A miniaturized 1536-well format γ-secretase assay Journal Article


Authors: Shelton, C. C.; Tian, Y.; Shum, D.; Radu, C.; Djaballah, H.; Li, Y. M.
Article Title: A miniaturized 1536-well format γ-secretase assay
Abstract: γ-Secretase is an aspartyl protease that cleaves multiple substrates including the amyloid precursor protein (APP) and the Notch proteins. Abnormal proteolysis of APP is involved in the pathogenesis of Alzheimer's disease (AD) and overactive Notch signaling plays an oncogenic role in a variety of cancers. γ-Secretase has emerged as a promising target for drug development in the treatment of AD and cancer. Assays with increased capacity for high-throughput screening would allow for quicker screening of chemical libraries and facilitate inhibitor development. We have developed a homogeneous time-resolved fluorescence (HTRF)-based assay that makes use of a novel biotinylated recombinant APP substrate and solubilized membrane preparation as the source of the γ-secretase enzyme. The assay was miniaturized to a 1536-well format and validated in a pilot screen against a library of ∼3,000 compounds. The overall assay performance was robust due to a calculated Z′ factor of 0.74 and its demonstrated ability to identify known γ-secretase inhibitors such as pepstatin A. This validated assay can readily be used for primary screening against large chemical libraries searching for novel inhibitors of γ-secretase activity that may represent potential therapeutics for AD and a variety of neoplasms. © Mary Ann Liebert, Inc.
Keywords: unclassified drug; human cell; cisplatin; nonhuman; validation process; enzyme inhibition; fluorescence; dose-response relationship, drug; drug discovery; enzyme activity; drug evaluation, preclinical; hela cells; automation; luminescence; recombinant protein; cost-benefit analysis; enzyme assay; amyloid precursor protein; amyloid precursor protein secretases; gamma secretase; biotinylation; biotin; 7 oxocholesteryl acetate; acetic acid derivative; colistin; digitonin; pepstatin; library; performance measurement system; indicators and reagents; miniaturization
Journal Title: Assay and Drug Development Technologies
Volume: 7
Issue: 5
ISSN: 1540-658X
Publisher: Mary Ann Liebert, Inc  
Date Published: 2009-01-01
Start Page: 461
End Page: 470
Language: English
DOI: 10.1089/adt.2009.0202
PUBMED: 19715456
PROVIDER: scopus
PMCID: PMC3096545
DOI/URL:
Notes: --- - "Cited By (since 1996): 4" - "Export Date: 30 November 2010" - "CODEN: ADDTA" - "Source: Scopus"
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MSK Authors
  1. Hakim Djaballah
    101 Djaballah
  2. Constantin Radu
    28 Radu
  3. Yueming Li
    132 Li
  4. David Shum
    54 Shum
  5. Yuan Tian
    16 Tian
  6. Christopher C Shelton
    12 Shelton
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