KR20200084569A - Peptide Inhibiting TLR4 Signaling Pathway and Uses Thereof - Google Patents

Abstract

The present invention relates to a peptide that inhibits toll-like receptor (TLR4) signaling pathway. More specifically, the present invention relates to a peptide that binds to the TIR domain of TLR4 to inhibit the secretion of proinflammatory cytokines and inhibits the activation of NF-κB and MAPKs, a fusion peptide in which a cell-penetrating peptide is bound to the peptide, a TLR4 antagonist comprising the peptide or fusion peptide, and a composition for preventing or treating autoimmune diseases or inflammatory diseases comprising the peptide or fusion peptide. The peptide according to the present invention exhibits an inhibitory effect on TLR4 and TLR3 signaling, and blocks MyD88- and TRIF-dependent TLR4- pathways in a number of human and murine cell lines. Since the peptide has a substantial disease alleviation effect in rheumatoid arthritis, psoriasis and systemic lupus erythematosus mouse models, the peptide can be used as a therapeutic agent for immune-related diseases requiring negative control of TLR.

Description

Peptide Inhibiting TLR4 Signaling Pathway and Uses Thereof}

The present invention relates to a peptide that inhibits the TLR4 (Toll-like receptor 4) signaling pathway, and more specifically, binds to the TIR domain of TLR4, inhibits the secretion of pro-inflammatory cytokines, and activates NF-κB and MAPKs A peptide for inhibiting, a fusion peptide having a cell permeable peptide bound to the peptide, a TLR4 antagonist comprising the peptide or fusion peptide, and a composition for preventing or treating an autoimmune disease or inflammatory disease comprising the peptide or fusion peptide will be.

Toll-like receptors (TLRs) are membrane-bound that recognize exogenous pathogen-associated molecular patterns (PAMPs) and/or endogenous damage-associated molecular patterns. These are membrane-bound pattern recognition receptors. Recognizing pathogen-associated molecular patterns or damage-associated molecular patterns, TLRs are dimerization-induced complexes leading to activation of nuclear factor kappa light-chain enhancer of activated B cells (NF-κB) or interferon-regulatory factors (IRFs). Causes signaling cascade (Kawai T & Akira S (2010) Nature immunology 11(5):373-384). These transcription factors spontaneously migrate to the nucleus, pro-inflammatory cytokines (interleukin-6 (IL-6), IL-1β and IL-10), tumor necrosis factor α; TNF- α) and antiviral interferon (type I IFNs) (O'Neill LA & Bowie AG (2007) Nature reviews . Immunology 7(5):353-364). Myeloid differentiation primary response 88 (MyD88) is the first adapter recruited to activated TLRs other than TLR3, and TIR domain-containing adapter-inducing interferon β (TRIF) through intermolecular TIR (Toll/interleukin-1 receptor) domain interaction ) (Gay NJ, Symmons MF, Gangloff M, & Bryant CE (2014) Nature reviews. Immunology 14(8):546-558). In the MyD88-dependent pathway, interleukin receptor-associated kinase 4 (IRAK4) is recruited, and IRAK1 or IRAK2 is recruited to complete a supramolecular assembly called'myddosome' (Lin SC, Lo YC, & Wu H (2010) Nature 465 (7300):885-890), which is essential for subsequent signaling. TLR4 can induce lipopolysaccharide (LPS)-induced TRIF-dependent signaling, compensating for the production of TLR3 IFNs. Thus, activation of TLR4 produces significant amounts of chemokines, interferons and interleukins (this is called a cytokine storm). This phenomenon can also lead to a fatal autoinflammatory condition called sepsis.

Since secreted cytokines and interferons activate the components of the adaptive immune system for the production of specific antibodies against specific infections, the nature of normal activation of intrinsic immunity lies in defense (Iwasaki A & Medzhitov R (2015)Nature immunology 16(4):343-353). Nevertheless, overactivation or dysregulation of intrinsic immunity can often be detrimental to survival. Due to autoantibodies expressed in response to auto-antigens, systemic lupus erythematosus (SLE) (Wu YW, Tang W, & Zuo JP (2015)Acta pharmacologica Sinica 36(12):1395-1407), rheumatoid arthritis (RA) (Wang Y, et al . (2017)Pediatric rheumatology online journal 15(1):12), psoriatic arthritis (SA) (Jimenez-Dalmaroni MJ, Gerswhin ME, & Adamopoulos IE (2016)Autoimmunity reviews 15(1):1-8), atherosclerosis (Feldman N, Rotter-Maskowitz A, & Okun E (2015)Ageing research reviews 24(Pt A):29-39), sepsis (Liew FY, Xu D, Brint EK, & O'Neill LA (2005)Nature reviews . Immunology 5(6):446-458) and the like, leading to autoimmune diseases and inflammatory diseases. Thus, unregulated TLRs are recognized as desirable drug targets for the treatment of autoimmune and inflammatory diseases (O'Neill LA (2003)Current opinion in pharmacology 3(4):396-403).

In recent years, many advances have been made in peptide-based preclinical agents that target cell surface-expressing TLRs (TLR2 and TLR4) by targeting TIR domains as well as extracellular domains (Piao W , et. al . (2015) Cell reports 11(12):1941-1952).

TIRAP (TIR domain adapter protein) is a unique adapter that acts as a bridge between TLR2 and/or TLR4 and MyD88 activated during signal transduction (Bonham KS , et. al . (2014) Cell 156(4):705-716). Since TIRAP is fixed to the phosphatidylinositol 4,5-bisphosphate molecule through the phosphatidylinositol 4,5-bisphosphate-binding domain, it continuously binds to the cytoplasmic membrane (Kagan JC & Medzhitov R (2006) Cell 125(5):943-955) . Nevertheless, the TIR domain of TIRAP is exposed to a solvent to facilitate recruitment of MyD88 to the TIR domain of TLR2 or TLR4 (Valkov E , et. al . (2011) Proceedings of the National Academy of Sciences of the United States of America 108(36):14879-14884). The essential role of TIRAP in downstream signaling of TLR2 and TLR4 has been previously identified (Zhao X , et. al . (2017) Scientific reports 7:43043).

Accordingly, the present inventors have tried diligently to develop a new peptide capable of inhibiting the TLR4 signaling pathway. As a result, a peptide combining a peptide derived from β-sheet of TIRAP with a cell permeable peptide successfully ligand-induced TLR signaling. It was confirmed that it inhibits, alleviates the inflammatory response in a mouse model of arthritis, alleviates psoriasis symptoms in the mouse, and confirms that it alleviates the autoimmune response observed in the systemic lupus erythematosus mouse model, and completed the present invention.

An object of the present invention is to provide a peptide that inhibits the TLR signaling pathway and a fusion peptide having a cell permeable peptide coupled to the peptide.

Another object of the present invention is to provide a TLR4 antagonist (antagonist) comprising the peptide or the fusion peptide.

Another object of the present invention is to provide a composition for the prevention or treatment of autoimmune diseases or inflammatory diseases comprising the peptide or the fusion peptide.

In order to achieve the above object, the present invention provides a peptide represented by the amino acid sequence of SEQ ID NO: 1.

The present invention also provides a fusion peptide having a cell permeable peptide bound to the peptide.

The present invention also provides a TLR4 antagonist comprising the peptide or the fusion peptide.

The present invention also provides a composition for preventing or treating autoimmune diseases comprising the peptide or the fusion peptide.

The present invention also provides a composition for preventing or treating inflammatory diseases comprising the peptide or the fusion peptide.

The peptide according to the present invention exhibits an inhibitory effect on TLR4 and TLR3 signaling, blocks MyD88- and TRIF-dependent TLR4- pathways in many human and mouse cell lines, and is a substantial disease in rheumatoid arthritis, psoriasis and systemic lupus erythematosus mouse models Since it has a relaxing effect, it can be used as a therapeutic agent for immune-related diseases that require negative regulation of TLR.

