KR102092843B1 - Peptides for prevention and treatment for cancer and use thereof - Google Patents

Abstract

The present invention relates to a peptide for preventing or treating cancer and uses thereof, and more particularly, to a peptide for preventing or treating cancer, and a pharmaceutical composition and a reagent composition comprising the same. The peptide represented by the amino acid sequence of SEQ ID NO: 1 according to the present invention contains a PKB substrate motif and can be useful as a peptide for prevention and treatment of various cancers. In addition, it is a peptide with increased safety and high cell permeability, which can effectively suppress tumor progression, and can be used in various ways, such as medicines and functional foods that can prevent, improve or treat cancer. In addition, it regulates the pathways involved in the development of cancer, so it can be usefully used to study cancer progression inhibition related to the pathways.

Description

Peptides for prevention and treatment for cancer and use thereof

The present invention relates to a peptide for preventing or treating cancer and uses thereof, and more particularly, to a peptide for preventing or treating cancer, and a pharmaceutical composition and a reagent composition comprising the same.

Molecular profiling by The Cancer Genome Atlas (TCGA) research network identified a very frequent mutation for oncogene KRAS (33%) in 230 lung adenocarcinoma tumors. However, these KRAS proteins were considered uncontrollable for many years. In addition, the KRAS protein did not have a pocket suitable for binding the drug except for the GDP / GTP binding site. In order to avoid direct targeting for these KRAS proteins, alternative studies of KRAS sub-paths have been conducted. KRAS signaling through PI3K / PKB and RAF / MEK / ERK pathways regulates processes such as survival and cell growth.

Protein kinase B (PKB), also known as AKT, is the most active in lung cancer, among other cancers. This PKB is a signal protein that phosphorylates other proteins to activate signals. In addition, nuclear factor-kappa B (NF-κB), a downstream effector of PKB, is an important transcription factor for cancer development preventing cell proliferation, cell death, onset and metastasis. Transmembrane Protein 39A (TMEM39A) is one of the family of TMEM proteins containing helical domains.

In recent years, peptides defined as 40 or fewer amino acids have attracted attention as new drugs that can cure cancer. In 2010, Sipuleucel-T became the first commercially available peptide-based vaccine against prostate cancer as a cancer treatment. In addition, recent research has focused on improving the biological stability of peptides to overcome the problems of low biological stability and rapidly degrading peptides.

Accordingly, the present inventors completed the present invention by confirming that the human TMEM39A PKB substrate motif-derived peptide TMEM39AS41, which has N-terminus-bonded myristic acid, is an important factor for use as a drug and exhibits high cell permeability and effective tumor progression inhibitory effect. Became.

Accordingly, an object of the present invention is to provide a cancer prevention or treatment peptide consisting of the amino acid sequence represented by SEQ ID NO: 1, a pharmaceutical composition and a reagent composition for preventing or treating cancer comprising the peptide as an active ingredient.

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

In addition, the present invention provides a pharmaceutical composition for preventing or treating cancer, comprising the peptide as an active ingredient.

In addition, the present invention includes the peptide as an active ingredient, in vitro (in vitro) NFκB (nuclear factor kappa-light-chain-enhancer of activated B cells) p65 expression suppression or LC3B (light chain 3 B) Ⅱ / LC3BI Provided is a reagent composition for reducing levels.

The peptide represented by the amino acid sequence of SEQ ID NO: 1 according to the present invention contains a PKB substrate motif and can be useful as a peptide for prevention and treatment of various cancers. In addition, the peptide of the present invention is a peptide having increased safety and high cell permeability, which can effectively inhibit tumor progression, and can be used in various ways as medicines, functional foods, etc., to prevent, improve or treat cancer. have. In addition, it regulates the pathways involved in the development of cancer, so it can be usefully used to study cancer progression inhibition related to the pathways.

