Development of a cellular tau enzyme-linked immunosorbent assay method for screening GSK-3β inhibitors.

Abstract

Glycogen synthase kinase-3β (GSK-3β), a serine/threonine kinase also known as tau protein kinase I, has been implicated in the pathogenic conditions of Alzheimer's disease. Many investigators have focused on GSK-3 inhibitor as a therapeutic drug. In this study, we established a cell-based assay for the screening of novel GSK-3β inhibitors. For this purpose, four-repeat tau cDNAs were stably expressed in human embryonic kidney 293 (HEK293) cells (HEK293-Tau). The proliferation of HEK293-Tau cells was no different from that of HEK293 cells, as measured by the bromodeoxyuridine enzyme-linked immunosorbent assay (BrdU ELISA). The concentration-dependent reduction of tau phosphorylation by GSK-3 inhibitors, LiCl, Chir98023, and SB415286, was examined by immunoblot analysis and Tau ELISA (in situ ELISA). Highly consistent data were obtained, suggesting that this novel ELISA method is highly reproducible. Using this ELISA strategy, we isolated a few candidate compounds, including compounds 114 and 149, from several hundreds of synthetic agents and demonstrated that such candidates protect nerve growth factor-differentiated PC12 cells against amyloid-β-induced cell death. These data indicate that this Tau ELISA method in HEK293-Tau cells may be a suitable cell-based assay system to screen for GSK-3β inhibitors.

• Authors: Goang-Won Cho, Min-Young Noh, Byung Yong Kang, Il-Whea Ku, Jiseon Park, Yoon-Ho Hong, Myung-Hwa Kim, Seung Hyun Kim
• Journal: Assay and drug development technologies (Assay Drug Dev Technol) Vol. 9 Issue 5 Pg. 503-13 (Oct 2011) ISSN: 1557-8127 [Electronic] United States
• PMID: 21561378 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)

Chemical References

• 3-(3-chloro-4-hydroxyphenylamino)-4-(4-nitrophenyl)-1H-pyrrole-2,5-dione
• Aminophenols
• CHIR98023
• Enzyme Inhibitors
• Imidazoles
• Maleimides
• Neuroprotective Agents
• Pyridines
• Pyrimidines
• tau Proteins
• Glycogen Synthase Kinase 3
• tau-protein kinase

Topics

• Alzheimer Disease (enzymology, metabolism, pathology)
• Aminophenols (analysis, metabolism, pharmacology, toxicity)
• Animals
• Cell Culture Techniques
• Cell Survival (drug effects)
• Dose-Response Relationship, Drug
• Drug Discovery (methods)
• Drug Evaluation, Preclinical
• Enzyme Inhibitors (analysis, metabolism, pharmacology, toxicity)
• Enzyme-Linked Immunosorbent Assay
• Glycogen Synthase Kinase 3 (analysis, antagonists & inhibitors, physiology)
• HEK293 Cells
• Humans
• Imidazoles (metabolism, pharmacology)
• Immunoblotting
• Maleimides (analysis, metabolism, pharmacology, toxicity)
• Molecular Targeted Therapy
• Neuroprotective Agents (analysis, metabolism, pharmacology, toxicity)
• PC12 Cells
• Phosphorylation (physiology)
• Plasmids
• Pyridines (metabolism, pharmacology)
• Pyrimidines (metabolism, pharmacology)
• Rats
• tau Proteins (genetics, metabolism)