JPH07149795A - Peptide having activity to inhibit release of glutamic acid - Google Patents

Abstract

PURPOSE:To obtain a new peptide having glutamic acid release inhibiting action and/or calcium channel inhibiting action. CONSTITUTION:A new peptide containing 48 amino acid residues is isolated from the venom of a spider called as FTX. The peptide has glutamic acid release inhibiting action and/or calcium channel inhibiting action and is useful as a preventing and treating agent for cranial nervous diseases.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel peptide having glutamate release inhibitory activity and useful as a medicine.

[0002]

BACKGROUND OF THE INVENTION and Prior Art In recent years, studies on excitatory amino acids have become more and more interesting because they are closely related to delayed neuronal necrosis and memory after cerebral ischemia. In particular, regarding neuronal necrosis due to cerebral ischemia, it is thought that ischemia causes abnormal release of glutamate from nerve endings, which triggers a series of cell necrosis reactions [SMRothman et al., Ann.Neur.
ol., 19 , 105-111 (1986), DWChoi, Neuron, 1 , 623-63
4 (1988)]. Therefore, if the release of glutamic acid is suppressed,
It is considered to be extremely useful for the treatment of ischemic cranial nerve disorders, and there is a strong demand for the development of drugs having such a mechanism of action.

On the other hand, in 1992 Mintz et al.
ω-Agatoxin IV A (ω), a peptide that suppresses glutamate release from rat nerve terminals from the venom of el web spider, scientific name Agelenopsisaperta (hereinafter referred to as FTX)
-Agatoxin IV A Hereinafter, simply referred to as agatoxin. ) For the first time and determined its structure. [I.Min
tzet al., Nature, 355 , 827-829 (1992)]. Agatoxin is a peptide consisting of 48 amino acid residues, and is believed to inhibit the release of glutamate by suppressing P-type calcium channels at nerve endings [T.
J. Turner et al., Science, 258, 310-313 (1992)]. Further, JP-A-3-14551 discloses a novel peptide separated from FTX, and describes that it has a pharmacological action similar to that of agatoxin.

[0004]

However, at present, the number of compounds known to have an action of inhibiting glutamate release from nerve endings is extremely small, and various studies have been conducted to actually use it as a drug. The current situation is that there are challenges. Therefore, an object of the present invention is to provide a novel peptide having glutamate release inhibitory activity.

[0005]

That is, the present invention provides a peptide containing the amino acid sequences of SEQ ID NO: 1 and SEQ ID NO: 2, and a prophylactic / therapeutic agent and calcium containing the amino acid sequence of SEQ ID NO: 2 and having an effective glutamate inhibitory action. It is intended to provide a prophylactic / therapeutic agent for diseases in which a channel inhibitory action is effective.

The present inventors have found that the components in FTX are
Further detailed examination was conducted. First, an attempt was made to separate and purify the active substance from the crude fraction FTX which inhibits the release of glutamic acid. Second, the structural analysis of the active substance was performed. As a result, an active substance that markedly inhibits the release of glutamic acid from rat synaptosomes was found in FTX. The present inventors further conducted structural analysis of the peptide,
It was clarified that the peptide had a double chain structure, the amino acid sequence of the peptide having this double chain structure was determined, and the present invention was completed here.

That is, the present invention is a peptide including a part or all of the amino acid sequence shown in SEQ ID NO: 1 or a peptide essentially equivalent thereto, and including a part or all of the amino acid sequence shown in SEQ ID NO: 2. A peptide or a peptide essentially equivalent thereto, which includes a part or all of the amino acid sequence of SEQ ID NO: 1 or a peptide essentially equivalent thereto and a part of the amino acid sequence of SEQ ID NO: 2. Alternatively, a peptide including all or a peptide essentially equivalent thereto is a peptide forming a double chain, a peptide consisting of a part thereof, or a peptide essentially equivalent thereto.

In the present invention, a "part" of the amino acid sequence
The term "SEQ ID NO: 1, SEQ ID NO: 2, or both in a peptide in which both are bound by a disulfide bond" means at least 3-5 consecutive amino acid residues, preferably 6-8 amino acid residues, and Preferably, it shows 9-12 amino acid residues. Furthermore, “essentially equivalent” means that the constituent amino acids have been modified, added, mutated, substituted or partially deleted by a known method. Further, in the present invention, another amino acid sequence or other compound bound to the C-terminal and / or N-terminal of the continuous amino acid sequence disclosed in the present invention, or some of the peptides disclosed in the present invention are attached. It goes without saying that it also includes ones that are linked by a bond to form a peptide chain of two or more chains.

