KR102553070B1 - Peptide with voltage-gated potassium ion channel inhibitory activity from Carybdea brevipedalia - Google Patents

Abstract

The present invention relates to a ShK domain-like peptide derived from the venom of the small box jellyfish ( Carybdea brevipedalia ). It can be usefully used for the prevention or treatment of autoimmune diseases by inhibiting it.

Description

Peptide with voltage-gated potassium ion channel inhibitory activity from Carybdea brevipedalia}

The present invention relates to a ShK domain-like peptide derived from the venom of Carybdea brevipedalia .

A voltage-gated potassium ion channel is a membrane protein that exists in the cell membrane and opens and closes according to the membrane voltage to pass potassium. It mainly exists in the membrane of excitable cells such as nerve cells and muscle cells and extinguishes the action voltage.

In human T cells, the Kv1.3 potassium ion channel plays an essential role in regulating resting membrane potential and Ca ²⁺ signaling. When autoimmune disease-related CCR7 effector memory T cells are activated in inflammatory sites, the expression of Kv1.3 channels in these cells greatly increases from 250 channels per cell to about 1,500 to 2,000 channels. This increase in Kv1.3 channel protein does not occur in central memory T cells or naive T cells in lymphoid organs. These findings suggest that selective inhibition of responder memory T cells with specific Kv1.3 inhibitors can effectively suppress the immune response to alleviate disease. In addition, the therapeutic effect of Kv1.3 channel blockade was confirmed by in vitro analysis and in multiple sclerosis, type 1 diabetes, rheumatoid arthritis, psoriasis, asthma, lupus nephritis, acute coronary syndrome, delayed-type hypersensitivity, bone resorption after periodontitis, contact dermatitis and It has been verified by in vivo animal models of T cell-mediated autoimmune diseases such as skin transplantation (Chandy, KG and Norton, RS, 2017. Peptide blockers of Kv1.3 channels in T cells as therapeutics for autoimmune disease. Curr. Opin. Chem .Biol., 38:97-107.).

Representative peptides that inhibit voltage-gated potassium ion channels include the ShK domain. The ShK domain is a 35-residue basic peptide extracted from the sea anemone Stichodactyla helianthus that blocks multiple potassium channels.

Research on the ShK domain-like peptides has been conducted in various ways. In Korean Patent Registration No. 10-1524517, peptides (Vm23 and Vm24) isolated from the venom of the Mexican scorpion Vaejovis mexicanus smithi have a potassium ion channel (Kv1.3) It has been disclosed that it can be used for the treatment of autoimmune diseases by inhibiting. WO 2010/108154 discloses that toxin peptide analogues related to ShK, HmK and AETX-K can be used for the treatment of autoimmune diseases by inhibiting potassium ion channels (Kv1.3), and Korean Patent Publication No. 10-2014 In No. -0038501, a radioactive isotope and a chelator were attached to the ShK domain to disclose a superior potassium ion channel (Kv1.3) inhibitory effect than the existing ShK domain.

The present inventor analyzed the genome information of the small box jellyfish, compared the analyzed gene set with the Tox-Prot database, obtained 27 poison-related domains, and identified peptides that inhibit the hKv1.3 potassium ion channel among them. , completed the present invention.

The present invention provides a peptide comprising the amino acid sequence of SEQ ID NO: 1, and since the peptide inhibits the hKv1.3 potassium ion channel, it can be used to improve, prevent or treat autoimmune diseases.

The present invention provides a peptide comprising the amino acid sequence of SEQ ID NO: 1.

The present invention provides a composition for inhibiting Kv1.3 potassium ion channels containing the peptide as an active ingredient.

The present invention provides a pharmaceutical composition for preventing or treating autoimmune diseases containing the peptide as an active ingredient.

The present invention provides a preventive or health functional food composition for autoimmune diseases containing the peptide as an active ingredient.

The peptide of SEQ ID NO: 1 of the present invention can be usefully used for preventing or treating autoimmune diseases by inhibiting the hKv1.3 potassium ion channel, which plays an important role in activating T lymphocytes in the early stage.

Figure 1 shows the amino acid sequence (SEQ ID NO: 1) of the CBRV1_10493 peptide.
Figure 2 shows the amino acid sequence (SEQ ID NO: 1) of the CBRV1_10493 peptide including three disulfide bonds formed by six cysteines.
3 is a graph showing the voltage clamp results of Psora4 in the hKv1.3 potassium ion channel.
4 is a graph showing the voltage clamp results of the CBRV1_10493 peptide in the hKv1.3 potassium ion channel.
5 is a graph showing the voltage clamp results of the CBRV1_05766-1 peptide in the hKv1.3 potassium ion channel.

