WO2024096677A1 - Tlr signaling inhibitory peptide and composition including same for prevention or treatment of inflammatory diseases - Google Patents
Tlr signaling inhibitory peptide and composition including same for prevention or treatment of inflammatory diseases Download PDFInfo
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- WO2024096677A1 WO2024096677A1 PCT/KR2023/017529 KR2023017529W WO2024096677A1 WO 2024096677 A1 WO2024096677 A1 WO 2024096677A1 KR 2023017529 W KR2023017529 W KR 2023017529W WO 2024096677 A1 WO2024096677 A1 WO 2024096677A1
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Images
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
-
- C—CHEMISTRY; METALLURGY
- C40—COMBINATORIAL TECHNOLOGY
- C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
- C40B30/00—Methods of screening libraries
- C40B30/04—Methods of screening libraries by measuring the ability to specifically bind a target molecule, e.g. antibody-antigen binding, receptor-ligand binding
Definitions
- the present invention relates to a peptide library constructed from the sequence of a TIR-like domain, a TLR signal inhibitory peptide involved in the interaction of the TIR domain selected therefrom, and its use in the prevention or treatment of inflammatory diseases.
- TLR Toll-like receptor
- TIR Toll/interleukin-1 receptor
- a protein family that recognizes external pathogens or substances secreted from internal damage or aged tissues has a TIR (Toll/interleukin-1 receptor) domain inside the cell.
- TIR Toll/interleukin-1 receptor
- homologous/heterologous binding to the TIR domain of adapter proteins expressed within cells is a key mechanism that protects the human body by transmitting activated signals. Excessive activation and failure to regulate TLR signaling is not only a major cause of chronic inflammatory diseases such as autoimmune diseases and sepsis, but also, during infection with viruses or pathogens, the interaction of the TIR domain is used as a target for immune evasion of the pathogen. .
- peptide inhibitors derived from human TIR domain sequences have been developed to inhibit TIR-TIR interactions inside cells and have shown efficacy in immunomodulation and treatment of inflammatory diseases.
- most of them are approached only in a low-throughput method through peptide synthesis. Therefore, there is a limitation in sufficiently utilizing the TIR domain sequences that exist in various biological species.
- TIR domains of various biological species in the natural world have been reported to have many heterologous bonds between species (bacteria-human, bacteria-plant, etc.)
- a method of searching for peptide inhibitors that can regulate TIR-TIR interactions from the vast TIR domain sequences in the natural world has been developed. It is necessary, and new peptide inhibitors that can inhibit this TIR-TIR interaction can be used to treat various diseases through immune regulation.
- the present inventors designed TIR surface sequences from the vast TIR domains possessed by various biological species, produced approximately 190,000 peptide sequences, constructed a phage display library using these, and used these to bind to the human TIR domain to transmit TRL signaling.
- the present invention regarding a novel peptide candidate sequence that regulates the pathway and its screening method has been completed.
- the purpose of the present invention is to provide a method for constructing a phage display library of peptides containing the surface sequence of the TIR domain.
- Another object of the present invention is to provide a method for screening peptides derived from the surface sequence of the TIR domain and having TLR signal transduction inhibitory activity.
- Another object of the present invention is to provide a TLR signal inhibitory peptide derived from the surface sequence of the TIR domain.
- Another object of the present invention is to provide a composition for preventing, improving or treating inflammatory diseases containing a TLR signal inhibitory peptide.
- the present invention provides a method for constructing a phage display library of peptides containing the surface sequence of the TIR domain.
- the present invention provides a method for screening peptides having TLR signal transduction inhibitory activity derived from the surface sequence of the TIR domain.
- the present invention provides a TLR signal inhibitory peptide derived from the surface sequence of the TIR domain.
- the present invention provides a composition for preventing, improving or treating inflammatory diseases containing a TLR signal inhibitory peptide.
- the present invention relates to a library of peptides containing the surface sequence of the TIR domain from the TIR domain possessed by various biological species and a method for constructing the same.
- the peptide library was constructed using a method including the following steps. First, in order to extract TIR domains from various species of organisms based on cross-species linkage of TIR-like domains existing in nature, full sequence alignment of the surface sequence of 13,644 TIR domains (PF01582) from PFAM was performed. was used, and the sequences of all surfaces excluding the inner (core) region of the TIR domain tertiary structure were designed into approximately 16 amino acid sequences to construct a library of 190,946 peptides.
- peptides with TLR inhibitory activity were screened from the constructed peptide library.
- the screening may be done through, for example, phage display techniques.
- phage display technology Through phage display technology, peptides are genetically linked, inserted, or replaced with the phage's coat protein and displayed on the outside of the phage, and the peptide can be encoded by genetic information within the virion.
- phage or “bacteriophage” are used interchangeably and may refer to a virus that infects bacteria and replicates within the bacteria.
- the phage or bacteriophage may be used to display a peptide belonging to the library of the present invention, and the peptide may be genetically engineered to be displayed on the phage coat protein or a fragment thereof.
- the genetic manipulation includes introducing a gene encoding the peptide.
- the term “phage display” may mean displaying a functional foreign peptide or protein on the surface of a phage or phagemid particle.
- the surface of the phage may refer to the phage coat protein or a fragment thereof.
- the phage may be formed in such a manner that the C-terminus of the functional foreign peptide is connected to the N-terminus of the phage coat protein, or the peptide is inserted between the contiguous amino acid sequences of the phage coat protein, or the contiguous amino acid sequence of the coat protein. It may be a phage that replaces a part of .
- the coat protein is not limited to its type and may be p3, p6, p8, p9, or a fusion thereof.
- peptides that specifically bind to MAL or MyD88, a key adapter protein of TLR signaling were screened.
- peptide sequences binding to large-scale MAL or MyD88 were obtained through NGS analysis of the base sequences encoding the selected peptides.
- the peptide screened through the phage display method was confirmed to have the function of regulating TLR signals and, in particular, inhibiting the signaling pathway.
- the screened TIR domain-binding peptide having TLR inhibitory activity may include a peptide consisting of the amino acid sequences shown in Tables 1 to 4, and may further include a sequence for intracellular penetration.
- the TIR domain binding peptide may further include a peptide sequence for cell immersion.
- peptides combining the TIR domain-binding peptide and the cell-penetrating peptide may be prepared as shown in SEQ ID NO: 1 to SEQ ID NO: 21, and cloning was performed using the prepared peptide.
- TLR signaling pathway is a signal transduction pathway regulated by Toll like receptor (TLR), and TLR is a protein or keystone of the defense mechanism that recognizes and removes external pathogens or internal damage and aged tissues.
- TLR Toll like receptor
- Excessive activation of TLR signaling can be a major cause of severe chronic inflammatory diseases such as infectious diseases, sepsis, autoimmune diseases, and degenerative brain diseases. Additionally, since TLRs play an important role in controlling inflammatory diseases, various drugs targeting them exist.
- the TIR domain-binding peptide of the present invention can be used as a treatment for various inflammatory diseases through its TRL signal inhibition effect.
- the inflammatory disease is an immune response caused by various causes such as infection by pathogens or tissue damage.
- the inflammatory disease herein is not limited to the type, and is more specifically dermatitis, allergy, atopy, asthma, and periodontitis.
- rhinitis allergic and non-allergic rhinitis, otitis media, pharyngitis, tonsillitis, pneumonia, gastric ulcer, chronic and acute gastritis, Crohn's disease, colitis, chronic and acute enteritis, hemorrhoids, gout, ankylosing spondylitis, osteoarthritis, rheumatoid arthritis, periarthritis, Tendinitis, tenosynovitis, myositis, acute and chronic hepatitis, cystitis, acute and chronic nephritis, Sjogren's syndrome, multiple sclerosis, rhinitis, chronic obstructive pulmonary disease, irritable bowel syndrome, viral infection, bacterial infection, fungal infection, burn, Any selected from the group consisting of surgical or dental wounds, atherosclerosis, arthritis, Hodgkin's disease, pancreatitis, ulceris, scleritis, uveitis, conjunctivitis,
- “sepsis” refers to a disease in which a serious inflammatory response occurs throughout the body. In particular, it is a disease that can cause serious organ damage to an individual due to immune system abnormalities caused by microbial infection. The condition in which these symptoms worsen and circulatory, cellular, and metabolic abnormalities occur is called septic shock, and septic shock is a serious disease with a high mortality rate.
- “preventing, ameliorating or treating sepsis” includes reducing, improving or eliminating all or part of the symptoms associated with sepsis and conditions associated with multi-organ dysfunction syndrome.
- macular degeneration is a chronic progressive degenerative disease of the macula, caused by excessive free radicals, mitochondrial dysfunction, inflammation, abnormalities in lipid metabolism, genetic factors, environmental factors (smoking, exposure to ultraviolet rays, etc.), etc. It is known to occur, but the exact cause has not been identified.
- the macular degeneration of the present invention may be particularly dry macular degeneration, and dry macular degeneration accounts for 85 to 90% of all macular degeneration, and end-stage macular degeneration may lead to wet macular degeneration. Dry macular degeneration is specifically characterized by medium-sized or larger yellow deposits called drusen, pigmentary abnormalities of the retinal pigment epithelium, and reticular pseudodrusen beneath the retina. It is characterized by an increase in deposits called , and gradual atrophy of the nerves in the macular area.
- prevention, improvement or treatment of macular degeneration refers to preventing the formation of waste products in the retinal pigment epithelium (RPE) and the outer nuclear layer (ONL) and the atrophy of photoreceptors, thereby causing macular degeneration and It involves reducing, eliminating and improving associated symptoms.
- RPE retinal pigment epithelium
- ONL outer nuclear layer
- prevention refers to all actions that can suppress or delay the onset of the disease by administering the pharmaceutical composition according to the present invention.
- treatment refers to any action in which the symptoms of the disease are improved or benefited by administration of the pharmaceutical composition according to the present invention.
- the pharmaceutical composition of the present invention can be formulated and used in the form of oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, external preparations, suppositories, and sterile injection solutions according to conventional methods. It may additionally contain carriers or excipients necessary for the formulation.
- Pharmaceutically acceptable carriers, excipients and diluents that may be additionally included in the active ingredients include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, These include calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, magnesium stearate and mineral oil. When formulated, it is prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants.
- solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and such solid preparations include the extract or compound with at least one excipient, at least cotton, starch, and calcium carbonate. It is prepared by mixing sucrose, lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium styrate talc are also used.
- Liquid preparations for oral administration include suspensions, oral solutions, emulsions, and syrups. 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. there is.
- Preparations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories.
- Non-aqueous solvents and suspensions include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate.
- injectable ester such as ethyl oleate.
- As a base for suppositories witepsol, macrogol, tween 61, cacao, laurin, glycerogeratin, etc. can be used.
- the pharmaceutical composition of the present invention can be administered orally or parenterally (intravenously, subcutaneously, intraperitoneally, or topically) depending on the desired method, and the dosage depends on the condition and weight of the patient, the degree of the disease, and the form of the drug. It varies depending on the route and time of administration, and an appropriate form can be selected by a person skilled in the art.
- the pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount.
- pharmaceutically effective amount means a reasonable amount applicable to medical treatment and an amount sufficient to treat a disease, and the criteria are the patient's disease, severity, drug activity, sensitivity to the drug, and administration time. , can be determined depending on the route of administration and excretion rate, treatment period, ingredients used together, and other matters.
- the pharmaceutical composition of the present invention can be administered individually or in combination with other therapeutic agents, and can be administered sequentially or simultaneously with conventional therapeutic agents. Considering all of the above factors, the dosage can be determined at a level that can minimize side effects, and this can be easily determined by a person skilled in the art.
- the dosage of the pharmaceutical composition may vary depending on the patient's age, weight, severity, gender, etc., and is generally administered in an amount of 0.001 to 150 mg per 1 kg of body weight, more preferably 0.01 to 100 mg per kg of body weight every day or every other day. It can be administered 1 to 3 times a day. However, this is an example, and the dosage can be set differently as needed.
- the present invention constructs a library of over 190,000 peptides derived from the TIR surface sequence from the vast TIR domains possessed by various biological species, and from this, new peptides that bind to the human TIR domain (MAL, MYD88) can be screened, and these peptides Can be used as a TLR signaling inhibitor.
- MAL, MYD88 human TIR domain
- Figure 1 is a schematic diagram explaining the process for constructing a peptide library that binds to the human TIR domain using the entire sequence of the TIR domain in PFAM.
- Figure 2 shows the results confirming the inhibitory efficacy of the peptide of the present invention against various TRLs.
- Figure 3 shows the results of selecting peptides that specifically bind to MAL and MyD88 and confirming their efficacy in inhibiting TLR4 activity.
- Figure 4 shows the results of confirming the concentration-dependent inhibitory effect of peptides confirmed to have TRL4 inhibitory activity.
- Figure 5 shows the results of confirming the phosphorylation inhibitory effect of IRAK4 and IRF3, which are downstream proteins of TLR4 signal, for four types of peptides whose TLR4 inhibitory activity was confirmed.
- Figure 6 shows the results of confirming the effect of four types of peptides confirmed to have TRL4 inhibitory activity on phosphorylation inhibition of ERK, p38, and JNK, which are downstream proteins of TLR4 signaling.
- Figure 7 shows the results of confirming the inhibitory effect of NF-kB nuclear localization of four types of peptides confirmed to have TRL4 inhibitory activity.
- Figure 8 confirms the inhibitory effect of a peptide having TRL4 inhibitory activity on cytokine secretion in macrophages.
- Figure 9 shows the sepsis treatment effect of a peptide with TRL4 inhibitory activity and the results of confirming the survival rate of mice.
- Figure 10 shows the results confirming the effect of suppressing inflammatory cytokine secretion in relation to the sepsis treatment effect of a peptide having TRL4 inhibitory activity.
- Figure 11 shows the results of confirming the effect on kidney cell death in relation to the sepsis treatment effect of a peptide with TRL4 inhibitory activity.
- Figure 12 shows the results of confirming the protective effect of retinal pigment epithelial cells (RPE) and outer nuclear layer (ONL) through SD-OCT and H&E staining in relation to the treatment effect of dry macular degeneration of a peptide with TRL4 inhibitory activity.
