Solid-phase immunoassays in mechanism-based drug discovery: their application in the development of inhibitors of the molecular chaperone heat-shock protein 90

High-throughput screening of chemical libraries and the subsequent rapid progress of hit compounds through an iterative developmental test cascade are essential parts of modern molecular mechanism-based drug discovery. These processes depend on the use of efficient assay technologies and equipment....

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Veröffentlicht in:Assay and drug development technologies 2005-06, Vol.3 (3), p.273-285
Hauptverfasser: Hardcastle, Anthea, Boxall, Kathy, Richards, Juliet, Tomlin, Peter, Sharp, Swee, Clarke, Paul, Workman, Paul, Aherne, Wynne
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container_end_page 285
container_issue 3
container_start_page 273
container_title Assay and drug development technologies
container_volume 3
creator Hardcastle, Anthea
Boxall, Kathy
Richards, Juliet
Tomlin, Peter
Sharp, Swee
Clarke, Paul
Workman, Paul
Aherne, Wynne
description High-throughput screening of chemical libraries and the subsequent rapid progress of hit compounds through an iterative developmental test cascade are essential parts of modern molecular mechanism-based drug discovery. These processes depend on the use of efficient assay technologies and equipment. Enzyme-linked immunosorbent assays have historically been carried out in 96-well microtitre plates. Improvements in reagents and assay technologies mean that solid-phase immunoassays can be adapted for higher throughput to play an important role in modern drug discovery. The molecular chaperone heat-shock protein (Hsp) 90 is an important anticancer drug target because it maintains the conformation, stability, and function of many important oncogenic client proteins, including those involved with signal transduction, cell proliferation, survival, differentiation, motility angiogenesis, and metastasis. Using the standard inhibitors of the adenosine triphosphatase (ATPase) activity of Hsp90, geldanamycin (GA) and 17-allylamino-17- demethoxygeldanamycin (17AAG), novel solid-phase immunoassays have been validated using a time-resolved fluorescence (TRF) end point. Their utility for confirming the mechanism of action of Hsp90 inhibition in secondary cell-based assays has been shown and applied to the novel Hsp90 inhibitor CCT018159. Adaptation of these assays for later studies using human tumour xenografts and samples obtained from a Phase 1 trial of 17AAG is also described. Finally, comparison is made between the use and applicability of this type of immunoassay and other techniques such as western blotting, immunohistochemistry, and flow cytometry analysis.
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subjects Animals
Benzoquinones
Blotting, Western
Carrier Proteins - antagonists & inhibitors
Carrier Proteins - metabolism
Cell Extracts - analysis
Dose-Response Relationship, Drug
Drug Design
eIF-2 Kinase - antagonists & inhibitors
eIF-2 Kinase - metabolism
Enzyme Inhibitors - pharmacology
HCT116 Cells
Heterocyclic Compounds, 2-Ring - pharmacology
HSP70 Heat-Shock Proteins - antagonists & inhibitors
HSP70 Heat-Shock Proteins - metabolism
HSP90 Heat-Shock Proteins - antagonists & inhibitors
HSP90 Heat-Shock Proteins - metabolism
HT29 Cells
Humans
Immunoassay - methods
Lactams, Macrocyclic
Lymphocytes - chemistry
Lymphocytes - drug effects
Lymphocytes - metabolism
Mice
Neoplasm Transplantation
Proto-Oncogene Proteins c-raf - antagonists & inhibitors
Proto-Oncogene Proteins c-raf - metabolism
Pyrazoles - pharmacology
Quinones - pharmacology
Rifabutin - analogs & derivatives
Rifabutin - pharmacology
Technology, Pharmaceutical - methods
Technology, Pharmaceutical - trends
Transplantation, Heterologous
title Solid-phase immunoassays in mechanism-based drug discovery: their application in the development of inhibitors of the molecular chaperone heat-shock protein 90
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