Hereinafter, the present invention will be described in detail.

The present invention provides a peptide comprising the amino acid sequence of SEQ ID NO: 1.

SEQ ID NO: 1 is Cys-Arg-Asp-Arg-Asn-Ser-Asn-Cys-Ala-Arg-Trp-Glu-Lys-Lys-Gly-Glu-Cys-Lys-Arg-Asn-Pro-Gly-Thr -Met-Leu-Ser-Thr-Cys-Arg-Lys-Ser-Cys-Lys-Val-Cys.

SEQ ID NO: 1 is shown in Table 1 and FIG. 1 below.

sequence number peptide name order SEQ ID NO: 1 CBRV1_10493 CKDHHTYGVYCKDWKSSGECKKNPKGMRHFCRKTCGFC

The peptide of SEQ ID NO: 1 includes 6 cysteines and includes 3 disulfide bonds. The disulfide bonds include the first Cys and the sixth Cys, the second Cys and the fourth Cys, and the third Cys and the fifth Cys, respectively. do. Specifically, in the peptide of SEQ ID NO: 1, Cys 1 and Cys 35, Cys 8 and Cys 28, and Cys 17 and Cys 32 may form disulfide bonds, respectively (FIG. 2).

As a method for synthesizing the peptide, it is preferable to synthesize it by a conventional chemical synthesis method of peptides in the art (W H Freeman and Co, Proteins; structures and molecular principles, 1983), specifically, a liquid phase peptide synthesis method (Solution Phase Peptide synthesis), solid-phase peptide syntheses, fragment condensation, and F-moc or T-BOC chemistry are more preferred, and more specifically, solid-phase peptide synthesis is most preferred.

The peptides of the present invention can be prepared by genetic engineering methods as follows. First, a DNA sequence encoding the peptide is constructed according to a conventional method. DNA sequences can be prepared by PCR amplification using appropriate primers.

Alternatively, DNA sequences may be synthesized by standard methods known in the art, such as using an automated DNA synthesizer (eg, from Biosearch or Applied iosystems). The DNA sequence is inserted into a vector containing one or more expression control sequences (eg, promoter, enhancer, etc.) operably linked thereto to control the expression of the DNA sequence, and the recombinant expression vector formed therefrom is used in host cells. After transformation, the resulting transformants are cultured in media and conditions appropriate to allow expression of the DNA sequence to obtain a substantially pure peptide encoded by the DNA sequence from the culture by methods known in the art (such as , chromatography) is used to recover.

The term 'substantially pure peptide' means that the peptide according to the present invention does not substantially contain any other host-derived proteins. For the genetic engineering method for synthesizing the peptide of the present invention, reference may be made to the following documents: Maniatis et al, Molecular Cloning; A laboratory Manual, Cold Spring Harbor laboratory, 1982; Sambrook et al, Molecular Cloning: A Laboratory Manual, ColdSpring Harbor Press, NY, Second (1998) and Third (2000) Edition; Gene Expression Technology, Method in Enzymology, Genetics and Molecular Biology, Method in Enzymology, Guthrie & Fink (eds), Academic Press, San Diego, Calif, 1991; and Hitzeman et al, J Biol Chem, 255:12073-12080, 1990.

The peptide is a component of the venom of Carybdea brevipedalia . The present inventor analyzed the genome information of the small box jellyfish, and analyzed the gene set to the Tox-Prot database (Jungo F, Bougueleret L, Xenarios I, Poux S. The UniProtKB/Swiss-Prot Tox-Prot program: a central hub of Compared with integrated venom protein data. Toxicon. (2012) 60(4): 551-7), 27 venom-related domains were obtained (Table 3).

Comparing the 27 poison-related domains with the three commonalities described in Example 1 below of the amino acid sequences of ShK and similar peptides found in cnidarians, SEQ ID NO: 1 and SEQ ID NO: 1 and SEQ ID NO: similar in structure to the amino acid sequence of the ShK domain 2 peptides (CBRV1_10493 and CBRV1_05766-1) were obtained (Table 4).

