WO2018203613A1 - Peptide for toll-like receptor (tlr) inhibition and pharmaceutical composition comprising same - Google Patents

Peptide for toll-like receptor (tlr) inhibition and pharmaceutical composition comprising same Download PDF

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Publication number
WO2018203613A1
WO2018203613A1 PCT/KR2018/004803 KR2018004803W WO2018203613A1 WO 2018203613 A1 WO2018203613 A1 WO 2018203613A1 KR 2018004803 W KR2018004803 W KR 2018004803W WO 2018203613 A1 WO2018203613 A1 WO 2018203613A1
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Prior art keywords
peptide
tip1
seq
disease
diseases
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PCT/KR2018/004803
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French (fr)
Korean (ko)
Inventor
최상돈
권혁권
신현준
계향애
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아주대학교산학협력단
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Priority claimed from KR1020180046715A external-priority patent/KR102024186B1/en
Application filed by 아주대학교산학협력단 filed Critical 아주대학교산학협력단
Priority to US16/610,278 priority Critical patent/US11352399B2/en
Priority to JP2020512344A priority patent/JP6906692B2/en
Priority to CN201880045288.0A priority patent/CN111094321B/en
Priority to EP18793867.5A priority patent/EP3647321A4/en
Publication of WO2018203613A1 publication Critical patent/WO2018203613A1/en

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    • 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
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof

Definitions

  • the present invention provides a fusion peptide that inhibits TLR1 / 2, TLR2 / 6, TLR7, TLR8 and TLR9 signaling pathways, including TLR4 (Toll-like receptor 4) and TLR3, and TLR pathway mediation including the same. It relates to a pharmaceutical composition for preventing or treating a disease.
  • Pattern recognition receptors such as toll-like receptors, are known as pathogen-associated molecular patterns (PAMPs) or risk-associated molecular patterns (danger). activated by recognizing an associated molecular pattern (DAMP).
  • PAMPs pathogen-associated molecular patterns
  • DAMP risk-associated molecular patterns
  • TLRs play an important role in the innate immune response, and cells such as endosomes, including extracellular TLRs and TLR3, TLR7, TLR8 and TLR9, which act on plasma membranes including TLR1, TLR2, TLR4, TLR5, TLR6 and TLR11. It can be divided into intracellular TLRs that act in vivo. Structurally, the TLR has a leucine-rich repeat (LRR) site that is recognized by a ligand or an accessory molecule at the N-terminus of the extracellular domain and signals at the C-terminus of the intracellular portion. It has a Toll / interleukin 1 receptor (TIR) domain to deliver.
  • LRR leucine-rich repeat
  • TIR Toll / interleukin 1 receptor
  • TLR4 is the first identified receptor in the TLR family, activating innate immune signals that are amplified through the MyDoid (myeloid differentiation 88) -dependent signaling pathway and the MyD88-independent signaling pathway.
  • TLR3 activates only the MyD88-independent signaling pathway. Due to the role of TLRs, interest in researches for using TLRs as a target for treating various immune diseases is increasing.
  • MyD88-dependent signaling of TLR4 is initiated by LPS recognition through accessory molecules such as cluster of differentiation 14 (CD14) and myeloid differentiation factor 2 (MD2). After LPS binding, TLR4 forms a dimer and the TIR domain of TLR4 and the TIR domain of TIR domain adapter protein (TIRAP or MyD88 adapter-like (MAL)) bind to each other and form a complex with MyD88. Thereby activating the signaling pathway. Activated TLR4 induces initial activation of NF- ⁇ B, migration into the nucleus, and activation of MAPK through Myd88-dependent signaling.
  • CD14 cluster of differentiation 14
  • MD2 myeloid differentiation factor 2
  • TLR4 and TLR3 The MyD88-independent signaling process of TLR4 and TLR3 is initiated by the binding between the TIR domain of each TLR and the TIR domain of the TRAM domain-containing adapter-inducing interferon- ⁇ (TRIF) -related adapter molecule (TRAM) complex. And secrete type 1 interferon by activation of an interferon-regulatory factor (IRF).
  • IRF interferon-regulatory factor
  • TLRs may be targets for treating various diseases such as autoimmune diseases, inflammatory diseases and cancer
  • various diseases such as autoimmune diseases, inflammatory diseases and cancer
  • researches on substances targeting TLRs and medical compositions for treating diseases related to TLRs have been made.
  • TLR4 promoters and antagonists have been obtained, eritoran, lipid A and Rhodobacter.
  • RsLA sphaeroids lipid A
  • the TLR4 signaling pathway by LPS is closely related to degenerative neurological diseases such as Alzheimer's disease and Parkinson's disease due to the involvement of NLRP3 inflamasome formation.
  • PAMPs pathogen-associated molecular patterns
  • the inventors have described a novel peptide (decoy peptide 1) consisting of the amino acid sequence of SEQ ID NO: 1 and a fusion peptide (Toll-like receptor inhibitory peptide 1, TIP1) in which the novel peptide is linked to the N-terminus of a cell-penetrating peptide.
  • decoy peptide 1 consisting of the amino acid sequence of SEQ ID NO: 1 and a fusion peptide (Toll-like receptor inhibitory peptide 1, TIP1) in which the novel peptide is linked to the N-terminus of a cell-penetrating peptide.
  • TLR4 signaling pathways that inhibit cytokine (IL-6, TNF- ⁇ , IFN- ⁇ ), NO and ROS secretion, and activation of NF- ⁇ B and MAPK, as well as in animals It has been shown to alleviate renal and hepatic damage, sepsis and rheumatoid arthritis due to overactivity of TLR4.TLR3 signaling pathway induced by Poly (I: C) and NLRP3 inflamasome formation induced by LPS / ATP were also identified. Inhibition was demonstrated to complete the present invention. In addition, it was confirmed in cell experiments that TLR1 / 2, TLR2 / 6, TLR7, TLR8 and TLR9 were also partially inhibited.
  • an object of the present invention is to provide a fusion peptide in which a peptide consisting of the amino acid sequence of SEQ ID NO: 1 and a peptide consisting of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2 is connected to the N-terminus of the cell permeable peptide.
  • Still another object of the present invention is to mediate one or more TLR pathways selected from the group consisting of autoimmune diseases, inflammatory diseases and neurodegenerative diseases comprising a peptide consisting of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2 as an active ingredient It provides a pharmaceutical composition for the prevention or treatment of sexual diseases.
  • the present invention provides a peptide consisting of the amino acid sequence of SEQ ID NO: 1.
  • the present invention also provides a fusion peptide wherein a peptide consisting of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2 is linked to the N-terminus of the cell permeable peptide.
  • the present invention also provides for the prevention of one or more TLR pathway mediated diseases selected from the group consisting of autoimmune diseases, inflammatory diseases and neurodegenerative diseases comprising peptides consisting of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2 as an active ingredient Or it provides a pharmaceutical composition for treatment.
  • the present invention from the group consisting of autoimmune diseases, inflammatory diseases and neurodegenerative diseases comprising a fusion peptide linked to the peptide consisting of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2 at the N-terminal end of the cell permeable peptide as an active ingredient
  • autoimmune diseases inflammatory diseases
  • neurodegenerative diseases comprising a fusion peptide linked to the peptide consisting of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2 at the N-terminal end of the cell permeable peptide as an active ingredient
  • pharmaceutical compositions for the prophylaxis or treatment of one or more selected TLR pathway mediated diseases are provided.
  • the fusion peptide of the present invention is excellent in inhibiting TLR4 and various TLR pathways, thereby inhibiting the TLR mediated immune response, thereby causing various TLR pathway mediated diseases caused by the signaling pathway, such as autoimmune diseases and inflammatory diseases. Or it can be usefully used to prevent and treat degenerative neurological diseases.
  • FIG. 1 (a) shows that SEAP (secreted alkaline phosphatase) activity is decreased when TIPs (TIP1, TIP2; TIP2 is used as a control) at different concentrations in HEK-Blue TM hTLR4 (human TLR4) cells. It is a figure which shows decreasing by TIP1.
  • Figure 1 (b) is a diagram showing the results of measuring the SEAP activity of TIP1 (TIP1 W / O CPP) and TIP2 (TIP2 W / O CPP) to which the CPP sequence is not linked.
  • Figure 2 (a) is a diagram showing the results confirmed by Western blotting NF- ⁇ B (p65) and IRF3 activity when TIP1 was treated with LPS to RAW 264.7 cells, which are mouse macrophages.
  • Figure 2 (b) is a diagram showing the results confirmed by Western blotting MAPK activity under the same conditions.
  • NF- ⁇ B activity (p-p65, green) was confirmed by confocal microscopy when TIP1 was treated with LPS in RAW 264.7 cells, which are mouse macrophages.
  • Fig. shows that the nuclear transfer of p65 is blocked.
  • FIG. 4 is a diagram showing the result of confirming the cytokine secretion when treated with LPS TIP1 of different concentrations in RAW 264.7 cells, which are mouse macrophages.
  • 4 (a) shows TNF- ⁇
  • FIG. 4 (b) shows IL-6
  • FIG. 4 (c) shows IFN- ⁇ secretion.
  • FIG. 5 is a diagram showing the results of confirming the NO and ROS generation amount when TIP1 was treated with LPS in RAW 264.7 cells, which are mouse macrophages.
  • Figure 5 (a) shows the amount of NO generated in the cytoplasm
  • Figure 5 (b) shows the amount of ROS generated in the cytoplasm
  • Figure 5 (c) shows the amount of NO secreted out of the cells.
  • FIG. 6 is a diagram showing the results of confirming the cytokine and NO generation amount when TIP1 was treated with LPS in mBMDM (mouse bone marrow derived macrophage) cells, which are mouse bone marrow-derived macrophages.