- RPE retinal pigment epithelial cells
- ONL outer nuclear layer
- Figure 13 shows the results of a quantitative comparative analysis of the thickness of the retinal pigment epithelial cells and the outer nuclear layer observed in Figure 12.
- a 16 mer amino acid sequence (187,814) was designed with all surface sequences, excluding the core region of the tertiary structure of the TIR domain belonging to the above species, cut to overlap about 7 amino acids.
- the human TIR domain was designed as a 16 mer amino acid sequence.
- a list of 3,132 peptide sequences was constructed from the surface sequences of 14 types of bacterial TIR domains, 2 types of bacterial TIR domains, and 3 non-TIR domains known to bind to TIR. As a result, a peptide library of approximately 190,946 16mers was constructed.
- phage display was performed as follows.
- DNA sequences for expression of each peptide belonging to the library constructed in Example 1 were synthesized and vector cloning was performed.
- Vector cloning was performed using p3/p8 phagemid.
- the vector was introduced into M13 bacteriophage, and the peptide derived from the TIR domain was displayed on the phage surface.
- peptides that inhibit the activity of MAL and MyD88 which are key proteins of the TLR signaling pathway, were selected through specific binding.
- MAL and MyD88 proteins produced in E. coli were fixed to a 96-well plate, and the phage-displayed peptide library fused to the P8 or P3 envelope protein was mixed, then non-binding phages were removed, and the remaining phages were amplified in E. coli again. A total of 4 rounds of combining process were performed. After the fourth round, the remaining phages were obtained by binding to the final immobilized target protein, and through NGS analysis of the phagemid in the phage, the peptide sequence binding to the target and the relative distribution of each sequence were confirmed.
- P3_MyD88 Peptide sequence NGS count Population SSFREGLWRLLELSKK 79510 23.15% NQWRNALTAAANLSGW 28662 8.35% DHHLLDLPRGKAIKPEILG 26722 7.78% YTALTNSGFHTFRDND 17600 5.12% LVAHEISKKMKIFATI 17452 5.08% LKVESWRQALEKAGKL 16847 4.91% EWIREQLRPALKERLP 16821 4.90% YAFTGHLHRYLVQRGV 13420 3.91% NELVHVWSSKANSLVG 8816 2.57% KNLFNELVSKNIRTFM 8543 2.49% LKWGDPYFWSKLRYAM 8310 2.42% YLKWGYPWFWDRLRFA 7321 2.13% SENYATSPWALDELVL 6494 1.89% KEVEGWRDALTKVASL 6187 1.80% SELRVVDDDGSVSPLEM 4872 1.42% AVDKETDKIQTPLKNA 4790
- a sequence for passing through the cell membrane (CPP; RQIKIWFQNRRMKWKK, SEQ ID NO. 23) was fused to the above sequence and synthesized, and a peptide consisting of the sequences shown in Table 5 below was produced.
- TLR TLR2, TLR3 of 21 synthetic peptides using HEK-Blue hTLR2, 3, 4, 5, 7, 8, 9 reporter cell lines overexpressing TLRs targeting the screened peptides as shown in Tables 1, 2, 3, and 4 above.
- TLR4, TLR 5, TLR7, TLR8, and TLR9 TLR2, TLR4, TLR7, and TLR9 inhibitory activity was confirmed for most peptides.
- inhibitory activity against TLR3 and TLR5 was confirmed in some peptides.
- Figure 2 In addition, as shown in Figure 3, the inhibitory effect on TLR4 was confirmed through SEAP reporter assay using HEK-Blue hTLR4, and TLR4 inhibitory activity was confirmed in most peptides compared to the control group.
- mice In order to confirm that the TLR inhibitory activity peptide of the present invention is effective in treating sepsis, an experiment was conducted on mice.
- the TLR inhibitory peptides (TDIP1, TDIP5, TDIP6, TDIP20) of the present invention were intraperitoneally injected into mice in which sepsis was induced with LPS at an amount of 10 nmol per g of mouse body weight, and the mouse survival rate and 16 hours after LPS and peptide administration were injected intraperitoneally.
- Cytokines in serum (IL-23, IL-1 ⁇ , IFN ⁇ , TNF- ⁇ , MCP-1, IL-12p70, IL-1 ⁇ , IL-10, IL-6, IL-27, IL-17A, IFN ⁇ ) ELISA analysis was performed to confirm expression level, and TUNEL assay was performed to confirm cell death in kidney tissue.
- the TLR inhibitory active peptide of the present invention has a therapeutic effect on dry macular degeneration
- the TLR inhibitory active peptide of the present invention (TDIP1, TDIP5, TDIP6, After directly administering 5 ⁇ l of TDIP20) at a concentration of 20 ⁇ M to the upper corneal surface of the eye once a day for 21 days, the outer nuclear layer (ONL) and retinal cell epithelial thickness (RPE) of the diseased mice were checked, The effect on retinal cell death was confirmed.
- the present invention provides a surface-exposed sequence involved in inter-domain binding excluding the core region of the tertiary structure of the TIR domain from various biological species, with a length of 16 to 21 amino acids in which some sequences overlap.
- the overlapping sequence of the peptide fragment is 5 to 8 amino acids long.
- the present invention provides a TLR signal transduction inhibitory activity comprising screening for a peptide that specifically binds to TIR domains including human MAL or MyD88 from a phage display library constructed by the above method. This is about a screening method for peptides having .
- the peptide having the TLR signal transduction inhibitory activity specifically binds to the TIR domain contained in humans, bacteria, or plants, including human MAL or MyD88, and inhibits the binding between TIR domains to TLR. It is characterized by suppressing signals.
- the present invention relates to a TLR (Toll like receptor) signal inhibitory peptide comprising a peptide fragment derived from the surface sequence of the TIR domain.
- TLR Toll like receptor
- the TLR may be selected from the group consisting of TLR2, TLR3, TLR4, TLR5, TLR7, and TLR9.
- the peptide is characterized as having been screened by the method of claim 4.
- the peptide is characterized in that it includes one or more peptides consisting of the amino acid sequences of SEQ ID NO: 1 to SEQ ID NO: 21.
- the peptide is characterized as being fused with a peptide consisting of the sequence of SEQ ID NO: 23.
- the present invention relates to a pharmaceutical composition for preventing, improving or treating inflammatory diseases comprising the peptide of claim 5.
- the inflammatory disease includes dermatitis, allergy, atopy, asthma, periodontitis, allergic and non-allergic rhinitis, otitis media, pharyngitis, tonsillitis, pneumonia, gastric ulcer, chronic and acute gastritis, Crohn's disease, and colitis.
- chronic obstructive pulmonary disease irritable bowel syndrome
- viral infections bacterial infections, fungal infections, burns, surgical or dental wounds
- atherosclerosis arthritis
- Hodgkin's disease pancreatitis
- scleritis uveitis
- conjunctivitis conjunctivitis.
- dry macular degeneration wet macular degeneration, sepsis, or septic shock.
- the inflammatory disease may be sepsis, septic shock, or symptoms associated with sepsis and multi-organ dysfunction syndrome.
- the inflammatory disease may be macular degeneration.
- the macular degeneration may be dry macular degeneration.
- the composition can reduce the thickness of the retinal cell epithelium (RPE) or outer nuclear layer (ONL).
- RPE retinal cell epithelium
- ONL outer nuclear layer
- the peptide may include one or more of the peptides consisting of the amino acid sequences of SEQ ID NOs: 1, 5, 6, or 20.
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Abstract
The present invention relates to a peptide library derived from the vast TIR domains present across various biological species, a construction method therefor, and a novel peptide, screened from this library, which is involved in the TLR signaling pathway by binding to the human TIR domain, and a use thereof as a TLR4 signaling inhibitor.
Description
본 발명은 TIR 유사 도메인의 서열로 구축한 펩타이드 라이브러리 및 이들로부터 선별된 TIR 도메인의 상호 작용에 관여하는 TLR 신호 억제 펩타이드 및 이의 염증성 질환에 대한 예방 또는 치료 용도에 대한 것이다. The present invention relates to a peptide library constructed from the sequence of a TIR-like domain, a TLR signal inhibitory peptide involved in the interaction of the TIR domain selected therefrom, and its use in the prevention or treatment of inflammatory diseases.
외부의 병원체 또는 인체 내부의 손상, 노화된 조직으로부터 분비되는 물질을 인식하는 단백질 패밀리인 톨-유사 수용체(Toll-like receptor, TLR)는 세포 내부의 TIR(Toll/interleukin-1 receptor) 도메인을 가지고 있으며, 세포 내에 발현되는 어댑터 단백질의 TIR 도메인과 동종/이종 결합은 활성화된 신호를 전달하여 인체를 보호하는 핵심 메커니즘이다. 과도한 TLR 신호의 활성화 및 조절의 실패는 자가면역질환, 패혈증과 같은 만성염증질환의 주요 원인이 될 뿐만 아니라, 바이러스 또는 병원균의 감염시 TIR 도메인의 상호작용은 병원체의 면역회피를 위한 타겟으로 활용된다. 이에 따라 세포 내부의 TIR-TIR 상호작용을 억제하기 위한, 인간 TIR 도메인 서열 유래의 펩타이드 저해제가 개발되어 면역조절 및 염증질환 치료 효능을 보여준 바가 있으나, 대부분 펩타이드 합성을 통한 low-throughput 방식으로만 접근하여, 다양한 생물종에 존재하는 TIR 도메인의 서열을 충분히 활용하지 못하는 한계가 존재한다. Toll-like receptor (TLR), a protein family that recognizes external pathogens or substances secreted from internal damage or aged tissues, has a TIR (Toll/interleukin-1 receptor) domain inside the cell. In addition, homologous/heterologous binding to the TIR domain of adapter proteins expressed within cells is a key mechanism that protects the human body by transmitting activated signals. Excessive activation and failure to regulate TLR signaling is not only a major cause of chronic inflammatory diseases such as autoimmune diseases and sepsis, but also, during infection with viruses or pathogens, the interaction of the TIR domain is used as a target for immune evasion of the pathogen. . Accordingly, peptide inhibitors derived from human TIR domain sequences have been developed to inhibit TIR-TIR interactions inside cells and have shown efficacy in immunomodulation and treatment of inflammatory diseases. However, most of them are approached only in a low-throughput method through peptide synthesis. Therefore, there is a limitation in sufficiently utilizing the TIR domain sequences that exist in various biological species.
자연계의 다양한 생물종이 가진 TIR 도메인은 종간(박테리아-인간, 박테리아-식물 등) 이종 결합이 다수 보고되고 있어, 자연계의 방대한 TIR 도메인 서열로부터 TIR-TIR 상호작용을 조절할 수 있는 펩타이드 저해제의 탐색 방법이 필요하며, 이러한 TIR-TIR 상호작용을 억제할 수 있는 새로운 펩타이드 저해제는 면역 조절을 통해 다양한 질환의 치료에 활용할 수 있다.As the TIR domains of various biological species in the natural world have been reported to have many heterologous bonds between species (bacteria-human, bacteria-plant, etc.), a method of searching for peptide inhibitors that can regulate TIR-TIR interactions from the vast TIR domain sequences in the natural world has been developed. It is necessary, and new peptide inhibitors that can inhibit this TIR-TIR interaction can be used to treat various diseases through immune regulation.
이에 본 발명자들은 다양한 생물종이 가지고 있는 방대한 TIR 도메인으로부터 TIR 표면의 서열들을 디자인하여 19만여개의 펩타이드 서열을 제작하고, 이를 통한 파지 디스플레이 라이브러리를 구축하였으며, 이를 이용하여 인간 TIR 도메인에 결합하여 TRL 신호전달 경로를 조절하는 신규 펩타이드 후보 서열 및 이의 스크리닝 방법에 대한 본 발명을 완성하였다. Accordingly, the present inventors designed TIR surface sequences from the vast TIR domains possessed by various biological species, produced approximately 190,000 peptide sequences, constructed a phage display library using these, and used these to bind to the human TIR domain to transmit TRL signaling. The present invention regarding a novel peptide candidate sequence that regulates the pathway and its screening method has been completed.
따라서, 본 발명의 목적은 TIR 도메인의 표면 서열을 포함하는 펩타이드의 파지 디스플레이 라이브러리 구축 방법을 제공하는 것이다. Therefore, the purpose of the present invention is to provide a method for constructing a phage display library of peptides containing the surface sequence of the TIR domain.
본 발명의 다른 목적은, TIR 도메인의 표면 서열로부터 유래된, TLR 신호 전달 억제 활성을 가지는 펩타이드의 스크리닝 방법을 제공하는 것이다. Another object of the present invention is to provide a method for screening peptides derived from the surface sequence of the TIR domain and having TLR signal transduction inhibitory activity.
본 발명의 또 다른 목적은 TIR 도메인의 표면 서열로부터 유래된 TLR 신호 억제 펩타이드를 제공하는 것이다. Another object of the present invention is to provide a TLR signal inhibitory peptide derived from the surface sequence of the TIR domain.
본 발명의 또 다른 목적은 TLR 신호 억제 펩타이드를 포함하는 염증성 질환의 예방, 개선 또는 치료용 조성물을 제공하는 것이다. Another object of the present invention is to provide a composition for preventing, improving or treating inflammatory diseases containing a TLR signal inhibitory peptide.
상기와 같은 목적을 달성하기 위하여, 본 발명은 TIR 도메인의 표면 서열을 포함하는 펩타이드의 파지 디스플레이 라이브러리 구축 방법을 제공한다. In order to achieve the above object, the present invention provides a method for constructing a phage display library of peptides containing the surface sequence of the TIR domain.
본 발명의 다른 목적을 달성하기 위하여 본 발명은, TIR 도메인의 표면 서열로부터 유래된, TLR 신호 전달 억제 활성을 가지는 펩타이드의 스크리닝 방법을 제공한다. In order to achieve another object of the present invention, the present invention provides a method for screening peptides having TLR signal transduction inhibitory activity derived from the surface sequence of the TIR domain.
본 발명의 또 다른 목적을 달성하기 위하여 본 발명은 TIR 도메인의 표면 서열로부터 유래된 TLR 신호 억제 펩타이드를 제공한다. In order to achieve another object of the present invention, the present invention provides a TLR signal inhibitory peptide derived from the surface sequence of the TIR domain.
