JP5344462B2 - Analgesics using sodium channel inhibitory peptides - Google Patents

Description

  The present invention relates to the use of SKTX peptides having sodium channel inhibitory activity as analgesics.

The puffer venom tetrodotoxin (TTX), which has been conventionally known as a sodium channel inhibitor, functions by inhibiting the activity of the sodium channel, and is widely used for research on sodium channels and the like. There are nine known subfamily Na _v 1.1-1.9 and low homology Nax in mammalian sodium channels. Among them, TTX is Na _v 1.1, Na _v 1.2, Na _v 1.3, It inhibits the activity of Na _v 1.4, Na _v 1.6, Na _v 1.7 (Non-patent Document 1). These TTX-acting sodium channel subfamilies are distributed in the central nervous system, peripheral nerves, and muscle tissues. Reflecting these tissue distributions, TTX affects a wide range of life activities and causes serious damage. Cause to people.
Meanwhile, some of the subfamily of sodium channels work in transmission of pain in nerve, among them _{Na v 1.3, Na v 1.8,} Na v 1.9 In the peripheral nervous system, the central role in the transmission of pain Plays. If it is possible to specifically inhibit this sodium channel subfamily, it is believed that only pain can be removed without causing damage to humans.
In particular, it has been desired to provide an analgesic that specifically acts on Na _v 1.3, which is closely related to neuropathic pain that occurs when nerves are damaged (Non-patent Document 3).
Ogata, Ohishi Y. (2002) Molecular Diversity of Structure and Function f the Voltage-Gated Na + Channels. Jpn J Pahrmacol. 88: 365-377. Craik DJ, Daly NL, Waine C. (2001) The cystine knot motif in toxins and implications for drug design.Toxicon. 39 (1): 43-60. Toshio Katsuo (2006) Basics of Pain Remedies-Basic Pain and New Pain Targets-Nihon Pharmacological Journal. 128: 321-325. Yoshimura, M., Onoyama, K. (2007) A new sibling species of the genus Strumigenys, with a redefinition of S. lewisi Cameron, pp. 664-690. In Snelling, RR, BL Fisher, and PS Ward (eds) Advances in antystematics (Hymenoptera: Formicidae): homage to EO Wilson-50 years of contributions. Memoirs of the American Entomological Institute, 80. Bosmans F, Rash, Zhu S, Diochot S, Lazdunski M, Escoubas P, Tytgat J. (2006) Four novel tarantula toxins as selective modulators of voltage-gated sodium channel subtypes. Mol Pharmacol. 69 (2): 419-29. Xiao YC, Liang SP. (2003) Purification and characterization of Hainantoxin-V, a tetrodotoxin-sensitive sodium channel inhibitor from the venom of the spider Selenocosmia hainana. Toxicon. 41 (6): 643-50. Peng K, Chen XD, Liang SP. (2001) The effect of Huwentoxin-I on Ca (2+) channels in differentiated NG108-15 cells, a patch-clamp study.Toxicon. 39 (4): 491-8. Ebbinghaus J, Legros C, Nolting A, Guette C, Celerier ML, Pongs O, Baehring R. (2004) Modulation of Kv4. 2 channels by a peptide isolated from the venom of the giant bird-eating tarantula Theraphosa leblondi. (8): 923-32. Conticello SG, Gilad Y, Avidan N, Ben-Asher E, Levy Z, and Fainzilber M. (2001) Mechanisms for evolving hypervariability: the case of conopeptides. Mol. Biol. Evol. 18 (2): 120-131. Abstracts of Annual Meeting 2008 Agricultural Chemical Society of Japan P223 Published March 5, 2008

  The present invention is intended to provide an analgesic comprising a sodium channel inhibitor peptide as an active ingredient.

