WO2024096677A1 - Tlr signaling inhibitory peptide and composition including same for prevention or treatment of inflammatory diseases - Google Patents

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

TLR signal inhibitory peptide and composition containing the same for preventing or treating inflammatory diseases

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Hereinafter, the present invention will be described in detail.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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. 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.

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.

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.

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.

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.

Example 1. Construction of TIR domain binding peptide library

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.

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.

Example 2. Screening of TLR inhibitory activity peptides

2-1. Phage display using phagemid

To screen peptides with TLR inhibitory activity from the peptide library constructed in Example 1, phage display was performed as follows.

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. Confirmation of TLR signaling pathway inhibition activity

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.

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_MAL Peptide sequence NGS count Population WFYERLVSLILLRDTKV 79000 24.40% AFLYTFLINLTHPTNF 61063 18.86% AFFWAQLKSVLGNPTM 28801 8.90% RNFVAYLFQALANKGI 27950 8.63% TFLGHLYNALVQAGIH 24732 7.64% LFFWESLKQLLRSDGR 22560 6.97% YFLHFLLFSRPETEYI 17198 5.31% AFFWEQLRTVLGKPTM 11826 3.65% QLIQQWIELHLHQLHL 9394 2.90% DTKLKKGESIAPELLR 5617 1.74% KLFWEKFKFFLPSLSR 4888 1.51% SEFIQKIILWMNPKIV 4458 1.38% HFFWIQLKSILGETSF 3624 1.12% WFLFTVRSAIYAMSPA 3156 0.97% RNNFIGLYQALYRIG 3120 0.96% SLLYDHLRWLGIRAFL 2576 0.80% WFMMKFRTVLDHVNDV 2269 0.70% KLIQGWFLYRLLKIEQ 2068 0.64% DFLNWFIALKSTVIEN 867 0.27% NQWRNALTAAANLSGW 848 0.26% FTLAVHVRNWLVGGFI 620 0.19% YLKWGYPWFWDRLRFA 617 0.19% AYANFRQGLLRLLELA 466 0.14% WFLTAFRMAMDQVADT 353 0.11% YLISYIFNELARAGFS 295 0.09% AWALHQLQIINWHET 232 0.07% WALQELVKILQAKIFL 227 0.07% KFIWDIINAISSQIRV 146 0.05%

P3_MAL Peptide sequence NGS count Population YAFTGHLHRYLVQRGV 43764 16.44% SSFREGLWRLLELSKK 29955 11.26% PNLRTKVLNYLMRTKT 26931 10.12% NQWRNALTAAANLSGW 25831 9.71% RGFLSHLRHHLQAKDH 15065 5.66% RLLKWKEALHQFHSWV 14924 5.61% LKVESWRQALEKAGKL 6180 2.32% LKWGDPYFWSKLRYAM 5128 1.93% DWFLSKLSQHILKREK 4580 1.72% KMQKWKDALYEAANLS 4427 1.66% KEVEGWRDALTKVASL 3758 1.41% ESILNQLRRSSQSIAN 3680 1.38% SLRKNLVSNLIEEFKR 3569 1.34% KNLFNELVSKNIRTFM 3259 1.22% HHKGSYGEALAEHEKR 3056 1.15% FLNFRGLDVRNYFLGH 2855 1.07% KWFWDKLIYALSRNPH 2796 1.05% YNFISHLYDALHRNRI 2678 1.01% EWIREQLRPALKERLP 2354 0.88% SFFWNKLRYHLPAPQH 2297 0.86% LQNQWKLALTKVANLV 2285 0.86% VEPTQVRKQSGAVENA 2283 0.86% DHHLLDLPRGKAIKPEILG 2280 0.86% HQAFNRLWAKLKPPT 1932 0.73% NELVHVWSSKANSLVG 1836 0.69% GFLYQAFAREGFKIFM 1764 0.66% YLKWGYPWFWDRLRFA 1680 0.63% LVAHEISKKMKIFATI 1671 0.63%