1 is a diagram showing the results confirming that TMEM39A mRNA expression is increased in most cancer types including lung adenocarcinoma through TCGA RNA sequencing data.
2 is a view showing a schematic diagram for the TMEM39AS41 peptide of the present invention. The underlined S represents the phosphorylated site, and h represents the helical region.
3 is a diagram showing the results of confirming that phosphorylation is significantly increased in serine 41 of the present invention TMEM39AS41 peptide as a result of ELISA-based in vitro kinase analysis.
4 is a view showing the result of confirming that the fluorescent signal of the biotin-labeled TMEM39AS41 peptide as a control result is not detected as a result of cell permeability.
5 is a view showing the results of confirming the cell permeability confirmed Myristic acid-TMEM39AS41 peptide of the present invention shows excellent cell permeability.
6 is a view showing the results of real-time cell analysis confirming that the Myristic acid-TMEM39AS41 peptide of the present invention inhibits cell proliferation compared to the control group.
7 is a view showing the results of confirming the color of the culture medium after 167 hours of peptide treatment, and confirming that cell growth is inhibited when treating the Myristic acid-TMEM39AS41 peptide of the present invention.
8 is a diagram showing the result of confirming that sudden cell death occurs during Myristic acid-TMEM39AS41 peptide treatment of the present invention as a result of JuLI ™ analysis for 180 hours after peptide treatment.
9 is a diagram showing the results of immunoblot analysis confirming that the Myristic acid-TMEM39AS41 peptide of the present invention significantly reduces the expression level of NFκB p65 and the level of light chain 3 B (LC3B) Ⅱ / LC3BI in A549 cells.
10 is a diagram showing the result of numerically quantifying the results in FIG. 9 and showing the relative protein content in a graph.
11 is a diagram schematically showing an administration protocol for administering the Myristic acid-TMEM39AS41 peptide of the present invention diluted with DPBS to a KRAS-mutated NSCLC mouse model (KRAS ^LA1 mouse).
12 is a view showing an image of a lung tumor obtained on the day of sacrifice of the KRAS ^LA1 mouse.

The present invention provides a peptide for preventing or treating cancer, consisting of the amino acid sequence represented by SEQ ID NO: 1.

The peptide of the present invention is a peptide derived from human TMEM39A . In addition, as a peptide containing a motif represented by the amino acid sequence of RXRXXS containing Ser 41 (serine 41, S41), it was named “TMEM39AS41”. The X may be selected from the group consisting of A, K, N, D, C, H, I, M, S, V, G, L, P, T, F, R, Y, W, E and Q. have.

In addition, the motif is a substrate motif of PKB and by containing it, it can have a higher binding affinity with PKB and can be used as a peptide for prevention and treatment of various cancers. In addition, the motif is PKB (protein kinase B), ROCK (rho-associated protein kinase), RSK (ribosomal protein kinase), S6K (S6 kinase) and SGK (serum glucocorticoid kinase) selected from the group consisting of one or more kinases Can be phosphorylated, but is not limited thereto. In addition to these PKBs, phosphorylation by various cancer-related kinases can act on various cancers through various pathways.

The peptide may be a peptide in which the N-terminus is myristoylated. By combining the N-terminus with myristic acid through the myristoylation, without increasing the sequence of the peptide for imparting cell permeability, the TMEM39AS41 peptide of the present invention increases the cell permeability, which is an important factor for use as a drug in the future. You can.

In addition, the peptide may be a C-terminal amidated peptide. By amidating the C-terminus, it is possible to prevent the degradation of the TMEM39AS41 peptide of the present invention to improve biological stability.

Therefore, the peptide of the present invention may preferably be a peptide in which the N-terminus is myristoylated and the C-terminus is amidated.

The cancer of the present invention is lung cancer, eye cancer, skin cancer, uterine cancer, thyroid cancer, thymoma, testicular cancer, stomach cancer, sarcoma, prostate cancer, pancreatic cancer, adrenal cancer, mesothelioma, malignant melanoma, uveal melanoma, liver cancer, head and neck cancer, brain cancer, nerve Glioblastoma, glioblastoma, esophageal cancer, malignant lymphoma, bile duct cancer, cervical cancer, breast cancer, bladder cancer, leukemia, oral cancer, laryngeal cancer, small intestine cancer, and colon cancer may be one or more selected from the group, but is not limited thereto.

