WO2020141704A1 - Peptide inhibiteur de la signalisation tlr4 et utilisation associée - Google Patents

Peptide inhibiteur de la signalisation tlr4 et utilisation associée Download PDF

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WO2020141704A1
WO2020141704A1 PCT/KR2019/014195 KR2019014195W WO2020141704A1 WO 2020141704 A1 WO2020141704 A1 WO 2020141704A1 KR 2019014195 W KR2019014195 W KR 2019014195W WO 2020141704 A1 WO2020141704 A1 WO 2020141704A1
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peptide
tip3
tlr4
composition
disease
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PCT/KR2019/014195
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Korean (ko)
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최상돈
아첵아스마
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주식회사 젠센
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/10—Fusion polypeptide containing a localisation/targetting motif containing a tag for extracellular membrane crossing, e.g. TAT or VP22

Definitions

  • 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- ⁇ B and MAPKs
  • TLR4 Toll-like receptor 4
  • 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.
  • TLRs Toll-like receptors
  • PAMPs exogenous pathogen-associated molecular patterns
  • IRFs interferon-regulatory factors
  • Myeloid differentiation primary response 88 is the first adapter recruited to activated TLRs other than TLR3, and TIR domain-containing adapter-inducing interferon ⁇ (TRIF) through intermolecular TIR (Toll/interleukin-1 receptor) domain interaction ) (Gay NJ, Symmons MF, Gangloff M, & Bryant CE (2014) Nature reviews.
  • TLR4 interleukin receptor-associated kinase 4
  • LPS lipopolysaccharide
  • TLR2 and TLR4 cell surface-expressing TLRs
  • TIRAP TIR domain adapter protein
  • TIRAP 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) .
  • 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).
  • the present inventors have tried diligently to develop a new peptide capable of inhibiting the TLR4 signaling pathway.
  • a peptide combining a peptide derived from ⁇ -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.
  • 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 present invention also provides a method for preventing or treating an autoimmune disease comprising the step of administering the peptide or the fusion peptide to an individual in need thereof.
  • the present invention also provides the use of the composition for the prevention or treatment of autoimmune diseases.
  • the present invention also provides the use of the composition for preparing a composition for the prevention or treatment of autoimmune diseases.
  • the present invention also provides a method of preventing or treating inflammatory diseases, including administering the peptide or the fusion peptide to an individual in need thereof.
  • the present invention also provides the use of the composition for the prevention or treatment of inflammatory diseases.
  • the present invention also provides the use of the composition for preparing a composition for the prevention or treatment of inflammatory diseases.
  • 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
  • TIP TLR-inhibiting peptides
  • 1B is a cell viability analysis result showing that 100 ⁇ M of TIP3 (TLR-inhibitory peptide 3) and TIP2 slightly affect cell proliferation.
  • 1C is a graph showing substantial inhibition (measured by SEAP analysis) of NF- ⁇ B 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).
  • FIG. 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 ⁇ M TIP3 is safe in subsequent experiments against RAW264.7 cells and other cell lines.
  • 2B and 2C show Western blot analysis of the protein expression levels of p-p65, I ⁇ -B ⁇ , p-IRF3, ATF3, p-ERK, ERK, p-JNK, JNK, p-p38 and p38 in pre-protein extracts. As measured by, ⁇ -actin was used as a loading control.
  • 2D shows the phosphorylation of NF- ⁇ B (p-p65) by immunofluorescence staining and confocal microscopy, and Hoechst was used for nuclear staining (scale bar indicates 20 ⁇ m).
  • 2E-2H show the secretion levels of TNF- ⁇ , IL-6, IFN- ⁇ and IFN- ⁇ by ELISA, and the secretion of four cytokines was inhibited concentration-dependently by TIP3.
  • 2 I is a graph showing NO secretion levels evaluated using a standard NO secretion kit.
  • 2J is a graph showing the expression levels of iNOS and COX2 measured by Western blotting, and ⁇ -actin was used as a loading control.
  • 2K and 2L are graphs showing that intracellular NO and ROS were quantified by DAF-FM and DCF-DA staining, respectively, and substantially down-regulated by 50 ⁇ M TIP3.
  • TLR2/1 shows TNF- ⁇ levels when cells were activated with PAM 3 CSK 4 (TLR2/1), FSL-1 (TLR2/6), R848 (TLR7/8) or CpG-ODN (TLR9) at various concentrations.
