Peptide binding to OTUB1 and use thereof

Abstract

The present invention relates to a peptide binding to OTUB1 and a use thereof. A peptide of a specific sequence having the property of binding to OTUB1 is observed to exhibit an anticancer effect on cancer cells without affecting the cell growth of normal cells, and thus can be usefully used as a composition for preventing, treating, or alleviating cancer diseases.

Description

Peptides that bind to OTUB1 and their uses

[0001] The present invention relates to a peptide binding to OTUB1 and uses thereof.

[0002] OTUB1 (Ubiquitin Aldehyde Binding 1), a deubiquitinating enzyme implicated as a potential oncogene in various tumors, regulates tumorigenesis, invasion, and metastasis in various cancers. OTUB1 removes ubiquitin chains, but protein stability regulation by OTUB1 is sometimes independent of its catalytic activity. OTUB1 typically inhibits ubiquitination by inhibiting E2 ligase activity, which noncanonically regulates substrate stabilization. We screened peptides capable of binding to OTUB1 for their apoptotic effects in cancer therapy using a phage-displayed peptide library. The OTUB1-targeting peptide inactivated the noncanonical activity of OTUB1 by dephosphorylating tyrosine 26. Interestingly, the OTUB1-targeting peptide killed more OTUB1-low-expressing cancer cells than OTUB1-high-expressing cells. Furthermore, overexpression of OTUB1 prevented peptide-induced apoptosis in OTUB1-low-expressing cancer cells, whereas OTUB1 depletion enhanced apoptosis in OTUB1-high-expressing cells through regulation of the anti-apoptotic protein Bcl-xL. In tumor-bearing mice, the OTUB1-targeting peptide reduced tumor volume, increased apoptosis, and downregulated Bcl-xL expression. These results suggest that OTUB1-targeting peptides are promising peptide therapeutics that induce apoptosis.

[0003] An object of the present invention is to provide a peptide comprising an amino acid sequence represented by sequence number 1.

[0004] Another object of the present invention is to provide a polynucleotide encoding the above peptide.

[0005] Another object of the present invention is to provide a recombinant vector comprising the polynucleotide.

[0006] Another object of the present invention is to provide a transformant transformed with the recombinant vector.

[0007] Another object of the present invention is to provide a pharmaceutical composition for preventing or treating cancer, comprising the peptide as an active ingredient.

[0008] Another object of the present invention is to provide a health functional food composition for preventing or improving cancer, which contains the peptide as an active ingredient.

[0009] To achieve the above purpose, the present invention provides a peptide consisting of an amino acid sequence represented by SEQ ID NO: 1.

[0010] In addition, the present invention provides a polynucleotide encoding the peptide.

[0011] In addition, the present invention provides a recombinant vector comprising the polynucleotide.

[0012] In addition, the present invention provides a transformant transformed with the recombinant vector.

[0013] In addition, the present invention provides a pharmaceutical composition for preventing or treating cancer, comprising the peptide as an active ingredient.

[0014] In addition, the present invention provides a health functional food composition for preventing or improving cancer, which contains the peptide as an active ingredient.

[0015] According to the present invention, it has been confirmed that a peptide of a specific sequence having a property of binding to OTUB1 exhibits an anticancer effect on cancer cells without affecting the cell growth of normal cells, and thus can be usefully utilized as a composition for preventing, treating, or improving cancer.

[0016] Figure 1 shows the results of bio-panning analysis for OTUB1 (Ubiquitin Aldehyde Binding 1) protein (Example 1). Specifically, Figure 1A is a schematic diagram of bio-panning using OTUB1 protein-labeled GST beads; Figure 1B shows the results of measuring phage titers (pfu) at each bio-panning run after performing six rounds; Figure 1C shows the results of ELISA analysis for the binding of phage clones displaying a peptide (CPAMMTLMC) to OTUB1 protein-labeled and unlabeled GST beads; Figure 1D shows the results of fluorescence analysis for the binding of the peptide (CPAMMTLMC) synthesized in Experimental Example 3 and the OTUB1 protein; and Figure 1E shows proteins having a similar sequence to the peptide using the number of repetitions of the peptide (CPAMMTLMC) and the protein database of the National Institutes of Health. *P<0.05.

