Peptide that inhibits activity of toll-like receptors (TLRS)

Abstract

The present invention relates to a peptide that inhibits the activity of toll-like receptors (TLRs). The peptide according to the present invention can significantly reduce the expression of TNF-α in macrophages treated with lipoteichoic acid (LTA) or lipopolysaccharide (LPS), and thus has an excellent effect in preventing or treating inflammatory diseases, immune diseases, and cancer by effectively inhibiting the signaling pathway of TLRs, and significantly improves a skin antioxidant effect, moisture retention, elasticity, and thickness, thereby exhibiting an excellent skin improvement effect.

Description

Peptides that inhibit the activity of TLR (TOLL-LIKE RECEPTORS)

[0001] The present invention was made with the support of the Ministry of Science and ICT under the task identification number 1711140272 and the detailed task number 2021R1A2B5B02086271. The research management specialized institution of the said task is the National Research Foundation of Korea, the research project name is "Individual Basic Research (MSIT) (R&D)", the research project name is "Discovery of Bone Metastatic Cancer Specific Genes and Identification of Mechanism", the main institution is the Samsung Life Public Welfare Foundation, and the research period is from September 1, 2021 to February 28, 2026.

[0002] This patent application claims priority to Republic of Korea Patent Application No. 10-2024-0061601, filed with the Korean Intellectual Property Office on May 10, 2024, the disclosure of which is incorporated herein by reference.

[0003] The present invention relates to a peptide that inhibits the activity of TLR (Toll-like receptors), and more particularly, to a novel peptide that can be used as a composition for preventing or treating inflammatory diseases, immune diseases and cancer by inhibiting the TLR (Toll-like receptors) signaling pathway, which is a major mechanism of inflammatory diseases, immune diseases and cancer, or as a composition for improving skin having skin antioxidant effects, moisture retention, elasticity and thickness improvement effects.

[0004] Innate immunity is the first defense mechanism in the mammalian immune system against bacterial infections and other diseases. Pattern recognition receptors such as Toll-like receptors (TLR) are activated by recognizing pathogen-associated molecular patterns (PAMPs) or damage-associated molecular patterns (DAMPs).

[0005] In particular, TLRs are proteins that play a crucial role in the innate immune response, and are mainly expressed on the surface of various immune cells such as macrophages or dendritic cells. They recognize pathogen-associated molecular patterns (PAMPs) or damage-associated molecular patterns (DAMPs) derived from microorganisms and activate signaling pathways. They then induce the secretion of inflammatory cytokines, anti-inflammatory cytokines, or chemokines and an immune response, thereby regulating adaptive immunity. There are a total of 13 types of TLRs (TLR1 to TLR13), and TLR11, TLR12, and TLR13 are known to not be expressed in humans.

[0006] The structure of TLR is that the ectodomain of TLR with respect to the cell membrane has a repetitive structure called leucine-rich repeats (LRRs), and PAMPs or DAMPs bind as ligands to that region, and through this, dimerization occurs, in which two TLR molecules form a dimer. Some TLRs can form heterodimers by binding to different types of TLR molecules, but in other cases, TLR molecules of the same type bind to form homodimers.

[0007] Based on the cell membrane, the internal domain of TLR is called the TIR (Toll / IL-1 receptor) domain, and when a ligand binds to the external domain and a signal molecule such as MyD88 (Myeloid differentiation factor 88) binds to the TIR domain, it is activated and induces a chain reaction within the cell. Except for TLR3 and TLR4, the signal molecule is MyD88 in all cases, and TRIF (TIR-containing adapter-inducing IFN-beta factor) is known to play that role in TLR3, and MyD88, TRIF, TRAM, and TICAM are known to play that role in TLR4.

[0008] Innate immune cells that recognize specific PAMPs through TLRs produce and secrete substances with antibiotic or antiviral properties to directly kill pathogens, or produce and secrete cytokines to induce an inflammatory response and other immune responses that help kill pathogens. Due to this role of TLRs, extensive research is being conducted to utilize them as targets for treating various diseases.

[0009] Meanwhile, steroid preparations, which are widely used for the purpose of alleviating inflammation and treating immune diseases, cause serious side effects due to their abuse, such as tissue damage and bone necrosis. Therefore, the demand for inhibitors that can precisely target and suppress only the cause of inflammation is increasing.

[0010] In addition, the importance of cellular immunity in the creation and proliferation of tumors has been revealed through recent studies, and as the role of TLRs in tumor cells has been elucidated, the development of anticancer drugs targeting TLRs is actively underway. Therefore, inhibitors that can effectively suppress TLR activity are expected to have a high possibility of being used as anticancer drugs in the future.

[0011] Meanwhile, as we age, internally, the secretion of various hormones that regulate metabolism decreases, and immune cell function and activity decline. Furthermore, externally, prolonged exposure to environmental factors like UV rays increases free radicals and reactive oxygen species, accelerating skin aging and causing skin damage. Efforts to prevent skin aging and damage caused by these internal and external factors are ongoing, and demand for cosmetics and topical skin products is growing.

[0012] Against this backdrop, the inventors of the present invention have conducted extensive research on peptides that inhibit the activity of Toll-like receptors (TLRs), and have established a novel peptide having an optimal amino acid sequence that inhibits the activity of TLRs by linking an amino acid sequence prediction algorithm using a reinforcement learning bandit model with a docking system, and have confirmed that the peptide according to the present invention has the effect of significantly reducing the expression of TNF-α in macrophages treated with LTA (Lipoteichoic acid) or LPS (Lipopolysaccharide).

[0013] In addition, when the peptide according to the present invention was treated at a high concentration to macrophages treated with LPS (Lipopolysaccharide), it was confirmed that it had a more excellent effect in reducing TNF-α expression than dexamethasone, a potent immunosuppressant.

[0014] In addition, it was confirmed that when the peptide according to the present invention was treated in an animal model treated with LPS (Lipopolysaccharide), an excellent exercise ability recovery effect was observed.

[0015] In addition, it was confirmed that when the peptide according to the present invention was treated on human dermal fibroblasts (Normal Huma Dermal Fibroblasts) treated with TNF-α, an excellent antioxidant effect was observed.

[0016] In addition, when the peptide according to the present invention was treated in an animal model irradiated with UV-B, it was confirmed that it had the effects of maintaining skin moisture, improving skin elasticity, and improving skin thickness.

[0017] Accordingly, the purpose of the present invention is to provide a peptide consisting of an amino acid sequence represented by sequence number 1.

[0018] Another object of the present invention is to provide a pharmaceutical composition for preventing or treating inflammatory diseases, which comprises a peptide consisting of an amino acid sequence represented by sequence number 1 as an active ingredient.

[0019] Another object of the present invention is to provide a pharmaceutical composition for preventing or treating an immune disease, which comprises a peptide consisting of an amino acid sequence represented by sequence number 1 as an active ingredient.

[0020] Another object of the present invention is to provide a pharmaceutical composition for preventing or treating cancer, which comprises as an active ingredient a peptide consisting of an amino acid sequence represented by SEQ ID NO: 1.

[0021] Another object of the present invention is to provide a composition for improving skin, which comprises as an active ingredient a novel peptide consisting of an amino acid sequence represented by sequence number 1.