Figure 1 shows the selection and initial evaluation of TIRAP-derived TLR-inhibiting peptide,
FIG. 1A shows TLR-inhibiting peptides (TIP) 2 and 3 selected from the core β-sheet structures βD (brown box) and βA (blue box), respectively. FIG. 1B shows 100 μM TIP3 (TLR-inhibitory peptide 3) and It is a result of cell viability analysis showing that TIP2 slightly affects cell proliferation. 1C is a graph showing substantial inhibition (measured by SEAP analysis) of NF-κB activation by TIP3, and unlike TIP2, shows TIP3's TLR-inhibiting ability. Data shown are from at least 3 independent experiments (n ≥ 3), and bars mean mean±SEM (* P <0.05, ** P <0.01).
Figure 2 shows that TIP3 down-regulates transcription factors and pro-inflammatory cytokines to inhibit the TLRs signaling pathway,
2A is a graph showing that 50 μM TIP3 is safe in subsequent experiments for RAW264.7 cells and other cell lines, and FIGS. 2B and 2C are p-p65, Iκ-Bα, p-IRF3, ATF3, p-ERK, ERK, Protein expression levels of p-JNK, JNK, p-p38 and p38 were measured by Western blot analysis of the pre-protein extract, β-actin was used as loading control. 2D shows the phosphorylation of NF-κB (p-p65) by immunofluorescence staining and confocal microscopy, and Hoechst was used for nuclear staining (scale bar indicates 20 μm). 2E-2H show the secretion levels of TNF-α, IL-6, IFN-α and IFN-β by ELISA, and the secretion of four cytokines was inhibited concentration-dependently by TIP3. Figure 2I is a graph showing the level of NO secretion evaluated using a standard NO secretion kit, Figure 2J is a graph showing the expression level of iNOS and COX2 measured by Western blotting, β-actin was used as a loading control. 2K and 2L are graphs showing that NO and ROS in cells were quantified by DAF-FM and DCF-DA staining, respectively, and substantially down-regulated by 50 μM TIP3, and FIG. 2M shows that cells have PAM ₃ at various concentrations. When activated with CSK ₄ (TLR2/1), FSL-1 (TLR2/6), R848 (TLR7/8) or CpG-ODN (TLR9), the inhibitory effect of TIP3 on multiple TLRs by measuring TNF-α levels As a graph evaluating, the degree of inhibition by TIP3 was not as high as that of TLR4. 2N to 2P are TNF-α (FIG. 2N), IL-6 (FIG. 2O) and IFN-β (FIG. 2P) after activation by poly(I:C)(TLR3) for 24 hours in RAW264.7 cells. ) Is a graph examining the TLR3-inhibitory effect of TIP3 through secretion. 2Q to 2U are 3 hours after TIP3 treatment, tnf -α , il -6 , ifn -β , cxcl -10 and It is a graph showing the effect of TIP3 on the LPS-induced mRNA expression level of il - 1β . Values are expressed as fold changes (relative quantification) at the mRNA level, normalized to β-actin, and the data shown are from at least 3 independent experiments (n ≥ 3), and the bars are mean±SEM (* P <0.05). , ** P <0.01).
Figure 3 shows the TLR-inhibitory effect characteristics of TIP3 on primary cells,
3A is a graph showing that TIP3 is safe for THP-1 cells at various concentrations (≤ 50 μM) for 24 hours, and FIGS. 3B and 3C show protein expression measured by Western blot analysis using hPBMC. The amounts of p-p65, Iκ-Bα, p-IRF3, ATF3, p-ERK, ERK, p-JNK, JNK, p-p38 and p38 were evaluated in the pre-protein extract, and β-actin was used as loading control. Became. 3D and 3E are graphs showing the secretion levels of TNF-α and IL-6 measured by ELISA after cells were stimulated with LPS and then treated with TIP3. 3F to 3I are graphs showing the effect of TIP3 on LPS-induced mRNA expression levels of il -6 , tnf -α , il -8 and il - 1β after 4 hours of TIP3 treatment, mRNA levels of these genes It was substantially down-regulated by TIP3, and the value is expressed as fold changes (relative quantification) at the mRNA level. Standardized as gapdh . 3J to 3M show the secretion of IL-6 (FIG. 3J), TNF-α (FIG. 3K), and IFN-β (FIG. 3L) measured by ELISA after 1 hour treatment of mBMDMs stimulated by LPS with TIP3. It is a graph showing the level, and a graph showing the NO production evaluated using the NO secretion kit (Fig. 3M). 3N is a cell viability analysis result of hMNCs treated with TIP3, and FIGS. 3O to 3P are IL-6 (FIG. 3O) and TNF-α (FIG. 3P) evaluated by ELISA using hMNCs stimulated by LPS. It is a graph showing the level of secretion. 3Q is a graph showing the TLR3-inhibitory effect of TIP3 evaluated by measuring TNF-α secretion by ELISA using poly(I:C)-stimulated hMNCs, showing similar effects to those observed in RAW264.7 cells. Data shown are from at least 3 independent experiments (n ≥ 3), and bars mean mean±SEM (* P <0.05, ** P <0.01).
FIG. 4 shows the protective effect of TIP3 evaluated by monitoring anatomical and behavioral parameters in the CIA mouse model.
Figure 4A is a summary of the experimental protocol, CIA was induced by subcutaneous injection of type II collagen into mice, daily after the second injection of collagen (day 22), or daily after the postarthritis phase (PAP) (day 35). TIP3 was treated and methotrexate was used as a positive control. 4B is a photograph of the right rear foot enlarged on day 45. 4C to 4E are graphs showing body weight (FIG. 4C), foot volume (FIG. 4D), and RA index (FIG. 4E) as a result of observing and analyzing mice. The black and blue arrows indicate the two viewpoints of the TIP3 treatment mentioned in Figure 4A. 4F is a 3D image of the knee joint taken by Micro-CT to predict joint corrosion and cartilage loss, and FIG. 4G is a 2D image of the spongy bone corresponding to the sagittal section of the tibia upper part taken using Micro-CT 4H is a 2D image of the cortical bone corresponding to the horizontal cross-section of the tibial middle. Figure 4I shows the bone density (BMD) of the right or left knee joint measured by Micro-CT, similar to MTX, it can be seen that TIP3 (10nmol/g) restores bone density. 4J is a histogram showing the quantification of synovial hyperplasia scores, and it can be seen that TIP3 treatment substantially reduces inflammation. 4K shows the histological evaluation of CIA synovial tissue and the effect of TIP3 treatment, the image was taken at x40 magnification, and the scale bar is 200 μm in the normal case. C is cartilage; F is the femur; M is meniscus; S is the subchondral bone; T stands for tibia. Numerical data are presented as means±SEM: ^# P <0.05, ^## P <0.01, and ^### P <0.001 CIA versus Normal; * P <0.05, ** P <0.01, and *** P <0.001 TIP3 versus CIA.
5 is a picture of a mouse taken on day 45.
6 is a 3D image of the knee joint taken using Micro-CT to estimate joint corrosion and cartilage loss.
7 is a 2D image of the spongy bone corresponding to the sagittal section of the tibia upper part measured by Micro-CT.
Figure 8 is a 2D image of the cortical bone corresponding to the horizontal cross-section of the middle part of the tibia measured by Micro-CT.
Figure 9 shows the treatment efficacy of TIP3 in the psoriasis mouse model,
9A summarizes the experimental procedure for evaluating the therapeutic effect of TIP3 in a psoriasis rat model. Psoriasis was induced in C57BL/6 male mice by topical application of imiquimod (IMQ), TIP3 was administered intraperitoneally prior to IMQ application, and Methotrexate (MTX) was used as a positive control for the relative treatment evaluation of TIP3. 9B is a photograph of the back skin on day 4, showing that TIP3 has a therapeutic effect compared to the untreated or MTX treated group. 9C is a graph showing disease severity scores based on the clinical psoriasis area and severity index (PASI), in which light purple or dark purple lines are 10 nmol/g or 50 nmol/g TIP3 treated mice, respectively. PASI score. 9D is a graph showing the effect of TIP3 on spleen weight, and FIG. 9E is a graph showing the body weight dynamics of mice during treatment. 9F shows the effect of TIP3 on the thickness of the epidermis (yellow arrow) and dermis (green arrow), and FIGS. 9G and 9H are graphs showing the skin thickness of each group measured by a Leica DMi8 fluorescence microscope. 9I shows the immunohistochemical analysis of back skin lesions of each group evaluated by immunohistochemical analysis, the meaning of the scale bar is as follows: low magnification image at the top of 250 μm; High magnification image 75μm at the bottom. Data represent mean±SEM obtained from 5 skin tissue samples from each group (Two-tailed Student's t-test( ^### P <0.001 between normal and PBS, **P <0.01, *P <0.05 between PBS and TIP3 (at 10 or 50 nmol) or MTX)).
Figure 10 shows the inhibitory effect of TIP3 on SLE in a mouse model,
10A summarizes the experimental validation of the inhibitory effect of TIP3 on SLE in a mouse model, and FIG. 10B shows C57BL6 male mice and lupus-prone mice. 10C is a photograph showing the improvement effect of TIP3 on lymphatic proliferation in the spleen and lymph nodes. 10D is a graph showing albumin content in urine, FIG. 10E is an anti-dsDNA antibody, and FIG. 10F is a C3 complement level in serum measured by ELISA. To compare the mean values of the two groups, a Wilcoxon Rank Sum test (numerically equivalent to the Mann-Whitney U test) was performed.
Figure 11 shows the intermolecular interaction between the TIR domain of TIP3 and TLR4,
11A shows the overall docking model of TIP3 (red cartoon) together with the entire TLR4 model, and chain A and chain B of TLR4 are shown in green and blue, respectively. MD2 is shown in gray and LPS is shown in red stick model. Phospholipid bilayer phosphate groups (purple beads) were used to indicate the membrane area, the TIR domain was represented by a gray space-fill model, and the BB loop was magenta. Figure 11B shows the surface of TIP3 (red) bound to the TIR domain (grey) of TLR4, Figure 11C shows the interaction of the TIP3 residue and TLR4 TIR domain in detail, the TIP3 residue is red, the TIR residue is gray , BB loop residues are magenta. The residue of the B chain of TLR4 is indicated by the residue number after "B".

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by a person skilled in the art to which the present invention pertains. In general, the nomenclature used herein is well known in the art and commonly used.

The unregulated response of Toll-like receptors (TLLs) is extensively associated with the development of various inflammatory and autoimmune diseases. For this reason, TLR is one of the drug targets for treating immune-related diseases. In recent years, with increasing awareness of TLR structural biology and signaling pathways, numerous therapeutic agents have been developed, ranging from low molecular organic compounds to giant polypeptide and nucleic acid mediators, and peptides have better efficacy, selectivity, safety and efficacy, and in the future It can be an attractive candidate for treatment strategies (Bovijn C , et. al . (2013) The Journal of biological chemistry 288(17):12054-12066).

The present inventors synthesized TIP3 (TLR-inhibitory peptide 3) derived from the core β-sheet of TIRAP (TIR domain adaptor protein). TIP3 substantially blocks TLR4-mediated cytokine production through both the MyD88-dependent and TRIF-dependent pathways. An inhibitory effect was observed on TLR3-mediated signaling, and was partially observed on other TLRs. It has been found that the arthritis mouse model successfully relieves the inflammatory response and improves psoriasis and systemic lupus erythematosus in In vivo treatment effects were observed. These results suggest that TIP3 can be an effective therapeutic candidate for TLR-mediated diseases.

Ligand-induced homo- and heterotypic TIR-TIR domain interactions are indispensable for initiating TLR signaling (Bovijn C , et. al . (2013) The Journal of biological chemistry 288(17):12054-12066). This highly compliant set of adapter molecules is critical for the initiation of related signals, and blocking of these interfaces can prevent TLR signaling. Although it is difficult to target transient interactions of the adapter proteins involved in signaling, it offers several advantages because some adapter molecules act important in different pathways in a stoichiometric manner. By targeting cytoplasmic proteins or domains, relatively high efficacy and broader specificity can be expected from signal transduction inhibition as opposed to antagonism of agonist function.

