An intramolecular folding sensor for imaging estrogen receptor-ligand interactions

Department of Radiology, Stanford University School of Medicine, James H. Clark Center, 318 Campus Drive, East Wing, First Floor, Stanford, CA 94305-5427, USA.
Proceedings of the National Academy of Sciences (Impact Factor: 9.67). 11/2006; 103(43):15883-8. DOI: 10.1073/pnas.0607385103
Source: PubMed


Strategies for high-throughput analysis of interactions between various hormones and drugs with the estrogen receptor (ER) are crucial for accelerating the understanding of ER biology and pharmacology. Through careful analyses of the crystal structures of the human ER (hER) ligand-binding domain (hER-LBD) in complex with different ligands, we hypothesized that the hER-LBD intramolecular folding pattern could be used to distinguish ER agonists from selective ER modulators and pure antiestrogens. We therefore constructed and validated intramolecular folding sensors encoding various hER-LBD fusion proteins that could lead to split Renilla/firefly luciferase reporter complementation in the presence of the appropriate ligands. A mutant hER-LBD with low affinity for circulating estradiol was also identified for imaging in living subjects. Cells stably expressing the intramolecular folding sensors expressing wild-type and mutant hER-LBD were used for imaging ligand-induced intramolecular folding in living mice. This is the first hER-LBD intramolecular folding sensor suited for high-throughput quantitative analysis of interactions between hER with hormones and drugs using cell lysates, intact cells, and molecular imaging of small living subjects. The strategies developed can also be extended to study and image other important protein intramolecular folding systems.

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    • "Gene or protein targets related to disease development and therapeutic response can be exploited through molecular imaging and dynamics of the ligands in vivo and allows screening for agonistic and antagonistic ligand candidates for the receptor (34). Protein-protein interaction can be in vivo imaged with PET technology using thymidine kinase, therefore it can be applied to human subjects (35). "
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    ABSTRACT: The process of drug discovery and development requires substantial resources and time. The drug industry has tried to reduce costs by conducting appropriate animal studies together with molecular biological and genetic analyses. Basic science research has been limited to in vitro studies of cellular processes and ex vivo tissue examination using suitable animal models of disease. However, in the past two decades new technologies have been developed that permit the imaging of live animals using radiotracer emission, Xrays, magnetic resonance signals, fluorescence, and bioluminescence. The main objective of this review is to provide an overview of small animal molecular imaging, with a focus on nuclear imaging (single photon emission computed tomography and positron emission tomography). These technologies permit visualization of toxicodynamics as well as toxicity to specific organs by directly monitoring drug accumulation and assessing physiological and/or molecular alterations. Nuclear imaging technology has great potential for improving the efficiency of the drug development process.
    Toxicological Research 03/2013; 29(1):1-6. DOI:10.5487/TR.2013.29.1.001
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    • "With the advance of molecular imaging techniques, more sophisticated strategies have been adapted for the design of reporter gene system to broaden their biomedical applications. One category of reporter genes can be activated at the post-translational level with protein-protein interaction, enzymatic reaction, phosphorylation or tertiary structure changes 45. In order to distinguish these reporter systems from those inducible reporter gene imaging, we named them here as “activatable reporter gene imaging”. "
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    ABSTRACT: Molecular imaging is a newly emerged multiple disciplinary field that aims to visualize, characterize and quantitatively measure biological processes at cellular and molecular levels in humans and other living systems. A reporter gene is a piece of DNA encoding reporter protein, which presents as a readily measurable phenotype that can be distinguished easily from the background of endogenous protein. After being transferred into cells of organ systems (transgenes), the reporter gene can be utilized to visualize transcriptional and posttranscriptional regulation of gene expression, protein-protein interactions, or trafficking of proteins or cells in living subjects. Herein, we review previous classification of reporter genes and regroup the reporter gene based imaging as basic, inducible and activatable, based on the regulation of reporter gene transcription and post-translational modification of reporter proteins. We then focus on activatable reporters, in which the signal can be activated at the posttranslational level for visualizing protein-protein interactions, protein phosphorylation or tertiary structure changes. The applications of several types of activatable reporters will also be summarized. We conclude that activatable reporter imaging can benefit both basic biomedical research and drug development.
    Theranostics 04/2012; 2(4):413-23. DOI:10.7150/thno.3940 · 8.02 Impact Factor
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    • "Although the compounds regarded as environmental contaminants that were examined here led to fluorescence at concentrations much higher than found in the environment, it is conceivable that multiple such compounds could lead to a cumulative estrogenic effect. The data presented here is from transiently transfected cells, a technique which has been used in other sensor studies such as (Paulmurugan and Gambhir 2006) and (Umezawa 2005), gives reproducible results, and allows for the easy choice of different target cells. Alternatively, stable integration into a human cell line such as HEK 293 would simplify screening by avoiding the need for transfection. "
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    ABSTRACT: Estrogenic compounds are an important class of hormonal substances that can be found as environmental contaminants, with sources including pharmaceuticals, human and animal waste, the chemical industry, and microbial metabolism. Here we report the creation of a biosensor useful for monitoring such compounds, based on complementation of fluorescent protein fragments. A series of sensors were made consisting of fragments of a split mVenus fluorescent protein fused at several different N-terminal and C-terminal positions flanking the ligand binding domain of the estrogen receptor alpha. When expressed in HeLa cells, sensor 6 (ERα 312-595) showed a nine-fold increase in fluorescence in the presence of estrogen receptor agonists or antagonists. Sensor 2 (ERα 281-549) discriminated between agonists and antagonists by showing a decrease in fluorescence in the presence of agonists while being induced by antagonists. The fluorescent signal of sensor 6 increased over a period of 24 h, with a two-fold induction visible at 4 h and four-fold at 8 h of ligand incubation. Ligand titration showed a good correlation with the known relative binding affinities of the compound. The sensor could detect a number of compounds of interest that can act as environmental endocrine disruptors. The lack of a substrate requirement, the speed of signal development, the potential for high throughput assays, and the ability to distinguish agonists from antagonists make this an attractive sensor for widespread use.
    Biotechnology and Bioengineering 12/2011; 108(12):2794-803. DOI:10.1002/bit.23254 · 4.13 Impact Factor
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