JPH06239889A - Neurotrophic activity suppressive substance - Google Patents

Abstract

PURPOSE:To provide a non-natural-type polypeptide having neurotrophic activity- suppressive action effective for the treatment of Alzheimer's disease. CONSTITUTION:This non-natural-type polypeptide having neurotrophic activity- suppressive action, contains an amino acid sequence of the formula Thr-Cys-Pro- Cys-Pro-Ser-Gly-Gly-Ser-Cys-Thr-Cys-Ala-Asp-Ser-Cys-Lys-Cys-Glu.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a polypeptide having a neurotrophic activity inhibitory action.

[0002]

2. Description of the Related Art Senile dementia has been of great interest in an aging society, and many efforts have been made for its prevention and treatment. In particular, senile dementia called Alzheimer's disease often occurs in the premature period (50 to 60 years old), and the cause of it and the establishment of a therapeutic method are urgently needed. According to the findings obtained to date, Alzheimer's disease has pathological features such as senile plaque and neurofibrillary degeneration,
It is an organic disease having a clinical feature of progressive dementia, and is considered to be involved in enhancement of neuronal metabolism and abnormal regeneration. JP-A-4-18100, European Patent Application Publication No. 458,673 and P
In CTWO92 / 10568, a novel protein found in the process of studying the components in the brain of Alzheimer's disease patients, which is present in the brain of normal humans but disappears from the brain of Alzheimer's disease patients. , The extraction of a natural protein (GIF) having a neurotrophic activity suppressing action, and the method of producing GIF by a genetic engineering method are shown.

[0003]

If the active center site of GIF can be known in detail, the action of GIF can be further investigated, and the possibility of its use as a medicine will be expanded. Further, if a polypeptide containing the polypeptide at the active center site can be used as a medicine, it is advantageous in terms of production, formulation, etc. of the polypeptide.

[0004]

Means for Solving the Problems The present inventors have studied the activity of GIF as a fragment for the purpose of exploring the action mechanism of GIF.
A polypeptide containing the amino acid sequence at position 23 has an excellent G
The present invention was completed as a result of finding that it has IF activity and conducting further research based on it.

The present invention provides the formula (I): Thr Cys Pro Cys Pro Ser Gly Gly Ser Cys Thr Cys Al
a Asp Ser Cys Lys CysGlu (SEQ ID NO: 1) which is a non-naturally occurring polypeptide having a neurotrophic activity inhibitory action (hereinafter sometimes referred to as the polypeptide of the present invention).

The polypeptide of the present invention is a non-naturally occurring polypeptide containing the amino acid sequence represented by the formula (I),
Therefore, the natural GIF is excluded. Examples of the polypeptide of the present invention include those having the amino acid sequence represented by the formula (I), and the N-terminal and / or the amino acid sequence represented by the formula (I) as long as it has a neurotrophic activity suppressing action. It may have one to several amino acids linked to the C-terminus. Furthermore, the polypeptide of the present invention may have at least one disulfide bond. The number of amino acids to be linked is, for example, 1 to 10
The number is preferably 1 to 5, more preferably 1 to 3. Specific examples of the amino acids to be linked include, for example,
Examples of amino acids linked to the N-terminal include those linked to one or more of methionine, tyrosine, serine, and threonine, and those linked to C-terminals linked to one or more of tyrosine, serine, and threonine. Is mentioned.

The polypeptide of the present invention is disclosed in, for example, JP-A-4-18100, EP-458,673,
It can be produced by subjecting a neurotrophic activity inhibitor (GIF) obtained by the method described in PCT WO92 / 10568 to a peptide chain cleavage reaction. For example, the cleavage can be performed by subjecting the GIF to a cleavage reaction using an enzyme.

Examples of the enzyme include protease, which may be either exo or endopeptidase. Examples of the protease include serine protease (eg, trypsin, chymotrypsin, thrombin, plasmin, elastase, V8 etc.); thiol protease (eg: cysteine protease, papain, ficin, bromelain, cathepsin B etc.);
Acidic protease (eg aspartic protease,
Pepsin, cathepsin D, renin, chymosin, etc.); metalloproteases (eg, carboxyprotease, collagenase, thermolysin, etc.) and the like. Among them, endopeptidase (eg, trypsin, V8, metalloprotease, etc.) can be preferably used.