[0022] Another object of the present invention is to provide a novel peptide comprising an amino acid sequence represented by SEQ ID NO: 1 for use in preventing or treating inflammatory diseases, immune diseases and cancer, or for improving skin.

[0023] The present invention relates to a peptide that inhibits the activity of TLR (Toll-like receptors), and the inventors of the present invention have invented a novel peptide that can be used as a composition for preventing or treating inflammatory diseases, immune diseases and cancer by inhibiting the TLR (Toll-like receptors) signaling pathway, which is a major mechanism of inflammatory diseases, immune diseases and cancer, or as a composition for improving skin having skin antioxidant effects, moisture retention, elasticity and thickness improvement effects, and confirmed that the peptide according to the present invention has the effect of significantly reducing the expression of TNF-α in macrophages treated with LTA (Lipoteichoic acid) or LPS (Lipopolysaccharide).

[0024] In addition, when the peptide according to the present invention was treated at a high concentration to macrophages treated with LPS (Lipopolysaccharide), it was confirmed that it had a more excellent effect in reducing TNF-α expression than dexamethasone, a potent immunosuppressant.

[0025] In addition, when the peptide according to the present invention was treated in an animal model treated with LPS (Lipopolysaccharide), it was confirmed that it had an effect of improving, treating, or preventing degenerative neurological diseases through an excellent effect of restoring motor ability.

[0026] In addition, it was confirmed that when the peptide according to the present invention was treated on human dermal fibroblasts (Normal Huma Dermal Fibroblasts) treated with TNF-α, an excellent antioxidant effect was observed.

[0027] In addition, when the peptide according to the present invention was treated on an animal model irradiated with UV-B, it was confirmed that there was a skin improvement effect through excellent skin moisture retention, improvement in skin elasticity, and improvement in skin thickness, thereby completing the present invention.

[0028] Hereinafter, the present invention will be described in more detail.

[0029] One aspect of the present invention is a peptide comprising an amino acid sequence represented by sequence number 1.

[0030] The term "peptide" as used herein refers to a molecule formed by amino acid residues bonded together by peptide bonds. The peptide may be prepared using chemical synthesis methods known in the art, and for example, preferably, but not limited to, solid-phase synthesis techniques.

[0031] In the present invention, the peptide may be a peptide having a homology of about 60% or more, about 70% or more, about 80% or more, about 90% or more, about 95% or more, about 98% or more, or about 99% or more to a peptide consisting of an amino acid sequence represented by SEQ ID NO: 1, but is not limited thereto.

[0032] In the present invention, the peptide may inhibit the TLR (Toll-like receptors) signaling pathway.

[0033] The term "TLR (Toll-like receptors)" in this specification may refer to a receptor that is a protein that plays an important role in innate immunity, recognizes pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs), activates a signaling pathway that promotes the secretion of inflammatory cytokines or chemokines, and regulates adaptive immunity.

[0034] The term "TLR (Toll-like receptors) signaling pathway" in this specification refers to two separate signaling pathways, one is a MyD88-dependent pathway that induces the production of inflammatory cytokines, and the other is a MyD88-independent pathway that is associated with IFN-β stimulation and maturation of dendritic cells.

[0035] The MyD88-dependent pathway is common to all TLRs except TLR3, and when activated by microbial antigens, TLRs induce the recruitment of MyD88 through the TIR domain, which in turn initiates a cascade of events that activates several signaling molecules, leading to the nuclear localization of NF-κB and the production of inflammatory cytokines such as TNF-α, IL-1, and IL-12. For example, TLR2 and TLR4 signaling require TIRAP / Mal, an adapter involved in the MyD88-dependent pathway.

[0036] The above MyD88 independent pathway is a TLR3 signaling process, and TLR3 can induce IFN-β production by reacting with double-stranded RNA (dsRNA) in a MyD88-independent manner through the adapter TRIF / TICAM-115.

[0037] The term "inhibition" as used herein may mean a phenomenon in which biological activity or activity is reduced due to deficiency, disharmony, or many other causes, and may mean, for example, but is not limited to, partially or completely inhibiting, reducing, preventing, delaying activation, inactivating, or down-regulating the activity of a TLR.

[0038] In the present invention, the TLR (Toll-like receptors) may be TLR4 (Toll-like receptor 4) or TLR6 (Toll-like receptor 6), but is not limited thereto.

[0039] The term "TLR4 (Toll-like receptor 4)" in this specification refers to a protein encoded by the TLR4 gene, which is a trans-membrane protein family that functions as a surveillance for pathogen infection, and is also called CD284 (cluster of differentiation 284). Since the TLR4 recognizes various PAMPs (Pathogen-associated molecular patterns) including LPS of Gram-negative bacteria, it may refer to a protein that is very important for the activation of the innate immune system.

[0040] The term "TLR6 (Toll-like receptor 6)" in this specification refers to a protein encoded by the TLR6 gene, which is a protein belonging to the TLR family of trans-membrane proteins that play a fundamental role in pathogen recognition and innate immunity activation. The TLR6 can recognize diacyl lipopeptides derived from sugars present in the cell walls of Gram-positive bacteria, mycoplasma, and various fungi as ligands, and in particular, can refer to a protein that can recognize pathogen-associated molecular patterns (PAMPs) expressed in infectious agents and mediate the production of cytokines necessary for an effective immune response.

[0041] In the present invention, the TLR (Toll-like receptors) signaling pathway may be induced by LTA (Lipoteichoic acid) or LPS (Lipopolysaccharide), but is not limited thereto.

[0042] The term "LTA (Lipoteichoic acid)" used herein refers to a major component of the cell wall of Gram-positive bacteria. The structure of LTA varies across different bacterial species and may be anchored to the cell membrane via diacylglycerol. Furthermore, LTA possesses antigenic properties capable of stimulating specific immune responses, and can bind to target cells nonspecifically via membrane phospholipids or specifically bind to CD14 or TLR.

[0043] The term "LPS (Lipopolysaccharide)" used herein refers to a major component of the outer membrane of Gram-negative bacteria, and LPS is structured with lipids and polysaccharides. LPS can bind to CD14 or MD2, particularly TLR4, in many cell types, such as monocytes, dendritic cells, macrophages, and B cells, and induce the secretion of inflammatory cytokines, nitric oxide, and the like.

[0044] In the present invention, the peptide may be one that inhibits TNF-α (Tumor necrosis factor-α), but is not limited thereto.

[0045] The term "TNF-α (Tumor necrosis factor-α)" in this specification refers to a type of cytokine associated with inflammatory responses and acute-phase responses, and can be mainly secreted by activated macrophages. The most important role of TNF-α is the regulation of immune cells, and since abnormal regulation of TNF-α is found in various diseases including inflammatory diseases, immune diseases, and cancer, it is known that regulation of TNF-α is very important in the prevention and treatment of the above diseases.

[0046] In the present invention, the substances recognized by the TLR and activating the TLR signaling pathway may include, but are not limited to, pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs).