[0017] Figure 2 shows the results of a pull-down (precipitation) analysis of the binding of OTUB1 protein and the peptide (CPAMMTLMC) synthesized in Experimental Example 3 (Example 2).

[0018] Figure 3 shows the results of analyzing the effect of peptide (CPAMMTLMC) on cell viability (Example 3).

[0019] Figure 4 shows the results of analyzing the effect of peptide (CPAMMTLMC) on the expression of OTUB1 target substrate protein in cancer cells (Example 4).

[0020] Figure 5 shows the results of analyzing the difference in anticancer effects of OTUB1 binding peptides according to the level of OTUB1 expression (Example 5). Specifically, Figure 5A shows the OTUB1 expression pattern in various cancer cells; Figure 5B shows the results of PARP {Poly (ADP-ribose) polymerase} protein expression analysis after OTUB1 overexpression was induced in cells with low OTUB1 expression and peptide (CPAMMTLMC) treatment; and Figure 5C shows the results of PARP protein expression analysis after OTUB1 deficiency was induced in cells with high OTUB1 expression and peptide (CPAMMTLMC) treatment.

[0021] Figure 6 shows the results of animal experiments and immunofluorescence analysis performed to confirm the anticancer effect of the peptide (CPAMMTLMC) on lung cancer (Example 6). Specifically, Figure 6A is a schematic diagram for the production of an A549 lung cancer cell xenograft model; Figure 6B is the result of body weight analysis of the model treated with the peptide; Figure 6C is the result of tumor volume analysis of the model treated with the peptide; Figures 6D and 6E are the result of tumor size analysis of the model treated with the peptide; and Figure 6F is the result of immunofluorescence analysis of tumor tissues extracted from the model on the last day of peptide treatment in the model (scale bar: 30 μm). n=7, *P<0.05; ***P<0.0005 Student t-test / one-way ANOVA.

[0022] Figure 7 shows the results of an animal experiment conducted to confirm the anticancer effect of a peptide (CPAMMTLMC) on breast cancer (Example 6). Specifically, Figure 7A is a schematic diagram of the production of an MDA-MB231 breast cancer cell xenograft model; Figure 7B is the result of body weight analysis of the model treated with the peptide; Figure 7C is the result of tumor volume analysis of the model treated with the peptide; and Figures 7D and 7E are the result of tumor size analysis of the model treated with the peptide; n=5, *P<0.05, ***P<0.0005 Student t-test / one-way ANOVA.

[0023] Hereinafter, the present invention will be described in more detail.

[0024]

[0025] The present invention provides a peptide comprising an amino acid sequence represented by SEQ ID NO: 1.

[0026] The amino acid sequence of the above peptide may be as follows.

[0027] [Sequence number 1]

[0028] CPAMMTLMC

[0029] The above peptide can bind to OTUB1 (Ubiquitin Aldehyde Binding 1) and inhibit OTUB1 activity.

[0030] Additionally, the peptide can inhibit the expression of one or more selected from the group consisting of, but not limited to, PARP {Poly(ADP-ribose) polymerase}, Raptor, p53, cIAP1, p100, and snail.

[0031]

[0032] In addition, the present invention provides a polynucleotide encoding a peptide consisting of an amino acid sequence represented by SEQ ID NO: 1.

[0033]

[0034] In addition, the present invention provides a recombinant vector comprising a polynucleotide encoding a peptide consisting of an amino acid sequence represented by SEQ ID NO: 1.

[0035]

[0036] In addition, the present invention provides a transformant transformed with a recombinant vector comprising a polynucleotide encoding a peptide consisting of an amino acid sequence represented by SEQ ID NO: 1.

[0037]

[0038] In addition, the present invention provides a pharmaceutical composition for preventing or treating cancer, which comprises a peptide consisting of an amino acid sequence represented by sequence number 1 as an active ingredient.

