JP2000166572A - Peptide having neurotransmission inhibitory action - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a new peptide, a peptide having a specific amino acid sequence and having neurotransmission inhibitory action, useful as e.g. a preventive/therapeutic agent for cerebroneural diseases by isolation from the venom of a kind of scorpion, called CTX. SOLUTION: This new peptide is such one as to have an amino acid sequence of the formula and have neurotransmission inhibitory action, being useful as e.g. a preventive/therapeutic agent for cerebroneural diseases because of being effective in inhibiting neurotransmission. This new peptide is obtained by the following procedure: a dried product of CTX crude venom prepared from the venom of the above kind of scorpion is dissolved in water, the resulting aqueous solution is subjected to ion exchange chromatography to effect elution with varied pH or salt concentration of a buffer solution for elution; subsequently, the resulting elute is subjected to reversed-phase chromatography using a silica gel support modified with hydrophobic groups to effect elution through concentration gradient elution method using a hydrophilic solvent such as acetonitrile to carry out separation and purification.

Description

DETAILED DESCRIPTION OF THE INVENTION

TECHNICAL FIELD [0001] The present invention relates to a novel peptide having a nerve conduction inhibitory action and useful as a medicament.

[0001]

2. Description of the Related Art In recent years, it has been considered that various nerve diseases are associated with excessive nerve excitation, and in particular, as one of the main causes of nerve cell death and chronic pain after cerebral ischemia. Has been caught. In addition, it has been clarified that sodium channels and potassium channels are involved in nerve conduction transmitting nerve excitation. Therefore, a substance that acts on such an ion channel and blocks nerve conduction is considered to be extremely promising as an agent for preventing or treating the above diseases. In fact, some of these substances with nerve conduction inhibitory activity are used as local anesthetics and are currently being developed as neuroprotective agents for cerebral ischemia and head injury [CP Taylor et al. , Trends in Pharmacol.
Sci., 16 , 309-316 (1995)]. In 1973 McIntosh et al.
Scorpion as a peptide inhibitor [Centruroides sculp
venom from Turatus (Spider Pharm)]
Abbreviations), the amino acid sequences of multiple bioactive peptides represented by Toxin I have been determined [McIntosh
et al., Toxins of Animal and Plant Origin, III , 5
29 (1973)]. Subsequent reports by Wang et al. Have shown that these peptides act on neural sodium channels and nerve conduction [GK Wang et a
l., Mol. Pharmacol., 23 , 519-533 (1983)].

[0002]

DISCLOSURE OF THE INVENTION The present inventors have conducted detailed studies on substances contained in CTX which have an effect on nerve conduction, and as a result, have found a novel peptide.
Therefore, an object of the present invention is to provide a novel peptide which is useful for treating and preventing a disease for which it is effective to have inhibition of nerve conduction.

[0003]