[0047] The term "pathogen-associated molecular patterns (PAMPs)" in this specification refers to small molecular motifs that are conserved in microorganisms but not present in the host. These are recognized by TLRs and other pattern recognition receptors (PRRs) in both plants and animals, thereby enabling the innate immune system to recognize pathogens and protect the host from infection. Recognition of PAMPs by TLRs can promote the activation of various signal transduction pathways in the host's immune cells, including stimulation of interferons or various cytokines. In general, various types of molecules, including glycans and glycoconjugates, can function as PAMPs, and for example, viral glycoproteins present in the viral envelope, mycobacterial cell wall components, etc. can activate the TLR4 signaling pathway.

[0048] The term "damage-associated molecular patterns (DAMPs)" used herein refers to intracellular molecules that serve as warning signals for cell damage or infection in vivo, as components of the innate immune response released from cells damaged by trauma or pathogen infection. When DAMPs are released into the extracellular space in response to damage, they can be recognized by receptors such as TLRs to promote non-infectious inflammatory responses. These DAMPs can be derived from various sources, such as the extracellular matrix, nucleus, cytoplasm, mitochondria, and endoplasmic reticulum. For example, biglycan, decorin, fibrinogen, etc. derived from the extracellular matrix can activate the TLR4 signaling pathway, or versican, etc. can activate the TLR6 signaling pathway; S10 protein, heat-shock protein, etc. derived from the cytoplasm can activate the TLR4 signaling pathway; and syndecan, etc. derived from the cell membrane (syndecan), glypican, etc. can activate the TLR4 signaling pathway.

[0049] Another aspect of the present invention is a pharmaceutical composition for preventing or treating an inflammatory disease, comprising a peptide consisting of an amino acid sequence represented by sequence number 1 as an active ingredient.

[0050] The pharmaceutical composition for preventing or treating an inflammatory disease according to the present invention comprises the same peptide as the peptide consisting of the amino acid sequence represented by the above-described sequence number 1, and therefore, a detailed description thereof will be referred to above, and the unique composition of the composition for preventing or treating an inflammatory disease will be described below.

[0051] The term "including as an active ingredient" in this specification means including a peptide consisting of an amino acid sequence represented by SEQ ID NO: 1 in a sufficient amount in the composition to achieve the desired effect. In this case, the "desired effect" in the present invention may be preventing, treating, or improving inflammatory diseases, immune diseases, and cancer caused by TLR (Toll-like receptors) signaling, and the sufficient amount to achieve such a desired effect may be appropriately adjusted depending on the subject of administration, the form of administration, the route of administration, etc.

[0052] The term “prevention” in this specification may mean any act of inhibiting or delaying the occurrence of inflammatory diseases, immune diseases, and cancer by administering the composition.

[0053] The term "treatment" in this specification may mean any action that improves or beneficially changes the symptoms of inflammatory diseases, immune diseases, and cancers by administering the composition.

[0054] The pharmaceutical composition of the present invention may additionally comprise a pharmaceutically acceptable carrier.

[0055] The term "pharmaceutically acceptable" in this specification means, as commonly used in the pharmaceutical field, that it does not irritate the subject when administered and does not inhibit the biological activity and properties of the compound being administered.

[0056] The term "subject" in this specification may be a mammal including a human, for example, but not limited to, a human, monkey, cow, horse, sheep, pig, cat, dog, mouse, rat, rabbit or guinea pig.

[0057] The term "administration" in this specification means providing a given substance to a subject by any appropriate method, and the route of administration of the composition including the peptide consisting of the amino acid sequence represented by SEQ ID NO: 1 of the present invention may be oral or parenteral administration through any common route as long as it can reach the target tissue, for example, oral, dermal, intravenous, intramuscular, subcutaneous, etc.

[0058] The appropriate dosage of the pharmaceutical composition of the present invention varies depending on factors such as the formulation method, administration method, patient's age, weight, sex, pathological condition, food, administration time, administration route, excretion rate, and response sensitivity, and a skilled physician can easily determine and prescribe a dosage effective for the desired treatment or prevention.

[0059] In the present invention, any carrier commonly used in the art may be used as the carrier. Non-limiting examples of the carrier include saline solution, sterile water, Ringer's solution, buffered saline, albumin injection solution, lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, maltodextrin, glycerol, ethanol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, mineral oil, hyaluronic acid, and the like, or a combination thereof.

[0060] In the present invention, the pharmaceutical composition may be used by adding other pharmaceutically acceptable additives such as excipients, diluents, antioxidants, buffers or bacteriostatic agents, if necessary, and may be used by additionally adding fillers, bulking agents, wetting agents, disintegrating agents, dispersing agents, surfactants, binders or lubricants.

[0061] Oral preparations include tablets, pills, powders, granules, capsules, suspensions, solutions, emulsions, syrups, and ointments. In addition to the commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included.

[0062] Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations, suppositories, and transdermal preparations. Non-aqueous solutions and suspensions may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate.

[0063] In the present invention, the peptide may inhibit the TLR (Toll-like receptors) signaling pathway, but is not limited thereto.

[0064] In the present invention, the TLR (Toll-like receptors) may be TLR4 (Toll-like receptor 4) or TLR6 (Toll-like receptor 6), but is not limited thereto.

[0065] In the present invention, the TLR (Toll-like receptors) signaling pathway may be induced by LTA (Lipoteichoic acid) or LPS (Lipopolysaccharide), but is not limited thereto.

[0066] In the present invention, the peptide may be one that inhibits TNF-α (Tumor necrosis factor-α), but is not limited thereto.

[0067] In the present invention, the inflammatory disease may be at least one selected from the group consisting of asthma, eczema, psoriasis, allergy, rheumatoid arthritis, degenerative arthritis, psoriatic arthritis, tendonitis, periodontitis, hypersensitivity reaction due to vaccination, neuritis, degenerative neurological disease, atopic dermatitis, acne, atopic rhinitis, pulmonary inflammation, allergic dermatitis, chronic sinusitis, contact dermatitis, seborrheic dermatitis, gastritis, gout, gouty arthritis, ulcer, chronic bronchitis, Crohn's disease, ulcerative colitis, ankylosing spondylitis, sepsis, vasculitis, bursitis, lupus, polymyalgia rheumatica, temporal arteritis, multiple sclerosis, Alzheimer's disease, arteriosclerosis, obesity, and viral infection, but is not limited thereto.

[0068] Another aspect of the present invention is a pharmaceutical composition for preventing or treating an immune disease, comprising a peptide consisting of an amino acid sequence represented by SEQ ID NO: 1 as an active ingredient.

[0069] In the present invention, the peptide may inhibit the TLR (Toll-like receptors) signaling pathway, but is not limited thereto.

[0070] In the present invention, the TLR (Toll-like receptors) may be TLR4 (Toll-like receptor 4) or TLR6 (Toll-like receptor 6), but is not limited thereto.

[0071] In the present invention, the TLR (Toll-like receptors) signaling pathway may be induced by LTA (Lipoteichoic acid) or LPS (Lipopolysaccharide), but is not limited thereto.

[0072] In the present invention, the peptide may be one that inhibits TNF-α (Tumor necrosis factor-α), but is not limited thereto.

