KR101064915B1 - Fusion polypeptide for inhibiting neurotransmitter secretion and method for delivering it - Google Patents

Abstract

The present invention is to fusion polypeptide and protein transduction domain (PTD) of the three proteins constituting the SNARE complex in neurons in order to suppress the secretion of neurotransmitters to intracellular delivery efficiency and local site It relates to a method of improving the delivery of.

Description

Fusion peptide for blocking neurotransmitter and delivery method thereof {FUSION POLYPEPTIDE FOR INHIBITING NEUROTRANSMITTER SECRETION AND METHOD FOR DELIVERING IT}

The present invention is to fusion polypeptide and protein transduction domain (PTD) of the three proteins constituting the SNARE complex in neurons in order to suppress the secretion of neurotransmitters to intracellular delivery efficiency and local site It relates to a method of improving the delivery of.

For secretion of neurotransmitters, synaptobrevin (Vesicle-associate membrane protein (VAMP)) and pre-synaptic plasma membranes present on the surface of vesicles carrying neurotransmitters Three proteins of SNAP25, Syntaxin, which are present in, form a SNARE complex and are secreted extracellularly by stimulation of Ca ²⁺ (FIG. 1).

Botolinium neurotoxins, which are most commonly used as inhibitors of neurotransmitters, have sub-types such as A, B, C, D, E, and F. These are specific to VAMP-2, SNAP25, and syntaxin, respectively. The site is cleaved to interfere with the formation of the SNARE complex to inhibit the secretion of neurotransmitters (Schiavo et al. Nature 359, 832-835, 1992).

These botulinum neurotoxins are highly toxic, and as mentioned above, studies on the inhibition of secretion of neurotransmitters using peptide fragments cut by the botulinum neurotoxin have been reported, but the specificity is low because only a part of the whole protein is used. It is common to have lower activity than botulinum neurotoxin, and when the size of the peptide is increased, the intracellular delivery efficiency is lower than that of the botulinum neurotoxin which binds to the receptor and is induced intracellularly due to the low rate of intracellular delivery. (Antonio V. Ferror-Monitiel et al. FEBS letters, 435, 84-88, 1998).

There is a need for a general method for the biologically effective delivery of biologically active macromolecules without damaging cells both inside and outside the body (LA Sternson, Ann. NY Acad. Sci., 57, 19-21 (1987)). . Examples of such methods include chemical addition of lipid peptides (P. Hoffmann et al. Immunobiol., 177, 158-170 (1998)) or methods using basic polymers such as polylysine or polyarginine (WC. Chen et al., Proc. Natl. Acad. Sci., USA, 75, 1872-1876 (1978)), but these methods have not yet been validated. Folic acid (CP Leamon and Low, Proc. Natl. Acd. Sci., USA, 88, 5572-5576 (1991)), used as a transporter, has been reported to be transported into the cell as a folate-salt conjugate, but to the cytoplasm. It is not confirmed yet. Pseudomonas Exotoxin is also used as a type of transporter (T.I. Prior et al., Cell, 64, 1017-1023 (1991)). However, even in these methods, the effect on the transfer of biologically activated 'transferred' material into cells and its general applicability are not clear. Therefore, there is a continuous need for a method that can safely and effectively deliver biologically active substances into the cytoplasm or nucleus of living cells.

As a result of this study, PTD has been suggested, and the most studied is Tat protein, a transcription factor of human immunodeficiency virus-1 (HIV-1). It has been found that the protein is more effective in transcribing cell membranes when it is composed of parts of the 47th to 57th amino acids (YGRKKRRQRRR), which are concentrated in positively charged amino acids, rather than the complete form of 86 amino acids. (Fawell S. et al. Proc. Natl. Acad. Sci. USA 91, 664-668 (1994)). As another example of the effect as PTD, amino acids from 267 to 300 of VP22 protein of HSV-1 (Herpes Simplex Virus type 1) (Elliott G. et al. Cell, 88, 223-233 (1997)) And 339 to 355 amino acids (Schwarze SR et al. Trends Pharmacol Sci. 21, 45-48 (2000)) of the Drosophila ANTP (Antennapedia) protein, including artificial peptides that list electrically positive amino acids. The effect was confirmed (Laus R. et al. Nature Biotechnol. 18, 1269-1272 (2000)).

Technical Challenge

Accordingly, the present inventors attempted to inhibit the secretion of neurotransmitters by compromising the binding of proteins competitively by increasing the transfer efficiency into cells using domains in which three proteins forming the SNARE complex are bound to each other. In the present invention, a fusion polypeptide is prepared by conjugating the binding domains of the protein transduction domain (PTD) and the three proteins that form the SNARE complex in order to enhance the delivery efficiency into cells. By using this, it suppresses the secretion of neurotransmitters.

The amino acid sequences of Synaptobrevin (VAMP-2), SNAP25, and Syntaxin constituting the SNARE complex are as follows.