The present inventors focused on a neurotoxin peptide derived from a venom producing organism (Non-patent Document 3), which has been attracting attention as a substance that inhibits the sodium channel activity of insects, and was discovered as a new species of ant in 2007. As a result of detailed examination of highly toxic neurotoxin peptides produced by Strumigenys kumadori, we succeeded in cloning three kinds of novel neurotoxin peptides (SKTX peptides). They found that these SKTX peptides markedly inhibit the activity of Drosophila sodium channel, filed a patent application (Japanese Patent Application No. 2008-130862), and reported it at the 2008 Agricultural Chemical Society of Japan (Non-patent Document 10).
As a result of intensive studies on the possibility of mammalian sodium channel inhibitory activity for these SKTX peptides, the present inventors have found that mammalian sodium plays a central role in neuropathic pain. It has been found that the activity of the channel subfamily Na _v 1.3 is inhibited, and the present invention relating to an analgesic with few side effects has been completed.

That is, the present invention is as follows.
[1] An analgesic comprising a peptide having sodium channel inhibitory activity, the peptide containing any one of the following amino acid sequences (a) to (e) as an active ingredient;
(A) any one of the amino acid sequences shown in SEQ ID NOs: 1 to 3;
(B) In the amino acid sequence of (a), an amino acid sequence in which one or several amino acid residues are deleted, substituted, or added at positions other than the six cysteine residues in the sequence,
(C) an amino acid sequence having 70% or more homology with the amino acid sequence of (a), wherein 6 cysteine residues in the sequence are conserved;
(D) an amino acid sequence described in the following formula (I),
Formula (I)
DX ₁ CX ₂ GWX ₃ X ₄ GCDPSX ₅ X ₆ GX ₇ CCX ₈ GYX ₉ CSYKYPWCRYX ₁₀ LF
(In the formula, X _{1 to} X ₁₀ each represents an arbitrary amino acid. However, X ₁ may be deleted.)
(E) an amino acid sequence encoded by any one of the nucleotide sequences shown in SEQ ID NOs: 4 to 6 or a nucleotide sequence that hybridizes with a complementary sequence under stringent conditions.
[2] In the amino acid sequence of (d), X ₁ in formula (I) is deleted or T, X ₂ is T, L or G, X ₃ is M or F, X ₄ is G or S, X ₅ is K or Q, X ₆ is T or K, X ₇ is E or Q, X ₈ is S or E, and X ₉ is V or The analgesic according to [1], which is T and X ₁₀ is D, E, or K.

Peptides having sodium channel inhibitory activity of the present invention, among the subfamily of Na _v 1.1- Na _v 1.9 and Nax least 10 types of sodium channels, the activity of the Na _v 1.3 which is considered to be related to neuropathic pain Since it inhibits, an effective analgesic action with fewer side effects can be expected.

Next, the present invention will be described in more detail.
The SKTX peptide having sodium channel inhibitory activity of the present invention is a peptide with a high insecticidal effect having sodium channel inhibitory activity obtained from the poison gland of Kitaurocoari.
The present invention, Strumigenys kumadori, is an ant belonging to the genus Urocoari of the Subfamily Phafficaria, and hunts for these insects by injecting a venom into soil insects such as flying beetles and gymkade. It has been classified as the same species as the related species Urocoari, but it was described as a new species by Masayoshi Yoshimura of Kyushu University Tropical Agricultural Research Center in 2007 because it has distinctly different morphological features. Reference 4).

The sodium channel is on the cell membrane, and permeates Na ions into the cells in response to stimulation, thereby generating and transmitting action potentials and controlling information transmission in the nervous system in the living body. The term “sodium channel inhibitory activity” includes any activity that binds to a channel molecule and inhibits the normal activity of the channel, and the sodium channel inhibitor refers to an agent having the sodium channel inhibitory activity. A typical drug having sodium channel inhibitory activity is TTX, a puffer venom. TTX inhibits the activity of Na _v 1.1, Na _v 1.2, Na _v 1.3, Na _v 1.4, Na _v 1.6, Na _v 1.7. These TTX-acting sodium channels are distributed in the central nervous system, peripheral nerves, and muscle tissues. Reflecting these tissue distributions, TTX affects a wide range of biological tissue activities and causes serious disabilities. To cause.
On the other hand, the SKTX peptide of the present invention acts on Na _v 1.3, which works mainly on the nervous system related to pain sensation, among mammalian sodium channels, and therefore is expected to have a low risk of causing serious damage. Is done.