Means for Solving the Problems The present inventors tried to separate and purify an active substance from crude venom CTX which affects nerve conduction, and analyzed the structure of the active substance. as a result,
A novel bioactive peptide that markedly inhibits nerve conduction was found in CTX. The present inventors further analyzed the structure of the peptide, determined the amino acid sequence, and completed the present invention. That is, the present invention relates to a peptide having the amino acid sequence of any one of SEQ ID NOs: 1 to 4, which suppresses neurotransmission, and to the deletion of one or several amino acids in the amino acid sequence of any of SEQ ID NOs: 1 to 4. It is intended to provide a peptide having an amino acid sequence which has been lost, substituted or added and which has an activity of suppressing neurotransmission. Further, the present invention provides a DN encoding the peptide.
A, a vector into which the DNA has been inserted, a transformant retaining the DNA so that it can be expressed, a method for producing the peptide including a step of culturing the transformant, and a medicament containing the peptide as an active ingredient. To provide. The peptide according to the present invention can be separated and purified from CTX by a known method, similarly to the Mono-S column, Vydac C18, and the like described in Examples below. For example, the target active substance can be isolated and purified by changing the pH or salt concentration of the elution buffer by ion exchange chromatography using an ion exchange carrier having a sulfonic acid group or a quaternary ammonium group. As the eluate in this case, all that can be used in this field are usually used,
Examples include acetate buffer, phosphate buffer, Tris buffer, ammonium formate, ammonium bicarbonate, and the like. In addition, the target substance can be obtained by performing a gradient elution with a hydrophilic organic solvent such as ethanol, propyl alcohol, or acetonitrile by reverse phase chromatography using a silica gel carrier modified with a hydrophobic group. Furthermore, purification can be performed using gel filtration chromatography, hydrophobic chromatography, isoelectric point chromatography, or the like. As described above, the peptide of the present invention can be produced by separating and purifying CTX as a raw material. Further, the peptide according to the present invention can be obtained by using a conventionally used peptide chemical synthesis technique. In this case, a modified amino acid may be inserted for the purpose of metabolic stabilization. Furthermore, the peptide according to the present invention can also be produced using a gene recombination technique. For example, a DNA designed to encode the peptide is inserted into an expression vector suitable for the host cell in which the peptide is to be produced. The host cell into which the vector of the present invention is introduced is not particularly limited as long as it is a cell compatible with the vector of the present invention, and includes various animal cells, bacteria, yeast, insect cells and the like. At this time, it is permissible to add a sequence other than that encoding the peptide, such as expression as a fusion protein or addition of a signal for secretion to facilitate the production of the peptide. Further, a gene for drug resistance or the like may be inserted into the expression vector in order to facilitate selection of a transformant or to improve production of the peptide. The expression vector is introduced into a microorganism or cell to obtain a transformant.
Introduction of a vector into a host cell is described, for example, in the literature (Sambru
ck, J., Fritsch, EF, and Maniatis, T. (1989) Molecular
Cloning: A Laboratory, Manual, Cold Spring Harbor La
boratory, Cold Spring Harbor, NY). Then, the transformant is cultured, and the peptide can be purified from the transformant or from the culture supernatant according to the method described above. When the protein of the present invention is expressed as a fusion protein with GST or Hisb, it can be purified using a glutathione sepharose column and a nickel sepharose column, respectively.

[0004] Tracking of the target fraction in separation / purification may be performed using an extracted nerve fiber specimen and using the nerve conduction inhibitory activity as an index. That is, as shown in the examples below, in a nerve conduction model using bullfrog sciatic nerve, the nerve conduction inhibitory action when a purified fraction is added may be recorded and observed electrically. Structural analysis of the peptide of the present invention can be performed by a usual method. For example, using an amino acid sequence automatic analyzer (protein sequencer), sequential decomposition from the N-terminal by Edman degradation,
It can be performed by combining amino acid sequence analysis of an enzyme digested peptide and C-terminal amino acid analysis using carboxypeptidase. The results are shown in SEQ ID NOs: 1, 2, 3 and 4. In many cases, peptides retain their activity even when other amino acids or modified amino acids are deleted, added, or substituted in addition to sites essential for their activity, and this is used to make other properties necessary for pharmaceuticals. (E.g., metabolic profile)
It is often done. Therefore, a peptide having a neurotransmission inhibiting activity among the converted peptides or modified peptides prepared by partially deleting, adding, or substituting amino acids based on the present sequence is claimed in the present invention. It is included in the range.

[0005]