[0073] In the present invention, the immune disease may be at least one selected from the group consisting of insulin-dependent diabetes, multiple sclerosis, autoimmune encephalomyelitis, rheumatoid arthritis, autoimmune arthritis, myasthenia gravis, thyroiditis, uveitis, Hashimoto's thyroiditis, primary myxedema, thyrotoxicosis, pernicious anemia, autoimmune atrophic gastritis, Addison's disease, premature menopause, male infertility, juvenile diabetes, Goodpasture's syndrome, pemphigoid, pemphigoid, sympathetic ophthalmia, phacoemulsifying uveitis, autoimmune hemolytic anemia, idiopathic leukopenia, primary biliary tract cirrhosis, cryptogenic cirrhosis, ulcerative colitis, Sjogren's syndrome, scleroderma, Wegener's granulomatosis, polymyositis, dermatomyositis, and systemic lupus erythematosus, but is not limited thereto.

[0074] Another aspect of the present invention is a pharmaceutical composition for preventing or treating cancer, comprising a peptide consisting of an amino acid sequence represented by SEQ ID NO: 1 as an active ingredient.

[0075] In the present invention, the peptide may inhibit the TLR (Toll-like receptors) signaling pathway, but is not limited thereto.

[0076] In the present invention, the TLR (Toll-like receptors) may be TLR4 (Toll-like receptor 4) or TLR6 (Toll-like receptor 6), but is not limited thereto.

[0077] In the present invention, the TLR (Toll-like receptors) signaling pathway may be induced by LTA (Lipoteichoic acid) or LPS (Lipopolysaccharide), but is not limited thereto.

[0078] In the present invention, the peptide may be one that inhibits TNF-α (Tumor necrosis factor-α), but is not limited thereto.

[0079] In the present invention, the cancer may be a solid cancer or a blood cancer, but is not limited thereto.

[0080] The term "cancer" in this specification may be used interchangeably with "malignant tumor" or "malignant neoplasm," and may refer to a disease that can occur in any tissue due to uncontrolled cell cycles and continuous cell division. Cancer is known to occur when mutations occur in the genes or tumor suppressor genes of normal cells due to various causative agents (carcinogens).

[0081] In particular, recent studies on the role of TLR in immunotherapy for tumors have been conducted, revealing the effects of TLR stimulation in cancer and specific TLRs expressed in various types of tumors. For example, it is known that TLR2 / 4 is specifically expressed and activated in melanoma, bladder cancer, etc., and TLR4 is specifically expressed and activated in cervical cancer, etc.

[0082] In the present invention, the cancer may be at least one selected from the group consisting of liver cancer, kidney cancer, brain tumor, urinary tract cancer, bone tumor, bile duct cancer, non-small cell lung cancer, small cell lung cancer, breast cancer, ovarian cancer, colon cancer, melanoma, pancreatic cancer, cervical cancer, multiple myeloma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, B-lymphoma, endometrial cancer, prostate cancer, testicular cancer, and sarcoma, but is not limited thereto.

[0083] Another aspect of the present invention is a composition for improving skin, which comprises, as an active ingredient, a peptide consisting of an amino acid sequence represented by sequence number 1.

[0084] In the present invention, the peptide may inhibit the TLR (Toll-like receptors) signaling pathway, but is not limited thereto.

[0085] In the present invention, the TLR (Toll-like receptors) may be TLR4 (Toll-like receptor 4) or TLR6 (Toll-like receptor 6), but is not limited thereto.

[0086] In the present invention, the TLR (Toll-like receptors) signaling pathway may be induced by LTA (Lipoteichoic acid) or LPS (Lipopolysaccharide), but is not limited thereto.

[0087] In the present invention, the peptide may be one that inhibits TNF-α (Tumor necrosis factor-α), but is not limited thereto.

[0088] In the present invention, the skin improvement may be at least one selected from the group consisting of skin antioxidant, skin moisture retention, skin elasticity improvement, and skin thickness improvement, but is not limited thereto.

[0089] The present invention relates to a peptide that inhibits the activity of TLR (Toll-like receptors), and the peptide according to the present invention can significantly reduce the expression of TNF-α in macrophages treated with LTA (lipoteichoic acid) or LPS (lipopolysaccharide), thereby effectively inhibiting the signal transduction pathway of TLR, and thus has an excellent effect in preventing or treating inflammatory diseases, immune diseases, and cancer, and has a remarkable effect in improving skin antioxidant effects, moisture retention, elasticity, and thickness, and thus has an excellent skin improvement effect.

[0090] FIGS. 1A and 1B are graphs illustrating the stages in which the remax-score, which is an evaluation criterion, is stabilized during the production of two peptide fragments (v1, v2) using a new drug development platform created to establish a novel peptide having an amino acid sequence for inhibiting TLR signaling according to an example of the present invention.

[0091] FIG. 2 illustrates the structure of a novel peptide predicted to effectively inhibit the TLR (Toll-like receptors) signaling pathway based on artificial intelligence, according to one embodiment of the present invention.

[0092] Figure 3 shows the results of simulating the binding site between TLR6 (pink) and the novel peptide according to the present invention, according to one embodiment of the present invention.

[0093] FIG. 4 shows the results of simulating the binding site between TLR6 and a novel peptide according to the present invention when present as a TLR2 (green) / TLR6 (pink) heterodimer, according to one embodiment of the present invention.

[0094] Figure 5 shows the results of simulating the binding site between TLR4 (pink) and a novel peptide according to the present invention, according to one embodiment of the present invention.

[0095] FIG. 6 is a graph showing a comparison of the expression level of TNF-α by treating Raw264.7 macrophages with a composition containing a novel peptide according to the present invention at different concentrations for 2 hours, and then treating them with LTA (Lipoteichoic acid), a ligand of TLR, at a concentration of 10 μg / ml to induce an inflammatory response, according to one embodiment of the present invention.

[0096] FIG. 7 is a graph showing a comparison of the expression level of TNF-α by treating Raw264.7 macrophages with a composition containing a novel peptide according to the present invention at different concentrations for 2 hours, and then treating them with LPS (Lipopolysaccharide), a ligand of TLR, at a concentration of 100 ng / ㎖ to induce an inflammatory response, according to one embodiment of the present invention.

[0097] FIG. 8 is a graph showing a comparison of the expression level of TNF-α after treating Raw 264.7 macrophages with a composition containing a novel peptide according to the present invention and dexamethasone at different concentrations for 2 hours, and then treating them with LPS (Lipopolysaccharide), a ligand of TLR, at a concentration of 100 ng / ㎖ to induce an inflammatory response, according to one embodiment of the present invention.

[0098] FIG. 9 is a graph showing the effect of recovery of exercise ability of a mouse through a Rotarod performance test after treating a mouse animal model with a novel peptide and LPS according to the present invention, according to one embodiment of the present invention.

[0099] FIG. 10 is a graph showing the amount of reactive oxygen generated by treating human dermal fibroblasts (NHDF; Normal Human Dermal Fibroblast) with a composition containing a novel peptide according to the present invention at different concentrations, and then treating with TNF-α at a concentration of 20 ng / mL to induce the generation of reactive oxygen species, according to one embodiment of the present invention.

[0100] FIG. 11 is a schematic diagram showing an experimental method for confirming the skin improvement effect of a mouse animal model by treating it with UV-B irradiation and a novel peptide according to the present invention, according to one embodiment of the present invention.

[0101] FIG. 12 is a graph showing the skin moisture content of a mouse after UV-B irradiation and treatment with a novel peptide according to the present invention in a mouse animal model according to one embodiment of the present invention.

