This Article Full Text (PDF) All Versions of this Article: 1087057108330114v1 14/3/273    most recent References Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Add to Saved Citations Download to citation manager Request Permissions Request Reprints Add to My Marked Citations Citing Articles Citing Articles via Google Scholar Citing Articles via Scopus Google Scholar Articles by Fang Yi Articles by Regan, L. Search for Related Content PubMed PubMed Citation Articles by Fang Yi, Articles by Regan, L. Social Bookmarking                         What's this?

An AlphaScreen^TM-Based High-Throughput Screen to Identify Inhibitors of Hsp90-Cochaperone Interaction

Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut

Pingjun Zhu

National Institutes of Health, National Human Genome Research Institute, NIH Chemical Genomics Center, Bethesda, Maryland

Noel Southall

National Institutes of Health, National Human Genome Research Institute, NIH Chemical Genomics Center, Bethesda, Maryland

James Inglese

National Institutes of Health, National Human Genome Research Institute, NIH Chemical Genomics Center, Bethesda, Maryland

Christopher P. Austin

National Institutes of Health, National Human Genome Research Institute, NIH Chemical Genomics Center, Bethesda, Maryland

Wei Zheng

National Institutes of Health, National Human Genome Research Institute, NIH Chemical Genomics Center, Bethesda, Maryland, wzheng{at}mail.nih.gov

Lynne Regan

Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut, Department of Chemistry, Yale University, New Haven, Connecticut

Hsp90 has emerged as an important anticancer drug target because of its essential role in promoting the folding and maturation of many oncogenic proteins. The authors describe the development of the first high-throughput screen, based on AlphaScreen^TM technology, to identify a novel type of Hsp90 inhibitors that interrupt its interaction with the cochaperone HOP. The assay used the 20-mer C-terminal peptide of Hsp90 and the TPR2A domain of HOP. Assay specificity was demonstrated by measuring different interactions using synthetic peptides, with measured IC₅₀s in good agreement with reported values. The assay was stable over 12 h and tolerated DMSO up to 5%. The authors first validated the assay by screening against 20,000 compounds in a 384-well format. After further optimization into a 1536-well format, it was screened against an NIH Chemical Genomics Center library of 76,134 compounds, with a signal-to-background ratio of 78 and Z' factor of 0.77. The present assay can be used for discovery of novel small-molecule Hsp90 inhibitors that can be used as chemical probes to investigate the role of cochaperones in Hsp90 function. Such molecules have the potential to be developed into novel anticancer drugs, for use alone or in combination with other Hsp90 inhibitors. (Journal of Biomolecular Screening 2009:273-281)

Key Words: heat shock protein 90 (Hsp90) • Hsp organizing protein (HOP) • tetratricopeptide repeat (TPR) • AlphaScreen^TM • high-throughput screening (HTS)

This version was published on March 1, 2009

Journal of Biomolecular Screening, Vol. 14, No. 3, 273-281 (2009)
DOI: 10.1177/1087057108330114


CiteULike    Complore    Connotea    Del.icio.us    Digg    Reddit    Technorati    Twitter    What's this?