본 발명의 또 다른 목적을 달성하기 위하여 본 발명은 TLR 신호 억제 펩타이드를 포함하는 염증성 질환의 예방, 개선 또는 치료용 조성물을 제공한다. In order to achieve another object of the present invention, the present invention provides a composition for preventing, improving or treating inflammatory diseases containing a TLR signal inhibitory peptide.
이하, 본 발명을 상세하게 설명한다. Hereinafter, the present invention will be described in detail.
본 발명의 일 양태로서, 본 발명은 다양한 생물종이 가지고 있는 TIR 도메인으로부터 상기 TIR 도메인의 표면 서열을 포함하는 펩타이드의 라이브러리 및 이의 구축 방법에 대한 것이다. In one aspect of the present invention, the present invention relates to a library of peptides containing the surface sequence of the TIR domain from the TIR domain possessed by various biological species and a method for constructing the same.
상기 펩타이드 라이브러리는 하기와 같은 단계를 포함하는 방법으로 구축하였다. 먼저, 자연계에 존재하는 TIR 유사 도메인들의 종간 교차(cross-species) 결합을 바탕으로 다양한 종의 생물로부터 TIR 도메인을 추출하기 위해, PFAM으로부터 13,644개의 TIR 도메인(PF01582)의 표면 서열에 대한 full sequence alignment을 이용하였고, 상기 TIR 도메인 3차 구조의 안쪽(core) 부위를 제외한 모든 표면의 서열을 대상으로 이를 약 16개의 아미노산 서열로 디자인하여, 190,946개의 펩타이드 라이브러리를 구축하였다. The peptide library was constructed using a method including the following steps. First, in order to extract TIR domains from various species of organisms based on cross-species linkage of TIR-like domains existing in nature, full sequence alignment of the surface sequence of 13,644 TIR domains (PF01582) from PFAM was performed. was used, and the sequences of all surfaces excluding the inner (core) region of the TIR domain tertiary structure were designed into approximately 16 amino acid sequences to construct a library of 190,946 peptides.
또한, 상기 구축된 펩타이드 라이브러리로부터 TLR 억제 활성을 가지는 펩타이드를 스크리닝하였다. 상기 스크리닝은 예를 들어, 파지 디스플레이 기법을 통해 선별될 수 있다. 파지 디스플레이 기법을 통해 펩타이드가 유전적으로 파지의 외피 단백질에 연결, 삽입 또는 치환되어 파지의 외부에 디스플레이되고, 펩타이드는 비리온 내의 유전 정보에 의해 암호화될 수 있다. Additionally, peptides with TLR inhibitory activity were screened from the constructed peptide library. The screening may be done through, for example, phage display techniques. Through phage display technology, peptides are genetically linked, inserted, or replaced with the phage's coat protein and displayed on the outside of the phage, and the peptide can be encoded by genetic information within the virion.
본 발명에서 용어 "파지(phage)" 또는 "박테리오파지(bacteriophage)"는 호환적으로 사용되며, 박테리아를 감염시키고, 박테리아 내에서 복제되는 바이러스를 의미할 수 있다. 상기 파지 또는 박테리오 파지는 상기 본 발명의 라이브러리에 속하는 펩타이드를 디스플레이 하기 위하여 사용될 수 있으며, 상기의 펩타이드가 파지의 외피 단백질 또는 이의 단편에 디스플레이 되도록 유전적으로 조작된 것일 수 있다. 상기 유전적 조작은 상기의 펩타이드를 코딩하는 유전자가 도입되는 것을 포함한다. In the present invention, the terms “phage” or “bacteriophage” are used interchangeably and may refer to a virus that infects bacteria and replicates within the bacteria. The phage or bacteriophage may be used to display a peptide belonging to the library of the present invention, and the peptide may be genetically engineered to be displayed on the phage coat protein or a fragment thereof. The genetic manipulation includes introducing a gene encoding the peptide.
본 발명에서 용어 "파지 디스플레이(phage display)"는 파지 또는 파지미드(phagemid) 입자의 표면에 기능적 외래 펩타이드 또는 단백질이 표시(display)되는 것을 의미할 수 있다. 상기 파지의 표면은 파지의 외피 단백질 또는 그의 단편을 의미할 수 있다. 또한 상기 파지는 상기 기능적 외래 펩티드의 C-말단이 파지의 외피 단백질의 N-말단에 연결되거나, 또는 상기 펩타이드가 파지의 외피 단백질의 연속되는 아미노산 서열 사이에 삽입되거나 또는 외피단백질의 연속되는 아미노산 서열의 일 부분을 치환한 것인 파지일 수 있다. 또한, 상기 외피 단백질은 그 종류에 제한되지 않으며, p3, p6, p8, p9 또는 이들이 융합된 것 일 수 있다. In the present invention, the term “phage display” may mean displaying a functional foreign peptide or protein on the surface of a phage or phagemid particle. The surface of the phage may refer to the phage coat protein or a fragment thereof. In addition, the phage may be formed in such a manner that the C-terminus of the functional foreign peptide is connected to the N-terminus of the phage coat protein, or the peptide is inserted between the contiguous amino acid sequences of the phage coat protein, or the contiguous amino acid sequence of the coat protein. It may be a phage that replaces a part of . Additionally, the coat protein is not limited to its type and may be p3, p6, p8, p9, or a fusion thereof.
상기 파지 디스플레이된 펩타이드 중에서, TLR 신호전달 경로에 대한 억제 활성을 가지는 펩타이드를 스크리닝 하기 위하여, TLR 신호의 핵심 어댑터 단백질인 MAL 또는 MyD88에 특이적으로 결합하는 펩타이드를 스크리닝 하였다. 상기 결과, 선별된 펩타이드를 코딩하는 염기 서열에 대한 NGS 분석을 통해 대규모 MAL 또는 MyD88에 결합하는 펩타이드 서열을 확보하였다. Among the phage-displayed peptides, in order to screen for peptides with inhibitory activity on the TLR signaling pathway, peptides that specifically bind to MAL or MyD88, a key adapter protein of TLR signaling, were screened. As a result, peptide sequences binding to large-scale MAL or MyD88 were obtained through NGS analysis of the base sequences encoding the selected peptides.
또한, 본 발명의 일 실시예에서, 상기 파지디스플레이 방식을 통해 스크리닝된 펩타이드는 TLR 신호를 조절하며, 특히 상기 신호전달 경로를 억제하는 기능을 가지는 것을 확인하였다.Additionally, in one example of the present invention, the peptide screened through the phage display method was confirmed to have the function of regulating TLR signals and, in particular, inhibiting the signaling pathway.
이에 제한되는 것은 아니나, 상기 스크리닝된 TLR 억제 활성을 가지는 TIR 도메인 결합 펩타이드는 표 1 내지 4에 기재된 아미노산 서열로 이루어진 펩타이드를 포함할 수 있으며, 세포 내 침투를 위한 서열을 추가로 포함할 수 있다. 본 발명의 일 실시예에서는 상기 TIR 도메인 결합 펩타이드는 세포 침두를 위한 펩타이드 서열을 추가로 포함할 수 있다. 이에 제한되는 것은 아니나, 상기 TIR 도메인 결합 펩타이드와 세포 침투 펩타이드가 결합된 펩타이드는 서열번호 1 내지 서열번호 21과 같이 제작될 수 있으며, 상기의 제작된 펩타이드를 이용하여 클로닝을 진행하였다. Although not limited thereto, the screened TIR domain-binding peptide having TLR inhibitory activity may include a peptide consisting of the amino acid sequences shown in Tables 1 to 4, and may further include a sequence for intracellular penetration. In one embodiment of the present invention, the TIR domain binding peptide may further include a peptide sequence for cell immersion. Although not limited thereto, peptides combining the TIR domain-binding peptide and the cell-penetrating peptide may be prepared as shown in SEQ ID NO: 1 to SEQ ID NO: 21, and cloning was performed using the prepared peptide.
TLR 신호 경로는 톨 유사 수용체(Toll like receptor; TLR)에 의해 조절되는 신호 전달 경로이며, TLR은 외부의 병원체 또는 인체 내부의 손상, 노화된 조직 등을 인식하여 제거하는 방어 메커니즘의 핵심인 단백질이나, 과도한 TLR 신호의 활성화는 감염병, 패혈증, 자가면역질환, 퇴행성뇌질환과 같은 중증 만성염증질환의 주요 원인이 될 수 있다. 또한 TLR은 염증 질환의 조절에 중요한 역할을 하므로, 이를 타겟으로 하는 다양한 약물이 존재한다. The TLR signaling pathway is a signal transduction pathway regulated by Toll like receptor (TLR), and TLR is a protein or keystone of the defense mechanism that recognizes and removes external pathogens or internal damage and aged tissues. , Excessive activation of TLR signaling can be a major cause of severe chronic inflammatory diseases such as infectious diseases, sepsis, autoimmune diseases, and degenerative brain diseases. Additionally, since TLRs play an important role in controlling inflammatory diseases, various drugs targeting them exist.
본 발명의 TIR 도메인 결합 펩타이드는 TRL 신호 억제 효과를 통해 다양한 염증성 질환에 대한 치료제로서 활용될 수 있다. 상기 염증성 질환은 병원체에 의한 감염 또는 조직의 손상 등 다양한 원인에 의해 유발되는 면역 반응으로서, 본 명세서의 염증성 질환은 그 종류에 제한되는 것은 아니며, 보다 구체적으로는 피부염, 알레르기, 아토피, 천식, 치주염, 알레르기성 및 비-알레르기성 비염, 중이염, 인후염, 편도염, 폐렴, 위궤양, 만성 및 급성 위염, 크론병, 대장염, 만성 및 급성 장염, 치질, 통풍, 강직성 척추염, 골관절염, 류마티스관절염, 견관절주위염, 건염, 건초염, 근육염, 급성 및 만성 간염, 방광염, 급성 및 만성 신장염, 쇼그렌 증후군(sjogren's syndrome), 다발성 경화증, 비염, 만성 폐쇄성 폐질환, 과민성 대장 증후군, 바이러스 감염, 박테리아 감염, 곰팡이 감염, 화상, 외과적 또는 치과적 수술에 의한 상처, 아테롬성 동맥 경화증, 관절염, 호지킨병, 췌장염, 홍채염, 공막염, 포도막염, 결막염, 건성황반변성, 습성황반변성, 패혈증 또는 패혈증성 쇼크로 구성된 군에서 선택되는 어느 하나의 질환일 수 있으며, 바람직하게는 건성황반변성, 패혈증 또는 패혈증성 쇼크일 수 있다. The TIR domain-binding peptide of the present invention can be used as a treatment for various inflammatory diseases through its TRL signal inhibition effect. The inflammatory disease is an immune response caused by various causes such as infection by pathogens or tissue damage. The inflammatory disease herein is not limited to the type, and is more specifically dermatitis, allergy, atopy, asthma, and periodontitis. , allergic and non-allergic rhinitis, otitis media, pharyngitis, tonsillitis, pneumonia, gastric ulcer, chronic and acute gastritis, Crohn's disease, colitis, chronic and acute enteritis, hemorrhoids, gout, ankylosing spondylitis, osteoarthritis, rheumatoid arthritis, periarthritis, Tendinitis, tenosynovitis, myositis, acute and chronic hepatitis, cystitis, acute and chronic nephritis, Sjogren's syndrome, multiple sclerosis, rhinitis, chronic obstructive pulmonary disease, irritable bowel syndrome, viral infection, bacterial infection, fungal infection, burn, Any selected from the group consisting of surgical or dental wounds, atherosclerosis, arthritis, Hodgkin's disease, pancreatitis, iritis, scleritis, uveitis, conjunctivitis, dry macular degeneration, wet macular degeneration, sepsis, or septic shock. It may be one disease, preferably dry macular degeneration, sepsis, or septic shock.
특히 본 발명에서 상기 "패혈증(sepsis)"은 전신에 심각한 염증 반응이 나타나는 질환을 의미하는 것으로, 특히 미생물 감염에 의한 면역계 이상으로 개체에 심각한 장기 손상을 유발할 수 있는 질환이다. 이러한 증상이 악화되어, 순환성, 세포성, 대사성 이상이 발생된 상태를 패혈성 쇼크라고 하며, 패혈성 쇼크는 사망률이 높은 심각한 질환이다. In particular, in the present invention, “sepsis” refers to a disease in which a serious inflammatory response occurs throughout the body. In particular, it is a disease that can cause serious organ damage to an individual due to immune system abnormalities caused by microbial infection. The condition in which these symptoms worsen and circulatory, cellular, and metabolic abnormalities occur is called septic shock, and septic shock is a serious disease with a high mortality rate.
본 발명의 일 양태로서 "패혈증 예방, 개선 또는 치료"란, 패혈증과 연관된 증상 및 다기관 기능 부전 증후군에 연관된 상태의 전부 또는 일부 증상을 감소, 개선 또는 제거하는 것을 포함한다. In one aspect of the present invention, “preventing, ameliorating or treating sepsis” includes reducing, improving or eliminating all or part of the symptoms associated with sepsis and conditions associated with multi-organ dysfunction syndrome.
또한 본 발명에서 "황반 변성"은 황반의 만성 진행성 퇴행성 질환으로서, 과도한 활성산소, 미토콘드리아의 기능장애, 염증, 지질 대사의 이상, 유전적 요소, 환경적 요소(흡연, 자외선 노출 등) 등에 의해 유발되는 것으로 알려져 있으나, 명확한 원인이 밝혀진 것은 아니다. 본 발명의 상기 황반변성은 특히 건성 황반변성일 수 있으며, 상기 건성 황반변성은 전체 황반변성 중 85 내지 90%를 차지하며, 말기 황반변성은 습성 황반변성으로 이어지기도 한다. 건성 황반변성은 특히 망막 색소 상피 층 아래에 존재하는 드루젠(drusen)으로 불리는 중간 크기 이상의 노란색 침착물, 망막 색소 상피의 색소이상(pigmentary abnormalities), 망막 아래에 존재하는 망상 슈도드루젠(reticular pseudodrusen)으로 불리는 침착물이 증가되며, 황반부의 신경이 점점 위축되는 특징을 가진다. In addition, in the present invention, “macular degeneration” is a chronic progressive degenerative disease of the macula, caused by excessive free radicals, mitochondrial dysfunction, inflammation, abnormalities in lipid metabolism, genetic factors, environmental factors (smoking, exposure to ultraviolet rays, etc.), etc. It is known to occur, but the exact cause has not been identified. The macular degeneration of the present invention may be particularly dry macular degeneration, and dry macular degeneration accounts for 85 to 90% of all macular degeneration, and end-stage macular degeneration may lead to wet macular degeneration. Dry macular degeneration is specifically characterized by medium-sized or larger yellow deposits called drusen, pigmentary abnormalities of the retinal pigment epithelium, and reticular pseudodrusen beneath the retina. It is characterized by an increase in deposits called , and gradual atrophy of the nerves in the macular area.