  • FIG. 6A illustrates TNF- ⁇
  • FIG. 6B IL-6 FIG. 6C NO
  • FIG. 6D show IFN- ⁇ generation amount.
  • FIG. 7 shows p-ERK, ERK, p-JNK, JNK, pp38, p38, p-p65 and I ⁇ in cells when TIP1 was treated with LPS in human peripheral blood mononuclear cells (hPBMCs).
  • -B ⁇ expression is confirmed by Western blotting as a result showing that it is inhibited by TIP1.
  • FIG. 8 shows TIP1 in mouse macrophages RAW 264.7 cells, PAM 3 CSK 4 (TLR1 / 2), FSL-1 (TLR2 / 6), Poly (I: C) (TLR3), When treated with R848 (TLR7 / 8) or CpG-ODN (TLR9), it is a measure of TNF- ⁇ secretion in cells and shows that it is inhibited by TIP1.
  • FIG. 9 is a diagram showing that when TIP1 was treated with Poly (I: C) in RAW 264.7 cells, which are mouse macrophages, the secretion amount of IFN- ⁇ was confirmed by ELISA as a result of TIP1.
  • FIG. 10 (a) shows that the molecules involved in the formation of NLRP3 (NACHT, LRR and PYD domains-containing protein 3) -mediated inflammasomes induced by LPS and ATP are inhibited by TIP1. Is confirmed in THP1 cells.
  • Figure 10 (b) is a diagram showing the results of the experiment in the mBMDM cells under the same conditions.
  • Figure 11 (a) is a diagram confirming the THP1 cells that IL-1 ⁇ secretion is suppressed when TIP1 treatment in the NLRP3-mediated inflamasome state induced by treatment with LPS and ATP.
  • Figure 11 (b) is a diagram showing the results of performing the experiment in the mBMDM cells under the same conditions.
  • FIG. 12 is a diagram showing the results of confirming the NF- ⁇ B activity and the amount of NO secretion of the peptide having a full or partial amino acid sequence of TIP1.
  • 12 (a) shows SEAP activity
  • FIG. 12 (b) shows NO secretion amount, respectively.
  • FIG. 13 shows TIP1 (TIP1-FITC) or TIP1-FITC combined with fluorescein isothiocyanate (FITC) or THS1 cells, which are human monocytes, between TIP1 (green) and TLR4 (red) and MyD88 (blue). Interactions were observed by confocal laser scanning microscopy to confirm that TIP1 binds to TRL4.
  • TIP1-FITC fluorescein isothiocyanate
  • THS1 cells which are human monocytes
  • FIG. 14 is a diagram showing the results of confirming the cytokine secretion through blood collected after 1 hour or 2 hours after treatment with PBS (phosphate buffered saline) and LPS or TIP1 and LPS in C57BL / 6J mice.
  • FIG. 14A illustrates TNF- ⁇
  • FIG. 14B shows IL-12p40
  • FIG. 14C shows IL-6 secretion.
  • FIG. 15 is a diagram showing the results of confirming the cytokine secretion in liver tissue of rats when C57BL / 6J mice were treated with PBS and LPS or TIP1 and LPS.
  • Figure 15 (a) shows the result of confirming the secretion amount of TNF- ⁇ and IL-6 through Western blotting
  • Figure 15 (b) is a graph showing the quantification of the band intensity according to the Westin blotting .
  • FIG. 16 is a diagram showing the results obtained through blood collected after 24 hours the effect of TIP1 on kidney and liver damage when PBS and LPS or TIP1 and LPS were treated together in C57BL / 6J mice.
  • A) and (b) of FIG. 16 show TNF- ⁇ and IL-6 secretion, respectively.
  • C) and (d) of FIG. 16 show secretion amounts of blood urea nitrogen (BUN) and creatin (creatinine, Cr), which are renal damage markers, respectively.
  • Figure 16 (e) and (f) is a diagram showing the secretion amount of the liver damage markers AST (aspartate aminotransferase) and ALT (alanine aminotransferase).
  • FIG. 17 shows TUNEL (terminal deoxynucleotidyl transferase dUTP nick end) using renal tissue harvested after 24 hours of treatment with PBS and LPS or TIP1 and LPS in C57BL / 6J mice. As a result of the labeling staining method, it is shown that TIP1 reduces apoptosis.
  • FIG. 18 is a diagram showing that survival of LPS-induced sepsis model rats is increased when LPS or TIP1 and LPS are treated in BALB / c mice.
  • Figure 19 (a) is a diagram showing an experimental design for confirming the therapeutic effect of TIP1 on rheumatoid arthritis using a collagen-induced arthritis (collagen-induced rheumatoid arthritis) model of DAB-1J mice.
  • Figure 19 (b) is a visual observation of the therapeutic effect when treated with TIP1 is a diagram showing that rheumatoid arthritis is being treated.
  • FIG. 20 is a diagram showing the results of confirming the therapeutic effect when TIP1 treatment in the CIA rheumatoid arthritis model of DAB-1J mice.
  • Figure 20 (a) is the weight
  • Figure 20 (b) is squeaking (rat rat squeaking)
  • Figure 20 (c) is a foot swelling degree
  • Figure 20 (d) is a result of observing the arthritis index Respectively.
  • FIG. 21 shows that arthritis is being treated by measuring 3D image and bone mineral density (BMD) using Micro-CT (micro-computed tomography) after TIP1 treatment in a CIA model of DAB-1J mice. to be.
  • BMD 3D image and bone mineral density
  • Micro-CT micro-computed tomography
  • FIG. 23 is a diagram schematically showing a signaling process induced by TLR4 and a region controlled by TIP1.
  • FIG. 23 is a diagram schematically showing a signaling process induced by TLR4 and a region controlled by TIP1.
  • the present invention provides a peptide consisting of the amino acid sequence of SEQ ID NO.
  • peptide refers to a linear molecule formed by binding amino acid residues to each other by peptide bonds.
  • the peptide may be prepared according to chemical synthesis methods known in the art, and preferably may be prepared according to a solid phase synthesis technique, but is not limited thereto.
  • TLR4 refers to a protein belonging to Toll-like receptors (TLRs), a family of transmembrane proteins that function as a monitor for pathogen infection. It is also called CD284 (cluster of differentiation 284).
  • TLR4 is very important for the activation of the innate immune system because it recognizes a variety of pathogen-associated molecular patterns (PAMPs), including LPS of Gram-negative bacteria.
  • PAMPs pathogen-associated molecular patterns
  • TLR3 refers to a protein that belongs to TLRs, a family of transmembrane proteins that function as a monitor for pathogen infection, and refers to a protein encoded by the TLR3 gene, and is named CD283 (cluster of differentiation 283). Sometimes. The TLR3 is very important for activation of the innate immune system because it recognizes a variety of PAMPs, including double-strand RNA (dsRNA) of the virus.
  • dsRNA double-strand RNA
  • NLRP3 inflammasome is a receptor and sensor of the intrinsic immune system that regulates the activation of caspase-1, and is a type of inflammasome that causes inflammation in response to microbial infections. Is activated in response. NLRP3 requires priming, for example the binding of LPS to TLR4. The TLR4 signaling pathway by LPS, along with ATP, promotes NLRP3 inflammasome formation. Abnormally activated NLRP3 inflammasomes lead to the development of a variety of inflammatory diseases, particularly neurodegenerative diseases. Recent studies have shown a direct link between the accumulation of beta amyloid (amyloid ⁇ ), a major cause of Alzheimer's disease, and NLRP3 inflammasomes, and has also been linked to Parkinson's disease.
  • beta amyloid amyloid ⁇
  • TLR4 mediated signaling pathway refers to a signaling pathway through TLR4, which may be an LPS response that depends on the TLR4 / MD2 complex formed by TLR4 and MD2, through which signals are transmitted. do. TLR4 carries signals by several adapter proteins and works through Mal (also called TIRAP), MyD88, and TRAM and TRIF. Activated TLR4 activates NF- ⁇ B to the nucleus through Myd88-dependent signaling, leading to the activation of MAPK.
  • NF- ⁇ B and MAPK Due to the activation of NF- ⁇ B and MAPK, inflammatory cytokines such as TNF- ⁇ , IL-1 ⁇ and IL-6 are secreted, and nitric oxide (hereinafter referred to as NO) and free radicals (hereinafter referred to as ROS) in macrophages To produce oxidative stressors.
  • NO nitric oxide
  • ROS free radicals
  • TRAM / TRIF, interferon-regulator (IRF), and NF- ⁇ B induces MyD88-independent signaling and secretes Type 1 interferon.
  • TIR domain is a domain for intracellular signaling, which has three highly conserved regions and mediates the interaction between TLRs and other signaling molecules. Activated TIR domains induce binding of MyD88 and activate the TLR signaling pathway.
  • the term “inhibition” refers to a phenomenon in which biological activity or signaling activity is deteriorated due to deficiency, incompatibility, and many other causes, and partially, completely blocks, reduces, prevents, or activates TLR activity. May be delayed, inactivated or down regulated.
  • the peptide or fusion peptide according to the present invention provides TLR4 and TLR3 signaling pathways and NLRP3 inflamasome inhibition. It also provides partial inhibition of TLR1 / 2, TLR2 / 6, TLR7, TLR8 and TLR9.
  • the SHCR sequence (SEQ ID NO: 2) of the peptide consisting of the amino acid sequence of SEQ ID NO: 1 is characterized in that it specifically binds to the TIR (Toll / interleukin-1 receptor) domain of the TLR (Toll-like receptor) can do.
  • the peptide consisting of the amino acid sequence of SEQ ID NO: 1 has sequence specificity.
