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Rapamycin (Rap), a small-molecule inhibitor of mTOR, is an immunosuppressant, and several Rap analogues are cancer chemotherapeutics. Further pharmacologic development will be significantly facilitated if in vivo reporter models are available to enable monitoring of molecular-specific pharmacodynamic actions of Rap and its analogues. Herein we present the use of a Gal4→Fluc reporter mouse for the study of Rap-induced mTOR/FKBP12 protein-protein interactions in vivo with the use of a mouse two-hybrid transactivation strategy, a derivative of the yeast two-hybrid system applied to live mice. Upon treatment with Rap, a bipartite transactivator was reconstituted, and transcription of a genomic firefly luciferase reporter was activated in a concentration-dependent (K(d) = 2.3 nmol/L) and FK506-competitive (K(i) = 17.1 nmol/L) manner in cellulo, as well as in a temporal and specific manner in vivo. In particular, after a single dose of Rap (4.5 mg/kg, i.p.), peak Rap-induced protein-protein interactions were observed in the liver at 24 hours post treatment, with photon flux signals 600-fold over baseline, which correlated temporally with suppression of p70S6 kinase activity, a downstream effector of mTOR. The Gal4→Fluc reporter mouse provides an intact physiologic system to interrogate protein-protein interactions and molecular-specific pharmacodynamics during drug discovery and lead characterization. Imaging protein interactions and functional proteomics in whole animals in vivo may serve as a basic tool for screening and mechanism-based analysis of small molecules targeting specific protein-protein interactions in human diseases.

Monitoring Molecular-Specific Pharmacodynamics of Rapamycin In vivo with Inducible Gal4→Fluc Transgenic Reporter Mice