EXAMPLES The present invention will be described in more detail with reference to the following Examples, but it should not be construed that the present invention is limited thereto. Example 1 Purification of Peptide According to the Present Invention A dried product of crude CTX toxin was dissolved in water, and this was subjected to ion exchange chromatography, and a Mono-S column (inner diameter) equilibrated with 10 mM ammonium acetate (pH 3.5) was used. 5mmX length 50m
m). Elution was performed with ammonium acetate and 500
The active component was separated and eluted by a concentration gradient elution method using mM ammonium acetate (pH 6.95). FIG. 1 shown below shows the elution pattern, in which the horizontal axis shows the elapsed time after the concentration gradient elution, and the vertical axis shows the absorbance. Fractions 1, 2 and 3 in the blacked-out portions showed remarkable nerve conduction inhibitory activity. Each of these three fractions was subjected to a second stage purification as described below. Fraction 1 was subjected to purification by reverse phase chromatography, and an octadecyl 4PW column (4.6 mm ID × 150 mm length, equilibrated with 67 mM phosphate buffer (pH 7.4))
(Tosoh), and the components were eluted by a concentration gradient elution method using the same solution and acetonitrile. As shown by the elution pattern in FIG. 2, a fraction A having activity was obtained. The molecular weight of this fraction was measured using a laser ionization time-of-flight mass spectrometer, and it was found that the fraction contained a single peptide having a molecular weight of about 7467 daltons. Fraction 2
Was also purified by reverse phase chromatography,
After adsorbing on a Vydac C18 column (4.6 mm ID x 250 mm length, manufactured by Separation Science) equilibrated with a 0.1% trifluoroacetic acid aqueous solution, the components were eluted by a concentration gradient elution method using the same solution and acetonitrile. FIG. 3 shows the elution pattern. As the active fractions, two fractions, fraction B and fraction C, were obtained. The measurement of both fractions by a mass spectrometer was carried out in the same manner as above, and it was confirmed that fraction B contained a single peptide having a molecular weight of about 7358 dalton and fraction C contained a single peptide having a molecular weight of about 7138 dalton. Fraction 3 was also subjected to reverse phase chromatography,
The solution was adsorbed on a Vydac C18 column (4.6 mm ID x 250 mm length) equilibrated with a 0.1% trifluoroacetic acid aqueous solution, and each component was eluted by a concentration gradient elution method using the same solution and acetonitrile. As shown by the elution pattern in FIG. 4, fraction D was activated. As a result of measuring the molecular weight of this fraction, a single peptide having a molecular weight of about 6704 dalton was contained.

Example 2 Determination of Structure Regarding the four fractions A, B, C and D obtained by the above-mentioned purification, an equivalent of 500 pmol was applied to a PSQ-1 type protein sequencer (Shimadzu Corporation) to give amino acids. The amino acid sequence of the terminal region was analyzed. As a result, fraction A is SEQ ID NO: 1,
It was identified that fraction B had an amino acid sequence corresponding to SEQ ID NO: 2, fraction C had an amino acid sequence corresponding to SEQ ID NO: 3, and fraction D had an amino acid sequence corresponding to SEQ ID NO: 4. As described above, the peptide of the present invention was isolated and purified and its structure was determined, and its main body was clarified.

Example 3 Measurement of nerve conduction i) Experimental method In order to examine the effect of the peptide of the present invention on nerve conduction, a paper [J. Kobayashi et al., Neurosci.
t., 162 , 93-96 (1993)] with reference to the following method. Ringer's solution used for the experiment [Composition (mM): NaCl 11
2, KCl 2, CaCl ₂ 2, HEPES 10, pH 7.4] is 9
The mixture was sufficiently saturated with a 5% O ₂ + 5% CO ₂ mixed gas before use. The bullfrog was allowed to stand in ice water for 5 minutes or more, and anesthetized with ice, and the sciatic nerve was immediately removed. The removed sciatic nerve was incubated in Ringer's solution for 1 hour or more. This was transferred to a measurement chamber, and platinum electrodes were set at both ends of the nerve. One side was used as a stimulating electrode, and a square wave stimulus of 1 msec · 1 mA was applied before and after the addition of the sample, and the other was used as a recording electrode and recorded on a computer (Macintosh Quadra840AV) from an oscilloscope via an A / D converter (CED1401plus). The sample was added to the Ringer's solution in the measurement chamber, incubated at room temperature for 10 minutes, and then stimulated with a square wave to observe the nerve conduction inhibitory activity. Experimental Results By applying electrical stimulation, nerve conduction is observed as a change in potential as shown in FIG. When fraction A containing the peptide of SEQ ID NO: 1 according to the present invention was added thereto, the size of the waveform was reduced. It is thought that this inhibits nerve conduction and suppresses nerve excitation. When fraction B containing the peptide of SEQ ID NO: 2 was added, a burst-like fine waveform was observed as shown in FIG. Similar waveforms were observed for the fraction C containing the peptide of SEQ ID NO: 3 and the fraction D containing the peptide of SEQ ID NO: 4. It is thought that these peptides inhibit normal conduction of nerve excitation by inducing fine nerve conduction in this way, resulting in suppression of excessive nerve excitation. Based on the above results, the peptide according to the present invention can be used for the prevention and treatment of diseases effective in affecting nerve conduction. To give a concrete example,
Prevention and treatment of cerebral nerve diseases typified by acute encephalopathy occurring in diseased bodies such as cerebral ischemia and status epilepticus, chronic diseases such as Alzheimer's disease and Huntington's disease, and abnormal sensory treatments such as acute and chronic pain In addition to being able to be used as an agent, it can be said that it is also very useful as a drug for elucidating cerebral nerve physiology.

