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High-Throughput Fluorescence Polarization Method for Identification of FKBP12 Ligands

Department of Neuroscience, Pharmaceutical Research Institute, Bristol-Myers Squibb, Inc., Wallingford, CT.

J. J. Herbst

Department of Lead Discovery, Pharmaceutical Research Institute, Bristol-Myers Squibb, Inc., Wallingford, CT.

G. T. Gaughan

Department of Neuroscience, Pharmaceutical Research Institute, Bristol-Myers Squibb, Inc., Wallingford, CT.

T. A. Verdoorn

Department of Neuroscience, Pharmaceutical Research Institute, Bristol-Myers Squibb, Inc., Wallingford, CT.

J. Ditta

CNS Chemistry, Pharmaceutical Research Institute, Bristol-Myers Squibb, Inc., Wallingford, CT.

G. M. Dubowchik

CNS Chemistry, Pharmaceutical Research Institute, Bristol-Myers Squibb, Inc., Wallingford, CT.

A. Vinitsky

Department of Neuroscience, Pharmaceutical Research Institute, Bristol-Myers Squibb, Inc., Wallingford, CT.

FKBP12 is best known as the target of the widely used immunosuppressive drug FK506 but may also play a role in neuronal survival. Nonimmunosuppressive ligands of FKBP12 have been shown to have neuroprotective and neuroregenerative activity both in vitro and in vivo, stimulating interest in the development of high-throughput screens to rapidly identify novel ligands. FKBP12 was expressed as a His₆-fusion in bacteria and purified by metal ion affinity and gel filtration chromatography. A high-throughput fluorescence polarization assay was developed to identify novel ligands of FKBP12. Dissociation constant values of known FKBP12 ligands measured by the new method agreed closely with K_i values obtained by assaying inhibition of the rotamase activity of the enzyme. The fluorescence polarization assay is rapid, robust, and inexpensive and does not generate radioactive waste. It is very well suited for high-throughput screening efforts.

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