The cnidarians are a phylum belonging to the metazoa, and are heterozygous animals, including corals, anemones, hydras, jellyfish, and the like. The body is radially symmetrical, there is no head, and the body wall is composed of two cell layers. The pouch-shaped tonic has one mouth. It is composed of distinct tissues, but does not form organs. They are called cnidocytes because they have cnidocytes on the surface of their body and inside these cnidocytes are stinging sacs called cnidae. They mainly live in the sea and some live in freshwater. There are several forms of cnidae, the most common being the nematocyst, which has venomous, spring-shaped filaments that fire when it comes into contact with foreign objects such as prey or predators, killing or harming them. put on There is a polyp type that adheres to the bottom, such as sea anemones and corals, and a jellyfish type (or medusa) that freely swims in the water and moves around, such as a jellyfish.

The present invention provides a composition for inhibiting Kv1.3 potassium ion channels containing the peptide of SEQ ID NO: 1 as an active ingredient.

It was confirmed that the peptide of SEQ ID NO: 1 inhibited the activity of hKV1.3 by about 70.4% based on the median value at +50 mV (FIG. 4).

The peptide of SEQ ID NO: 2 inhibited the activity of hKV1.3 by about 5% based on the median value at +50 mV, and it was confirmed that there was no inhibitory activity (FIG. 5).

The present invention provides a pharmaceutical composition for preventing or treating autoimmune diseases containing the peptide of SEQ ID NO: 1 as an active ingredient.

The autoimmune disease is multiple sclerosis, rheumatoid arthritis, type 1 diabetes, psoriasis, inflammatory bowel disease, contact-mediated dermatitis, psoriatic arthritis, asthma, allergy, restenosis, systemic sclerosis, fibrosis, scleroderma, glomerulonephritis, Sjogren's syndrome , inflammatory bone resorption, graft rejection, graft versus host disease, and lupus.

Kv1.3 channel inhibitors are known to suppress autoimmune diseases by reducing the activation of effector memory T cells (TEM cells) and preventing tissue damage caused by T cells (Beeton et al., 2006. Kv1.3 channels are a therapeutic target for T cell-mediated autoimmune diseases. PNAS, 103: 17414-17419). It is also known to reduce the inflammatory response of autoimmune diseases by preventing secondary damage to neurons by blocking the respiratory burst of activated microglia (Fordyce et al., 2005. Microglia Kv1.3 channels contribute to their ability to kill neurons. J. Neurosci., 25:7139-7149). According to a recent review, Kv1.3 blockers are emerging as the best drugs for the treatment of autoimmune diseases and chronic inflammation (Perez-Verdaguer et al., 2015. The voltage-gated potassium channel Kv1.3 is a promising multitherapeutic target against human pathologies. Expert Opin. Ther. Targets, 20:5, 577-591). In addition, many experiments have verified Kv 1.3 blocking peptides as therapeutic agents for autoimmune diseases (Table 2, Chandy, KG and Norton, RS, 2017. Peptide blockers of Kv1.3 channels in T cells as therapeutics for autoimmune disease. Curr. Opin Chem. Biol., 38:97-107).