본 발명의 일 양태로서 "황반변성의 예방, 개선 또는 치료"란, 망막색소 상피(Retinal Pigment Epithelium; RPE)와 외부과립층(outer nuclear layer; ONL)의 노폐물 형성 및 시세포의 위축을 방지하여, 황반변성과 관련된 증상을 감소, 제거 및 개선하는 것을 포함한다. In one aspect of the present invention, "prevention, improvement or treatment of macular degeneration" refers to preventing the formation of waste products in the retinal pigment epithelium (RPE) and the outer nuclear layer (ONL) and the atrophy of photoreceptors, thereby causing macular degeneration and It involves reducing, eliminating and improving associated symptoms.
본 명세서에서 본 명세서에서 용어 "예방"은 본 발명에 따른 상기 약학적 조성물의 투여에 의해 상기의 질환을 억제시키거나 발병을 지연시킬 수 있는 모든 행위를 의미한다. The term “prevention” herein refers to all actions that can suppress or delay the onset of the disease by administering the pharmaceutical composition according to the present invention.
본 명세서에서 용어 "치료"는 본 발명에 따른 상기 약학적 조성물의 투여에 의해 상기 질환에 대한 증세가 호전되거나 이롭게 되는 모든 행위를 말한다.As used herein, the term “treatment” refers to any action in which the symptoms of the disease are improved or benefited by administration of the pharmaceutical composition according to the present invention.
본 발명의 상기 약학적 조성물은 통상의 방법에 따라 산제, 과립제, 정제, 캡슐제, 현탁액, 에멀젼, 시럽, 에어로졸 등의 경구형 제형, 외용제, 좌제 및 멸균 주사용액의 형태로 제형화하여 사용될 수 있으며, 상기 제형화에 필요한 담체 혹은 부형제 등을 추가로 포함할 수 있다. 상기 유효 성분에 추가로 포함될 수 있는 약학적으로 허용 가능한 담체, 부형제 및 희석제로는 락토즈, 덱스트로즈, 수크로스, 솔비톨, 만니톨, 자일리톨, 에리스리톨, 말티톨, 전분, 아카시아 고무, 알지네이트, 젤라틴, 칼슘 포스페이트, 칼슘 실리케이트, 셀룰로즈, 메틸 셀룰로즈, 미정질 셀룰로스, 폴리비닐 피롤리돈, 물, 메틸히드록시벤조에이트, 프로필히드록시벤조에이트, 마그네슘 스테아레이트 및 광물유 등이 포함된다. 제제화할 경우에는 보통 사용하는 충진제, 증량제, 결합제, 습윤제, 붕해제, 계면활성제 등의 희석제 또는 부형제를 사용하여 조제된다. The pharmaceutical composition of the present invention can be formulated and used in the form of oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, external preparations, suppositories, and sterile injection solutions according to conventional methods. It may additionally contain carriers or excipients necessary for the formulation. Pharmaceutically acceptable carriers, excipients and diluents that may be additionally included in the active ingredients include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, These include calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, magnesium stearate and mineral oil. When formulated, it is prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants.
예를 들어, 경구 투여를 위한 고형 제제에는 정제, 환제, 산제, 과립제, 캡슐제 등이 포함되며, 이러한 고형제제는 상기 추출물 또는 화합물에 적어도 하나 이상의 부형제 적어도 면, 전분, 칼슘카보네이트 (calcium carbonate), 수크로스 (sucrose) 또는 락토오스 (lactose), 젤라틴 등을 섞어 조제된다. 또한 단순한 부형제 이외에 마그네슘 스티레이트 탈크 같은 윤활제들도 사용된다. 경구 투여를 위한 액상제제로는 현탁제, 내용액제, 유제, 시럽제 등이 해당되는데 흔히 사용되는 단순희석제인 물, 리퀴드 파라핀 이외에 여러 가지 부형제, 예를 들면 습윤제, 감미제, 방향제, 보존제 등이 포함될 수 있다. For example, solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and such solid preparations include the extract or compound with at least one excipient, at least cotton, starch, and calcium carbonate. It is prepared by mixing sucrose, lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium styrate talc are also used. Liquid preparations for oral administration include suspensions, oral solutions, emulsions, and syrups. 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. there is.
비경구 투여를 위한 제제에는 멸균된 수용액, 비수성용제, 현탁제, 유제, 동결건조제제, 좌제가 포함된다. 비수성용제, 현탁제로는 프로필렌글리콜 (propylene glycol), 폴리에틸렌 글리콜, 올리브 오일과 같은 식물성 기름, 에틸올레이트와 같은 주사 가능한 에스테르 등이 사용될 수 있다. 좌제의 기제로는 위텝솔 (witepsol), 마크로골, 트윈 (tween) 61, 카카오지, 라우린지, 글리세로제라틴 등이 사용될 수 있다.Preparations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Non-aqueous solvents and suspensions include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate. As a base for suppositories, witepsol, macrogol, tween 61, cacao, laurin, glycerogeratin, etc. can be used.
본 발명의 약학적 조성물은 목적하는 방법에 따라 경구 또는 비경구 투여(정맥주사, 피하, 복강 내 또는 국소에 적용)될 수 있으며, 투여량은 환자의 상태 및 체중, 질병의 정도 및 약물 형태, 투여 경로 및 시간에 따라 다르며, 당업자에 의해 적절한 형태로 선택될 수 있다. The pharmaceutical composition of the present invention can be administered orally or parenterally (intravenously, subcutaneously, intraperitoneally, or topically) depending on the desired method, and the dosage depends on the condition and weight of the patient, the degree of the disease, and the form of the drug. It varies depending on the route and time of administration, and an appropriate form can be selected by a person skilled in the art.
본 발명의 상기 약학적 조성물은, 약학적으로 유효한 양으로 투여한다. 본 발명에서 "약학적으로 유효한 양"이란 의학적 치료에 적용 가능한 합리적인 양으로, 질병을 치료하기에 충분한 양을 의미하며, 그 기준은 환자의 질환, 중증도, 약물 활성, 약물에 대한 민감도, 투여 시간, 투여 경로 및 배출 비율, 치료 기간, 병용되는 성분 및 기타 사항에 따라 결정될 수 있다. 본 발명의 상기 약학적 조성물은, 개별 치료제 혹은 다른 치료제와 병용하여 투여될 수 있고, 종래의 치료제와는 순차적 또는 동시에 투여될 수 있다. 상기 요소들을 모두 고려하여 부작용을 최소화할 수 있는 수준으로 투여량을 결정할 수 있고, 이는 당업자에 의해 용이한 수준으로 결정될 수 있다. 구체적으로 상기 약학적 조성물의 투여량은 환자의 연령, 체중, 중증도, 성별 등에 따라 달라질 수 있으며, 일반적으로 체중 1kg 당 0.001 내지 150 mg, 보다 바람직하게는 0.01 내지 100mg의 양을 매일 또는 격일, 1일 1 내지 3회 투여할 수 있다. 다만, 이는 예시적인 것으로, 상기 투여량은 필요에 달리 설정할 수 있다.The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. In the present invention, "pharmaceutically effective amount" means a reasonable amount applicable to medical treatment and an amount sufficient to treat a disease, and the criteria are the patient's disease, severity, drug activity, sensitivity to the drug, and administration time. , can be determined depending on the route of administration and excretion rate, treatment period, ingredients used together, and other matters. The pharmaceutical composition of the present invention can be administered individually or in combination with other therapeutic agents, and can be administered sequentially or simultaneously with conventional therapeutic agents. Considering all of the above factors, the dosage can be determined at a level that can minimize side effects, and this can be easily determined by a person skilled in the art. Specifically, the dosage of the pharmaceutical composition may vary depending on the patient's age, weight, severity, gender, etc., and is generally administered in an amount of 0.001 to 150 mg per 1 kg of body weight, more preferably 0.01 to 100 mg per kg of body weight every day or every other day. It can be administered 1 to 3 times a day. However, this is an example, and the dosage can be set differently as needed.
본 발명은 다양한 생물종이 가지고 있는 방대한 TIR 도메인으로부터 TIR 표면의 서열 유래의 19만여개의 펩타이드 라이브러리를 구축하여, 이로부터 인간 TIR 도메인(MAL, MYD88)에 결합하는 신규 펩타이드를 스크리닝 할 수 있으며, 이들 펩타이드는 TLR 신호 전달 억제제로서의 용도로 사용될 수 있다. The present invention constructs a library of over 190,000 peptides derived from the TIR surface sequence from the vast TIR domains possessed by various biological species, and from this, new peptides that bind to the human TIR domain (MAL, MYD88) can be screened, and these peptides Can be used as a TLR signaling inhibitor.
도 1은 PFAM에서 TIR domain에 대한 전체 시퀀스를 이용하여, 이로부터 인간 TIR 도메인과 결합하는 펩타이드 라이브러리를 구축하기 위한 과정을 설명한 모식도이다. Figure 1 is a schematic diagram explaining the process for constructing a peptide library that binds to the human TIR domain using the entire sequence of the TIR domain in PFAM.
도 2는 다양한 TRL에 대한 본 발명의 펩타이드의 억제 효능을 확인한 결과이다. Figure 2 shows the results confirming the inhibitory efficacy of the peptide of the present invention against various TRLs.
도 3은 MAL 및 MyD88 과 특이적 결합을 이루는 펩타이드를 선별하여, TLR4 활성 억제 효능을 확인한 결과이다. Figure 3 shows the results of selecting peptides that specifically bind to MAL and MyD88 and confirming their efficacy in inhibiting TLR4 activity.
도 4는 TRL4 억제 활성이 확인된 펩타이드들의 농도 의존적 억제 효과를 확인한 결과이다. Figure 4 shows the results of confirming the concentration-dependent inhibitory effect of peptides confirmed to have TRL4 inhibitory activity.
도 5는 TLR4 억제 활성이 확인되 4종의 펩타이드에 대한 TLR4신호의 하위 단백질인 IRAK4와 IRF3의 인산화 억제 효과를 확인한 결과이다.Figure 5 shows the results of confirming the phosphorylation inhibitory effect of IRAK4 and IRF3, which are downstream proteins of TLR4 signal, for four types of peptides whose TLR4 inhibitory activity was confirmed.
도 6은 TRL4 억제 활성이 확인된 4종의 펩타이드에 대한 TLR4신호의 하위 단백질인 ERK, p38, JNK의 인산화 억제 효과를 확인한 결과이다. Figure 6 shows the results of confirming the effect of four types of peptides confirmed to have TRL4 inhibitory activity on phosphorylation inhibition of ERK, p38, and JNK, which are downstream proteins of TLR4 signaling.
도 7은 TRL4 억제 활성이 확인된 4종의 펩타이드의 NF-kB nuclear localization 억제 효과를 확인한 결과이다. Figure 7 shows the results of confirming the inhibitory effect of NF-kB nuclear localization of four types of peptides confirmed to have TRL4 inhibitory activity.
도 8은 TRL4 억제 활성을 가지는 펩타이드의 대식세포에서의 사이토카인 분비 억제 효과를 확인한 것이다. Figure 8 confirms the inhibitory effect of a peptide having TRL4 inhibitory activity on cytokine secretion in macrophages.
도 9는 TRL4 억제 활성을 가지는 펩타이드 패혈증 치료 효과로서, 마우스의 생존률을 확인한 결과이다. Figure 9 shows the sepsis treatment effect of a peptide with TRL4 inhibitory activity and the results of confirming the survival rate of mice.
도 10은 TRL4 억제 활성을 가지는 펩타이드의 패혈증 치료 효과와 관련하여, 염증성 사이토카인 분비 억제 효과를 확인한 결과이다. Figure 10 shows the results confirming the effect of suppressing inflammatory cytokine secretion in relation to the sepsis treatment effect of a peptide having TRL4 inhibitory activity.
도 11은 TRL4 억제 활성을 가지는 펩타이드의 패혈증 치료 효과와 관련하여, 신장세포 사멸에 미치는 영향을 확인한 결과이다. Figure 11 shows the results of confirming the effect on kidney cell death in relation to the sepsis treatment effect of a peptide with TRL4 inhibitory activity.
도 12는 TRL4 억제 활성을 가지는 펩타이드의 건성 황반변성의 치료 효과와 관련하여, 망막색소상피세포(RPE) 및 외핵층(ONL) 보호 효과를 SD-OCT 및 H&E 염색을 통해 확인한 결과이다. Figure 12 shows the results of confirming the protective effect of retinal pigment epithelial cells (RPE) and outer nuclear layer (ONL) through SD-OCT and H&E staining in relation to the treatment effect of dry macular degeneration of a peptide with TRL4 inhibitory activity.
도 13은 상기 도 12에서 관찰된 망막색소상피세포와 외핵층의 두께를 정량적으로 비교분석한 결과이다. Figure 13 shows the results of a quantitative comparative analysis of the thickness of the retinal pigment epithelial cells and the outer nuclear layer observed in Figure 12.
이하, 본 명세서를 구체적으로 설명하기 위해 실시예를 들어 상세히 설명한다. 그러나, 본 명세서에 따른 실시예들은 여러 가지 다른 형태로 변형될 수 있으며, 본 명세서의 범위가 아래에서 상술하는 실시예들에 한정되는 것으로 해석되지는 않는다. 본 명세서의 실시예들은 당업계에서 평균적인 지식을 가진 자에게 본 명세서를 보다 완전하게 설명하기 위해 제공되는 것이다.Hereinafter, examples will be given in detail to explain the present specification in detail. However, the embodiments according to the present specification may be modified into various other forms, and the scope of the present specification is not to be construed as being limited to the embodiments described in detail below. The embodiments of this specification are provided to more completely explain the present specification to those with average knowledge in the art.
