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

Area of Science:

• Biochemistry
• Molecular Biology
• Drug Discovery

Background:

• Heat shock protein 90 (Hsp90) is a crucial molecular chaperone implicated in cancer and various other diseases.
• Developing novel Hsp90 inhibitors with drug-like properties is a significant area of research.

Purpose of the Study:

• To develop and optimize a novel fluorescence polarization assay for identifying Hsp90 inhibitors.
• To validate the assay's performance in a high-throughput screening (HTS) format.

Main Methods:

• Utilized fluorescence polarization to measure competitive binding of fluorescently labeled geldanamycin (GM-cy3B) to cellular Hsp90.
• Employed NCI-N417 small-cell lung carcinoma cells and optimized assay conditions in a 384-well format.
• Validated the assay using over 15,000 compounds from the NIH Molecular Libraries Screening Center Network.

Main Results:

• GM-cy3B demonstrated high affinity and specificity for cellular Hsp90.
• The assay exhibited robust performance with excellent signal-to-noise ratios (>10) and Z' values (>0.75).
• The assay is a simple, cost-effective mix-and-read format, stable for up to 24 hours.

Conclusions:

• The developed fluorescence polarization assay is a robust and efficient tool for Hsp90 inhibitor screening.
• This assay facilitates the discovery of novel therapeutic agents targeting Hsp90 for various diseases.
• The assay's performance in HTS format was successfully validated, supporting its utility in drug discovery pipelines.