SNAP25 (SEQ ID.NO .: 1)

MAEDADMRNE LEEMQRRADQ LADESLESTR RMLQLVEESK DAGIRTLVML DEQGEQLERI

EEGMDQINKD MKEAEKNLTD LGKFCGLCVC PCNKLKSSDA YKKAWGNNQD GVVASQPARV

VDEREQMAIS GGFIRRVTND ARENEMDENL EQVSGIIGNL RHMALDMGN E IDTQNRQIDR

IMEKAQANKT RIDEANQRAT KMLGSG

VAMP2 (SEQ ID.NO .: 2)

MSATAATAPP AAPAGEGGPP APPPNLTSV NR RLQQTQAQVD EVVDIMRVNV DKVLERDQKL

SELDDRADAL QAGASQFETS AAKLKR KYWW KNLKMMIILG VICAIILIII IVYFSS

Taxin (SEQ ID. NO .: 3)

MKDRTQELRT AKDSDDDDDV TVTVDRDRFM DEFFEQVEEI RGFIDKIAEN VEEVKRKHSA

ILASPNPDEK TKEELEELMS DIKKTANKVR SKLKSIEQSI EQEEGLNRSS ALDRIRKTQH

STLSRKFVEV MSEYNATQSD YRERCKGRIO RQLEITGRTT TSEELEDMLE SGNPAIFASG

IIMDSSISKQ ALSEIETR HS EIIKLENSIR ELHDMFMD MA MLVESQGEMI DRIEYNVEHA

VDYVERAVSD TKKAVKYQSK ARRKKIMIII CCVILGIIIA STIGGIFG

From the 170th amino acid of SNAP25 (SEQ ID NO .: 1), the 206th amino acid (bold and underlined) is the part which is combined with VAMP-2 (SEQ ID NO .: 4, SBD), VAMP-2 (SEQ The 86 amino acid (bold and underlined) at the 29th amino acid of ID NO .: 2) is the domain (SEQ ID NO .: 5, VBD) that binds to SNAP25 / syntaxin. In the present invention, PTD, SBD (SEQ ID NO .: 4) and VBD (SEQ ID NO .: 5) are conjugated and used as inhibitors of neurotransmitters.

Technical solution

In order to achieve the above object, the present invention provides a domain for binding SNAMP25 to VAMP-2, and a domain for binding VAMP-2 to syntax, SNAP25, that is, SNARE formed for the secretion of neurotransmitters in neurons. The present invention provides a fusion polypeptide of a peptide that inhibits complex composition and a protein transduction domain (PTD) having high efficiency in cell permeability, skin, and eyes. The fusion peptide according to the present invention inhibits the secretion of neurotransmitters such as catecholamine, acetcholine, and greatly increases the secretion inhibitory effect of the neurotransmitter according to the binding of PTD, and the cytotoxicity does not change.

The fusion peptide according to the present invention is a fusion polypeptide in which a PTD peptide is bound to a peptide derived from human SNAP-25 (referred to as SBD) and a peptide derived from human VAMP-2 (referred to as VBD) (sometimes used herein) (Also referred to as 'PTD-SBD or VBD'), the peptide sequence thereof may be represented by the following Chemical Formula 1:

Formula 1

In the above formula, [] _PTD is a self-penetrating PTD peptide, R ₁ is at least one selected from the group consisting of tyrosine, alanine, arginine, valine, glycine and proline, and n is 5-30 It is an integer and at least 30% of all amino acids are arginine. Examples of PTD include Hph-1 (SEQ ID. NO .: 6-YARVRRRGPRR), Sim-2 (SEQ ID. NO .: 7-AKAARQAAR), Tat (SEQ ID. NO .: 8-YGRKKRRQRRR), Antp (SEQ ID.NO .: 9-RQIKIWFQNRRMKWKK), VP22 (SEQ ID.NO.10-DAATATRGRSAASRPTERPRAPARSAS RPRRPVE), R7 (SEQ ID.NO. 11-RRRRRRR), MTS (SEQ ID.NO .: 12-AAVALLPAVLLALLAPAAADQNQLMP), pe 1 (SEQ ID. NO. 13-KETWWETWWTEWSQPKKKRKV).

[] _Gly connects autologous cell penetrating PTD peptides with SBD and VBD peptides and enhances the fluidity of fusion peptides to enhance the binding efficiency between the infiltrated PTD-SBD or VBD and SNAPE complexes. m is an integer of 0-5. In Formula 1, Gly is used as a linker, but various linkers may be used according to the purpose of the present invention.

SBD (SEQ ID. NO .: 4) and VBD (SEQ ID. NO .: 5) are peptides derived from human SNAP-25 and VAMP-2 and include fragments of these domains in the present invention. It will be understood by those skilled in the art that any modifications, ie substitutions, insertions and deletions, etc. to the sequences of SEQ ID NOS: 4 and 5 can be used as the SBD and VBD moieties of the fusion peptides according to the invention.

According to the present invention, by inserting about three glycine residues between the peptides of SBD or VBD and PTD, the efficiency of delivery and uptake into cells can be enhanced.

On the other hand, the method for producing a fusion peptide according to the present invention, methods known in the art, for example, a solid phase synthesis method or a synthetic method using a recombinant expression system may be used. However, the method for preparing the fusion peptide according to the present invention is not limited only by this method.

