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Development of a Fluorescence Polarization Assay for the Molecular Chaperone Hsp90

Program in Cell Biology and Department of Medicine, Memorial Sloan-Kettering Cancer Center, New York, NY 10021.

Sara Felts

Department of Biochemistry and Molecular Biology, Mayo College of Medicine, Rochester, MN 55905.

Laura Llauger

Program in Cell Biology and Department of Medicine, Memorial Sloan-Kettering Cancer Center, New York, NY 10021.

Huazhong He

Program in Cell Biology and Department of Medicine, Memorial Sloan-Kettering Cancer Center, New York, NY 10021.

Henri Huezo

Program in Cell Biology and Department of Medicine, Memorial Sloan-Kettering Cancer Center, New York, NY 10021.

Neal Rosen

Program in Cell Biology and Department of Medicine, Memorial Sloan-Kettering Cancer Center, New York, NY 10021.

Gabriela Chiosis

Program in Cell Biology and Department of Medicine, Memorial Sloan-Kettering Cancer Center, New York, NY 10021.

Heat shock protein 90 (Hsp90) is a molecular chaperone with essential functions in maintaining transformation, and there is increasing interest in developing Hsp90 inhibitors as cancer therapeutics. In this study, the authors describe the development and optimization of a novel assay for the identification of Hsp90 inhibitors using fluorescence polarization. The assay is based on the competition of fluorescently (BODIPY) labeled geldanamycin (GM) for binding to purified recombinant Hsp90 (GM is a natural product that binds to the ATP/ADP pocket in the amino terminal of Hsp90). The authors show that GM-BODIPY binds Hsp90 with high affinity. Even at low Hsp90 concentrations (30 nM), the measured polarization value is close to the maximum assay range of 160 mP, making measurements very sensitive. Its performance, as judged by signal-to-noise ratios (> 10) and Z and Z' values (> 0.5), suggests that this is a robust and reliable assay. GM, PU24FCl, ADP, and ATP, all known to bind to the Hsp90 pocket, compete with GM-BODIPY for binding to Hsp90 with EC₅₀s in agreement with reported values. These data demonstrate that the Hsp90-FP-based assay can be used for high-throughput screening in aiding the identification of novel Hsp90 inhibitors.

Key Words: fluorescence polarization • Hsp90 • competitive assay • high-throughput screening

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