P8_MyD88 Peptide sequence NGS count Population DPQDLDWFYERLVSLF 54757 21.10% LGYHWIDWEYFVGIED 25216 9.72% DWVWDYLLPNLEGPAG 19394 7.47% WALDELLQILHARELF 14404 5.55% DPQDLDWFYERLVSLL 13137 5.06% PQDLEWFYERLVSLIL 10568 4.07% WALDELLKILYANETM 10515 4.05% DSLFWDRLLYSLHVTE 7586 2.92% WDFASNMEQFWDRLYY 7352 2.83% YLLLSVFQTIFARRTE 7165 2.76% YASYDDNDYYWVTHVLLRHL 6893 2.66% DPQDLQWFYERLVSLI 5730 2.21% SAAYDDFWDRLIRMIE 5335 2.06% VQWDDPWFWDKLAYAM 4902 1.89% DPQDLEWFYERLFSQM 4488 1.73% DYASSTWLRELLQIA 4111 1.58% YLRADDSWFWEKLLYA 4067 1.57% YAASPWWFDELVKIVG 3433 1.32% HWIDWEYFVGIEDHIK 3422 1.32% IDDDHLTYLVRLLLSR 3059 1.18% YASSSWALDELLRILY 2327 0.90% VANILGYHWIDWEYFV 2029 0.78% GLSWGPYEWWMGLYDA 1965 0.76% SPQEQDGFWVRLIEAL 1842 0.71% QLIQQWIELHLHQLHL 1605 0.62% DWVWEHFFPMEEKDQT 1538 0.59% MFGGDTFHDFLDLISM 1489 0.57% LEANDSLLFWDRLLYSL 1465 0.56%

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 1.39% NGHESEIIKEIVDTIV 4233 1.23% FLSYRALEKRFGPIGQ 3289 0.96% YNFISHLYDALHRNRI 2891 0.84% GFLYQAFAREGFKIFM 2579 0.75% YYLDPSHLRKQSGEFG 2538 0.74% PEKDNGGENAFINNIV 2498 0.73% DTKLKKGESIAPELLR 2465 0.72% RKGDLISDELLIAIEE 2452 0.71% HLLKEFDKKLITAFKD 2399 0.70% PSEVRKQTGSLAKAFA 2253 0.66% TSHLHHALRLNKIETF 2076 0.60% ESILNQLRRSSQSIAN 1976 0.58%

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.

synthetic peptides peptide sequence
(Cell penetrating peptide + TIR-binding motif) sequence number TDIP1 RQIKIWFQNRRMKWKK WFYERLVSLILLRDTKV SEQ ID NO: 1 TDIP2 RQIKIWFQNRRMKWKK AFLYTFLINLTHPTNF SEQ ID NO: 2 TDIP3 RQIKIWFQNRRMKWKK AFFWAQLKSVLGNPTM SEQ ID NO: 3 TDIP4 RQIKIWFQNRRMKWKK RNFVAYLFQALANKGI SEQ ID NO: 4 TDIP5 RQIKIWFQNRRMKWKK TFLGHLYNALVQAGIH SEQ ID NO: 5 TDIP6 RQIKIWFQNRRMKWKK LFFWESLKQLLRSDGR SEQ ID NO: 6 TDIP7 RQIKIWFQNRRMKWKK YAFTGHLHRYLVQRGV SEQ ID NO: 7 TDIP8 RQIKIWFQNRRMKWKK SSFREGLWRLLELSKK SEQ ID NO: 8 TDIP9 RQIKIWFQNRRMKWKK PNLRTKVLNYLMRTKT SEQ ID NO: 9 TDIP10 RQIKIWFQNRRMKWKK NQWRNALTAAANLSGW SEQ ID NO: 10 TDIP11 RQIKIWFQNRRMKWKK RGFLSHLRHHLQAKDH SEQ ID NO: 11 TDIP12 RQIKIWFQNRRMKWKK KEVEGWRDALTKVASL SEQ ID NO: 12 TDIP13 RQIKIWFQNRRMKWKK DPQDLDWFYERLVSLF SEQ ID NO: 13 TDIP14 RQIKIWFQNRRMKWKK LGYHWIDWEYFVGIED SEQ ID NO: 14 TDIP15 RQIKIWFQNRRMKWKK DWVWDYLLPNLEGPAG SEQ ID NO: 15 TDIP16 RQIKIWFQNRRMKWKK WALDELLQILHARELF SEQ ID NO: 16 TDIP17 RQIKIWFQNRRMKWKK DPQDLDWFYERLVSLL SEQ ID NO: 17 TDIP18 RQIKIWFQNRRMKWKK DHHLLDLPRGKAIKPEILG SEQ ID NO: 18 TDIP19 RQIKIWFQNRRMKWKK YTALTNSGFHTFRDND SEQ ID NO: 19 TDIP20 RQIKIWFQNRRMKWKK LVAHEISKKMKIFATI SEQ ID NO: 20 TDIP21 RQIKIWFQNRRMKWKK EWIREQLRPALKERLP SEQ ID NO: 21 CP
(Negative control) RQIKIWFQNRRMKWKK GGAASSTTGGSSAATT SEQ ID NO: 22

