Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Peptide containing doai
Reexamination Certificate
2000-03-29
2002-12-24
Kemmerer, Elizabeth (Department: 1647)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Peptide containing doai
C514S014800, C530S300000, C530S350000, C530S326000, C435S007100, C435S007800
Reexamination Certificate
active
06498139
ABSTRACT:
The present application is a 371 of PCT/JP98/04293 filed Sep. 25, 1998.
TECHNICAL FIELD
The present invention relates to a drug, particularly a remedy for diseases caused by insulin resistance, such as diabetes, as well as to a screening method for the remedy.
BACKGROUND ART
Insulin is a hormone which regulates the concentration of blood sugar and blood lipid through the promotion of glucose and lipid intake into cells and utilization and storage of them. Insulin resistance indicates the condition in which insulin does not act normally on cells, and this condition causes elevation of the concentration of blood sugar or blood lipid. Examples of diseases caused by insulin resistance include diabetes, diabetic microangiopathies (diabetic nephropathy, diabetic neuropathy, and diabetic retinopathy), impaired glucose tolerance, hyperinsulinemia, hyperlipemia, arteriosclerosis, hypertension, obesity, ischemic heart diseases, ischemic brain disorders, and peripheral arterial embolism (Tamio Teramoto, et al., (1995) Biomedicine & Therapeutics 29, 8-96). The cause of insulin resistance has not yet been fully elucidated, and causal therapy thereof has not been developed.
Recently, abnormality of intracellular signal transduction induced by insulin has become of interest as a cause of insulin resistance. In signal transduction of insulin, the first response induced by insulin is activation of insulin receptor tyrosine kinase. Subsequently, several intracellular substrates including insulin receptor substrate-1 (IRS-1) (Sun, X. et al., (1991) Nature 352, 73-77) and insulin receptor substrate-2 (IRS-2) (Sun, X. et al., (1995) Nature 377, 173-177) are phosphorylated. IRS-1 and IRS-2 have potential tyrosine-phosphorylated sites in amounts of 21 and 23, respectively, and they function as “docking protein” which transmits insulin signals to several proteins having Src-homology 2 domains (SH2-protein) (Sun, X. et al., (1993) Mol. Cell. Biol. 13, 7418-7428).
However, the function of IRS-1 and IRS-2 relating to insulin signal transduction in the aforementioned action is not necessarily fully elucidated, and elucidation of novel function thereof and development of drugs on the basis of the function are demanded.
An object of the present invention is to elucidate novel function of IRS-1 and IRS-2, and to provide a drug based on the function.
DISCLOSURE OF THE INVENTION
In view of the foregoing, the present inventors have focused on the relation between IRS-1 or IRS-2 and 14-3-3 protein.
14-3-3 Protein is widely distributed in eucaryotes such as animals, plants, ahd yeast, and is a protein family which is supposed to act as a regulatory factor by binding to a particular target protein in a variety of signal transductions depending on phosphorylation and dephosphorylation of proteins (Fumiko Shinkai, et al., (1996) Protein Nucleic Acid Enzyme 41, 313-326). Recently, it has been reported that 14-3-3 protein binds to phosphatidylinositol 3-kinase (PI3K) and inhibits its activity in T lymphocytes (Bonnefoy-Berard, N. et al., (1995) Proc. Natl. Acad. Sci. 92, 10142-10146). PI3K plays an important role in signal transduction of insulin (Masato Kasuga, (1996) Saishin-Igaku 51, 1564-1572), and thus 14-3-3 protein has been supposed to effect some type of regulation against signal transduction of insulin (Humiko Shinkai, et al., (1996) Protein Nucleic Acid Enzyme 41, 313-326). In addition, very recently, it has been reported that the &egr; isoform of 14-3-3 protein binds to IRS-1, but the physiological significance has not been elucidated (Craparo, A. (1997) J. Biol. Chem. 272. 11663-11669).
