JPH05968A - Proteasome inhibitor - Google Patents

Abstract

PURPOSE:To obtain an inibitor capable of powerfully and specifically acting on proteasomes. CONSTITUTION:A proteasome inhibitor containing a polypeptide expressed by sequence No.1 in the sequence table expressed by the formula. Calpastatin and its related compounds are cited as specific examples of the polypeptide. The aforementioned inhibitor is useful as a therapeutic agent, etc., for various diseases to which the proteasomes participate.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a drug that inhibits proteasome, and more particularly to a drug that potently and highly specifically inhibits proteasome.

[0002]

2. Description of the Related Art The major site of intracellular proteolytic system is in the lysosomal fraction, and it is known that enzymes such as cathepsin B, H, D and L are involved in the degradative system [Harshko A. (Hershko, A.) et al., Annual Review of Biochemistry
Biochemistry) 51, 335 (1982)].
On the other hand, it is known that the proteolytic system also operates in the cytoplasm [Pontremoli S. (Pontremol
i, S.) et al., said magazine, Vol. 55, p. 455 (198).
6)]. The main one is calpain, whose activity is regulated by a specific inhibitor called calpastatin [Murachi T. et al. (Murachi, T.), Advances in Enzyme R
egulation), vol. 19, p. 407 (1981), Suzuki K. et al. (Suzuki, K.), Trends in Biochemical Scienc
es), Vol. 12, p. 103 (1987)]. More recently, a multifunctional protease complex called proteasome having a molecular weight of 450,000 to 1,000,000 has been isolated from many animal tissues [Dahlmann B. (Dahlmann, B.), Biochemical Journal (Biochem. J.), Volume 228,
161 (1985), Ishiura S.H. (Ishiura,
S.), Febbs Letters (FEBS Lett.), 18th
9, p. 119 (1985), Tanaka K .; (Tanak
a.K.), Journal of Biological Chemistry (J. Biol. Chem.), Vol. 261, 15204 (1986), Rose I. et al. A. (Rose, IA), Journal of Biological Chemistry, No. 254
Vol., 8135 (1979)].

[0003]

It is known that the biosynthesis and secretion of this proteasome vary depending on various pathological conditions. For example, human leukemia cells have increased biosynthesis, and a significant increase has been detected in the blood of various cancer patients. Furthermore, attention has been paid to the relationship between Alzheimer's disease and abnormal regulation of proteasome activity. In this way, the proteasome is deeply involved in all biological control systems such as cell proliferation, differentiation, senescence, and canceration.
The development of proteasome inhibitors is an extremely important issue as a drug. As known proteasome inhibitors, antipain, chymostatin, elastatinal, leupepsin, cystatin, and kininogen have been known [Oakbo I. (Ohkubo, I.), Nagoya Medical Journal, 34th
Vol. 199 (1990)].

However, the above-mentioned known proteasome inhibitors have low specificity and their inhibitory activity has not been strong. In view of the above situation, an object of the present invention is to provide an inhibitor that strongly and specifically acts on the proteasome.

[0005]

Means for Solving the Problems The present invention will be summarized. The present invention relates to a proteasome inhibitor, which is characterized by containing the polypeptide represented by SEQ ID NO: 1 in the sequence listing.

As a result of diligent research, the present inventors have surprisingly found that calpastatin and its related compounds, which were considered to be specific inhibitors of calpain, inhibit the proteasome with high specificity. The present invention has been completed.

The compound used in the present invention may include the polypeptide represented by SEQ ID NO: 1 in the sequence listing. That is, any substance such as calpastatin containing the polypeptide in the molecule may be used.

[0008] Calpastatin is, for example, muscle type calpastatin or erythrocyte type calpastatin, which may be purified from mammalian tissues / cells by a conventional method or may be produced by a genetic engineering method. Further, the compound related to calpastatin is, for example, calpastatin domain I.
Related compounds, which may be produced by genetic engineering or may be synthesized by peptide synthesis method.

The above compounds used in the present invention are, for example, JP-A-1-283300, JP-A-2-56498 and JP-A-2-56498.
It can be manufactured and used by the method described in JP-A-30700, and the compound may be blended, for example, in a standard pharmaceutical composition, and may be a pharmaceutical composition comprising a pharmaceutically acceptable carrier.

[0010]

EXAMPLES The present invention will now be specifically described with reference to examples, but the present invention is not limited to these examples.