The peptide having a sodium channel inhibitory activity (SKTX peptide) in the present invention includes not only SKTX 1 to 3 peptide (SKTX peptide) but also sodium in the venom of 35 or 36 amino acids shown in SEQ ID NOs: 1 to 3. This includes a case where a part of the amino acid is deleted, substituted with another amino acid, or has an amino acid sequence inserted or added to such an extent that the channel inhibitory activity is not lost. At that time, the six cysteine residues common to other biotoxins having sodium channel inhibitory activity need to be conserved.
Specifically, it is a peptide having the following sodium channel inhibitory activity, and can be expressed as an isolated peptide containing an amino acid sequence such as (a) to (d) below. However, in any peptide, six cysteine residues in the amino acid sequence of the SKTX mature peptide include an amino acid sequence that is not changed.
(A) An amino acid sequence corresponding to the mature peptide of SKTX1 to 3 shown in SEQ ID NOs: 1 to 3.
(B) In the amino acid sequence of (a) above, one or several amino acid residues other than the cysteine residues at 6 positions are deleted, that is, 1 to 5, preferably 1 to 3, more preferably 1 amino acid residue. A substituted amino acid sequence, or 1 to 5, preferably 1 to 3, more preferably 1 amino acid residue inserted in the amino acid sequence, or 1 to 100 amino acid residues in the amino acid sequence, An amino acid sequence to which 1 to 30, more preferably 1 to 5 amino acid residues are added.
(C) 70% or more, preferably 80% or more, more preferably 85% or more, more preferably 90% or more, most preferably 95% or more homology with the amino acid sequence shown in any one of SEQ ID NOs: 1 to 3 Amino acid sequence having sex. The homology herein refers to “identity” calculated by the homology calculation method (FIG. 3) by the Clastal W method (analysis software DNASTAR Version 5.0 (Lasergene)).
(D) Here, the amino acid sequences of SKTX1 to 3 peptides represented by SEQ ID NOs: 1 to 3 have many common sequences other than 6 cysteine residues, and have 72% homology.
Therefore, a typical SKTX peptide in the present invention can be represented as a one-letter amino acid sequence represented by the following formula (I).

Formula (I)
DX ₁ CX ₂ GWX ₃ X ₄ GCDPSX ₅ X ₆ GX ₇ CCX ₈ GYX ₉ CSYKYPWCRYX ₁₀ LF

In the formula (I), X _{1 to} X ₁₀ may be any amino acid, and ₁ to 3 of them may be deleted. Preferably, X ₁ in formula (I) is missing or T, X ₂ is T, L or G, X ₃ is M or F, X ₄ is G or S, X ₅ is K or Q, X ₆ is T or K, X ₇ is E or Q, X ₈ is S or E, X ₉ is V or T, X ₁₀ is D, E or K. (SEQ ID NOs: 7, 8)
(E) Along with an amino acid sequence including the amino acid sequence (SEQ ID NO: 1 to 3) encoded by the base sequence shown in SEQ ID NO: 4 to 6, a complementary sequence and stringent of the base sequence shown in SEQ ID NO: 4 Amino acid sequence encoded by a base sequence that hybridizes under various conditions. Here, examples of stringent conditions include 6M urea, 0.4% SDS, 0.5 × SSC conditions, or hybridization conditions with stringency equivalent thereto. Isolation of DNA with higher homology can be expected by using conditions with higher stringency, for example, conditions of 6M urea, 0.4% SDS, and 0.1 × SSC.

