Assays for identification of Hsp90 inhibitors and biochemical methods for discriminating their mechanism of action

Robert L. Matts, Jacob R. Manjarrez

Research output: Contribution to journalReview articlepeer-review

19 Scopus citations


The Hsp90-dependence of many oncogenic proteins has precipitated a great deal of interest in Hsp90 as a drug target, as evidence mounts that Hsp90 inhibitors may be effective chemotherapeutic agents for the treatment of cancer. In addition, Hsp90-inhibitors have shown promise for the treatment of neurodegenerative diseases characterized by the accumulation of toxic denatured protein aggregates. The development of assays for the identification of novel Hsp90 inhibitors began in earnest when it became apparent that the Hsp90 inhibitors available at the time had less than ideal pharmacological properties. This review summarizes what is known about Hsp90's structure and function, its ATPase cycle, its interactions with co-chaperones and clients, and the effect of Hsp90-inhibitors on these processes. It further summarizes various high throughput assays (and secondary confirmatory assays) developed to identify new Hsp90 inhibitors from chemical libraries based on the inhibitors ability to: inhibit Hsp90's ATPase activity; compete for ligand binding to Hsp90 and its N-terminal ATP-binding domain; inhibit Hsp90-dependent refolding of denatured luciferase; and deplete culture cells of Hsp90-dependnet client protein or induce Hsp70 expression. In addition, in vitro assays are described that help determine the site of inhibitor binding to Hsp90 (N- or C-terminal domain) and there mechanism of action based on effects of inhibitors on Hsp90/ co-chaperone and client interactions, and Hsp90 conformation characterized by proteolytic fingerprinting.

Original languageEnglish
Pages (from-to)1462-1478
Number of pages17
JournalCurrent Topics in Medicinal Chemistry
Issue number15
StatePublished - 2009
Externally publishedYes


  • Geldanamycin
  • High-throughput screens
  • Hsp90
  • Hsp90 co-chaperones
  • Hsp90 inhibitor assays
  • Novobiocin


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