[0102] FIG. 13 is a graph showing the skin elasticity of a mouse after UV-B irradiation and treatment with a novel peptide according to the present invention in a mouse animal model according to one embodiment of the present invention.

[0103] FIG. 14 is a graph showing the transepidermal water loss of a mouse after UV-B irradiation and treatment with a novel peptide according to the present invention in a mouse animal model according to one embodiment of the present invention.

[0104] FIG. 15 is a graph showing the skin thickness of a mouse after UV-B irradiation and treatment with a novel peptide according to the present invention in a mouse animal model, according to one embodiment of the present invention.

[0105] The present invention relates to a peptide comprising an amino acid sequence represented by sequence number 1.

[0106] Hereinafter, embodiments of the present invention will be described in detail with reference to the drawings so that those skilled in the art can easily implement the present invention. Unless otherwise defined, terms used herein should be interpreted as generally understood by those skilled in the art.

[0107] The drawings and examples of this specification are provided to enable those skilled in the art to easily understand and practice the present invention. Contents that may obscure the gist of the invention may be omitted from the drawings and examples, and the present invention is not limited to the drawings and examples.

[0108] Throughout this specification, whenever a part is said to "include" a component, this means that it may include other components, but not to the exclusion of other components, unless specifically stated otherwise.

[0109] Throughout this specification, "%" used to indicate the concentration of a particular substance is (wt / wt)% for solid / solid, (wt / vol)% for solid / liquid, and (vol / vol)% for liquid / liquid, unless otherwise stated.

[0110]

[0111] Example 1: Establishment of an optimal novel peptide for inhibiting TLR (Toll-like receptor) signaling.

[0112] The present invention utilizes a new drug development platform created to establish a novel peptide having an amino acid sequence for inhibiting TLR signaling, and produces two fragments having a 10 amino acid sequence that exhibit a stabilized graph, which are shown in FIGS. 1A and 1B.

[0113] At this time, the x-axis of each graph represents the new drug development platform cycle step, and the y-axis represents the remax-score (Schneider et al, 2022). The remax-score is one of the scores calculated in the above paper as a weighted average of the f-nat score prediction values, and was used as an evaluation criterion when producing this new peptide.

[0114] As shown in FIGS. 1A and 1B, a fragment having the amino acid sequence of VEQEIIVIID (v1, FIG. 1A) and a fragment having the amino acid sequence of KVIDLIDLLF (v2, FIG. 1B) were prepared, and then v1 and v2 were combined in reverse order to prepare v1f and v2f, respectively.

[0115] Then, a novel peptide (v1fR10v2f; peptide 1) was produced by linking 10 arginine (R) amino acids between the produced v1f and v2f, which is shown in Table 1 and Figure 2 below and used in subsequent experiments.

[0116] Sequence number distinction sequence (N'→C')1v1fR10v2f, peptide 1DIIVIIEQEVRRRRRRRRRRFLLDILDIVK

[0117]

[0118] Example 2: Confirmation of binding of novel peptides to TLR (Toll-like receptors)

[0119] Protein folding and binding simulations were performed using the Alphafold multimer model, a protein structure prediction program, based on the amino acid sequence of the novel peptide (peptide 1) produced in Example 1 and the amino acid sequence extracted from the structure of TLR6 (3A79) existing in the PDB database. Binding was confirmed based on the results obtained after the simulation, which are shown in Figure 3. Furthermore, the amino acid sequences of the TLRs used in the simulations are shown in Table 2.

[0120] As shown in Fig. 3, it was confirmed that the novel peptide according to the present invention can effectively bind to TLR6.

[0121] 서열번호구분서열 (N'→C')2TLR2 (PDB;3A79,2Z7X)SLSCDRNGICKGSSGSLNSIPSGLTEAVKSLDLSNNRITYISNSDLQRCVNLQALVLTSNGINTIEEDSFSSLGSLEHLDLSYNYLSNLSSSWFKPLSSLTFLNLLGNPYKTLGETSLFSHLTKLQILRVGNMDTFTKIQRKDFAGLTFLEELEIDASDLQSYEPKSLKSIQNVSHLILHMKQHILLLEIFVDVTSSVECLELRDTDLDTFHFSELSTGETNSLIKKFTFRNVKITDESLFQVMKLLNQISGLLELEFDDCTLNGVGNFRASDNDRVIDPGKVETLTIRRLHIPRFYLFYDLSTLYSLTERVKRITVENSKVFLVPCLLSQHLKSLEYLDLSENLMVEEYLKNSACEDAWPSLQTLILRQNHLASLEKTGETLLTLKNLTNIDISKNSFHSMPETCQWPEKMKYLNLSSTRIHSVTGCIPKTLEILDVSNNNLNLFSLNLPQLKELYISRNKLMTLPDASLLPMLLVLKISRNQLKSVPDGIFDRLTSLQKIWLHTNPWDCSCPRIDYLSRWLNKNSQKEQGSAKCSGSGKPVRSIICP3TLR4 (PDB;3FXI)EPCVEVVPNITYQCMELNFYKIPDNLPFSTKNLDLSFNPLRHLGSYSFFSFPELQVLDLSRCEIQTIEDGAYQSLSHLSTLILTGNPIQSLALGAFSGLSSLQKLVAVETNLASLENFPIGHLKTLKELNVAHNLIQSFKLPEYFSNLTNLEHLDLSSNKIQSIYCTDLRVLHQMPLLNLSLDLSLNPMNFIQPGAFKEIRLHKLTLRNNFDSLNVMKTCIQGLAGLEVHRLVLGEFRNEGNLEKFDKSALEGLCNLTIEEFRLAYLDYYLDDIIDLFNCLTNVSSFSLVSVTIERVKDFSYNFGWQHLELVNCKFGQFPTLKLKSLKRLTFTSNKGGNAFSEVDLPSLEFLDLSRNGLSFKGCCSQSDFGTTSLKYLDLSFNGVITMSSNFLGLEQLEHLDFQHSNLKQMSEFSVFLSLRNLIYLDISHTHTRVAFNGIFNGLSSLEVLKMAGNSFQENFLPDIFTELRNLTFLDLSQCQLEQLSPTAFNSLSSLQVLNMSHNNFFSLDTFPYKCLNSLQVLDYSLNHIMTSKKQELQHFPSSLAFLNLTQNDFACTCEHQSFLQWIKDQRQLLVEVERMECATPSDKQGMPVLSLNITC4TLR6 (PDB;3A79);

[0122] Then, protein folding and binding simulations were performed using the Alphafold multimer model, a protein structure prediction program, based on the amino acid sequence of the novel peptide (peptide 1) produced in Example 1 and the amino acid sequence extracted from the structure of the TLR2 / 6 heterodimer (3A79) existing in the PDB database. The degree of protein folding and binding was confirmed from the results obtained after the simulation, and is shown in Fig. 4.

[0123] As shown in Fig. 4, it was confirmed that the novel peptide according to the present invention can effectively bind to TLR6 in a TLR2 / 6 heterodimer structure.

[0124] In addition, protein folding and binding simulations were performed using the Alphafold multimer model, a protein structure prediction program, for the amino acid sequence of the novel peptide (peptide 1) produced in Example 1 and the amino acid sequence extracted from the structure of TLR4 (3FX1) existing in the PDB database. Binding was confirmed from the results obtained after the simulation, and this is shown in Figure 5.