In the present invention, it was confirmed that the strong TLR-inhibitory peptide 3 (TIP3) derived from the first β-sheet (βA) of TIRAP successfully reduced the secretion of pro-inflammatory cytokines by TLR4. . First, the TLR4-inhibiting effect was confirmed by evaluating LPS-induced NF-κB activation in HEK-Blue ^™ hTLR4 cells. Since TLR4 signals through the MyD88 and TRIF adapter molecules, it was confirmed whether TIP3 blocks both TLR4 signaling. TIP3 inhibited not only pro-inflammatory cytokine production in LPS-stimulated mouse and human macrophages, but also type I interferon induction (Figures 2 and 3), which TIP3 inhibits both MyD88-dependent and TRIF-dependent signaling. It means you can. Next, TNF-α secretion activated by other TLR ligands in RAW264.7 cells was measured, and then treated with TIP3 to evaluate the effect of TIP3 on other TLRs. It was confirmed that TNF-α secretion dependent on TLR was substantially inhibited by TIP3 (FIGS. 2E, 2M and 2N). The low inhibitory effect of TIP3 on TLR2, TLR7, TLR8 and TLR9 signaling in addition to the strong inhibitory ability on TLR4 and TLR3 signaling is partially explained by the fact that the TIR domains in the adapter protein and TLR protein share sequence and structural similarity. Can (Dunne A &O'Neill LA (2003) Science's STKE : signal transduction knowledge environment 2003(171):re3). The affinity of TIP3 for the TIR domain of TLR4 can be explained by structural studies. In secondary, primary human and mouse cell lines, the results were confirmed by Western blot analysis, mRNA quantification of various multiple transcription factors and pro-inflammatory cytokines and Enzyme-linked immunosorbent assay (ELISA). As a result, the protective effect of TIP3 was confirmed in the TLR-mediated autoimmune disease model.

The inventors tested the protective effect of TIP3 against RA in a rat CIA model. TIP3 significantly reduced induction of RA in CIA mice, reversed weight loss, reduced foot volume, and decreased arthritis index. The peptide also partially promoted reversal of bone and cartilage erosion by helping the bone recover microstructure and mineral components (FIG. 4 ). Furthermore, articular tissue sections have shown that TIP3 attenuates histopathological abnormalities by reducing intensive immune cell penetration, reducing synovial tissue thickness, and restoring normal spacing within the joint.

Next, the effect of TIP3 on chronic inflammatory skin disease was confirmed using a mouse model of IMQ-induced psoriasis-like disease, and the effect of TIP3 on systemic autoimmune disease in the MRL/lpr mouse model of SLE was confirmed. . It was confirmed that the peptides according to the present invention have protective effects against both diseases in these models. In the psoriasis model, TIP3 significantly improved skin lesions, prevented hypertrophy, and improved the overall physiological condition of mice. TIP3 also mitigated histological symptoms of the disease by attenuating the increase in the thickness of the epidermis and dermis and suppressing the hyperproliferative state of the skin. Despite the concentration-dependent protective effect of the peptides according to the invention, weight loss was observed at 50 nmol/g administration of TIP3. However, this is not entirely due to peptide treatment. The reason is that significant weight recovery was observed in the CIA model when treated with 10 nmol/g (FIG. 4C ). In addition, TIP3 prevented lymphatic proliferation in the mouse model of SLE and inhibited glomerulonephritis index by reducing albumin concentration in urine. Furthermore, after treatment of mice with the peptides according to the invention, the levels of complement components C4 and C3 were restored, while the production of anti-dsDNA autoantibodies was reduced.

Numerous researchers have found that the TIR domain in the TLR family shares a high degree of structural similarity, and bacteria (Alaidarous M , et. al . (2014) The Journal of biological chemistry 289(2):654-668) and plants (Zhang X , et al . (2017) Proceedings of the National Academy of Sciences of the United States of America 114(10):E2046-E2052) has been proposed to belong to one of the most evolutionarily conserved families of structural folds. In particular, the BB loop and its contiguous regions are highly conserved and are believed to represent adapter recruitment sites as well as major sites for TIR dimerization. In the present invention, it was confirmed through computational docking simulation that TIP3 has excellent complementarity of form and charge to the TIR domain of TLR4. The peptide according to the present invention penetrates the cell membrane and inhibits the MyD88-dependent and non-dependent pathways by binding to the adapter docking site of the TLR4 TIR domain. Homology modeling in the absence of the experimental structure of a given TIR domain has been widely considered in constructing 3D models based on hypotheses about homodimerization, receptor binding and adapter-binding interfaces (Li C, Zienkiewicz J, & Hawiger J (2005) The Journal of biological chemistry 280(28):26152-26159). In the present invention, as a result of computational docking analysis, it was found that TIP3 can bind to the TLR4 TIR domain of the BB loop region and block downstream signaling.

Taken together, these results suggest that TIP3 can ameliorate their symptoms by stopping the progression of RA, psoriasis and SLE and reducing the inflammatory response mediated by TLR signaling.

Accordingly, the present invention, in one aspect, relates to a peptide represented by the amino acid sequence of SEQ ID NO: 1.

In the present invention, the peptide is a signal of any one or more of the TLR (Toll-like receptor) selected from the group consisting of TLR4 (Toll-like receptor 4), TLR3, TLR2/1, TLR2/6, TLR7/8 and TLR9 It may be characterized by suppressing the delivery pathway.

In the present invention, the TLR4 signaling pathway may be characterized by being induced by lipopolysaccharide (LPS).

In the present invention, the peptide may be characterized by binding to the TLR4's TIR (Toll/interleukin-1 receptor) domain.

In the present invention, the term "peptide" refers to a linear molecule formed by bonding amino acid residues to each other by peptide bonding. The peptide may be prepared according to chemical synthesis methods known in the art, and preferably solid phase synthesis technology. It may be prepared according to, but is not limited to.

In the present invention, the term "TLR4" is a protein belonging to the TLRs, a family of transmembrane protein that functions as a monitor for pathogen infection, and refers to a protein encoded by the TLR4 gene, as CD 284 (cluster of differentiation 284) It is also named. The TLR4 recognizes various pathogen-associated molecular patterns (PAMPs) including LPS of Gram-negative bacteria, which is very important for the activation of the innate immune system.

The term "TLR4 signaling pathway" in the present invention refers to the signaling pathway through TLR4. TLR4 is signaled by several adapter proteins, and the signaling pathways work with TIRAP (also referred to as Mal) and MyD88 and TRAM and TRIF. The TLR4-mediated signaling pathway induces activation of MyD88-dependent and MyD88-independent signaling through various sub- signaling molecules. Initiation of the MyD88-independent pathway induces early stage activation of NF-κB and secretion of pro-inflammatory cytokines such as TNF-α and IL-6, and initiation of the MyD88-independent pathway activates IRF3 and 7, IFNs Induces secretion and late-stage activation of NF-κB (Park, BS et al ., Exp . Mol . Med . 45:e66, 2013; Nijland, R et al., Mar. Drugs 12: 4260-4273, 2014). In addition, TLR4 causes activation of MAPK, including ERK, JNK and p38, and secretes inflammatory cytokines and IFNs (Achek, A et. al ., Arch . Pharmacal Res . 39:1032-1049, 2016; Kawai, T et al., Nat. Immunol . 11:373-384, 2010). Stimulation of TLR4 by related ligands in macrophages induces COX2 and iNOS, induces NO production, and produces mitochondria and intracellular ROS (Kim, JY et al., Eur . J. Pharmacol . 584:175- 184, 2008; Liu, K.-L. et al. J. Agric . Food Chem . 54:3472-3478, 2006; West, AP et al . Nature 472:476, 2011).

In the present invention, the term "inhibition" refers to a phenomenon in which biological activity or activity decreases due to deficiency, disharmony, and many other causes, partially or completely blocking, reducing, preventing, or preventing TLR4 activity. It can be delayed, inactivated or down regulated.

In the present invention, inhibition of the expression of TNF-α, IL-6, IFN-α or IFN-β by blocking the TLR4 signaling pathway of the peptide; Inhibition of NO or ROS secretion; Alternatively, the activity of NF-κB or MAPK may be inhibited, and the peptide may be characterized by inhibiting both MyD88-dependent and MyD88-independent TLR4 signaling pathways.

In another aspect, the present invention relates to a fusion peptide in which a cell penetrating peptide is bound to the peptide.

In the present invention, the fusion peptide may be characterized by inhibiting the TLR4 or TLR3 signaling pathway, and the TLR4 signaling pathway may be characterized by being induced by LPS.

In addition, in the present invention, the expression of TNF-α, IL-6, IFN-α or IFN-β is inhibited by blocking the TLR4 signaling pathway of the fusion peptide; Inhibition of NO or ROS secretion; Alternatively, the activity of NF-κB or MAPK may be inhibited, and the fusion peptide may be characterized by inhibiting both MyD88-dependent and MyD88-independent TLR4 signaling pathways.

In the present invention, the term, “cell penetrating peptide (CPP)” is a kind of signal peptide, which is a combination of specific amino acid sequences used for the purpose of delivering polymer substances such as proteins, DNA, and RNA into cells. It is a phosphorus peptide. To date, it has been used for intracellular delivery of various low-molecular compounds, proteins, peptides, RNA, DNA, and other high-molecular substances.

The fusion peptide of the present invention uses a cell-permeable peptide, and the cell-permeable peptide is not particularly limited as long as it has a feature that enters the cell by a mechanism of cell internalization (endocytosis), but is preferably cell permeability listed in Table 1 below. It can be selected from peptides or variants thereof. More preferably, the cell permeable peptide may be characterized by being represented by the amino acid sequence of SEQ ID NO: 3.