  • the present inventors searched for the amino acid minimum of TIP1 that binds to the TIR domain of TLR4 and examined whether this region could effectively inhibit the signaling pathway of TLR4. That is, according to one embodiment of the present invention, decoy peptides are selected from the TIR domain of TIRAP and SHCR (decoy peptide 1-2, SEQ ID NO: 2) and VLLI (decoy peptide 1-3, VLLI) using the amino acid sequence SHCRVLLI.
  • SEQ ID NO: 7 Binding the sequence to the N-terminus of the same CPP sequence as used in Example 1-1, respectively, to treat HEK-Blue TM -hTLR4 cells and hPBMC cells with LPS, followed by NF- ⁇ B activity and NO secretion was measured. As a result, it was confirmed that the SHCR (decoy peptide 1-2) sequence is important for producing an inhibitory effect.
  • the present invention also provides a fusion peptide wherein a peptide consisting of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2 is linked to the N-terminus of the cell permeable peptide.
  • the fusion peptide may inhibit the signaling pathway mediated by TLR (Toll-like receptor), the TLR (Toll-like receptor) is TLR1 / 2 (Toll-like receptor 1/2) , Toll-like receptor 2/6 (TLR2 / 6), toll-like receptor 3 (TLR3), toll-like receptor 4 (TLR4), toll-like receptor 7 (TLR7), toll-like receptor 8 (TLR8), and It may be any one selected from the group consisting of TLR9 (Toll-like receptor 9), preferably TLR4 (Toll-like receptor 4) or TLR3 (Toll-like receptor 3), more preferably TLR4 ( Toll-like receptor 4) can be characterized.
  • TLR9 Toll-like receptor 9
  • TLR4 Toll-like receptor 4
  • TLR3 Toll-like receptor 3
  • TLR4 Toll-like receptor 4
  • the activity of NF- ⁇ B, MAPK or NLRP3 inflamasomes may be inhibited, and the fusion peptide may be characterized by inhibiting both MyD88-dependent and MyD88-independent TLR4 signaling pathways.
  • cell penetrating peptide is a kind of signal peptide (Signal Peptide) of a kind of specific amino acid sequence used for the purpose of delivering a polymer material such as protein, DNA, RNA, etc. into the cell Peptides in combination.
  • signal peptide Signal Peptide
  • various low molecular compounds, proteins, peptides, RNA, DNA, etc. have been used for intracellular delivery of high molecular materials.
  • the fusion peptide of the present invention uses a cell-penetrating peptide, and the cell-penetrating peptide is not particularly limited as long as it has a characteristic of entering into a cell by a mechanism of cell internalization, but preferably the cell permeability listed in Table 1 below. It can be selected and used among the peptides or variants thereof.
  • Peptide origin order SEQ ID NO: Penetratin Drosophila Antennapedia homeodomain RQIKIWFQNRRMKWKK 9 TAT (48-60) Human immunodeficiency virus type 1 (HIV-1) TAT GRKKRRQRRRPPQ 10 pVEC VE-Cadherin (615-632) LLIILRRRIRKQAHAHSK 11 Transportan 10 / TP10 Galanin-lys-mastoparan GWTLNSAGYLLGKINLKALAALAKKIL 12 MPG A hydrophobic domain from the fusion sequence of HIV gp41 and NLS of SV40 T-antigen GALFLGFLGAAGSTMGAWSQPKKKRKV 19 Pep-1 NLS from Simian Virus 40 large T antigen and reverse transcriptase of HIV-1 KETWWETWWTEWSQPKKKRKV 20 MAP Amphipathic model peptide KLALKLALKALKAALKLA 21 R 6 / W 3 Based on penetratin
  • the transportan of the cell permeable peptides of Table 1 includes those used in the form of the following variants: AGYLLGKINLKALAALAKKIL-NH 2 (TP10, PepFect 3, SEQ ID NO: 13), AGYLLGKINLKALAALAKKIL-NH 2 (TP10, PepFect 6, sequence No.
  • amino acid sequence consisting of SEQ ID NO: 37 of Table 1
  • r means d -Arginine
  • the second and sixth amino acid in the amino acid sequence of SEQ ID NO: 37 corresponds to this.
  • SEQ ID NO: 41 and 42 of Table 1 constitutes one peptide (TATp-D) which is branched to each other, the 14th Lys residue (K) in the amino acid sequence of SEQ ID NO: 41 in the amino acid sequence of SEQ ID NO: 42 It is characterized in that the side chain is linked to the 13th Gln residue (Q).
  • r means d -Arginine
  • the 2nd, 4, 6, 10th amino acid in the amino acid sequence of SEQ ID NO: 43 corresponds to this.
  • k means d- Lysine, which corresponds to the eighth amino acid in the amino acid sequence of SEQ ID NO.
  • SEQ ID NO: 43 is characterized in that the cyclic peptide.
  • the experiment was performed by selecting the Penetratin sequence (RQIKIWFQNRRMKWKK, SEQ ID NO: 9) among the cell permeable peptides of Table 1, and other cell permeable peptides in addition to the actually used cell permeable peptides were fused with the peptides of the present invention.
  • the Penetratin sequence RQIKIWFQNRRMKWKK, SEQ ID NO: 9
  • the fusion peptide is linked to the peptide consisting of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2 at the N-terminal end of the cell permeable peptide, characterized in that consisting of the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 4 can do.
  • variants of the amino acid sequence may also be included within the scope of the present invention, and specifically, the variants may be 70% or more, preferably 80% or more, more preferably 90% or more, and even more, each of SEQ ID NO: 3 or 4, respectively. Preferably at least 95%, even more preferably at least 98%, most preferably at least 99% of all peptides having sequence homology.
  • the term "homology" refers to a degree of similarity with a wild type amino acid sequence and a wild type nucleic acid sequence.
  • the fusion peptide according to the present invention by inhibiting the TLR4 signaling pathway induced by lipopolysaccharide (LPS) cytokines (IL-6, TNF- ⁇ , IFN- ⁇ ), TLR3 signaling induced by Poly (I: C), which inhibits NO and ROS secretion and activation of NF- ⁇ B and MAPK, as well as relieving kidney and liver damage, sepsis and rheumatoid arthritis due to overactivation of TLR4 in animals It is excellent in inhibiting the pathway and LPS / ATP-induced NLRP3 inflamasome formation, preventing and treating autoimmune diseases, inflammatory diseases or neurodegenerative diseases caused by NLRP3 inflamasome caused by the signaling pathway It can be useful to
  • the fusion peptide according to the present invention can be used as TLR4, TLR3 and NLRP3 inflammasome inhibitor.
  • inhibitor refers to a molecule that, by any mechanism, partially or completely inhibits the effects of other molecules, such as receptors or intracellular mediators.
  • TLR4, TLR3 and NLRP3 inflammasome inhibitor refers to a substance that can directly, indirectly, or substantially interfere with, reduce or inhibit the biological activity of TLR4, TLR3 and NLRP3 inflammasomes, preferably Peptides reactive with TLR4, TLR3 bind directly to the TIR domains of TLR4, TLR3 and neutralize the activity of TLR4, TLR3, thereby blocking TLR4, TLR3 signaling pathways, thereby inhibiting NF- ⁇ B and MAPK and NLRP3 inflammasome activation.
  • the present invention also provides for the prevention of one or more TLR pathway mediated diseases selected from the group consisting of autoimmune diseases, inflammatory diseases and neurodegenerative diseases comprising peptides consisting of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2 as an active ingredient Or it provides a pharmaceutical composition for treatment.
  • the present invention from the group consisting of autoimmune diseases, inflammatory diseases and neurodegenerative diseases comprising a fusion peptide linked to the peptide consisting of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2 at the N-terminal end of the cell permeable peptide as an active ingredient
  • a fusion peptide linked to the peptide consisting of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2 at the N-terminal end of the cell permeable peptide as an active ingredient Provided are pharmaceutical compositions for the prophylaxis or treatment of one or more selected TLR pathway mediated diseases.
  • the fusion peptide is preferably characterized in that consisting of the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 4, variants of the amino acid sequence may also be included within the scope of the present invention.
  • pathogenic disease refers to any pathological condition in which the activation of one or more TLRs and the TLR mediated signaling pathway are contributing factors.
  • the condition is not limited thereto, but may be preferably one or more selected from the group consisting of autoimmune diseases, inflammatory diseases and degenerative diseases.
  • autoimmune disease is caused by a process in which a problem occurs in inducing or maintaining self-tolerance to cause an immune response to an autoantigen, thereby attacking its own tissue.
  • the self-tolerance refers to immunologic unresponsiveness that does not deleteriously react with the antigenic material constituting self.
  • Autoimmune diseases of the present invention include insulin-dependent diabetes mellitus, multiple sclerosis, experimental autoimmune encephalomyelitis, rheumatoid arthritis, experimental autoimmune arthritis, myasthenia gravis, thyroiditis, experimental form of uveitis, Hashimoto thyroiditis, primary myxedema, thyroid poisoning, malignant Anemia, autoimmune atrophy gastritis, Addison's disease, premature menopause, male infertility, childhood diabetes, Goodpasture syndrome, common pemphigus, leiomyelitis, sympathetic ophthalmitis, lens uveitis, autoimmune hemolytic anemia, idiopathic leukocyte reduction, primary cholangiovascular sclerosis , Chronic active hepatitis Hbs-ve, latent cirrhosis, ulcerative colitis, Sjogren's syndrome, scleroderma, Wegener's granulomatosis, polymyositis / skin myositis, discoid
  • the term "inflammatory disease” refers to TNF- ⁇ , IL-1, IL-6, which are secreted by immune cells such as macrophages by excessively promoting the immune system due to harmful stimuli such as inflammation-inducing factors or irradiation.