실시예 1. TIR 도메인 결합 펩타이드 라이브러리 구축 Example 1. Construction of TIR domain binding peptide library
다양한 종의 생물에 존재하는 TIR 도메인을 추출하기 위해, PFAM으로부터 13,644개의 TIR 도메인(PF01582)의 표면 서열에 대한 full sequence alignment(#13644)를 이용하였다. To extract TIR domains present in various species of organisms, full sequence alignment (#13644) of the surface sequence of 13,644 TIR domains (PF01582) from PFAM was used.
상기 생물종에 속하는 TIR 도메인의 3차 구조 안쪽(core) 부위를 제외한 모든 표면 서열을 대상으로 약 7개 아미노산이 오버랩되도록 자른 16 mer의 아미노산 서열(187,814개)로 디자인하였으며, 추가로 인간 TIR 도메인 14종과 2종의 bacterial TIR 도메인, TIR과 결합하는 것으로 알려진 3개의 non-TIR 도메인의 표면 서열로 3,132개의 펩타이드 서열 리스트를 구축하였다. 그 결과, 약 190,946개의 16mer의 펩타이드 라이브러리를 구축하였다.A 16 mer amino acid sequence (187,814) was designed with all surface sequences, excluding the core region of the tertiary structure of the TIR domain belonging to the above species, cut to overlap about 7 amino acids. In addition, the human TIR domain was designed as a 16 mer amino acid sequence. A list of 3,132 peptide sequences was constructed from the surface sequences of 14 types of bacterial TIR domains, 2 types of bacterial TIR domains, and 3 non-TIR domains known to bind to TIR. As a result, a peptide library of approximately 190,946 16mers was constructed.
실시예 2. TLR 억제 활성 펩타이드 스크리닝Example 2. Screening of TLR inhibitory activity peptides
2-1. 파지미드를 이용한 파지 디스플레이2-1. Phage display using phagemid
실시예 1로부터 구축된 펩타이드 라이브러리로부터 TLR 억제 활성을 가지는 펩타이드를 스크리닝 하기 위하여, 아래와 같이 파지디스플레이를 수행하였다. To screen peptides with TLR inhibitory activity from the peptide library constructed in Example 1, phage display was performed as follows.
먼저, 실시예 1로부터 구축된 라이브러리에 속하는 펩타이드 각각의 발현을 위한 DNA 서열을 합성하여 벡터 클로닝을 수행하였다. 벡터 클로닝은 p3/p8 파지미드를 이용하여 수행하였다. 상기 벡터는 M13 bacteriophage에 도입되었으며, TIR 도메인으로부터 유래된 상기 펩타이드는 파지 표면에 디스플레이되었다. First, DNA sequences for expression of each peptide belonging to the library constructed in Example 1 were synthesized and vector cloning was performed. Vector cloning was performed using p3/p8 phagemid. The vector was introduced into M13 bacteriophage, and the peptide derived from the TIR domain was displayed on the phage surface.
2-2. TLR 신호 경로 억제 활성 확인 2-2. Confirmation of TLR signaling pathway inhibition activity
상기 2-1의 결과 파지 디스플레이된 펩타이드를 대상으로, TLR 신호경로의 핵심 단백질인 MAL및 MyD88과의 특이적 결합을 통해 이의 활성을 억제하는 펩타이드를 선별하였다. 대장균에서 생산된 MAL및 MyD88 단백질을 96-well 플레이트에 고정하고, P8 또는 P3 외피단백질에 융합되어 파지 디스플레이된 펩타이드 라이브러리를 섞어준 다음, 결합하지 않는 파지를 제거하고 남은 파지를 대장균에서 증폭시켜 다시 결합시키는 과정을 총4라운드 진행하였다. 4라운드 이후 최종적으로 고정된 타겟 단백질에 결합하여 남아있는 파지들을 얻고, 파지 속 파지미드에 대한 NGS 분석을 통해서, 타겟에 결합하는 펩타이드 서열과 각 서열의 상대적인 분포를 확인하였다. For the phage-displayed peptides as a result of 2-1 above, peptides that inhibit the activity of MAL and MyD88, which are key proteins of the TLR signaling pathway, were selected through specific binding. MAL and MyD88 proteins produced in E. coli were fixed to a 96-well plate, and the phage-displayed peptide library fused to the P8 or P3 envelope protein was mixed, then non-binding phages were removed, and the remaining phages were amplified in E. coli again. A total of 4 rounds of combining process were performed. After the fourth round, the remaining phages were obtained by binding to the final immobilized target protein, and through NGS analysis of the phagemid in the phage, the peptide sequence binding to the target and the relative distribution of each sequence were confirmed.
P8 또는 P3 외피 단백질을 이용하여 파지디스플레이된 라이브러리를 이용하여 human MAL과 MYD88 도메인에 각각 결합하는 펩타이드 서열을 약 500여개 씩 스크리닝하였으며, 이 중 상위 30개씩의 펩타이드 서열을 하기 표 1 내지 표 4와 같이 나타내었다. Approximately 500 peptide sequences binding to the human MAL and MYD88 domains were screened using a phage-displayed library using the P8 or P3 coat protein, and the top 30 peptide sequences are shown in Tables 1 to 4 below. shown together.
| P8_MALP8_MAL | ||
| Peptide sequencePeptide sequence | NGS countNGS count | PopulationPopulation |
| WFYERLVSLILRDTKVWFYERLVSLILLRDTKV | 7900079000 | 24.40%24.40% |
| AFLYTFLINLTHPTNFAFLYTFLINLTHPTNF | 6106361063 | 18.86%18.86% |
| AFFWAQLKSVLGNPTMAFFWAQLKSVLGNPTM | 2880128801 | 8.90%8.90% |
| RNFVAYLFQALANKGIRNFVAYLFQALANKGI | 2795027950 | 8.63%8.63% |
| TFLGHLYNALVQAGIHTFLGHLYNALVQAGIH | 2473224732 | 7.64%7.64% |
| LFFWESLKQLLRSDGRLFFWESLKQLLRSDGR | 2256022560 | 6.97%6.97% |
| YFLHFLLFSRPETEYIYFLHFLLFSRPETEYI | 1719817198 | 5.31%5.31% |
| AFFWEQLRTVLGKPTMAFFWEQLRTVLGKPTM | 1182611826 | 3.65%3.65% |
| QLIQQWIELHLHQLHLQLIQQWIELHLHQLHL | 93949394 | 2.90%2.90% |
| DTKLKKGESIAPELLRDTKLKKGESIAPELLR | 56175617 | 1.74%1.74% |
| KLFWEKFKFFLPSLSRKLFWEKFKFFLPSLSR | 48884888 | 1.51%1.51% |
| SEFIQKIILWMNPKIVSEFIQKIILWMNPKIV | 44584458 | 1.38%1.38% |
| HFFWIQLKSILGETSFHFFWIQLKSILGETSF | 36243624 | 1.12%1.12% |
| WFLFTVRSAIYAMSPAWFLFTVRSAIYAMSPA | 31563156 | 0.97%0.97% |
| RNNFIGHLYQALYRIGRNNFIGLYQALYRIG | 31203120 | 0.96%0.96% |
| SLLYDHLRWLGIRAFLSLLYDHLRWLGIRAFL | 25762576 | 0.80%0.80% |
| WFMMKFRTVLDHVNDVWFMMKFRTVLDHVNDV | 22692269 | 0.70%0.70% |
| KLIQGWFLYRLLKIEQKLIQGWFLYRLLKIEQ | 20682068 | 0.64%0.64% |
| DFLNWFIALKSTVIENDFLNWFIALKSTVIEN | 867867 | 0.27%0.27% |
| NQWRNALTAAANLSGWNQWRNALTAAANLSGW | 848848 | 0.26%0.26% |
| FTLAVHVRNWLVGGFIFTLAVHVRNWLVGGFI | 620620 | 0.19%0.19% |
| YLKWGYPWFWDRLRFAYLKWGYPWFWDRLRFA | 617617 | 0.19%0.19% |
| AYANFRQGLLRLLELAAYANFRQGLLRLLELA | 466466 | 0.14%0.14% |
| WFLTAFRMAMDQVADTWFLTAFRMAMDQVADT | 353353 | 0.11%0.11% |
| YLISYIFNELARAGFSYLISYIFNELARAGFS | 295295 | 0.09%0.09% |
| AWALHQLQQIINWHETAWALHQLQIINWHET | 232232 | 0.07%0.07% |
| WALQELVKILQAKIFLWALQELVKILQAKIFL | 227227 | 0.07%0.07% |
| KFIWDIINAISSQIRVKFIWDIINAISSQIRV | 146146 | 0.05%0.05% |
| P3_MALP3_MAL | ||
| Peptide sequencePeptide sequence | NGS countNGS count | PopulationPopulation |
| YAFTGHLHRYLVQRGVYAFTGHLHRYLVQRGV | 4376443764 | 16.44%16.44% |
| SSFREGLWRLLELSKKSSFREGLWRLLELSKK | 2995529955 | 11.26%11.26% |
| PNLRTKVLNYLMRTKTPNLRTKVLNYLMRTKT | 2693126931 | 10.12%10.12% |
| NQWRNALTAAANLSGWNQWRNALTAAANLSGW | 2583125831 | 9.71%9.71% |
| RGFLSHLRHHLQAKDHRGFLSHLRHHLQAKDH | 1506515065 | 5.66%5.66% |
| RLLKWKEALHQFHSWVRLLKWKEALHQFHSWV | 1492414924 | 5.61%5.61% |
| LKVESWRQALEKAGKLLKVESWRQALEKAGKL | 61806180 | 2.32%2.32% |
| LKWGDPYFWSKLRYAMLKWGDPYFWSKLRYAM | 51285128 | 1.93%1.93% |
| DWFLSKLSQHILKREKDWFLSKLSQHILKREK | 45804580 | 1.72%1.72% |
| KMQKWKDALYEAANLSKMQKWKDALYEAANLS | 44274427 | 1.66%1.66% |
| KEVEGWRDALTKVASLKEVEGWRDALTKVASL | 37583758 | 1.41%1.41% |
| ESILNQLRRSSQSIANESILNQLRRSSQSIAN | 36803680 | 1.38%1.38% |
| SLRKNLVSNLIEEFKRSLRKNLVSNLIEEFKR | 35693569 | 1.34%1.34% |
| KNLFNELVSKNIRTFMKNLFNELVSKNIRTFM | 32593259 | 1.22%1.22% |
| HHKGSYGEALAEHEKRHHKGSYGEALAEHEKR | 30563056 | 1.15%1.15% |
| FLNFRGLDVRNYFLGHFLNFRGLDVRNYFLGH | 28552855 | 1.07%1.07% |
| KWFWDKLIYALSRNPHKWFWDKLIYALSRNPH | 27962796 | 1.05%1.05% |
| YNFISHLYDALHRNRIYNFISHLYDALHRNRI | 26782678 | 1.01%1.01% |
| EWIREQLRPALKERLPEWIREQLRPALKERLP | 23542354 | 0.88%0.88% |
| SFFWNKLRYHLPAPQHSFFWNKLRYHLPAPQH | 22972297 | 0.86%0.86% |
| LQNQWKLALTKVANLVLQNQWKLALTKVANLV | 22852285 | 0.86%0.86% |
| VEPTQVRKQSGAVENAVEPTQVRKQSGAVENA | 22832283 | 0.86%0.86% |
| DHHLDLPRGKAIKPEILGDHHLLDLPRGKAIKPEILG | 22802280 | 0.86%0.86% |
| HQAFWNRLWAKLKPPTHQAFNRLWAKLKPPT | 19321932 | 0.73%0.73% |
| NELVHVWSSKANSLVGNELVHVWSSKANSLVG | 18361836 | 0.69%0.69% |
| GFLYQAFAREGFKIFMGFLYQAFAREGFKIFM | 17641764 | 0.66%0.66% |
| YLKWGYPWFWDRLRFAYLKWGYPWFWDRLRFA | 16801680 | 0.63%0.63% |
| LVAHEISKKMKIFATILVAHEISKKMKIFATI | 16711671 | 0.63%0.63% |
| P8_MyD88P8_MyD88 | ||
| Peptide sequencePeptide sequence | NGS countNGS count | PopulationPopulation |
| DPQDLDWFYERLVSLFDPQDLDWFYERLVSLF | 5475754757 | 21.10%21.10% |
| LGYHWIDWEYFVGIEDLGYHWIDWEYFVGIED | 2521625216 | 9.72%9.72% |
| DWVWDYLLPNLEGPAGDWVWDYLLPNLEGPAG | 1939419394 | 7.47%7.47% |
| WALDELLQILHARELFWALDELLQILHARELF | 1440414404 | 5.55%5.55% |
| DPQDLDWFYERLVSLLDPQDLDWFYERLVSLL | 1313713137 | 5.06%5.06% |
| PQDLEWFYERLVSLILPQDLEWFYERLVSLIL | 1056810568 | 4.07%4.07% |
| WALDELLKILYANETMWALDELLKILYANETM | 1051510515 | 4.05%4.05% |
| DSLFWDRLLYSLHVTEDSLFWDRLLYSLHVTE | 75867586 | 2.92%2.92% |
| WDFASNMEQFWDRLYYWDFASNMEQFWDRLYY | 73527352 | 2.83%2.83% |
| YLLLSVFQTIFARRTEYLLLSVFQTIFARRTE | 71657165 | 2.76%2.76% |
| YASYDDNDYYWVTHVLLRHLYASYDDNDYYWVTHVLLRHL | 68936893 | 2.66%2.66% |
| DPQDLQWFYERLVSLIDPQDLQWFYERLVSLI | 57305730 | 2.21%2.21% |