The peptide according to the present invention is Brownlee
Similar to RP-4, RP-8 and RP-18, it can be separated and purified from FTX by a known method. As a method for separating and purifying from FTX, for example, a silica gel carrier modified with a hydrophobic group is used and eluted with a hydrophilic organic solvent having a concentration gradient,
This can be performed by a method of collecting the target fraction. As the hydrophobic organic solvent in this case, any one that can be used in this field is usually used.
Examples thereof include lower aliphatic alcohols such as ethanol and propyl alcohol, and lower aliphatic nitriles such as acetonitrile. It can also be purified by ion exchange chromatography, gel filtration chromatography, hydrophobic chromatography and the like. Thus, the peptide of the present invention can also be produced by separating and purifying FTX as a raw material. in this case,
The peptides shown in SEQ ID NO: 1 and SEQ ID NO: 2 have a double chain structure due to a disulfide bond. The target fraction in the separation / purification in the above method may be traced with the activity of suppressing the release of calcium-dependent glutamate from the prepared synaptosome as an index. That is,
As will be shown in Examples below, a synaptosome fraction from rat forebrain, NADP ^+, and glutamate dehydrogenase (hereinafter abbreviated as GDH) were added to a sample, and 3
After incubating at 7 ° C. for 20 minutes, depolarized with 30 mM potassium chloride, and measured as NADPH purified by glutamate released from synaptosome.

The peptide according to the present invention can be produced by a gene recombination technique by a known method or a chemical peptide synthesis technique, in addition to the method of separating and purifying from FTX as described above. In short, those obtained by any method are included in the present invention.

The peptide of the present invention is mainly extracted from FTX as a double chain, but a single chain alone is included in the present invention. In this case, the extracted 2
The single-chain peptide may be made single-stranded by a commonly used method such as reduction of 2-mercaptoethanol with 2-mercaptoethanol, or a single-chain single peptide may be obtained by a known gene recombination technique or peptide chemical synthesis technique. You may get it. That is, the method for obtaining a single-chain peptide is not particularly limited. The structural analysis of the peptide according to the present invention can be carried out by a known method. For example, as shown in the examples below, an amino acid sequence automatic analyzer is used and sequential decomposition from the N terminus is performed by the Edman decomposition method. And the amino acid sequence can be determined.
The results are shown in SEQ ID NOS: 1 and 2.

[0012]

The effects of the present invention will be shown below.
Examples of pharmacological experiments are listed. Example of Pharmacological Experiment (1) Glutamic Acid Release Inhibitory Activity i) Experimental Method Preparation of Synaptosome Fraction Preparation of synaptosome fraction is carried out by the conventional method [E.
G. Gray et al., JAnat., 96 , 79-88 (1962)]. All the solutions used for the preparation of synaptosomes were sufficiently saturated with a mixed gas of 95% O ₂ + 5% CO ₂ before the experiment. Brains were immediately removed after slaughtering 6-week-old male Wistar rats, and cold calcium-free Tyrode's solution [composition (mM): NaCl 140, KCl 5, MgCl ₂ 1, D-glucos
e 24, Na-HEPES 10, pH 7.4] was used for cooling, and the forebrain was divided into two parts between the inferior colliculus and the cerebellum. 20 times the volume of 0.
A 32 M sucrose solution was added, and the mixture was slowly homogenized with a glass homogenizer under ice cooling. 1000 × g, 1 at 4 ℃
After centrifuging for 0 minutes, the supernatant was centrifuged at 20000 xg and 4 ° C for 20 minutes. A calcium-free Tyrode's solution was added to the precipitate, and the mixture was stirred and then centrifuged again at 20000 × g at 4 ° C. for 20 minutes.

Calcium-free Tyrode's solution was added to the precipitate and suspended at a concentration of 3.5 mg protein / ml to give 10
Calcium chloride was added to 0 μM. A calcium-added sample and a calcium-free sample to which calcium chloride was not added were prepared, and each was incubated at 37 ° C. for 1 hour. The synaptosome fraction obtained as described above was used in the experiment of glutamate derivative inhibitory activity shown below.

Measurement of Glutamic Acid Release Inhibitory Activity 80 μl (final concentration 1.4 mg) of the sample and the prepared synaptosome were added to each well of a 96-well microplate for oral measurement.
protein / ml), 1 mM NADP ⁺ , 50 V
GDH was added to a final volume of 200 μl and incubated for 20 minutes at 37 ° C. Note that 0.2 mM EGTA was added to the calcium-free sample. Next, 30m
Depolarize by adding M potassium chloride, and 10 minutes later, 96
355 with automatic fluorimeter for well microplate
Excitation at nm and relative fluorescence intensity at 460 nm was measured. In the experimental system, the difference in fluorescence value between the calcium-added sample and the calcium-free sample 0.2 mM EGTA is the amount of calcium-dependent release of glutamic acid, and therefore the inhibition rate of the glutamic acid release of the sample is calculated by the formula shown below. Calculated.

