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. 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.
Haddow Laboratories, Cancer Research UK Centre for Cancer Therapeutics at Institute of Cancer Research, Sutton, Surrey, UK.
Anthea
A
Hardcastle
Kathy
K
Boxall
Juliet
J
Richards
Peter
P
Tomlin
Swee
S
Sharp
Paul
P
Clarke
Paul
P
Workman
Wynne
W
Aherne
D000818:Animals; D016227:Benzoquinones; D015153:Blotting, Western; D002352:Carrier Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D002457:Cell Extracts / Q000032:analysis; D004305:Dose-Response Relationship, Drug; D015195:Drug Design; D004791:Enzyme Inhibitors / Q000494:pharmacology; D045325:HCT116 Cells; D018840:HSP70 Heat-Shock Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D018841:HSP90 Heat-Shock Proteins / Q000037:antagonists & inhibitors* / Q000378:metabolism; D019073:HT29 Cells; D006574:Heterocyclic Compounds, 2-Ring / Q000494:pharmacology; D006801:Humans; D007118:Immunoassay / Q000379:methods*; D047908:Intracellular Signaling Peptides and Proteins; D047029:Lactams, Macrocyclic; D008214:Lymphocytes / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism; D051379:Mice; D009368:Neoplasm Transplantation; D019908:Proto-Oncogene Proteins c-raf / Q000037:antagonists & inhibitors / Q000378:metabolism; D011720:Pyrazoles / Q000494:pharmacology; D011809:Quinones / Q000494:pharmacology; D017828:Rifabutin / Q000031:analogs & derivatives / Q000494:pharmacology; D013678:Technology, Pharmaceutical / Q000379:methods / Q000639:trends; D014183:Transplantation, Heterologous; D019892:eIF-2 Kinase / Q000037:antagonists & inhibitors / Q000378:metabolism
D016227:Benzoquinones; D002352:Carrier Proteins; D002457:Cell Extracts; D004791:Enzyme Inhibitors; D018840:HSP70 Heat-Shock Proteins; D018841:HSP90 Heat-Shock Proteins; D006574:Heterocyclic Compounds, 2-Ring; D047908:Intracellular Signaling Peptides and Proteins; D047029:Lactams, Macrocyclic; D019908:Proto-Oncogene Proteins c-raf; D011720:Pyrazoles; D011809:Quinones; D017828:Rifabutin; D019892:eIF-2 Kinase; C506244:CCT018159; C498804:Erbb2ip protein, mouse; C112765:tanespimycin; C001277:geldanamycin