  • TLR2/6 shows TNF- ⁇ levels when cells were activated with PAM 3 CSK 4 (TLR2/1), FSL-1 (TLR2/6), R848 (TLR7/8) or CpG-ODN (TLR9) at various concentrations.
  • TLR9 shows TNF- ⁇ levels when cells were activated with PAM 3 CSK 4 (TLR2/1), FSL-1 (TLR2/6), R848 (TLR7/8) or CpG-ODN (TLR9) at various concentrations.
  • TLR9 shows TNF- ⁇ levels when cells were activated with PAM 3 CSK 4 (TLR2/1), FSL-1 (TLR2/6), R848 (TLR7/8) or CpG-ODN (TLR9) at various concentrations.
  • TNF- ⁇ (FIG. 2N), IL-6 (FIG. 2O) and IFN- ⁇ (FIG. 2P) after activation by poly(I:C)(TLR3) for 24 hours in RAW264.7 cells.
  • Figure 2Q to Figure 2U is a graph showing the effect of TIP3 for TIP3 treatment after 3 hours, tnf- ⁇ , il-6, ifn- ⁇ , LPS- induced mRNA expression level of cxcl-10 and il-1 ⁇ . Values are expressed as fold changes (relative quantification) at the mRNA level, normalized to ⁇ -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).
  • FIG. 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 ⁇ M) for 24 hours.
  • 3B and 3C show the protein expression measured by Western blot analysis using hPBMC, p-p65, I ⁇ -B ⁇ , p-IRF3, ATF3, p-ERK, ERK, p-JNK in the pre-protein extract. , JNK, p-p38 and p38 were evaluated and ⁇ -actin was used as a loading control.
  • 3D and 3E are graphs showing the secretion levels of TNF- ⁇ 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- ⁇ , il-8 and il-1 ⁇ after 4 hours of TIP3 treatment, mRNA levels of these genes was substantially down-regulated by TIP3, values were expressed as fold changes (relative quantification) at the mRNA level and normalized to gapdh .
  • 3J to 3M show the secretion of IL-6 (FIG. 3J), TNF- ⁇ (FIG. 3K), and IFN- ⁇ (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 result of analyzing the cell viability of hMNCs treated with TIP3.
  • 3O and 3P are graphs showing the secretion levels of IL-6 (FIG. 3O) and TNF- ⁇ (FIG. 3P) evaluated by ELISA using hMNCs stimulated by LPS.
  • 3Q is a graph showing the TLR3-inhibitory effect of TIP3 evaluated by measuring TNF- ⁇ 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.
  • FIG. 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 left and right arrows indicate the two viewpoints of the TIP3 treatment mentioned in FIG. 4A above.
  • 4F is a 3D image of the knee joint taken by Micro-CT to predict joint corrosion and cartilage loss.
  • Figure 4G is a 2D image of the spongy bone corresponding to the sagittal section of the upper tibia taken using Micro-CT.
  • 4H is a 2D image of the cortical bone corresponding to the horizontal cross-section of the tibial middle part.
  • 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 ⁇ m 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.
  • 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
  • FIG. 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)
  • IMQ imiquimod
  • TIP3 was administered intraperitoneally prior to IMQ application
  • MTX Methotrexate
  • 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 clinical psoriasis area and severity index (PASI).
  • FIG. 9D is a graph showing the effect of TIP3 on spleen weight
  • 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 (top arrow) and dermis (bottom arrow).
  • 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 ⁇ m; High magnification image 75 ⁇ m 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.
  • 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.
  • FIG. 10D is a graph showing albumin content in urine
  • FIG. 10E is an anti-dsDNA antibody
  • FIG. 10F is a C3 complement level in serum measured by ELISA.
  • a Wilcoxon Rank Sum test (numerically equivalent to the Mann-Whitney U test) was performed.
  • FIG. 11 shows the intermolecular interaction between the TIR domain of TIP3 and TLR4,
  • 11A shows the overall docking model of TIP3 (red cartoon) along with the entire TLR4 model.
  • 11B shows the surface of TIP3 bound to the TIR domain of TLR4.
  • FIG. 11C details the interaction of TIP3 residues with the TLR4 TIR domain.
