Chemistry: analytical and immunological testing – Biospecific ligand binding assay
Reexamination Certificate
2000-04-11
2002-09-17
Whisenant, Ethan C. (Department: 1655)
Chemistry: analytical and immunological testing
Biospecific ligand binding assay
C435S007100, C435S069100, C530S300000, C530S350000
Reexamination Certificate
active
06451617
ABSTRACT:
TECHNICAL FIELD
The present invention relates to a method for screening substances that inhibit binding between TAK1 and TAB1. The present invention also relates to a method for screening substances that inhibit the signal transduction of transforming growth factor-&bgr; (TGF-&bgr;). The present invention further relates to substances and uses thereof obtainable by the method for screening substances that inhibit binding between TAK1 and TAB1.
BACKGROUND ART
Transforming growth factor-&bgr; (TGF-&bgr;) is a multilfunctional factor that controls various aspects of cell functions. As one such function, TGF-&bgr; is responsible for the repair and regeneration of tissues associated with various injuries (Border, W. A. & Noble, N. A., The New England Journal of Medicine (1994) 331, 1286-1292).
An abnormal production of TGF-&bgr; in chronic injuries can sometimes disturb balances in the repair and regeneration of tissues resulting in pathological fibrosis. As a pathological condition in which the balance of TGF-&bgr; production has been disturbed, hepatic fibrosis is known. It has been elucidated that TGF-&bgr; acts as a main causative agent of fibrosis of various organs such as the liver, by enhancing the production of extracellular matrix protein that can cause fibrosis, inhibiting the synthesis of proteolytic enzymes of extracellular matrix, and by inducing substances that inhibit proteolytic enzymes of extracellular matrix (Border, W. A. & Noble, N. A., The New England Journal of Medicine (1994) 331, 1286-1292).
Other known functions of TGF-&bgr; include the activity of inhibiting cellular growth (Moses, H. L. et al., Cell (1990) 63, 245-247), the activity of migrating monocytes (Wahl, S. M. et al., Proc. Natl. Acad. Sci. U.S.A. (1987) 84, 5788-5792), the activity of inducing biologically active substances (Wahl, S. M. et al., Proc. Natl. Acad. Sci. U.S.A. (1987) 84, 5788-5792), the activity of facilitating the deposition of amyloid &bgr; protein (Wyss-Coray, T. et al., Nature (1997) 389, 603-606), and the like.
TGF-&bgr; transduces its signals through heteromer complexes of type I and type II TGF-&bgr; receptors and transmembrane proteins containing the serine- and threonine-specific kinase domains at the side of cytoplasm (Wrana, J. L. et al., Nature (1994) 370, 341; Kingsley, D. M. et al., Genes Dev. (1994) 8, 133). However, much of the mechanism of signaling downward from the TGF-&bgr; receptor into the cell on the molecular level remains to be elucidated.
As a series of systems involved in the signal transduction of the TGF-&bgr; superfamily, mitogen-activated protein kinase (MAPK) is known.
The MAPK system is a conserved eukaryotic signaling system that converts signals of a receptor into various functions. The MAPK system contains three types of protein kinases, i.e. mitogen-activated protein kinase kinase kinase (MAPKKK), mitogen-activated protein kinase kinase (MAPKK), and mitogen-activated protein kinase (MAPK). MAPK is activated through phosphorylation by MAPKK. MAPKK is activated through phosphorylation by MAPKKK (Nishida, E. et al., Trends Biochem. Sci. (1993) 18, 128; Blumer, K. J. et al., Trends Biochem. Sci. (1993) 19, 236; David R. J. et al., Trends Biochem. Sci. (1993) 19, 470; Marchall, C. J. et al., Cell (1995) 80, 179).
TAK1 (TGF-&bgr;-activated kinase 1), that is a member of the MAPKKK family that functions in the signaling system of biologically active substances and that belongs to the TGF-&bgr; superfamily, was identified by Yamaguchi, K. et al. (Yamaguchi, K. et al., Science (1995) 270, 2008).
TAB1 (TAK1 binding protein 1), a protein involved in the signaling system of TGF-&bgr; that binds to and activates TAK1, was identified by Shibuya, H. et al. (Shibuya, H. et al., Science (1996) 272, 1179-1182).