disease name cell model animal model Blocker or knockout model multiple sclerosis MBP-specific Kv1.3 high TEM cells in patient's peripheral blood.
Kv1.3 blockers inhibit antigen-induced proliferation of these cells. Experimental Autoimmune Encephalomyelitis (Rat) ShK; ShK-Dap22; ShK-186; ShK-192; 20 kDa PEG-ShK; 20 kDa PEG-[Lys16]ShK; ADWX-1 Kv1.3 CCR7+CCR5 T+EM cells in post-mortem MS plaques. Experimental Autoimmune Encephalomyelitis (Mouse) Kv1.3 knockout mouse type 1 diabetes GAD65 and insulin-specific Kv1.3 high TEM cells in the patient's peripheral blood. Experimental Autoimmune Diabetes (Rat) PAP-1 ShK-186 inhibits antigen-induced proliferation of these cells. rheumatoid arthritis Patient's synovial fluid Kv1.3 high TEM cells Pristan-Induced Arthritis (Rat) ShK-186 CD3CCR7+Kv1.3 T cells+ in synovial tissue psoriasis Kv1.3high TEM cells from psoriatic skin lesions. SCID-Hu mouse model transplanted human psoriasis skin into mice PAP-1 A twice-weekly dose of 60 μg of ShK-186 resulted in an improvement in psoriasis area and severity index (PASI) in 9/10 patients with plaque psoriasis.
Improvements were sustained for 4 weeks after treatment was discontinued. asthma Kv1.3 high Th2TEM cells from sputum of asthmatic patients. Ovalbumin-induced asthma model (rat) ShK-186 ShK-186 inhibited allergen-stimulated Th2T cell proliferation and cytokine production in the patient's peripheral blood. lupus nephritis TEM cells in the blood of active SLE patients have higher Kv1.3 than those of inactive SLE or controls. Experimental autoimmune glomerulonephritis (rat) Psora4, ShK-186 ShK-186 inhibits IL-17 and IFN-γ production by TEM cells from active lupus more than T cells from inactive lupus or controls. acute coronary syndrome Kv1.3 high TEM cells from patient's peripheral blood. - - PAP-1 inhibited interferon-γ and perforin by TEM cells. delayed-type hypersensitivity - DTH (pig) margatoxin and correolide KV261-HSA-34 (does not inhibit DTH but reduces cellularity of draining LNs). DTH (rat) Calliotoxin (Kaliotoxin); ShK; ShK-186; PAP-1; ShK-192; Vm24; AcK1; BmK1; BmKTX-D33H; Syn-Vm24-CDR3L; Fc-ShK Bone resorption after periodontitis - Experimental Periodontitis (Rat) Kaliotoxin contact dermatitis - Allergic contact dermatitis (mouse) PAP-1, Psora4 skin graft - Skin allograft (mouse) Clofazimine

In particular, Psora4, which was used as a positive control in the present invention, was also verified as a therapeutic agent for autoimmune diseases (Table 2). It was confirmed that it can be used for the prevention or treatment of (FIG. 4).

The pharmaceutical composition according to the present invention may include 10 to 95% by weight of the peptide of SEQ ID NO: 1, which is an active ingredient, based on the total weight of the composition.

In addition, the pharmaceutical composition of the present invention may further include at least one active ingredient exhibiting the same or similar function in addition to the above active ingredient.

The pharmaceutical composition of the present invention may further contain pharmaceutically acceptable additives.

The additives may further include suitable carriers, excipients and diluents commonly used in the preparation of pharmaceutical compositions.

The pharmaceutical composition of the present invention may include a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier is one commonly used in formulation, and includes lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate , microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, but are not limited thereto no.

The pharmaceutical composition of the present invention can be formulated in the form of oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, external preparations, suppositories or sterile injection solutions according to conventional methods. can Specifically, when formulated, it may be prepared using diluents or excipients such as commonly used fillers, weighting agents, binders, wetting agents, disintegrants, and surfactants.

Solid preparations for oral administration include, but are not limited to, tablets, pills, powders, granules, capsules, and the like. Such a solid preparation may be prepared by mixing at least one or more excipients, for example, starch, calcium carbonate, sucrose, lactose, gelatin, etc., in addition to the active ingredient. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. It may be prepared by adding various excipients, for example, wetting agents, sweeteners, aromatics, and preservatives, in addition to liquids and liquid paraffin for oral use.

Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized formulations and tablets. Propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used as non-aqueous solvents and suspending agents. As a base for suppositories, Witepsol, Macrosol, Tween 61, cacao butter, laurin paper, glycerogelatin, and the like may be used.

The pharmaceutical composition of the present invention may further include a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, and the like in addition to the above components.

Suitable pharmaceutically acceptable carriers and agents are described in detail in Remington's Pharmaceutical Sciences (19th ed, 1995).

The pharmaceutical composition of the present invention can be administered orally or parenterally, and in the case of parenteral administration, intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, transdermal administration, etc. can be administered.

A suitable dosage of the pharmaceutical composition of the present invention is determined by factors such as formulation method, administration method, patient's age, weight, sex, pathological condition, food, administration time, route of administration, excretion rate and reaction sensitivity, and the degree of bone loss of the patient. Depending on the field, it may be appropriately selected by a person skilled in the art, specifically 0.001 mg/kg to 500 mg/kg, and may be administered once or several times a day as needed.

The pharmaceutical composition of the present invention is prepared in unit dosage form by formulation using a pharmaceutically acceptable carrier and/or excipient according to a method that can be easily performed by those skilled in the art. or it may be prepared by incorporating into a multi-dose container. At this time, the formulation may be in the form of a solution, suspension or emulsion in an oil or aqueous medium, or in the form of an extract, powder, granule, tablet or capsule, and may additionally contain a dispersing agent or stabilizer.