The peptide represented by the amino acid sequence of SEQ ID NO: 1 according to the present invention contains a PKB substrate motif and can be useful as a peptide for prevention and treatment of various cancers. In addition, as a peptide having increased safety and high cell permeability, it can be effectively used as a drug.

The present invention also provides a pharmaceutical composition for preventing or treating cancer, comprising the peptide as an active ingredient.

The pharmaceutical composition of the present invention may further include suitable carriers, excipients and diluents commonly used in the manufacture of pharmaceutical compositions. In addition, additives for the preparation of a solid or liquid may be used in the preparation of the pharmaceutical composition. The additive for formulation may be either organic or inorganic.

Examples of excipients include lactose, sucrose, white sugar, glucose, corn starch, starch, talc, sorbit, crystalline cellulose, dextrin, kaolin, calcium carbonate, silicon dioxide, and the like. Examples of the binder include polyvinyl alcohol, polyvinyl ether, ethyl cellulose, methyl cellulose, gum arabic, tragacanth, gelatin, shellac, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, calcium citrate, Dextrin, pectin, and the like. Examples of the lubricant include magnesium stearate, talc, polyethylene glycol, silica, and cured vegetable oil. Any colorant can be used as long as it is normally permitted to be added to pharmaceuticals. These tablets and granules can be appropriately coated according to sugar content, gelatin coating, or other needs. In addition, preservatives, antioxidants and the like can be added as necessary.

In addition, the pharmaceutical composition of the present invention may be prepared in any formulation conventionally prepared in the art (e.g., Remington's Pharmaceutical Science, latest edition; Mack Publishing Company, Easton PA), and the form of the formulation is not particularly limited. However, it can be used in the form of oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, external preparations, suppositories, and sterile injectable solutions according to conventional methods, but is not limited thereto. .

The pharmaceutically effective amount of the present invention may vary depending on the type of wound, the application site, the number of treatments, the treatment time, the formulation, the patient's condition, and the type of adjuvant. The amount of use is not particularly limited, but when the daily effective amount of the pharmaceutical composition of the present invention is applied to a patient, it may be 0.00001 to 10000 μg. The above daily amount may be administered once a day or divided into 2-3 times a day at appropriate intervals, or may be administered intermittently at intervals of several days.

However, the amount of the pharmaceutical composition of the present invention may include the route of administration, the age of the patient, the sex, the weight, the severity of the patient, the type of wound, the application site, the number of treatments, the treatment time, the formulation, the patient's condition, and the type of adjuvant. As determined in light of a number of relevant factors, the effective amount should not be construed as limiting the scope of the invention in any aspect.

When using a pharmaceutical composition comprising a peptide for preventing or treating cancer consisting of the amino acid sequence represented by SEQ ID NO: 1 of the present invention as an active ingredient, it is possible to effectively suppress the progression of a tumor, and prevent, improve or treat cancer. It can be useful for the manufacture of pharmaceutical products.

The present invention also provides a method for preventing or treating cancer, comprising administering the peptide to an individual.

In the present invention, "individual" means a mammal including humans, cows, dogs, pigs, chickens, sheep, and horses, but is not limited thereto. The subject of the present invention includes a patient in need of cancer treatment, a patient who has undergone cancer treatment, and a patient in need of cancer treatment as a patient in need of prevention or treatment of cancer. Patients administered may also be included.

 In addition, the peptide of the present invention may be treated in combination with other existing drugs or treatment methods for cancer treatment. When the peptide of the present invention is used in combination, it may be treated simultaneously or sequentially with other drugs or treatment methods for the treatment of cancer.