In the present invention, the nucleic acid capable of expressing a peptide having sodium channel inhibitory activity and having six cysteine residues as a recombinant peptide is a DNA encoding a mature SKTX peptide shown in SEQ ID NOs: 4 to 6, It includes not only DNA encoding the SKTX peptide precursor in SEQ ID NO: 13, 15 or 17, but also nucleic acids containing the following base sequences (a) to (g). However, the expressed recombinant peptide is a peptide in which six cysteine residues common to SEQ ID NOs: 1 to 3 are conserved.
(A) a nucleic acid comprising the base sequence shown in SEQ ID NOs: 4 to 6, a nucleic acid encoding a SKTX peptide precursor in SEQ ID NOs: 13, 15, and 17 (positions 70 to 246 in SEQ ID NO: 13, 82 to SEQ ID NO: 15) 267, corresponding to positions 2 to 178 of SEQ ID NO: 17), or a nucleic acid contained in the nucleotide sequence shown in SEQ ID NO: 13, 15, 17 and having a reading frame aligned with SEQ ID NOs: 4-6 .
(B) a base sequence that hybridizes with a complementary sequence of the base sequence of (a) under stringent conditions;
(C) A nucleotide sequence having a homology of 70% or more, preferably 80% or more, more preferably 85% or more, still more preferably 90% or more, and most preferably 95% or more with the nucleotide sequence of (a). Here, the homology means “identity” calculated by the homology calculation method by the Clastal W method (analysis software DNASTAR Version 5.0 (Lasergene)).
(D) A base sequence encoding the amino acid sequence of the SKTX mature peptide shown in SEQ ID NOs: 1 to 3, or a base sequence encoding the SKTX precursor peptide shown in SEQ ID NOs: 14, 16, and 18.
(E) an amino acid sequence in which one or several amino acid residues are deleted, substituted, or added at positions other than the six cysteine residues in the amino acid sequence shown in SEQ ID NOs: 1 to 3; The base sequence to encode. Here, the amino acid sequence in which one or several amino acid residues are deleted / substituted / added is 1 to 5, preferably 1 to 3, more preferably 1 deleted / substituted / inserted. An array. Alternatively, it refers to an amino acid sequence in which 1 to 100, preferably 1 to 30, more preferably 1 to 5 amino acid residues are added to the amino acid sequence shown in SEQ ID NOs: 1 to 3.
(F) A base sequence encoding an amino acid sequence having 70% or more homology with the amino acid sequence shown in SEQ ID NOs: 1 to 3 and conserving six cysteine residues in the sequence.
(G) a base sequence encoding the amino acid sequence described in the following formula (I),
Formula (I)
DX ₁ CX ₂ GWX ₃ X ₄ GCDPSX ₅ X ₆ GX ₇ CCX ₈ GYX ₉ CSYKYPWCRYX ₁₀ LF
(In the formula, X _{1 to} X ₁₀ each represents an arbitrary amino acid, provided that X ₁ may be deleted).

Using an expression vector containing these nucleic acids, the SKTX1-3 peptide or a similar peptide can be expressed alone or as a fusion recombinant protein in a transformed host, but depending on the host selected, the mature peptide A corresponding nucleotide sequence or a nucleotide sequence corresponding to a precursor peptide containing a signal sequence can be used.
And these nucleic acid sequences or their complementary sequences, or nucleic acids consisting of partial sequences containing 15 or more base sequences, preferably 17 or more, more preferably 20 or more consecutive base sequences in these sequences are SKTX1-3 peptides and It can be used as a probe or primer for obtaining a nucleic acid encoding a peptide having similar sodium channel inhibitory activity. In particular, as a primer, a base sequence corresponding to a non-coding region on the 5 ′ side or 3 ′ side is also usefully used.

  The peptide of the present invention can be chemically synthesized, but can be obtained as a recombinant peptide from a transformed host using a gene recombination technique. In this case, as a host / vector system to be used, any ordinary host / vector system using Escherichia coli, insect cells, plant cells, or mammalian cells as hosts can be used. For example, although mass production is possible using an E. coli host, mass production is further possible particularly by using a cold shock system (Takara Bio Inc.). Further, it can be obtained as a fusion protein. For example, if expressed as a fusion protein (GE Healthcare) with glutathione S transferase, a large amount of peptides with a highly accurate three-dimensional structure can be obtained. In that case, depending on the host to be expressed, either DNA encoding a mature peptide or DNA containing a signal sequence can be used as appropriate.

When the present invention is used as an analgesic, it may be used as a purified peptide, an unpurified peptide, or a mixed peptide of a plurality of types, and may be used in combination with other known analgesics. In this case, the dosage form of the SKTX peptide or a pharmacologically acceptable salt thereof is preferably a parenteral dosage form such as an injection, a suppository, a coating, or a patch. These preparations are produced by known methods using additives such as excipients, binders, stabilizers, and diluents. An appropriate amount may be dissolved in a normal aqueous solvent containing ethanol (eg 0.00001-10mg / ml) and applied to the skin surface, or the aqueous peptide solution may be administered by inserting a catheter directly under the spinal cord. Can do.
The amount of the SKTX peptide of the present invention and a pharmacologically acceptable salt thereof varies depending on symptoms, age, administration method, etc., for example, 0.00001-100 mg / kg, preferably 0.001-10 mg / kg per day for an adult. It is desirable to administer the dose according to the symptoms in several or several times.