The "administration target" of the present invention includes humans, monkeys, cows, horses, pigs, sheep, chickens, cats, dogs, mice, rabbits, etc., but is not particularly limited thereto.

The present invention provides a health functional food composition for preventing or improving autoimmune diseases containing the peptide of SEQ ID NO: 1 as an active ingredient.

Matters mentioned in the pharmaceutical composition and health functional food composition of the present invention are equally applied unless they contradict each other.

Hereinafter, the present invention will be described in detail by the following Examples and Experimental Examples.

However, the following examples and experimental examples are only for exemplifying the present invention, and the present invention is not limited thereto.

<Example 1> Derivation of ShK domain-like peptides

1-1. Derivation of poison-related domain

The present inventor analyzed the genome information of the small box jellyfish and compared the analyzed gene set with the Tox-Prot database (Jungo et al., 2012) to obtain 27 poison-related domains (Table 3).

Pfam ID Pfam Description Pfam ID Pfam Description PF12947.9 EGF domain PF01823.21 MAC/Perforin domain PF00008.29 EGF-like domain PF04103.17 CD20-like family PF07974.15 EGF-like domain PF05826.14 Phospholipase A2 PF07645.17 Calcium-binding EGF domain PF00068.21 Phospholipase A2 PF12661.9 Human growth factor-like EGF PF06103.13 Bacterial protein of unknown function (DUF948) PF12662.9 Complement Clr-like EGF-like PF08686.13 PLAC (protease and lacunin) domain PF14670.8 Coagulation Factor Xa inhibitory site PF10242.11 Lipoma HMGIC fusion partner-like protein PF01549.26 ShK domain-like PF01456.19 Mucin-like glycoproteins PF01400.26 Astacin (Peptidase family M12A) PF12044.10 Putative peptidase family PF00053.26 Laminin EGF domain PF17189.6 Glycosyl hydrolase family 30 beta sandwich domain PF00413.26 Matrixin PF10218.11 SREBP regulating gene protein PF00397.28 WW domain PF17333.4 Beta-defensins 136 PF00014.25 Kunitz/Bovine pancreatic trypsin inhibitor domain PF03243.17 Alkylmercury lyase PF17855.3 MCM AAA-lid domain

1-2. Derivation of ShK domain-like peptides The 27 venom-related domains obtained in 1-1 above were compared with the following three commonalities of amino acid sequences of ShK and similar peptides found in cnidarians.

1) The length of the peptide is less than 40 amino acids.

2) There are 6 cysteine inside the peptide.

3) The structure of the C-terminal side of the peptide is CXXXCXXC (C is cysteine and X is any amino acid.)

As a result of comparing the three points in common, two peptides (CBRV1_10493 and CBRV1_05766-1) having similar structures of the ShK domain and amino acid sequence were obtained (Table 4).

sequence number peptide name Sequence (N-terminus -> C-terminus) length SEQ ID NO: 1 CBRV1_10493 CRDRNSNCARWEKKGECKRNPGYMLSYCRKSCKVC 35 aa SEQ ID NO: 2 CBRV1_05766-1 CDKKKELPFCNIVDAKTFCRDINQAKQCPLKCKLC 35 aa SEQ ID NO: 3 ShK CIDTIPKSRCTAFQCKHSMKYRLSFCRKTCGTC 33 aa

<Example 2> Preparation of Peptide Three peptides derived from Example 1 (CBRV1_10493 and CBRV1_05766-1) were prepared by the solid phase peptide synthesis method below.

DMF (15 ml/g) and resin were put in a test tube and the resin was swollen for 30 minutes. DMF was removed, and a 20% piperidine DMF solution (15ml/g) was added to deprotection reaction for 5 minutes. After removing the 20% piperidine DMF solution in the same way, a new 20% piperidine DMF solution (15 ml/g) was added to deprotection reaction for 15 minutes. The resin subjected to the deprotection reaction was washed twice with DMF (10ml/g), twice with methanol (10ml/g), and twice with DMF (10ml/g). Then, amino acids (3eq) and HOBt (3eq) to be reacted were dissolved in as little DMF as possible, put into a test tube, and DIC (3eq) was added to conduct a coupling reaction for 30 minutes. It was washed with DMF (10ml/g) once, methanol (10ml/g) twice, and DMF (10ml/g) twice. The deprotection reaction, coupling reaction, and washing were repeated to obtain the desired sequence. After reacting the desired sequence, a dissolution solution (10ml/g) of TFA 94.0%, water 2.5%, EDT 2.5%, TIS 1% was added to the test tube and reacted for 120 minutes to obtain a lysate dissolved from the resin (Note: g is based on the unit of resin). Finally, the lysate was dried with nitrogen, washed with ether 6 times, and dried at room temperature to obtain a peptide.

