486 DEVELOPMENT OF A HIGH-THROUGHPUT SCREENING ASSAY FOR THE IDENTIFICATION OF CANCER CELL-SPECIFIC HSP90

Abstract

You have accessJournal of UrologyProstate Cancer: Basic Research III1 Apr 2012486 DEVELOPMENT OF A HIGH-THROUGHPUT SCREENING ASSAY FOR THE IDENTIFICATION OF CANCER CELL-SPECIFIC HSP90 Daniel Woodruff, Takrima Sadikot, Jeff Eskew, Brian Blagg, George Vielhauer, and Jeffrey Holzbeierlein Daniel WoodruffDaniel Woodruff Kansas City, KS More articles by this author , Takrima SadikotTakrima Sadikot Kansas City, KS More articles by this author , Jeff EskewJeff Eskew Kansas City, KS More articles by this author , Brian BlaggBrian Blagg Kansas City, KS More articles by this author , George VielhauerGeorge Vielhauer Kansas City, KS More articles by this author , and Jeffrey HolzbeierleinJeffrey Holzbeierlein Kansas City, KS More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2012.02.555AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES The 90 kDa heat shock protein (Hsp90) in complex with co-chaperones allows proper folding of nascent or misfolded proteins, preventing degradation and aiding cell survival. Since Hsp90 folds many oncogenic proteins that are drug targets, discovery and development of drugs that inhibit Hsp90 is a priority. Many assays have been developed into high-throughput screening (HTS) strategies for the identification of Hsp90 inhibitors but the only validated functional assay is based on refolding of thermally denatured luciferase in rabbit reticulocyte lysates (RRL). While quite sensitive and robust, this assay may result in compound hits that are less relevant for cancer cells. We sought to develop a functional based HTS bioassay that directly measures Hsp90 dependent refolding activity in the cancer cell milieu. METHODS Prostate cancer cells (PC3-MM2) that stably express luciferase were created by lentivirus transduction under puromycin selection. Thermal denaturation of luciferase was optimized so as not to affect cell viability while resulting in decreased activity by >99% within 6 minutes. Cells were then incubated for 60 minutes with well known Hsp90 inhibitors, 17AAG, radicicol and KU-174, along with other test articles demonstrating activity in the RRL to test their ability to inhibit refolding of denatured luciferase. RESULTS 17AAG, radicicol and KU-174 exhibited dose-dependent inhibitory responses of luciferase refolding at 60 minutes yielding an IC50 of 300nM, 300 nM and 7 ìM, respectively. This represents a 10 fold increase in potency for these compounds compared to the RRL assay. KU298, our most active Hsp90 inhibitor in the RRL assay showed no activity in PC3-MM2 cells suggesting off target toxicity. Western blot for luciferase in PC3-MM2 cells treated with 17AAG or KU-174 showed a dose-dependent decrease in luciferase expression. CONCLUSIONS These data support the feasibility of using thermally denatured luciferase in cancer cells for screening Hsp90 inhibitors. They also suggest this assay can distinguish highly active compounds that might otherwise be false negatives in the RLL assays. This assay can be used in future screening of Hsp90 inhibitors to develop lead candidates for further drug development. © 2012 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 187Issue 4SApril 2012Page: e199 Advertisement Copyright & Permissions© 2012 by American Urological Association Education and Research, Inc.MetricsAuthor Information Daniel Woodruff Kansas City, KS More articles by this author Takrima Sadikot Kansas City, KS More articles by this author Jeff Eskew Kansas City, KS More articles by this author Brian Blagg Kansas City, KS More articles by this author George Vielhauer Kansas City, KS More articles by this author Jeffrey Holzbeierlein Kansas City, KS More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...