The present inventors have performed extensive studies on the relation between 14-3-3 protein and IRS-1 or IRS-2; have elucidated that IRS-1 or IRS-2 binds to 14-3-3 protein at a particular site and that the binding effects negative regulation against insulin signal transduction; and have found that a substance inhibiting the binding is useful for a remedy for diseases caused by insulin resistance. The present invention has been accomplished on the basis of these findings.
Accordingly, the present invention provides a remedy for diseases caused by insulin resistance, which comprises, as an active ingredient, a substance exhibiting activity for inhibiting the binding of the full-length IRS-1 or IRS-2 or a portion of the same to the full-length 14-3-3 protein or a portion of the same.
The present invention also provides a screening method for a remedy for diseases caused by insulin resistance, which comprises assaying activity for inhibiting the binding of the full-length IRS-1 or IRS-2 or a portion of the same to the full-length 14-3-3 protein or a portion of the same.
The present invention also provides a pharmaceutical composition for diseases caused by insulin resistance, which comprises a substance exhibiting activity for inhibiting the binding of the full-length IRS-1 or IRS-2 or a portion of the same to the full-length 14-3-3 protein or a portion of the same, and a pharmaceutically acceptable carrier.
The present invention also provides use of a substance exhibiting activity for inhibiting the binding of the full-length IRS-1 or IRS-2 or a portion of the same to the full-length 14-3-3 protein or a portion of the same for producing a remedy for diseases caused by insulin resistance.
The present invention also provides a method for treating diseases caused by insulin resistance, which comprises administering to a patient in need thereof an effective dose of a substance exhibiting activity for, inhibiting the binding of the full-length IRS-1 or IRS-2 or a portion of the same to the full-length 14-3-3 protein or a portion of the same.
BEST MODE FOR CARRYING OUT THE INVENTION
Active ingredients of the remedy of the present invention include a substance exhibiting activity for inhibiting the binding of the full-length IRS-1 or IRS-2 or a portion of the same to the full-length 14-3-3 protein or a portion of the same in screening for assaying the inhibiting activity.
As described below, the present inventors were the first to elucidate that the binding of 14-3-3 protein to IRS-1 or IRS-2 effects negative regulation against insulin signal transduction.
Firstly, in order to identify a unique protein that binds to IRS-1, the present inventors used
32
P-labeled recombinant IRS-1 as a probe in order to screen a cDNA library derived from human heart, to thereby obtain two isoforms (&egr; and &zgr;) which belong to a 14-3-3 protein family. In addition, they found that 14-3-3 protein associates with IRS-1 in L6 muscular cells, HepG2 hepatoma cells, and Chinese hamster ovary cells, in which IRS-1 is overexpressed by means of an adenovirus expression system, as well as in the brain tissue of cow in a natural state.
The present inventors also elucidated that 14-3-3 protein associates with IRS-1 or IRS-2 in SF9 cells in which 14-3-3 protein and IRS-1 or IRS-2 are overexpressed by means of a baculovirus expression system.
The present inventors also elucidated, by use of HepG2 hepatoma cells in which IRS-1 is overexpressed in the same manner as described above, that the amount of 14-3-3 protein binding to IRS-1 is not changed by insulin stimulation, and that the amount is significantly increased by okadaic acid, which is an inhibitor of serine/threonine phosphatase.
The present inventors also elucidated that IRS-1 has three putative binding sites (Ser-270, Ser-374, and Ser-641) for 14-3-3 protein, on the basis of the finding that, in a cell lysate of L6 muscular cells, the binding of IRS-1 to 14-3-3 protein fused with glutathione S-transferase (GST) is inhibited by three types of 15-residue oligopeptide shown in sequence Nos. 2-4 which contains a serine residue and several amino acid residues in the vicinity of it corresponding to the amino acid sequence of IRS-1, and the serine residue is phosphorylated. Of the above three binding sites, the motif around of Ser-270 are located in the phosphotyrosine binding domain (PTB domain) of IRS-1, and the domain is known to play an important role in interaction with insulin receptors (Wolf, G. (19
Asano Tomoichiro
Kanda Akira
Kubo Hideo
Yazaki Yoshio
Daiichi Pharmaceutical Co. Ltd.
DeBerry Regina M.
Kemmerer Elizabeth
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