First, the synthesis of pure fusion peptides can be performed using an organic synthesis device for peptide synthesis. This method is based on Merrifield's solid phase peptide synthesis ( J. Am. Chem. Soc . 85, 2149-21, 54 (1963)) and amino acid using a peptide semi-automatic synthesizer (Peti-Syzer Model PSS-510). A fusion peptide is synthesized by condensation reaction of a monomer having a C-terminus with an N-terminus of.

Solid phase synthesis begins by coupling an amino acid having an α-amino group protected to a suitable resin from the amino acid at the carboxyl end. At this time, the amino acid in which the α-amino group is protected is attached to the hydroxy methyl resin or the chloromethylated resin as an ester bond. As a group protecting the α-amino group, Fmoc (9-fluorenyl methoxycarbonyl) is used. (Fmoc-protected amino acids can be purchased from Bidtech Co., Ltd.). For example, the amino group of Arg is reactive. Reactive moieties of Gly, Lys, Gln are Fmoc-amino acids protected with appropriate groups such as tert-butyl (t-Bu), Pmc (2,2,5,7,8-pentamethylchroman-6-sulfonyl) Use Peptide synthesis proceeds by sequentially removing protecting groups with reagents such as trifluoroacetic acid (TFA), phenol, on the amino terminus of the peptide chain attached to a solid support resin. Peptides can be isolated from TFA solution by extraction with diethyl ether and purified by high performance liquid chromatography (HPLC).

Second, the method for producing a fusion peptide using a biological method, (a) to produce a recombinant vector that can be expressed in the form of a fusion peptide by cloning the gene of the fusion peptide in the expression vector pPET that can express a specific peptide. And (b) mass expression in the form of recombinant transporter-PTD-SBD (VBD) peptide in E. coli using the pPET-PTD-SBD (VBD) expression vector, followed by pure separation and purification. Include. At this time, in order to prepare a recombinant vector, a base sequence encoding a fusion peptide can be easily inferred from the amino acid sequence disclosed by those skilled in the art, a cloning vector for inserting such a base sequence, prepared using a cloning technique A recombinant expression vector, a host cell for transforming with the recombinant vector to express the desired fusion peptide, a method for transforming the recombinant vector into a host, a method for expressing the desired fusion peptide from the transformed host cell, and a target from the final product Selection and general technical matters, such as how to obtain the fusion peptide, will be readily appreciated by those skilled in the art.

On the other hand, the present invention provides a neurotransmitter inhibitor including a fusion peptide in which the protein transporter PTD peptide is coupled to human-derived SBD, VBD peptide.

Such neurotransmitter inhibitors can be applied to various fields such as pain relief, wrinkle improvement, and square jaw improvement.

Favorable effect

The fusion peptide according to the present invention is excellent in intracellular delivery efficiency and local delivery efficiency, and thus effectively inhibits the secretion of neurotransmitters by the fusion peptide.

1 is a diagram showing the secretion mechanism of neurotransmitters.

2 shows the HPLC results of the synthetic peptides.

3 shows a DNA cleavage map of the PTD conjugate.

Figure 4 shows agarose gel pictures of PTD-SBD, PTD-VBD.

5 shows the cell permeation efficiency according to the conjugation of PTD.

Figure 6 is the average of the compound muscle action potential recorded by stimulating the sciatic nerve 50 times at 2Hz. From this, the amplitude of the action potential (dY) indicating the amount of muscle contraction, and the delay time (tC) until the action potential of the muscle after the stimulation is shown.

7 shows Amplitude of compound muscle action potential.

DETAILED DESCRIPTION OF THE INVENTION

Example 1 Preparation of PTD-SBD (VBD) Conjugates

(1) Preparation of PTD-SBD (VBD) conjugate

YARVRRRGPRRGGGEIDTQNRQIDRIMEKAQANKTRIDEANQRATKMLGSG (PTD-GGG-SBD polypeptide)

A fusion polypeptide having the amino acid sequence of YARVRRRGPRRGGGNRRLQQTQAQVDEVVDIMRVNVDKVLERDQKLSELDDRADALQAGASQFETSAAKLKR (PTD-GGG-VBD polypeptide) was synthesized in a solid phase synthesis using a peptide semiautomatic synthesizer (Peti-Syzer Model PSS-510). 0.1 mmol of Rink Amide MBHA resin was placed in a standard reaction vessel, and the synthesis was started by adding 0.5 mmol of Fmoc-G and Fmoc-R, the first amino acids of C-terminal of the peptide to be synthesized. From the C-terminal amino acid to the N-terminal amino acid, the corresponding amino acid residues were reacted three times with 0.5 mmol each. Deprotection of Fmoc was performed three times for 10 minutes using 20% piperidine diluted with dimethyl formamide (DMF), washed for 10 minutes with DMF and coupling for 100 minutes. Coupling used N-hydroxybenzotriazole (HOBT) and 1,3-diisopropylcarbodiimide (DIC), and the system which wash | cleaned with DMF for 10 minutes was used.