  • TLR Toll-like receptors
  • TIP3 TLR-inhibitory peptide 3
  • TIRAP TIR domain adaptor protein
  • TIP3 TLR-inhibitory peptide 3
  • ⁇ A first ⁇ -sheet
  • TIRAP TLR-inhibitory peptide 3
  • the TLR4-inhibiting effect was confirmed by evaluating LPS-induced NF- ⁇ B activation in HEK-Blue TM 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.
  • TNF- ⁇ 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- ⁇ secretion dependent on TLR was substantially inhibited by TIP3 (FIGS. 2E, 2M and 2N).
  • TIP3 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.
  • 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.
  • 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 ).
  • 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.
  • TIP3 chronic inflammatory skin disease
  • MRL/lpr mouse model of SLE systemic autoimmune disease
  • the peptides according to the present invention have protective effects against both diseases in these models.
  • 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.
  • 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 also suggested that belongs to one of the found structural folds of the most evolutionarily conserved families of that. 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.
  • 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).
  • TIP3 can ameliorate their symptoms by stopping the progression of RA, psoriasis and SLE and reducing the inflammatory response mediated by TLR signaling.
  • the present invention in one aspect, relates to a peptide represented by the amino acid sequence of SEQ ID NO: 1.
  • 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.
  • TLR Toll-like receptor
  • the TLR4 signaling pathway may be characterized by being induced by lipopolysaccharide (LPS).
  • LPS lipopolysaccharide
  • the peptide may be characterized by binding to the TLR4's TIR (Toll/interleukin-1 receptor) domain.
  • 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.
  • 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.
  • PAMPs pathogen-associated molecular patterns
  • 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.
  • TIRAP also referred to as Mal
  • 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- ⁇ B and secretion of pro-inflammatory cytokines such as TNF- ⁇ and IL-6, and initiation of the MyD88-independent pathway activates IRF3 and 7, IFNs Induces secretion and late-stage activation of NF- ⁇ B (Park, BS et al., Exp. Mol. Med. 45:e66, 2013; Nijland, R et al., Mar. Drugs 12:4260-4273, 2014) .
  • TLR4 causes activation of MAPKs 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).
  • 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.
  • inhibition of the expression of TNF- ⁇ , IL-6, IFN- ⁇ or IFN- ⁇ by blocking the TLR4 signaling pathway of the peptide; Inhibition of NO or ROS secretion;
  • the activity of NF- ⁇ B or MAPK may be inhibited, and the peptide may be characterized by inhibiting both MyD88-dependent and MyD88-independent TLR4 signaling pathways.
  • the present invention relates to a fusion peptide in which a cell penetrating peptide is bound to the peptide.
  • 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.
  • the expression of TNF- ⁇ , IL-6, IFN- ⁇ or IFN- ⁇ is inhibited by blocking the TLR4 signaling pathway of the fusion peptide; Inhibition of NO or ROS secretion;
  • the activity of NF- ⁇ B or MAPK may be inhibited, and the fusion peptide may be characterized by inhibiting both MyD88-dependent and MyD88-independent TLR4 signaling pathways.
  • cell penetrating peptide (cell penetrating peptide; CPP) is a kind of signal peptide (signal peptide), a combination of a specific amino acid sequence used for the purpose of delivering a polymer material such as protein, DNA, RNA into the cell It is a phosphorus peptide. To date, it has been used for intracellular delivery of various low-molecular-weight compounds, proteins, peptides, RNA, DNA, and other polymer materials.
  • 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.
  • 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).
  • an experiment was performed by selecting the Penetratin sequence (RQIKIWFQNRRMKWKK; SEQ ID NO: 3) among the cell permeable peptides in Table 1, 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.
  • the fusion peptide may be characterized in that the cell-permeable peptide is bound to the N-terminal or C-terminal of the peptide.
  • the cell-permeable peptide may be bound to the N-terminus of the peptide, but is not limited thereto.
  • 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.
  • 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.
  • the present invention relates to a TLR4 antagonist comprising the peptide or the fusion peptide.
  • 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.
  • 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- ⁇ B and MAPKs, thereby reducing the secretion of inflammatory cytokines, NO and ROS.
  • 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- ⁇ B 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.
  • PPI protein-protein interaction
  • 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.
  • the development of peptide-based drugs has low stability to host proteolytic enzymes, thus low activity in vivo and low bioavailability.
  • the present invention relates to a composition for preventing or treating autoimmune diseases comprising the peptide or the fusion peptide.
  • 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.