Although TAB1 transduces the signal of TGF-&bgr; by binding to TAK1 and activating TAK1 kinase activity, no attempts have been made so far to search for substances that inhibit binding between TAK1 and TAB1 in order to suppress or activate signal transduction of TGF-&bgr; by focusing on the binding between TAK1 and TAB1.
DISCLOSER OF THE INVENTION
The present invention is intended to provide a method for screening substances that inhibit binding between TAK1 and TAB1. The present invention is also intended to provide a method for screening substances that suppress or activate the signal transduction of TGF-&bgr;. The present invention further is intended to provide substances that are obtainable by a method for screening substances that inhibit binding between TAK1 and TAB1.
Thus, the present invention provides (1) a method for screening substances that inhibit binding between a TAK1 polypeptide and a TAB1 polypeptide, which method comprises contacting the TAB1 polypeptide to the TAK1 polypeptide and a test sample and then detecting or determining the TAK1 polypeptide that is bound to the TAB1 polypeptide. Preferably, the TAB1 polypeptide is a TAB1 polypeptide that has been bound to a support. A preferred support is beads or a plate. In another preferred embodiment, the contact between a TAK1 polypeptide, a TAB1 polypeptide and a test sample is carried out in a homogeneous system.
The present invention also provides (2) a method for screening substances that inhibit binding between a TAK1 polypeptide and a TAB1 polypeptide, which method comprises contacting the TAK1 polypeptide to the TAB1 polypeptide and a test sample, and then detecting or determining the TAB1 polypeptide that is bound to the TAK1 polypeptide. Preferably, the TAK1 polypeptide is a TAK1 polypeptide that has been bound to a support. A preferred support is beads or a plate. In another preferred embodiment, the contact between a TAK1 polypeptide, a TAB1 polypeptide and a test sample is carried out in a homogeneous system.
The present invention also provides (3) a screening method described in the above (1) and (2), which method comprises using a TAB1 polypeptide having an amino acid sequence comprising Met at amino acid position 1 to Pro at amino acid position 504 of the amino acid sequence as set forth in SEQ ID NO: 2, or having an amino acid sequence modified by the substitution, deletion and/or addition of one or a plurality of amino acid residues of the amino acid sequence as set forth in SEQ ID NO: 2 and maintaining the biological activity of the TAB1 polypeptide; and/or
a TAK1 polypeptide having an amino acid sequence comprising Met at amino acid position 1 to Ser at amino acid position 579 of the amino acid sequence as set forth in SEQ ID NO: 4, or having an amino acid sequence modified by the substitution, deletion and/or addition of one or a plurality of amino acid residues of the amino acid sequence as set forth in SEQ ID NO: 4 and maintaining the biological activity of the TAK1 polypeptide.
The present invention also provides (4) a screening method described in the above (1) to (3), which comprises using a TAK1 polypeptide fused to another peptide or polypeptide and/or a TAB1 polypeptide fused to another peptide or polypeptide.
The present invention also provides (5) a method for screening substances that inhibit binding between a TAK1 polypeptide and a TAB1 polypeptide, which method comprises contacting the TAB1 polypeptide to the labeled TAK1 polypeptide and a test sample, and then detecting or determining the labeled TAK1 polypeptide that is bound to the TAB1 polypeptide. Preferably, the TAB1 polypeptide is a TAB1 polypeptide that has been bound to a support. A preferred support is beads or a plate. In another preferred embodiment, the contact between a TAK1 polypeptide, a TAB1 polypeptide and a test sample is carried out in a homogeneous system.
The present invention also provides (6) a method for screening substances that inhibit binding between a TAK1 polypeptide and a TAB1 polypeptide, which method comprises contacting the TAK1 polypeptide to the labeled TAB1 polypeptide and a test sample, and then detecting or determining the labeled TAB1 polypeptide that is bound to the TAK1 polypeptide. Preferably, the TAK1 polypeptide is a TAK1 polypeptide that has been bound to a suppo
Ohtomo Toshihiko
Ono Koichiro
Tsuchiya Masayuki
Chugai Seiyaku Kabushiki Kaisha
Lu Frank W
Whisenant Ethan C.
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