Compositions Comprising P53-Activating Peptides

Abstract

The present invention provides a cosmetic composition for suppressing or ameliorating seborrhea and a pharmaceutical composition for preventing, ameliorating, or treating seborrhea, each of which contains a specific peptide as an active ingredient. This peptide significantly reduces the interaction between p53 and MDM2 and significantly increases the interaction between p53 and RNA polymerase II (POLII), thereby inducing the expression of cytokines that cause apoptosis of sebaceous gland cells.

Description

[Technical field]

[0001] The present invention relates to a composition comprising a peptide that activates p53. More specifically, the present invention relates to a cosmetic composition for suppressing or improving seborrhea, and a pharmaceutical composition for preventing, improving or treating seborrhea. These compositions comprise, as an active ingredient, a peptide having an activity of significantly increasing the interaction between p53 and RNA polymerase II (POLII) by significantly decreasing the interaction between p53 and MDM2. [Background technology]

[0002] Sebum secreted from sebaceous gland cells in the sebaceous gland has beneficial functions such as preventing the intrusion of harmful substances from the outside of the skin and regulating the oil and moisture of the skin. However, excessive secretion of sebum due to hormonal changes during puberty and stress can cause dysfunction and lead to various skin diseases. Seborrhea is a disease of the sebaceous gland or skin trouble characterized by excessive secretion of sebum on the skin, the skin being covered with a sebum film, and the formation of crusts and scales on the skin.

[0003] Seborrhea can be suppressed or improved mainly by self-washing with soap, shampooing, and other hygienic care. Drug therapy includes antifungal agents such as selenium sulfide, azole compounds, sodium sulfacetamide, and terbinafine, and anti-inflammatory agents such as steroids. However, such therapy is only intended to alleviate the symptoms of seborrhea, and does not fundamentally suppress, improve, or treat seborrhea. Therefore, there is a need in the art for the development of a method that can fundamentally suppress, improve, or treat seborrhea.

[0004] CD99 is a type 1 transmembrane protein consisting of a glycosylated extracellular domain, a transmembrane domain, and a short intracellular domain (Banting, GS, Pym, B., Darling, SM, and Goodfellow PN (1989). The MIC2 gene product: epitope mapping and structural prediction analysis define an integral membrane protein. Mol. Immunol. 26: 181). It has been reported that binding of CD99 molecules expressed in Ewing sarcoma (EWS) cells with anti-CD99 monoclonal antibodies results in loss of MDM2 (E3 ubiquitin ligase) and inhibition of binding of MDM2 to p53 (Guerzoni C. et. al. (2015). CD99 triggering in Ewing sarcoma delivers a lethal signal through p53 pathway reactivation and cooperates with doxorubicin. Clin Cancer Res 21:146-156). p53 released from MDM2 activates RNA polymerase complexes containing RNA polymerase II (POLII) in the cell nucleus, increasing the expression of downstream target genes such as TRAIL (tumor necrosis factor-related apoptosis-inducing ligand), resulting in apoptosis of sebaceous cells (Melnik BC (2017). p53: key conductor of all anti-acne therapies. J Transl Med 15:195-206). In addition, the proliferation and differentiation of sebaceous cells are induced by IGF-1 and c-Myc, but p53 suppresses IGF-1 signaling and c-Myc function.

[0005] Therefore, by inhibiting the binding of MDM2 and p53 and increasing the interaction between p53 and POLII, it is expected that the proliferation and differentiation of sebaceous cells will be inhibited and apoptosis of sebaceous cells will be induced, resulting in the fundamental inhibition, improvement, or treatment of seborrhea. Summary of the Invention [Problem to be solved by the invention]

[0006] The present inventors have found that a specific peptide can be used to significantly increase the interaction between PKA (protein tyrosine kinase) and SHP2 (Src homology region 2 domain-containing phosphatase 2), thereby suppressing the metastasis of cancer cells via β1 integrin signaling, and that this peptide can be usefully applied to the prevention or treatment of inflammatory diseases (Korean Patent Publication No. 10-2018-0099092). Surprisingly, this specific peptide was found to exhibit inhibitory, ameliorative or therapeutic activity against seborrhea by significantly decreasing the interaction between p53 and MDM2 and significantly increasing the interaction between p53 and POLII.

[0007] Therefore, an object of the present invention is to provide a cosmetic composition for suppressing or ameliorating seborrhea, which contains this specific peptide as an active ingredient.