  • 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, polymyos
  • composition for preventing or treating an autoimmune disease 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.
  • pharmaceutically acceptable means that when physiologically acceptable and administered to a human, it does not usually cause an allergic reaction such as gastrointestinal disorders, dizziness or similar reactions.
  • 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.
  • fillers, anti-coagulants, lubricants, wetting agents, fragrances, emulsifiers and preservatives may be further included.
  • 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.
  • compositions 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.
  • the present invention relates to a composition for preventing or treating inflammatory diseases comprising the peptide or the fusion peptide.
  • inflammatory disease is TNF- ⁇ , 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 prostaglandin
  • leukotriene leukotriene
  • inflammatory cytokine inflammatory cytokine
  • 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.
  • 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.
  • the present invention relates to a method for preventing or treating an autoimmune disease, comprising administering the peptide or the fusion peptide to an individual in need thereof.
  • the present invention relates to the use of the composition for the prevention or treatment of autoimmune diseases.
  • the present invention also provides the use of the composition for preparing a composition for the prevention or treatment of autoimmune diseases.
  • the present invention relates to a method of preventing or treating inflammatory diseases, including administering the peptide or the fusion peptide to an individual in need thereof.
  • the present invention relates to the use of the composition for the prevention or treatment of inflammatory diseases.
  • the present invention relates to the use of the composition for preparing a composition for the prevention or treatment of inflammatory diseases.
  • Example 1 Selection of TIRAP-derived TLR-inhibiting peptides and preparation of fusion peptides combining cell permeable peptides
  • Protein-protein interfaces are widely used to interfere with specific signaling pathways by manipulating the interface.
  • TIP3 TLR-inhibitory peptide 3
  • TIP2 TIPLLS; SEQ ID NO: 2
  • CPP Drosophila antennapedia cell penetrating peptide
  • TIP3 and TIP2 did not show significant cytotoxicity in the range of 12.5 to 50 ⁇ M, while they showed some cytotoxicity at high concentrations of 100 ⁇ M.
  • concentration-dependent survival data confirmed that TIP3 did not affect cell viability at a concentration of 50 ⁇ M or less, and thus was assumed to be safe within the above range (FIG. 1B ).
  • NF- ⁇ B activity was measured by analyzing secreted embryonic alkaline phosphatase (SEAP) activity based on HEK-Blue TM hTLR4 cells stimulated by LPS.
  • SEAP embryonic alkaline phosphatase
  • DMEM Dulbecco's modified Eagle's medium
  • FBS fetal bovine serum
  • HEK-Blue TM hTLR4 cells InvivoGen, San Diego, CA, USA
  • Human peripheral blood monocyte cells hPBMCs and human monocyte cells hMNCs were purchased from Lonza Inc.
  • RPMI 1640 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.
  • PMA phorbol 12-myristate 13-acetate
  • PAM 3 CSK 4 poly(I:C), R848 and CpG-ODN were purchased from Thermo Fisher Scientific, Inc., FSL-1 from InvivoGen, LPS ( Escherichia coli 0111:B4) from Sigma-Aldrich. All peptides used in the experiment were synthesized from Peptron, Inc. (Daejeon, Korea) and BioStem (Ansan, Korea).
  • Example 2-2 mouse bone marrow derived macrophages (mBMDM) culture optimization
  • 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.
  • M-CSF macrophage colony stimulating factor
  • mBMDMs were isolated with trypsin-EDTA and plated in 60 mm dishes. TIP3-related experiments were performed after 24 hours.
  • HEK-Blue TM hTLR4 cells were dispensed at a density of 5 ⁇ 10 4 /well, and RAW264.7 and THP-1 cells were dispensed at 2 ⁇ 10 5 /well. All cells were cultured overnight in 96-well plates (BD Biosciences, San Jose, CA, USA).
  • Example 3-2 Analysis of secreted embryonic alkaline phosphatase (SEAP) activity
  • 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 ⁇ l) 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 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.
  • 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 ⁇ M) 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.).
  • Example 3-5 TNF- ⁇ , IL-6, IFN- ⁇ and IFN- ⁇ detection assay
  • IFN- ⁇ secretion was measured using a LEGEND MAX TM Mouse IFN- ⁇ pre-coated ELISA Kit (BioLegend, San Diego, CA, USA), and Mouse IL-6 ELISA MAX TM Deluxe (BioLegend) or IL-6 secretion was evaluated using Mouse IL-6 Platinum ELISA (eBioscience, San Diego, CA, USA), and Mouse TNF alpha ELISA Ready-SET-Go! TNF- ⁇ production was evaluated using a kit (eBioscience, San Diego, CA, USA).
