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J Biomol Screen. 2007 Oct;12(7):915-24.

High-throughput screening fluorescence polarization assay for tumor-specific Hsp90.

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Department of Pharmacology, Emory University School of Medicine and Emory Chemical Biology Discovery Center, Atlanta, Georgia, USA.


Heat shock protein 90 (Hsp90) is a molecular chaperone that has emerged as an important target in cancer and several other diseases, such as neurodegenerative diseases, nerve injuries, inflammation, and infection. Discovery of novel agents that inhibit Hsp90 and have druglike properties is therefore a major focus in several academic and industrial laboratories. In this study, the authors describe the development and optimization in a 384-well format of a novel assay for the identification of Hsp90 inhibitors using fluorescence polarization, which measures competitive binding of red-shifted fluorescently labeled geldanamycin (GM-cy3B) to Hsp90 found in the NCI-N417 small-cell lung carcinoma cells. The authors demonstrate that GMcy3B binds with high affinity and specificity to cellular Hsp90. The assay results in excellent signal-to-noise ratios (>10) and Z' values (>0.75) at tracer concentrations greater than 4 nM and 1 microg/well of total NCI-N417 protein, indicating a robust assay. It also equilibrates after 5 h of incubation at room temperature and remains stable for up to 24 h. Furthermore, it is a simple mix-and-read format that is cost-effective and uses only low amounts of fluorophore and cell lysates. A study using more than 15,000 compounds from the National Institutes of Health Molecular Libraries Screening Center Network was performed to validate its performance in a high-throughput screening format.

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