| SAAYDDFWDRLIRMIESAAYDDFWDRLIRMIE | 53355335 | 2.06%2.06% |
| VQWDDPWFWDKLAYAMVQWDDPWFWDKLAYAM | 49024902 | 1.89%1.89% |
| DPQDLEWFYERLFSQMDPQDLEWFYERLFSQM | 44884488 | 1.73%1.73% |
| DYASSTWWLRELLQIADYASSTWLRELLQIA | 41114111 | 1.58%1.58% |
| YLRADDSWFWEKLLYAYLRADDSWFWEKLLYA | 40674067 | 1.57%1.57% |
| YAASPWWFDELVKIVGYAASPWWFDELVKIVG | 34333433 | 1.32%1.32% |
| HWIDWEYFVGIEDHIKHWIDWEYFVGIEDHIK | 34223422 | 1.32%1.32% |
| IDDDHLTYLVRLLLSRIDDDHLTYLVRLLLSR | 30593059 | 1.18%1.18% |
| YASSSWALDELLRILYYASSSWALDELLRILY | 23272327 | 0.90%0.90% |
| VANILGYHWIDWEYFVVANILGYHWIDWEYFV | 20292029 | 0.78%0.78% |
| GLSWGPYEWWMGLYDAGLSWGPYEWWMGLYDA | 19651965 | 0.76%0.76% |
| SPQEQDGFWVRLIEALSPQEQDGFWVRLIEAL | 18421842 | 0.71%0.71% |
| QLIQQWIELHLHQLHLQLIQQWIELHLHQLHL | 16051605 | 0.62%0.62% |
| DWVWEHFFPMEEKDQTDWVWEHFFPMEEKDQT | 15381538 | 0.59%0.59% |
| MFGGDTFHDFLDLISMMFGGDTFHDFLDLISM | 14891489 | 0.57%0.57% |
| LEANDSLFWDRLLYSLLEANDSLLFWDRLLYSL | 14651465 | 0.56%0.56% |
| P3_MyD88P3_MyD88 | ||
| Peptide sequencePeptide sequence | NGS countNGS count | PopulationPopulation |
| SSFREGLWRLLELSKKSSFREGLWRLLELSKK | 7951079510 | 23.15%23.15% |
| NQWRNALTAAANLSGWNQWRNALTAAANLSGW | 2866228662 | 8.35%8.35% |
| DHHLDLPRGKAIKPEILGDHHLLDLPRGKAIKPEILG | 2672226722 | 7.78%7.78% |
| YTALTNSGFHTFRDNDYTALTNSGFHTFRDND | 1760017600 | 5.12%5.12% |
| LVAHEISKKMKIFATILVAHEISKKMKIFATI | 1745217452 | 5.08%5.08% |
| LKVESWRQALEKAGKLLKVESWRQALEKAGKL | 1684716847 | 4.91%4.91% |
| EWIREQLRPALKERLPEWIREQLRPALKERLP | 1682116821 | 4.90%4.90% |
| YAFTGHLHRYLVQRGVYAFTGHLHRYLVQRGV | 1342013420 | 3.91%3.91% |
| NELVHVWSSKANSLVGNELVHVWSSKANSLVG | 88168816 | 2.57%2.57% |
| KNLFNELVSKNIRTFMKNLFNELVSKNIRTFM | 85438543 | 2.49%2.49% |
| LKWGDPYFWSKLRYAMLKWGDPYFWSKLRYAM | 83108310 | 2.42%2.42% |
| YLKWGYPWFWDRLRFAYLKWGYPWFWDRLRFA | 73217321 | 2.13%2.13% |
| SENYATSPWALDELVLSENYATSPWALDELVL | 64946494 | 1.89%1.89% |
| KEVEGWRDALTKVASLKEVEGWRDALTKVASL | 61876187 | 1.80%1.80% |
| SELRVVDDGSVSPLEMSELRVVDDDGSVSPLEM | 48724872 | 1.42%1.42% |
| AVDKETDKIQTPLKNAAVDKETDKIQTPLKNA | 47904790 | 1.39%1.39% |
| NGHESEIIKEIVDTIVNGHESEIIKEIVDTIV | 42334233 | 1.23%1.23% |
| FLSYRALEKRFGPIGQFLSYRALEKRFGPIGQ | 32893289 | 0.96%0.96% |
| YNFISHLYDALHRNRIYNFISHLYDALHRNRI | 28912891 | 0.84%0.84% |
| GFLYQAFAREGFKIFMGFLYQAFAREGFKIFM | 25792579 | 0.75%0.75% |
| YYLDPSHLRKQSGEFGYYLDPSHLRKQSGEFG | 25382538 | 0.74%0.74% |
| PEKDNGGENAFINNIVPEKDNGGENAFINNIV | 24982498 | 0.73%0.73% |
| DTKLKKGESIAPELLRDTKLKKGESIAPELLR | 24652465 | 0.72%0.72% |
| RKGDLISDELLIAIEERKGDLISDELLIAIEE | 24522452 | 0.71%0.71% |
| HLLKEFDKKLITAFKDHLLKEFDKKLITAFKD | 23992399 | 0.70%0.70% |
| PSEVRKQTGSLAKAFAPSEVRKQTGSLAKAFA | 22532253 | 0.66%0.66% |
| TSHLHHALRLNKIETFTSHLHHALRLNKIETF | 20762076 | 0.60%0.60% |
| ESILNQLRRSSQSIANESILNQLRRSSQSIAN | 19761976 | 0.58%0.58% |
또한 상기의 펩타이드의 효능을 확인하기 위하여, 상기 서열에 세포막 통과를 위한 서열(CPP; RQIKIWFQNRRMKWKK, 서열번호 23)을 융합하여 합성하였으며, 하기의 표 5의 서열로 이루어진 펩타이드를 제작하였다. In addition, in order to confirm the efficacy of the above peptide, a sequence for passing through the cell membrane (CPP; RQIKIWFQNRRMKWKK, SEQ ID NO. 23) was fused to the above sequence and synthesized, and a peptide consisting of the sequences shown in Table 5 below was produced.
| 합성 펩타이드 synthetic peptides |
펩타이드 서열 (Cell penetrating peptide + TIR-binding motif) peptide sequence (Cell penetrating peptide + TIR-binding motif) |
서열번호 sequence number | |
| TDIP1TDIP1 | RQIKIWFQNRRMKWKKRQIKIWFQNRRMKWKK | WFYERLVSLILRDTKV WFYERLVSLILLRDTKV | 서열번호 1SEQ ID NO: 1 |
| TDIP2TDIP2 | RQIKIWFQNRRMKWKKRQIKIWFQNRRMKWKK | AFLYTFLINLTHPTNF AFLYTFLINLTHPTNF | 서열번호2SEQ ID NO: 2 |
| TDIP3TDIP3 | RQIKIWFQNRRMKWKK RQIKIWFQNRRMKWKK | AFFWAQLKSVLGNPTM AFFWAQLKSVLGNPTM | 서열번호 3SEQ ID NO: 3 |
| TDIP4TDIP4 | RQIKIWFQNRRMKWKKRQIKIWFQNRRMKWKK | RNFVAYLFQALANKGI RNFVAYLFQALANKGI | 서열번호 4SEQ ID NO: 4 |
| TDIP5TDIP5 | RQIKIWFQNRRMKWKKRQIKIWFQNRRMKWKK | TFLGHLYNALVQAGIH TFLGHLYNALVQAGIH | 서열번호 5SEQ ID NO: 5 |
| TDIP6TDIP6 | RQIKIWFQNRRMKWKKRQIKIWFQNRRMKWKK | LFFWESLKQLLRSDGR LFFWESLKQLLRSDGR | 서열번호 6SEQ ID NO: 6 |
| TDIP7TDIP7 | RQIKIWFQNRRMKWKKRQIKIWFQNRRMKWKK | YAFTGHLHRYLVQRGV YAFTGHLHRYLVQRGV | 서열번호 7SEQ ID NO: 7 |
| TDIP8TDIP8 | RQIKIWFQNRRMKWKKRQIKIWFQNRRMKWKK | SSFREGLWRLLELSKK SSFREGLWRLLELSKK | 서열번호 8SEQ ID NO: 8 |
| TDIP9TDIP9 | RQIKIWFQNRRMKWKKRQIKIWFQNRRMKWKK | PNLRTKVLNYLMRTKT PNLRTKVLNYLMRTKT | 서열번호 9SEQ ID NO: 9 |
| TDIP10TDIP10 | RQIKIWFQNRRMKWKKRQIKIWFQNRRMKWKK | NQWRNALTAAANLSGW NQWRNALTAAANLSGW | 서열번호 10SEQ ID NO: 10 |
| TDIP11TDIP11 | RQIKIWFQNRRMKWKKRQIKIWFQNRRMKWKK | RGFLSHLRHHLQAKDH RGFLSHLRHHLQAKDH | 서열번호 11SEQ ID NO: 11 |
| TDIP12TDIP12 | RQIKIWFQNRRMKWKKRQIKIWFQNRRMKWKK | KEVEGWRDALTKVASL KEVEGWRDALTKVASL | 서열번호 12SEQ ID NO: 12 |
| TDIP13TDIP13 | RQIKIWFQNRRMKWKKRQIKIWFQNRRMKWKK | DPQDLDWFYERLVSLF DPQDLDWFYERLVSLF | 서열번호 13SEQ ID NO: 13 |
| TDIP14TDIP14 | RQIKIWFQNRRMKWKKRQIKIWFQNRRMKWKK | LGYHWIDWEYFVGIED LGYHWIDWEYFVGIED | 서열번호 14SEQ ID NO: 14 |
| TDIP15TDIP15 | RQIKIWFQNRRMKWKKRQIKIWFQNRRMKWKK | DWVWDYLLPNLEGPAG DWVWDYLLPNLEGPAG | 서열번호 15SEQ ID NO: 15 |
| TDIP16TDIP16 | RQIKIWFQNRRMKWKKRQIKIWFQNRRMKWKK | WALDELLQILHARELF WALDELLQILHARELF | 서열번호 16SEQ ID NO: 16 |
| TDIP17TDIP17 | RQIKIWFQNRRMKWKKRQIKIWFQNRRMKWKK | DPQDLDWFYERLVSLL DPQDLDWFYERLVSLL | 서열번호 17SEQ ID NO: 17 |
| TDIP18TDIP18 | RQIKIWFQNRRMKWKKRQIKIWFQNRRMKWKK | DHHLDLPRGKAIKPEILG DHHLLDLPRGKAIKPEILG | 서열번호 18SEQ ID NO: 18 |
| TDIP19TDIP19 | RQIKIWFQNRRMKWKKRQIKIWFQNRRMKWKK | YTALTNSGFHTFRDND YTALTNSGFHTFRDND | 서열번호 19SEQ ID NO: 19 |
| TDIP20TDIP20 | RQIKIWFQNRRMKWKKRQIKIWFQNRRMKWKK | LVAHEISKKMKIFATI LVAHEISKKMKIFATI | 서열번호 20SEQ ID NO: 20 |
| TDIP21TDIP21 | RQIKIWFQNRRMKWKKRQIKIWFQNRRMKWKK | EWIREQLRPALKERLP EWIREQLRPALKERLP | 서열번호 21SEQ ID NO: 21 |
|
CPCP
(Negative control)(Negative control) |
RQIKIWFQNRRMKWKKRQIKIWFQNRRMKWKK | GGAASSTTGGSSAATT GGAASSTTGGSSAATT | 서열번호 22SEQ ID NO: 22 |
상기 표 1, 2, 3 ,4와 같이 스크리닝된 펩타이드를 대상으로 TLR 과발현 HEK-Blue hTLR2, 3, 4, 5, 7, 8, 9 리포터 세포주를 이용하여 21종 합성 펩타이드의 TLR(TLR2, TLR3, TLR4, TLR 5, TLR7, TLR8, TLR9)에 대한 억제 효능을 확인한 결과, 대부분의 펩타이드에서 TLR2, TLR4, TLR7, TLR9 억제 활성이 확인되었다. 또한 일부 펩타이드에서 TLR3, TLR5에 대한 억제 활성이 확인되었다. (도 2)또한, 도 3에서 보듯이, TLR4에 대한 억제 효능을 HEK-Blue hTLR4를 이용한SEAP 리포터 어세이를 통해 확인한 결과, 대조군에 비해 대부분의 펩타이드에서 TLR4 억제 활성이 확인되었다. 또한, 도 4와 같이 상기의 TLR4 억제 활성이 각 펩타이드(TDIP1, TDIP2, TDIP3, TDIP5, TDIP6, TDIP7, TDIP8, TDIP10, TDIP16, TDIP20)의 농도에 따라 변화하는지 여부를 확인한 결과, 실험 대상이 된 모든 펩타이드에서 농도 의존적으로 억제 활성이 높아지는 것을 확인할 수 있었다. TLR (TLR2, TLR3) of 21 synthetic peptides using HEK-Blue hTLR2, 3, 4, 5, 7, 8, 9 reporter cell lines overexpressing TLRs targeting the screened peptides as shown in Tables 1, 2, 3, and 4 above. , TLR4, TLR 5, TLR7, TLR8, and TLR9), TLR2, TLR4, TLR7, and TLR9 inhibitory activity was confirmed for most peptides. In addition, inhibitory activity against TLR3 and TLR5 was confirmed in some peptides. (Figure 2) In addition, as shown in Figure 3, the inhibitory effect on TLR4 was confirmed through SEAP reporter assay using HEK-Blue hTLR4, and TLR4 inhibitory activity was confirmed in most peptides compared to the control group. In addition, as shown in Figure 4, as a result of confirming whether the TLR4 inhibitory activity changes depending on the concentration of each peptide (TDIP1, TDIP2, TDIP3, TDIP5, TDIP6, TDIP7, TDIP8, TDIP10, TDIP16, TDIP20), the test subjects were It was confirmed that the inhibitory activity increased in a concentration-dependent manner for all peptides.
2-3. 마우스 면역 세포를 이용한 면역 조절 효능 검증 2-3. Verification of immune regulation efficacy using mouse immune cells
상기의 펩타이드 중, TDIP1, TDIP5, TDIP6, TDIP20 펩타이드에 대한 TLR4 억제 효과를 검증하기 위하여, 마우스의 면역 세포(macrophage, RAW264.7)를 이용하여 LPS 처리 후 펩타이드를 처리했을 때, IRAK4의 인산화와 IRF3의 인산화 억제 효과를 확인하였다. 그 결과 도 5에서 보듯이, 본 발명의 펩타이드들은 MyD88-의존적 및 TRIF-의존적 TLR4 신호전달을 모두 억제하고 있음을 확인할 수 있었다.Among the above peptides, in order to verify the TLR4 inhibitory effect on TDIP1, TDIP5, TDIP6, and TDIP20 peptides, when mouse immune cells (macrophages, RAW264.7) were treated with the peptides after LPS treatment, phosphorylation of IRAK4 and The effect of suppressing phosphorylation of IRF3 was confirmed. As a result, as shown in Figure 5, it was confirmed that the peptides of the present invention inhibited both MyD88-dependent and TRIF-dependent TLR4 signaling.