  • hMNC was dispensed into 96-well plates (BD Biosciences) at a density of 2 x 10 5 /well and grown overnight.
  • 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).
  • 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 3-8 Histological analysis and 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 ⁇ m 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.
  • TLR4 exceptionally activates the downstream signaling network 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).
  • TRIF adapter proteins Schott G, Dayer JM, & Manger B (2016) Nature reviews.Rheumatology 12(1):14 -24.
  • TIP3 did not show a toxic effect on RAW264.7 cells at 50 ⁇ M (FIG. 2A ), confirming the HEK-Blue TM hTLR4 survival rate results mentioned above.
  • TPS3 inhibits I ⁇ -B ⁇ degradation and inhibits phosphorylation (p-p65) of subunit p-65 of NF- ⁇ B and phosphorylation of IRF3 (p-IRF3), thereby causing LPS-induced MyD88-dependent and independent signaling pathways was confirmed through Western blot data (FIG. 2B ).
  • 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 prevented the in vitro production of pro-inflammatory cytokines (TNF- ⁇ and IL-6) and IFN- ⁇ , IFN- ⁇ concentration-dependently (FIGS. 2E-2H ).
  • TLR4 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).
  • COX2 cyclooxygenase 2
  • iNOS inducible nitric oxide synthase
  • 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.
  • TLR3 TLR ligands
  • PAM 3 CSK 4 TLR1/2
  • FSL-1 TLR2/6
  • poly(I:C)(TLR3) TLR7/8
  • TLR9 CpG-ODN
  • TIP3 is the same as the suppression of the protein expression level, in the macrophages of the rat ⁇ tnf-, il-6, ⁇ ifn-, cxcl-10 and l i-1 to that significantly inhibited the mRNA expression of ⁇ genes qRT-PCR It was confirmed through (Fig. 2Q to 2U).
  • TIP3 showed a significant concentration-dependent inhibitory effect on TNF- ⁇ and IL-6 secretion in THP-1 cells (FIGS. 3D and 3E ). Infectious cytokines (IL-6, TNF- ⁇ , IL-1 ⁇ ) and chemokines (IL-8) in THP-1 cells when TIP3 (50 ⁇ M) 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 ).
  • TIP3 primary mouse bone marrow-derived macrophages
  • mBMDMs primary mouse bone marrow-derived macrophages
  • Example 6-1 CIA (collagen-induced arthritis; collagen-induced arthritis) model preparation and TIP3 treatment
  • mice 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 protocol previously described (Xin W , et al. (2014) British journal of pharmacology 171(14):3526-3538).
  • mice 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.
  • CII chicken II collagen
  • Sigma-Aldrich complete Freund's adjuvant solution
  • 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 was measured as the volume change of the electrolyte solution in a water displacement volumetric flowmeter (Ugo-Basil Biological), as previously described (Bang JS , et al.
  • Bone specimens were scanned from the knee joint using an in vivo Micro-CT (NFR Polaris-G90; NanoFocus Ray Co., JeonJu, Korea). In addition, it was set as follows: X-ray voltage of 55 kV, X-ray current of 105 ⁇ A, X-ray spot size of 8 ⁇ m, and exposure time of 80 ms for each 180° rotational steps.
  • 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.
  • body weight body weight
  • foot volume arthritis index
  • arthritis symptoms were evident on all limbs between day 22 and day 35.
  • induction of RA in TIP3-injected mice was significantly attenuated compared to CIA mice (FIGS. 4B and 5 ).
  • the weight of CIA mice decreased (mean 18.2 g), while the weight of normal (control) mice increased (mean 21.9 g).
  • Example 7-1 Psoriasis mouse model and TIP3 treatment
  • MTX 10 ⁇ g/g; cat. # M9929, Sigma-Aldrich
  • mice were euthanized by respiratory anesthesia, and skin lesions, spleen and serum samples were collected for analysis.
  • 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 Confirmation of significant treatment effect of TIP3 in the psoriasis mouse model
  • 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.
  • 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).
  • the histopathological changes and thickness of skin in each group were evaluated by H&E staining.
  • 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).