The present invention also includes the peptide as an active ingredient, in vitro (in vitro) NFκB (nuclear factor kappa-light-chain-enhancer of activated B cells) p65 expression suppression or LC3B (light chain 3 B) Ⅱ / LC3BI Provided is a reagent composition for reducing levels.

The NFκB and LC3B are important factors for cancer development related to cell proliferation and cell death. The peptide represented by the amino acid sequence of SEQ ID NO: 1 of the present invention can inhibit the expression of NFκB and effectively inhibit the level of LC3B (light chain 3 B) Ⅱ / LC3BI, so the reagent containing the peptide of the present invention as an active ingredient When the composition is used, it can be confirmed that the expression of NFκB p65 in vitro is suppressed or the level of light chain 3 B (LC3B) II / LC3BI is reduced, which can be useful in cancer progression inhibition studies.

The present invention also includes the steps of processing the peptide; Including, in vitro (NFKB) (nuclear factor kappa-light-chain-enhancer of activated B cells) p65 expression suppression or LC3B (light chain 3 B) Ⅱ / LC3BI level reduction method.

The present invention also provides a food composition for preventing or improving cancer, comprising the peptide as an active ingredient.

The "food" of the present invention can be prepared in all forms, such as functional food, nutritional supplement, health food, and food additives. For example, as a health food, sialyl lactose of the present invention may be prepared in the form of tea, juice, and drink for drinking or granulated, encapsulated, and powdered. In addition, functional foods include beverages (including alcoholic beverages), fruits and processed foods (e.g. canned fruits, canned foods, jams, marmalade, etc.), fish, meat, and processed foods (e.g. ham, sausage, corn beef, etc.) , Breads and noodles (e.g. udon, buckwheat noodles, ramen, spaghetti, macaroni, etc.), juice, various drinks, cookies, syrup, dairy products (e.g. butter, cheats, etc.), edible vegetable oils, margarine, vegetable protein, retort foods , Frozen food, various seasonings (eg, miso, soy sauce, sauce, etc.) can be prepared by adding the peptide represented by the amino acid sequence of SEQ ID NO: 1 of the present invention.

The present invention also provides a quasi-drug composition for preventing or improving cancer, comprising the peptide as an active ingredient.

The term “quasi-drug” may be an article that has a weak effect on the human body or does not directly act on the human body, and is an apparatus, machine, or device among articles used for the purpose of diagnosing, treating, reducing, treating, or preventing diseases of a human or animal. This refers to items other than those that are not utensils, machines, or devices that are used for the purpose of having a pharmacological effect on the structure and function of a person or animal.

When the quasi-drug composition of the present invention is used as a quasi-drug additive, the composition may be added as it is or used with other quasi-drugs or quasi-drug components, and may be suitably used according to a conventional method. The mixing amount of the active ingredient can be appropriately determined according to the purpose of use.

When using a food composition or quasi-drug composition comprising a peptide for preventing or treating cancer consisting of the amino acid sequence represented by SEQ ID NO: 1 of the present invention as an active ingredient, it is possible to safely and effectively suppress the progression of cancer. It can be useful for the production of functional foods and quasi-drugs that can be improved or treated.

Hereinafter, the present invention will be described in more detail based on examples. The examples are only intended to illustrate the present invention in more detail, and it will be apparent to those skilled in the art that the scope of the invention is not limited by these examples according to the gist of the present invention.

Materials-peptides, compounds and antibodies.

All peptides were purchased from GL Biochem (Shanghai) Ltd (Shanghai, China). Each peptide was N-terminal biotinylated, N-terminal myristic acid bound, or C-terminal FITC bound and C-terminal amidated. The peptides were more than 90% pure and stored under minus 20 ° C under 10 mM or 100 mM DMSO (dimethylsulphoxide) storage solution. The myristic acid-binding peptide for intravenous infusion in 100 mM DMSO stock solution was diluted with DPBS (Dulbecco's phosphate buffered saline), boiled at 100 ° C. for 5 minutes, and then dissolved by vortexing. Alexafluor 488 binding streptavidin (s11223) was purchased from Life Technologies.