[0125] As shown in Fig. 5, it was confirmed that the novel peptide according to the present invention can effectively bind to TLR4.

[0126]

[0127] Example 3: Confirmation of the inhibitory effect of the TLR (Toll-like receptors) signaling pathway in mouse macrophages.

[0128] Using a composition containing the novel peptide (peptide 1) produced in Example 1, the TLR signal pathway inhibitory effect was confirmed as follows.

[0129] 3-1. LTA (lipoteichoic acid) induction

[0130] 2 x 10 Raw264.7 mouse macrophage cells 5Cells / well (48-well) were cultured overnight at 37°C in an incubator. The novel peptide (peptide 1) was diluted in DMEM medium at concentrations of 0, 10, 25, 50, and 100 μM, respectively, and treated on Raw 264.7 cells, followed by incubation at 37°C for 2 hours. Then, each cell was treated with LTA at a concentration of 10 μg / ml, followed by incubation at 37°C for 3 hours to obtain only the medium. Afterwards, the expression level of TNF-α was measured using the Mouse TNF-alpha Quantikine ELISA Kit (R&D systems, MTA00B-1), and the results are shown in Table 3 and Figure 6 below.

[0131] LTA (10 ㎍ / ㎖)peptide 1 (uM)Control102550100TNF-α (pg / ml)1844.8891314.889617.9444412.9444179.6111

[0132] As shown in Table 3 and Figure 6, in the control group that was not treated with the novel peptide (peptide 1) according to the present invention, TNF-α was expressed at about 1,800 pg / ㎖ or more by LTA treatment, whereas in the group treated with the novel peptide (peptide 1), the expression level of TNF-α was significantly suppressed, and in particular, it was confirmed that the expression level of TNF-α was effectively suppressed in a concentration-dependent manner.

[0133]

[0134] 3-2. LPS (lipopolysaccharide) induction

[0135] 2 x 10 Raw264.7 mouse macrophage cells 5Cells / well (48-well) were cultured overnight at 37°C in an incubator. The novel peptide (peptide 1) was diluted in DMEM medium at concentrations of 0, 10, 25, 50, and 100 μM, respectively, and treated on Raw 264.7 cells, followed by incubation at 37°C for 2 hours. Then, each cell was treated with LPS at a concentration of 100 ng / mL, followed by incubation at 37°C for 3 hours to obtain only the medium. Afterwards, the expression level of TNF-α was measured using the Mouse TNF-alpha Quantikine ELISA Kit (R&D systems, MTA00B-1), and the results are shown in Table 4 and Figure 7 below.

[0136] LPS (100 ng / ml)peptide 1 (uM)Control102550100TNF-α (pg / ml)2250.303766.060649054.2424217.87879

[0137] As shown in Table 4 and Figure 7, in the control group that was not treated with the novel peptide (peptide 1) according to the present invention, TNF-α was expressed at about 2,300 pg / ㎖ or more by LPS treatment, whereas in the group treated with the novel peptide (peptide 1), the expression level of TNF-α was significantly suppressed, and in particular, it was confirmed that the expression level of TNF-α was effectively suppressed in a concentration-dependent manner.

[0138]

[0139] 3-3. Confirmation of LPS-induced and dexamethasone-induced inhibitory effects

[0140] The expression levels of TNF-α were compared after inducing an inflammatory response with LPS in mouse macrophages treated with a composition containing the novel peptide (peptide 1) produced in Example 1 and dexamethasone (Dex), one of the potent immunosuppressants as a steroid preparation.

[0141] Specifically, 2 x 10 Raw264.7 cells, which are mouse macrophages 5 Cells / well (48-well) were cultured overnight at 37°C in an incubator. The novel peptide (peptide 1) and dexamethasone (Dex) were diluted in DMEM medium at concentrations of 0, 1, and 10 μM, respectively, and treated with Raw 264.7 cells, followed by incubation at 37°C for 2 hours. Then, each cell was treated with LPS at a concentration of 100 ng / mL, followed by incubation at 37°C for 3 hours to obtain only the medium. Afterwards, the expression level of TNF-α was measured using the Mouse TNF-alpha Quantikine ELISA Kit (R&D systems, MTA00B-1), and the results are shown in Table 5 and Fig. 8 below.

[0142] Treatment substance LPS (100 ng / ㎖) Dexamethasone (uM) Peptide 1 (uM) Group Control 110 110 TNF-α (pg / ㎖) 1959.161069.68857.3161889.68669.421

[0143] As shown in Table 5 and Fig. 8, it was confirmed that the expression level of TNF-α induced by LPS decreased with dexamethasone treatment. Similarly, it was confirmed that the expression level of TNF-α induced by LPS also decreased with treatment with the novel peptide (peptide 1) according to the present invention. In particular, when treated with the same concentration (10 uM), the novel peptide (peptide 1) of the present invention showed a more excellent TNF-α expression inhibitory effect than dexamethasone, thereby confirming that the novel peptide of the present invention can be used as an effective immunosuppressant.

[0144]

[0145] Example 4: Confirmation of the effect of restoring motor ability in a mouse animal model.

[0146] The effects of the novel peptide (peptide 1) produced in Example 1 above on recovery of motor ability in a mouse animal model treated with LPS were measured as follows.

[0147] Specifically, the mouse model was divided into three groups: PBS treatment group (control, PBS 20 μl, n=3), LPS treatment group (LPS 10 μg / mouse, n=3), and peptide 1 + LPS treatment group (peptide 1 200 nmol / mouse, LPS 10 μg / mouse, n=3).

[0148] The LPS-treated mouse model was divided into two groups. The peptide 1 + LPS-treated group was injected with peptide 1 into the knee joint capsule area by intra-articular injection 1 hour before LPS injection, and the LPS-treated group and PBS-treated group were injected with the same amount (20 μl) of PBS.

[0149] To determine the effect of LPS on motor ability in C57BL / 6 mice in each group, the Rotarod performance test was performed 0, 1, 3, and 6 hours after injection. The mice were placed in the center of the rotating rod and the time from the start of rotation until the mouse fell off the rotating rod (latency time) was measured. This test was performed three times for each mouse in each group, and the average of the three measurements was calculated.

[0150] Furthermore, mice in each group underwent identical Rotarod training before the start of the experiment. For the first two days, they performed isokinetic exercise at 4 rpm and 10 rpm, and for the next three days, they performed isokinetic exercise at 4 rpm and 10 rpm and accelerated exercise at 4-40 rpm. The specific training method is shown in Table 6 below.

[0151] Day 1, 2Day 3, 4, 5Step TimeMethodStep TimeMethodTraining1 min 4 rpm isokinetic exercise on RotarodTraining1 min 4 rpm isokinetic exercise on RotarodRecovery10 min recovery in cageRecovery10 min recovery in cageTraining5 min 10 rpm isokinetic exercise on RotarodTraining5 min 10 rpm isokinetic exercise on RotarodRecovery10 min recovery in cageTraining5 min 4 - 40 rpm acceleration exercise on Rotarod

[0152] And, this experiment was evaluated by measuring the time from the time the Rotarod's rotating rod started to rotate until the mouse fell off the rotating rod by performing an acceleration movement that increased from 4 rpm to 40 rpm for 270 seconds (accelerating by 4 rpm every 30 seconds). The measurement method is shown in Table 7 below, and the measurement results are shown in Table 8 and Fig. 9 below.