[0008] Another object of the present invention is to provide a pharmaceutical composition for preventing, ameliorating or treating seborrhea, which comprises this specific peptide as an active ingredient. [Means for solving the problem]

[0009] According to one aspect of the present invention, there is provided a cosmetic composition for suppressing or ameliorating seborrhea, comprising a compound represented by formula 1: <Expression 1> Leu-X-Asp (In the formula, X is glutamic acid (Glu), serine (Ser), glycine (Gly), alanine (Ala), glutamine (Gln), arginine (Arg), lysine (Lys), leucine (Leu), tyrosine (Tyr), aspartic acid (Asp), phenylalanine (Phe), asparagine (Asn), cysteine ​​(Cys), histidine (His), isoleucine (Ile), methionine (Met), proline (Pro), threonine (Thr), tryptophan (Trp), or valine (Val); - is a peptide bond. The present invention provides a cosmetic composition comprising, as an active ingredient, a peptide consisting of three amino acids represented by the formula:

[0010] According to another aspect of the present invention, there is provided a pharmaceutical composition for preventing, ameliorating or treating seborrhea, comprising a peptide consisting of three amino acids represented by the above formula 1 as an active ingredient.

[0011] The peptide contained in the cosmetic composition and pharmaceutical composition according to the present invention may preferably be a peptide consisting of the amino acid sequence of SEQ ID NO: 2, 3, 4, 6, 7, 9 or 18. Effect of the Invention

[0012] In the present invention, it has been found that the peptide of formula 1, i.e., a peptide consisting of three amino acids represented by formula 1, significantly reduces the interaction between p53 and MDM2 and significantly increases the interaction between p53 and POLII. Therefore, since this peptide induces the expression of cytokines (e.g., TRAIL (tumor necrosis factor-related apoptosis-inducing ligand)) that cause apoptosis of sebaceous gland cells, it can be usefully applied to cosmetic compositions for suppressing or ameliorating seborrhea and pharmaceutical compositions for preventing, ameliorating or treating seborrhea. [Brief description of the drawings]

[0013] [Figure 1]1 shows changes in the interaction between p53 and MDM2 depending on the treatment concentration when human keratinocytes (HaCaT cells) are treated with the peptide of the present invention. [Diagram 2] 1 shows changes in p53 phosphorylation and MDM2 expression levels depending on the treatment time when human keratinocytes (HaCaT cells) were treated with the peptide of the present invention. [Diagram 3] 1 shows the results of evaluating the effect on the interaction between p53 and POLII when human keratinocytes (HaCaT cells) are treated with the peptides of the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0014] As used herein, "seborrhea" refers to a sebaceous gland disease or skin trouble characterized by excessive secretion of sebum, including acne caused by excessive secretion of sebum.

[0015] The present invention relates to a cosmetic composition for suppressing or ameliorating seborrhea, comprising a compound represented by formula 1: <Expression 1> Leu-X-Asp (In the formula, X is glutamic acid (Glu), serine (Ser), glycine (Gly), alanine (Ala), glutamine (Gln), arginine (Arg), lysine (Lys), leucine (Leu), tyrosine (Tyr), aspartic acid (Asp), phenylalanine (Phe), asparagine (Asn), cysteine ​​(Cys), histidine (His), isoleucine (Ile), methionine (Met), proline (Pro), threonine (Thr), tryptophan (Trp), or valine (Val); - is a peptide bond. A cosmetic composition comprising, as an active ingredient, a peptide consisting of three amino acids represented by the formula: to provide.

[0016] The present invention also provides a pharmaceutical composition for preventing, ameliorating or treating seborrhea, which comprises a peptide consisting of three amino acids represented by the above formula 1 as an active ingredient.

[0017] In the cosmetic and pharmaceutical compositions according to the present invention, it has been found that a peptide consisting of the amino acid sequence of SEQ ID NO: 2, 3, 4, 6, 7, 9 or 18 has particularly excellent activity in decreasing the interaction between p53 and MDM2 and increasing the interaction between p53 and POLII. Thus, in one embodiment, the peptide may be a peptide consisting of the amino acid sequence of SEQ ID NO: 2, 3, 4, 6, 7, 9 or 18.

[0018] The cosmetic composition according to the present invention may be prepared, for example, in the form of a functional cosmetic composition. The cosmetic composition according to the present invention may be prepared in various forms according to a conventional method in the field of cosmetic compositions. For example, the cosmetic composition according to the present invention may be prepared in the form of a cosmetic, a skin lotion, a cream, a lotion, etc., and when used, these may be diluted with a cleansing water, an astringent, or a moisturizing liquid. The cosmetic composition according to the present invention may also contain conventional additives such as stabilizers, solubilizers, vitamins, pigments, and fragrances that are commonly used in the field of cosmetic compositions. In the cosmetic composition according to the present invention, the peptide may be contained in an amount effective for exerting an effect of suppressing or improving seborrhea, for example, about 0.00001 to 1% by weight, preferably about 0.001 to 0.1% by weight, based on the total weight of the cosmetic composition according to the present invention.