[0039] The above cancer disease may be one or more selected from the group consisting of brain cancer, melanoma, skin cancer, lung cancer, liver cancer, stomach cancer, pancreatic cancer, bone cancer, head or neck cancer, uterine cancer, ovarian cancer, breast cancer, fallopian tube carcinoma, endometrial cancer, rectal cancer, anal cancer, endocrine cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, chronic or acute leukemia, lymphocytic lymphoma, bladder cancer, kidney or ureter cancer, renal cell carcinoma, renal pelvic carcinoma, central nervous system tumor, primary central nervous system lymphoma, spinal cord tumor, brainstem glioma, and pituitary adenoma, but is not limited thereto.

[0040] The pharmaceutical composition of the present invention can be manufactured in a unit dose form or can be manufactured by placing it in a multi-dose container by formulating it using a pharmaceutically acceptable carrier according to a method that can be easily performed by a person having ordinary skill in the art to which the present invention pertains.

[0041] The pharmaceutically acceptable carriers mentioned above are those commonly used in formulations, and include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, mineral oil, and the like. In addition to the above components, the pharmaceutical composition of the present invention may further include a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifier, a suspending agent, a preservative, and the like.

[0042] In the present invention, the content of the additive included in the pharmaceutical composition is not particularly limited and can be appropriately adjusted within the content range used in conventional formulations.

[0043] The above pharmaceutical composition may be formulated in the form of one or more external skin preparations selected from the group consisting of injectable formulations such as aqueous solutions, suspensions, emulsions, pills, capsules, granules, tablets, creams, gels, patches, sprays, ointments, ointments, lotions, liniments, pastes, and cataplasmas, but is not limited thereto.

[0044] The pharmaceutical composition of the present invention may further comprise pharmaceutically acceptable carriers and diluents for formulation. The pharmaceutically acceptable carriers and diluents include, but are not limited to, excipients such as starch, sugar, and mannitol; fillers and extenders such as calcium phosphate; cellulose derivatives such as carboxymethylcellulose and hydroxypropylcellulose; binders such as gelatin, alginates, and polyvinyl pyrrolidone; lubricants such as talc, calcium stearate, hydrogenated castor oil, and polyethylene glycol; disintegrants such as povidone and crospovidone; and surfactants such as polysorbates, cetyl alcohol, and glycerol. The pharmaceutically acceptable carriers and diluents may be biologically and physiologically compatible with the subject. Examples of diluents include, but are not limited to, saline, aqueous buffers, solvents, and / or dispersion media.

[0045] The pharmaceutical composition of the present invention may be administered orally or parenterally (e.g., intravenously, subcutaneously, intraperitoneally, or topically) depending on the intended method. In the case of oral administration, it may be formulated as tablets, troches, lozenges, aqueous suspensions, oily suspensions, prepared powders, granules, emulsions, hard capsules, soft capsules, syrups, elixirs, etc. In the case of parenteral administration, it may be formulated as injections, suppositories, powders for respiratory inhalation, aerosols for sprays, ointments, powders for application, oils, creams, etc.

[0046] The dosage of the pharmaceutical composition of the present invention may vary depending on the patient's condition, weight, age, sex, health status, dietary constitution, nature of the formulation, severity of the disease, administration time of the composition, administration method, administration period or interval, excretion rate, and drug form, and may be appropriately selected by a person skilled in the art. For example, it may be in the range of about 0.1 to 10,000 mg / kg, but is not limited thereto, and may be administered once or several times a day in divided doses.

[0047] The pharmaceutical composition may be administered orally or parenterally (e.g., intravenously, subcutaneously, intraperitoneally, or topically) depending on the intended method. The pharmaceutically effective amount and effective dosage of the pharmaceutical composition of the present invention may vary depending on the formulation method, administration method, administration time, administration route, etc. of the pharmaceutical composition. A person skilled in the art can easily determine and prescribe an effective dosage for the intended treatment. The pharmaceutical composition of the present invention may be administered once a day or divided into several doses.