The source of the antibody used is as follows. LC3B (L7543) was purchased from Sigma-Aldrich, NFκB p65 (sc8008) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA), and phospho-AKT substrate (RXRXXS / Tp) (# 10001) was Cell Signaling Technologies (Beverly) , MA, USA).

Experimental Example 1. Cell culture and transfection

A549 cells were added with 10% fetal bovine serum (Biowest, Kansas City, MO, USA) and 1% antibiotic-antibacterial (Life Technologies, Inc., Carlsbad, CA, USA) Dulbecco's modified Eagle medium / high-glucose (WELGENE) Inc., Daegu, South Korea) at 37 ° C., 5% CO ₂ . Cells were then transduced using the Neon® transformation system (Invitrogen, Carlsbad, CA, USA) for electroporation.

Experimental Example 2. Immunofluorescence

A549 cells were cultured in 8-well chamber slides (Thermo Fisher Scientific, Pittsburgh, PA, USA), treated with peptide, and then 3% paraformaldehyde (Electron Microscopy Sciences, Hatfield, PA, USA) for 10 minutes at room temperature. Was fixed on. Permeation with 0.5% Triton X-100 (Sigma-Aldrich, St Louis, MO, USA) for 5 minutes, then blocking for 30 minutes, 0.1% saponin (Sigma-Aldrich) and 3% bovine serum albumin (Biosesang, Seongnam, Korea) was incubated with alexafluor 488 bound streptavidin (s11223, Life Technologies) in a blocking solution contained in PBS (phosphate-buffered saline). The slides were then washed with PBS and covered with cover glass.

Experimental Example 3. Immunoblot analysis

For immunoblot analysis, cells were 50 mM Tris-HCl, 1% v / v Nonidet P-40, 40 mM β-glycerol phosphate, 120 mM NaCl, 25 mM sodium fluoride, 0.1 mM sodium orthovanadate, 1 mM After dissolving in pH 7.5 lysis buffer containing 2 mM microcystin-LR, 1 mM benzamidine was treated at 4 ° C. for 30 minutes. Thereafter, centrifugation was performed at 13,000 × g for 30 minutes, protein concentration was measured, and the same amount of lysate was used for immune blocking. Western blotting was performed with labeled primary antibody and anti-rabbit antibody bound to horseradish peroxide (KOMA Biotechnology, Seoul, Korea) and chemiluminescence through X-ray film exposure (Agfa-Gevaert NV, Morstel, Antwerp, Belgium) And visually confirmed.

Experimental Example 4. in vitro (Ex vivo) Kinase assay-based ELISA

Biotin bound TMEM39AS41 peptide was immobilized on streptavidin coated plate for 1 hour at room temperature. The peptide-coated plate was washed 3 times with a washing solution (PBS containing 0.05% Tween20 (Sigma-Aldrich)). The coated peptide was incubated with A549 cell lysate at 30 ° C for 30 minutes. The plate was washed 3 times and anti-phospho-AKT substrate antibody (3: 1000) was added at room temperature for 30 minutes. The plate was then washed 3 times and HRP-bound secondary antibody (3: 1000) was added at room temperature for 30 minutes. After the last 3 washes, 100 μl of TMB substrate solution (Pierce, Rockford, IL, USA) was incubated at 30 ° C. for 6 minutes, and the absorbance was 650 using an EZ Read 800 microplate reader (Biochrom, Berlin, Germany). nm.

