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

Lakshmi Galam, M. Kyle Hadden, Zeqiang Ma, Qi Zhuang Ye, Bo Geon Yun, Brian S.J. Blagg, Robert L. Matts

Research output: Contribution to journalArticlepeer-review

65 Scopus citations


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 μg/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 μM and represent a number of new lead structures with the potential for further development and optimization as potent Hsp90 inhibitors.

Original languageEnglish (US)
Pages (from-to)1939-1946
Number of pages8
JournalBioorganic and Medicinal Chemistry
Issue number5
StatePublished - Mar 1 2007


  • High-throughput screening
  • Hsp90
  • Inhibitors
  • Luciferase refolding

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Medicine
  • Molecular Biology
  • Pharmaceutical Science
  • Drug Discovery
  • Clinical Biochemistry
  • Organic Chemistry

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