[0048]

[0049] In addition, the present invention provides a health functional food composition for preventing or improving cancer, which comprises a peptide consisting of an amino acid sequence represented by sequence number 1 as an active ingredient.

[0050] The present invention can be generally used as a commonly used food.

[0051] The food composition of the present invention can be used as a health functional food. The term "health functional food" refers to a food manufactured and processed using raw materials or ingredients with functional properties useful to the human body as defined in the Health Functional Food Act. "Functionality" refers to ingestion for the purpose of obtaining beneficial effects for health purposes, such as regulating nutrients for the structure and function of the human body or physiological effects.

[0052] The above health functional food composition may contain conventional food additives, and its suitability as the above “food additive” is determined by the specifications and standards for the relevant item in accordance with the general provisions and general test methods of the Food Additive Code approved by the Ministry of Food and Drug Safety, unless otherwise specified.

[0053] Items listed in the above “Food Additives Code” include, for example, chemical compounds such as ketones, glycine, potassium citrate, nicotinic acid, and cinnamic acid; natural additives such as persimmon pigment, licorice extract, crystalline cellulose, high-molecular-weight pigment, and guar gum; and mixed preparations such as sodium L-glutamate preparations, alkaline agents for noodles, preservative preparations, and tar color preparations.

[0054] The food composition of the present invention can be manufactured and processed in the form of tablets, capsules, powders, granules, liquids, pills, etc. For example, among health functional foods in capsule form, hard capsules can be manufactured by mixing and filling a composition according to the present invention with additives such as excipients into a conventional hard capsule, and soft capsules can be manufactured by mixing a composition according to the present invention with additives such as excipients and filling a capsule base such as gelatin. The soft capsules may contain a plasticizer such as glycerin or sorbitol, a coloring agent, a preservative, etc., as necessary.

[0055] The definitions of terms for the above excipients, binders, disintegrants, lubricants, flavoring agents, etc. are described in documents known in the art and include those with the same or similar functions. There is no particular limitation on the type of food, and all health functional foods in the conventional sense are included.

[0056] In the present invention, the term “prevention” refers to any act of suppressing or delaying cancer disease by administering a composition according to the present invention.

[0057] In the present invention, the term “treatment” refers to any act of improving or beneficially changing the symptoms of a cancer disease by administering a composition according to the present invention.

[0058] In the present invention, the term “improvement” refers to any act of improving the bad condition of a cancer disease by administering a composition according to the present invention.

[0059] Hereinafter, to aid understanding of the present invention, examples will be given in detail. However, the following examples are intended only to illustrate the scope of the present invention and are not intended to limit its scope. These examples are provided to more fully explain the present invention to those of average skill in the art.

[0060]

[0061] [Experimental Example 1] Experimental Preparation

[0062] A549, HCC15, and SNU1330 cells (hereinafter referred to as human lung cancer cells); MDA-MB231, MDA-MB361, and MCF7 cells (hereinafter referred to as human breast cancer cells); U87MG, U251MG, and T98G cells (hereinafter referred to as human brain cancer cells); TCMK-1 cells (mouse kidney normal cells); MC cells (mouse glomerular normal cells); and EA.hy926 cells (human vascular endothelial cells) were purchased from ATCC (American Type Culture Collection, Manassas, VA, USA). Among the above cells, human lung cancer cells were cultured using RPMI-1640 (Gibco, Carsbad, CA, USA) medium, and the remaining cells, excluding human lung cancer cells, were cultured using DMEM (DMEM high glucose) medium containing high glucose (Gibco, Carsbad, CA, USA). All culture media were supplemented with 10% fetal bovine serum (FBS, Gibco), 100 U / ml penicillin, and 100 μg / ml streptomycin. All cells were cultured at 37°C in a CO2 incubator.

[0063] Human OTUB1 (Ubiquitin Aldehyde Binding 1) antibody was purchased from Santa Cruz Biotechnology and used, and the TUNEL Assay Kit was purchased from BrdU-Red (Abcam).