Cell permeable peptide Peptide origin Sequence (5'->3 ') Penetratin Drosophila Antennapedia homeodomain RQIKIWFQNRRMKWKK (SEQ ID NO: 3) TAT _(48-60) Human immunodeficiency virus type 1 (HIV-1) TAT GRKKRRQRRRPPQ pVEC VE-Cadherin (615-632) LLIILRRRIRKQAHAHSK Transportan 10
/TP10 Galanin-Lys-mastoparan GWTLNSAGYLLGKINLKALAALAKKIL MPG A hydrophobic domain from the fusion sequence of HIV gp41 and NLS of SV40 T-antigen GALFLGFLGAAGSTMGAWSQPKKKRKV Pep-1 NLS from Simian Virus 40 large T antigen and reverse transcriptase of HIV-1 KETWWETWWTEWSQPKKKRKV MAP Amphipathic model peptide KLALKLALKALKAALKLA R ₆ /W ₃ Based on penetratin RRWWRRWRR Polyarginine(R ₉ ,R ₈ ) Positively charged sequence R _n , n = 8 or 9 VP22 Herpes simplex virus NAKTRRHERRRKLAIER YTA2 MMP cleavage site as seeding sequence YTAIAWVKAFIRKLRK YTA4 MMP cleavage site as seeding sequence IAWVKAFIRKLRKGPLG M918 The tumor suppressor protein p14ARF MVTVLFRRLRIRRACGPPRVRV CADY Derived from PPTG1 peptide, W and charged amino acids GLWRALWRLLRSLWRLLWRA SAP Designed based on a natural protein of maize, γ-zein VHL (PPP) ₈ (VRLLPPP) ₃ SAP(E) Design inspired by SAP; Arg residue replaced by Glu Ac-CGGW(VELPPP)3 CyLoP-1 Derived from crotamine toxin found in snake venom, crot (27-39) CRWRWKCCKK gH 625 Based on the 625-644 residues of the glycoprotein HSV 1 HGLASTLTRWAHYNALIRAF GALA Glu-rich an containing His (imidazole group) in order to be pH responsive (endosomes) (Designed to efficiently escape endosomes) WEAALAEALAEALAEHLAEALAEALEALAA CADY Designed; based on chimeric peptide carrier PPTG1 derived from the fusion peptide JTS1 Ac-GLWRALWRLLRSLWRLLWRA-cysteamide L17E Inspired by the spider venom M-lycotoxin IWLTALKFLGKHAAKHEAKQQLSKL MPPs Designed to contain un-natural, cyclohexylalanine (F _x ) residues and to have differential intracellular localization Mitochondria-penetrating peptides (example: F _X rF _X KF _X rF _X K) RR ₅ -APPRR ₄ -APP
RR ₃ -APP Small proteins (36-residue polypeptides) RPRRPRRPRRPGRRAPVEDLIRFYNDLQQYLNVVTRHRYC
RRPRRPRRPGRRAPVEDLIRFYNDLQQYLNVVTRHRYC
GPRRPRRPGRRAPVEDLIRFYNDLQQYLNVVTRHRYC TATp-D Analogue of TAT

Cyclic Tat Lys- and Glu- amino acids added to the linear Tat sequence to obtain a ring with the same overall charge as the native form c[K-rRrQrRkKrG-E]c K ₁₀ (QW) ₆ The design was based on combining W and K at the primary structure level to obtain self-assembly into a variety of nanostructures KKKKKKKKKKQWQWQWQWQWQW TAT _(49-57) HIV-1 TAT protein RKKRRQRRR DPV1047 Chemically synthesized VKRGLKLRHVRPRVTRMDV ARF _(1-22) p14ARF protein MVRRFLVTLRIRRACGPPRVRV BPrPr _(1-28) N terminus of unprocessed bovine prion protein MVKSKIGSWILVLFVAMWSDVGLCKKRP p28 Azurin LSTAADMQGVVTDGMASGLDKDYLKPDD VT5 Chemically synthesized DPKGDPKGVTVTVTVTVTGKGDPKPD C105Y α1-Antitrypsin CSIPPEVKFNKPFVYLI PFVYLI Derived from synthetic C105Y PFVYLI Pep-7 CHL8 peptide phage clone SDLWEMMMVSLACQY

Meanwhile, among the cell permeable peptides of Table 1, Transportan includes those used in the form of the following variants: AGYLLGKINLKALAALAKKIL-NH ₂ (TP10, PepFect 3), AGYLLGKINLKALAALAKKIL-NH ₂ (TP10, PepFect 6), AGYLLGKLLOOLAAAALOOLL-NH ₂ (TP10, PepFect 14), AGYLLGKTNLKALAALAKKIL-NH ₂ (NickFect 1), AGYLLGKTNLKALAALAKKIL-NH ₂ (NickFect 2) and AGYLLGKTNLKALAALAKKIL-NH ₂ (Nickfect 3).

In an embodiment of the present invention, an experiment was performed by selecting the Penetratin sequence (RQIKIWFQNRRMKWKK; SEQ ID NO: 3) among the cell permeable peptides of Table 1 above, and other cell permeable peptides other than the cell permeable peptides actually used were fused with the peptides of the present invention. It will be apparent to those skilled in the art that similar effects to the present invention are exhibited even in the case of prescribing.

In the present invention, the fusion peptide may be characterized in that the cell-permeable peptide is bound to the N-terminal or C-terminal of the peptide. Preferably, the cell-permeable peptide is bound to the N-terminus of the peptide, but is not limited thereto.

In the present invention, the fusion peptide to which the cell-permeable peptide is connected to the N-terminus of the peptide may preferably be characterized by consisting of the amino acid sequence of SEQ ID NO:4. In addition, variants of the amino acid sequence may also be included within the scope of the present invention, specifically, the variants are SEQ ID NO: 4 and 70% or more, preferably 80% or more, more preferably 90% or more, and even more preferably, respectively. May include all peptides having a sequence homology of 95% or more, even more preferably 98% or more, and most preferably 99% or more. The term "homology" refers to the degree of similarity to wild type amino acid sequences and wild type nucleic acid sequences.

In another aspect, the present invention relates to a TLR4 antagonist comprising the peptide or the fusion peptide.

As used herein, the term “antagonist” refers to a molecule that partially or completely inhibits the influence of other molecules, such as receptors or mediators within cells, by any mechanism.

In the present invention, the term "TLR4 antagonist" refers to a substance capable of directly, indirectly, or substantially interfering, reducing or inhibiting the biological activity of TLR4, preferably a peptide reactive with TLR4 is directly bound to the TLR4 or TIR domain And, by neutralizing the activity of TLR4, blocking the TLR4 signaling pathway, causing a decrease in the activation of NF-κB and MAPKs, thereby reducing the secretion of inflammatory cytokines, NO and ROS.

According to one embodiment of the present invention, the peptide represented by SEQ ID NO: 1 of the present invention inhibits the TLR4 signaling pathway induced by LPS, thereby secreting IL-6, NO and ROS and activating NF-κB and MAPKs It is excellent in suppressing the effect, it can be useful as a composition for the prevention or treatment of autoimmune diseases and inflammatory diseases caused by the TLR4 signaling pathway.

The biological complexity of the organism is expressed by the size of the protein-protein interaction (PPI) interaction (Stumpf, MP). et al ., Proc . Natl . Acad . Sci . USA 105:6959-6964, 2008). Approximately, half of the medicinal proteins (total ~ 3000-5000) are expected to be associated with disease states (Hambly, K et. al ., Mol . Divers . 10:273-281, 2006). The role of PPI in understanding biological processes is essential, and the characteristic of'druggability' is crucial for finding a strong regulator of deregulate PPI. In addition, it is a difficult problem to inhibit the PPI interface with small molecules.

Thus, peptide-based therapeutics can overcome the effects of proteins on PPI treatment due to protein properties and similar binding methods. The development of peptides or peptide-like drugs is a key way to extend the'druggable genome', targeting PPI interfaces that are less suitable for small molecule based therapies. Peptide drugs have lower toxicity and improved target selectivity compared to small molecules. Despite the promising effect as a therapeutic agent, the development of peptide-based drugs has low stability to host proteolytic enzymes, thus low activity in vivo and low bioavailability.

In another aspect, the present invention relates to a composition for preventing or treating autoimmune diseases comprising the peptide or the fusion peptide.

In the present invention, the term, "autoimmune disease" is caused by a problem in inducing or continuing self-tolerance, resulting in an immune response to the self-antigen, thereby attacking his or her tissue. Means disease. The self-tolerance refers to immunological unresponsiveness that does not adversely react to the antigenic substances constituting self. The autoimmune diseases of the present invention include psoriasis, rheumatoid arthritis, psoriatic arthritis, experimental autoimmune arthritis, asthma, Crohn's disease, multiple sclerosis, experimental autoimmune encephalomyelitis, myasthenia gravis, thyroiditis, experimental form of uveitis, Hashimoto's thyroiditis, primary myxedema, Thyroid poisoning, malignant anemia, autoimmune atrophic gastritis, Addison's disease, early menopause, male infertility, childhood diabetes, Goodpasture's syndrome, normal celestial ulcer, erythematosus, sympathetic ophthalmitis, capsular uveitis, autoimmune hemolytic anemia, idiopathic leukocyte reduction , Primary cholangiosclerosis, chronic active hepatitis, latent cirrhosis, ulcerative colitis, Sjogren's syndrome, scleroderma, Wegener's granulomatosis, polymyositis, dermatomyositis, discoid lupus and systemic lupus erythematosus, It is not limited to this.

The composition for preventing or treating an autoimmune disease according to the present invention may contain the pharmaceutically effective amount of the peptide or fusion peptide alone, or may include one or more pharmaceutically acceptable carriers, excipients or diluents. The pharmaceutically effective amount in the above refers to an amount sufficient to prevent, ameliorate and treat the symptoms of an autoimmune disease.

The term "pharmaceutically acceptable" means that when physiologically acceptable and administered to humans, it does not usually cause an allergic reaction such as gastrointestinal disorders, dizziness, or similar reactions. Examples of the carrier, excipients and diluents include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, Polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. In addition, fillers, anti-coagulants, lubricants, wetting agents, fragrances, emulsifiers and preservatives may be further included.

In addition, the pharmaceutical composition of the present invention may include one or more known active ingredients having an autoimmune disease treatment effect together with the peptide or fusion peptide.

The pharmaceutical composition of the present invention can be formulated using methods known in the art to provide rapid, sustained or delayed release of the active ingredient after administration to a mammal other than humans. Formulations may be in the form of powders, granules, tablets, emulsions, syrups, aerosols, soft or hard gelatin capsules, sterile injectable solutions, sterile powders.

The pharmaceutical composition of the present invention may be administered through various routes including oral, transdermal, subcutaneous, intravenous, or intramuscular, and the dosage of the active ingredient may include various factors such as the route of administration, the patient's age, gender, weight, and the severity of the patient. It may be appropriately selected according to the composition for preventing or treating autoimmune diseases according to the present invention may be administered in combination with a known compound having an effect of preventing, improving or treating symptoms of an autoimmune disease.

In another aspect, the present invention relates to a composition for preventing or treating inflammatory diseases comprising the peptide or the fusion peptide.