[0019] The pharmaceutical composition of the present invention may contain pharma- ceutically acceptable carriers, such as excipients, such as lactose and corn starch, lubricants, such as magnesium stearate, currently available emulsifiers, suspending agents, buffers, and isotonicity agents. The pharmaceutical composition of the present invention may be formulated into oral or parenteral dosage forms, and is preferably formulated into parenteral dosage forms for transdermal, subcutaneous, intramuscular, and the like. If necessary, additives, such as buffers and isotonicity agents, may be used in parenteral dosage forms. The pharmaceutical composition of the present invention may be in the form of an aqueous solution containing a carrier, such as phosphate buffered saline (PBS). The pharmaceutical composition of the present invention may be administered to a patient in an amount suitable for preventing, ameliorating, or treating seborrhea, for example, at a dose of about 0.01 to 10 mg / kg per day, although the daily dose may usually vary depending on the age, weight, and condition of the patient.

[0020] The present invention will be described in more detail with reference to the following examples and test examples. However, these examples and test examples are for the purpose of explaining the present invention, and the scope of the present invention is not limited to these examples and test examples. EXAMPLES

[0021] Example 1: Synthesis of peptides Peptides of SEQ ID NOs: 1 to 20 (see Table 1 below) were synthesized by the FMOC solid-phase method using an automated peptide synthesizer (PeptrEx-R48, Peptron, Daejeon, Republic of Korea). The synthesized peptides were purified and analyzed using reversed-phase high-performance liquid chromatography (reversed-phase HPLC) (Prominence LC-20AB, Shimadzu, Japan) using a C18 analytical RP column (Shiseido Capsule Pack), and identified using a mass spectrometer (HP1100 series LC / MSD, Hewlett-Packard, Roseville, USA). [Table 1]

[0022] Example 2: Preparation of a Composition Containing a Peptide The peptides of SEQ ID NOs: 1 to 20 were each dissolved in phosphate buffered saline (PBS) to a concentration of 1 M. Using the obtained protein solutions, the following test examples were carried out.

[0023] Test Example 1: Evaluation of the effect of peptides on the physical binding of p53 and MDM2 After cells were treated with the peptide of the present invention, the effect of the peptide of the present invention on the physical binding of p53 and MDM2 was evaluated by in situ PLA (proximity ligation assay). 4.5 × 10 HaCaT cells (human keratinocytes, CLS) were placed in each well of a 24-well microplate together with DMEM containing 10% fetal bovine serum. 4 The cells were added at a density of 1000 / well and cultured at 37°C in a 5% CO2 incubator for 24 hours. The peptide solution of Example 2 was added so that the peptide concentration in the medium was 1 μM, 5 μM, 10 μM or 20 μM, and then incubated under the same conditions for 30 minutes. The control group was not treated. The cells in each well were washed with PBS, fixed by treatment with 2% formaldehyde for 15 minutes, and then treated with 0.1% TritonX-100 for 5 minutes to improve the permeability of the antibody into the cells. Anti-p53 polyclonal antibody (Abcam, UK) and anti-MDM2 polyclonal antibody (Santa Cruz, CA, USA) were added. After adding the PLA probe using an in situ PLA kit (Sigma-Aldrich), the hybridization, ligation, amplification and encapsulation steps were performed according to the manufacturer's protocol. The physical interaction between p53 and MDM2 was quantified by measuring the luminescence signal (PLA signal) detected in each cell using a confocal laser microscope (Olympus fluoview FW1000; Olympus, Tokyo, Japan). The results are shown in Figure 1.

[0024] From the results shown in FIG. 1, it can be confirmed that the interaction between p53 and MDM was significantly reduced in the group treated with the peptide of the present invention, compared to the control group that was not treated.

[0025] Test Example 2: Evaluation of peptide activity on p53 phosphorylation and MDM2 depletion Phosphorylation of p53 and expression of MDM2 were evaluated using Western blotting. 6 The peptide solution of Example 2 was added to each well at a density of 1000 / well, and the peptide concentration in the medium was 20 μM, and then incubated for 15, 30 or 60 minutes under the same conditions as above. The cells in each well were washed three times with PBS and then lysed in 1% NP40 lysis buffer (1% Nonidet P40, 0.1 M NaCl, 0.05 M Tris (pH 8.0), 5 mM EDTA) containing 0.1 μM PMSP (phenylmethylsulfonyl fluoride), 1 μg / ml pepstatin A, 10 μg / ml leupeptin, 1 μg / ml aprotinin and 1 mM Na3VO4. The cell lysate was quantified using the Bradford assay, and then samples were prepared for electrophoresis. Electrophoresis of the immunoprecipitates was performed on a 10% polyacrylamide gel. After transferring the developed proteins to a nitrocellulose membrane, blocking solution (i.e., Tris-buffered saline (TBS) containing 0.05% Tween 20 and 3% bovine serum albumin) was added and treated at room temperature for about 1 hour. Then, reaction was performed for 2 hours in TBS (containing 0.5% bovine serum albumin) containing anti-p53 polyclonal antibody (Santa Cruz, California, USA) or anti-MDM2 polyclonal antibody (Santa Cruz, California, USA), and then washed with TBS containing 0.05% Tween 20. Horseradish peroxidase-labeled anti-rabbit IgG antibody (Santa Cruz, California, USA) was added and treated at room temperature for 1 hour, followed by washing five times with TBS containing 0.05% Tween 20 and color development using an antibody detection kit (Ab frontier, Korea). To confirm that the amount of cell lysate electrophoresed was the same, actin was detected using anti-β-actin monoclonal antibody (Santa Cruz, California, USA). The results are shown in Figure 2.