  • the inflammatory diseases of the present invention are asthma, eczema, psoriasis, allergy, rheumatoid arthritis , Psoriatic arthritis, atopic dermatitis, acne, atopic rhinitis, pneumonia, allergic dermatitis, chronic sinusitis, contact dermatitis, seborrheic dermatitis, gastritis, gout, gouty arthritis, ulcer, chronic bronchitis , Crohn's disease, ulcerative colitis, ankylosing spondylitis, sepsis, vasculitis, bursitis, lupus, rheumatoid polymyalgia, temporal arteritis, multiple sclerosis One including solid tumors, Alzheimer's disease, atherosclerosis, obesity and viral infection, but is not limited thereto.
  • the term "degenerative neuropathy” refers to a motor dysfunction, cognitive function, perceptual function, sensory function, and autonomic nerve abnormalities due to a decrease or loss of neuronal function, mainly classified as a clinical feature. And involvement sites.
  • Neurodegenerative diseases of the present invention include Alzheimer's disease, frontal temporal dementia, Louis dementia, cortical hypodegeneration, Parkinson's disease, multiple system atrophy, Huntington's disease, eukaryotic nucleus palsy, Lou Gehrig's disease, primary lateral sclerosis, spinal muscular atrophy
  • the present invention is not limited thereto.
  • the pharmaceutical composition of the present invention may include a pharmaceutically effective amount of the peptide alone or may include one or more pharmaceutically acceptable carriers, excipients or diluents.
  • the pharmaceutically effective amount herein refers to an amount sufficient to prevent, ameliorate and treat the symptoms of the autoimmune disease.
  • pharmaceutically acceptable refers to a composition that is physiologically acceptable and, when administered to a human, typically does not cause gastrointestinal disorders, allergic reactions such as dizziness or the like.
  • carriers, 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.
  • the pharmaceutical composition of the present invention may include at least one known active ingredient having an effect of treating autoimmune diseases, inflammatory diseases or neurodegenerative diseases with the peptide.
  • compositions of the 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.
  • the 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 vary depending on the route of administration, the age, sex, weight and severity of the patient. It may be appropriately selected depending on factors, and may be administered in combination with known compounds having the effect of preventing, ameliorating or treating symptoms of autoimmune disease, inflammatory disease or neurodegenerative disease.
  • the present invention comprises the steps of administering a peptide consisting of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2 to a subject in need thereof; It provides a method for preventing or treating a TLR pathway mediated disease comprising a.
  • the present invention comprises the steps of administering to a subject in need thereof a fusion peptide linked to a peptide consisting of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2 at the N-terminal end of the cell permeable peptide; It provides a method for preventing or treating a TLR pathway mediated disease comprising a.
  • the subject is a mammal including a human, a patient in need of treatment for a TLR pathway mediated disease, a patient being treated for a TLR pathway mediated disease, a patient who has been treated for a TLR pathway mediated disease, a TLR pathway mediated disease
  • a mammal including a human, a patient in need of treatment for a TLR pathway mediated disease, a patient being treated for a TLR pathway mediated disease, a patient who has been treated for a TLR pathway mediated disease, a TLR pathway mediated disease
  • peptide consisting of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2 of the present invention may be simultaneously / sequentially used in combination with other existing drugs or methods for treating TLR pathway mediated diseases. Can be processed.
  • HEK-Blue TM -hTLR4 cells (InvivoGen, San Diego, CA, USA) were treated with 1% penicillin / streptomycin, 10% fetal bovine serum (FBS) (Thermo Fisher Scientific Inc., Waltham, MA, USA) and 0.2% normocin (InvivoGen) added to DMEM (Dulbecco's modified Eagle's medium) (Thermo Fisher Scientific Inc.) medium.
  • Rat macrophages, RAW 264.7 cells were added to low-glucose DMEM (DMEM) supplemented with 1% penicillin / streptomycin and 10% FBS (Thermo Fisher Scientific, Inc.). Put incubated.
  • THP1 cells which are human monocytes, were cultured in RPMI 1640 supplemented with 1% penicillin / streptomycin and 10% FBS (Thermo Fisher Scientific, Inc.), followed by 10 nM of phorbol 12-myristate 13-acetate (PMA) ( Sigma-Aldrich Co. LLC., St. Louis, MO, USA) was used to induce differentiation into macrophages for 24 hours.
  • HPBMC cells PromoCell, Heidelberg, Germany
  • HPBMC cells which are human peripheral blood monocytes, were cultured in RPMI 1640 (Thermo Fisher Scientific Inc.) with 2.05 mM L-glutamine, 1% penicillin / streptomycin and 10% FBS.
  • MBMDM cells which are mouse bone marrow-derived macrophages, were cultured in DMEM (Thermo Fisher Scientific Inc.) with 1% penicillin / streptomycin and 10% FBS. All cells were incubated in a humidified culture system (Thermo Fisher Scientific Inc.) at 5 ° C. CO 2 , 37 ° C., and the medium was changed every 16 hours.
  • PAM 3 CSK 4 , Poly (I: C), R848 and CpG-ODN are from Thermo Fisher Scientific, Inc.
  • FSL-1 is from InvivoGen
  • LPS Escherichia coli 0111: B4
  • ATP Sigma-Aldrich Co. St. Louis, MO, USA
  • HEK-Blue TM- hTLR4 cells are dispensed in 96-well plates (BD Biosciences, San Jose, CA, USA) to a cell number of 5 x 10 4 cells / well, RAW264.7 cells are 2 x 10 5 cells / well The cells were aliquoted and cultured overnight. Thereafter, MTT analysis was performed using 1- (4,5-dimethylthiazol-2-yl) -3,5-diphenylformazan (MTT) solution (Sigma-Aldrich Co. LLC).
  • MTT 1- (4,5-dimethylthiazol-2-yl) -3,5-diphenylformazan
  • HEK-Blue TM -hTLR4 cells were aliquoted into 24-well plates (BD Biosciences) to a cell number of 2 ⁇ 10 5 cells / well and incubated overnight. The next day, after removing the culture medium and changing the medium, a portion of the culture medium (200 ⁇ l) was transferred to a microcentrifuge tube and heated at 65 ° C. for 10 minutes using a heating block (FINEPCR. Co., Seoul, Korea). . The cultures were then transferred to 96-well plates (BD Biosciences) and absorbance was measured at 405 nm using a HEK-Blue TM detection kit (InvivoGen) and a microplate reader spectrophotometer (Molecular Devices Inc., Silicon Valley, CA, USA). SEAP (Secreted Alkaline phosphatase) expression levels were analyzed.
  • Pre-protein extraction solutions (M-PER, Thermo Fisher Scientific Inc.) were mixed with protease and phosphatase inhibition mixtures and added to RAW 264.7 cells or hPBMC cell pellets to perform western blotting. The pellet was cooled for 10 minutes and the lysate was centrifuged at 16000 X g for 10 minutes. Then, the proteins of the cytoplasm and the nucleus were extracted using NE-PER nuclear and cytoplasmic extraction reagents (Thermo Fisher Scientific Inc.), and the concentration of the protein was measured using a BCA kit (Sigma-Alderich Co. LLC). It was.
  • the same amount of protein was then developed on SDS-polyacrylamide gels, and electrophoresis was performed using a Mini-PROTEAN Tetra Cell two-dimensional electrophoresis system (Bio-Rad Laboratories, Hercules, CA, USA).
  • the membranes were immunoblotted by light shaking with primary antibody overnight at a temperature of 4 ° C.
  • the primary antibodies were p-p65, p-JNK, p-IRF3, ERK, p38 and human IL-1 ⁇ (Cell Signaling Technology).
  • the membrane was incubated with anti-rat / rabbit HRP-conjugated secondary antibody (Thermo Fisher Scientific Inc.) for 2 hours and the SuperSignal West Pico ECL solution (Thermo Fisher Scientific Inc.) Protein was detected and the detected protein was visualized using the Fuji LAS-3000 system (Fujifilm, Tokyo, Japan).
  • RAW 264.7 cells and THP1 cells were seeded in 24-well plates to a cell number of 2 ⁇ 10 5 cells / well and grown in incubator overnight. After 24 hours of treatment with TIP1 and LPS together, the RAW 264.7 cells and THP1 cells were fixed with 3.7% formaldehyde (Sigma-Aldrich Co. LLC) for 15 minutes and 0.2% Triton X-100 (AMRESCO) for 15 minutes. , Solon, OH, USA), washed three times with PBS and blocked with 2% BSA solution.
  • the blocked cells were treated with TIP1-FITC (25 ⁇ M; Peptron, Inc., Daejeon, Korea), p-p65, TLR4, Myd88, TOM20 (1: 1000; Santa Cruz Biotechnology Inc.) and NLRP3 (Adipogen) antibodies. Incubated for hours, then washed three times with PBS. The cells were then incubated with AlexaFluor 408 and / or 488 and / or 546-conjugated secondary antibodies (Invitrogen, Carlsbad, CA, USA) for 1 hour and washed three times with PBS.
  • the cells were stained at room temperature for 15 minutes using 5 ⁇ M of Hoechst 33258 (Sigma-Aldrich Co.), and the number of the fluorescently stained cells was counted using a confocal laser scanning microscope (LSM-700, Carl Zeiss MicroImaging GmbH). The image was analyzed using Zen 2009 software.
  • RAW 264.7 cells were aliquoted into 6-cm dishes (SPL Life Sciences., Pochun, Korea) to a cell number of 1 ⁇ 10 6 cells / well and incubated overnight. TIP1 was then treated and stained with DAF-FM and DCF-DA (Thermo Fisher Scientific, Inc.), respectively, and incubated for 1 hour. Thereafter, the cells were collected by centrifugation at 200 X g per 5 minutes, transferred to a brown tube, and stored in PBS at a temperature of 4 ° C. The intensity of DAF-FM, DCF-DA phosphors was measured and quantified using FACSAria III with Diva software (BD Biosciences).
