Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
2000-07-28
2001-09-18
Horlick, Kenneth R. (Department: 1656)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S007800, C435S008000
Reexamination Certificate
active
06291194
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to methods for determining androgenic or anti-androgenic activity of a sample, wherein the sample is either mammalian serum or a solution of a compound to be tested. The invention further concerns a cell useful in said methods.
BACKGROUND OF THE INVENTION
The publications and other materials used herein to illuminate the background of the invention, and in particular, cases to provide additional details respecting the practice, are incorporated by reference.
Androgens are required for the masculinization of male genitalia in utero, the development of secondary sex characteristics in boys, and the maintenance of male sexual function in adult life. After entering the target cell, androgens bind to androgen receptor (AR)—a ligand-dependent transcription factor. After binding of the hormone, AR enters the nucleus and binds to the regulatory region of the target gene as a homodimer. AR belongs to the nuclear receptor superfamily comprising receptors for various forms of vitamin D
3
, thyroid hormones, retinoids, and steroid hormones (
1
). These receptors have conserved DNA—and ligand-binding domains (DBD and LBD, respectively), and variable hinge and N-terminal regions (
1
). In the case of AR, the N-terminal region encompasses the primary transcriptional activation domain. Upon androgen binding, LBD and the N-terminal region of AR have been shown to interact, which is suggested to facilitate AR dimerization (
2
,
3
), modulate transcriptional activity (
4
), and stabilize the receptor at low ligand concentrations (
5
).
AR-interacting protein 3 (ARIP3), a 572-amino acid nuclear protein expressed primarily in the testis, represents a potential coregulator of AR-dependent transcription (
6
). Although the exact physiologic role of ARIP3 is not yet known, we have observed that it can considerably facilitate the androgen-dependent interaction between the AR LBD and N-terminal region (
6
). Herein, we report the development of a bioassay that is based on ARIP3-facilitated interaction between the LBD and N-terminal region of AR. This assay appeared useful for quantitation of circulating androgen bioactivity in pediatric patients. We expect that the assay will have wide ramifications in clinical endocrinology.
SUMMARY OF THE INVENTION
Thus, according to one aspect this invention concerns a method for determining the androgenic activity of a sample, comprising the steps of
a) contacting the sample with a cell comprising
a luciferase reporter plasmid,
a fusion protein comprising a ligand-binding domain of the androgen receptor and a Gal4 DNA-binding domain, said fusion protein being able to bind to binding sites in said luciferase reporter plasmid,
a fusion protein comprising an N-terminal region of the androgen receptor and a transcriptional activation domain, and
an androgen receptor-interacting protein 3,
b) allowing the sample to incubate with said cell,
c) lysing said cell,
d) measuring the luciferase activity of the lysate,
e) comparing the measured luciferase activity to that obtained by repeating the steps a) to d) above except that a control is added instead of the sample, to give the relative luciferase activity of the sample, and
f) using the relative luciferase activity to detect or quantify an active androgen in the sample.
According to another aspect, the invention concerns a method for determining the anti-androgenic activity of a compound, comprising the steps of
a) measuring the luciferase activity emitted from a first sample comprising a first compound having androgenic activity as described above,
b) measuring the luciferase activity, according to the previous step, emitted from a second sample comprising said first compound having androgenic activity and either I) a second compound which shall be tested in respect of anti-androgenic activity, or II) mammalian serum,
c) comparing the luciferase activities obtained in steps a) and b) above, and
d) using a decreased luciferase activity emitted from said second sample to detect or quantify the anti-androgenic activity of said second compound or serum.
According to a third aspect, the invention concerns a cell comprising
a luciferase reporter plasmid,
a fusion protein comprising a ligand-binding domain of the androgen receptor and a Gal4 DNA-binding domain, said fusion protein being able to bind to binding sites in said luciferase reporter plasmid,
a fusion protein comprising an N-terminal region of the androgen receptor and a transcriptional activation domain, and
an androgen receptor-interacting protein 3.
REFERENCES:
patent: 5789170 (1998-08-01), Chang et al.
David J. Mangelsdorf, et al., “The Nuclear Receptor Superfamily: The Second Decade”, Dec. 15, 1995, Cell vol. 83, 835-839.
Elizabeth Langley, et al., “Evidence for an Anti-parallel Orientation of the Ligand-activated Human Androgen Receptor Dimer”, Dec. 15, 1995, Journal Biological Chemistry vol. 270, pp. 29983-29990.
Tarja Ikonen, et al., Interaction between the Amino-and Carboxyl-terminal Regions of the Rat Androgen Receptor Modulates Transcriptional Activity and Is Influnced by Nuclear Receptor Coactivators, Nov. 21, 1997, Journal Biological Chemistry vol. 272, pp. 29821-29828.
Jon A. Kemppainen, et al., “Distinguishing Androgen Receptor Agonists and Antagonists: Distinct Mechanisms of Activation by Medroxyprogesterone Acetate and Dihydrotestosterone”, 1999, Molecular Endocrinology vol. 13 No. 3 pp. 440-454.
Anu-Maarit Moilanen, et al., “A Testis-specific Androgen Receptor Coregulator That Belongs to a Novel Family of Nuclear Proteins”, Feb. 5, 1999, Journal Biological Chemistry vol. 274 No. 6, pp. 3700-3704.
A. Moilanen, et al., “The presence of a transcription activation function in the hormone-binding domain of androgen receptor is revealed by studies in yeast cells”, Jun. 6, 1997, FEBS Letters vol. 412 pp. 355-358.
Ralph I. Dorfman, “Androgens and Anabolic Agents”, 1962, Chapter 6 pp. 275-313, Method in Hormone Research, vol. II. New York, Academic Press.
Jänne Olli A.
Palvimo Jorma J.
Raivio Taneli
Horlick Kenneth R.
Rothwell Figg Ernst & Manbeck p.c.
Strzelecka Teresa
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