또한, TRL4-의존적 MAPK 신호의 억제 효과를 확인하였다. RAW264.7세포에 상기 펩타이드를 농도 의존적으로 처리하고, 처리군 및 비 처리군에서의 ERK, JNK 및 p38의 인산화 억제 효과를 확인하였다. 그 결과, 도 6에서 보듯이, 본 발명의 펩타이드를 처리한 경우, MAPK 신호 전달경로에 관여하는 단백질들의 인산화가 현저히 감소된 것을 확인할 수 있었다.Additionally, the inhibitory effect of TRL4-dependent MAPK signaling was confirmed. RAW264.7 cells were treated with the above peptide in a concentration-dependent manner, and the inhibitory effect on phosphorylation of ERK, JNK, and p38 in the treated and non-treated groups was confirmed. As a result, as shown in Figure 6, when treated with the peptide of the present invention, it was confirmed that phosphorylation of proteins involved in the MAPK signaling pathway was significantly reduced.
또한, 상기의 펩타이드가 NF-kB의 핵내 이동에 영향을 주는지 여부를 확인하였다. 마우스 면역 세포에 상기 4종의 펩타이드를 각각 처리하고, NF-kB의 핵 이동에 미치는 영향을 확인하였다. 그 결과, 도 7에서 보듯이, 펩타이드를 처리한 경우에는 NF-kB의 핵내 이동이 저해되는 것을 알 수 있었다. In addition, it was confirmed whether the above peptide affects the intranuclear movement of NF-kB. Mouse immune cells were treated with each of the four peptides above, and their effects on the nuclear movement of NF-kB were confirmed. As a result, as shown in Figure 7, it was found that the intranuclear movement of NF-kB was inhibited when treated with the peptide.
또한, 상기 펩타이드 처리를 통해 마우스 대식세포에서 사이토카인 분비 억제 효과가 있음을 확인하였다. 마우스의 대식세포에서의 IL-6 및 TNF-α 분비량은 대조군과 비교하여 상기의 펩타이드 처리 시, 감소되는 경향이 있음을 확인하였다. (도 8) In addition, it was confirmed that the peptide treatment had an inhibitory effect on cytokine secretion in mouse macrophages. It was confirmed that the secretion amount of IL-6 and TNF-α from mouse macrophages tended to decrease when treated with the above peptide compared to the control group. (Figure 8)
실시예 3. TLR 억제 활성 펩타이드의 패혈증 치료 효과 확인 Example 3. Confirmation of sepsis treatment effect of TLR inhibitory active peptide
본 발명의 TLR 억제 활성 펩타이드가 패혈증 치료에 효과가 있음을 확인하기 위하여 마우스를 대상으로 실험을 진행하였다. LPS로 패혈증을 유도한 마우스에 본 발명의 TLR 억제 활성 펩타이드(TDIP1, TDIP5, TDIP6, TDIP20)를 마우스 체중 g당 10 nmol 양으로 각각 복강 내 주사하고, 마우스 생존률과 LPS와 펩타이드 투여 후 16시간 후 혈청 내 사이토카인(IL-23, IL-1α, IFNγ, TNF-α, MCP-1, IL-12p70, IL-1β, IL-10, IL-6, IL-27, IL-17A, IFN β) 발현량 확인을 위한 ELISA 분석을 수행하였으며, 신장 조직의 세포 사멸 확인을 위한 TUNEL assay을 진행하였다.In order to confirm that the TLR inhibitory activity peptide of the present invention is effective in treating sepsis, an experiment was conducted on mice. The TLR inhibitory peptides (TDIP1, TDIP5, TDIP6, TDIP20) of the present invention were intraperitoneally injected into mice in which sepsis was induced with LPS at an amount of 10 nmol per g of mouse body weight, and the mouse survival rate and 16 hours after LPS and peptide administration were injected intraperitoneally. Cytokines in serum (IL-23, IL-1α, IFNγ, TNF-α, MCP-1, IL-12p70, IL-1β, IL-10, IL-6, IL-27, IL-17A, IFN β) ELISA analysis was performed to confirm expression level, and TUNEL assay was performed to confirm cell death in kidney tissue.
그 결과, 도 9에서 보듯이, 본 발명의 펩타이드를 투여한 질환 마우스는 비 투여군 및 CP 투여군이 48 내지 96 시간 사이에 사망한 것과 비교하여, 생존률이 크게 증가된 것을 확인할 수 있었다. 또한, 도 10에서 보듯이 패혈증이 유발된 마우스에서 분비량이 증가된 사이토카인이 본 발명의 TLR 활성 억제 펩타이드 투여에 의해 분비량이 대부분 감소하였음을 알 수 있었다. 또한, 신장 조직의 세포 사멸 확인을 위한 TUNEL assay 에서도 비 투여군 및 CP 투여군에서 TUNEL positive cell이 증가한 반면 본 발명의 펩타이드 투여군에서는 TUNEL positive cell이 감소되어, 세포 사멸이 감소되었다는 점을 확인할 수 있었다. (도 11)As a result, as shown in Figure 9, it was confirmed that the survival rate of diseased mice administered with the peptide of the present invention was significantly increased compared to the non-administered group and the CP administered group, which died between 48 and 96 hours. In addition, as shown in Figure 10, it was found that the secretion amount of cytokines increased in sepsis-induced mice was mostly reduced by administration of the TLR activity inhibitory peptide of the present invention. In addition, in the TUNEL assay to confirm cell death in kidney tissue, it was confirmed that TUNEL positive cells increased in the non-administered group and CP-administered group, while TUNEL positive cells decreased in the peptide-administered group of the present invention, resulting in reduced cell death. (Figure 11)
실시예 4. TLR 억제 활성 펩타이드의 건성황반변성 치료 효과 확인Example 4. Confirmation of dry macular degeneration treatment effect of TLR inhibitory activity peptide
본 발명의 TLR 억제 활성 펩타이드가 건성 황반변성에 치료 효과가 있음을 확인하기 위하여, NaIO3를 복강에 투여하여 건성 황반변성을 유도한 마우스 모델에 본 발명의 TLR 억제 활성 펩타이드(TDIP1,TDIP5, TDIP6, TDIP20)를 각각 20 μM농도로 5 μl씩 눈의 상부 각막 표면에 21일 동안 1일 1회 직접투여한 후, 상기 질환 마우스의 외핵층(ONL) 및 망막세포 상피 두께(RPE)를 확인하고, 망막 세포 사멸에 미치는 영향을 확인하였다.In order to confirm that the TLR inhibitory active peptide of the present invention has a therapeutic effect on dry macular degeneration, the TLR inhibitory active peptide of the present invention (TDIP1, TDIP5, TDIP6, After directly administering 5 μl of TDIP20) at a concentration of 20 μM to the upper corneal surface of the eye once a day for 21 days, the outer nuclear layer (ONL) and retinal cell epithelial thickness (RPE) of the diseased mice were checked, The effect on retinal cell death was confirmed.
그 결과, 도 12 및 13과 같이 NaIO3를 통해 건성황반변성을 유발한 경우, 망막색소상피세포 및 외핵층에 노폐물이 증가되며 두께가 감소되는 것을 확인하였으나, TDIP1, TDIP5, TDIP6 투여군에서는 노폐물이 없이 두께가 유지되는 것을 확인하였다. As a result, as shown in Figures 12 and 13, when dry macular degeneration was induced through NaIO 3 , it was confirmed that waste products increased and the thickness of the retinal pigment epithelial cells and outer nuclear layer decreased. However, in the TDIP1, TDIP5, and TDIP6 administration groups, waste products were decreased. It was confirmed that the thickness was maintained.
이제까지 본 발명에 대하여 그 바람직한 실시예들을 중심으로 살펴보았다. 본 발명이 속하는 기술 분야에서 통상의 지식을 가진 자는 본 발명이 본 발명의 본질적인 특성에서 벗어나지 않는 범위에서 변형된 형태로 구현될 수 있음을 이해할 수 있을 것이다. 그러므로 개시된 실시예들은 한정적인 관점이 아니라 설명적인 관점에서 고려되어야 한다. 본 발명의 범위는 전술한 설명이 아니라 청구범위에 나타나 있으며, 그와 동등한 범위 내에 있는 모든 차이점은 본 발명에 포함된 것으로 해석되어야 할 것이다.So far, the present invention has been examined focusing on its preferred embodiments. A person skilled in the art to which the present invention pertains will understand that the present invention may be implemented in a modified form without departing from the essential characteristics of the present invention. Therefore, the disclosed embodiments should be considered from an illustrative rather than a restrictive perspective. The scope of the present invention is indicated in the claims rather than the foregoing description, and all differences within the equivalent scope should be construed as being included in the present invention.
본 발명의 일 양태로서, 본 발명은 다양한 생물 종 유래의 TIR 도메인 3차 구조 코어 부위를 제외한 도메인간 결합에 관여하는 표면에 노출되어있는 서열에 대하여, 일부 서열이 오버랩되는 16 내지 21개 아미노산 길이의 펩타이드 단편을 제작하는 단계; 상기 펩타이드 단편을 암호화하는 각각의 DNA를 합성하는 단계; 상기 합성된 DNA를 파지미드(phagemid) 벡터를 이용하여 클로닝하는 단계; 및 상기 클로닝된 벡터를 박테리오파지에 도입하여 파지 디스플레이(phage display)를 위한 라이브러리를 제작하는 단계를 포함하는 TIR 도메인 표면 서열 유래 펩타이드 단편 파지 디스플레이 라이브러리 구축 방법에 대한 것이다. In one aspect of the present invention, the present invention provides a surface-exposed sequence involved in inter-domain binding excluding the core region of the tertiary structure of the TIR domain from various biological species, with a length of 16 to 21 amino acids in which some sequences overlap. Producing a peptide fragment of; Synthesizing each DNA encoding the peptide fragment; Cloning the synthesized DNA using a phagemid vector; and a method of constructing a phage display library of peptide fragments derived from the TIR domain surface sequence, including the step of introducing the cloned vector into a bacteriophage to create a library for phage display.
본 발명의 일 실시예에서, 상기 펩타이드 단편의 오버랩되는 서열은 5 내지 8개 아미노산 길이인 것이다. In one embodiment of the present invention, the overlapping sequence of the peptide fragment is 5 to 8 amino acids long.
본 발명의 일 양태로서, 본 발명은 상기 방법에 의해 구축된 파지 디스플레이 라이브러리로부터 인간 MAL 또는 MyD88등을 포함하는 TIR 도메인들과 특이적 결합을 이루는 펩타이드를 스크리닝하는 단계를 포함하는 TLR 신호 전달 억제 활성을 가지는 펩타이드의 스크리닝 방법에 대한 것이다. In one aspect of the present invention, the present invention provides a TLR signal transduction inhibitory activity comprising screening for a peptide that specifically binds to TIR domains including human MAL or MyD88 from a phage display library constructed by the above method. This is about a screening method for peptides having .
본 발명의 일 실시예에서, 상기 TLR 신호 전달 억제 활성을 가지는 펩타이드는 인간 MAL 또는 MyD88을 포함하는 인간, 박테리아 또는 식물에 포함된 TIR 도메인에 특이적으로 결합하여, TIR 도메인간의 결합을 저해하여 TLR 신호를 억제하는 것을 특징으로 한다. In one embodiment of the present invention, the peptide having the TLR signal transduction inhibitory activity specifically binds to the TIR domain contained in humans, bacteria, or plants, including human MAL or MyD88, and inhibits the binding between TIR domains to TLR. It is characterized by suppressing signals.
본 발명의 일 양태로서, 본 발명은 TIR 도메인의 표면 서열로부터 유래된 펩타이드 단편을 포함하는 TLR(Toll like receptor) 신호 억제 펩타이드에 대한 것이다. In one aspect of the present invention, the present invention relates to a TLR (Toll like receptor) signal inhibitory peptide comprising a peptide fragment derived from the surface sequence of the TIR domain.
본 발명의 일 실시예에서, 상기 TLR은 TLR2, TLR3, TLR4, TLR5, TLR7 및 TLR9 로 이루어진 군에서 선택될 수 있다. In one embodiment of the present invention, the TLR may be selected from the group consisting of TLR2, TLR3, TLR4, TLR5, TLR7, and TLR9.
본 발명의 일 실시예에서, 상기 펩타이드는 제4항의 방법에 의해 스크리닝 된 것임을 특징으로 한다. In one embodiment of the present invention, the peptide is characterized as having been screened by the method of claim 4.
본 발명의 일 실시예에서 상기 펩타이드는 서열번호 1 내지 서열번호21의 아미노산 서열로 이루어진 펩타이드 중 하나 이상을 선택하여 포함하는 것을 특징으로 한다. In one embodiment of the present invention, the peptide is characterized in that it includes one or more peptides consisting of the amino acid sequences of SEQ ID NO: 1 to SEQ ID NO: 21.
본 발명의 일 실시예에서, 상기 펩타이드는 서열번호 23의 서열로 이루어진 펩타이드와 융합된 것임을 특징으로 한다. In one embodiment of the present invention, the peptide is characterized as being fused with a peptide consisting of the sequence of SEQ ID NO: 23.
본 발명의 일 양태로서, 본 발명은 제5항의 펩타이드를 포함하는 염증성 질환의 예방, 개선 또는 치료용 약학적 조성물에 대한 것이다. In one aspect of the present invention, the present invention relates to a pharmaceutical composition for preventing, improving or treating inflammatory diseases comprising the peptide of claim 5.