[0153] Step Time Method Training 1 min 4 rpm constant speed exercise on the Rotarod Recovery 10 min Recovery in the cage Measurement 5 min 4 - 40 rpm acceleration exercise on the Rotarod

[0154] As shown in Fig. 9, the Rotarod performance test before LPS treatment (baseline) was evaluated, and the PBS treatment group was measured at 165.2 ± 8.1 seconds, the LPS treatment group at 163.6 ± 12.9 seconds, and the peptide 1 + LPS treatment group at 179.7 ± 25.1 seconds, showing no difference between the groups.

[0155] On the other hand, 1 hour after LPS treatment, the PBS treatment group showed no difference at 173.2 ± 11.6 seconds, but the LPS treatment group showed a significant decrease in motor ability at 125.2 ± 7.8 seconds, and the peptide 1 + LPS treatment group showed a recovery in motor ability at 193.7 ± 6.9 seconds, showing a significant increase of 54.7% in maintenance time compared to the LPS treatment group.

[0156] And, 3 hours after LPS treatment, the retention time was measured to be 150.2 ± 8.5 seconds for the PBS treatment group, 125.6 ± 23.4 seconds for the LPS treatment group, and 211.2 ± 5.5 seconds for the peptide 1 + LPS treatment group, showing a significant increase of 68.2% in the retention time compared to the LPS treatment group.

[0157] Additionally, 6 hours after LPS treatment, the retention time was measured to be 151.4 ± 9.6 seconds for the PBS treatment group, 103.1 ± 18.1 seconds for the LPS treatment group, and 186.5 ± 9.8 seconds for the peptide 1 + LPS treatment group, showing a significant increase of 80.9% in retention time compared to the LPS treatment group.

[0158] Therefore, it was confirmed that the novel peptide (peptide 1) according to the present invention has an effect of improving or preventing degenerative neurological diseases such as Alzheimer's by restoring motor control ability.

[0159] Latency time (s)0 hours later1 hour later3 hours later6 hours laterPBS treatment group165.2 ± 8.1173.2 ± 11.6150.2 ± 8.5151.4 ± 9.6LPS treatment group163.6 ± 12.9125.2 ± 7.8125.6 ± 23.4103.1 ± 18.1peptide 1 + LPS treatment group179.7 ± 25.1193.7 ± 6.9211.2 ± 5.5186.5 ± 9.8

[0160]

[0161] Example 5: Confirmation of antioxidant effect in human dermal fibroblasts

[0162] The effect of the novel peptide (peptide 1) produced in Example 1 above on reducing reactive oxygen species (ROS) in human dermal fibroblasts was measured as follows.

[0163] Specifically, 1 x 10 NHDF (Normal Human Dermal Fibroblast) cells were seeded in a 96-well black plate. 4 After seeding at a density of 10 cells / well, the cells were cultured overnight in an incubator at 37°C and 5% CO2 using DMEM medium (10% FBS, 1% penicillin / streptomycin). Afterwards, the medium was replaced with DMEM without FBS to induce cell cycle arrest, and the cells were cultured for an additional 24 hours.

[0164] Afterwards, the cultured cells were pretreated for 1 hour in DMEM medium (1% penicillin / streptomycin) containing the novel peptide (peptide 1) at concentrations of 0, 1.56, 3.13, 6.25, and 12.5 uM, respectively, and then simultaneously treated with TNF-α (20 ng / mL) and DCFDA (2',7'-dichlorodihydrofluorescein diacetate 10 μM, Sigma-Aldrich, St. Louis, MO, USA; CAT No. 35845-1G) for 15 minutes. After the reaction was completed, the supernatant was removed, and the cells were washed with PBS (pH 7.4).

[0165] The level of intracellular ROS production was measured by fluorescence intensity at an excitation wavelength of 485 nm and an emission wavelength of 535 nm using a microplate reader (SPARK 10M, Tecan), and the results are shown in Table 9 and Figure 10 below.

[0166] Intracellular ROS generationpeptide 1 (uM)Control01.563.136.2512.5Fold change12.041.931.851.441.32

[0167] As shown in Table 9 and Figure 10, in the negative control group not treated with the novel peptide (peptide 1) according to the present invention, the amount of reactive oxygen species generated increased by about two times or more compared to the control group not treated with TNF-α, whereas in the case of the group treated with the novel peptide (peptide 1), the amount of reactive oxygen species generated was significantly suppressed, and in particular, it was confirmed that the amount of reactive oxygen species generated was effectively suppressed in a concentration-dependent manner.

[0168]

[0169] Example 6: Confirmation of skin improvement effect in a mouse animal model

[0170] The effect on skin improvement in a mouse animal model treated with the novel peptide (peptide 1) produced in Example 1 above was measured as follows.

[0171] Specifically, 6-week-old female SKH-1 mice weighing 18-20 g were purchased from Orient Bio, acclimatized for 1 week, and then used in the experiment. The mice were divided into three groups: a control group that did not receive any treatment, a UV-B + 70% PEG (Polyethylene Glycol; 200 uL) treatment group, and a UV-B + peptide 1 (250 uM; 200 uL) treatment group, and the experiment was conducted as shown in Fig. 11.

[0172] UV-B was exposed using a self-made UV-B lamp, and the concentration of irradiated UV-B was measured using a UV radiometer (RMX-3W, Vilber Lourmat, France). UV-B irradiation was performed three times a week for 10 weeks, for a total of 30 times, and the initial irradiation concentration was 50 mJ / cm 2 was set to 10 mJ / cm after every two investigations. 2The irradiation concentration was increased by increments. In addition, 70% PEG and peptide 1 were applied transdermally daily from week 1 to week 10 of UV-B irradiation. Thereafter, skin moisture content, elasticity, transepidermal water loss (TEWL), and skin thickness were measured weekly.

[0173]

[0174] 6-1. Measuring skin moisture content

[0175] Skin moisture content is AramoSG ® Using ASG 200F (Aram HuvisCo., Ltd., Seognam-si, Gyeonggi-do, Republic of Korea), the sensor of the device was brought into contact with the mouse's back and the measurement was repeated three times, and the results are shown in Table 10 and Fig. 12 below.

[0176] Moisture (%)Week12345678910Control13.210.212.615.812.613.816.015.415.415.2UVB18.29.48.09.08.07.210.610.610.610.6UVB + peptide 112.05.213.810.013.812.414.615.015.013.8

[0177] As shown in Table 10 and Figure 12, in the experimental group not treated with the novel peptide (peptide 1) according to the present invention, the skin moisture content was reduced by about 30% or more due to UV-B irradiation, but in the group treated with the novel peptide (peptide 1), the skin moisture content was confirmed to be almost the same as that of the control group.

[0178]

[0179] 6-2. Skin elasticity measurement

[0180] Skin elasticity is AramoSG ®Using ASG 200F (Aram HuvisCo., Ltd., Seognam-si, Gyeonggi-do, Republic of Korea), the sensor of the device was brought into contact with the mouse's back and the measurement was repeated three times, and the results are shown in Table 11 and Fig. 13 below.

