High-throughput screening fluorescence polarization assay for tumor-specific Hsp90 Journal Article


Authors: Du, Y.; Moulick, K.; Rodina, A.; Aguirre, J.; Felts, S.; Dingledine, R.; Fu, H.; Chiosis, G.
Article Title: High-throughput screening fluorescence polarization assay for tumor-specific Hsp90
Abstract: Heat shock protein 90 (Hsp90) is a molecular chaperone that has emerged as an important target in cancer and several other diseases, such as neurodegenerative diseases, nerve injuries, inflammation, and infection. Discovery of novel agents that inhibit Hsp90 and have druglike properties is therefore a major focus in several academic and industrial laboratories. In this study, the authors describe the development and optimization in a 384-well format of a novel assay for the identification of Hsp90 inhibitors using fluorescence polarization, which measures competitive binding of red-shifted fluorescently labeled geldanamycin (GM-cy3B) to Hsp90 found in the NCI-N417 small-cell lung carcinoma cells. The authors demonstrate that GMcy3B binds with high affinity and specificity to cellular Hsp90. The assay results in excellent signal-to-noise ratios (>10) and Z′ values (>0.75) at tracer concentrations greater than 4 nM and 1 μg/well of total NCI-N417 protein, indicating a robust assay. It also equilibrates after 5 h of incubation at room temperature and remains stable for up to 24 h. Furthermore, it is a simple mix-and-read format that is cost-effective and uses only low amounts of fluorophore and cell lysates. A study using more than 15,000 compounds from the National Institutes of Health Molecular Libraries Screening Center Network was performed to validate its performance in a high-throughput screening format. © 2007 Society for Biomolecular Sciences.
Keywords: controlled study; human cell; binding affinity; neoplasms; protein analysis; infection; signal noise ratio; inflammation; high throughput screening; fluorescence polarization; molecular library; cell line, tumor; cost effectiveness analysis; lung small cell cancer; heat shock protein 90; hsp90 heat-shock proteins; nerve injury; degenerative disease; room temperature; tumor diagnosis; cell lysate; geldanamycin; chaperone; fluorine; 384-well format; gm-cy3b; small-cell lung carcinoma; tumor hsp90
Journal Title: Journal of Biomolecular Screening
Volume: 12
Issue: 7
ISSN: 1087-0571
Publisher: Sage Publications  
Date Published: 2007-09-01
Start Page: 915
End Page: 924
Language: English
DOI: 10.1177/1087057107306067
PUBMED: 17942784
PROVIDER: scopus
DOI/URL:
Notes: --- - "Cited By (since 1996): 12" - "Export Date: 17 November 2011" - "CODEN: JBISF" - "Source: Scopus"
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MSK Authors
  1. Kamalika Moulick
    16 Moulick
  2. Gabriela Chiosis
    218 Chiosis
  3. Julia Aguirre
    15 Aguirre