Experimental Example 5. In vivo  (In vivo) KRAS lung cancer mouse model experiment

All animals used in this experiment were maintained with the animal protocol according to the guidelines of Chungnam National University, and this experiment was approved by the Animal Experiment and Use Committee (IACUC) of Chungnam National University. KRAS ^LA1 mice, a model approved as a model of human non-small cell lung cancer, were purchased from the Human Cancer Consortium-National Cancer Institute (Frederick, MD, USA). Animals were then maintained in a laboratory animal facility under a light and dark cycle of 12 hours while maintaining temperature and relative humidity at 12 ± 2 ° C. and 40 ± 20% respectively. The experiment was performed with 5 week old male KRAS ^LA1 mice (each n = at least 4 per group). All mouse groups, DMSO group and peptide (30 mg / kg body weight) group were injected with DMSO once per mouse by tail vein injection for 3 weeks and injected with 150 μl of DPBS diluted peptide three times a week. Mice were sacrificed approximately 24 hours after the final injection and individual tumors were numbered and collected for further study.

Experimental Example 6. Statistical Analysis

Data are expressed as mean ± SEM. Student's t- test was used for comparison between the two groups. All statistical analyzes were performed using GraphPad Software version 5.01 (GraphPad Software, San Diego, CA, USA).

Example 1. TMEM39A  Confirm the relationship between expression and cancer progression

First, in order to confirm whether TMEM39A plays a pivotal role in human cancer, TMEM39A mRNA expression was monitored in multiple malignancies, and this is shown in FIG. 1.

1, TMEM39A mRNA expression through cancer sequencing data of Cancer Genome Atlas (TCGA) (cBioPortal: http://www.cbioportal.org/index.do.) It was confirmed that the increase in cancer type.

Example 2. TMEM39AS41 peptide design and phosphorylation confirmation

Ser 41 (serine 41, S41) of human TMEM39A is included in the PKB binding motif (RXRXXS). Also, many kinases such as ROCK, RSK, S6K and SGK that recognize and phosphorylate the motif are known. Accordingly, in the present invention, a peptide comprising the RXRXXS motif in TMEM39A and consisting of the amino acid sequence GLRNRNGSAIGLPVP was designed and named as TMEM39AS41. The schematic diagram of the peptide TMEM39AS41 of the present invention is shown in FIG. 2.

In addition, TMEM39AS41 peptide phosphorylation in Serine 41 was performed with A549 cell lysates containing various kinases including PKB, ROCK, RSK, S6K and SGK, and the ELISA based in vitro kinase analysis results are shown in FIG. 3.

As shown in Figure 3, it was confirmed that the phosphorylation of the TMEM39AS41 peptide of the present invention in serine 41 is significantly increased compared to the control group.

These results indicate that the TMEM39AS41 peptide of the present invention is phosphorylated by a kinase that recognizes the RXRXXS motif.

Example 3. Confirmation that Myristic acid binding of TMEM39AS41 peptide increases intracellular uptake

In this example, in order to examine the cell permeability of the TMEM39AS41 peptide, the N-terminal biotin-labeled peptide was cultured in A549 cells for 90 minutes. The results of the CLSM analysis are shown in FIG. 4.

4, it was confirmed that the fluorescent signal of the biotin-labeled TMEM39AS41 peptide was not detected at all concentrations.

Next, the N-terminal of the TMEM39AS41 peptide was combined with myristic acid, and the result of CLSM analysis for this was treated in A549 cells for 15 minutes, and the results of this analysis are shown in FIG. 5.

As shown in Figure 5, Myristic acid-TMEM39AS41 peptide coupled with myristic acid was confirmed that the intracellular uptake was increased in a concentration-dependent manner. These results indicate that binding of the N-terminal myristic acid increased cell permeability of the peptide.

Example 4. Confirm that the TMEM39AS41 peptide inhibits cell growth in A549 lung cancer cell line

To investigate the effect of the TMEM39AS41 peptide on cell growth, the proliferation of A549 cells was analyzed using a real-time cell analyzer, and the results are shown in FIG. 6.

6, it was confirmed that the Myristic acid-TMEM39AS41 peptide of the present invention inhibits cell proliferation in a concentration-dependent manner compared to the control and biotin-TMEM39AS41 peptides. In particular, when the Myristic acid-TMEM39AS41 peptide of the present invention was treated with a 30 μM dose for 48 hours, the cell growth rate was significantly reduced compared to the control group, and it was confirmed that cell death was observed after 120 hours.

