KR101990437B1 - Peptide from Parapolybia varia having antitumor activity and uses thereof - Google Patents

Abstract

The present invention relates to peptibodies derived from snake-bite monkeys having anticancer activity and uses thereof.
The peptide according to the present invention possesses excellent anticancer activity without hemolytic activity against normal cells and thus can be usefully used as an anticancer therapeutic agent.

Description

Peptides derived from snake-bite monkeys having anticancer activity and uses thereof -

The present invention relates to a peptidic-derived peptibulant peptide having anticancer activity and an anticancer composition containing the same as an effective ingredient.

Some of the substances that play an important role in maintaining the homeostasis of organisms are physiologically active substances derived from various organisms. Many researches on many physiologically active substances have been carried out so far. Among them, researches on anti-cancer peptides are very important areas in life sciences and medicine.

Animal poisons contain a variety of functional substances including amines, lipids, proteins, peptides and other small molecules. Among them, peptides are considered to be important resources for drug development because they have high specificity and low nonspecific toxicity to the target substance (Kirk et al., 2013; Fosgerau and Hoffmann, 2015). Therefore, the development of peptide drugs for diseases such as cancer, diabetes, cardiovascular diseases, pain and epilepsy is an important goal of drug development research utilizing these advantages. The global peptides therapeutic market is expected to grow from $ 14.1 billion in 2011 to $ 25.4 billion in 2018 (Fosgerau and Hoffmann, 2015), and approximately 140 peptide therapeutics are in clinical use and approximately 500 peptide therapeutics are expected to be available in 2015 It is presumed to be in pre-clinical stage (Fosgerau and Hoffmann, 2015).

Recent studies have shown that more than 100 functional peptides are contained in single species species (Fosgerau and Hoffmann, 2015), and the venom of arthropods is known to contain a wide range of diseases such as inflammation, arthritis, multiple sclerosis and pain It is known to provide novel peptides that can provide a variety of clinically beneficial treatments (Cherniack, 2010). Most studies of functional peptides contained in animal poisons have been focused on frogs, snakes, scorpions, spiders, gastropods, etc., because their toxins do not adequately treat human health and life threatening, Because they produce enough poisons enough to be analyzed in a biochemical way.

On the other hand, ants, bees, hornets, and wasps belonging to the family Hymenoptera are known to produce poisons for various purposes such as defending themselves from predators or hunting for food Lee et al., 2016). There are various kinds of peptides such as mastoparan, chemotactic peptide and quinine which are known to share the amphipathic alpha-helix structure (Lee et al. et al., 2016). The mastoparan is the most secreted peptide in wasps belonging to the wasp and Vespidae. The gene encoding primasol blue is the most transcribed in the wasp (Vespacrabro) and is known to be the second most transcribed in the wasp (Vespa analis), and the wasp and the wasp mastoparan are known as antimicrobial, Tumor, and hemolytic activity (Yoon et al., 2015b).

Vespid chemotactic precursor (VCP) and vespakinin (abbreviated as Vespk), together with premastoparan, are known to be one of the three most widely expressed proteins in the wasps and wasps, .

However, the toxic peptides of very closely related species exhibit some mutations in the amino acid sequence, and if there is a difference in only one amino acid or some amino acid sequence, the physicochemical and chemical properties of the expressed final peptide are considerable (Kaas and Craik, 2015).

Thus, there is a need to study the diverse activities of peptides contained in the bee-secreting venom of various species.

On the other hand, the lesser paper wasp (Parapolybia varia) is a medium-sized wasp with a population living in widespread populations in Central Asia, India-Papua New Guinea, and East Asia (Katada et al., 2007; Saito-Morooka et al., 2015).

Although the snake-fawn twins are abundant in nature, they have an advantage in using peptides contained in the toxins secreted by the peaks in the production of therapeutic agents for various diseases. However, in the case of the types of peptides constituting the snake venom of snake- There is no known information about the type of VCP peptide or Vespakinin (Vespks) peptides belonging to the genus of the snake cats.

