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Monitoring molecular-specific pharmacodynamics of rapamycin in vivo with inducible Gal4->Fluc transgenic reporter mice.

ABSTRACT: 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.

SUBMITTER: Pan MH

PROVIDER: S-EPMC2953587 | biostudies-literature | 2010 Oct

REPOSITORIES: biostudies-literature

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Monitoring molecular-specific pharmacodynamics of rapamycin in vivo with inducible Gal4->Fluc transgenic reporter mice.

Pan Mei-Hsiu MH Lin Jeffrey J Prior Julie L JL Piwnica-Worms David D

Molecular cancer therapeutics 20100921 10

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-hy ...[more]

PMID: 20858726

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Project description:Reliable and objective markers of neuronal function and pathology that can directly assess the effects of neuroprotective treatments in the brain are urgently needed for clinical trials in neurodegenerative diseases. Here we assessed the sensitivity of high field proton magnetic resonance spectroscopy ((1)H MRS) to monitor reversal of neurodegeneration by taking advantage of a well characterized conditional mouse model of spinocerebellar ataxia type 1 (SCA1), where the cerebellar pathology and ataxic phenotype are reversible by doxycycline administration. Transgene expression was suppressed by feeding the mice with chow that contains doxycycline from 6 to 12 weeks of age in an early stage group and from 12 to 24 weeks in a mid-stage group. Cerebellar neurochemical profiles of treated and untreated conditional mice were measured at 9.4 tesla (T) before and after treatment and compared to those of wild type (WT) controls, as well as to histology measures (molecular layer thickness in the primary fissure and a global pathological severity score). Concentrations of N-acetylaspartate (NAA) and myo-inositol in the treated mice trended toward normalization to WT levels in both the early and mid-stage groups. The NAA-to-myo-inositol ratio was significantly different between the treated vs. untreated SCA1 mice and demonstrated partial reversal to WT values both at early and mid-stage, consistent with the histological measures. Taurine and total creatine levels were completely normalized in early and mid-stage treatment groups, respectively. The MRS markers were a more sensitive measure of treatment response than the histological measures from the same volume-of-interest in the early stage group. NAA, myo-inositol and taurine levels were significantly correlated with the histology measures in data combined from all groups. These data demonstrate that MRS markers reliably detect rescue from neuronal pathology and imply that the neurochemical levels measured by MRS accurately reflect treatment efficacy. Therefore this study presents an important step in validating MRS biomarkers as potential surrogate markers to evaluate therapeutics in pre-clinical and clinical trials in SCA1.

Dataset Information

Monitoring molecular-specific pharmacodynamics of rapamycin in vivo with inducible Gal4->Fluc transgenic reporter mice.

Publications

Monitoring molecular-specific pharmacodynamics of rapamycin in vivo with inducible Gal4->Fluc transgenic reporter mice.

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