High-throughput fluorescence polarization method for identification of FKBP12 ligands

Area of Science:

• Biochemistry
• Neuroscience
• Drug Discovery

Background:

• FKBP12 protein is recognized as a target for the immunosuppressive drug FK506.
• Non-immunosuppressive FKBP12 ligands exhibit neuroprotective and neuroregenerative properties in vitro and in vivo.
• There is a growing need for efficient methods to discover novel FKBP12 ligands for therapeutic development.

Purpose of the Study:

• To develop and validate a high-throughput fluorescence polarization (FP) assay for identifying novel FKBP12 ligands.
• To assess the utility of the FP assay for screening potential neuroprotective compounds.

Main Methods:

• Bacterial expression and purification of His(6)-tagged FKBP12 protein.
• Development of a high-throughput fluorescence polarization assay to detect FKBP12 ligand binding.
• Validation of the FP assay by comparing dissociation constant (Kd) values with known FKBP12 ligand inhibition constants (Ki) for rotamase activity.

Main Results:

• A robust and inexpensive high-throughput fluorescence polarization assay for FKBP12 was successfully established.
• The FP assay demonstrated high accuracy, with measured Kd values closely correlating to established Ki values.
• The assay is rapid, requires no radioactive materials, and is suitable for large-scale screening.

Conclusions:

• The developed fluorescence polarization assay is a powerful tool for high-throughput screening of FKBP12 ligands.
• This assay facilitates the discovery of novel compounds with potential neuroprotective and neuroregenerative activities.
• The method offers an efficient, cost-effective, and environmentally friendly approach for drug discovery targeting FKBP12.