The enzyme reaction is carried out by reacting a purified product or a partially purified product of GIF with an enzyme. The reaction is preferably carried out using a buffer solution. Examples of the buffer solution include an inorganic acid (phosphoric acid etc.) or an organic acid (acetic acid etc.) and an inorganic base (sodium hydroxide, potassium hydroxide, ammonia etc.). Any salt may be used as long as it does not inhibit the enzyme reaction. The pH in the enzyme reaction is preferably about 2 to 10, more preferably about 6 to 9 in terms of stability of the enzyme and the polypeptide. The reaction time is preferably about 1 to 100 hours, especially about 18 to 24 hours. The reaction temperature is about 4
~ 70 ° C, especially about 20-37 ° C is preferred. The amount of peptidase used in the reaction may be about 1/1000 to 1 amount (molar ratio) with respect to the substrate GIF,
About 1/300 to 1/20 amount (molar ratio) is preferable in terms of yield, reaction time, and economy. In the enzymatic reaction, aminopeptidase was added to agarose, dextran,
It can be immobilized on cellulose, polyacrylamide, or a derivative thereof or a copolymer thereof, and used in the reaction.

In addition, the action of aminopeptidase causes Me
Methionine in t-Asp-Pro may be specifically cleaved. The method is described in European Patent Application Publication No. 204,52.
It is carried out according to the method described in JP-A-7.

The polypeptide of the present invention can also be produced by a synthetic method. Chemical synthesis of the polypeptide of the present invention can be performed by an automatic peptide synthesizer. RB Merr
ifield [Advances in Enzymology (Adva
nces in Enzymology) 32, 221-296 (1969)]. In this method, the amino acid at the carboxyl terminal is covalently bound to the resin carrier, the removal of the protecting group of the α-amino group and the condensation of the protected amino acid are sequentially repeated, and the peptide chain is extended toward the amino terminal. The principle is to obtain a protected peptide resin having an amino acid sequence. Condensation of each amino acid and removal of the protecting group for the α-amino group are performed under substantially the same conditions, and no intermediate purification is performed. Therefore, generally no high skill is required in the synthesis. Moreover, this method is rapid and is a very convenient method for synthesizing various peptides. The protected peptide resin thus obtained is reacted with, for example, anhydrous hydrogen fluoride, trifluoromethanesulfonic acid or trifluoroacetic acid in the presence of various additives to remove the peptide from the resin and remove all protecting groups. Can be done in one step.

To isolate and purify the target peptide, generally known peptide purification methods may be used. For example,
Gel filtration, ion exchange chromatography, high performance liquid chromatography, affinity chromatography, hydrophobic chromatography, thin layer chromatography, electrophoresis and the like can be appropriately combined and performed.

The thus-obtained polypeptide having a neurotrophic activity-suppressing action of the present invention is useful as a drug for treating Alzheimer's disease. Moreover, the toxicity of this polypeptide is low. In order to use the polypeptide having a neurotrophic activity-suppressing action of the present invention as a drug, it can be used as a powder as it is, or with other pharmacologically acceptable carriers, excipients and diluents in a pharmaceutical composition (for example, injection , Tablets, capsules, solutions, ointments) can be safely administered parenterally or orally to warm-blooded animals (eg, humans, mice, rats, hamsters, rabbits, dogs, cats). The injection is formulated according to a conventional method using, for example, physiological saline or an aqueous solution containing glucose or other auxiliary agent. Pharmaceutical compositions such as tablets and capsules can also be prepared according to a conventional method. When the polypeptide having a neurotrophic activity-suppressing action of the present invention is used as the above-mentioned drug, for example, in the above-mentioned warm-blooded animal, the daily dose of about 1 ng / kg to 1 mg / Select an appropriate amount from kg and administer.