[0064]

[0065] [Experimental Example 2] Bio-panning using the T7 phage peptide library

[0066] Biopanning was performed using a T7 phage peptide library. The T7 phage peptide library was constructed using a phage vector purchased from Novagen. OTUB1 protein was labeled on GST (glutathione-S-transferase) beads and used. For the subtraction procedure, 1 × 10 9 A phage library of plaque-forming units (pfu) was incubated with unlabeled GST beads for 1 h at 4°C. Unbound phages were recovered and incubated with OTUB1-labeled GST beads for 1 h at 4°C. Unbound phages were then washed with phosphate-buffered saline (PBS) containing 10 mg / ml bovine serum albumin (BSA). Protein-bound phages were eluted with BL21 cell (Escherichia coli BL21) (optical density: 1.0) culture medium for 10 min at room temperature. The eluate was used for titration, and the remaining eluted phage clones were added to 10 ml BL21 cells (optical density: 0.5) and reacted for 2–3 h until the T7 phage underwent a lysis cycle and became transparently lysed. The above process was repeated five times, followed by serial dilutions. The lysate was inoculated into LB medium Petri plates and incubated overnight at 37°C. The phage titer was determined by counting the number of colonies.

[0067]

[0068] [Experimental Example 3] Sequence Analysis and Peptide Synthesis

[0069] After six rounds of bio-panning (Experimental Example 2), the inserted clones were confirmed by PCR analysis. Each of the 95 clones containing inserted DNA was sequenced (Macrogen, Seoul, South Korea). Sequence similarity was analyzed using the Clustal W program, and the most promising OTUB1-binding peptide (sequence: CPAMMTLMC) was synthesized (Peptron Inc. Daejeon, Korea). Fluorescein isothiocyanate (FITC) green fluorescence and biotin were conjugated to the N-terminus of the peptide. The lyophilized peptide was reconstituted with distilled water or dimethyl sulfoxide (DMSO) to a concentration of 50 or 10 mM, respectively. It was then further diluted to a concentration of 10 μM with PBS. A peptide present in the phage coat protein (sequence: NSSSVDK) was used as a control peptide.

[0070]

[0071] [Experimental Example 4] Phage and Peptide Binding ELISA Analysis

[0072] For phage protein binding ELISA assay, OTUB1-labeled GST beads (2 μg) were blocked with 1 mg / ml BSA for 30 min at room temperature. They were then washed three times with PBS-T (0.1% Tween-20) buffered saline, and individual phage clones (1 × 10 8After adding 100 μL of (pfu / tube), incubated at 4°C for 1 hour, washed, and then HRP (horseradish peroxidase)-conjugated anti-T7 antibody (ThermoFisher Scientific) diluted 1:10,000 in blocking buffer was added to the electron tube and incubated at 4°C for 1 hour. After washing with HBS-T, TMB (3,3´, 5,5´-tetramethylbenzidine) substrate was added to the electron tube and incubated at room temperature for 10–15 minutes. After that, the reaction was stopped by adding 100 μL of 2 M sulfuric acid (H2SO4), and transferred to a microplate for detection. The optical density was measured at 450 nm using a multiscan Sky microplate spectrophotometer (Thermo Fisher Scientific).

[0073] For peptide binding assays, OTUB1-labeled GST beads (2 μg) were blocked with 1 mg / ml BSA for 30 min at room temperature and washed three times with PBS-T (0.1% Tween-20) buffered saline. 100 μL of the synthesized FITC-labeled peptide (10 μM) was added, incubated for 1 h at 4°C, washed, and then transferred to a black, light-shielding microplate for detection. FITC (green fluorescence) was measured using a Plex Station 3 microplate reader.