[0015]

[Equation 1]

Ii) Experimental Results The results of the above method are shown in Table 1.

[Table 1] The purified peptide markedly suppressed the release of calcium-dependent glutamate at concentrations equivalent to 10000-fold diluted solution and 1000-fold diluted solution in terms of FTX.
It was concentration-dependent.

From the above results, it became clear that the peptide of the present invention has a glutamate release inhibitory action. Therefore, the peptide according to the present invention can be used as a therapeutic agent for cranial nerve disease represented by acute brain injury or Alzheimer's disease, chronic disease such as Huntington's chorea, etc. that occur in conditions such as cerebral ischemia and status epilepticus. Besides, it can be said that it is also very useful as a reagent for clarifying the neurophysiology of the brain.

Furthermore, the peptide according to the present invention has low toxicity and high safety, so that the present invention is also useful in that sense. When the peptide of the present invention is used as a medicine, it is administered parenterally, but usually it is administered as an injection or the like. The dose depends on the degree of symptoms, age, sex, weight of the patient, difference in sensitivity to the drug,
It varies depending on the timing of administration, the interval of administration, the nature of the pharmaceutical preparation, etc., but is not particularly limited, but usually 0.1 μg per adult
/ Kg to 300 μg / kg is preferable.

When preparing an injection, a pH adjusting agent, a buffering agent, a suspending agent, a solubilizing agent, a stabilizing agent, an isotonicity agent, a preservative and the like are added to the main drug as required, and the conventional method is used. For intravenous, subcutaneous and intramuscular injection. At that time, if necessary, a freeze-dried product can be prepared by a conventional method.

Examples of the suspending agent include methyl cellulose, polysorbate 80, hydroxyethyl cellulose, gum arabic, tragacanth powder, sodium carboxymethyl cellulose, polyoxyethylene sorbitan monolaurate and the like.

Examples of the solubilizer include polyoxyethylene hydrogenated castor oil, polysorbate 80, nicotinic acid amide, polyoxyethylene sorbetane monolaurate, macrogol, castor oil fatty acid ethyl ester and the like.

Examples of the stabilizer include sodium sulfite, sodium metasulfite and ether, and examples of the preservative include methyl paraoxybenzoate, ethyl paraoxybenzoate, sorbic acid, phenol, cresol and chlorocresol. be able to.

[0023]

EXAMPLES In order to describe the present invention in more detail, examples of the present invention will be given below, but it goes without saying that the present invention is not limited thereto.

Example 1 (1) Purification of Peptide According to the Present Invention 20 μl of FTX was acidified by adding 2 μl of 1% trifluoroacetic acid, and then subjected to high performance chromatography.
It was adsorbed on a Brownley RP-8 column (4.6 mm diameter × 25 cm length) equilibrated with a 0.1% trifluoroacetic acid aqueous solution. For elution, the active ingredient was eluted by a concentration gradient elution method using the same solution and a 60% acetonitrile aqueous solution. FIG. 1 shown below is the elution pattern, in which the horizontal axis shows the elapsed time after elution of the concentration gradient, and the vertical axis shows the absorbance. Fractions having glutamic acid release inhibitory activity, which are filled in black in FIG. 1, were collected and the solvent was removed by freeze-drying. When the obtained substance was analyzed by a laser desorption time-of-flight mass spectrometer, it was recognized as a single component having a molecular weight of about 30,000 daltons.

