL)-pretreated flasks (48-well plates; 3×104 cells per culture well). The cultures were incubated in a tissue culture incubator at 37°C in 5% CO2. On days
The neuronal survival and morphology of the hippocampal primary culture were evaluated by immunocytochemical staining with antibodies against NeuN (neuron-specific RNA binding nuclear protein) and Map2 (microtubule-associated protein 2, for neurite morphology). Cells were fixed and stained with primary antibodies (1:1000; NeuN and Map2; Millipore) and then fluorescence tagged secondary antibodies and 4’,6-diamidino-2-phenylindole (DAPI) for nuclei. Mature neuron, neurite length and branching were analyzed by Metamorph image analysis software (ImageXpress Micro, Molecular Devices) using images from evenly distributed 16 arenas in each well (totally 64 arenas was set) from independent three experiments. Each neurite length longer than 2 folds of neuronal soma diameter was included.
For each set of values, data were expressed as the mean ± standard deviation (SD). Three independent tests in two or three biological replicates were performed in each experiment and differences between groups were evaluated by Student’s
A large number of structurally diverse potent GSK-3β inhibitors have been reported (Xu
Table 1 Prediction of bioavailability and BBB permeation
Compound | VB-030 | VB-031 | VB-032 | VB-035 | VB-036 | VB-037 | VB-041 |
---|---|---|---|---|---|---|---|
HBD | 0 | 2 | 3 | 2 | 1 | 0 | 2 |
HBA | 2 | 5 | 5 | 4 | 3 | 7 | 8 |
cLogP | 5.1 | 4.6 | 5.0 | 5.0 | 6.1 | 4.5 | 3.4 |
PSA (Å2) | 25 | 71 | 61 | 44 | 46 | 89 | 112 |
BBB score | 0.341 | 0.095 | 0.193 | 0.237 | 0.181 | 0.096 | 0.060 |
MW, molecular weight; HBD, hydrogen bond donor; HBA, hydrogen bond acceptor; cLogP, calculated octanol-water partition coefficient; PSA, polar surface area; BBB, blood-brain barrier.
Table 2 Inhibition potency against GSK-3β kinase
Compound | VB-030 | VB-031 | VB-032 | VB-035 | VB-036 | VB-037 | VB-041 |
---|---|---|---|---|---|---|---|
Residual GSK-3β activity at 0.018 μM (%) | 36.1 ± 1.1 | 63.1 ± 1.2 | 82.0 ± 8.8 | 79.9 ± 3.8 | 75.2 ± 4.8 | 90.9 ± 6.6 | 79.2 ± 6.2 |
SB-216763 was used as a positive inhibitor control for GSK-3β activity assay which gave an IC50 of 0.018 μM. The data were obtained from three independent experiments (n=3).
Tet-On ∆K280 TauRD-DsRed SH-SY5Y cells with DsRed fluorescence reflecting Tau aggregation status (Lin
We then examined the effect of SB-415286, VB-030 and VB-037 on the levels of p-GSK-3α at Ser21 (to inactivate GSK-3α) and p-GSK-3β at Ser9 (to inactivate GSK-3β) (Fig. 3A) in ∆K280 TauRD-DsRed-expressing SH-SY5Y cells by Western blotting. No significant difference was detected in the level of total GSK-3α (92-99% versus 100%,
The effects of SB-415286, VB-030 and VB-037 on the levels of kinases which phosphorylate Tau including ERK, AKT, P38 and JNK were then examined (Fig. 3B). Treatment of SB-415286 increased the level of p-ERK (Thr202/Tyr204) (135% versus 103%,
We then examined whether SB-415286, VB-030 or VB-037 treatment could inhibit endogenous Tau phosphorylation at Ser202, Thr231, Ser396 and Ser404 in ∆K280 TauRD-DsRed-expressing SH-SY5Y cells. As shown in Fig. 3C, the steady-state levels of total Tau showed no significant difference between test compound-treated and untreated cells (100-106% versus 100%,
To evaluate whether compounds VB-030 and VB-037 are with neuroprotective activity, we further applied 10 μM compounds to mouse hippocampal primary culture under Tau toxicity, given that both compounds at 10 μM reduced Tau protein aggregation in SH-SY5Y cells. It has been reported that co-treatment of wortmannin (WT) (inhibitor of PI3K) and GF109203X (GFX) (inhibitor of PKC) increased GSK-3 activity and decreased cell viability (Xu
Effective treatments to slow AD neurodegeneration are still unavailable. GSK-3β, a key player in AD pathophysiology, influences all the major hallmarks of the disease including: amyloid-β production, Tau phosphorylation, memory formation and storage, neuronal growth, and synaptic plasticity (Lauretti
Similar to the previously described mouse N2a ∆K280 TauRD cell model (Khlistunova
Tau phosphorylation is regulated by a balance between Tau kinase and phosphatase activities. In the case where the high number of Tau phosphorylation sites is involved in AD, GSK-3β is a promising target involved with 70% pathological Tau phosphorylation sites (Martin
Through coordinating multiple proliferation and differentiation signals, GSK-3 is a master regulator of neural progenitor homeostasis (Kim
Increasing evidences have suggested that GSK-3β activation-mediated hyperphosphorylation of Tau can induce neuronal apoptosis (Gao
Both VB-030 and VB-037 are nitrogen-containing heterocyclic aromatic compounds with quinoline scaffold. Biologically active quinoline derivatives possessing dual effects of inhibition of Aβ toxicity in MC65 neuroblastoma cells and GSK-3β enzyme have been documented (Lu
In addition to quinoline scaffold, VB-037 also contains a heterocycle morpholine moiety featuring both amine and ether functional groups. Morpholine-containing compounds with potent
In summary, through combinations of virtual screening, GSK-3β enzyme assay, and models of human SH-SY5Y cells expressing Tau folding reporter and mouse hippocampal primary neuron culture under Tau cytotoxicity, two quinoline compounds VB-030 and VB-037 were identified and experimental results showed that they effectively inhibit GSK-3β kinase activity or p-P38 (Thr180/Tyr182) (Fig. 5). Further study will be needed to confirm that this also works in AD animal models and eventually humans.
We thank the Molecular Imaging Core Facility of National Taiwan Normal University for the technical assistance. This work was supported by the grants 103-2321-B-182-008, 103-2321-B-003-003, 104-2325-B-003-001 and 104-2325-B-003-003 from the Ministry of Science and Technology, and 104T3040B05 and 104T3040B07 from National Taiwan Normal University, Taipei, Taiwan.
All authors have no conflicts of interest to declare.
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