[0008]

[Sequence List] <110> Eisai Co., Ltd. <120> Peptides which inhibit neurotransmission <130> EP99TH0801 <150> JP Japanese Patent Application No. 10-276275 <151> 1998-9-30 <160> 4 <170> PatentIn Ver. 2.0 <210> 1 <211> 69 <212> PRT <213> Centruroides sculpturatus <400> 1 Lys Leu Arg Asp Gly Tyr Pro Leu Ala Ser Asn Gly Cys Lys Phe Gly 1 5 10 15 Cys Ser Gly Leu Gly Glu Asn Asn Pro Thr Cys Asn His Val Cys Glu 20 25 30 Lys Lys Ala Gly Ser Asp Tyr Gly Tyr Cys Tyr Trp Trp Thr Cys Tyr 35 40 45 Cys Glu His Val Ala Glu Gly Thr Val Leu Trp Gly Asp Ser Gly Thr 50 55 60 Gly Pro Cys Lys Ser 65 69 <210> 2 <211> 40 <212> PRT <213> Centruroides sculpturatus <400> 2 Lys Asp Gly Tyr Leu Val Asn Lys Lys Thr Gly Cys Lys Tyr Gly Cys 1 5 10 15 Trp Asp Ser Lys Asp Asn Lys Tyr Cys Asn Lys Glu Cys Gln Ala Lys 20 25 30 Asn Gln Gly Gly Thr Tyr Gly Tyr 35 40 <210> 3 <211> 40 <212> PRT <213> Centruroides sculpturatus <400> 3 Lys Asp Gly Tyr Pro Leu Gly Arg Glu Gln Cys The Ile Pro Cys Leu 1 5 10 15 Phe Asp Asn Asp Phe Cys Asn Arg Lys Cys Val Glu Leu Lys Gly Lys 20 25 30 Ser Gly Tyr Cys Gln Phe Trp Lys 35 40 <210> 4 <211> 40 <212> PRT <213> Centruroides sculpturatus <400> 4 Lys His Gly Tyr Pro Val Asp Ser Lys Gly Cys Lys Leu Ser Cys Val 1 5 10 15 Ala His Asn Tyr Cys Asp Asp Glu Cys Leu Lys Arg Lys Ala Ser Gly 20 25 30 Gly Tyr Cys Lys Asp Met Ser Ser 35 40

[0009]

[Brief description of the drawings]

FIG. 1. Purification of CTX crude poison by ion exchange chromatography

FIG. 2. Purification of fraction 1 by reverse phase chromatography

FIG. 3. Purification of fraction 2 by reverse phase chromatography

FIG. 4. Purification of fraction 3 by reverse phase chromatography

FIG. 5 shows changes in nerve conduction when a peptide represented by SEQ ID NO: 1 (fraction A) is added.

FIG. 6 shows changes in nerve conduction when a peptide represented by SEQ ID NO: 2 (fraction B) is added.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) // A61K 35/56 A61K 37/02 AED

Claims (10)

[Claims] 1. A peptide having the amino acid sequence of SEQ ID NO: 1. 2. A peptide having the amino acid sequence of SEQ ID NO: 2. 3. A peptide having the amino acid sequence of SEQ ID NO: 3. 4. A peptide having the amino acid sequence of SEQ ID NO: 4. 5. A peptide having an amino acid sequence according to claim 1 wherein one or several amino acids have been deleted, substituted or added, and having an activity of suppressing neurotransmission. 6. A DNA having a nucleotide sequence encoding the peptide according to any one of claims 1 to 5. A vector into which the DNA according to claim 6 has been inserted. (8) a transformant which retains the DNA of (6) in an expressible manner; 9. A method for producing a peptide according to any one of claims 1 to 5, comprising a step of culturing the transformant according to claim 8. 10. A medicament comprising a peptide containing the amino acid sequence according to any one of claims 1 to 5 as an active ingredient.