Drug – bio-affecting and body treating compositions – In vivo diagnosis or in vivo testing – Testing efficacy or toxicity of a compound or composition
Reexamination Certificate
2000-09-20
2003-07-01
Yucel, Remy (Department: 1636)
Drug, bio-affecting and body treating compositions
In vivo diagnosis or in vivo testing
Testing efficacy or toxicity of a compound or composition
C435S006120, C435S018000, C435S029000, C435S325000
Reexamination Certificate
active
06585954
ABSTRACT:
TECHNICAL FIELD
The present invention provides a method of screening for compounds which inhibit steroid sulfatase activity related to the biosynthesis of steroid hormones with the aim of healing hormone-dependent diseases such as breast cancer.
BACKGROUND ART
It has been known for long that anti-hormone agents are effective against hormone-dependent cancers, and anti-male sex hormone agents and anti-female sex hormone agents have been used for treating them. Recently, tamoxifen, which is an anti-hormone agent, has been developed as an anti-tumor agent for hormone-dependent cancers (Iacobelli, S., et al. , The Role of Tamoxifen in Breast Cancer, Raven Press, NY, 1982). For the biosynthesis of steroid hormones, specifically, estrogen, two pathways are known; i.e., the aromatase pathway through which androstenedione is converted into estrone and the sulfatase pathway through which estrone-sulfate is converted into estrone. In recent years, rapid progress has been made in the research and development of aromatase inhibitors which suppress the estrone biosynthesis by blocking the aromatase pathway.
For example, there have been reported screening systems using, as markers, the inhibition of the growth of cells carrying an introduced aromatase gene [Cancer Res., 50, 6949 (1990); J. Steroid Biochem. Molec. Biol., 44, 611 (1993); PCT National Publication No. 502261/92] and the reduction in the volume of a tumor established by transplanting cells carrying an introduced aromatase gene into a nude mouse [Cancer Res., 55, 3073 (1995)].
However, with the development of analytical research on the steroid hormone biosynthetic systems, it has become clear that the steroid sulfatase pathway plays a more important role than the aromatase pathway in the estrone biosynthesis [J. Clin. Endocrinol. Metab., 59, 29 (1984); Ann. NY Acad. Sci., 464, 126 (1986); J. Steroid Biochem., 34, 155 (1989)].
Recently, reports have been made of attempts to screen steroid sulfatase inhibitors by assay systems using a fraction having steroid sulfatase activity obtained by fractionating human placenta rich in steroid sulfatase [Steroids, 58, 106 (1993); J. Steroid Biochem. Molec. Biol., 48, 523 (1994); J. Steroid Biochem. Molec. Biol., 59, 41 (1996); Biochemistry, 36, 2586 (1997)]. It is also reported that steroid sulfatase inhibitors can be screened and evaluated by not only the inhibiting activity on a fraction having the enzyme activity, but also the growth inhibition against cells, showing hormone-dependent growth [J. Steroid Biochem. Molec. Biol., 59, 83 (1996); Cancer Res., 57, 702 (1997)]. A steroid sulfatase gene derived from human is known [J. Biol. Chem., 264, 13865 (1989)].
However, the screening systems using a fraction having the enzyme activity and cells showing hormone-dependent growth are not sufficient in sensitivity. Further, there exists a need for a screening system using animals which can be models of actual conditions of diseases.
DISCLOSURE OF THE INVENTION
An object of the present invention is to provide a method of efficiently screening for compounds inhibiting steroid sulfatase activity which are useful for the treatment of hormone-dependent diseases such as breast cancer.
The present invention relates to a method of screening for steroid sulfatase inhibitors by using the inhibiting activity on the growth of cells carrying an introduced steroid sulfatase gene as a marker. More specifically, the present invention relates to a method of screening for steroid sulfatase inhibitors by causing a test compound to act on cells carrying an introduced steroid sulfatase gene and estimating the inhibiting activity of the compound on the growth of the cells. The present invention also relates to a method of screening for steroid sulfatase inhibitors by transplanting cells carrying an introduced steroid sulfatase gene into an animal, causing a test compound to act on the animal, and estimating the inhibiting activity of the compound on the growth of the cells. Further, the present invention relates to steroid sulfatase inhibitors obtained by the above screening methods. However, known steroid sulfatase inhibitors are excluded from the above-mentioned steroid sulfatase inhibitors which are one of the aspects of the present invention.
