High-throughput assay for the identification of Hsp90 inhibitors based on Hsp90-dependent refolding of firefly luciferase

Bioorg Med Chem. 2007 Mar 1;15(5):1939-46. doi: 10.1016/j.bmc.2007.01.004. Epub 2007 Jan 4.


Previously, we have demonstrated that the renaturation of heat denatured firefly luciferase is dependent upon the activity of Hsp90 in rabbit reticulocyte lysate. Here, we demonstrate that this assay may identify inhibitors that obstruct the chaperone activity of Hsp90 either by direct binding to its N-terminal or C-terminal nucleotide binding sites or by interference with the ability of the chaperone to switch conformations. The assay was adapted and optimized for high-throughput screening. Greater than 20,000 compounds were screened to demonstrate the feasibility of using this assay on a large scale. The assay was reproducible (av Z-factor=0.62) and identified 120 compounds that inhibited luciferase renaturation by greater than 70% at a concentration of 12.5 microg/mL. IC50 values for twenty compounds with varying structures were determined for inhibition of luciferase refolding and in cell-based assays for Hsp90 inhibition. Several compounds had IC50 values <10 microM and represent a number of new lead structures with the potential for further development and optimization as potent Hsp90 inhibitors.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cell Division / drug effects
  • Cell Line, Tumor
  • Enzyme-Linked Immunosorbent Assay
  • HSP90 Heat-Shock Proteins / antagonists & inhibitors*
  • HSP90 Heat-Shock Proteins / metabolism
  • Humans
  • Luciferases, Firefly / metabolism*
  • Protein Folding
  • Reproducibility of Results


  • HSP90 Heat-Shock Proteins
  • Luciferases, Firefly