  • the NO level of the culture supernatant was determined using the NO detection kit (iNtRON Biotechnology Inc.). , Seongnam, Korea). Absorbance was measured at 550 nm using a microplate reader spectrophotometer (Molecular Devices In.) And the results were analyzed using Softmax Pro 5.3 software (Molecular Devices Inc.).
  • the activity of NF- ⁇ B was measured in HEK-Blue TM -hTLR4 cells cultured in Example 1-2.
  • the IL-12 p40 minimal promoter IL-12 p40 containing a site where DNA of NF- ⁇ B and AP-1 (activator protein 1) binds to the activation of NF- ⁇ B and AP-1 after stimulation of TLR4.
  • SEAP inducible secreted embryonic alkaline phosphatase
  • TIP or CPP-linked TIP TIP W / O CPP
  • concentration of TIP or CPP-linked TIP TIP W / O CPP
  • concentration of TIP or CPP-linked TIP TIP W / O CPP
  • TIP1 of the present invention once associated with CPP, interferes with adapter molecules downstream of the signaling pathway once translocated into the cell, and blocks the activation of the TLR4-mediated signaling pathway induced by LPS, thus providing an effective TLR4. It was confirmed that it can be used as an inhibitor.
  • TLR4-mediated responses induced by LPS induce direct interactions between the TIR domain of TIRAP and MyD88 to activate MyD88-dependent signaling pathways. It also induces interactions between the TIR domain of the TRAM and TRIF, activating the MyD88-independent signaling pathway.
  • the initial activity of NF- ⁇ B induces the secretion of pro-inflammatory cytokines such as tumor necrosis factor alpha (TNF- ⁇ ) and interleukin 6 (IL-6).
  • TNF- ⁇ tumor necrosis factor alpha
  • IL-6 interleukin 6
  • MyD88-independent signaling pathways induce late secretory responses of type I interferons (IFNs) such as IFN- ⁇ and IFN- ⁇ , as well as the activity of IRF3 and 7.
  • IFNs type I interferons
  • the activated NF- ⁇ B (p-p65) is expressed in the nucleus when only LPS is treated, but the phosphorylation level of p65 induced by LPS in the nucleus is reduced when TIP1 is treated together. It was confirmed that the activity of NF- ⁇ B decreases through.
  • Cytokines (TNF- ⁇ , IL-6, IFN- ⁇ ) and NO (Nitric) when TIP1 prepared in Example 1-1 were treated to RAW 264.7 cells, which are mouse macrophages cultured in Example 1-2 Oxygen secretion and the experiment to determine whether the generation of reactive oxygen species (ROS) in the cytoplasm is inhibited.
  • ROS reactive oxygen species
  • TNF- ⁇ , IL-6, IFN- ⁇ levels of the culture supernatant of RAW 264.7 cells were determined according to the method described in Example 2-5.
  • TIP1 and LPS were treated together, it was confirmed that the secretion of TNF- ⁇ , IL-6, IFN- ⁇ decreased in a concentration-dependent manner. This confirmed that TIP1 according to the present invention inhibits the secretion of cytokines induced by LPS.
  • RAW 264.7 cells were treated with TIP1 and DAF-FM (Invitrogen Corp., CA, respectively) , USA) and DCF-DA (Invitrogen Corp.) to quantify cytosolic NO and ROS, respectively.
  • the NO value of the culture supernatant of RAW 264.7 cells was measured using the NO detection kit. The results are shown in FIG. 5.
  • TIP1 effectively reduced NO and ROS generation in the cytoplasm as well as extracellular NO secretion.
  • TIP1 according to the present invention inhibits the oxidative stress induced by LPS.
  • Cytokines (TNF- ⁇ , IL-6, IFN- ⁇ ) when TIP1 prepared in Example 1 were treated with mBMDM, a mouse bone marrow-derived macrophage, and hPBMC cells, which are human peripheral blood mononuclear cells, ) And to confirm whether NO (Nitric Oxide) secretion and activation of TLR signaling proteins are inhibited.
  • TNF- ⁇ , IL-6, IFN- ⁇ levels of the culture supernatant of mBMDM cells were measured according to the method described in Example 2-5 in the rat TNF- ⁇ , IL-6, IFN- ⁇ ELISA kit Ready-SET-Go! Measured using the kit, the NO value of the supernatant was measured according to the method described in Example 2-7, and the results are shown in FIG. 6.
  • TIP1 reduces the secretion amount of TNF- ⁇ , IL-6 and NO induced by LPS, and IFN- ⁇ induced by Poly (I: C). It was confirmed to inhibit the production of. It was confirmed that TIP1 according to the present invention inhibits the secretion of cytokines and NO induced by LPS or Poly (I: C) in rat bone marrow-derived macrophages.
  • NF- ⁇ B activity was increased to degrade I ⁇ -B ⁇
  • TIP1 was treated together
  • NF- Inhibition of ⁇ B activity reduced the level of I ⁇ -B ⁇ degradation.
  • the activity of MAPK was increased to phosphorylate ERK, JNK, and p38, but when TIP1 was treated together, it was confirmed that the activity of MAPK was inhibited to decrease the phosphorylated degree of the enzymes.
  • TIP1 according to the present invention inhibits TLR4 signaling pathways not only in immortalized cell lines, but also in primary cells directly extracted from animals.
  • TNF- ⁇ levels induced by different TLR ligands in the culture supernatant of RAW 264.7 cells were measured in Example 2-5. It measured using the method of, and the result is shown in FIG.
  • IFN- ⁇ levels in the culture supernatant of RAW 264.7 cells were determined by ELISA kit Ready-SET-Go! It measured using, and the result is shown in FIG.
  • TIP1 and PAM3CSK4 (TLR1 / 2) or FSL-1 (TLR2 / 6) or Poly (I: C) (TLR3) or R848 (TLR7 / 8) or CpG-ODN (TLR9) together
  • TLR3 TLR3 signaling
  • TLR7 / 8 TLR7 / 8
  • TLR9 TLR9
  • NF- ⁇ B NOD-like receptor (NLR), NLRP3 (NACHT, LRR and PYD-domains-containing) in macrophages. protein 3) and IL-1 ⁇ .
  • NLR NOD-like receptor
  • NLRP3 NACHT, LRR and PYD-domains-containing
  • ATP and potassium efflux agent reduce potassium levels in cells and induce the production of mature IL-1 ⁇ by NLRP3 inflammasomes.
  • THP1 cells which are human monocytes
  • mBMDM THP1 cells which are mouse bone marrow-derived macrophages.
  • pro-caspase-1 45 kDa
  • active capase-1 10 kDa
  • pro-IL-1 ⁇ 35 kDa
  • mature IL Protein expression of -1 ⁇ (17 kDa) was visualized by Western blotting. The results are shown in FIG.
  • THP1 cells and mBMDM cells were treated with LPS / ATP or TIP1 and LPS / ATP, and then IL-1 ⁇ of the culture supernatant.
  • the values were determined according to the method described in Example 2-5.
  • NLRP3 inflamasome is abnormally activated, it can cause various inflammatory diseases, especially degenerative neurological diseases. If NLRP3 inflamasome can be effectively inhibited, it can be used as a therapeutic agent for neurodegenerative diseases through inhibition of inflammatory response. You can expect Accordingly, the present inventors confirmed that the TIP1 according to the present invention may have a prophylactic or therapeutic effect on neurodegenerative diseases.
  • SHCR decoy peptide 1-2
  • SHCRVLLI SEQ ID NO: 1
  • SEQ ID NO: 2 amino acid sequence of the decoy peptide prepared in Example 1-1
  • VLLI amino acid minimum region of TIP1 that binds to the TIR domain of TLR4.
  • Decoy peptide 1 TIP1 W / O CPP
  • SHCRVLLI One Decoy peptide 1-2
  • SHCR 2 TIP1 RQIKIWFQNRRMKWKK
  • SHCRVLLI 3 TIP1-2 RQIKIWFQNRRMKWKK
  • SHCR 4 Decoy peptide 1-3 VLLI 7 TIP1-3 RQIKIWFQNRRMKWKK VLLI 8
  • HEK-Blue TM -hTLR4 in which TIP1 (CPP-SHCRVLLI), TIP1-1 (CPP), TIP1-2 (CPP-SHCR) and TIP1-3 (CPP-VLLI) of Table 3 were cultured in Examples 1-2.
  • TIP1 CPP-SHCRVLLI
  • TIP1-1 CPP
  • TIP1-2 CPP-SHCR
  • TIP1-3 CPP-VLLI
  • NF- ⁇ B activity was measured using the method of Experimental Example 1 above.
  • the hPBMC cells cultured in Example 1-2 were treated with TIP1 (CPP-SHCRVLLI), TIP1-1 (CPP), TIP1-2 (CPP-SHCR) and TIP1-3 (CPP-VLLI) together with LPS.
  • the amount of NO secretion was measured using the method of Example 2-7. The results are shown in FIG.
  • TIP1 had the highest inhibitory effect on NF- ⁇ B activity and TIP1-2 also decreased NF- ⁇ B activity, whereas TIP1-1 and TIP1-3 were not changed or insignificant. It was confirmed.
  • TIP1 had the highest NO secretion reducing effect and TIP1-2 also suppressed NO secretion, whereas TIP1-1 and TIP1-3 were not changed or were insignificant. Confirmed. This confirmed that the S-H-C-R sequence of the sequence of TIP1 according to the present invention is important for producing an inhibitory effect.
