Engineered In Vivo Biosensors for Nuclear Hormone Receptor Ligands

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Princeton University Invention # 04-2095



Researchers in the Chemical Engineering Department at Princeton University have developed a new type of biosensor using Escherichia coli that can report ligand binding to eukaryotic nuclear hormone receptors.  This system has been optimized to report the presence of endocrine-active compounds through changes in growth rates of bacterial (Escherichia coli) cells.  Similar assays have been constructed in yeast Saccharomyces cerevisiae, but this new bacterial system is simple, faster, and more cost efficient.


Over 2% of all the drug targets of current therapies belong to the nuclear hormone receptor superfamily.  Notable examples of drugs that target these receptors include tamoxifen, raloxifene, estrogen replacement therapies and several thyroid hormone therapies.  The nuclear hormone receptors control the expression of several genes in response to the presence of small-molecule hormones or hormone-like compounds, and include the estrogen, androgen, thyroid hormone, progesterone and vitamin D receptors among others.  Their function has been linked to a broad spectrum of diseases, including breast, endometrial and prostate cancer, leukemia, cardiovascular diseases, osteoporosis and inflammations1, 2.  Therefore the discovery of novel compounds with the ability to modulate these targets could lead to the development of valuable therapeutics against serious pathological conditions.  Typical methods for identifying these compounds have included in vivo reporter systems based on engineered cell lines3 and whole animal assays4, as well as in vitro receptor binding assays5.


In vivo approaches, however, are generally complex, time-consuming and expensive, and thus not appropriate for the construction of high-throughput screening systems6In vitro binding assays, although simpler, often cannot accurately predict the effect of a given ligand on the function of the receptor target (e.g. agonist vs. antagonist).  Thus simple in vivo assays based on receptor function in yeast or bacteria might greatly accelerate the lead identification process, allowing new drugs to be discovered more rapidly and cheaply.


We have developed a novel sensor of nuclear hormone binding in Escherichia coli by constructing a gene fusion that combines the ligand binding domain of the a and b subtypes of the human estrogen receptor with a thymidylate synthase reporter enzyme (TS)7.  Expression of this fusion in TS-deficient bacterial cells results in estrogen-dependent cell growth, which can be used to detect and identify estrogenic compounds in the growth medium.  Subsequent replacement of the estrogen receptor with the ligand-binding domain of the human thyroid hormone receptor leads to thyroid hormone-dependent growth.  By successfully incorporating the b subtype of the estrogen NHR, we have been able to differentiate agonist from antagonist activities in a wide variety of drug-like compounds8, as well as detect subtype-selective estrogen receptor modulators9, endocrine disrupting pollutant compounds that affect humans and animals (in preparation), and even complex mixtures associated with home products and cosmetics (submitted).  Most recently, we have developed thyroid hormone sensors, using both the a and b subtypes, and have shown the ability to detect subtype-selective thyroid hormone modulators as well (in preparation).


These biosensors have been validated with a significant number of estrogen and thyroid hormone analogues, where it was observed that levels of cell growth correlate well with reported ligand-binding affinities (i.e. potency).  Discrimination between agonistic and antagonistic activities is trivial through the use of combinations of known agonists with test compounds - antagonists counteract the effects of the agonist.  Remarkably, we have used our system to identify three new estrogenic compounds from a recently synthesized small-molecule library, and the activities of all three of the compounds have been confirmed in human breast cancer and endometrial cells.  One of them appears to be one of the strongest estrogen antagonists yet discovered, and all of the compounds are being patented by their inventor.  Estrogen antagonists are particularly important in the treatment of breast cancer, and include such compounds as Tamoxifen and Raloxifene.  Thus this system has proven utility for screening libraries of novel compounds for potential therapeutics, and we have recently adapted it to an automated microtiter format (submitted).  The appeal of this system will increase with the development of additional accessory technologies that apply our sensor to the rapid identification or evolution of potential therapeutic compounds.


Princeton is currently seeking industrial collaboration to commercialize this technology. Patent protection is pending.


For more information on Princeton University Invention # 04-2095 please contact:


                        Laurie Tzodikov

                        Office of Technology Licensing and Intellectual Property

                        Princeton University

                        4 New South Building

                        Princeton, NJ 08544-0036

                        (609) 258-7256

                        (609) 258-1159 fax



 Publications and  References


Gawrys, M; Hartman, I; Landweber, L; Wood, DW, Use of engineered Escherichia coli cells to detect estrogenicity in everyday consumer products, J Chem Technol Biotechnol 2009;84: 1834-1840, early view



Hartman, I; Gillies, A ; Arora, S; Andaya, C; Royapet, N; Welsh,W;Wood,D W; Zauhar, R J, Application of Screening Methods, Shape Signatures and Engineered Biosensors in Early Drug Discovery, Pharmaceutical Research, Vol 26,No.10, October2009, pg 2247-2258.


1.         Bourguet, W.; Germain, P.; Gronemeyer, H., Nuclear receptor ligand-binding domains: three-dimensional structures, molecular interactions and pharmacological implications. Trends Pharmacol Sci 2000, 21, (10), 381-8.

2.         Riggs, B. L.; Hartmann, L. C., Selective estrogen-receptor modulators -- mechanisms of action and application to clinical practice. N Engl J Med 2003, 348, (7), 618-29.

3.         Joyeux, A.; Balaguer, P.; Germain, P.; Boussioux, A. M.; Pons, M.; Nicolas, J. C., Engineered cell lines as a tool for monitoring biological activity of hormone analogs. Anal Biochem 1997, 249, (2), 119-30.

4.         Ramamoorthy, K.; Wang, F.; Chen, I. C.; Norris, J. D.; McDonnell, D. P.; Leonard, L. S.; Gaido, K. W.; Bocchinfuso, W. P.; Korach, K. S.; Safe, S., Estrogenic activity of a dieldrin/toxaphene mixture in the mouse uterus, MCF-7 human breast cancer cells, and yeast-based estrogen receptor assays: no apparent synergism. Endocrinology 1997, 138, (4), 1520-7.

5.         Zacharewski, T., In vitro bioassays for assessing estrogenic substances. Environ. Sci. & Technol. 1997, 31, 613-623.

6.         O'Connor, J. C.; Cook, J. C.; Marty, M. S.; Davis, L. G.; Kaplan, A. M.; Carney, E. W., Evaluation of Tier I screening approaches for detecting endocrine-active compounds (EACs). Crit Rev Toxicol 2002, 32, (6), 521-49.

7.         Skretas, G.; Wood, D. W., A bacterial biosensor of endocrine modulators. J Mol Biol 2005, 349, (3), 464-74.

8.         Skretas, G.; Meligova, A. K.; Villalonga-Barber, C.; Mitsiou, D. J.; Alexis, M. N.; Micha-Screttas, M.; Steele, B. R.; Screttas, C. G.; Wood, D. W., Engineered Chimeric Enzymes as Tools for Drug Discovery: Generating Reliable Bacterial Screens for the Detection, Discovery, and Assessment of Estrogen Receptor Modulators. J Am Chem Soc 2007, 129, (27), 8443-8457.

9.         Skretas, G.; Wood, D. W., Rapid Detection of Subtype-Selective Nuclear Hormone Receptor Binding with Bacterial Genetic Selection. Appl Environ Microbiol 2005, 71, (12), 8995-8997.


Patent Information:
For Information, Contact:
Laurie Tzodikov
Licensing Associates
Princeton University
David Wood
Georgios Skretas