[0074]

[0075] [Experimental Example 5] Western Blot Analysis

[0076] For Western blot experiments, cells were washed with cold phosphate-buffered saline (PBS) and lysed in tris-HCl (pH 7.4) supplemented with protease inhibitors consisting of 100 μM phenylmethanesulfonylfluoride (PMSF), 10 μg / ml leupeptin, 10 μg / ml pepstatin, and 2 mM ethylenediaminetetraacetic acid (EDTA); 1% nonyl phenoxypolyethoxylethanol-40 (NP-40); 0.25% sodium deoxycholate; 150 mM sodium chloride (NaCl); 1 mM sodium orthovanadate (Na3VO4); And 1 mM sodium fluoride (NaF) buffer was used to lyse the cells on ice using Radioimmunoprecipitation (RIPA) buffer. The lysate was centrifuged at 10,000 × g for 10 min at 4°C and the supernatant was collected. Western blot analysis was performed according to the instructions of the Western blotting kit manufacturer, and the proteins were separated by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to immobilon-P membranes. After that, specific proteins were detected using the enhanced chemiluminescence western blotting detection system.

[0077]

[0078] [Experimental Example 6] Pull-down technique using magnetic beads

[0079] Recombinant OTUB1 protein and OTUB1 binding peptide (Experimental Example 3) were reacted at room temperature for 1 hour, the protein was eluted using BcMag™ Monomeric Avidin Magnetic Kit, and the binding between the protein and peptide was confirmed through Western blot analysis.

[0080]

[0081] [Experimental Example 7] Cell Viability Analysis

[0082] To confirm the effect of OTUB1 binding peptide (Experimental Example 3) on cell viability, cells were treated with the peptide (0 to 2 μM), and cell viability was confirmed through XTT{2,3-Bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carbox anilide} analysis.

[0083]

[0084] [Experimental Example 8] OTUB1 overexpression and knockdown using plasmid and siRNA

[0085] Non-targeting control and OTUB1 siRNA (sequence: GACAACAUCUAUCAACAGA) were obtained from Bioneer (Daejeon, Korea). siRNA was transfected into cancer cells using Lipofectamine RNAiMAX (Thermo Fisher Scientific, Waltham, MA, USA). Plasmids were transfected into cancer cells using Lipofectamine 2000 (Thermo Fisher Scientific, Waltham, MA, USA).

[0086]

[0087] [Experimental Example 9] Analysis of anticancer effects using animal models

[0088] To confirm the anticancer effect of OTUB1 binding peptide (Experimental Example 3), animal experiments were performed. All animal experiments were performed under the guidelines of the Institutional Animal Care and Use Committee (IACUC) of Kyungpook National University (Approval Number: 2021-0143, 2022-0332). Lung cancer cells (A549 cells, 5 × 10) were injected into the lower right flank of 4-5 week-old Balb / c nude mice (purchased from Orient Bio). 6 , Figure 6A) or breast cancer cells (MDAMB-231, 1 × 10 6 , Fig. 7A) was injected. After that, mice were randomly selected, and tumors with a size of approximately 100 mm 3 When reached, they were grouped. After that, saline and peptides were injected intravenously every other day for a total of 5 times (5 mg / kg per session for a total of 25 mg / kg for the lung cancer model (Fig. 6A), and 10 mg / kg per session for a total of 50 mg / kg for the breast cancer model (Fig. 7A)). Tumor size was measured using a digital caliper, and tumor volume was calculated using the following mathematical formula 1. Mice were then sacrificed for tumor size and histological analysis. The experimental groups were set as follows.

[0089] 1) Experimental group 1 (normal group; Saline): Saline intravenous administration

[0090] 2) Experimental group 2 (control group; R7-Con Pep): intravenous administration of R7-NSSSVDK peptide

[0091] 3) Experimental group 3 (R7-OTUB1 Pep): Intravenous administration of R7-CPAMMTLMC peptide

[0092] [Mathematical Formula 1]

[0093] Tumor volume = 0.52 × (L × W × W)

[0094] * L: Length; longest dimension

[0095] * W: Width; Short dimension; Parallel to the mouse body

[0096]

[0097] [Experimental Example 10] Immunofluorescence analysis of tissues

[0098] To determine whether OTUB1 induces cell death in tissues, paraffin sections (4 μm thick) were prepared from tumor tissues of mice injected with lung cancer cells (A549). Tissue sections were subjected to TUNEL Assay Kit reaction for cell death detection and observed using a confocal microscope.