Under these circumstances, the present inventors have made intensive efforts to find a novel peptide having anticancer activity. As a result, the novel peptide isolated from the venom of the snake slough monkey showed a very low hemolytic activity in normal cells, but showed excellent antitumor effect on tumor cells And thus the present invention can be utilized effectively as an anticancer drug.

Patent Publication No. 10-2012-0062328

Kyungjae Andrew Yoon at. al., Selective anti-tumor activities of venom peptides from the lesser paper wasp Parapolybia varia, Journal of Asia-Pacific Entomology 19 (2016) 821.828

Therefore, it is a main object of the present invention to provide a peptide derived from snake-bite monkeys having anticancer activity.

It is another object of the present invention to provide an anticancer composition using the snake slough-derived pea-derived peptide.

Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention and claims.

In order to achieve the above object, the present invention provides a peptide for treating or preventing cancer, which is represented by the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2.

In the present invention, the peptide for treating and preventing cancer, which is represented by the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2, is characterized in that it is derived from a serpentine double-sheep bean genome.

For the development of a novel anticancer agent, the present inventors have investigated and analyzed the expression genome and the protein body of the functional peptide existing in the insect's poisonous liquid for many years. As a result, the whole expression genome and the protein body of the Korean native snake- The functionality of the peptide having the amino acid sequence of SEQ ID NO: 2 was verified.

In the present invention, the term " peptide " means a polymer composed of amino acids linked by an amide bond (or peptide bond). For purposes of the present invention, it is meant peptides that exhibit high tumor suppressive capacity against cancer cells.

The peptide represented by the amino acid sequence of SEQ ID NO: 1 of the present invention ( Parapolybia The term varia vespid chemotactic precursor (hereinafter abbreviated as 'PvVCP') is a virulent predatory protein isolated from the snake monkeys and translated into 76 premature peptides when first translated, post-transcription modification) are generated through the process to the final 13 amino acids long life hornets (Vespa magnifica ) with 76% sequence homology with the VCP peptide (see Figures 1A-1C).

As a result of sequencing of the immature PvVCP peptide, the molecular weight was 7.72 kDa and the pI value was 3.96. The 1 st to 25 th amino acid sequence of the immature PvVCP peptide was presumed to be a signal peptide, and glycosylation was not observed in the immature PvVCP peptide .

In addition, the mature PvVCP peptide of SEQ ID NO: 1 consists of a tridecapeptide (FWPGLILKGLGAL) comprising a positively charged amino acid residue and a plurality of hydrophobic residues. In addition, the precursor of the PvVCP peptide has a repeating sequence of several AXP (X = leucine, glutamic acid, glycine or aspartic acid) residues. PvVCP peptides were found to have high α-helix content (> 29.0%) along with various β-sheet structure contents (0.2% to 27.6%) in a hydrophobic environment.

The peptide represented by the amino acid sequence of SEQ ID NO: 2 of the present invention ( Parapolybia varia vespakinin, hereinafter abbreviated as 'PvVespk') is a vespakinin protein isolated from the snake monkeys. First translated immature PvVespk peptide is composed of 38 amino acids, bumblebee (Vespa tropica ) vespakinin peptide (see Figures 1D-1F).

Sequence analysis of the immature PvVCP peptide revealed that it was 4.12 kDa and the pI value was 12.11. The 1-21 amino acid sequence of the PvVespk peptide was presumed to be a signal peptide, and the N-glycosylation site was present in the signal peptide region of the PvVespk peptide .

In addition, the mature PvVespk peptide is composed of a dodecapeptide (GRPPGFSPFRSG) containing two positively charged amino acid residues.

On the other hand, since the conventional studies bar for the anti-cancer activity of the peptides and VCP vespakinin peptides, in the present invention longevity wasp (Vespa mandarina ) VCP and vespakinin peptides.

As a result, the PvVCP peptide of SEQ ID NO: 1 and the PvVespk peptide of SEQ ID NO: 2 of the present invention showed strong anticancer activity against the ovarian cancer cell line SK-OV-3 cell line as compared with the Vespa mandarina VCP and vespakinin peptide .