In the present invention, the term, "inflammatory disease" is TNF-α, IL-1, IL-6, prostaglandin secreted from immune cells such as macrophages by excessively enhancing the immune system due to harmful stimuli such as inflammatory factors or irradiation. (prostaglandin), leukotriene (leukotriene) or refers to a disease caused by an inflammatory substance (inflammatory cytokine) such as NO. Inflammatory diseases of the present invention include insulin-dependent diabetes mellitus, eczema, allergy, atopic dermatitis, acne, atopic rhinitis, pneumonia, allergic dermatitis, chronic sinusitis, contact dermatitis, seborrheic dermatitis, Choose from the group consisting of gastritis, gout, gouty arthritis, ulcer, chronic bronchitis, ulcerative colitis, ankylosing spondylitis, sepsis, vasculitis, bursitis, temporal arteritis, solid cancer, Alzheimer's disease, arteriosclerosis, obesity and viral infections It is characterized by being, but is not limited to this.

Since the composition for preventing or treating the inflammatory disease includes the pharmaceutical agent containing the above-described peptide as an active ingredient, the description overlapped with the above-described composition of the present invention is omitted.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention, it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as limited by these examples.

Example One: TIRAP -origin TLR - Inhibitory Peptide Screening and cell permeability Peptides Combined fusion Peptide Produce

Protein-protein interfaces are widely used to interfere with specific signaling pathways by manipulating the interface. We chose a short peptide fragment of TIRAP that is believed to disrupt myddosome assembly by interfering with TIR-TIR interaction. Specifically, two β-sheet structures, βA and βB, were selected from the TIRAP core region through a structural bioinformatics approach (FIG. 1A ). The designed peptides were named TIP3 (TLR-inhibitory peptide 3) (DYDVCVCH; SEQ ID NO: 1) and TIP2 (TIPLLS; SEQ ID NO: 2), for intracellular translocation and efficient delivery to target sites, Drosophila antennapedia homeodomain (Derossi D, Joliot AH, Chassaing G, & Prochiantz A (1994) The Journal of biological Cell penetrating peptide (CPP) (RQIKIWFQNRRMKWKK; SEQ ID NO: 3) derived from chemistry 269(14):10444-10450) was fused to the N-terminus of TIP3 and TIP2.

TIP3 and TIP2 did not show significant cytotoxicity in the range of 12.5 to 50 μM, while they showed some cytotoxicity at high concentrations of 100 μM. The concentration-dependent survival data confirmed that TIP3 did not affect cell viability at a concentration of 50 μM or less, and thus was assumed to be safe within the above range (FIG. 1B ).

In order to study the inhibitory effect of the selected peptide, NF-κB activity was measured by analyzing secreted embryonic alkaline phosphatase (SEAP) activity based on HEK-Blue ^TM hTLR4 cells stimulated by LPS. As a result, it was confirmed that unlike TIP2, TIP3 can interfere with LPS-induced SEAP activity in a concentration-dependent manner (FIG. 1C).

Example 2: Sample preparation

Example 2-1: Reagent and cell line optimization

A rat macrophage in a hypoglycemic Dulbecco's modified Eagle's medium (DMEM) (Thermo Fisher Scientific, Inc., Waltham, MA, USA) containing 1% penicillin/streptomycin solution and 10% fetal bovine serum (FBS) RAW264.7 cells (Korean Cell Line Bank, Seoul, Korea) were cultured. HEK-Blue ^TM hTLR4 cells (InvivoGen, San Diego, CA, USA) were cultured in high sugar DMEM containing 1% penicillin/streptomycin solution, 10% FBS and 0.2% normocin. Human peripheral blood monocyte cells hPBMCs and human monocyte cells hMNCs were purchased from Lonza Inc. (Allendale, NJ, USA), 2.05 mM L-glutamine, 1% penicillin/streptomycin solution and 10% FBS It was cultured in RPMI 1640 containing. Human monocyte cells THP-1 cells (ATCC, Manassas, VA, USA) were cultured in RPMI 1640 medium containing 1% penicillin/streptomycin solution and 10% FBS, and 80 nM phorbol 12-myristate 13-acetate (PMA; Sigma-Aldrich, St. Louis, MO, USA) was used to induce differentiation into macrophages for 24 hours. All cells were cultured in a 5% CO ₂ , 37° C. humid conditions culture system (Thermo Fisher Scientific, Inc.) and the medium was changed every 18 hours. PAM ₃ CSK ₄ , poly(I:C), R848 and CpG-ODN from Thermo Fisher Scientific, Inc., FSL-1 from InvivoGen, LPS ( Escherichia coli 0111:B4) was purchased from Sigma-Aldrich. All peptides used in the experiment were synthesized from Peptron, Inc. (Daejeon, Korea) and BioStem (Ansan, Korea).

Example 2-2: mBMDM ( mouse bone marrow derived macrophages ) Culture optimization

6-7 weeks old C57BL/6J mice (RAONBIO, Seoul, Korea) grown in aseptic conditions for 6-7 weeks were used for cell extraction. Femurs and tibias were collected from mice euthanized under aseptic conditions. The end of the bone was separated and a bone marrow cavity was flowed into DMEM medium using a sterile 26-gauge needle. Suspended cells were cultured in complete medium (DMEM, 10% FBS, 100 U/ml penicillin and 100 μg/ml streptomycin; Thermo Fisher Scientific, Inc.) at 37° C. and 5% CO ₂ for 1 hour. Thereafter, bone marrow cells were cultured with macrophage colony stimulating factor (M-CSF, 25 ng/ml; R&D Systems Inc., Minneapolis, MN, USA) and obtained after 3-4 days. mBMDMs were isolated with trypsin-EDTA and plated in 60 mm dishes. TIP3-related experiments were performed after 24 hours.

Example 3: Analysis method

Example 3-1: Cell viability ( Cell viability ) analysis

Cell viability was measured using colorimetric 1-(4,5-dimethylthiazol-2-yl)-3,5-diphenylformazan (MTT) analysis (Sigma-Aldrich), which was performed as previously described (Kwon HK , et al . (2015) Toxicological sciences: an official journal of the Society of Toxicology 148(1):204-219). HEK-Blue ^TM hTLR4 cells were dispensed at a density of 5Х10 ⁴ /well, and RAW264.7 and THP-1 cells were dispensed at 2Х10 ⁵ /well. All cells were cultured overnight in 96-well plates (BD Biosciences, San Jose, CA, USA).

Example 3-2: SEAP ( secreted embryonic alkaline phosphatase ) Activity analysis

HEK-Blue ^TM hTLR4 cells were dispensed at a density of 2 x 10 ⁵ /well and cultured overnight in 24-well plates (BD Biosciences). Cells were treated with different concentrations of TIP3 in the presence and/or absence of LPS. A portion of the supernatant (200 μl) was transferred from the treated cells to a microcentrifuge tube and heated at 65° C. for 10 minutes using a heating block (FINEPCR Co., Seoul, Korea). Then, the supernatant was transferred to new 96-well plates (BD Biosciences), and SEAP production was detected using a HEK-Blue ^TM detection kit (InvivoGen). Absorbance was measured at 620 nm using a microplate reader spectrophotometer system (Molecular Devices Inc., Silicon Valley, CA, USA).

Example 3-3: Western Blot ( western blot ) analysis

Pre-protein extraction was performed using M-PER mammalian protein extraction reagent (Thermo Fisher Scientific, Inc.). The protein concentration was measured with a bicinchoninic acid (BCA) assay kit (Sigma-Aldrich). Western blot analysis including gel electrophoresis and development was performed using Mini-PROTEAN Tetra Cell and mini trans-blot electrophoretic transfer cell system (Bio-Rad Laboratories, Hercules, CA, USA) (Kwon HK, et. al . (2015) Toxicological sciences: an official journal of the Society of Toxicology 148(1):204-219). Membranes were immunoblotted by gently shaking overnight with the primary antibody at a temperature of 4° C. (above primary p-p65, p-JNK, JNK, p-IRF3, p-ERK, ERK, p-p38, p38 and Iκ). -Bα (Cell Signaling Technology Inc., Danvers, MA, USA); p-ERK, p-p38, JNK, ATF3, COX2 and β-actin (Santa Cruz Biotechnology Inc., Dallas, TX, USA); and iNOS ( BD Biosciences)). Subsequently, the membrane was thoroughly washed with phosphate buffered saline with Tween 20 (PBST) and incubated for 2 hours with peroxidase-binding anti-mouse or anti-rabbit IgG antibody (1:1000). Proteins were detected with SuperSignal West Pico ECL solution (Thermo Fisher Scientific, Inc.) and proteins detected with ChemiDoc ^TM Touch Imaging System (Bio-Rad Laboratories) were visualized.

Example 3-4: Confocal microscope( Confocal microscopy ) analysis

RAW264.7 cells were dispensed to 24-well plates (BD Biosciences) containing coverslips at a density of 2 x 10 ⁵ /well and grown overnight. Cells were treated with TIP3 (50 μM) for 1 hour prior to LPS stimulation (100 ng/ml). Cells were then fixed with 3.7% formaldehyde (Sigma-Aldrich Co. LLC) and immersed with 0.2% Triton X-100 solution (AMRESCO, Solon, OH, USA) for 15 minutes. Cells were then washed with PBS and blocked using 2% BSA solution (Thermo Fisher Scientific, Inc.). Then, these cells were incubated with an anti-p-p65 antibody (1:1000; Santa Cruz Biotechnology Inc.) for 2 hours, and washed thoroughly with PBS. Next, the cells were incubated with Alexa Fluor 546 secondary antibody (Invitrogen, Carlsbad, CA, USA) for 1 hour, and washed 3 times with PBS. Nuclei were stained using Hoechst 33258 solution (5 μM; Sigma-Aldrich). Fluorescence intensity was measured with a confocal microscope (LSM-700; Carl Zeiss Microscopy GmbH, Munich, Germany), and images were analyzed using Zen 2009 software.

Example 3-5: TNF -α, IL -6, IFN -α and IFN -β detection analysis

RAW264.7 cells, hMNCs, mBMDMs and THP-1- derived from the macrophage at a density of 2Х10 ⁵ / well in 96-well plates (BD Biosciences) , or 5Х10 in 24-well plates (BD Biosciences) density of ⁵ / well And then grew overnight. 24 hours after treatment, IFN-β secretion was measured using a LEGEND MAX ^TM Mouse IFN-β pre-coated ELISA Kit (BioLegend, San Diego, CA, USA), and Mouse IL-6 ELISA MAX ^TM Deluxe (BioLegend) or IL-6 secretion was assessed using Mouse IL-6 Platinum ELISA (eBioscience, San Diego, CA, USA), and Mouse TNF alpha ELISA Ready-SET-Go!® kit (eBioscience, San Diego, CA, USA) was used. TNF-α production was evaluated. hMNC was dispensed into 96-well plates (BD Biosciences) at a density of 2 x 10 ⁵ /well and grown overnight. 24 hours after TIP3 treatment, secretion levels of IL-6 and TNF-α were evaluated using Human IL-6 and TNF alpha ELISA MAX ^TM Deluxe (BioLegend) kits, respectively. Plates were then analyzed at each wavelength using a microplate spectrophotometer system (Molecular Devices).