Hereinafter, the present invention will be specifically described with reference to production examples, but the present invention is not limited thereto.
Unless otherwise specified, the details of the genetic engineering method were in accordance with the method described in Molecular Cloning 2nd edition. Cold Spring Harvor Laboratory Press, Cold Spring Harvor, NY. (1989).

(Production Example 1) Isolation of cDNA Encoding Kitaurocoli-Derived Bioactive Peptide SKTX1 (1-1) Production of Synthetic Primer Common to Kitaurocoari Derived and Bioactive Peptide The amino acid sequences of bioactive peptides Oxyopinins from a species of Oxyopes kitabensis were compared. A base sequence corresponding to an amino acid sequence having high homology among these sequences and close to the action site of the signal peptide was synthesized as a primer Pilosulin / Oxyopinin (5′-ACYTTNGGIAGIACYTT-3 ′) (SEQ ID NO: 9).

(1-2) Isolation of RNA Total RNA was extracted from Strumigenys kumadori using TRIZOL LS solution (GIBCO BRL) according to the method of use. From this total RNA, poly A ⁺ RNA was purified using Oligotex-dT30 (Super) (Takara Bio Inc.) according to the method of use.

(1-3) Construction of cDNA Library Using the thus obtained poly A ⁺ RNA as a template, a cDNA synthesis kit (Stratagene) was used to obtain a double-stranded cDNA mixture. An EcoR I adapter (Takara Bio Inc.) was added thereto, phosphorylated with polynucleotide kinase, treated with restriction enzyme with Xho I, and then a 0.8 to 2 kb DNA fragment was extracted. The DNA fragment thus obtained was placed on the EcoR I and Xho I sites of the pSD64TR vector (Nucleic Acids Res., 12: 7057 (1984) and Arch. Biochem. Biophys., 428 (2): 170-178 (2004)). Inserted. This was subsequently used as a cDNA library.

(1-4) Isolation of SKTX1-3 cDNA By PCR reaction using the cDNA library obtained in (1-1) as a template, primer Pilosulin / Oxyopinin and primer SDA (5'-TTATGTAGCTTAGAGACT-3 ') (sequence) The DNA fragment was amplified using No. 10). The amplified DNA fragment was subcloned using the cloning vector pCR2.1 (invitrogen). After determining the base sequence of the partial length cDNA and comparing it with a known cDNA base sequence, one encoding a novel peptide was selected. In order to isolate the full-length cDNA of the new peptide, a primer conoR (5'-TTAAAACAGGTCATAGCG-3 ') (SEQ ID NO: 11) is designed from a part of the base sequence of the partial-length cDNA of the new peptide, and the primer and vector primer Using SP6 (5′-TATTTAGGTGACACTATAG-3 ′) (SEQ ID NO: 12), PCR was performed again using the same cDNA library as a template. After obtaining the full-length cDNA, the entire nucleotide sequence was determined, and this clone was named SKTX1, SKTX2, and SKTX3.
The DNA base sequence was reacted with the BigDye Terminator Cycle Sequencing FS Ready Reaction Kit (Applied Biosystems) and SP6 primer, and analyzed with a fluorescence automatic sequencer ABI Sequencer377 (Applied Biosystems).
The base sequence of SKTX1 cDNA isolated and sequenced as described above and the amino acid sequence deduced from the base sequence are shown in FIG. (SKTX1 corresponds to SEQ ID NOS: 13, 14, and 1, 4. SKTX2 corresponds to SEQ ID NOS: 15, 16, and 2, 5. SKTX3 corresponds to SEQ ID NOS: 17, 18, and 3, 6.)
The mature peptide encoded by SKTX1-3 (Fig. 2) is a homology calculation method using the Clastal W method (analysis software DNASTAR Version 5.0). The spider neurotoxin CcoTX (Non-patent Document 5), HNTX (Non-patent Document) 6), HWTX (Non-patent document 7), TLTX (Non-patent document 8), conotoxin of mussels (Non-patent document 9), but the homology is limited to cysteine residues, It was found that this is a new type of neurotoxin peptide that shows only 30% to 50% homology at the amino acid sequence level of the mature peptide (FIG. 3).