In particular, the PvVCP peptide of SEQ ID NO: 1 of the present invention showed a hemolytic activity 12.4 times lower than that of the VmVCP peptide at a concentration of 200 mM. In addition, at 24 hours and 72 hours after the elapse of 24 hours and 72 hours, the PvVCP peptides were compared with the SK-OV-3 ovarian cancer cell line and the NIH-OVCAR-3 cell line, which is an anticancer drug resistant ovarian cancer cell line, All of which had strong anticancer activities.

Based on the above results, it was confirmed that the PvVCP peptide of SEQ ID NO: 1 and the PvVespk peptide of SEQ ID NO: 2 of the present invention have a strong anticancer activity and can be effectively used as a therapeutic agent for cancer treatment.

On the other hand, the peptides of the present invention can additionally be linked to an internalization sequence or a protein transduction domain to effectively enter cells.

Recent reports have shown that a variety of cellular internalization sequences, such as the TAT transactivation domain of HIV virus, transportan, which can transport antennapedia, across the plasma membrane, are known. In addition, polyarginine is known to be an attractive tool for transporting peptides into cells by transporting peptides and proteins across the plasma membrane much more efficiently.

Thus, the peptides of the present invention may comprise a cellular internalization-transporting substance or sequence. The cellular internalization sequence may be any internalization sequence known or newly found in the art, or a conservative variant thereof. Non-limiting examples of cell internalizing transport materials and sequences include, but are not limited to, polyarginine (such as R9), Antennapedia sequence, TAT, HIVTat, Penetratin, Antp-3A (Antp mutant), Buforin II, MAP (model amphipathic peptide), KFGF, Ku70, prion, pVEC, Pep-1, SynB1, Pep-7, HN-1, BGSC (bis- guanidinium- Aniline-threonine-cholesterol).

In addition, the peptides of the invention may also include additional amino acid sequences designed for specific purposes to increase the stability of the targeting sequence, capping motif, tag, labeled residue, half-life or peptide. In addition, the peptides of the present invention can be linked to coupling partners such as effectors, drugs, prodrugs, toxins, peptides, and delivery molecules.

In addition, the peptide of the present invention can be prepared in the form of a pharmaceutically acceptable salt. Specifically, a salt can be formed by adding an acid, and examples thereof include salts with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, nitric acid, sulfuric acid and the like, organic carboxylic acids such as acetic acid and halo such as trifluoroacetic acid (Such as acetic acid, propionic acid, maleic acid, succinic acid, malic acid, citric acid, tartaric acid, salicylic acid) and acidic sugars (glucuronic acid, galacturonic acid, gluconic acid, ascorbic acid), acidic polysaccharides (such as hyaluronic acid, chondroitin sulfate, Arginic acid), and organic sulfonic acids (e.g., methane, sulfonic acid, and p-toluenesulfonic acid) including sulfonic acid esters such as chondroitin sulfate may be added to form a salt.

The peptides of the present invention can be obtained by a variety of methods well known in the art. Specifically, it can be produced by chemical synthesis such as in vitro synthesis, cell-free protein synthesis, or genetic engineering using a protein expression system. However, as described through the production of the following peptides, Is preferably used.

In addition, the peptide of the present invention can be produced by a genetic engineering method using the PvVCP peptide obtained through genome information, the amino acid sequence derived from PvVespk peptide, and the nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2.

As used herein, the term "expression vector" refers to a recombinant vector capable of expressing a peptide of interest in a suitable host cell, including a necessary regulatory element operatively linked to the expression of the gene insert. Expression vectors of the present invention include those elements normally present in a suitable expression vector, including expression regulatory elements such as promoters, operators, and initiation codons. The initiation codon and termination codon are generally considered to be part of the nucleotide sequence encoding the polypeptide and must be operative in the individual when the gene product is administered and in the coding sequence and in frame. The promoter of the vector may be constitutive or inducible.