Example 3-6: intracellular NO , ROS Evaluation and NO Secretion analysis

RAW264.7 cells were dispensed into 1 cm 10 ⁶ in a ⁶ cm culture dish (SPL Life Sciences, Pochun, Korea) and cultured overnight. After LPS stimulation and TIP3 treatment, intracellular NO (Nitric Oxide) and reactive oxygen species (ROS) were quantified using DAF-FM and DCF-DA dyes (Thermo Fisher Scientific, Inc.) (Kwon HK, Lee JH, Shin HJ, Kim JH, & Choi S (2015) Scientific reports 5:15623). The fluorescence intensity of the cells was analyzed using a FACSAria III instrument with Diva software (BD Biosciences).

To evaluate extracellular NO production, RAW264.7 cells and mBMDMs were dispensed in 96-well plates (BD Biosciences) at a density of 2 x 10 ⁵ /well and cultured overnight. The secretion of NO was measured with a Nitric Oxide Detection Kit (iNtRON Biotechnology Inc., Seongnam, Korea). Absorbance was measured at 550 nm using a microplate spectrophotometer system (Molecular Devices).

Example 3-7: Quantitative RT- PCR ( quantitative reverse transcription polymerase chain reaction ) analysis

RAW264.7 cells and THP-1 cells were treated with TIP3 (50 μM) for 1 hour before LPS stimulation (100 ng/ml) for 3 or 4 hours. Total RNA was isolated from cells using TRIzol ^® Reagent (Invitrogen). Total RNA concentration was measured using a Micro UV-Vis fluorescence spectrophotometer (e-spect, Malcom, Japan), iScript ^TM cDNA was synthesized using the cDNA Synthesis Kit (Bio-Rad Inc.). Light Cycler ^® 480 SYBER Green I Master kit (Roche Life-science Inc.,) and pre-designed Il - 1β , Il-6, Il- 8, Tnf- α, Inf- β, Cxcl- 10, β- actin and Quantitative RT-PCR was performed with Gapdh primers (Bionics, Seoul, Korea) (Table 2). Using Real-Time PCR detection system (Qiagen , Hilden, Germany) with SYBR Green method was measured gene expression levels, and analysis data to ΔΔ _t C method.

Primer sequence Target gene Primer Sequence(5'->3 ') Sequence number Interleukin 1 beta IL-1β(F) GAAAGCTCTCCACCTCAATG 5 IL-1β(R) GCCGTCTTTCATTACACAGG 6 Interleukin 6 IL-6(F) CCAGAGATACAAAGAAATGATGG 7 IL-6(R) ACTCCAGAAGACCAGAGGAAA 8 Chemokine (C-X-C motif) ligand 10 CXCL-10(F) GCCGTCATTTTCTGCCTCAT 9 CXCL-10(R) GCTTCCCTATGGCCCTCATT 10 Interferon beta IFN-β(F) CGTGGGAGATGTCCTCAACT 11 IFN-β(R) AGATCTCTGCTCGGACCACC 12 Tumor necrosis factor alpha TNF-α(F) CACCACCATCAAGGACTCAA 13 TNF-α(R) AGGCAACCTGACCACTCTCC 14 Beta actin β-actin(F) GTTACAGGAAGTCCCTCACCC 15 β-actin(R) CAGACCTGGGCCATTCAGAAA 16 Glyceraldehyde-3-phosphate dehydrogenase GAPDH(F) CATGTAGGCCATGAGGTCCACCAC 17 GAPDH(R) TGAAGCAGGCATCTGAGGG 18

Example 3-8: Histological analysis ( histological analysis ) And immunochemical staining (immunochemical staining )

The skin sample obtained from the dorsal lesion of the mouse was fixed in a 4% paraformaldehyde solution, embedded in paraffin, and then sliced to a thickness of 7 μm on a glass slide. Sections were stained with H&E to confirm the thickness of the epidermis and dermis. Skin thickness was measured using a Leica DMi8 fluorescence microscope (Leica microsystems gmbh, Wetzlar, Germany) using a Leica LAS X Hardware Configurator. IMQ-mediated inflammation in the skin was assessed using a mouse specific HRC/DAB Detection IHC Kit (Abcam, cat. # AB64259) using a primary antibody that recognizes CD68.

Example 4: TIP3 MyD88 -Dependent and MyD88 -Independent TLR4 And TLR3 Signal transmission suppression

Of the TLRs, TLR4 exceptionally activates downstream signaling networks simultaneously in a time-dependent manner via MyD88 and TRIF adapter proteins (Schett G, Dayer JM, & Manger B (2016) Nature reviews . Rheumatology 12(1):14-24). Thus, the inhibitory effect of TIP3 on both signaling of the TLR4 pathway in LPS-stimulated murine RAW264.7 cells was investigated.

TIP3 did not show a toxic effect on RAW264.7 cells at 50 μM (FIG. 2A ), confirming the HEK-Blue ^TM hTLR4 survival rate results mentioned above. TPS3 inhibits Iκ-Bα degradation and inhibits phosphorylation (p-p65) of subunit p-65 of NF-κB and phosphorylation of IRF3 (p-IRF3), thereby causing LPS-induced MyD88-dependent and independent signaling pathways Was confirmed through Western blot data (FIG. 2B ). Furthermore, TIP3 inhibits phosphorylation of extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and p38, thereby expressing LPS-induced activating transcription factor 3 (ATF3) and mitogen-activated MAPKs. protein kinases) (Fig. 2B and Fig. 2C). It was confirmed by confocal microscopy analysis that TIP3 reduced the migration of LPS-induced p-p65 to the nucleus (FIG. 2D). TIP3 is a proinflammatory cytokine (TNF-α and IL-6) and IFN-α, IFN-β in In vitro production was prevented in a concentration-dependent manner (FIGS. 2E-2H ).

Stimulation of TLR4 by ligands similar to TLR4 leads to nitric oxide (NO) production in RAW264.7 cells, as well as induction of cyclooxygenase 2 (COX2) and inducible nitric oxide synthase (iNOS) (Kim JY , et. al . (2008) European journal of pharmacology 584(1):175-184). In addition, stimulation of primary macrophages by LPS has been reported to cause mitochondria and intracellular ROS (reactive oxygen species) production induction (West AP , et. al . (2011) Nature 472 (7344):476-480). TIP3 inhibited LPS-mediated extracellular secretion of NO and expression of iNOS and COX2 in a concentration-dependent manner (FIGS. 2I and 2J). Intracellular production of NO and ROS was significantly inhibited (Figures 2K and 2L). These results indicate that TIP3 inhibits LPS-induced MyD88-dependent and MyD88-independent TLR4 signaling in RAW264.7 cells.

To confirm the effect of TIP3 on other TLRs, other TLR ligands (PAM ₃ CSK ₄ (TLR1/2), FSL-1 (TLR2/6), poly(I:C)(TLR3), resiquimod (TLR7/8) ) Or CpG-ODN (TLR9)), followed by measuring the level of TNF-α secretion in RAW264.7 cells. TIP3 somewhat inhibited TNF-α secretion mediated by TLR1/2, TLR2/6, TLR7, TLR8 and TLR9 at 50 μM, and TLR3-mediated TNF-α secretion was significantly decreased in a concentration-dependent manner (FIG. 2M And Figure 2N). Similarly, poly(I:C)-induced secretion of IL-6, IFN-α and IFN-β in RAW264.7 cells was also inhibited (FIGS. 2O and 2P ). QRT-PCR showed that TIP3 significantly inhibits mRNA expression of tnf -α, il -6, ifn -β, cxcl -10 and i l-1β genes in rat macrophages, the same as the inhibition of protein expression levels. It was confirmed through (Fig. 2Q to 2U).

Example 5: primary cell line ( primary cell lines )in TIP3 TLR4 -Inhibition and TLR3 -Check the inhibitory effect

Similar experiments were performed on THP-1 derived macrophages to further evaluate the inhibitory effect of TIP3 observed in RAW264.7 cells. In the absence of any cytotoxicity (FIG. 3A ), the effect of the peptides on LPS-induced activation of NF-κB and MAPK was evaluated by Western blot analysis. As a result, it was confirmed that TIP3 successfully interferes with the activation of NF-κB, ATF3 and phosphorylation of IRF3 in human peripheral blood mononuclear cells (hPBMCs) (FIG. 3B ). TIP3 also inhibited phosphorylation of MAPK, including ERK, JNK and p38 (Figure 3C). Furthermore, TIP3 showed a significant concentration-dependent inhibitory effect on TNF-α and IL-6 secretion in THP-1 cells (FIGS. 3D and 3E ). Infectious cytokines (IL-6, TNF-α, IL-1β) and chemokines (IL-8) in THP-1 cells when TIP3 (50 μM) was treated for 1 hour and LPS was treated simultaneously for 4 hours. MRNA expression was observed. As a result, it was confirmed that TIP3 successfully inhibited mRNA expression of these cytokines and IL-8 induced by LPS (FIGS. 3F to 3I ).

Similarly, the inhibitory effect of TIP3 on primary mouse bone marrow-derived macrophages (mBMDMs) was evaluated. Cells were pretreated with TIP3 for 1 hour followed by LPS treatment for 24 hours. Like RAW264.7 cells, TIP3 inhibited secretion of TLR4-mediated IL-6, TNF-α and secretion of TLR3-mediated IFN-β in mBMDMs (Figures 3J to 3L). In addition, a significant reduction in the extracellular production of NO was observed (Figure 3M). When tested in human mononuclear cells (hMNC), TIP3 showed similar effects, and substantially reproducible results were observed in terms of cell viability (FIG. 3N) and cytokine secretion. TIP3 significantly inhibited TLR4-mediated IL-6 secretion and TLR4-mediated and TLR3-mediated TNF-α secretion (FIGS. 3O-3Q ).