Example 1 The inhibitory activity of various protease inhibitors was examined using the proteasome purified from human erythrocytes by the method of Oak Bo et al. [Previously described Nagoya Medical Journal, Vol. 34, p. 199 (1990)]. . The proteasome activity was obtained by reacting the substrate represented by SEQ ID NO: 2 in the sequence listing [Peptide Research Institute Co., Ltd.] as a substrate with the composition shown in Table 1 below at 37 ° C. for 30 minutes at 0.2 ml of 2 ml.
It was determined by adding N-acetic acid to stop the reaction and measuring the fluorescence intensity at excitation 380 nm and emission 460 nm.

[0012]

[Table 1] Table 1 ──────────────────────────────── 0.2 mM substrate 50 μl 0.5 M Tris-HCl buffer (PH 7.5) 100 μl Proteasome 2-20 μl H ₂ O Suitable amount ──────────────────────────────── 1 ml

In the above reaction system, calpastatin prepared by the genetic engineering method described in JP-A-2-56498, and the calpastatin domain shown in SEQ ID NO: 1 of the sequence listing described in JP-A-2-307000. The residual activity when the I-related compound HCSd42 was added and reacted at a molar ratio of 1: 4 and 1: 2 with the enzyme (3.3 pmol) was examined. The results are shown in Table 2.

[0014]

[Table 2] Table 2 ──────────────────────────────── Calpastatin HCSd42 Addition ratio 1: 4 1: 2 1 : 4 1: 2 Residual activity (%) 13 22 32 45 ─────────────────────────────────

As shown in Table 2, calpastatin exhibited remarkably strong proteasome inhibitory activity, and HCSd42, a compound related to calpastatin domain I, also exhibited inhibitory activity.

Example 2 Next, the proteasome residual activity was examined by varying the concentration of the inhibitor. The proteasome activity was measured in the same manner as in Example 1. The result is shown in FIG. That is, FIG. 1 is a diagram showing the relationship between inhibitor concentration and inhibitory activity,
The abscissa represents the concentration (pmol) of calpastatin and the calpastatin domain I-related compound HCSd42, and the ordinate represents the proteasome residual activity (%). As shown in Fig. 1, calpastatin and calpastatin domain I-related compound HC
Sd42 shows inhibitory activity on the proteasome, respectively.

[0017]

INDUSTRIAL APPLICABILITY The present invention provides a novel proteasome inhibitor. The inhibitor is useful as a therapeutic agent for various diseases associated with proteasome.

[Sequence Listing] SEQ ID NO: 1 Sequence Length: 133 Sequence Type: Amino Acid Number of Chains: Single Strand Topology: Linear Sequence Type: Peptide Sequence: Lys Ser Leu Thr Pro Ala Val Pro Val Glu Ser Lys Pro Asp Lys 1 5 10 15 Pro Ser Gly Lys Ser Gly Met Asp Ala Ala Leu Asp Asp Leu Ile 20 25 30 Asp Thr Leu Gly Gly Pro Glu Glu Thr Glu Glu Glu Asn Thr Thr 35 40 45 Tyr Thr Gly Pro Glu Val Ser Asp Pro Met Ser Ser Thr Tyr Ile 50 55 60 Glu Glu Leu Gly Lys Arg Glu Val Thr Ile Pro Pro Lys Tyr Arg 65 70 75 Glu Leu Leu Ala Lys Pro Ile Gly Pro Asp Asp Ala Ile Asp Ala 80 85 90 Leu Ser Ser Asp Phe Thr Cys Gly Ser Pro Thr Ala Ala Gly Lys 95 100 105 Lys Thr Glu Lys Glu Glu Ser Thr Glu Val Leu Lys Ala Gln Ser 110 115 120 Ala Gly Thr Val Arg Ser Ala Ala Pro Pro Gln Glu Lys 125 130 SEQ ID NO: : 2 Sequence length: 4 Sequence type: Amino acid Number of chains: Single strand Topology: Linear Sequence type: Peptide Sequence characteristics: 1 L-succinylleucine 4 L-tyrosine 4-Methyl-co umaryl-7-amide sequence:

[Brief description of drawings]

FIG. 1 is a graph showing the relationship between inhibitor concentration and residual activity of proteasome.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Tadao Asano 3-4-1 Seta, Otsu-shi, Shiga Prefecture Central Research Institute, Inc. (72) Inventor Asada Kizozo 3-chome Seta, Otsu, Shiga Prefecture No. 1 in the Central Research Institute of Tao Shuzo Co., Ltd. (72) Inventor Ikuno Kato 3-4-1, Seta, Otsu City, Shiga Prefecture In the Central Research Laboratory of Tao Shuzo Co., Ltd.

Claims (1)

What is claimed is: 1. A proteasome inhibitor, which comprises the polypeptide represented by SEQ ID NO: 1 in the sequence listing.