In the present specification, amino acids, peptides,
When an abbreviation is used for a protecting group, an active group, etc., they are IUPAC-IUB (Commission on Biochemica
l Nomenclature) or an abbreviation commonly used in this field, examples of which are given below. In addition, when there may be optical isomers of amino acids,
Unless otherwise specified, the L form is shown. A, Ala: Alanine C, Cys: Cysteine D, Asp: Aspartic acid E, Glu: Glutamic acid F, Phe: Phenylalanine G, Gly: Glycine H, His: Histidine I, Ile: Isoleucine K, Lys: Lysine L, Leu: Leucine M, Met: Methionine N, Asn: Asparagine P, Pro: Proline Q, Gln: Glutamine R, Arg: Arginine S, Ser: Serine T, Thr: Threonine V, Val: Valine W, Trp: Tryptophan Y, Tyr: Tyrosine

[0015]

EXAMPLES The present invention will be described in more detail with reference to the following Reference Examples and Examples. Reference Example 1 Separation and Purification of GIF GIF was produced by the method described in JP-A-4-18100. That is, 60 ml of water was added to 20 g of gray matter of normal human cerebral cortex, homogenized and centrifuged at 20,000 xg for 1 hour, and then 55 ml of the supernatant was obtained. 55 ml of the obtained supernatant was subjected to ultrafiltration using an Amicon YM-10 membrane (trade name), and a fraction having a molecular weight of 10 kilodaltons or more was subjected to DE filtration.
Place on an AE-Sephacel column (1.6 cmφ x 16 cm, Pharmacia) and wash buffer [50 mM NaC
1, 50 mM Tris-Cl (pH 7.6)] 200 ml, and then a 20 mM Tris-Cl (pH 7.6) buffer 32 having a linear concentration gradient of 50 mM to 300 mM NaCl.
Extracted with 0 ml. The chromatogram obtained by the DEAE-Sephacel column is shown in FIG. Fraction No. 31
Fractions with inhibitory activity from 38 to 38 were collected (40 ml)
After dialysis, it was concentrated using Ficoll 400. afterwards,
Gel filtration (column size 7.5 mmφ × 6 cm) with TSK G2000SW (manufactured by Tosoh Corporation), fraction N
o. Collect active fractions from 30 to 32 (2.5 ml), 5 mM
It was dialyzed in phosphate buffer (pH 7.4). Above TS
The result of gel filtration chromatography using KG2000SW is shown in FIG. After concentrating the volume to 550 μl,
It was applied to a Cl8 reverse-phase HPLC column (4.6 mmφ × 25 cm, manufactured by Senshu Chemical Co., Ltd.). 0% to 80% for elution
A 5 mM ammonium formate solution with a linear gradient of acetonitrile was used. The results of this Cl8 reverse phase HPLC chromatography are shown in FIG. As shown in FIG. 3, C
18 sharp reversed peaks were obtained by 18 reverse phase HPLC chromatography and GIF was isolated.

Example 1 Digestion of GIF with Protease 50 μg of GIF obtained in Reference Example 1 was pyridylethylated by a conventional method, and 0.5 μg of trypsin (manufactured by Sigma) was added to 100 μl of 0.1 M Tris-Cl (pH 8.0) solution.
Was added and reacted at room temperature for 18 hours.
Separated on an A30 gel filtration column. The separation pattern by GFA30 column chromatography is shown in FIG.
As shown in FIG. 4, three peaks (TG1, TG2
And TG3) were obtained. TG out of these peaks
The three products were collected and digested with V8 protease, and then the reaction products were separated by GFA30 gel filtration column. as a result,
Two peaks (VG1 and VG2) were obtained (Fig. 5). Separately, 400 μg of the GIF obtained in Reference Example 1 was added to 0.5 μg of trypsin (manufactured by Sigma) in 100 μl of phosphate-buffered saline of Dulbecco, and the same procedure as above was followed. A digest was obtained.

Example 2 Analysis of Amino Acid Sequence Five types of degradation products (TG1, TG2, obtained in Example 1)
TG3, VG1 and VG2) were separately collected and applied to a gas phase protein sequencer (model 477A; manufactured by Applied Biosystems) to determine the amino acid sequence of each peptide. As a result, each peptide was found to have the following amino acid sequence. GIF: MDPETCPCPS GGSCTCADS
C KCEGCKCTSC KKSCCSCCPA E
CEKCAKDCV CKGGGEAAEAE AEKCSCCQ (SEQ ID NO: 2) TG1: MDPETCPCPS GGSCTCADS
C KCEGCKCTSC KKSCCSCCPA E
CEKCAKDCV CKGGGEAAEAE AEKCSCCQ (SEQ ID NO: 2) TG2: MDPETCPCPS GGSCTCADS
C KCEGCKCTSC KKSCCSCCPA E
CEKCAKDCV CKGGGEAAEAE AEKCSCCQ (SEQ ID NO: 2) TG3: MDPETCPCPS GGSCTCADS
C KCEGCK (SEQ ID NO: 3) VG1: MDPETCPCPS GGSCTCADS
CKCEGCK (SEQ ID NO: 3) VG2: TCPCPSGGGSC TCADSCKCE (SEQ ID NO: 1)