(Production Example 2) Expression / Purification of Recombinant SKTX1-3 Peptide SKTX1-3 peptide comprising the amino acid sequence shown in SEQ ID NOs: 1 to 3 is obtained by using a cold shock system of E. coli (Takara Bio Inc.), and It was expressed in Escherichia coli Rossetta Gami2 (Merck) using two types of methods, a fusion protein with glutathione S-transferase (GE Healthcare Biosciences). In either case, the fusion protein is purified, treated with the protease PreScission ^™ (GE Healthcare Biosciences), and eluted with 40% acetonitrile containing 0.1% trifluoroacetic acid with SepPakC18 to produce recombinant SKTX1 peptide. Was purified. This was freeze-dried and then dissolved in distilled water.

(2-1) When SKTX1-3 peptide is expressed using E. coli cold shock system
The base sequence (SEQ ID NO: 4-6) encoding the mature peptide of SKTX1-3 cDNA was connected to the BamHI site together with the pCOLD II (Takara Bio Inc.) histidine hexamer sequence and the PreScission ^TM recognition sequence. SKTX1-3 were created. This was co-transformed with E. coli and pGro7 (Takara Bio Inc.), which has an E. coli chaperonin sequence in BL21. This transformed Escherichia coli was cultured in LB (containing 0.02% arabinose) medium at 37 ° C for 3 hours, then IPTG was added to reduce the temperature to 15 ° C to 1 µM, and the E. coli was cultured overnight. did. Escherichia coli was recovered from this culture solution, and the recombinant peptide was purified using Ni-NTA (Qiagen) and SepPakC18 (Waters). Since this peptide had the sequence of histidine hexamer and the recognition sequence of PreScission ^™ on the N-terminal side, it was reacted overnight 4 times using the protease PreScission ^™ to obtain the SKTX1 peptide having no extra sequence. Protease treatment was able to obtain the SKTX1 peptide without the same extra sequence even when the reaction was carried out at 37 degrees for 1 hour using Tagzyme (Qiagen).

(2-2) When SKTX1-3 peptides are expressed using the E. coli expression vector pGEX
Between the BamH I site and EcoR I site of pGEX-2T (GE Healthcare Bioscience Co., Ltd.), together with the PreScission ^TM recognition sequence, the base sequence encoding the mature peptide of SKTX1-3 cDNA (SEQ ID NOs: 4-6) PGEX-SKTX1 was created. This was transformed into E. coli Rossetta Gami2. This transformed Escherichia coli was cultured in LB medium at 37 ° C. for 3 hours, IPTG was added to 1 μM by lowering the temperature to 20 ° C., and E. coli was cultured overnight. Escherichia coli was recovered from this culture solution, and the recombinant peptide was purified using glutathione sepharose (GE Healthcare Biosciences) and SepPakC18 (Waters). Since this peptide had a glutathione S transferase and PreScission ^™ recognition sequence on the N-terminal side, it was reacted overnight four times using the protease PreScission ^™ to obtain SKTX1-3 peptides having no extra sequence.

(Examples) Sodium Channel / Inhibitory Activity of SKTX1-3 Peptide A conventional double-membranous voltage fixation method using Xenopus oocytes is described in, for example, “Ion Channels. Edited by: Bernardo Rudy, Methods in Enzymology Volume 207, Pages 3-917 (1992) ".
A typical method is a method of electrophysiological analysis of ion channel activity according to the following method (FIG. 4).
(1) Synthesize ion channel cRNA,
(2) Injecting the cRNA into the oocyte to express the channel molecule on the cell membrane surface,
(3) Two to four days later, two microelectrodes are inserted into an oocyte, and the outflow or inflow of ions induced by changing the voltage inside or outside the membrane is replaced with a change in current amount and measured. .
In this example, rat sodium channel α subunit Na _v 1.3 was used as the ion channel cRNA in the step (1) of the above method. In step (3), the SKTX1 peptide obtained in (Preparation Example 2) was added, and the activity of the ion channel was measured after 5 minutes.
This result was compared with the activity of the ion channel without addition of SKTX1-3 peptide. As a result, SKTX1 peptide almost completely inhibited rat sodium channel activity at 1 μM (FIG. 4A). At this time, the voltage dependence of the sodium channel was not changed (FIG. 4B).