In addition, the color of the culture medium was confirmed after 167 hours of peptide treatment, and the results are shown in FIG. 7.

As shown in FIG. 7, in the Myristic acid-TMEM39AS41 peptide (30 μM) group, it was confirmed that the peptide inhibited cell growth by showing a red color indicating a higher pH than the control group.

In addition, JuLI ™ analysis was performed for 180 hours after peptide treatment, and the results are shown in FIG. 8.

As shown in FIG. 8, in the Myristic acid-TMEM39AS41 peptide (30 μM) group, rapid cell death was observed compared to the control group.

These results indicate that the Myristic acid-TMEM39AS41 peptide of the present invention causes cell death in A549 cells.

Example 5. Confirmation that Myristic acid-TMEM39AS41 peptide inhibits the inflammatory / autophagy pathway

Previous studies have shown that auto-digestion is essential for inducing inflammation, and this auto-digestion plays a pivotal role in the pathogenesis of immune diseases, including cancer. Therefore, in order to confirm whether inflammation and autodigestion signals are affected by TMEM39AS41 peptide treatment, immunoblot analysis was performed using lysates of A549 cells treated with Myristic acid-TMEM39AS41 peptide for 48 hours. The results for this are shown in FIG. 9, and the results are shown in FIG. 10 by graphing the relative protein content.

9 and 10, it was confirmed that the Myristic acid-TMEM39AS41 peptide significantly reduced the levels of NFκB p65 and LC3B (light chain 3 B) II / LC3BI in A549 cells.

These results indicate that the Myristic acid-TMEM39AS41 peptide inhibits cancer progression through the inflammatory / autogenous pathway.

Example 6. Myristic acid-TMEM39AS41 peptide is KRAS ^LA1  Confirmed to suppress tumor progression in lung cancer mice

In this example, a KRAS-mutated NSCLC mouse (murine) model was used to confirm the effect of the Myristic acid-TMEM39AS41 peptide on tumor growth in vivo . The Myristic acid-TMEM39AS41 peptide was modified with C-terminal amidation to improve biological stability in vivo. Thereafter, the Myristic acid-TMEM39AS41 peptide on 100 mM DMSO (dimethylsulphoxide) stock solution was completely diluted with DPBS (Dulbecco's phosphate buffered saline) and injected intravenously into the tumor for 3 weeks 3 times a week. The administration protocol is schematically shown in FIG. 11.

In addition, the image of the lung tumor obtained on the day of sacrifice of the KRAS ^LA1 mouse is shown in FIG. 12.

As shown in FIG. 12, when the 30 mg / kg Myristic acid-TMEM39AS41 peptide was treated, it was confirmed that the size of the excised tumor decreased compared to the control group.

These results indicate that the Myristic acid-TMEM39AS41 peptide of the present invention inhibits tumor progression.

Formulation Example 1. Preparation of pharmaceutical products

1.1 Preparation of powder

Myristic acid-TMEM39AS41 peptide 200mg

Lactose 100mg

Talc 10mg

The above ingredients are mixed and filled in an airtight fabric to prepare a powder.

1.2 Preparation of tablets

Myristic acid-TMEM39AS41 peptide 100mg

Corn starch 100mg

Lactose 100mg

Magnesium stearate 2mg

After mixing the above ingredients, tablets are prepared by tableting according to a conventional tablet manufacturing method.

1.3 Preparation of capsules

Myristic acid-TMEM39AS41 peptide 100mg

Corn starch 100mg

Lactose 100mg

Magnesium stearate 2mg

According to a conventional capsule preparation method, the above ingredients are mixed and filled into gelatin capsules to prepare tablets.

1.4 Preparation of injections

Myristic acid-TMEM39AS41 peptide 10μg / ml

Suitable amount of sterile distilled water for injection

pH Adjuster

It is prepared with the above-mentioned ingredient content per ampoule (2 ml) according to the manufacturing method of a conventional injection.