Example 6: Arthritis ( arthritis )for TIP3 Confirmation of treatment effect

Example 6-1: CIA ( collagen - induced arthritis ; Collagen-induced arthritis) model preparation and TIP3 process

All animal care and experimental procedures were performed in accordance with the NIH Laboratory Animal Use and Care Guidelines and approved by the Kyung Hee University Animal Care and Use Committee (Permit No.: KHUASP(SE)-15-115). Male DBA/1J mice weighing 20-23 g (6-7 weeks old) were purchased from Central Lab Animal Inc. (Seoul, Korea). The mice were placed in a rodent facility with limited access at 22-24°C, and up to four animals per polycarbonate cage were allowed to freely consume food and water from pellets in a light/dark cycle for 12 hours. CIA was derived according to the previously described protocol (Xin W , et. al . (2014) British journal of pharmacology 171(14):3526-3538). Mouse type 100μg chicken II collagen (CII, Sigma-Aldrich) and an equal amount of complete Freund's adjuvant solution (Sigma-Aldrich) were immunized at the base of the tail; This time point was designated as day 0. Next day 20, mice were injected with a booster (secondary) of the mixture.

All mice were randomly subdivided into 6 experimental groups, each containing 7 (n=7). Each group was marked as follows: (1) Normal group not treated (Normal); (2) Vehicle-treated arthritis group (CIA); (3) 2.5 nmol/g TIP3-treated group (designated CIA-TIP3-2.5), (4) 10 nmol/g TIP3-treated group (designated CIA-TIP3-10) and (5) PAP TIP3-10 group ( TIP3-treated arthritis group such as 10nmol/g TIP3-injected mice at PAP)); (6) methotrexate (MTX)-treated arthritis group (designated MTX (2 mg/kg)). TIP3 or methotrexate dissolved in physiological saline was injected intraperitoneally (i.p.) once a day from day 22.

Body weight, foot volume and arthritis index were measured to assess arthritis progression in CIA mice. Mice were weighed using a digital balance (Mettler-Toledo Inc., Columbus, OH, USA). The hindpaw joint pain was assessed by squeaking (mouse squeaking) scores during the forced flexion and extension of the ankle joint 10 times every 5 seconds. A score of 0 (no speech) or 1 (speech) was given to each hind paw during all flexion and extension procedures. The total number of utterances observed was calculated as the squeaking score. Foot volume is as previously described (Bang JS , et. al . (2009) Arthritis research & therapy 11(2):R49), measured by volume change of electrolyte solution in a water displacement volumetric flow meter (Ugo-Basil Biological Research Apparatus Co., Comerio-Varese, Italy). Foot volume was expressed as a value relative to day 0, defined as 1.0 (100%). The arthritis index was assessed by rating the apparent arthritis severity of all joints of the limbs per mouse on a 4-point scale: 0 = no erythema or swelling in any joint of one leg, 1 = erythema or swelling in at least one joint per leg. Yes, 2 = erythema or swelling in less than 3 joints per leg, 3 = erythema or swelling in all joints on one leg, 4 = ankyloses and deformity on all joints on one leg. The maximum score for each mouse was 16. Behavioral testing was performed twice a week for each animal.

For imaging via micro-CT analysis, bone samples were fixed with 10% formalin solution. In In vivo micro-CT (NFR Polaris-G90; NanoFocus Ray Co., JeonJu, Korea) was used to scan bone samples from the knee joint. In addition, it was set as follows: X-ray voltage of 55 kV, X-ray current of 105 μA, X-ray spot size of 8 μm, and exposure time of 80 ms for each 180° rotational steps. Reconstruction of the 3D image of the knee joint and the 2D image of the trabecular bones for the sagittal section above the tibia and the cortical bones for the horizontal section in the middle of the tibia is supported by Data viewer, CTVox and CTAn software packages ( SkyScan, Kontich, Belgium) at 500 slices. The bone density of each reconstructed knee joint was measured in 1 mm increments from the top of the tibia after setting the region of interest.

Example 6-2: CIA In the model TIP3 Confirmation of treatment effect

Many researchers are involved in TLR4 in the inflammatory response associated with rheumatoid arthritis (RA) and osteoarthritis, and hope to establish a new treatment strategy accordingly (Roelofs MF , et. al . (2009) Annals of the rheumatic diseases 68(9):1486-1493).

In this example, the CIA mouse model was used to investigate the anti-inflammatory effect of TIP3 in chronic inflammatory diseases such as RA. The TIP3 treatment plan is summarized in Figure 4A. To evaluate the relative therapeutic effect of TIP3 on RA, several parameters were monitored during treatment, including body weight, foot volume and arthritis index. According to images showing swelling of the knees, ankles, and feet, arthritis symptoms were evident on all limbs between day 22 and day 35. In contrast, induction of RA in TIP3-injected mice was significantly attenuated compared to CIA mice (FIGS. 4B and 5 ). During the period before arthritis, the weight of CIA mice decreased (mean 18.2 g), while the weight of normal (control) mice increased (mean 21.9 g). No weight loss was observed in TIP3 (10 nmol/g)-injected and methotrexate (MTX)-injected mice (average 20.5 g and 21.0 g, respectively; FIG. 4C). The relative paw volume was measured in normal, CIA and TIP3-injected mice. Compared to normal mice, the volume of the untreated CIA group paws was large, but the TIP3 (10 nmol/g)-injected and MTX injected mice did not (Fig. 4D). Arthritis index was also increased in CIA mice, but significantly decreased when treated with TIP3 (10 nmol/g) or MTX (Fig. 4E).

In the post-arthritis phase (PAP) TIP3 (10 nmol/g)-treated mice (starting treatment from day 35), the development of arthritis, expressed in foot volume and arthritis index, was significantly inhibited when compared to CIA mice. However, no significant change in body weight was observed. To assess the extent of bone erosion reversal, in vivo During micro-computed tomography (Micro-CT) scanning, 3D (3D) images of the knee joints of normal, CIA and TIP3 (10 nmol/g)-injected mice and 2D of trabecular bones and cortical bones The image was confirmed. Compared to normal mice, severe corrosion was observed in the bones and cartilage of the knee joint in the CIA mice, and bone microstructure loss, cortical thickness reduction, and bone density reduction were observed (FIGS. 4F to 4H). On the other hand, in TIP3-treated mice, bone loss was significantly recovered, and the inflammation score was significantly reduced (FIGS. 4F to 4J and FIGS. 6 to 8 ). In histological examination of joint tissue sections stained with hematoxylin and eosin (H&E), infiltration of immune cells in CIA mice, thicker synovial tissues, synovial hyperplasia, pannus formation and joint gap reduction It appeared clearly. This phenomenon was significantly reversed in mice treated with 10 nmol/g TIP3, and the histopathological aberrations were effectively prevented in a concentration-dependent manner (FIG. 4K). The histological observations were consistent with the data obtained from the micro-CT images.

Example 7: Psoriasis About TIP3 Confirmation of treatment effect

Example 7-1: psoriasis Mouse model and TIP3 process

TIP3 in vivo It was intended to confirm the therapeutic effect. 6-7 week old C57BL/6 mice were purchased from Orient Bio Inc. (Seongnam, Korea). Mice were stored under conditions free of specific pathogens and optionally provided with a standard experimental diet (STD). All animal experiments were approved by the Ajou University Animal Experiment and Use Committee (IACUC) (approval number 2017-0002). 62.5 mg Aldara cream (5%) with IMQ (Aldara; 3M, Maplewood, MN, USA) was used for 5 consecutive days to produce symptoms similar to psoriasis. IMQ was not applied to the control group. One day before application of Aldara cream (5%) to mice, TIP3 (10 or 50 nmol/g) or PBS (control) was administered. As a positive control, MTX (10 μg/g; cat. # M9929, Sigma-Aldrich) was also administered one day before application of the IMQ cream. After 5 days, mice were euthanized by respiratory anesthesia, and skin lesions, spleen and serum samples were collected for analysis. To evaluate the degree of inflammation of the back skin, an objective scoring system was developed based on the clinical PSI (Psoriasis Area and Severity Index). Erythema, scaling, wrinkles and thickening were scored independently from 0 to 4 as follows: 1, slightly; 2, medium; 3, significant; 4, very remarkable. Erythema, scaling and thickness were scored under close supervision of skilled researchers. Cumulative scores (erythema + scaling + thickening, grades 0-12) indicate the severity of psoriasis symptoms.

Example 7-2: psoriasis In mouse model TIP3 Significant treatment effect confirmation

Psoriasis is a chronic inflammatory skin disease characterized by amplified proliferation of epidermal cells, altered differentiation and inflammatory cell penetration (Schon MP, Boehncke WH, & Brocker EB (2005) Discovery medicine 5(27):253-258). It has been found that TLRs are involved in the development and progression of psoriasis (Baker BS, Ovigne JM, Powles AV, Corcoran S, & Fry L (2003) The British journal of dermatology 148(4):670-679). Therefore, the present inventors attempted to confirm possible inhibition of TIP3-mediated TLR and fundamental relief of symptoms in the psoriasis model.

Psoriasis was induced by topical application of 62.5 mg of imiquimod (IMQ) cream (5%) to the skin for 5 days in a row, shaved in 6-7 week old C57BL/6 mice (FIG. 9A). Inflammation reversal was clearly seen on the back skin of TIP3-treated mice (FIG. 9B ). Mice treated with 10 nmol/g TIP3 showed a relatively weak effect than the MTX-treated group. In addition, according to the PASI results, TIP3 did not significantly reduce the PASI score starting from day 2 (FIG. 9C), but significantly prevented the splenomegaly in a concentration-dependent manner (FIG. 9D). Treatment with a concentration of 50 nmol/g significantly reduces the spleen weight (compared to the normal rat spleen), showing a much better effect. The 50 nmol/g-treated group showed weight loss compared to the MTX-treated and TIP3 (10 nmol/g)-treated groups (FIG. 9E). Furthermore, the histopathological changes and thickness of skin in each group were evaluated by H&E staining. As a result, it was confirmed that treatment with 50 nmol/g and 10 nmol/g TIP3 significantly reduced the thickness of the epidermis (yellow arrow) and dermis (green arrow) in mice with IMQ-induced psoriasis-like disease (FIG. 9F). To Figure 9H).