(Reference Example) Actions of SKTX1-3 peptides on phlebet crickets Insecticidal activity using conventional phleet crickets is “Scorpion toxins affecting insects: Loret. Are described in detail.
The typical method is
(1) Use a 10 μl Hamilton syringe between the middle and hind limbs (average 0.2 g)
Inject 80 μM peptide solution dissolved in 5 μl PBS,
(2) A method of observing changes in insects after 5, 15, 60 minutes and 18 hours.
In the reference example, the insecticidal activity of the peptide of the present invention was evaluated according to this method. As a result, SKTX1 exhibited a paralytic action in 50% crickets, and the paralysis persisted for 30 minutes or more. In SKTX2, 60% crickets were paralyzed and the paralysis persisted for over 18 hours.
Therefore, the insecticidal activity of the peptides of the present invention was demonstrated.

SKTX cDNA base sequence and amino acid sequence A: SKTX1 (positions 25-59 are mature peptides) B: SKTX2 (positions 27-62 are mature peptides) C: SKTX3 (positions 25-59 are mature peptides) Amino acid sequence of mature peptide of SKTX (It is presumed that the 1-4th, 2-5th and 3-6th cysteine residues are cross-linked by disulfide bonds.) A: SKTX1, B: SKTX2, C : SKTX3 Comparison with SKTX peptide and homologous toxic peptide: SKTX shows about 30-50% homology with spider venom, conotoxin and the like. It is known that peptides with ICK have significantly different physiological activities due to subtle differences in amino acid sequences. An asterisk indicates C-terminal amidation. Inhibitory activity of SKTX1 peptide on rat sodium channel A: Concentration-dependent change B: Voltage-dependent change Reference figure 1: Drosophila sodium channel inhibitory activity of SKTX1 peptide A: Concentration-dependent change B: Voltage-dependent change Reference figure 2: Drosophila sodium channel inhibitory activity of SKTX2 peptide A: Concentration-dependent change B: Voltage-dependent change Reference figure 3: Drosophila sodium channel inhibitory activity of SKTX3 peptide A: Concentration-dependent change B: Voltage-dependent change

Claims (2)

A sodium channel Na _v 1.3 activity inhibitor comprising a peptide having sodium channel inhibitory activity, the peptide containing any one of the following amino acid sequences (a) to (e) as an active ingredient;
(A) any one of the amino acid sequences shown in SEQ ID NOs: 1 to 3;
(B) In the amino acid sequence of (a), an amino acid sequence in which one or several amino acid residues are deleted, substituted, or added at positions other than the six cysteine residues in the sequence,
(C) an amino acid sequence having 90% or more homology with the amino acid sequence of (a) , wherein 6 cysteine residues in the amino acid sequence of (a) are conserved;
(D) an amino acid sequence described in the following formula (I),
Formula (I)
DX ₁ CX ₂ GWX ₃ X ₄ GCDPSX ₅ X ₆ GX ₇ CCX ₈ GYX ₉ CSYKYPWCRYX ₁₀ LF
(In the formula, X _{1 to} X ₁₀ each represents an arbitrary amino acid. However, X ₁ may be deleted.)
(E) any one of the nucleotide sequences shown in SEQ ID NOs: 4 to 6, or an amino acid sequence encoded by a nucleotide sequence that hybridizes with a complementary sequence under stringent conditions , and the amino acid of (a) An amino acid sequence in which 6 cysteine residues in the sequence are conserved . In the amino acid sequence of (d), X ₁ in formula (I) is deleted or T, X ₂ is T, L or G, X ₃ is M or F, and X ₄ is G or S, X ₅ is K or Q, X ₆ is T or K, X ₇ is E or Q, X ₈ is S or E, and X ₉ is V or T The sodium channel Na _v 1.3 activity inhibitor according to claim 1, wherein X ₁₀ is D, E or K.