1.5 Preparation of liquid

Myristic acid-TMEM39AS41 peptide 100mg

20 g sugar

Iseonghwadang 20g

Lemon flavor

Purified water was added to make a total of 1,00 ml. After mixing the above components according to the manufacturing method of a conventional liquid formulation, a brown bottle is filled and sterilized to prepare a liquid formulation.

Formulation Example 2. Preparation of food

Myristic acid-TMEM39AS41 peptide 100mg

Vitamin mixture

Vitamin A Acetate 70 μg

Vitamin E 1.0 mg

Vitamin B1 0.13 mg

Vitamin B2 0.15 mg

Vitamin B6 0.5 mg

Vitamin B12 0.2 μg

Vitamin C 10 mg

Biotin 10 μg

Nicotinic acid amide 1.7 mg

50 ㎍ folic acid

Calcium Pantothenate 0.5 mg

Suitable amount of mineral mixture

Ferrous sulfate 1.75 mg

Zinc oxide 0.82 mg

Potassium monophosphate potassium 15 mg

Dibasic calcium phosphate 55 mg

Potassium citrate 90 mg

Calcium carbonate 100 mg

Magnesium chloride 24.8 mg

The composition ratio of the vitamin and mineral mixture is a composition suitable for relatively functional health food in a preferred embodiment, but the mixing ratio may be arbitrarily modified, and the above ingredients are mixed according to a general health functional food manufacturing method. Then, it can be used in the preparation of a health functional food composition (eg, nutritional candy, etc.) according to a conventional method.

Formulation Example 3. Preparation of beverage

Myristic acid-TMEM39AS41 peptide 100mg

Citric acid 1000 mg

Oligosaccharide 100 g

2 g of plum concentrate

Taurine 1 g

900 ml total by adding purified water

After mixing the above ingredients according to the normal method for preparing a functional beverage, and stirring and heating at 85 ° C. for about 1 hour, the resulting solution is filtered, obtained by sterilization in a 2 liter container, sterilized and sealed, then refrigerated and stored. It is used for preparing the health functional beverage composition of the present invention.

Although the above composition ratio is a mixture of components suitable for a preference drink in a preferred embodiment, the composition ratio may be arbitrarily modified according to regional and ethnic preferences such as demand hierarchy, country of demand, and usage.

<110> The Industry & Academic Cooperation in Chungnam National University (IAC) <120> Peptides for prevention and treatment for cancer and use thereof <130> CNU1.60P <160> 1 <170> KoPatentIn 3.0 <210> 1 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> TMEM39AS41 <400> 1 Gly Leu Arg Asn Arg Asn Gly Ser Ala Ile Gly Leu Pro Val Pro   1 5 10 15

Claims (8)

A pharmaceutical composition for preventing or treating lung cancer, comprising a peptide consisting of the amino acid sequence represented by SEQ ID NO: 1.
According to claim 1,
The peptide is characterized in that it comprises a motif represented by the amino acid sequence of RNRNGS, lung cancer prevention or treatment pharmaceutical composition.
According to claim 2,
The motif is phosphorylated by one or more kinases selected from the group consisting of protein kinase B (PKB), rho-associated protein kinase (ROCK), ribosomal protein kinase (RSK), S6 kinase (S6K) and serum glucocorticoid kinase (SGK). It characterized in that, the pharmaceutical composition for preventing or treating lung cancer.
According to claim 1,
The peptide is characterized in that the N- terminal is myristoylated (myristoylation), pharmaceutical composition for preventing or treating lung cancer.
According to claim 1,
The peptide is characterized in that the C- terminal amidation (amidation), pharmaceutical composition for preventing or treating lung cancer.
delete delete In vitro, nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB) p65 expression inhibition or LC3B (light chain 3 B) II / LC3BI, which includes the peptide represented by SEQ ID NO: 1 as an active ingredient Reagent composition for level reduction.

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