  • TIP1 Protein-protein docking was performed to analyze the binding interface between the TIR domain of TLR4 and TIP1. As a result, TIP1 was expected to bind to the BB loop, the DD loop and the C-terminal tail in the TIR domain of TLR4, to confirm this by surface plasmon resonance according to the method described in Examples 2-8; SPR) analysis was performed.
  • TIP1-FITC was prepared by binding fluorescein isothiocyanate (FITC) to the N-terminus of TIP1 to confirm binding of TIP1 to TLR4.
  • FITC fluorescein isothiocyanate
  • TIP1 (TIP1-FITC) or LPS and TIP1 were treated together with THP1 cells of Example 1-2, and then fluorescently stained using immunofluorescent staining and confocal microscopy according to the method described in Examples 2-4. The number of cells was counted to analyze the interactions between the proteins. The results are shown in FIG.
  • TIP1-FITC-TLR4 strongly binds
  • MyD88 was found to form a weak bond to TIP1-FITC.
  • the inhibitory effect of TIP1 on the TLR4 signaling pathway is due to the binding of the C-terminal tail and the BB loop among TLR4's TIR domains.
  • the interaction between TIP1 and MyD88 is inhibited, ultimately leading to TLR4 signaling. It was confirmed to be inhibited.
  • TNF- ⁇ , IL-12p40 (diluted at 1: 100) and IL-6 (diluted at 1: 100) levels in plasma were determined using the mouse TNF- ⁇ , IL-12p40, IL-6 ELISA Kit ELISA MAX Deluxe (BioLegend, San Diego, CA, USA). Absorbance was measured at 450 nm using a microplate reader spectrophotometer (Molecular Devices In.) And the results were analyzed using Softmax Pro 5.3 software (Molecular Devices Inc.). The results are shown in FIG.
  • TIP1 As shown in (a) to (c) of FIG. 14, when TIP1 and LPS were treated together, the secretion of TNF- ⁇ , IL-12p40, and IL-6 was significantly reduced. Through this, it was confirmed that TIP1 according to the present invention inhibits TLR4 signaling pathway and inhibits cytokine secretion not only in vitro but also in vivo .
  • liver tissues were extracted and included with the Protease inhibitor cocktail (Thermo Fisher Scientific, Inc.) and M-PER mammalian protein extraction reagent. After homogenization using Kontes Pellet Pestle Cordless Motor (Thermo Fisher Scientific, Inc.), the protein was extracted according to the manufacturer's protocol. Then, protein expression of IL-6, TNF- ⁇ and ⁇ -actin were visualized by Western blotting according to the method described in Example 2-3, and the intensity of the band was graphically shown and the results are shown in FIG. 15. Indicated.
  • TIP1 and LPS were treated together, it was confirmed that the secretions of TNF- ⁇ and IL-6 and BUN, Cr, AST, and ALT were significantly reduced. Through this, it was confirmed that TIP1 according to the present invention inhibits the TLR4 signaling pathway and can mitigate kidney and liver damage as well as cytokine secretion.
  • ER-180A ER-180A, A & D Company, Tokyo, Japan
  • TUNEL-positive kidney cells were significantly increased when LPS was treated only, ie, cell death (green dot) in kidney tissues was increased, whereas when TIP1 and LPS were treated together, kidneys increased by LPS. It was confirmed that apoptosis (green dot) in the tissue was reduced.
  • arthritis was induced by applying collagen-induced arthritis (CIA) model to 6-7 week old DBA / 1J (20-23g) male mice.
  • CIA collagen-induced arthritis
  • emulsified by mixing chicken collagen type II (Sigma-Aldrich Co. LLC) and complete Freund's adjuvant (Sigma-Aldrich Co. LLC.) In a 1: 1 ratio.
  • 100 ⁇ g of the emulsified collagen solution was injected intravenously into the tail of the mouse to induce primary immunity and designated day 0.
  • the emulsified collagen solution was again injected intradermal into the mouse tail to induce secondary immunity.
  • TIP1 treatment plans were then designed: 1 TIP1 (2.5, 5 and 10 nmol / g) or 30 mg of positive control prednisolone, a well known treatment for conventional arthritis, after secondary immunization (day 15) plans to inject / kg daily; And 2 plan to inject 10 mmol / g of TIP1 for 10 days after fully inducing arthritis (day 35).
  • arthritis-induced DAB-1J rats were injected once daily with TIP1 and prednisolone every 30 days through the CIA model, and then the mice were sacrificed on day 45 to sample the joint tissue. Thereafter, 3D image and bone mineral density (BMD) of joint tissue were analyzed by micro-computed tomography (Micro-CT).
  • BMD bone mineral density
  • H & E hematoxylin and eosin staining was performed using the knee joints of rats, and the synovial excess in cartilage, subchondral bone, femur, tibia, and meniscus. Changes in synovial hyperplasia were observed. The results are shown in FIGS. 21 and 22.
  • TIP1 when treated with TIP1, bones damaged by the CIA model were alleviated similarly to normal rats, and cartilage (C), cartilage lower bone (S), femur (F), and tibia (T). , Histopathological modification of meniscus (M) was also reduced.
  • TIP1 according to the present invention effectively inhibits the TLR4 signaling pathway, it was confirmed that it can be usefully used as a therapeutic agent for various acute or chronic inflammatory diseases including rheumatoid arthritis.

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Abstract

The present invention relates to a fusion polypeptide that inhibits TLR1/2, TLR2/6, TLR7, TLR8 and TLR9 signaling pathways as well as Toll-like receptor 4 (TLR4) and TLR3, and a pharmaceutical composition for preventing or treating TLR pathway mediated diseases. The fusion peptide of the present invention has an excellent effect of inhibiting TLR4 and various TLR pathways and can be effectively used in preventing and treating various TLR pathway mediated diseases caused by the signaling pathways, such as autoimmune diseases, inflammatory diseases and degenerative neurological diseases, by inhibiting the TLR mediated immune responses.

Description

톨-유사 수용체(TLR) 억제를 위한 펩타이드 및 이를 포함하는 약학적 조성물Peptides for Toll-Like Receptor (TLR) Inhibition and Pharmaceutical Compositions Comprising the Same
본 발명은 TLR4(Toll-like receptor 4, 톨-유사 수용체 4)와 TLR3를 비롯한 TLR1/2, TLR2/6, TLR7, TLR8 및 TLR9 신호전달 경로를 억제하는 융합 펩타이드 및 이를 포함하는 TLR 경로 매개성 질환의 예방 또는 치료용 약학적 조성물에 관한 것이다.The present invention provides a fusion peptide that inhibits TLR1 / 2, TLR2 / 6, TLR7, TLR8 and TLR9 signaling pathways, including TLR4 (Toll-like receptor 4) and TLR3, and TLR pathway mediation including the same. It relates to a pharmaceutical composition for preventing or treating a disease.
선천 면역은 포유류의 면역 시스템에서 세균 감염에 대항하는 첫 번째 방어 작용으로 톨-유사 수용체와 같은 패턴 인식 수용체가 병원균-연관 분자 패턴(pathogen-associated molecular pattern, PAMP)이나 위험-연관 분자 패턴(danger-associated molecular pattern, DAMP)을 인식함으로써 활성화된다.Innate immunity is the first defense against bacterial infection in the mammalian immune system. Pattern recognition receptors, such as toll-like receptors, are known as pathogen-associated molecular patterns (PAMPs) or risk-associated molecular patterns (danger). activated by recognizing an associated molecular pattern (DAMP).
TLR는 선천 면역 반응에서 중요한 역할을 하며, TLR1, TLR2, TLR4, TLR5, TLR6 및 TLR11을 포함하는 원형질막에서 작용을 하는 세포 외 TLR와 TLR3, TLR7, TLR8 및 TLR9를 포함하는, 엔도솜과 같은 세포 내에서 작용을 하는 세포 내 TLR로 나눌 수 있다. 구조적으로, TLR는 세포외 도메인의 N-말단에 리간드 또는 부속분자(accessory molecule)에 의해 인식되는 루신고반복(leucine-rich repeat, LRR) 부위를 가지고 있으며, 세포내 부분의 C-말단에 신호를 전달하는 톨/인터루킨 1 수용체(Toll/interleukin 1 receptor,TIR) 도메인을 가지고 있다.TLRs play an important role in the innate immune response, and cells such as endosomes, including extracellular TLRs and TLR3, TLR7, TLR8 and TLR9, which act on plasma membranes including TLR1, TLR2, TLR4, TLR5, TLR6 and TLR11. It can be divided into intracellular TLRs that act in vivo. Structurally, the TLR has a leucine-rich repeat (LRR) site that is recognized by a ligand or an accessory molecule at the N-terminus of the extracellular domain and signals at the C-terminus of the intracellular portion. It has a Toll / interleukin 1 receptor (TIR) domain to deliver.
특히, TLR4는 TLR 패밀리에서 처음으로 규명된 수용체로서, MyD88(myeloid differentiation 88)-의존적 신호전달 경로 및 MyD88-비의존적 신호전달 경로를 통해 증폭되는 선천 면역신호를 활성화시킨다. 이에 반해 TLR3는 MyD88-비의존적 신호전달 경로만을 활성화시킨다. 이러한 TLR의 역할로 인해 여러 면역 질병을 치료하기 위한 타겟으로 TLR를 활용하기 위한 연구에 대한 관심이 높아지고 있다.In particular, TLR4 is the first identified receptor in the TLR family, activating innate immune signals that are amplified through the MyDoid (myeloid differentiation 88) -dependent signaling pathway and the MyD88-independent signaling pathway. In contrast, TLR3 activates only the MyD88-independent signaling pathway. Due to the role of TLRs, interest in researches for using TLRs as a target for treating various immune diseases is increasing.