It may also contain a signal sequence for the release of the fusion polypeptide to facilitate the separation of the protein from the cell culture. A specific initiation signal may also be required for efficient translation of the inserted nucleic acid sequence. These signals include the ATG start codon and adjacent sequences. In some cases, an exogenous translational control signal, which may include the ATG start codon, should be provided. These exogenous translational control signals and initiation codons can be of various natural and synthetic sources. Expression efficiency can be increased by the introduction of suitable transcription or translation enhancers.

The expression vector may be any conventional expression vector. For example, plasmid DNA, phage DNA, etc. may be used. Specific examples of plasmid DNA include commercial plasmids such as pUC18, pIDTSAMRT-AMP. Other examples of plasmids that can be used in the present invention include plasmids derived from E. coli (pYG601BR322, pBR325, pUC118 and pUC119), Bacillus subtilis-derived plasmids (pUB110 and pTP5), and yeast-derived plasmids (YEp13, YEp24, YCp50). Specific examples of phage DNA include lambda-phages (Charon4A, Charon21A, EMBL3, EMBL4, lambda gt10, lambda gt11 and lambda ZAP). In addition, animal viruses such as retrovirus, adenovirus or vaccinia virus, insect viruses such as baculovirus may also be used. Since the expression amount of the protein and the expression of the expression of the expression vector differ depending on the host cell, the host cell most suitable for the purpose may be selected and used.

Also, in the present invention, the term "transformation" refers to genetically stable genetics such that the nucleotide fragment migrates into the genome of the host organism to express the desired peptide.

Any transformation method of the present invention can be used, and can be easily carried out according to a conventional method in the art. Generally, the transformation methods include the CaCl ₂ precipitation method, the Hanahan method which uses a reducing material called DMSO (dimethyl sulfoxide) for the CaCl ₂ method, the electroporation method, the calcium phosphate precipitation method, the protoplasm fusion method, Agrobacterium-mediated transformation, transformation with PEG, dextran sulfate, lipofectamine, and drying / inhibition-mediated transformation methods.

The host is not particularly limited as long as it is capable of expressing the peptide of the present invention. Specific examples of hosts that can be used in the present invention include bacteria such as the bacterium Bacillus bacterium Pseudomonas putida , such as Escherichia genus Bacillus subtilis , such as E. coli , Pseudomonas (Pseudomonas) bacterium belonging to the genus Saccharomyces as MY process three Levy jiae (Saccharomyces S. cerevisiae , Schizosaccharomyces pombe , and insect cells.

According to still another aspect of the present invention, there is provided a pharmaceutical composition for preventing or treating cancer, which comprises, as an active ingredient, a peptide comprising the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2.

In the pharmaceutical composition for preventing or treating cancer according to the present invention, the cancer is selected from the group consisting of a carcinoma, a lymphoma, a blastoma, a sarcoma, a liposarcoma, a neuroendocrine tumor, The present invention relates to a method of treating a cancer selected from the group consisting of mesothelioma, schwanoma, meningioma, adenocarcinoma, melanoma, leukemia, lymphoid malignancy, squamous cell cancer, epithelial squamous cell cancer, lung cancer, small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, squamous carcinoma, of the lung, cancer of the peritoneum, hepatocellular cancer, gastric or stomach cancer, gastrointestinal cancer, pancreatic cancer, brain cancer, Glioblastoma, cervical cancer, ovarian cancer (ovar) ian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial or uterine cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney and renal cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma, , Anal carcinoma, penile carcinoma, testicular cancer, esophageal cancer, biliary tract, and head and neck cancer. Preferably, ovarian cancer can be mentioned.

The term "prevention" in the present invention means all actions that inhibit or delay the onset of cancer by administration of the peptide or composition according to the present invention.

The term "treatment" in the present invention means any action that improves or alters the cancer by the administration of the peptide or composition according to the present invention.

