Biomolecules & Therapeutics
Virtual Screening and Testing of GSK-3 Inhibitors Using Human SH-SY5Y Cells Expressing Tau Folding Reporter and Mouse Hippocampal Primary Culture under Tau Cytotoxicity
Chih-Hsin Lin1,†, Yu-Shao Hsieh2,†, Ying-Chieh Sun2,†, Wun-Han Huang1, Shu-Ling Chen1, Zheng-Kui Weng1, Te-Hsien Lin1, Yih-Ru Wu3, Kuo-Hsuan Chang3, Hei-Jen Huang4, Guan-Chiun Lee1,*, Hsiu Mei Hsieh-Li1,* and Guey-Jen Lee-Chen1,*
1School of Life Science, National Taiwan Normal University, Taipei 11677,
2Department of Chemistry, National Taiwan Normal University, Taipei 11677,
3Department of Neurology, Chang Gung Memorial Hospital, Taoyuan 33378,
4Department of Nursing, Mackay Junior College of Medicine, Nursing and Management, Taipei 11260, Taiwan
*E-mail: (Lee GC), (Hsieh-Li HM), (Lee-Chen GJ)
Tel: +886-2-7749-6351 (Lee GC), +886-2-7749-6354 (Hsieh-Li HM), +886-2-7749-6359 (Lee-Chen GJ)
Fax: +886-2-2931-2904 (Lee GC), +886-2-2931-2904 (Hsieh-Li HM), +886-2-2931-2904 (Lee-Chen GJ)
The first three authors contributed equally to this work.
Received: March 11, 2022; Revised: May 5, 2022; Accepted: May 24, 2022; Published online: July 5, 2022.
© The Korean Society of Applied Pharmacology. All rights reserved.

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Glycogen synthase kinase-3β (GSK-3β) is an important serine/threonine kinase that implicates in multiple cellular processes and links with the neurodegenerative diseases including Alzheimer’s disease (AD). In this study, structure-based virtual screening was performed to search database for compounds targeting GSK-3β from Enamine’s screening collection. Of the top-ranked compounds, 7 primary hits underwent a luminescent kinase assay and a cell assay using human neuroblastoma SH-SY5Y cells expressing Tau repeat domain (TauRD) with pro-aggregant mutation ΔK280. In the kinase assay for these 7 compounds, residual GSK-3β activities ranged from 36.1% to 90.0% were detected at the IC50 of SB-216763. In the cell assay, only compounds VB-030 and VB-037 reduced Tau aggregation in SH-SY5Y cells expressing ΔK280 TauRD-DsRed folding reporter. In SH-SY5Y cells expressing ΔK280 TauRD, neither VB-030 nor VB-037 increased expression of GSK-3α Ser21 or GSK-3β Ser9. Among extracellular signal-regulated kinase (ERK), AKT serine/threonine kinase 1 (AKT), mitogen-activated protein kinase 14 (P38) and mitogenactivated protein kinase 8 (JNK) which modulate Tau phosphorylation, VB-037 attenuated active phosphorylation of P38 Thr180/ Tyr182, whereas VB-030 had no effect on the phosphorylation status of ERK, AKT, P38 or JNK. However, both VB-030 and VB-037 reduced endogenous Tau phosphorylation at Ser202, Thr231, Ser396 and Ser404 in neuronally differentiated SH-SY5Y expressing ΔK280 TauRD. In addition, VB-030 and VB-037 further improved neuronal survival and/or neurite length and branch in mouse hippocampal primary culture under Tau cytotoxicity. Overall, through inhibiting GSK-3β kinase activity and/or p-P38 (Thr180/Tyr182), both compounds may serve as promising candidates to reduce Tau aggregation/cytotoxicity for AD treatment.
Keywords: GSK-3β kinase inhibitor, Alzheimer’s disease, Virtual screening, Enzyme assay, Cell assay, Mouse hippocampal primary culture

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