(2) Isolation and Purification of Fusion Peptides

After the synthesis was completed, the peptide was placed in a 50 mL corning tube, cooled to 0 ° C., 0.75 g of phenol, 0.25 mL of 1,2-ethanedithiol (EDT), 0.25 mL of tianisol, 0.5 mL of distilled water, and After cutting the cutting solvent having a mixing ratio of 8.25 mL of TFA was reacted for 4 hours at room temperature. After the reaction, the resin separated from the peptide was filtered and 30 ml of diethyl ether below 50 ° C. was added to the remaining solution to precipitate the peptide, followed by washing with two more diethyl ethers. The obtained precipitate was dried and then dissolved in MeOH and purified by HPLC. The columns used for purification were C18 analytical column 218TP54 (VyDac) and C18 preparative column 218TP1022 (VyDac). Purification conditions were 1 mL / min and 25 mL / min, respectively, and the peptide was eluted using A: 100% H ₂ O + 0.01% TFA and B: 100% acetonitrile + 0.01% TFA as a solvent. Acetonitrile of the eluted peptide was removed and lyophilized to obtain a high purity peptide. (Figure 2)

Example 2. Preparation and Purification of Fusion Peptide Expression Vectors Using a Microbial Expression System

(1) Expression Vector Preparation

To prepare a nucleotide sequence encoding the fusion peptide, Hind III at the N-terminus and BamHI at the C-terminus were put in pUC 19 (invitrogen) to prepare a template. In order to bind the poly His tag region for high expression and easy purification of the fusion peptide and the nucleotide sequence encoding the high expression protein, the template was separated through the restriction enzyme HindIII and BamH I reaction, and the quiaquick purification kit was used. Purification was carried out. The expression vector, pPET vector was processed using restriction enzymes HindIII, BamHI, purified using a Quiaquick purification kit, and then cloned the base template encoding the prepared fusion peptide to prepare a recombinant expression vector. The gene map of the schematic expression vector vector is as shown in FIG.

(2) Preparation of E. coli transformants and expression and purification of fusion peptides

E. coli BL21 (invitrogen) was transformed by a heat shock transformation method using the expression vector prepared above, and the transformed E. coli was transformed into 1 mL in 100 mL of LB medium in an amount of 1 mL. % Incubation and pre-culture with stirring at 37 ° C. for 12 hours. It was then inoculated again in 1,000 mL of LB medium and incubated at 37 ° C. for 4 hours, followed by 1 mM concentration of IPTG (isopropyl β-D-thiogalactopyranoside, GibcoBRL cat. # 15529-019). Was added to induce the expression of lac operon and incubated for 8 hours to induce the expression of the fusion peptide. The culture solution was centrifuged at 5,000 rpm for 20 minutes at 4 ° C. to remove the supernatant, leaving only the pellet. Then, 10 mL of buffer solution containing 1 mg / mL of lysozyme (Sigma, cat, # L-7651) (50 mM NaH) ₂ PO ₄ , 300 mM NaCl, 10 mM imidazole, pH 8.0), the pellet was left for 30 minutes on ice, and then subjected to an ultrasonic mill (Heat systems, ultrasonic processor XL) at a strength of 300 W for 10 seconds. The ultrasonic injection was repeated for 10 seconds and the cumulative ultrasonic injection time was 3 minutes. The eluate was centrifuged at 12,000 rpm for 20 minutes at 4 ° C. to remove the lysate of E. coli, and only pure eluate was separated. 2.5 mL of 50% Ni ²⁺ -NTA agarose slurry (Qiagen, cat # 30230) was added to the separated eluate, and stirred at 200 rpm at 4 ° C. for 1 hour to combine the fusion protein with Ni ²⁺ -NTA agarose. The mixed solution was poured into a 0.8 × 4 cm column for chromatography (BioRad, cat # 731-1550). After washing twice with 4 mL of Buffer 2 (50 mM NaH ₂ PO ₄ , 300 mM NaCl, 20 mM imidazole, pH 8.0), 0.5 mL of Buffer 3 (50 mM NaH ₂ PO ₄ , 300 mM NaCl, 250 mM Imidazole, pH 8.0) was divided into four times and the fusion protein was fractionated, and subjected to SDS-PAGE, and confirmed by the Coomassie blue staining method, the results are shown in FIG. In Figure 4, lane 1 is a standard molecular weight protein, lane 2 is PTD-SBD, lane 3 is PTD-VBD. 2 mL ice-cold 4 M NH ₂ OH, 0.4 MK ₂ CO ₃ , 6 M GuHCl, pH 9.0 was added to the fusion protein, resulting in 2 M NH ₂ OH, 0.2 MK ₂ CO ₃ , 3 M GuHCl, pH 9.0 After stirring, the mixture was stirred at 45 ° C. at 225 rpm for 5 hours to separate the fusion protein into the fusion peptide and the transporter protein, and 12 N HCl was added to adjust the pH to 2-3 and stirred for 10 minutes to complete the reaction. did. 12.5 N NaOH was added to neutralize the pH to 6.5, and the separated transport protein was separated from the packed Ni ₂₊ -NTA agarose column, and then gel-filtered to remove salts from the buffer solution and freeze-dried. Pure fusion peptides were obtained.