[0099]

[0100] [Experimental Example 11] Statistical Analysis

[0101] Statistical significance was measured using the unpaired Student's t test when comparing two experimental groups, and one- or two-way ANOVA when comparing multiple experimental groups. Values ​​are expressed as the mean ± standard deviation (SD or SEM) of three independent samples. A P value <0.05 was considered statistically significant.

[0102]

[0103] [Example 1] Bio-panning using the T7 phage library

[0104] As shown in Fig. 1A, bio-panning was performed 6 times on GST beads labeled with OTUB1 protein using a T7 phage library with random sequence peptides (Experimental Example 2). As shown in Fig. 1B, the phage titer increased approximately 40-fold in the 6th time compared to the first time, and the titer decreased in the subsequent 5 times and then increased again in the 6th time.

[0105] According to the above results, a total of 95 phage clones were randomly selected from among the phage clones produced in rounds 5 and 6, and PCR and DNA sequence analysis were performed on each selected clone to confirm the sequence of the inserted clone (Experimental Example 3). Based on the analyzed sequences and the sequences with a high repetitive frequency, the protein BLAST database was used to confirm that three clones had sequences similar to OTUB1-related protein sequences. In addition, OTUB1 protein binding was analyzed through a phage binding ELISA analysis (Experimental Example 4) targeting unlabeled GST beads (GST) among OTUB1 protein-labeled GST beads (GST-OTUB1). As a result, as shown in Fig. 1C, a candidate phage (sequence: CPAMMTLMC) showing a high level of binding compared to the control group was confirmed. In addition, as shown in Fig. 1D, the excellent binding ability of the synthesized candidate peptide, CPAMMTLMC peptide, to OTUB1 was confirmed by fluorescence analysis (Experimental Example 4).

[0106]

[0107] [Example 2] Binding analysis between OTUB1 protein and peptide

[0108] In order to confirm the binding between the peptide (CPAMMTLMC) and the OTUB1 protein, a pull-down technique was performed using monomeric avidin magnetic beads (Experimental Example 6). As shown in Fig. 2, when the recombinant OTUB1 protein and the CPAMMTLMC peptide were reacted together, binding between the two was confirmed in the sample eluted from the beads.

[0109]

[0110] [Example 3] Cell viability analysis

[0111] As a result of analyzing the effect of peptide (CPAMMTLMC) on cell viability (Experimental Example 7), as shown in Fig. 3, in the case of normal cells such as kidney cells (TCMK-1), glomerular cells (MC), and vascular endothelial cells (EA.hy926), no significant change in cell viability was observed in the control group (NC-peptide, NSSSVDK peptide treatment) and CPAMMTLMC peptide treatment group (R7-OTUB1 pep), whereas in the case of cancer cells such as breast cancer cells (MDA-MB231, MDA-MB361, and MCF7), brain cancer cells (U87MG, U251MG, and T98G), and lung cancer cells (A549, HCC15, and SNU1330), cell viability significantly decreased in a concentration-dependent manner in the CPAMMTLMC peptide treatment group compared to the control group. From the above results, it was confirmed that the CPAMMTLMC peptide exhibited an anticancer effect on cancer cells without affecting the growth of normal cells.

[0112]

[0113] [Example 4] Analysis of OTUB1 target substrate protein expression

[0114] As a result of analyzing the effect of peptide (CPAMMTLMC) on the expression of OTUB1 target substrate proteins in cancer cells (Experimental Example 5), as shown in Fig. 4, the expression of Raptor, p53, cIAP1, p100, and snail proteins was significantly reduced in a concentration-dependent manner in the CPAMMTLMC peptide-treated group compared to the control group (untreated peptide group). From the above results, it was confirmed that the CPAMMTLMC peptide inhibits the expression of target substrates by losing the function of OTUB1.

