Description:
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
systems6. In 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
tzodikov@princeton.edu
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.