The term "pharmaceutically effective amount " as used herein means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment, and the effective dose level will depend on the patient's sex, age, The activity of the drug, the sensitivity to the drug, the time of administration, the route of administration and the rate of release, the duration of the treatment, factors including co-administered drugs, and other factors well known in the medical arts. The pharmaceutical composition of the present invention may be administered as an individual therapeutic agent or in combination with another therapeutic agent, and may be administered sequentially or simultaneously with a conventional therapeutic agent. In addition, the pharmaceutical composition of the present invention can be administered singly or multiply. It is important to take into account all of the above factors and to administer the amount in which the maximum effect can be obtained in a minimal amount without adverse effect, and can be easily determined by those skilled in the art. Specifically, the pharmaceutical composition of the present invention is preferably administered orally or intravenously.

Examples of pharmaceutically acceptable carriers, excipients and diluents that can be used in the pharmaceutical composition of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, , Gelatin, calcium phosphate, calcium silicate, calcium carbonate, cellulose, methylcellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil and the like.

The pharmaceutical composition of the present invention may be formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like, oral preparations, suppositories and sterilized injection solutions according to a conventional method . In the case of formulation, it is prepared using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, surfactants and the like which are generally used. Solid formulations for oral administration include tablets, pills, powders, granules, capsules and the like, which may contain at least one of the above-mentioned turmeric or herbal extracts, a linalool compound isolated therefrom or a pharmaceutically acceptable salt thereof One or more excipients such as starch, calcium carbonate, sucrose or lactose, gelatin, and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral administration include suspensions, solutions, emulsions, syrups and the like. In addition to water and liquid paraffin which are commonly used simple diluents, various excipients such as wetting agents, sweeteners, fragrances and preservatives are included . Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. Examples of the suppository base include witepsol, macrogol, tween 61, cacao paper, laurin, glycerogelatin and the like.

In another aspect, the present invention provides a method for treating cancer in an animal other than human, comprising administering the peptide or a composition comprising the peptide as an active ingredient to a subject in need of treatment.

The method of treatment according to the present invention does not mean that a method of treating an animal other than a human, but such a treatment method is ineffective in humans. In addition, in the case of humans, considering the fact that the symptom can be improved by administration of the therapeutic composition according to the present invention, it can be sufficiently used in human therapy.

The term "animal excluding human beings" in the present invention means animals, such as horses, sheep, pigs, goats, camels, nutrition, dogs, etc., except for humans having cancer whose symptoms may be improved by administration of the therapeutic composition according to the present invention . By administering the therapeutic composition according to the present invention to an animal other than a human, colon cancer can be effectively prevented and treated.

The term "administering" in the present invention means introducing a predetermined substance into an animal by any appropriate method, and the administration route of the therapeutic composition according to the present invention may be administered orally, May be administered parenterally. In addition, the therapeutic composition according to the present invention may be administered by any device capable of moving the active ingredient into the target cell.

The preferable dosage of the therapeutic composition according to the present invention varies depending on the condition and body weight of the patient or the animal to be treated, the degree of disease, the drug form, the route of administration and the period of time, but can be appropriately selected by those skilled in the art. However, for the desired effect, the pharmaceutical composition of the present invention is preferably administered at 0.01 to 10 mg / kg, preferably 0.1 to 5 mg / kg per day. The administration may be carried out once a day or divided into several times.

As described above, the peptide of the present invention possesses excellent anticancer activity against cancer cells and can be usefully used as an anticancer therapeutic agent using the same.