[0115]

[0116] [Example 5] Analysis of differences in anticancer effects according to the level of OTUB1 expression.

[0117] As shown in Fig. 5A, OTUB1 protein expression is low in cancer cells compared to normal cells, but specifically, expression is high only in HCC95 lung cancer cells. To determine whether there is a difference in the anticancer effect of the peptide (CPAMMTLMC) depending on the level of OTUB1 expression in cancer cells, cancer cells with low OTUB1 expression (MDA-MB231, U87M G, and A549) were transfected with a plasmid to overexpress OTUB1, and cancer cells with high OTUB1 expression (HCC95) were transfected with OTUB1 siRNA to suppress OTUB1 expression (Experimental Example 8). After that, after treating cancer cells with CPAMMTLMC peptide (2 μM), Western blot analysis was performed to measure PARP {Poly (ADP-ribose) polymerase} protein expression. As a result, as shown in Figures 5B to 5C, PARP expression was significantly reduced by CPAMMTLMC peptide treatment in all cancer cells. In cancer cells that overexpressed OTUB1 expression, the PARP expression reduction effect by CPAMMTLMC peptide treatment was reduced by overexpression, and in cancer cells that suppressed OTUB1 expression, the PARP expression reduction effect by CPAMMTLMC peptide treatment was increased by suppression of expression. From the above results, it was confirmed that regulation of OTUB1 expression in cancer cells affects the anticancer effect of CPAMMTLMC peptide, and specifically, OTUB1 overexpression suppresses the anticancer effect of the peptide, and suppression of OTUB1 expression promotes the anticancer effect of the peptide.

[0118]

[0119] [Example 6] Analysis of anticancer effects using animal models

[0120] To confirm the anticancer effect of the peptide (CPAMMTLMC), animal experiments and immunofluorescence analysis were performed (Experimental Examples 9-10). As shown in Figures 6 and 7, no significant change in body weight was observed in any experimental group, and compared to the control group (Saline), the tumor volume and size of lung cancer (A549) and breast cancer (MDA-MB231) were significantly reduced in the CPAMMTLMC peptide treatment group (R7-OTUB1 Pep).

[0121]

[0122] While specific aspects of the present invention have been described in detail above, it should be apparent to those skilled in the art that these specific descriptions merely represent preferred embodiments and are not intended to limit the scope of the present invention. In other words, the substantial scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A peptide consisting of an amino acid sequence represented by sequence number 1.

2. The peptide according to claim 1, characterized in that the peptide binds to OTUB1 (Ubiquitin Aldehyde Binding 1) and inhibits OTUB1 activity.

3. The peptide according to claim 1, characterized in that the peptide inhibits the expression of one or more selected from the group consisting of PARP{Poly(ADP-ribose) polymerase}, Raptor, p53, cIAP1, p100, and snail.

4. A polynucleotide encoding the peptide of claim 1.

5. A recombinant vector comprising the polynucleotide of paragraph 4.

6. A transformant transformed with the recombinant vector of paragraph 5.

7. A pharmaceutical composition for the prevention or treatment of cancer, comprising the peptide of claim 1 as an active ingredient.

8. A pharmaceutical composition according to claim 7, wherein the cancer disease is one or more selected from the group consisting of brain cancer, melanoma, skin cancer, lung cancer, liver cancer, stomach cancer, pancreatic cancer, bone cancer, head or neck cancer, uterine cancer, ovarian cancer, breast cancer, fallopian tube carcinoma, endometrial cancer, rectal cancer, prostatic cancer, endocrine gland cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, chronic or acute leukemia, lymphocytic lymphoma, bladder cancer, kidney or ureteral cancer, renal cell carcinoma, renopelvic carcinoma, central nervous system tumor, primary central nervous system lymphoma, spinal cord tumor, brainstem glioma and pituitary adenoma.

9. A health functional food composition for preventing or improving cancer, comprising the peptide of claim 1 as an active ingredient.

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