Princeton University Office of Technology Licensing and Intellectual Property
Fourth Floor, New South Building
Post Office Box 36
Princeton, New Jersey 08544-0036
Phone: (609) 258-6762
FAX: (609) 258-1159
Engineered In Vivo Biosensors for Nuclear Hormone Receptor Ligands, Princeton University Invention # 04-2095
Researchers in the Chemical Engineering Department at Princeton University have developed a new in vivo sensor in Escherichia coli that can report ligand binding to eukaryotic nuclear hormone receptors. This system has been optimized to report the presence of an active compound by changes in growth levels of bacteria ( Escherichia coli) cells. Similar assays have been constructed in yeast Saccharomyces cerevisiae, but this new bacterial system is simple, faster, and more cost efficient.
Approximately 2% of the drug targets of current therapies belong to the nuclear hormone receptor superfamily². These transcription factors 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 inflammations2, 3. 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 engineered in vivo reporter systems4, as well as in vitro receptor binding assays5. More recently, small peptides which bind and modulate receptor activity have been identified using combinatorial methods6.
In vivo approaches, however, are generally complex, time-consuming and expensive, and thus not appropriate for the construction of high-throughput screening systems. In vitro binding assays, although simpler, cannot directly report the effect of the ligand on the function of the receptor target. 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 combined the ligand binding domain of the a-subtype of the human estrogen receptor with a thymidylate synthase enzyme (TS)7. Expression of this fusion in TS-deficient bacterial cells resulted in estrogen-dependent cell growth. Subsequent replacement of the estrogen receptor with the ligand-binding domain of the human thyroid hormone receptor led to thyroid hormone-dependent growth that was insensitive to estrogen.
We then challenged this biosensor with a small library of estrogen and thyroid hormone analogues, and it was observed that levels of cell growth correlated well with ligand-binding affinity. Remarkably, this simple biosensor was able to discriminate between agonistic and antagonistic activities, as combinations of estrogen agonists had an additive impact on cell growth, whereas known estrogen antagonists were found to neutralize agonist effects. More recently we have inserted the b-subtype of the human estrogen receptor, and have used this construct to detect estrogenic compounds with high reliability and sensitivity. We have further been able to detect compounds with subtype-selective activity, as well as a wide range of environmental and pollutant estrogens. Most significantly, our system was the first to identify three new estrogenic compounds from a recently synthesized small-molecule library, and all three of the compounds have new been confirmed in human breast cancer and endometrial cells. One of them has now been characterized as 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 in the screening of a small library of novel compounds for potential therapeutics.. The appeal of this system will increase with the development of accessory technologies that apply our sensor to the rapid identification or evolution of potential therapeutic compounds.
References and publications:
1. Drews, J. Drug Discovery: a historical perspective. Science 287, 1960-1964 (2000)
2. Bourguet, W., Germain, P. & Gronemeyer, H. Nuclear receptor ligand-binding domains: three dimensional structures, molecular interactions and pharmacological implications. Trends Pharmacol Sci 21, 381-388 (2000).
3. Riggs, B.L. & Hartmann,L.C. Selective estrogen-receptor modulators—mechanisms of action and application to clinical practice. N England J Med 348, 618-629 (2003)
4. Joyeux, A. et al. Engineered cell lines as a tool for monitoring biological activity of hormone analogs. Anal Biochem 249, 119-130 (1997).
5. Zacharewski, T. In vitro bioassays for assessing estrogenic substances. Environ Sci & Technol, 31, 613-623 (1997).’
6. Venkatesh, N. et al A synthetic Peptide with estrogen like activity derived from a phage-display peptide library. Peptides 23, 573-580 (2002)
7. Skretas, G. & Wood, D.W. A bacterial biosensor of endocrine modulators. J Mol. Biol, 349, 464-474 (2005).
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