<Experimental Example 1> Preparation of cells for measuring current (HK293 cells having hKv1.3 potassium ion channels)

1-1. Cloning of the hKv1.3 potassium ion channel and completion of the hKv1.3 vector

To amplify the hKv1.3 potassium ion channel gene, oriGENE human cDNA clones were used as templates, and PCR was performed using the primer sets shown in Table 5 below.

sequence number sequence name Sequence (5'→3') SEQ ID NO: 4 NheI-Kv1.3-F 5′-TTTGCTAGCGCCACCATGGACGAGCGC-3′ SEQ ID NO: 5 SaI1-Kv1.3-R 5'-TTTGTCGACCTAAACATCGGTGAATATCTTTT-3'

PCR was carried out under conditions of 1) 95 ° C 5 minutes, 2) 95 ° C 30 seconds, 58 ° C 30 seconds, 72 ° C 1 minute 45 seconds, 30 times, 3) 72 ° C 3 minutes. The hKv1.3 potassium ion channel gene obtained by performing the above PCR between the NhaI site and the SalI site of the AAV-IRES2-EGFP vector cloned using oriGENE human cDNA clones as a template Insertion completed the AAV-CMV-hK1.3-IRES2-EGFP vector. 1-2. Transfection of the hKv1.3 potassium ion channel gene into HEK 293 cells

2 µg of AAV-CMV-hKv1.3-IRES2-EGFP vector was transfected into HEK 293 cells (50% confluency, 7X105 or more) cultured in a 35 mm Petri dish using Qiagen Effectene. That is, the provided buffer was added to 2 μg of DNA, titrated to 100 μl, 8 μl of enhancer was added, and after waiting for 2-5 minutes, 20 μl of effectene was added and mixed for 1 second with a vortex mixer. The sample was centrifuged to precipitate (spin down), waited for 10 minutes, added 0.6 ml of DMEM+10% Fetal Bovine Serum (FBS), mixed well, and sprinkled on HEK 293 cells in a clockwise direction in the form of droplets. It was then stored in a 35 mm culture vessel for 17 hours.

1-3. Transfected HEK 293 cells confirmed that hKv1.3 is a voltage-dependent potassium ion channel

1-3-1. Preparation of Transfected HEK 293 Cells for Amperometric Measurements

Transfectants (Transfectants, HEK293+ AAV-CMV-hKv1.3-IRES2-EGFP) were removed from a 35 mm Petri dish and suspended in 12 ml DMEM + 10% FBS, and 0.5 ml per well of a 24 well plate. Twenty-four 0.01 mg ml PDL-coated coverslips (12 mm in diameter) were seeded in such a way that the number of cells reached 1X10 ⁵ . After waiting for an hour for the cells to adhere to the coverslip, a patch clamp method was performed for current measurement.

1-3-2. current measurement

Amplifier (amplifier, MultiClamp 700A) connected to patch clamp device, digitizer (digitizer, DIGIDATA 1322A), remote manipulator, bath application, cell and fluorescence expression using HCImageLive program in microscope and computer, cell and pipette was found, and whole-cell patches were recorded using Clampex software, MultiClamp 700A, and digidata 1550. An internal solution [140 mM k-gluconate, 10 mM HEPES, 0.2 mM ATP, 0.06 mM GTP] was prepared and stored in a syringe on ice, and an external solution [130 mM NaCl, 10 mM HEPES, 3 mM KCl, 1.5 mM D-Glucose, 10 mM sucrose, 24 mM CaCl ₂ , 1.5 mM MgCl ₂ 6H ₂ O, osmilarity 320 mmol/kg, pH 7.2] was placed in a reservoir and used as a basic buffer. The material was diluted with an external solution to a concentration of 100 nM in the bath chamber and stored in a drug reservoir. The current was measured by applying a voltage step (first step from -130mV to 50mV, delta 20mV 0.6s) in voltage clamp mode.