TLR4의 MyD88-의존적 신호전달은 CD14(cluster of differentiation 14) 및 MD2 (myeloid differentiation factor 2)와 같은 부속 분자를 통한 LPS 인식에 의해 개시된다. LPS 결합 이후 TLR4는 이량체(dimer)를 형성하고 TLR4의 TIR 도메인과 TIR 도메인 어댑터 단백질(TIR domain adaptor protein, TIRAP 또는 MyD88 adapter-like, MAL)의 TIR 도메인끼리 결합하며, MyD88과 함께 복합체를 형성함으로써 신호전달 경로를 활성화시킨다. 활성화된 TLR4는 Myd88 의존 신호전달 과정을 통해 NF-κB의 초기 활성화 및 핵으로의 이동, MAPK의 활성화를 유도한다. 상기 NF-κB와 MAPK의 활성화는 TNF-α(tumor necrosis factor α), IL-1β(interleukin 1β) 및 IL-6와 같은 염증성 사이토카인을 분비시킨다. TLR4와 TLR3의 MyD88-비의존적 신호전달 과정은 각 TLR의 TIR 도메인과 TRAM/TRIF(TIR domain-containing adapter-inducing interferon-β(TRIF)-related adaptor molecule, TRAM) 복합체의 TIR 도메인간의 결합으로 개시되며, IRF(interferon-regulatory factor)의 활성화에 의해 타입 1 인터페론을 분비시킨다. 또한, TLR4 활성은 대식세포에서 NO와 ROS와 같은 산화 스트레스성 물질을 생성한다.MyD88-dependent signaling of TLR4 is initiated by LPS recognition through accessory molecules such as cluster of differentiation 14 (CD14) and myeloid differentiation factor 2 (MD2). After LPS binding, TLR4 forms a dimer and the TIR domain of TLR4 and the TIR domain of TIR domain adapter protein (TIRAP or MyD88 adapter-like (MAL)) bind to each other and form a complex with MyD88. Thereby activating the signaling pathway. Activated TLR4 induces initial activation of NF-κB, migration into the nucleus, and activation of MAPK through Myd88-dependent signaling. The activation of NF-κB and MAPK secretes inflammatory cytokines such as tumor necrosis factor α (TNF-α), interleukin 1β (IL-1β) and IL-6. The MyD88-independent signaling process of TLR4 and TLR3 is initiated by the binding between the TIR domain of each TLR and the TIR domain of the TRAM domain-containing adapter-inducing interferon-β (TRIF) -related adapter molecule (TRAM) complex. And secrete type 1 interferon by activation of an interferon-regulatory factor (IRF). TLR4 activity also produces oxidative stressors such as NO and ROS in macrophages.
이와 같이 TLR는 자가면역 질환, 염증성 질환 및 암과 같은 다양한 질환을 치료하기 위한 타겟이 될 수 있으므로 TLR를 타겟으로 하는 물질과 TLR와 관련된 질병을 치료하기 위한 의학적 조성물에 대한 연구가 이루어지고 있다. 특히, 지질 A(lipid A)의 주요 골격 구조를 변형함으로써 많은 수의 TLR4 촉진제와 길항제를 얻은 바 있으며, 에리토란(eritoran), 지질 A 및 Rhodobacter sphaeroids 지질 A(RsLA)가 LPS와 MD2의 상호작용을 억제하고 쥐에서 LPS로 유도된 쇼크를 예방할 수 있음이 밝혀졌다. 또한, LPS에 의한 TLR4 신호전달 경로는 NLRP3 인플라마좀 형성에 관여하여 알츠하이머 병과 파킨슨 병 등 퇴행성 신경질환과도 밀접한 관련이 있다고 보고되었다.As such, since TLRs may be targets for treating various diseases such as autoimmune diseases, inflammatory diseases and cancer, researches on substances targeting TLRs and medical compositions for treating diseases related to TLRs have been made. In particular, by modifying the major skeletal structure of lipid A, a large number of TLR4 promoters and antagonists have been obtained, eritoran, lipid A and Rhodobacter. It has been shown that sphaeroids lipid A (RsLA) inhibits LPS-MD2 interactions and prevents LPS-induced shock in mice. In addition, it has been reported that the TLR4 signaling pathway by LPS is closely related to degenerative neurological diseases such as Alzheimer's disease and Parkinson's disease due to the involvement of NLRP3 inflamasome formation.
한편, 단백질의 결합도메인 일부를 이용하여 본래 상호작용하는 단백질에 대신 결합함으로써 신호전달을 제어하는 decoy 펩타이드를 이용해 병원균-연관 분자 패턴(PAMP)과 유사하거나 반대되는 작용을 하는 펩타이드에 관해 연구가 활발히 진행되고 있다. 펩타이드들은 일반적인 저분자 (small molecule) 치료제에 비해 부작용이 적고, 변형 및 품질관리가 수월한 것으로 알려져 있으며, 세포 투과성 펩타이드(cell penetrating peptide, CPP)의 개발로 인해 낮은 세포 침투성 역시 개선되고 있다.On the other hand, studies are actively conducted on peptides that act similar to or opposite to pathogen-associated molecular patterns (PAMPs) using decoy peptides that control signaling by binding to a protein that interacts with the original interacting domain using a portion of the binding domain of the protein. It's going on. Peptides are known to have fewer side effects, easier modification and quality control than general small molecule therapeutics, and low cell permeability is also improved due to the development of cell penetrating peptide (CPP).
본 발명자들은 서열번호 1의 아미노산 서열로 이루어진 신규 펩타이드(decoy peptide 1) 및 상기 신규 펩타이드가 세포 투과성 펩타이드의 N-말단에 연결된 융합 펩타이드(Toll-like receptor inhibitory peptide 1, TIP1)가 지질다당류(lipopolysaccharide, LPS)에 의해 유도된 TLR4 신호전달 경로를 억제하여 사이토카인(IL-6, TNF-α, IFN-β), NO 및 ROS의 분비와 NF-κB와 MAPK의 활성화를 억제함은 물론 동물에서 TLR4의 과활성에 의한 신장과 간 손상, 패혈증 및 류마티스 관절염을 완화시킴을 확인하였고, Poly(I:C)에 의해 유도된 TLR3 신호전달 경로 및 LPS/ATP에 의해 유도된 NLRP3 인플라마좀 형성 역시 억제시킴을 증명하여 본 발명을 완성하였다. 또한, TLR1/2, TLR2/6, TLR7, TLR8 및 TLR9도 일부 억제하고 있음을 세포 실험에서 확인하였다.The inventors have described a novel peptide (decoy peptide 1) consisting of the amino acid sequence of SEQ ID NO: 1 and a fusion peptide (Toll-like receptor inhibitory peptide 1, TIP1) in which the novel peptide is linked to the N-terminus of a cell-penetrating peptide. , LPS) -induced TLR4 signaling pathways that inhibit cytokine (IL-6, TNF-α, IFN-β), NO and ROS secretion, and activation of NF-κB and MAPK, as well as in animals It has been shown to alleviate renal and hepatic damage, sepsis and rheumatoid arthritis due to overactivity of TLR4.TLR3 signaling pathway induced by Poly (I: C) and NLRP3 inflamasome formation induced by LPS / ATP were also identified. Inhibition was demonstrated to complete the present invention. In addition, it was confirmed in cell experiments that TLR1 / 2, TLR2 / 6, TLR7, TLR8 and TLR9 were also partially inhibited.
따라서, 본 발명의 목적은 서열번호 1 의 아미노산 서열로 이루어진 펩타이드 및 세포 투과성 펩타이드의 N-말단에 서열번호 1 또는 서열번호 2의 아미노산 서열로 이루어진 펩타이드가 연결된 융합 펩타이드를 제공하는 것이다.Accordingly, an object of the present invention is to provide a fusion peptide in which a peptide consisting of the amino acid sequence of SEQ ID NO: 1 and a peptide consisting of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2 is connected to the N-terminus of the cell permeable peptide.
또한, 본 발명의 또 다른 목적은 서열번호 1 또는 서열번호 2의 아미노산 서열로 이루어진 펩타이드를 유효성분으로 포함하는 자가면역질환, 염증성 질환 및 퇴행성 신경질환으로 이루어진 군으로부터 선택되는 1종 이상의 TLR 경로 매개성 질환의 예방 또는 치료용 약학적 조성물을 제공하는 것이다.Still another object of the present invention is to mediate one or more TLR pathways selected from the group consisting of autoimmune diseases, inflammatory diseases and neurodegenerative diseases comprising a peptide consisting of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2 as an active ingredient It provides a pharmaceutical composition for the prevention or treatment of sexual diseases.
상기와 같은 목적을 달성하기 위하여, 본 발명은 서열번호 1의 아미노산 서열로 이루어진 펩타이드를 제공한다.In order to achieve the above object, the present invention provides a peptide consisting of the amino acid sequence of SEQ ID NO: 1.
또한, 본 발명은 세포 투과성 펩타이드의 N-말단에 서열번호 1 또는 서열번호 2의 아미노산 서열로 이루어진 펩타이드가 연결된 융합 펩타이드를 제공한다.The present invention also provides a fusion peptide wherein a peptide consisting of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2 is linked to the N-terminus of the cell permeable peptide.
또한, 본 발명은 서열번호 1 또는 서열번호 2의 아미노산 서열로 이루어진 펩타이드를 유효성분으로 포함하는 자가면역질환, 염증성 질환 및 퇴행성 신경질환으로 이루어진 군으로부터 선택되는 1종 이상의 TLR 경로 매개성 질환의 예방 또는 치료용 약학적 조성물을 제공한다.The present invention also provides for the prevention of one or more TLR pathway mediated diseases selected from the group consisting of autoimmune diseases, inflammatory diseases and neurodegenerative diseases comprising peptides consisting of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2 as an active ingredient Or it provides a pharmaceutical composition for treatment.