Example 3 Inhibitory Effect of Neurotransmitter Secretion by Fusion Peptides in Vitro

The secretion inhibitory effect of the neurotransmitters of the fusion peptides prepared in Examples 1 and 2 were analyzed. Two fragments of SBD and VBD were each prepared to compare their activity.

Sample 1: SBD (SEQ ID.NO.:4)

Sample 2: VBD (SEQ ID. NO.:5)

Sample 3: Hph-1-GGG-SBD (SEQ ID.NO.:14)

Sample 4: Hph-1-GGG-VBD (SEQ ID.NO.:15)

Sample 5: Tat-GGG-SBD (SEQ ID.NO.:16)

Sample 6: Hph-1-GGG-SBDF1 (SEQ ID.NO.:17)

Sample 7: Hph-1-GGG-SBDF2 (SEQ ID.NO.:18)

Sample 8: Hph-1-GGG-VBDF1 (SEQ ID.NO.:19)

Sample 9: Hph-1-GGG-VBDF2 (SEQ ID.NO.:20)

Hph-1-GGG-SBD (SEQ ID.NO .: 14)

YARVRRRGPRRGGGEIDTQNRQIDRIMEKAQANKTRIDEANQRATKMLGSG

Hph-1-GGG-VBD (SEQ ID.NO .: 15)

YARVRRRGPRRGGGNRRLQQTQAQVDEVVDIMRVNVDKVLERDQKLSELDDRADALQAGASQFETSAAKLKR

Tat-GGG-SBD (SEQ ID.NO .: 16)

YGRKKRRQRRRGGGEIDTQNRQIDRIMEKAQANKTRIDEANQRATKMLGSG

Hph-1-GGG-SBDF1 (SEQ ID.NO .: 17)

YARVRRRGPRRGGGIMEKAQANKTRIDEANQRATKMLGSG

Hph-1-GGG-SBDF2 (SEQ ID.NO .: 18)

YARVRRRGPRRGGGKTRIDEANQRATKM

Hph-1-GGG-VBDF1 (SEQ ID.NO .: 19)

YARVRRRGPRRGGGNRRLQQTQAQVDEVVDIMRVNVDKVLERDQK

Hph-1-GGG-VBDF2 (SEQ ID.NO .: 20)

YARVRRRGPRRGGGLSELDDRADALQAGASQFETSAAKLKR

PC12 cells ( Peochromocytoma , ATCC CRL-1721) were thawed in T75 (Nunc) in 15 mL of F-12K medium (Gibco) in 2 mM L-glutamine, 1.5 g / L sodium bicarbonate, 15% horse serum, 2.5% FBS. After culturing for 2 days, the cell density was 625,000 cells / cm 2, the supernatant was removed by centrifugation, and cultured for one day by adding F-12K medium and drugs of the same volume, respectively, at a concentration of Ca ²⁺ 10 uM. The amount of catecholamine stimulated and secreted was analyzed via fluorescence HPLC. The concentration of drugs was put in a concentration (M) from 10 ^-6 to 10 ^-5 . The results are shown in Table 1. As shown in Table 1, the compound represented by the formula (1) used in the present invention was found to be excellent in the secretion inhibitory effect of the neurotransmitter.

TABLE 1

Inhibitory effect of neurotransmitter on polypeptide

Negative: no stimulation

Positive: Ca ²⁺ stimulation and no drug

Example 4 Intracellular Delivery Effect by PTD Conjugation

In order to analyze the intracellular delivery effect of PTD binding, the fluorescent substance FITC (Sigma, Fluorescein 5-isothiocyanate, 27072-45-3) was applied to SBDF1 peptide conjugated with PTD and unconjugated SBDF1 peptide. Labeling was analyzed by Facs (FACSCalibur, Becton Dickinson) (FIG. 5). As a result, according to the conjugation of PTD, it was analyzed that the effect of improving delivery effect was about 50 times.

Example 5 Myofascial Effect Test by PTD Conjugate

Since botulinum neurotoxin and the peptide of the present invention block the neurotransmitter and cause muscle paralysis, the injection of botulinum neurotoxin and peptide into the forearm muscle of rats was applied to the electrical stimulation of the sciatic nerve at 1, 2 and 3 weeks. The amplitude (dY) of the compound muscle action potential (CMAP) of the forearm muscle was measured. First, 8-week-old SD rats (source: Hyochang) were injected with bolulinum neurotoxin A (manufacturer: allergan, purchased from Sigma) and samples 1, 3, and 6 into 5 μl of the right forearm muscle. In the method of injection of botulinum neurotoxin, 400 U of botulinum neurotoxin was dissolved in 1 ml of physiological saline to prepare a solution of 400 U / ml, and 5 µl of the rat was injected once. Sample 1, Sample 3, and Sample 6 were each administered 10 ng, 100 ng, and 500 ng per rat.