  • FIG. 9H To Figure 9H).
  • T cells or macrophages 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 the 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 8-1 Lupus model and TIP3 treatment
  • mice Three female wild type (C57BL/6) and lupus-prone mice (MRL/lpr) with initial weights of 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. .
  • ip intraperitoneal
  • TIP3 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 Confirmation of the therapeutic effect of TIP3 in a systemic lupus erythematosus (SLE) mouse model
  • TIP3 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.
  • Example 9 Construction of 3D model of TIP3 and simulation of molecular dynamics (MD)
  • TIP3 was conjugated with a cell-penetrating peptide (CPP-TIP3).
  • CPP-TIP3 The 3D model of CPP-TIP3 was produced 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 ⁇ 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 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 ⁇ 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.
  • TIP3 and TLR4 TIR domains were 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 (2016) Frontiers in immunology 9:489).
  • the complex docked with the highest score in the largest cluster was selected.
  • Example 10-2 Confirmation of binding of TIP3 to TIR domain of TLR4 through protein-protein docking simulation
  • a representative low-energy TIP3 model was docked to the TIR domain of TLR4 to confirm intermolecular interaction at the atomic level (FIG. 11A).
  • BB loop of the TIR domain of the 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).
  • amino acid residues K15 and D19 of TIP3 form two hydrogen bonds with R689 and A720 (BB loop) of the TIR domain (FIG. 11C).

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Abstract

La présente invention concerne un peptide permettant d'inhiber la signalisation du récepteur de type Toll 4 (TLR4) et, plus particulièrement, un peptide qui se lie au domaine TIR de TLR4 pour supprimer la sécrétion de cytokines pré-inflammatoires et inhiber l'activation de NF-κB et de MAPK, un peptide de fusion dans lequel le peptide est fusionné à un peptide de pénétration cellulaire, un antagoniste de TLR4 comprenant le peptide ou le peptide de fusion, et une composition comprenant le peptide ou le peptide de fusion pour prévenir ou traiter une maladie auto-immune ou une maladie inflammatoire. Le peptide selon la présente invention a un effet inhibiteur sur la signalisation de TLR4 et de TLR3, bloque la voie de signalisation de TLR4 dépendante de MyD88 et TRIF dans plusieurs lignées cellulaires humaines et de souris, et a des effets importants de soulagement de maladie dans la polyarthrite rhumatoïde, le psoriasis et des modèles de souris de lupus érythémateux disséminé. Ainsi, le peptide peut être utilisé en tant qu'agent thérapeutique pour des maladies liées au système immunitaire nécessitant un contrôle négatif du TLR pour sa thérapie.
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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20030023993A1 (en) * 2001-05-09 2003-01-30 Ruslan Medzhitov Toll/interleukin-1 receptor adapter protein (TIRAP)
US20130203649A1 (en) * 2012-02-07 2013-08-08 University Of Maryland, Baltimore Inhibitors of tlr signaling by targeting tir domain interfaces
US20140045759A1 (en) * 2010-11-04 2014-02-13 The Board Of Regents Of The University Of Oklahoma Peptide compositions that downregulate tlr-4 signaling pathway and methods of producing and using same
KR20160140031A (ko) * 2015-05-29 2016-12-07 아주대학교산학협력단 신규한 tlr4 길항제
KR20180122940A (ko) * 2017-05-04 2018-11-14 주식회사 젠센 톨-유사 수용체(tlr) 억제를 위한 펩타이드 및 이를 포함하는 약학적 조성물

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20030023993A1 (en) * 2001-05-09 2003-01-30 Ruslan Medzhitov Toll/interleukin-1 receptor adapter protein (TIRAP)
US20140045759A1 (en) * 2010-11-04 2014-02-13 The Board Of Regents Of The University Of Oklahoma Peptide compositions that downregulate tlr-4 signaling pathway and methods of producing and using same
US20130203649A1 (en) * 2012-02-07 2013-08-08 University Of Maryland, Baltimore Inhibitors of tlr signaling by targeting tir domain interfaces
KR20160140031A (ko) * 2015-05-29 2016-12-07 아주대학교산학협력단 신규한 tlr4 길항제
KR20180122940A (ko) * 2017-05-04 2018-11-14 주식회사 젠센 톨-유사 수용체(tlr) 억제를 위한 펩타이드 및 이를 포함하는 약학적 조성물

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