Development of a fluorescence polarization assay for the molecular chaperone Hsp90 Journal Article


Authors: Kim, J.; Felts, S.; Llauger, L.; He, H.; Huezo, H.; Rosen, N.; Chiosis, G.
Article Title: Development of a fluorescence polarization assay for the molecular chaperone Hsp90
Abstract: Heat shock protein 90 (Hsp90) is a molecular chaperone with essential functions in maintaining transformation, and there is increasing interest in developing Hsp90 inhibitors as cancer therapeutics. In this study, the authors describe the development and optimization of a novel assay for the identification of Hsp90 inhibitors using fluorescence polarization. The assay is based on the competition of fluorescently (BODIPY) labeled geldanamycin (GM) for binding to purified recombinant Hsp90α (GM is a natural product that binds to the ATP/ADP pocket in the amino terminal of Hsp90). The authors show that GM-BODIPY binds Hsp90α with high affinity. Even at low Hsp90α concentrations (30 nM), the measured polarization value is close to the maximum assay range of 160 mP, making measurements very sensitive. Its performance, as judged by signal-to-noise ratios (> 10) and Z and Z′ values (>0.5), suggests that this is a robust and reliable assay. GM, PU24FC1, ADP, and ATP, all known to bind to the Hsp90 pocket, compete with GM-BODIPY for binding to Hsp90α with EC50s in agreement with reported values. These data demonstrate that the Hsp90-FP-based assay can be used for high-throughput screening in aiding the identification of novel Hsp90 inhibitors. © 2004 The Society for Biomolecular Screening.
Keywords: unclassified drug; protein function; metabolism; fluorescent dye; signal noise ratio; fluorescent dyes; high throughput screening; fluorescence polarization; adenosine diphosphate; amino terminal sequence; protein purification; heat shock protein 90 inhibitor; heat shock protein 90; quinone derivative; hsp90 heat-shock proteins; quinones; binding site; reliability; drug labeling; adenosine triphosphate; natural product; protein determination; drug protein binding; concentration response; protein inhibitor; benzoquinones; lactams, macrocyclic; geldanamycin; hsp90; benzoquinone derivative; macrocyclic lactam; high-throughput screening; drug identification; boron compounds; fluorescence polarization immunoassay; priority journal; article; competitive assay; 4,4 difluoro 1,3,5,7 tetramethyl 4 bora 3a,4a diaza s indacene; 4,4 difluoro 4 bora 3a,4a diaza s indacene; 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene; boron derivative
Journal Title: Journal of Biomolecular Screening
Volume: 9
Issue: 5
ISSN: 1087-0571
Publisher: Sage Publications  
Date Published: 2004-08-01
Start Page: 375
End Page: 381
Language: English
DOI: 10.1177/1087057104265995
PROVIDER: scopus
PUBMED: 15296636
DOI/URL:
Notes: J. Biomol. Screen. -- Cited By (since 1996):48 -- Export Date: 16 June 2014 -- CODEN: JBISF -- Source: Scopus
Altmetric Score
MSK Authors
  1. Huazhong He
    9 He
  2. Neal Rosen
    366 Rosen
  3. Gabriela Chiosis
    218 Chiosis
  4. Joungnam Kim
    14 Kim
  5. Henri   Huezo
    11 Huezo