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Journal of Biomolecular Screening
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High Throughput Scintillation Proximity Assay for the Identification of FKBP-12 Ligands

Francesca Graziani

GlaxoWellcome Medicines Research Centre, Department of Lead Discovery, Verona, Italy

Laura Aldegheri

GlaxoWellcome Medicines Research Centre, Department of Lead Discovery, Verona, Italy

Georg C. Terstappen

GlaxoWellcome Medicines Research Centre, Department of Lead Discovery, Verona, Italy

A high throughput scintillation proximity assay (SPA) was developed to identify novel ligands of FKBP-12, an immunophilin with peptidyl prolyl isomerase (rotamase) activity. Recombinant histidine-tagged FKBP-12 was expressed in Escherichia coli, purified by metal ion affinity chromatography, and immobilized to SPA beads by an antibody that recognizes the histidine tag of the recombinant protein. Using 1 nM [3H] FK506, a well-known macrolid ligand of FKBP-12, specific binding was saturable and accounted for 95% of total binding. Analysis of saturation and homologous displacement isotherms indicated the existence of a single binding site with a KD value of 1.6 nM. The specificity of [3H] FK506 binding was demonstrated in displacement experiments and showed that rapamycin, another macrolid, was as active as FK506 (IC50 of 3.5 and 3.2 nM, respectively), whereas GPI-1046, a prototype of small molecular compounds with neurotrophic properties and affinity for FKBP-type immunophilins, was more than 1000-fold less active. The high signal-to-noise ratio of 30, together with small standard deviations, makes this novel assay well suited for automated high throughput screening.

Journal of Biomolecular Screening, Vol. 4, No. 1, 3-7 (1999)
DOI: 10.1177/108705719900400102
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