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Development of an Assay to Screen for Inhibitors of Tau Phosphorylation by Cdk5

Center for Neurologic Diseases, Brigham and Women’s Hospital, Harvard Medical School, Boston

Andrea Musacchio

Department of Experimental Oncology, European Institute of Oncology, Milan, Italy

Marina Mapelli

Department of Experimental Oncology, European Institute of Oncology, Milan, Italy

Jake Ni

Laboratory for Drug Discovery in Neurodegeneration, Brigham and Women s Hospital, Harvard Medical School, Boston

Leonard Scinto

Laboratory for Higher Cortical Functions, Department of Neurology, Brigham and Women s Hospital, Harvard Medical School, Boston

Ross Stein

Laboratory for Drug Discovery in Neurodegeneration, Brigham and Women s Hospital, Harvard Medical School, Boston

Kenneth S. Kosik

Center for Neurologic Diseases, Brigham and Women s Hospital, Harvard Medical School, Boston

Li-An Yeh

Laboratory for Drug Discovery in Neurodegeneration, Brigham and Women s Hospital, Harvard Medical School, Boston lyeh{at}rics.bwh.harvard.edu

A high-throughput assay for tau phosphorylation by cdk5/p25 is described. Full-length recombinant tau was used as a substrate in the presence of saturating adenosine triphosphate (ATP). Using PHF-1, an antibody directed specifically against 2 tau phosphorylation epitopes (serine 396 and serine 404), an enzyme-linked immunosorbent assay (ELISA)- based colorimetric assay was formatted in 384-well plates. The assay was validated by measuring kinetic parameters for cdk5/p25 catalysis and known inhibitors. Rate constants for the site-specific phosphorylations at the PHF-1 epitopes were determined and suggested preferential phosphorylation at these sites. The performance of this assay in a high-throughput format was demonstrated and used to identify inhibitors of tau phosphorylation at specific epitopes phosphorylated by cdk5/p25. (Journal of Biomolecular Screening 2004:122-131)

Key Words: HTS • cdk5 • tau phosphorylation • Alzheimer’s disease

Journal of Biomolecular Screening, Vol. 9, No. 2, 122-131 (2004)
DOI: 10.1177/1087057103260594


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