Figure 1 compares the primary structures of the PvVCP peptide of SEQ ID NO: 1 and the PvVespk peptide of SEQ ID NO: 2, comparing the primary structures of the PvVCP peptide precursors (Figure 1A) and comparing the primary structures of the PvVCP mature peptides (Fig. 1B), a schematic diagram of the PvVCP peptide (Fig. 1C), a comparison of the primary structure of the PvVespk peptide precursor (Fig. 1D), a comparison of the primary structure of the PvVespk mature peptide (Fig. 1E), a diagram of the PvVespk peptide 1C).
2 is a graph showing the hemolytic activity of PvVCP peptide and PvVespk peptide.
FIG. 3 is a graph showing anticancer activity of PvVCP peptide of SEQ ID NO: 1 acting on SK-OV-3 and NIH-OVCAR-3 cell lines at 24 hours and 72 hours.
4 is a graph showing the anticancer activity of PvVespk peptide of SEQ ID NO: 2 acting on SK-OV-3 and NIH-OVCAR-3 cell lines at 24 hours and 72 hours.
FIG. 5 is a graph showing the result of the circularly polarized light spectrum analysis of the PvVCP peptide of SEQ ID NO: 1 and the PvVespk peptide of SEQ ID NO: 2 (FIGS. 5A and 5D: 10 mM SPB; FIG. 5B and 5E: 30 mM SDS; 50% TFE).

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood that the scope of the present invention is not limited by these examples in accordance with the gist of the present invention, and it is to be understood by those skilled in the art that the present invention is not limited thereto It will be obvious.

Example  1. Active Of peptide  Separation, Identification, and Synthesis

1-1. Beetroot and polemic extraction, cDNA and gDNA  synthesis

( Parapolybia varia ) were collected from Gwangju Taehwasan in Gyeonggi Province, Surucsan in Gunpo, Kyonggi Province, Chiak in Gangwon Province, and Songdongdong in Andong, Gyeongbuk Province. The collected snake slings were transported to the laboratory in a live state, and immediately anesthetized with CO _{2. Then} , the slides were separated from the gastrointestinal tract by forceps with a 1X PBS buffer on a slide glass.

A total of 30 mature bees were directly isolated from the gastrocnemius for the extraction of total mRNA and synthesized total mRNA using Trizol reagent (200 μl, MRC, Cincinnati, OH, USA) according to the manufacturer's protocol.

Genomic DNA (gDNA) was extracted according to the manufacturer's protocol using a DNA extraction kit (DNeasyOBlood & Tissue Kit, Qiagen, Valencia, CA, USA) from the entire body of the snake slipper.

CDNA sequences of PvVCP and PvVespk genes were obtained based on the results of whole transcript analysis of the snake bifurcation.

CDNA was synthesized by using reverse transcriptase (SuperScript III reverse transcriptase (Invitrogen, Carlsbad, Calif., USA) for DNase I (Ambion, Austin, TA, USA)

PCR reaction was performed using the primer set of the following Table 1 using gDNA and cDNA of the snake mutant pair of beehives as a template.

[Table 1]

The PCR reaction was pre-incubated at 95 ° C for 1 min, followed by denaturation; 95 ° C, 30 sec, annealing; 50 캜 (PvVCP) or 45 캜 (PvVespk), 20 seconds, and polymeration; 35 cycles of PCR were carried out at 68 ° C for 1 minute according to the manufacturer's protocol (Advantage 2 Polymerase Mix, Clontech, Palo Alto, Calif., USA).

PCR products were separated using gels and extracted using Qiagen QIAquick gel extraction kit for sequencing.

1-2. Peptides  Synthesis and purification

The PvVCP peptide of SEQ ID NO: 1, the PvVespk peptide of SEQ ID NO: 2, and the mature peptides of VmVCP peptide and VmVespk peptide of longevity wasp used as a comparative example were synthesized using GenScript (GenScript, Piscataway, NJ, USA) , And stored at -80 [deg.] C until used for physiological activity experiments. The peptides were stored in 1% DMSO (dimethyl sulfoxide) at various concentrations.

1-3. PvVCP  and PvVespk Peptides  Character analysis

Analysis of gDNA and cDNA sequences of PvVCP and PvVespk genes revealed the intron structure contained in the gene.

The immature PvVCP peptide consists of 76 amino acids and has 76% sequence homology with the VCP of Vespamagnifica. The immature PvVespk peptide consists of 38 amino acids and the vespakinin of Vespatropica and 38 % Sequence homology.

Based on the results of the primary analysis of the PvVCP peptide and the PvVespk peptide, the immature PvVCP peptide has a molecular weight of 7.72 kDa, a pI value of 3.96, a molecular weight of 4.12 kDa of the PvVespk peptide, and a pI value of 12.11.