[0181] Elasticity (%)Week12345678910Control16.412.415.819.815.814.615.815.415.414.6UVB22.811.69.69.49.68.69.69.89.810.6UVB + peptide 114.86.217.012.217.013.214.615.215.214.4

[0182] As shown in Table 11 and Figure 13, in the experimental group that was not treated with the novel peptide (peptide 1) according to the present invention, skin elasticity was reduced by about 35% or more due to UV-B irradiation, but in the group treated with the novel peptide (peptide 1), skin elasticity was confirmed to be almost the same level as the control group.

[0183]

[0184] 6-3. Measurement of transepidermal water loss

[0185] Measurement of transepidermal water loss (TEWL) using GPSkinBarrier ® (GPOWER Inc., Seoul, Korea), the sensor of the device was brought into contact with the mouse's back and the measurement was repeated three times, and the results are shown in Table 12 and Figure 14 below.

[0186] TEWL (g / h / m 2)Week12345678910Control20.416.014.014.214.015.816.816.816.817.0UVB14.616.423.823.423.826.024.624.624.623.6UVB + peptide 112.819.421.617.021.621.822.621.221.221.0

[0187] As shown in Table 12 and Figure 14, in the experimental group not treated with the novel peptide (peptide 1) according to the present invention, the amount of transepidermal water loss increased by about 45% or more due to UV-B irradiation, but in the group treated with the novel peptide (peptide 1), it was confirmed that the amount of transepidermal water loss was significantly reduced compared to the PEG treatment group.

[0188]

[0189] 6-4. Skin thickness measurement

[0190] Skin thickness was measured using electronic calipers, and the measurements were repeated three times after fixing the mouse, and the results are shown in Table 13 and Figure 15 below.

[0191] Skin thickness (mm)Week12345678910Control0.91.01.01.00.91.01.01.01.11.1UVB1.11.21.41.71.71.71.71.61.71.7UVB + peptide 11.11.11.21.31.41.51.41.51.51.5

[0192] As shown in Table 13 and Figure 15, in the experimental group not treated with the novel peptide (peptide 1) according to the present invention, the skin thickness increased by about 50% or more due to UV-B irradiation, but in the group treated with the novel peptide (peptide 1), it was confirmed that the amount of skin thickness increase was significantly reduced compared to the PEG treatment group.

[0193]

[0194] Sintering.

[0195] In summary of the results of the above examples, the novel peptide (peptide 1) according to the present invention can effectively inhibit the TLR signaling pathway by binding to TLR, which plays an important role in inflammatory response, immune response, and tumor generation and proliferation through innate immune cells. In addition, in an animal experiment using mice, the novel peptide (peptide 1) according to the present invention was confirmed to effectively restore exercise ability reduced by LPS treatment, suggesting that it can be effectively used for the prevention or treatment of inflammatory diseases, immune diseases, and cancer.

[0196] In addition, the novel peptide (peptide 1) according to the present invention showed an excellent antioxidant effect in skin cells in which active oxygen generation was induced, and it was confirmed that it could effectively improve skin damaged by ultraviolet irradiation in an animal experiment using mice, suggesting that it can be effectively used for skin improvement purposes.

[0197]

[0198] While the present invention has been described in detail above, it should be apparent to those skilled in the art that these specific descriptions are merely preferred embodiments and are not intended to limit the scope of the present invention. Therefore, the substantial scope of the present invention is defined by the appended claims and their equivalents.

[0199] The present invention relates to a peptide that inhibits the activity of TLR (Toll-like receptors), and the peptide according to the present invention can significantly reduce the expression of TNF-α in macrophages treated with LTA (lipoteichoic acid) or LPS (lipopolysaccharide), thereby effectively inhibiting the signal transduction pathway of TLR, and thus has an excellent effect in preventing or treating inflammatory diseases, immune diseases, and cancer, and has a remarkable effect in improving skin antioxidant effects, moisture retention, elasticity, and thickness, and thus has an excellent skin improvement effect.

Claims

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

2. A peptide according to claim 1, wherein the peptide inhibits the TLR (Toll-like receptors) signaling pathway.

3. A peptide according to claim 2, wherein the TLR (Toll-like receptors) is TLR4 (Toll-like receptor 4) or TLR6 (Toll-like receptor 6).

4. A peptide according to claim 2, wherein the TLR (Toll-like receptors) signaling pathway is induced by LTA (lipoteichoic acid) or LPS (lipopolysaccharide).

5. A peptide according to claim 1, wherein the peptide inhibits TNF-α (tumor necrosis factor-α).

6. A pharmaceutical composition for preventing or treating an inflammatory disease, comprising a peptide according to any one of claims 1 to 5 as an active ingredient.

7. In paragraph 6, the inflammatory disease is at least one selected from the group consisting of asthma, eczema, psoriasis, allergy, rheumatoid arthritis, degenerative arthritis, psoriatic arthritis, tendonitis, periodontitis, hypersensitivity reaction due to vaccination, neuritis, neuritis due to degenerative nerve disease, atopic dermatitis, acne, atopic rhinitis, pulmonary inflammation, allergic dermatitis, chronic sinusitis, contact dermatitis, seborrheic dermatitis, gastritis, gout, gouty arthritis, ulcer, chronic bronchitis, Crohn's disease, ulcerative colitis, ankylosing spondylitis, sepsis, vasculitis, bursitis, lupus, polymyalgia rheumatica, temporal arteritis, multiple sclerosis, Alzheimer's disease, arteriosclerosis, obesity, and viral infection. A pharmaceutical composition for preventing or treating inflammatory diseases.

8. A pharmaceutical composition for preventing or treating an immune disease, comprising a peptide according to any one of claims 1 to 5 as an active ingredient.

9. A pharmaceutical composition for preventing or treating an immune disease, according to claim 8, wherein the immune disease is at least one selected from the group consisting of insulin-dependent diabetes, multiple sclerosis, autoimmune encephalomyelitis, rheumatoid arthritis, autoimmune arthritis, myasthenia gravis, thyroiditis, uveitis, Hashimoto's thyroiditis, primary myxedema, thyrotoxicosis, pernicious anemia, autoimmune atrophic gastritis, Addison's disease, premature menopause, male infertility, juvenile diabetes, Goodpasture's syndrome, pemphigoid, pemphigoid, sympathetic ophthalmia, phacoemulsifying uveitis, autoimmune hemolytic anemia, idiopathic leukopenia, primary biliary tract cirrhosis, cryptogenic cirrhosis, ulcerative colitis, Sjogren's syndrome, scleroderma, Wegener's granulomatosis, polymyositis, dermatomyositis, and systemic lupus erythematosus.

10. A pharmaceutical composition for preventing or treating cancer, comprising a peptide according to any one of claims 1 to 5 as an active ingredient.

11. A pharmaceutical composition for preventing or treating cancer, wherein the cancer is a solid cancer or a blood cancer in the 10th paragraph.

12. A composition for improving skin, comprising a peptide according to any one of claims 1 to 5 as an active ingredient.

13. A composition for improving skin, wherein the skin improvement in claim 12 is at least one selected from the group consisting of skin antioxidant, skin moisture retention, skin elasticity improvement, and skin thickness improvement.

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