The hyperproliferative condition of the skin and immune cell penetration (T cells or macrophages) are considered to be the main features of psoriasis. Therefore, the effect of TIP3 on skin penetration by macrophages was evaluated by immunohistochemical analysis of each group of back skin lesions by primary antibodies recognizing CD68 (macrophage markers). As a result, it was confirmed that TIP3 effectively reduced overexpression of CD68 (brown staining) in mice with IMQ-induced psoriasis-like disease, which was also found in the MTX-treated group (FIG. 9I ).

Example 8: Lupus ( lupus )for TIP3 Confirmation of treatment effect

Example 8-1: lupus model and TIP3 process

Three female wild-type (C57BL/6) and lupus-prone mice (MRL/lpr), initially weighing 18-20 and 38-40 g, respectively, were purchased from Jackson Laboratory (Bar Harbor, ME, USA). All animal experiments were reviewed and approved by the AAC University Medical Center IACUC (approval number 2017-0022). Mice were acclimatized for 1 week and then bred under pathogen-free conditions according to the approved guidelines of Ajou University School of Medicine. The mice in the vehicle group were injected with intraperitoneal (ip) 1% dimethyl sulfoxide, while others were injected with 10 nmol/g (dissolved in 1% dimethyl sulfoxide) TIP3 per day for 20 days, and their weight was monitored daily. . Mice were euthanized at the end of the experiment and blood, urine and tissues were collected. After incubation for 1 hour, blood samples were collected in a serum separation tube, centrifuged at 3000 rpm for 10 minutes at 20°C, and then stored at -80°C. Collected urine samples were immediately stored at -80°C, while tissue samples were thoroughly washed with PBS and immersed in RNA stabilization solution (Qiagen Sciences, Maryland, MD, USA). The concentrations of anti-dsDNA antibody and C3 complement were analyzed by ELISA. In addition, mouse urine albumin levels were analyzed using a Mouse Albumin ELISA Kit (41-ALBMS-E01, Alpco Diagnostics, Salem, NH, USA). Three independent measurements were performed per sample.

Example 8-2: Systemic lupus erythematosus ( systemic lupus erythematosus ; SLE ) In mouse model TIP3 Confirmation of treatment effect

The therapeutic efficacy of TIP3 for systemic autoimmunity was evaluated in a SLE mouse model. MRL/lpr mutant mice were injected intraperitoneally with 10 nmol/g TIP3 daily for 20 days (FIG. 10A ). Compared to vehicle-treated mice, TIP3-treated mice significantly improved the condition of the mice by significantly reducing lymphatic expansion and spleen volume (Figures 10B and 10C). Inflammation of the kidneys can lead to a specific type of kidney pathology known as glomerulonephritis. Glomerulonephritis is due to SLE, and its severity can be assessed through the detection of certain circulating proteins, such as albumin in urine. As shown in Figure 10D, albumin in urine is reduced by TIP3 treatment, indicating a therapeutic effect on kidney inflammation. In addition, the production of anti-double-stranded DNA (anti-double-stranded DNA (dsDNA) autoantibodies) remained low after TIP3 treatment (Figure 10E). The relatively low levels of complement components in serum, especially C4 and C3, are common indicators of SLE. When compared to the control mutant mice, it was confirmed that the C3 level was recovered in the serum of TIP3-treated mice (FIG. 10F ).

Example 9: TIP3 3D model building and molecular dynamics ( molecular dynamics ; MD ) simulation

Example 9-1: TIP3 Building 3D models

To facilitate membrane insertion and migration, TIP3 was conjugated with a cell-penetrating peptide (CPP-TIP3). The 3D model of CPP-TIP3 was created based on the predicted secondary structure (PSIPRED) in the Discovery Studio (DS) Visualizer (Dassault Systemses BIOVIA, San Diego, CA, USA) (Jones DT (1999) Journal of molecular biology 292(2):195-202). Peptide energy minimization and molecular dynamics (MD) simulations were performed through a dipalmitoyl phosphatidylcholine bilayer to model a rational side chain three-dimensional structure. The peptide was located 10 위 above the bilayer surface, and MD simulation was performed in GROMACS 5.0.7 (Van Der Spoel D , et. al . (2005) Journal of computational chemistry 26(16):1701-1718).

Example 9-2: MD Simulation parameters and protocol

MD simulation of TIP3 modeling was performed on a pre-equilibrated dipalmitoyl phosphatidylcholine bilayer composed of 288 phospholipids. A modified force field with GROMOS96 54A7 and Berger lipid parameters was selected. Orientation and placement of peptides on the membrane was performed in VMD 1.9.1 (Humphrey W, Dalke A, & Schulten K (1996) Journal of molecular graphics 14(1):33-38, 27-38). The counter ion (Na ⁺ / Cl ^-) charge point of a single water molecule and physiological concentrations (0.15M) was added to the charge neutralization. In order to remove the three-dimensional collision between atoms, a rapid reduction of the down energy was performed. MD simulation was first performed using NVT ensemble, then NPT ensemble was performed for 100 ps. Production was performed for 100 ns without resistance to backbone heavy atoms for NPU ensemble. Short-range (Lennard Jones) interaction was calculated with a 12 Å cutoff distance through the Verlet scheme, and long-range (Coulomb) interaction was calculated by the particle mesh Ewald algorithm. Periodic boundary conditions were applied to the simulation system. Temperature and pressure couplings were performed with Nose-Hoover (300K) and Parrinello-Rahman algorithm (1 bar), respectively. The LINCS algorithm was applied to the constraints on all bonds involving hydrogen atoms, and structural snapshots were stored every 2ps.

Example 10: TLR4 TIR For domain TIP3 Confirmation

Example 10-1: Protein-protein docking ( protein - protein docking )

The interaction of TIP3 and TLR4 TIR domains was calculated through protein-protein docking in the HADDOCK 2.2 web server (van Zundert GCP , et. al . (2016) Journal of molecular biology 428(4):720-725). The final snapshot of the TIP3 MD trajectory was docked in the TIR region of the recently reported TLR4 model (Patra MC, Kwon HK, Batool M, & Choi S (2018) Frontiers in immunology 9:489). For detailed structural analysis after energy minimization, the complex docked with the highest score in the largest cluster was selected.

Example 10-2: protein - protein docking Through simulation TLR4 TIR For domain TIP3 Confirmation

A representative low-energy TIP3 model was docked to the TIR domain of TLR4 to confirm intermolecular interaction at the atomic level (FIG. 11A). Considering that the BB loop of the TIR domain of TLR is an adapter recruitment site, a binding site was predicted. That is, it was expected to bind mainly to the BB loop residue. TIP3 fits perfectly into the BB loop pocket due to the complementary surface structure (Fig. 11B). Furthermore, it was confirmed that amino acid residues K15 and D19 of TIP3 form two hydrogen bonds with R689 and A720 (BB loop) of the TIR domain (FIG. 11C). It was confirmed that residues V22 and H24 of TIP3 are involved in hydrophobic interaction with V716 and I713 of the BB loop in the TIR domain. Because of the proximity between C23 of TIP3 and C664 of the TIR domain, it can be assumed that there is an intermolecular disulfide bond. These docking results indicate that TIP3 has a strong binding affinity for both the BB loop portion and the TIR domain.

Since specific parts of the present invention have been described in detail above, it will be apparent to those skilled in the art that this specific technique is only a preferred embodiment, and the scope of the present invention is not limited thereby. will be. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

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Claims (12)

A peptide represented by the amino acid sequence of SEQ ID NO: 1.
According to claim 1, The peptide is TLR4 (Toll-like receptor 4), TLR3, TLR2/1, TLR2/6, TLR7/8 and TLR9 selected from the group consisting of any one or more of the TLR (Toll-like receptor) A peptide characterized by inhibiting a signaling pathway.
3. The peptide according to claim 2, wherein the TLR4 signaling pathway is induced by lipopolysaccharide (LPS).
The peptide according to claim 1, wherein the peptide binds to a TIR (Toll/interleukin-1 receptor) domain of TLR4.
A fusion peptide in which a cell penetrating peptide is bound to the peptide of claim 1.
According to claim 5, wherein the cell permeable peptide is a fusion peptide, characterized in that represented by the amino acid sequence of SEQ ID NO: 3.
The fusion peptide of claim 5, wherein the fusion peptide is represented by the amino acid sequence of SEQ ID NO: 4.
A Toll-like receptor 4 (TLR4) antagonist comprising the peptide of claim 1 or the fusion peptide of claim 5.
A composition for the prevention or treatment of an autoimmune disease comprising the peptide of claim 1 or the fusion peptide of claim 5.
The method of claim 9, wherein the autoimmune disease is psoriasis, rheumatoid arthritis, psoriatic arthritis, experimental autoimmune arthritis, asthma, Crohn's disease, multiple sclerosis, experimental autoimmune encephalomyelitis, myasthenia gravis, thyroiditis, experimental forms of uveitis, Hashimoto's thyroiditis, Primary myxedema, thyroid poisoning, malignant anemia, autoimmune atrophy gastritis, Addison's disease, early menopause, male infertility, childhood diabetes, Goodpasture's syndrome, normal pemphigus, pylori pneumoniae, sympathetic ophthalmitis, lens uveitis, autoimmune hemolytic anemia , Idiopathic leukocyte reduction, primary cholangiosclerosis, chronic active hepatitis, latent cirrhosis, ulcerative colitis, Sjogren's syndrome, scleroderma, Wegener's granulomatosis, polymyositis, dermatomyositis, discoid lupus and systemic lupus erythematosus A composition for the prevention or treatment of autoimmune diseases.
A composition for preventing or treating an inflammatory disease comprising the peptide of claim 1 or the fusion peptide of claim 5.
The method of claim 11, wherein the inflammatory disease is insulin-dependent diabetes, eczema, allergy, atopic dermatitis, acne, atopic rhinitis, pulmonary inflammation, allergic dermatitis, chronic sinusitis, contact dermatitis, seborrheic dermatitis ( with seborrheic dermatitis, gastritis, gout, gouty arthritis, ulcers, chronic bronchitis, ulcerative colitis, ankylosing spondylitis, sepsis, vasculitis, bursitis, temporal arteritis, solid cancer, Alzheimer's disease, arteriosclerosis, obesity and viral infections A composition for the prevention or treatment of inflammatory diseases, which is selected from the group consisting of.

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