Also, the 1 st to 25 th amino acid sequences of the PvVCP peptides are presumed to be signal peptides, and the 1 st to 25 th amino acid sequences of the PvVespk peptides are presumed to be signal peptides. It is predicted that there is one predicted N-glycosylation site in the signal peptide region of the PvVespk peptide, and glycosylation is not expected to occur in the PvVCP peptide.

Primary structure analysis based on amino acid sequence revealed that the mature PvVCP peptide comprises a tridecapeptide (FWPGLILKGLGAL) containing a positively charged amino acid residue and a number of hydrophobic residues. In addition, the precursor of the PvVCP peptide has a repeating sequence of several AXP (X = leucine, glutamic acid, glycine or aspartic acid) residues.

In addition, the mature PvVespk peptide is composed of a dodecapeptide (GRPPGFSPFRSG) containing two positively charged amino acid residues.

Example  2. Hemolytic activity

The hemolytic activity of the peptides synthesized in Example 1-2 was analyzed using human erythrocytes purchased from Korean Red Cross. Erythrocytes were washed with an equal volume of PBS buffer and centrifuged at 960 rpm for 15 min. The above procedure was repeated three times and then re-suspended in 10% PBS buffer to use red blood cells at a final concentration of 2% (v / v). 10 [mu] l of synthetic peptides at various concentrations (10-200 mM) were incubated with 100 [mu] l of red blood cells washed for 30 min at 37 [deg.] C.

After the culture mixture was centrifuged at 960 rpm for 5 minutes, the OD value (540 nm) of the supernatant was measured using a microplate reader (VersaMax, Molecular Devices, Sunnyvale, CA, USA).

Relative hemolytic activity was measured by setting the activity of the 1% DMSO solution to 0% and the activity of the 0.1% Triton X-100 solution to 100%.

The experimental protocols were approved by the Clinical Research Committee (IRB # E1508 / 001-005, Seoul National University IRB) and the results were expressed as mean ± SE of 3 repeated measurements.

One of the known problems with functional dock peptides is hemolysis of blood cells that may lead to anaphylaxis (Lee and Vadas, 2011).

Therefore, we have tested whether synthetic PvVCP and PvVespk peptides cause hemolysis in human erythrocytes. In the hemolysis test, VmVCP and VmVespk peptides of longevity wasp known to cause hemolysis were used as a positive control.

As a result of the test, it was observed that PvVCP and VmVCP peptides showed very low hemolytic activity at low concentrations. In particular, the PvVCP peptide showed 12.4-fold lower hemolytic activity compared to the VmVCP peptide at a concentration of 200 mM (see FIG. 2).

On the other hand, unlike VCP peptides, both PvVespk and VmVespk peptides were observed to have very low hemolytic activity at a concentration of 200 mM. These results indicate that the PvVCP and VmVCP peptides of the present invention exhibit very low hemolytic activity and do not affect normal cells even when administered to a living body and do not induce anaphylaxis.

Example  3. Anticancer Activity Antitumor  activity)

SK-OV-3 strain (ATCC HTB-77), which is one of human ovarian cancer cell lines, and drug-resistant human ovarian adenocarcinoma cell line NIH-OVCAR-3 (ATCC HTB-161) strain, which is a drug-resistant human ovarian adenocarcinoma cell line, was treated with an active peptide and its anticancer effect was investigated.

The ovarian cancer cell line was prepared by seeding (1 × 10 ⁴ ) on the day before the experiment and culturing for about one day. Two kinds of prepared ovarian cancer cell lines were treated with the active peptides of the present invention at a concentration of 2.5 mM to 100 mM. The anticancer effect of the active protein of the present invention was observed using the untreated group treated with no treatment and the DMSO treated group treated with 0.001% DMSO as a control group.