(2) Structural analysis Using about 10 picomoles of this substance, it was confirmed from the amino terminal side by a gas phase protein sequencer (Shimadzu Corporation). Further, when this substance was reduced with 2-mercaptoethanol and analyzed by the above-mentioned mass spectrometer, two components having a molecular weight of about 28,000 daltons and about 2000 daltons were detected. That is, it was revealed that the double chain of this substance bound by a disulfide bond is another peptide. The amino acid sequence of each of the single-chain peptides obtained by the above method was determined by reducing carboxamide methylation according to the method of Crestfield, etc., and then using Baidac equilibrated with 0.1% trifluoroacetic acid aqueous solution. C4 column (4.6 mm diameter x 1
(5 cm length), and the substance was separated and purified into a light chain and a heavy chain by a concentration gradient elution method using the same solution and 80% acetonitrile as shown in FIG. Using a gas phase protein sequencer for each of the separated chains,
The amino acid sequence was analyzed from the amino terminal side. As a result, as shown in the sequence listing, 18 residues were
For the heavy chain, the amino acid sequence of 30 residues from the amino terminus was determined. The amino acid sequence of the light chain is shown in SEQ ID NO: 1. The structural analysis of the heavy chain was further conducted by preparing fragment peptides with lysyl endopeptidase, Staphylococcus aureus-derived V8 protease, and asparaginyl endopeptidase, and identifying the amino acid sequence of each peptide, as shown in SEQ ID NO: 2 below. The amino acid sequence of 161 residues from the amino terminus was identified. Before addition, the one subjected to depolarization under the above conditions is shown.

[0026]

[Sequence list]

 SEQ ID NO: 1 Sequence length: 18 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Val Glu Val Ala Thr Val Lys Asn Cys Gly Lys Lys Leu Leu Ala Thr 1 5 10 15 Pro Arg 18 sequence No .: 2 Sequence length: 161 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Ile Val Gly Gly Lys Thr Ala Lys Phe Gly Asp Tyr Pro Trp Met Val 1 5 10 15 Ser Ile Gln Gln Lys Asn Lys Lys Gly Thr Phe Asp His Ile Cys Gly 20 25 30 Gly Ala Ile Ile Asn Val Asn Trp Ile Leu Thr Ala Ala His Cys Phe 35 40 45 Asp Gln Pro Ile Val Lys Ser Asp Tyr Arg Ala Tyr Val Gly Leu Arg 50 55 60 Ser Ile Leu His Thr Lys Glu Asn Thr Val Gln Arg Leu Glu Leu Ser 65 70 75 80 Lys Ile Val Leu His Pro Gly Tyr Lys Pro Lys Lys Asp Pro Asp Asp 85 90 95 Ile Ala Leu Ile Lys Val Ala Lys Pro Ile Val Ile Gly Asn Tyr Ala 100 105 110 Asp Gly Ile Cys Val Pro Lys Gly Val Thr Asn Pro Glu Gly Xxx Ala 115 120 125 Thr Val Ile Gly Trp Gly Lys Ile Ser Ser Gly Gly Lys Gln Val Asn 130 135 140 Thr Leu Gln Glu Val Thr Ile Pro Ile Ile Pro Trp Lys Lys Cys Lys 145 150 155 160 Glu 161

[Brief description of drawings]

FIG. 1 shows a first-step purification chromatographic pattern of a peptide according to the present invention. Absorbance on the vertical axis is measured at 279 nm and is shown as 1.0 AuFS.

FIG. 2 shows a second-step purification chromatographic pattern of the peptide according to the present invention. Absorbance on the vertical axis is measured at 279 nm and is shown as 0.05 AuFS.

Continuation of the front page (51) Int.Cl. ⁶ Identification number Reference number within the agency FI Technical display area A61K 38/00 AAF AAM A61K 37/02 AAM (72) Inventor Yukio Nishizawa 4-6-32 Matsushiro Tsukuba, Ibaraki Prefecture

Claims (8)

[Claims] 1. A peptide comprising a part or all of the amino acid sequence of SEQ ID NO: 1 or a peptide essentially equivalent thereto. 2. A peptide comprising a part or all of the amino acid sequence of SEQ ID NO: 2 or a peptide essentially equivalent thereto. 3. A peptide containing part or all of the amino acid sequence of SEQ ID NO: 1 or a peptide essentially equivalent thereto and a peptide containing part or all of the amino acid sequence of SEQ ID NO: 2 or this A peptide in which an essentially equivalent peptide forms a double chain, a peptide consisting of a part thereof, or a peptide essentially equivalent thereto. 4. A peptide including a part or all of the amino acid sequence of SEQ ID NO: 1 or a peptide essentially equivalent thereto and a peptide including a part or all of the amino acid sequence of SEQ ID NO: 2 or the same. A peptide in which an essentially equivalent peptide forms a double chain by a disulfide bond, a peptide consisting of a part thereof, or a peptide essentially equivalent thereto. 5. The peptide having glutamate release inhibiting activity according to claim 1. 6. The peptide having glutamate release inhibiting activity according to claim 2. 7. The peptide having glutamate release inhibiting activity according to claim 3. 8. A preventive / therapeutic agent for a disease, which comprises the peptide of claim 1, claim 2 or claim 3 as an active ingredient and is effective for inhibiting glutamic acid release.