본 발명의 일 실시예에서, 상기 염증성 질환은 피부염, 알레르기, 아토피, 천식, 치주염, 알레르기성 및 비-알레르기성 비염, 중이염, 인후염, 편도염, 폐렴, 위궤양, 만성 및 급성 위염, 크론병, 대장염, 만성 및 급성 장염, 치질, 통풍, 강직성 척추염, 골관절염, 류마티스관절염, 견관절주위염, 건염, 건초염, 근육염, 급성 및 만성 간염, 방광염, 급성 및 만성 신장염, 쇼그렌 증후군(sjogren's syndrome), 다발성 경화증, 비염, 만성 폐쇄성 폐질환, 과민성 대장 증후군, 바이러스 감염, 박테리아 감염, 곰팡이 감염, 화상, 외과적 또는 치과적 수술에 의한 상처, 아테롬성 동맥 경화증, 관절염, 호지킨병, 췌장염, 홍채염, 공막염, 포도막염, 결막염, 건성황반변성, 습성황반변성, 패혈증 또는 패혈증성 쇼크로 구성된 군으로부터 선택될 수 있다. In one embodiment of the present invention, the inflammatory disease includes dermatitis, allergy, atopy, asthma, periodontitis, allergic and non-allergic rhinitis, otitis media, pharyngitis, tonsillitis, pneumonia, gastric ulcer, chronic and acute gastritis, Crohn's disease, and colitis. , chronic and acute enteritis, hemorrhoids, gout, ankylosing spondylitis, osteoarthritis, rheumatoid arthritis, periarthritis, tendinitis, tenosynovitis, myositis, acute and chronic hepatitis, cystitis, acute and chronic nephritis, Sjogren's syndrome, multiple sclerosis, rhinitis. , chronic obstructive pulmonary disease, irritable bowel syndrome, viral infections, bacterial infections, fungal infections, burns, surgical or dental wounds, atherosclerosis, arthritis, Hodgkin's disease, pancreatitis, iritis, scleritis, uveitis, conjunctivitis. , dry macular degeneration, wet macular degeneration, sepsis, or septic shock.
본 발명의 일 실시예에서, 상기 염증성 질환은 패혈증, 패혈성 쇼크 또는 패혈증과 연관된 증상 및 다기관 기능 부전 증후군일 수 있다. In one embodiment of the present invention, the inflammatory disease may be sepsis, septic shock, or symptoms associated with sepsis and multi-organ dysfunction syndrome.
본 발명의 일 실시예에서, 상기 염증성 질환은 황반변성일 수 있다. In one embodiment of the present invention, the inflammatory disease may be macular degeneration.
본 발명의 일 실시예에서, 상기 황반변성은 건성 황반변성일 수 있다. In one embodiment of the present invention, the macular degeneration may be dry macular degeneration.
본 발명의 일 실시예에서, 상기 조성물은 망막세포 상피(RPE) 또는 외핵층(ONL)의 두께를 감소시킬 수 있다. In one embodiment of the present invention, the composition can reduce the thickness of the retinal cell epithelium (RPE) or outer nuclear layer (ONL).
본 발명의 일 실시예에서, 상기 펩타이드는 서열번호 1, 5. 6 또는 20의 아미노산 서열로 이루어진 펩타이드 중 하나 이상을 포함할 수 있다. In one embodiment of the present invention, the peptide may include one or more of the peptides consisting of the amino acid sequences of SEQ ID NOs: 1, 5, 6, or 20.
Claims (16)
- 다양한 생물 종 유래의 TIR 도메인 3차 구조 코어 부위를 제외한 도메인간 결합에 관여하는 표면에 노출되어있는 서열에 대하여, 일부 서열이 오버랩되는 16 내지 21개 아미노산 길이의 펩타이드 단편을 제작하는 단계; For sequences exposed on the surface involved in inter-domain binding excluding the core region of the tertiary structure of TIR domains derived from various biological species, preparing a peptide fragment of 16 to 21 amino acids in length with some sequences overlapping;상기 펩타이드 단편을 암호화하는 각각의 DNA를 합성하는 단계;Synthesizing each DNA encoding the peptide fragment;상기 합성된 DNA를 파지미드(phagemid) 벡터를 이용하여 클로닝하는 단계; 및Cloning the synthesized DNA using a phagemid vector; and상기 클로닝된 벡터를 박테리오파지에 도입하여 파지 디스플레이(phage display)를 위한 라이브러리를 제작하는 단계를 포함하는 TIR 도메인 표면 서열 유래 펩타이드 단편 파지 디스플레이 라이브러리 구축 방법. A method of constructing a phage display library of peptide fragments derived from a TIR domain surface sequence, comprising the step of introducing the cloned vector into a bacteriophage to create a library for phage display.
- 제1항에 있어서, According to paragraph 1,상기 펩타이드 단편의 오버랩되는 서열은 5 내지 8개 아미노산 길이인 것을 특징으로 하는 라이브러리 구축 방법. A library construction method, characterized in that the overlapping sequence of the peptide fragment is 5 to 8 amino acids in length.
- 제1항의 방법에 의해 구축된 파지 디스플레이 라이브러리로부터 인간 MAL 또는 MyD88등을 포함하는 TIR 도메인들과 특이적 결합을 이루는 펩타이드를 스크리닝하는 단계를 포함하는 TLR 신호 전달 억제 활성을 가지는 펩타이드의 스크리닝 방법. A method for screening peptides having TLR signal transduction inhibitory activity, comprising the step of screening peptides that specifically bind to TIR domains including human MAL or MyD88 from a phage display library constructed by the method of claim 1.
- 제3항에 있어서, According to clause 3,상기 TLR 신호 전달 억제 활성을 가지는 펩타이드는 인간 MAL 또는 MyD88을 포함하는 인간 TIR 도메인들 또는 박테리아, 식물 등이 가지고 있는 TIR 패밀리 도메인에 특이적으로 결합하여, TIR 도메인간의 결합을 저해하여 TLR 신호를 억제하는 것을 특징으로 하는 스크리닝 방법. The peptide having the TLR signal transduction inhibitory activity specifically binds to human TIR domains including human MAL or MyD88 or the TIR family domain of bacteria, plants, etc., thereby inhibiting the binding between TIR domains and inhibiting TLR signaling. A screening method characterized by:
- TIR 도메인의 표면 서열로부터 유래된 펩타이드 단편을 포함하는 TLR(Toll like receptor) 신호 억제 펩타이드. A TLR (Toll like receptor) signaling inhibitory peptide containing a peptide fragment derived from the surface sequence of the TIR domain.
- 제5항에 있어서, According to clause 5,상기 TLR은 TLR2, TLR3, TLR4, TLR5, TLR7 및 TLR9 로 이루어진 군에서 선택되는 어느 하나 이상인 신호 억제 펩타이드. The TLR is any one or more signal inhibitory peptides selected from the group consisting of TLR2, TLR3, TLR4, TLR5, TLR7 and TLR9.
- 제5항에 있어서, According to clause 5,상기 펩타이드는 제4항의 방법에 의해 스크리닝 된 것임을 특징으로 하는 펩타이드. The peptide is characterized in that the peptide was screened by the method of claim 4.
- 제5항에 있어서, According to clause 5,상기 펩타이드는 서열번호 1 내지 서열번호21의 아미노산 서열로 이루어진 펩타이드 중 하나 이상을 선택하여 포함하는 것을 특징으로 하는 펩타이드. The peptide is a peptide characterized in that it contains one or more selected peptides consisting of the amino acid sequences of SEQ ID NO: 1 to SEQ ID NO: 21.
- 제5항에 있어서, According to clause 5,상기 펩타이드는 서열번호 23의 서열로 이루어진 펩타이드와 융합된 것임을 특징으로 하는 펩타이드. A peptide characterized in that the peptide is fused with a peptide consisting of the sequence of SEQ ID NO: 23.
- 제5항의 펩타이드를 포함하는 염증성 질환의 예방, 개선 또는 치료용 약학적 조성물. A pharmaceutical composition for preventing, improving or treating inflammatory diseases comprising the peptide of claim 5.
- 제10항에 있어서, According to clause 10,상기 염증성 질환은 피부염, 알레르기, 아토피, 천식, 치주염, 알레르기성 및 비-알레르기성 비염, 중이염, 인후염, 편도염, 폐렴, 위궤양, 만성 및 급성 위염, 크론병, 대장염, 만성 및 급성 장염, 치질, 통풍, 강직성 척추염, 골관절염, 류마티스관절염, 견관절주위염, 건염, 건초염, 근육염, 급성 및 만성 간염, 방광염, 급성 및 만성 신장염, 쇼그렌 증후군(sjogren's syndrome), 다발성 경화증, 비염, 만성 폐쇄성 폐질환, 과민성 대장 증후군, 바이러스 감염, 박테리아 감염, 곰팡이 감염, 화상, 외과적 또는 치과적 수술에 의한 상처, 아테롬성 동맥 경화증, 관절염, 호지킨병, 췌장염, 홍채염, 공막염, 포도막염, 결막염, 건성황반변성, 습성황반변성, 패혈증 또는 패혈증성 쇼크로 구성된 군으로부터 선택되는 질환인 약학적 조성물. The inflammatory diseases include dermatitis, allergy, atopy, asthma, periodontitis, allergic and non-allergic rhinitis, otitis media, pharyngitis, tonsillitis, pneumonia, gastric ulcer, chronic and acute gastritis, Crohn's disease, colitis, chronic and acute enteritis, hemorrhoids, Gout, ankylosing spondylitis, osteoarthritis, rheumatoid arthritis, periarthritis, tendonitis, tenosynovitis, myositis, acute and chronic hepatitis, cystitis, acute and chronic nephritis, Sjogren's syndrome, multiple sclerosis, rhinitis, chronic obstructive pulmonary disease, irritable bowel. Syndrome, viral infection, bacterial infection, fungal infection, burn, surgical or dental wound, atherosclerosis, arthritis, Hodgkin's disease, pancreatitis, iritis, scleritis, uveitis, conjunctivitis, dry macular degeneration, wet macular degeneration. , a pharmaceutical composition for a disease selected from the group consisting of sepsis or septic shock.
- 제10항에 있어서, According to clause 10,상기 염증성 질환은 패혈증, 패혈성 쇼크 또는 패혈증과 연관된 증상 및 다기관 기능 부전 증후군인 약학적 조성물. A pharmaceutical composition wherein the inflammatory disease is sepsis, septic shock, or symptoms associated with sepsis and multi-organ dysfunction syndrome.
- 제10항에 있어서, According to clause 10,상기 염증성 질환은 황반변성인 약학적 조성물. A pharmaceutical composition wherein the inflammatory disease is macular degeneration.
- 제13항에 있어서, According to clause 13,상기 황반변성은 건성 황반변성인 것을 특징으로 하는 약학적 조성물. A pharmaceutical composition, wherein the macular degeneration is dry macular degeneration.
- 제13항에 있어서, According to clause 13,상기 조성물은 망막세포 상피(RPE) 또는 외핵층(ONL)의 두께를 감소시키는 것을 특징으로 하는 약학적 조성물. A pharmaceutical composition characterized in that the composition reduces the thickness of the retinal cell epithelium (RPE) or outer nuclear layer (ONL).
- 제10항에 있어서, According to clause 10,상기 펩타이드는 서열번호 1, 5. 6 또는 20의 아미노산 서열로 이루어진 펩타이드 중 하나 이상을 포함하는 것인, 약학적 조성물. A pharmaceutical composition wherein the peptide includes one or more of the peptides consisting of the amino acid sequences of SEQ ID NOs: 1, 5, 6, or 20.
Applications Claiming Priority (4)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| KR10-2022-0144971 | 2022-11-03 | ||
| KR20220144971 | 2022-11-03 | ||
| KR10-2023-0150686 | 2023-11-03 | ||
| KR1020230150686A KR20240067017A (en) | 2022-11-03 | 2023-11-03 | TLR signal inhibitory peptide and composition containing the same for preventing or treating inflammatory diseases |
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| Publication Number | Publication Date |
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| WO2024096677A1 true WO2024096677A1 (en) | 2024-05-10 |
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| Application Number | Title | Priority Date | Filing Date |
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| PCT/KR2023/017529 WO2024096677A1 (en) | 2022-11-03 | 2023-11-03 | Tlr signaling inhibitory peptide and composition including same for prevention or treatment of inflammatory diseases |
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20070094802A (en) * | 2004-12-20 | 2007-09-21 | 시그마타우 인두스트리에 파르마슈티케 리우니테 에스.피.에이. | Myd88 homodimerization inhibitors |
| KR20210009197A (en) * | 2019-07-16 | 2021-01-26 | 아주대학교산학협력단 | Peptide Therapeutic Agent of Autoimmune Diseases and Inflammatory Diseases |
| KR102236773B1 (en) * | 2018-04-27 | 2021-04-06 | 주식회사 젠센 | TLR4 antagonizing peptide |
-
2023
- 2023-11-03 WO PCT/KR2023/017529 patent/WO2024096677A1/en unknown
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20070094802A (en) * | 2004-12-20 | 2007-09-21 | 시그마타우 인두스트리에 파르마슈티케 리우니테 에스.피.에이. | Myd88 homodimerization inhibitors |
| KR102236773B1 (en) * | 2018-04-27 | 2021-04-06 | 주식회사 젠센 | TLR4 antagonizing peptide |
| KR20210009197A (en) * | 2019-07-16 | 2021-01-26 | 아주대학교산학협력단 | Peptide Therapeutic Agent of Autoimmune Diseases and Inflammatory Diseases |
Non-Patent Citations (2)
| Title |
|---|
| VLADIMIR Y TOSHCHAKOV: "Targeting the TLR signalosome with TIR domain-derived cell-permeable decoy peptides: the current state and perspectives", INNATE IMMUNITY, SAGE PUBLICATIONS LTD., US, vol. 26, no. 1, 1 January 2020 (2020-01-01), Us , pages 35 - 47, XP093166319, ISSN: 1753-4259, DOI: 10.1177/1753425919844310 * |
| WENJI PIAO, KARI ANN SHIREY, LISA W RU, WENDY LAI, HENRYK SZMACINSKI , GREG A SNYDER , ERIC J SUNDBERG , JOSEPH R LAKOWICZ , STEFA: "A Decoy Peptide that Disrupts TIRAP Recruitment to TLRs Is Protective in a Murine Model of Influenza", CELL REPORTS, vol. 11, 30 June 2015 (2015-06-30), pages 1941 - 1952, XP055963630 * |
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