After 24 hours and 72 hours, the culture medium was removed, and 100 ml of culture medium and 10 ml of MTT (5 mg / ml) were added to a well plate. ₂ < / RTI > conditions for 4 hours. After the incubation, the supernatant was removed, 100 ul of DMSO was added, and the optical density was measured at 540 nm.

As a result, it was found that the PvVCP peptide of SEQ ID NO: 1 and the PvVespk peptide of SEQ ID NO: 2 of the present invention are more potent than the Vespa mandarina VCP and vespakinin peptide in ovarian cancer cell line SK-OV-3 It was observed to have anticancer activity.

In particular, the PvVCP peptide of SEQ ID NO: 1 of the present invention showed a hemolytic activity 12.4 times lower than that of the VmVCP peptide at a concentration of 200 mM.

In addition, at 24 hours and 72 hours after the elapse of 24 hours and 72 hours, the PvVCP peptides were compared with the SK-OV-3 ovarian cancer cell line and the NIH-OVCAR-3 cell line, which is an anticancer drug resistant ovarian cancer cell line, All of which had strong anticancer activities.

Based on the above results, it was confirmed that the PvVCP peptide of SEQ ID NO: 1 and the PvVespk peptide of SEQ ID NO: 2 of the present invention have a strong anticancer activity and can be effectively used as a therapeutic agent for cancer treatment.

Example  4. Circular polarization Dichroism  Spectroscopy dichroism  spectroscopy)

The circular polarization dichroism spectrum analysis for the secondary structure analysis of PvVCP, PvVespk, VmVCP, and VmVespk peptides was performed under three conditions of 10 mM SPB (sodium phosphate buffer, pH 7.2), 30 mM SDS, and 50% TFE (trifluoroethanol) Respectively.

The spectra were measured three times using a 0.1 cm path length quartz cuvette at 298 K using a JASCO J-715 Spectropholar Limiter (JASCO, Easton, MD, USA) and the mean value was used.

The protein sample concentration was 0.1 mg / ml and the data were measured at 0.1 nm bandwidth, 1 second response, and scan rate of 0.1 nm resolution, and the ratios of alpha helix, beta sheet, turn, and random coil structures were calculated.

FIG. 5 shows the results of the secondary structure analysis of the peptides synthesized in Example 1-2 through circular dichroism (CD) spectral analysis. As a result, all the peptides appeared to have mainly disordered structures in 10 mM SPB solution (see FIGS. 5A and 5D). On the contrary, it showed a distinct secondary structure under 30 mM SDS and 50% TFE solution.

VCP peptides were found to have high α-helix content (> 29.0%) along with various β-sheet structure contents (0.2% to 27.6%) in a hydrophobic environment. In addition, Vespk peptides have a low content of α-helix structure in a hydrophobic environment and have a high content of β-sheet structure (see Table 2).

[Table 2]

<110> NATIONAL INSTITUTE OF BIOLOGICAL RESOURCES          Industry Academic Cooperation Foundation, Hallym University <120> Peptide from Parapolybia varia having antitumor activity and uses          the <130> PN170006AN <160> 2 <170> KoPatentin 3.0 <210> 1 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> peptide of Parapolybia varia <400> 1 Phe Trp Pro Gly Leu Ile Leu Lys Gly Leu Gly Ala Leu   1 5 10 <210> 2 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> peptide of Parapolybia varia <400> 2 Gly Arg Pro Pro Gly Phe Ser Pro Phe Arg Ser Gly   1 5 10

Claims (7)

A peptide for the treatment and prevention of ovarian cancer comprising the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2. The method according to claim 1,
Wherein said peptide is derived from a serpentine monkey &lt; RTI ID = 0.0 &gt; genome. &Lt; / RTI &gt; delete A pharmaceutical composition for the prevention or treatment of ovarian cancer, which comprises a peptide consisting of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2 as an active ingredient. delete 5. The method of claim 4,
Wherein the ovarian cancer is an anticancer drug resistant ovarian cancer. 1. A method for treating ovarian cancer in an animal other than a human, comprising administering to a subject in need thereof a peptide consisting of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2.

Images