Example 3 Digestion of GIF with Protease 200 μg of GIF obtained in Reference Example 1 was dissolved in 200 ml of Dulbecco's phosphate buffered saline, 13 μg of trypsin (manufactured by Sigma) was added, and the mixture was reacted at room temperature for 18 hours. The reaction products were separated on a GFA30 gel filtration column. GFA
The separation pattern by 30 column chromatography is the same as that shown in FIG. 4, and three peaks (TG1, TG
2 and TG3) were obtained. T out of these peaks
The G3 product was collected and digested with V8 protease, and then the reaction product was separated on a GFA30 gel filtration column. As a result, the same two peaks (VG1 and VG2) as in FIG. 5 were obtained.

Example 4 Analysis of Amino Acid Sequence Five kinds of degradation products (TG1, TG2, obtained in Example 1)
Each of TG3, VG1 and VG2) was treated with cyanogen bromide and oxidized with formic acid according to a conventional method. N-terminal sequence analysis was performed with a PSQ-1 protein sequencer (Shimadzu). The amino acid composition was analyzed with a JLC-300 amino acid analyzer (manufactured by JEOL) after hydrolysis with 6N hydrochloric acid. As a result, each peptide was found to have the same amino acid sequence as SEQ ID NOS: 2 and 3.

Example 5 Measurement of neurotrophic activity suppressing activity 1.7 × 10 cells prepared from cerebral cortex of neonatal rat
⁴ pieces were spread on a 6 mm microplate coated with gelatin-polyornithine, and 1 part of Alzheimer's disease brain extract solution was spread.
Serum-free medium MEMN2 containing 100 μl of an aqueous solution adjusted to a concentration of 25 μg / ml (Eagle basal medium with insulin,
Transferrin, putrescine, progesterone, and sodium selenite were added), and each peptide obtained in Example 1 was added.
Cultured for a day. After fixing with paraformaldehyde and 90% methanol / 5% acetic acid solution, the amount of MAP2 was determined by ELISA using a microtubule-binding protein 2 (MAP2; protein specifically produced by nerve cells) antibody (manufactured by Amersham). It was quantified. On the other hand, as a control experiment, the amount of MAP2 when culturing by adding only the Alzheimer's disease brain extract without adding GIF was quantified, and the inhibitory activity was expressed by the% decrease in the amount of MAP2. Using the above method, the relationship between the amount of GIF peptides (TG3 and VG2) and the inhibition rate was measured. Results are shown in FIG. Figure 6
In the figure, ● indicates GIF, ◆ indicates TG3, and × indicates VG2. As shown in FIG. 6, TG3 and VG2 were found to have neurotrophic activity suppressing activity.

Example 6 The following experiment was carried out using TG3 (GIF1-26) obtained by digesting GIF with trypsin and having the same action. Metals were removed from TG3 by treatment with 0.1N hydrochloric acid, and gel filtration was performed with a GFA-30 column equilibrated with 0.01N hydrochloric acid to separate ApoTG3. This ApoTG3 had a neurotrophic activity suppressing activity.
P containing ApoTG3 in 2% β-mercaptoethanol
It was dissolved in BS and then β-mercaptoethanol was removed by gel filtration. This product, ApoTG3
+ ME did not have neurotrophic activity suppressing activity.
The results are shown in Fig. 7.