Psora 4 (100 nM), known as an inhibitor of Kv1.3, was used as a positive control. As a result of current measurement, it was confirmed that HEK 293 cells transfected with hKv1.3 were voltage-dependent potassium ion channels (FIG. 3).

<Experimental Example 2> Confirmation of inhibition of hKv1.3 potassium ion channel activity by Shk-like peptides derived from small box jellyfish

Using HEK293 cells having hKv1.3 potassium ion channels prepared in Experimental Example 1, the inhibition of hKv1.3 potassium ion channel activity by the peptides SEQ ID NO: 1 and SEQ ID NO: 2 (CBRV1_10493 and CBRV1_05766-1) was confirmed.

As a result, it was confirmed that the CBRV1_10493 peptide inhibited the activity of hKV1.3 by about 70.4% (pre-CBRV1_10493: 2285.8nA, post-CBRV1_10493: 676.5nA) based on the median value of hKV1.3 at +50 mV (FIG. 4).

On the other hand, the CBRV1_05766-1 peptide inhibits the activity of hKV1.3 by about 5.0% (pre-CBRV1_05766-1: 1.01nA, post- CBRV1_05766-1: 0.96nA) based on the median value of hKV1.3 at +50mV. It was confirmed that there was no (Fig. 5).

<110> Korea Institute of Ocean Science and Technology Korea Institute of Toxicology <120> Peptide with voltage-gated potassium ion channel inhibitory activity from Carybdea brevipedalia <130> 2022P-04-008 <160> 5 <170> KoPatentIn 3.0 <210> 1 <211> 35 <212> PRT <213> artificial sequence <220> <223> CBRV1_10493 <400> 1 Cys Arg Asp Arg Asn Ser Asn Cys Ala Arg Trp Glu Lys Lys Gly Glu 1 5 10 15 Cys Lys Arg Asn Pro Gly Tyr Met Leu Ser Tyr Cys Arg Lys Ser Cys 20 25 30 Lys Val Cys 35 <210> 2 <211> 35 <212> PRT <213> artificial sequence <220> <223> CBRV1_05766-1 <400> 2 Cys Asp Lys Lys Lys Glu Leu Pro Phe Cys Asn Ile Val Asp Ala Lys 1 5 10 15 Thr Phe Cys Arg Asp Ile Asn Gln Ala Lys Gln Cys Pro Leu Lys Cys 20 25 30 Lys Leu Cys 35 <210> 3 <211> 33 <212> PRT <213> artificial sequence <220> <223> ShK <400> 3 Cys Ile Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Gln Cys Lys 1 5 10 15 His Ser Met Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr Cys Gly Thr 20 25 30 Cys <210> 4 <211> 27 <212> DNA <213> artificial sequence <220> <223> NheI-Kv1.3-F <400> 4 tttgctagcg ccaccatgga cgagcgc 27 <210> 5 <211> 32 <212> DNA <213> artificial sequence <220> <223> SaI1-Kv1.3-R <400> 5 tttgtcgacc taaacatcgg tgaatatctt tt 32

Claims (7)

A peptide comprising the amino acid sequence of SEQ ID NO: 1.
According to claim 1,
The peptide has three disulfide bonds,
A peptide characterized in that the first Cys and the sixth Cys, the second Cys and the fourth Cys, and the third Cys and the fifth Cys form a disulfide bond, respectively.
According to claim 1,
The peptide is a small box jellyfish ( Carybdea brevipedalia ) Peptide, characterized in that the composition of the venom (venom).
A pharmaceutical composition for preventing or treating autoimmune diseases caused by inhibition of Kv1.3 potassium ion channels, containing the peptide of claim 1 as an active ingredient.

delete According to claim 4,
The autoimmune disease is multiple sclerosis, rheumatoid arthritis, type 1 diabetes, psoriasis, inflammatory bowel disease, contact-mediated dermatitis, psoriatic arthritis, asthma, allergy, restenosis, systemic sclerosis, fibrosis, scleroderma, glomerulonephritis, Sjogren's syndrome A pharmaceutical composition, characterized in that at least one selected from the group consisting of inflammatory bone resorption, graft rejection, graft versus host disease, and lupus.
A health functional food composition for preventing or improving autoimmune diseases caused by inhibition of Kv1.3 potassium ion channels, containing the peptide of claim 1 as an active ingredient.

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