The compound muscle action potential was measured by intraperitoneal injection of a ketamine / xylazine cocktail into the rat and anesthetized the limb with the rat placed on the abdomen on an examination table maintained at 30 ° C. The hip joint, knee joint and ankle joint of the lower limbs to be examined were fixed at 90 degrees, and the recording electrode, reference electrode, and ground electrode were attached to the forearm bone, calcaneus tendon, and sole of the foot, respectively. The stimulation electrode (+) needle was inserted into the hip, and the stimulation electrode (-) needle was inserted into the pubic bone of the posterior bone and the femoral femur. Stimulation of the sciatic nerve was induced by flowing a current in the range of 1 to 5 microA through the stimulation electrode to induce the complex muscle action potential of the forearm muscle. Repeated complex muscle action potential was induced by stimulating 50 times at 2Hz with ˜150% intensity of stimulus intensity indicating maximum amplitude. Repeated stimulation of 50 times at 20Hz after 5 minutes rest induced repeated complex muscle action potential. These signals are amplified 500 times in bioelectric amplifiers and signal processors (CyberAmp380, Axon Instruments, Inc.), and then filtered into a computer using a signal acquisition device (Digidata1320, Axon Instruments, Inc.) in the range 300 to 3000 Hz. Is indicated.

The signal stored in the computer was analyzed to calculate the difference between the amplitude of the negative peak and the positive peak (dY) of the 50 potential CMAP at 2 Hz and the delay time (tC) between the time points of the electrical stimulation and the negative peak (FIG. 6). .

The botulinum neurotoxin and the result of complex muscle action potential for the time of samples 1, 3, and 6 were completely paralyzed according to the injection of 5 units of botulinum neurotoxin, as shown in FIG. Weak muscle paralysis of about% was shown, and sample 3 and 6 showed the same effect as botulinum neurotoxin as the dose of substance increased.

Although the above has been described with reference to preferred embodiments of the present invention, those skilled in the art will be able to variously modify and change the present invention without departing from the spirit and scope of the invention as set forth in the claims below. It will be appreciated.

As described above, the fusion peptide according to the present invention is excellent in intracellular delivery efficiency and delivery efficiency to the local site, and thus effectively inhibits the secretion of neurotransmitters by the fusion peptide.