[0026] As can be seen from the results shown in Figure 2, p53 phosphorylation was maximized at 15 minutes after treatment with the peptide of Example 2. Furthermore, MDM2 expression disappeared at 60 minutes after treatment with the peptide of Example 2.

[0027] Test Example 3: Evaluation of the effect of peptides on the physical association of p53 and RNA polymerase II (POLII) After treating the cells with various peptides according to the present invention, the effect of the peptides according to the present invention on the physical binding of p53 and RNA polymerase II (POLII) was evaluated by in situ PLA (proximity ligation assay). 4.5 × 10 HaCaT cells (human keratinocytes, CLS) were inoculated into each well of a 24-well microplate with DMEM containing 10% fetal bovine serum. 4 The cells were added at a density of 10000 / well and cultured at 37°C in a 5% CO2 incubator for 24 hours. The various peptide solutions from Example 2 were added so that the concentration of each peptide in the medium was 20 μM, and then incubated under the same conditions for 1 hour. The control group was not treated. The cells in each well were washed with PBS, fixed by treatment with 2% formaldehyde for 15 minutes, and then treated with 0.1% TritonX-100 for 5 minutes to improve the permeability of the antibody into the cells. Anti-p53 polyclonal antibody (Abcam, UK) and anti-POLII polyclonal antibody (Santa Cruz, CA, USA) were added. After adding the PLA probe using an in situ PLA kit (Sigma-Aldrich), the hybridization, ligation, amplification and encapsulation steps were performed according to the manufacturer's protocol. The physical interaction between p53 and POLII was quantified by measuring the luminescence signal (PLA signal) detected in each cell using a confocal laser microscope (Olympus fluoview FW1000; Olympus, Tokyo, Japan). The results are shown in Figure 3.

[0028] 3, it can be seen that the interaction between p53 and POLII was significantly increased in the groups treated with each of the various peptides according to the present invention, compared to the control group that was not treated. In particular, it can be seen that the interaction between p53 and POLII was significantly increased (2.5-fold or more) in the groups treated with Pep2, Pep3, Pep4, Pep6, Pep7, Pep9 or Pep18 (i.e., peptides of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9 or SEQ ID NO:18) compared to the control group.

Claims

1. A cosmetic composition for suppressing or ameliorating seborrhea, comprising a compound represented by formula 1: <Formula 1> Leu-X-Asp (In the formula, X is glutamic acid (Glu), serine (Ser), glycine (Gly), alanine (Ala), glutamine (Gln), arginine (Arg), lysine (Lys), leucine (Leu), tyrosine (Tyr), aspartic acid (Asp), phenylalanine (Phe), asparagine (Asn), cysteine ​​(Cys), histidine (His), isoleucine (Ile), methionine (Met), proline (Pro), threonine (Thr), tryptophan (Trp), or valine (Val); - is a peptide bond. A cosmetic composition comprising, as an active ingredient, a peptide consisting of three amino acids represented by the formula:

2. The cosmetic composition according to claim 1 , wherein the peptide is a peptide consisting of the amino acid sequence of SEQ ID NO: 2, 3, 4, 6, 7, 9 or 18.

3. A pharmaceutical composition for preventing, ameliorating or treating seborrhea, comprising a compound of formula 1: <Formula 1> Leu-X-Asp (In the formula, X is glutamic acid (Glu), serine (Ser), glycine (Gly), alanine (Ala), glutamine (Gln), arginine (Arg), lysine (Lys), leucine (Leu), tyrosine (Tyr), aspartic acid (Asp), phenylalanine (Phe), asparagine (Asn), cysteine ​​(Cys), histidine (His), isoleucine (Ile), methionine (Met), proline (Pro), threonine (Thr), tryptophan (Trp), or valine (Val); - is a peptide bond. A pharmaceutical composition comprising, as an active ingredient, a peptide consisting of three amino acids represented by the formula:

4. The pharmaceutical composition according to claim 3 , wherein the peptide is a peptide consisting of the amino acid sequence of SEQ ID NO: 2, 3, 4, 6, 7, 9 or 18.

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