In the present invention, any gene can be used that encodes a polypeptide having steroid sulfatase activity. Examples of suitable genes include a gene encoding a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 1, and a gene encoding a polypeptide which has an amino acid sequence wherein one or more amino acid residues are deleted, substituted or added in the amino acid sequence of a polypeptide having the amino acid sequence shown in SEQ ID NO: 1 and which has steroid sulfatase activity.
The polypeptides having an amino acid sequence wherein one or more amino acid residues are deleted, substituted or added in the amino acid sequence of the polypeptide and having steroid sulfatase activity can be prepared according to the methods described in Nucleic Acids Research, 10, 6487 (1982); Proc. Natl. Acad. Sci., USA, 79, 6409 (1982); Proc. Natl. Acad. Sci., USA, 81, 5662 (1984); Science, 224, 1431 (1984); WO85/00817; Nature, 316, 601 (1985); Gene, 34, 315 (1985); Nucleic Acids Research, 13, 4431 (1985); Current Protocols in Molecular Biology, the 8th chapter, Mutagenesis of Cloned DNA, John Wiley & Sons, Inc.; 1989, etc.
Specific examples of the genes encoding a polypeptide having steroid sulfatase activity useful in the present invention include DNA comprising the nucleotide sequence shown in SEQ ID NO: 2, and DNA which hybridizes to such DNA under stringent conditions and which encodes a polypeptide having steroid sulfatase activity.
The “DNA which is hybridizable under stringent conditions” refers to DNA which is obtained by colony hybridization, plaque hybridization or Southern blot hybridization using the DNA encoding a polypeptide having steroid sulfatase activity as a probe. Such DNA can be identified, for example, by performing hybridization at 65° C. in the presence of 0.7-1.0 M sodium chloride using a filter with colony- or plaque-derived DNA immobilized thereon and then washing the filter at 65° C. using 0.1 to 2-fold concentrated SSC solution (SSC solution: 150 mM sodium chloride and 15 mM sodium citrate) The hybridizable DNA is, for example, DNA having at least 60% homology, preferably 80% or more homology, more preferably 95% or more homology to the nucleotide sequence of DNA encoding a polypeptide having an amino acid sequence included in the amino acid sequence shown in SEQ ID NO: 1.
Hybridization can be carried out according to the methods described in Sambrook, Fritsch and Maniatis, edit., Molecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, 1989 (hereinafter abbreviated as Molecular Cloning, Second Edition), etc.
As the host cell for the introduction of the steroid sulfatase gene of the present invention, any bacterial cells, yeast cells, animal cells, insect cells, etc. can be used so far as they are capable of introduction of a steroid sulfatase gene and expression of steroid sulfatase. Preferred are cells showing hormone-dependent growth.
Of steroid sulfatases of the present invention, human-derived steroid sulfatase is reported to require post-translational modifications for the expression of the activity [Cell, 82, 271 (1998)], and in this case, an animal cell is used as the host cell. Examples of animal cells useful as the host cell include Namalwa cells, HBT5637 (Japanese Published Unexamined Patent Application No. 299/88), COS1 cells, COS7 cells and CHO cells. When cells carrying an introduced steroid sulfatase gene are transplanted into an animal such as a mouse and fixed therein to form a tumor, cells which show hormone-dependent growth and which are easily fixed in an animal to form a tumor are used. Suitable cells include MCF-7 [Int. J. Cancer, 54, 119 (1993)], T47D [J. Clin. Endocrinol., 55, 276 (1982)]
Akinaga Shiro
Anazawa Hideharu
Ishida Hiroyuki
Kato Yoko
Nakata Taisuke
Fitzpatrick ,Cella, Harper & Scinto
Kyowa Hakko Kogyo Co. Ltd.
Loeb Bronwen M.
Yucel Remy
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