This experiment revealed that, although TG3, ApoTG3 and ApoTG3 + ME have the same amino acid sequence, only TG3 and ApoTG3 have neurotrophic activity suppressing activity. ApoTG3 + ME
Is treated with mercaptoethanol, and its S—S bond is cleaved and then recombined by autooxidation. The mercaptoethanol treatment is ApoTG3 +
The protein folding pattern of ME is changed to a pattern different from TG3. On the other hand, Apo
The S-S bond of TG3 was not cleaved, and the protein folding pattern of this peptide seems to be unchanged from that of TG3 with S-metal bond.

[0023]

INDUSTRIAL APPLICABILITY The polypeptide of the present invention can be used as a neurotrophic activity-suppressing substance for treating Alzheimer's disease. These neurotrophic activity-suppressing substances can be more easily produced by gene recombination technology or chemical synthesis than natural substances. Also, administration of the substances of the invention is easier. That is, the polypeptide of the present invention is easily soluble in water, easy to formulate, and easy to be absorbed in the living body. In particular, the polypeptide of the present invention seems to pass through the brain barrier more easily than the natural substance in view of its molecular size. Further, from the structural analysis of the substance of the present invention, it can be used as a raw material for synthesizing a compound having a similar action.

[0024]

[Sequence list]

 SEQ ID NO: 1 Sequence length: 19 Sequence type: Amino acid Topology: Linear Sequence type: Peptide Sequence: Thr Cys Pro Cys Pro Ser Gly Gly Ser Cys Thr Cys Ala Asp Ser Cys 1 5 10 15 Lys Cys Glu 19

SEQ ID NO: 2 Sequence length: 68 Sequence type: Amino acid Topology: Linear Sequence type: Peptide Sequence: Met Asp Pro Glu Thr Cys Pro Cys Pro Ser Gly Gly Ser Cys Thr Cys 1 5 10 15 Ala Asp Ser Cys Lys Cys Glu Gly Cys Lys Cys Thr Ser Cys Lys Lys 20 25 30 Ser Cys Cys Ser Cys Cys Pro Ala Glu Cys Glu Lys Cys Ala Lys Asp 35 40 45 Cys Val Cys Lys Gly Gly Glu Ala Ala Glu Ala Glu Ala Glu Lys Cys 50 55 60 Ser Cys Cys Gln 65 68

SEQ ID NO: 3 Sequence length: 26 Sequence type: Amino acid Topology: Linear Sequence type: Peptide Sequence: Met Asp Pro Glu Thr Cys Pro Cys Pro Ser Gly Gly Ser Cys Thr Cys 1 5 10 15 Ala Asp Ser Cys Lys Cys Glu Gly Cys Lys 20 25

[Brief description of drawings]

FIG. 1 is a chromatogram obtained by homogenizing normal human cerebral cortex, performing ultrafiltration, and applying a DEAE-Sephacel column to a fraction having a molecular weight of 10 kilodaltons or more.

FIG. 2 is a chromatogram obtained by gel filtration of a fraction having inhibitory activity in the purification process of GIF.

Figure 3: Chromatogram of GIF subjected to Cl8 reverse phase HPLC.

FIG. 4 is a separation pattern of the enzyme reaction product (trypsin digestion product) obtained in Example 1 by gel filtration column chromatography.

FIG. 5 is a pattern obtained by gel filtration column chromatography of the enzyme reaction product (V8 protease digestion product) obtained in Example 1.

FIG. 6 is a graph showing the neurotrophic activity inhibitory action of GIF and the polypeptide of the present invention obtained in Example 3.

FIG. 7 is a graph showing the neurotrophic activity inhibitory effect of GIF and the polypeptide of the present invention obtained in Example 6.

Claims (4)

[Claims] 1. A formula: Thr Cys Pro Cys Pro Ser Gly Gly Ser Cys Thr Cys Al
a Asp Ser Cys Lys CysGlu A non-naturally occurring polypeptide having an action of suppressing neurotrophic activity, which comprises the amino acid sequence shown. 2. An inhibitor of neurotrophic activity comprising an amino acid sequence represented by the formula: Met Asp Pro Glu Thr Cys Pro Cys Pro Ser Gly Gly Ser Cys Thr Cys Ala Asp 1 5 10 15 Ser Cys Lys Cys Glu Gly Cys Lys 20 25. A non-naturally occurring polypeptide having an action. 3. The polypeptide of claim 1 having at least one disulfide bond. 4. The polypeptide of claim 2 having at least one disulfide bond.