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. Verification of immune regulation efficacy using mouse immune cells

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.

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.

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.

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)

Example 3. Confirmation of sepsis treatment effect of TLR inhibitory active peptide

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.

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)

Example 4. Confirmation of dry macular degeneration treatment effect of TLR inhibitory activity peptide

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.

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.

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.

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.

In one embodiment of the present invention, the overlapping sequence of the peptide fragment is 5 to 8 amino acids long.

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 .

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.

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.

In one embodiment of the present invention, the TLR may be selected from the group consisting of TLR2, TLR3, TLR4, TLR5, TLR7, and TLR9.

In one embodiment of the present invention, the peptide is characterized as having been screened by the method of claim 4.

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.

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.

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.

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.

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.

In one embodiment of the present invention, the inflammatory disease may be macular degeneration.

In one embodiment of the present invention, the macular degeneration may be dry macular degeneration.

In one embodiment of the present invention, the composition can reduce the thickness of the retinal cell epithelium (RPE) or outer nuclear layer (ONL).

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)

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; 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 a TIR domain surface sequence, comprising the step of introducing the cloned vector into a bacteriophage to create a library for phage display. According to paragraph 1, A library construction method, characterized in that the overlapping sequence of the peptide fragment is 5 to 8 amino acids in length. 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. According to clause 3, 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: A TLR (Toll like receptor) signaling inhibitory peptide containing a peptide fragment derived from the surface sequence of the TIR domain. According to clause 5, The TLR is any one or more signal inhibitory peptides selected from the group consisting of TLR2, TLR3, TLR4, TLR5, TLR7 and TLR9. According to clause 5, The peptide is characterized in that the peptide was screened by the method of claim 4. According to clause 5, 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. According to clause 5, A peptide characterized in that the peptide is fused with a peptide consisting of the sequence of SEQ ID NO: 23. A pharmaceutical composition for preventing, improving or treating inflammatory diseases comprising the peptide of claim 5. According to clause 10, 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. According to clause 10, A pharmaceutical composition wherein the inflammatory disease is sepsis, septic shock, or symptoms associated with sepsis and multi-organ dysfunction syndrome. According to clause 10, A pharmaceutical composition wherein the inflammatory disease is macular degeneration. According to clause 13, A pharmaceutical composition, wherein the macular degeneration is dry macular degeneration. According to clause 13, A pharmaceutical composition characterized in that the composition reduces the thickness of the retinal cell epithelium (RPE) or outer nuclear layer (ONL). According to clause 10, 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.

Images