<110> FORHUMANTECH CO., LTD. <120> FUSION POLYPEPTIDE FOR INHIBITING NEUROTRANSMITTER SECRETION AND          METHOD FOR DELIVERING IT <130> IPN-34385 <160> 20 <170> KopatentIn 1.71 <210> 1 <211> 206 <212> PRT <213> Artificial Sequence <220> <223> SNAP25 <400> 1 Met Ala Glu Asp Ala Asp Met Arg Asn Glu Leu Glu Glu Met Gln Arg   1 5 10 15 Arg Ala Asp Gln Leu Ala Asp Glu Ser Leu Glu Ser Thr Arg Arg Met              20 25 30 Leu Gln Leu Val Glu Glu Ser Lys Asp Ala Gly Ile Arg Thr Leu Val          35 40 45 Met Leu Asp Glu Gln Gly Glu Gln Leu Glu Arg Ile Glu Glu Gly Met      50 55 60 Asp Gln Ile Asn Lys Asp Met Lys Glu Ala Glu Lys Asn Leu Thr Asp  65 70 75 80 Leu Gly Lys Phe Cys Gly Leu Cys Val Cys Pro Cys Asn Lys Leu Lys                  85 90 95 Ser Ser Asp Ala Tyr Lys Lys Ala Trp Gly Asn Asn Gln Asp Gly Val             100 105 110 Val Ala Ser Gln Pro Ala Arg Val Val Asp Glu Arg Glu Gln Met Ala         115 120 125 Ile Ser Gly Gly Phe Ile Arg Arg Val Thr Asn Asp Ala Arg Glu Asn     130 135 140 Glu Met Asp Glu Asn Leu Glu Gln Val Ser Gly Ile Gly Asn Leu 145 150 155 160 Arg His Met Ala Leu Asp Met Gly Asn Glu Ile Asp Thr Gln Asn Arg                 165 170 175 Gln Ile Asp Arg Ile Met Glu Lys Ala Gln Ala Asn Lys Thr Arg Ile             180 185 190 Asp Glu Ala Asn Gln Arg Ala Thr Lys Met Leu Gly Ser Gly         195 200 205 <210> 2 <211> 117 <212> PRT <213> Artificial Sequence <220> <223> VAMP2 <400> 2 Met Ser Ala Thr Ala Ala Thr Ala Pro Pro Ala Ala Pro Ala Gly Glu   1 5 10 15 Gly Gly Pro Pro Ala Pro Pro Pro Asn Leu Thr Ser Val Asn Arg Arg              20 25 30 Leu Gln Gln Thr Gln Ala Gln Val Asp Glu Val Val Asp Ile Met Arg          35 40 45 Val Asn Val Asp Lys Val Leu Glu Arg Asp Gln Lys Leu Ser Glu Leu      50 55 60 Asp Asp Arg Ala Asp Ala Leu Gln Ala Gly Ala Ser Gln Phe Glu Thr  65 70 75 80 Ser Ala Ala Lys Leu Lys Arg Lys Tyr Trp Trp Lys Asn Leu Lys Met                  85 90 95 Met Ile Ile Leu Gly Val Ile Cys Ala Ile Ile Leu Ile Ile Ile Ile             100 105 110 Val Tyr Phe Ser Ser         115 <210> 3 <211> 288 <212> PRT <213> Artificial Sequence <220> <223> Syntaxin <400> 3 Met Lys Asp Arg Thr Gln Glu Leu Arg Thr Ala Lys Asp Ser Asp Asp   1 5 10 15 Asp Asp Asp Val Thr Val Thr Val Asp Arg Asp Arg Phe Met Asp Glu              20 25 30 Phe Phe Glu Gln Val Glu Glu Ile Arg Gly Phe Ile Asp Lys Ile Ala          35 40 45 Glu Asn Val Glu Glu Val Lys Arg Lys His Ser Ala Ile Leu Ala Ser      50 55 60 Pro Asn Pro Asp Glu Lys Thr Lys Glu Glu Leu Glu Glu Leu Met Ser  65 70 75 80 Asp Ile Lys Lys Thr Ala Asn Lys Val Arg Ser Lys Leu Lys Ser Ile                  85 90 95 Glu Gln Ser Ile Glu Gln Glu Glu Gly Leu Asn Arg Ser Ser Ala Leu             100 105 110 Asp Arg Ile Arg Lys Thr Gln His Ser Thr Leu Ser Arg Lys Phe Val         115 120 125 Glu Val Met Ser Glu Tyr Asn Ala Thr Gln Ser Asp Tyr Arg Glu Arg     130 135 140 Cys Lys Gly Arg Ile Lys Arg Gln Leu Glu Ile Thr Gly Arg Thr Thr 145 150 155 160 Thr Ser Glu Glu Leu Glu Asp Met Leu Glu Ser Gly Asn Pro Ala Ile                 165 170 175 Phe Ala Ser Gly Ile Ile Met Asp Ser Ser Ile Ser Lys Gln Ala Leu             180 185 190 Ser Glu Ile Glu Thr Arg His Ser Glu Ile Ile Lys Leu Glu Asn Ser         195 200 205 Ile Arg Glu Leu His Asp Met Phe Met Asp Met Ala Met Leu Val Glu     210 215 220 Ser Gln Gly Glu Met Ile Asp Arg Ile Glu Tyr Asn Val Glu His Ala 225 230 235 240 Val Asp Tyr Val Glu Arg Ala Val Ser Asp Thr Lys Lys Ala Val Lys                 245 250 255 Tyr Gln Ser Lys Ala Arg Arg Lys Lys Ile Met Ile Ile Is Cys Cys             260 265 270 Val Ile Leu Gly Ile Ile Ile Ala Ser Thr Ile Gly Gly Ile Phe Gly         275 280 285 <210> 4 <211> 37 <212> PRT <213> Artificial Sequence <220> <223> SBD <400> 4 Glu Ile Asp Thr Gln Asn Arg Gln Ile Asp Arg Ile Met Glu Lys Ala   1 5 10 15 Gln Ala Asn Lys Thr Arg Ile Asp Glu Ala Asn Gln Arg Ala Thr Lys              20 25 30 Met Leu Gly Ser Gly          35 <210> 5 <211> 58 <212> PRT <213> Artificial Sequence <220> <223> VBD <400> 5 Asn Arg Arg Leu Gln Gln Thr Gln Ala Gln Val Asp Glu Val Val Asp   1 5 10 15 Ile Met Arg Val Asn Val Asp Lys Val Leu Glu Arg Asp Gln Lys Leu              20 25 30 Ser Glu Leu Asp Asp Arg Ala Asp Ala Leu Gln Ala Gly Ala Ser Gln          35 40 45 Phe Glu Thr Ser Ala Ala Lys Leu Lys Arg      50 55 <210> 6 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Hph-1 <400> 6 Tyr Ala Arg Val Arg Arg Arg Gly Pro Arg Arg   1 5 10 <210> 7 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Sim-2 <400> 7 Ala Lys Ala Ala Arg Gln Ala Ala Arg   1 5 <210> 8 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Tat <400> 8 Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg   1 5 10 <210> 9 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Antp <400> 9 Arg Gln Ile Lys Ile Trp Phe Gln Asn Arg Arg Met Lys Trp Lys Lys   1 5 10 15 <210> 10 <211> 34 <212> PRT <213> Artificial Sequence <220> <223> VP22 <400> 10 Asp Ala Ala Thr Ala Thr Arg Gly Arg Ser Ala Ala Ser Arg Pro Thr   1 5 10 15 Glu Arg Pro Arg Ala Pro Ala Arg Ser Ala Ser Arg Pro Arg Arg Pro              20 25 30 Val glu         <210> 11 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> R7 <400> 11 Arg Arg Arg Arg Arg Arg Arg   1 5 <210> 12 <211> 26 <212> PRT <213> Artificial Sequence <220> <223> MTS <400> 12 Ala Ala Val Ala Leu Leu Pro Ala Val Leu Leu Ala Leu Leu Ala Pro   1 5 10 15 Ala Ala Ala Asp Gln Asn Gln Leu Met Pro              20 25 <210> 13 <211> 21 <212> PRT <213> Artificial Sequence <220> <223> pep-1 <400> 13 Lys Glu Thr Trp Trp Glu Thr Trp Trp Thr Glu Trp Ser Gln Pro Lys   1 5 10 15 Lys Lys Arg Lys Val              20 <210> 14 <211> 51 <212> PRT <213> Artificial Sequence <220> <223> Hph-1-GGG-SBD <400> 14 Tyr Ala Arg Val Arg Arg Arg Gly Pro Arg Arg Gly Gly Glu Ile   1 5 10 15 Asp Thr Gln Asn Arg Gln Ile Asp Arg Ile Met Glu Lys Ala Gln Ala              20 25 30 Asn Lys Thr Arg Ile Asp Glu Ala Asn Gln Arg Ala Thr Lys Met Leu          35 40 45 Gly Ser Gly      50 <210> 15 <211> 72 <212> PRT <213> Artificial Sequence <220> <223> Hph-1-GGG-VBD <400> 15 Tyr Ala Arg Val Arg Arg Arg Gly Pro Arg Arg Gly Gly Gly Asn Arg   1 5 10 15 Arg Leu Gln Gln Thr Gln Ala Gln Val Asp Glu Val Val Asp Ile Met              20 25 30 Arg Val Asn Val Asp Lys Val Leu Glu Arg Asp Gln Lys Leu Ser Glu          35 40 45 Leu Asp Asp Arg Ala Asp Ala Leu Gln Ala Gly Ala Ser Gln Phe Glu      50 55 60 Thr Ser Ala Ala Lys Leu Lys Arg  65 70 <210> 16 <211> 51 <212> PRT <213> Artificial Sequence <220> <223> Tat-GGG-SBD <400> 16 Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Gly Gly Gly Glu Ile   1 5 10 15 Asp Thr Gln Asn Arg Gln Ile Asp Arg Ile Met Glu Lys Ala Gln Ala              20 25 30 Asn Lys Thr Arg Ile Asp Glu Ala Asn Gln Arg Ala Thr Lys Met Leu          35 40 45 Gly Ser Gly      50 <210> 17 <211> 40 <212> PRT <213> Artificial Sequence <220> <223> Hph-1-GGG-SBDF1 <400> 17 Tyr Ala Arg Val Arg Arg Arg Gly Pro Arg Arg Gly Gly Gly Ile Met   1 5 10 15 Glu Lys Ala Gln Ala Asn Lys Thr Arg Ile Asp Glu Ala Asn Gln Arg              20 25 30 Ala Thr Lys Met Leu Gly Ser Gly          35 40 <210> 18 <211> 28 <212> PRT <213> Artificial Sequence <220> <223> Hph-1-GGG-SBDF2 <400> 18 Tyr Ala Arg Val Arg Arg Arg Gly Pro Arg Arg Gly Gly Gly Lys Thr   1 5 10 15 Arg Ile Asp Glu Ala Asn Gln Arg Ala Thr Lys Met              20 25 <210> 19 <211> 45 <212> PRT <213> Artificial Sequence <220> <223> Hph-1-GGG-VDBF1 <400> 19 Tyr Ala Arg Val Arg Arg Arg Gly Pro Arg Arg Gly Gly Gly Asn Arg   1 5 10 15 Arg Leu Gln Gln Thr Gln Ala Gln Val Asp Glu Val Val Asp Ile Met              20 25 30 Arg Val Asn Val Asp Lys Val Leu Glu Arg Asp Gln Lys          35 40 45 <210> 20 <211> 41 <212> PRT <213> Artificial Sequence <220> <223> Hph-1-GGG-VBDF2 <400> 20 Tyr Ala Arg Val Arg Arg Arg Gly Pro Arg Arg Gly Gly Gly Leu Ser   1 5 10 15 Glu Leu Asp Asp Arg Ala Asp Ala Leu Gln Ala Gly Ala Ser Gln Phe              20 25 30 Glu Thr Ser Ala Ala Lys Leu Lys Arg          35 40

Claims (6)

 Protein transduction domain (PTD) having any one amino acid sequence selected from the group consisting of SEQ ID NO .: 6 to SEQ ID NO .: 13; And inhibits secretion of neurotransmitters in vivo, wherein the binding domain of SNAP25 (SEQ ID. NO .: 4) or VAMP-2 binding domain (SEQ ID. NO .: 5) constituting the SNARE complex is conjugated Fusion polypeptides for delete The method of claim 1, characterized in that the binding domain of the protein carrier and SNAP25 (SEQ ID. NO .: 4) or the binding domain of VAMP-2 (SEQ ID. NO .: 5) is conjugated by a linker. Fusion polypeptides. The fusion polypeptide of claim 3, wherein the linker consists of three glycines. A pharmaceutical composition for alleviating skin pain containing the fusion peptide of claim 1 as an active ingredient. A pharmaceutical composition for improving skin wrinkles comprising the fusion peptide of claim 1 as an active ingredient.

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