또한, 본 발명은 세포 투과성 펩타이드의 N-말단에 서열번호 1 또는 서열번호 2의 아미노산 서열로 이루어진 펩타이드가 연결된 융합 펩타이드를 유효성분으로 포함하는 자가면역질환, 염증성 질환 및 퇴행성 신경질환으로 이루어진 군으로부터 선택되는 1종 이상의 TLR 경로 매개성 질환의 예방 또는 치료용 약학적 조성물을 제공한다.In addition, the present invention from the group consisting of autoimmune diseases, inflammatory diseases and neurodegenerative diseases comprising a fusion peptide linked to the peptide consisting of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2 at the N-terminal end of the cell permeable peptide as an active ingredient Provided are pharmaceutical compositions for the prophylaxis or treatment of one or more selected TLR pathway mediated diseases.
본 발명의 융합 펩타이드는 TLR4 및 다양한 TLR 경로를 억제하는 효과가 우수하여, 상기 TLR 매개된 면역 반응을 저해함으로써 상기 신호전달 경로에 의해 발생하는 다양한 TLR 경로 매개성 질환, 예컨대 자가면역 질환, 염증성 질환 또는 퇴행성 신경질환을 예방 및 치료하는데 유용하게 활용할 수 있다.The fusion peptide of the present invention is excellent in inhibiting TLR4 and various TLR pathways, thereby inhibiting the TLR mediated immune response, thereby causing various TLR pathway mediated diseases caused by the signaling pathway, such as autoimmune diseases and inflammatory diseases. Or it can be usefully used to prevent and treat degenerative neurological diseases.
도 1의 (a)는 HEK-BlueTM hTLR4 (human TLR4) 세포에 TIPs(TIP1, TIP2; TIP2는 대조용으로 사용)의 농도를 달리하여 LPS와 함께 처리한 경우 SEAP (secreted alkaline phosphatase) 활성이 TIP1에 의해 감소하고 있음을 나타낸 도이다. 도 1의 (b)는 CPP 서열이 연결되지 않은 TIP1(TIP1 W/O CPP) 및 TIP2(TIP2 W/O CPP)의 SEAP 활성을 측정한 결과를 나타낸 도이다.FIG. 1 (a) shows that SEAP (secreted alkaline phosphatase) activity is decreased when TIPs (TIP1, TIP2; TIP2 is used as a control) at different concentrations in HEK-Blue TM hTLR4 (human TLR4) cells. It is a figure which shows decreasing by TIP1. Figure 1 (b) is a diagram showing the results of measuring the SEAP activity of TIP1 (TIP1 W / O CPP) and TIP2 (TIP2 W / O CPP) to which the CPP sequence is not linked.
도 2의 (a)는 쥐 대식세포인 RAW 264.7 세포에 TIP1을 LPS와 함께 처리한 경우 NF-κB(p65) 및 IRF3 활성을 웨스턴 블롯팅을 통해 확인한 결과를 나타낸 도이다. 도 2의 (b)는 동일 조건에서 MAPK 활성을 웨스턴 블롯팅을 통해 확인한 결과를 나타낸 도이다.Figure 2 (a) is a diagram showing the results confirmed by Western blotting NF-κB (p65) and IRF3 activity when TIP1 was treated with LPS to RAW 264.7 cells, which are mouse macrophages. Figure 2 (b) is a diagram showing the results confirmed by Western blotting MAPK activity under the same conditions.
도 3은 쥐 대식세포인 RAW 264.7 세포에 TIP1을 LPS와 함께 처리한 경우 NF-κB의 활성(p-p65, 녹색)을 공초점 레이저 주사 현미경(Confocal microscopy)으로 확인한 결과로 TIP1에 의해 p-p65의 핵내 이동이 차단되고 있음을 나타낸 도이다.3 shows that NF-κB activity (p-p65, green) was confirmed by confocal microscopy when TIP1 was treated with LPS in RAW 264.7 cells, which are mouse macrophages. Fig. shows that the nuclear transfer of p65 is blocked.
도 4는 쥐 대식세포인 RAW 264.7 세포에 농도를 달리한 TIP1을 LPS와 함께 처리한 경우 사이토카인 분비량을 확인한 결과를 나타낸 도이다. 도 4의 (a)는 TNF-α, 도 4의 (b)는 IL-6, 도 4의 (c)는 IFN-β 분비량을 각각 나타낸 것이다.Figure 4 is a diagram showing the result of confirming the cytokine secretion when treated with LPS TIP1 of different concentrations in RAW 264.7 cells, which are mouse macrophages. 4 (a) shows TNF-α, FIG. 4 (b) shows IL-6, and FIG. 4 (c) shows IFN-β secretion.
도 5는 쥐 대식세포인 RAW 264.7 세포에 TIP1을 LPS와 함께 처리한 경우 NO 및 ROS 발생량을 확인한 결과를 나타낸 도이다. 도 5의 (a)는 세포질에서의 NO 발생량, 도 5의 (b)는 세포질 내 ROS의 발생량, 도 5의 (c)는 세포 밖으로의 NO 분비량을 각각 나타낸 것이다.5 is a diagram showing the results of confirming the NO and ROS generation amount when TIP1 was treated with LPS in RAW 264.7 cells, which are mouse macrophages. Figure 5 (a) shows the amount of NO generated in the cytoplasm, Figure 5 (b) shows the amount of ROS generated in the cytoplasm, Figure 5 (c) shows the amount of NO secreted out of the cells.
도 6은 쥐 골수유래 대식세포인 mBMDM (mouse bone marrow derived macrophage) 세포에 TIP1을 LPS와 함께 처리한 경우 사이토카인 및 NO 발생량을 확인한 결과를 나타낸 도이다. 도 6의 (a)는 TNF-α, 도 6의 (b) IL-6, 도 6의 (c)는 NO, 도 6의 (d)는 IFN-β 발생량을 나타낸 것이다.6 is a diagram showing the results of confirming the cytokine and NO generation amount when TIP1 was treated with LPS in mBMDM (mouse bone marrow derived macrophage) cells, which are mouse bone marrow-derived macrophages. FIG. 6A illustrates TNF-α, FIG. 6B IL-6, FIG. 6C NO, and FIG. 6D show IFN-β generation amount.
도 7은 인간 말초혈액 단핵세포 (human peripheral blood mononuclear cells (hPBMCs)에 TIP1을 LPS와 함께 처리한 경우 세포에서의 p-ERK, ERK, p-JNK, JNK, pp38, p38, p-p65 및 Iκ-Bα 발현량을 웨스턴 블롯팅을 통해 확인한 결과로 TIP1에 의해 억제되고 있음을 보여주는 도이다.7 shows p-ERK, ERK, p-JNK, JNK, pp38, p38, p-p65 and Iκ in cells when TIP1 was treated with LPS in human peripheral blood mononuclear cells (hPBMCs). -Bα expression is confirmed by Western blotting as a result showing that it is inhibited by TIP1.
도 8은 쥐 대식세포인 RAW 264.7 세포에 TIP1을 각각의 TLR에 해당하는 리간드인 PAM3CSK4 (TLR1/2), FSL-1 (TLR2/6), Poly(I:C) (TLR3), R848 (TLR7/8) 또는 CpG-ODN (TLR9) 과 함께 처리한 경우 세포에서의 TNF-α 분비량을 측정한 것으로 TIP1에 의해 억제되고 있음을 보여주는 도이다.FIG. 8 shows TIP1 in mouse macrophages RAW 264.7 cells, PAM 3 CSK 4 (TLR1 / 2), FSL-1 (TLR2 / 6), Poly (I: C) (TLR3), When treated with R848 (TLR7 / 8) or CpG-ODN (TLR9), it is a measure of TNF-α secretion in cells and shows that it is inhibited by TIP1.
도 9는 쥐 대식세포인 RAW 264.7 세포에 TIP1을 Poly(I:C)와 함께 처리한 경우 IFN-β의 분비량을 ELISA를 통해 확인한 결과로 TIP1에 의해 감소하고 있음을 나타낸 도이다.9 is a diagram showing that when TIP1 was treated with Poly (I: C) in RAW 264.7 cells, which are mouse macrophages, the secretion amount of IFN-β was confirmed by ELISA as a result of TIP1.
도 10의 (a)는 LPS와 ATP를 처리하여 유도시킨 NLRP3 (NACHT, LRR 및 PYD domains-containing protein 3)-매개 인플라마좀 (inflammasome, 염증조절복합체) 형성에 관여하는 분자들이 TIP1에 의해 억제됨을 THP1 세포에서 확인한 도이다. 도 10의 (b)는 동일 조건의 실험을 mBMDM 세포에서 수행한 결과를 나타낸 도이다.FIG. 10 (a) shows that the molecules involved in the formation of NLRP3 (NACHT, LRR and PYD domains-containing protein 3) -mediated inflammasomes induced by LPS and ATP are inhibited by TIP1. Is confirmed in THP1 cells. Figure 10 (b) is a diagram showing the results of the experiment in the mBMDM cells under the same conditions.
도 11의 (a)는 LPS와 ATP를 처리하여 유도시킨 NLRP3-매개 인플라마좀 상태에서 TIP1을 처리할 경우 IL-1β 분비가 억제됨을 THP1 세포에서 확인한 도이다. 도 11의 (b)는 동일 조건의 실험을 mBMDM 세포에서 수행한 결과를 나타낸 도이다. Figure 11 (a) is a diagram confirming the THP1 cells that IL-1β secretion is suppressed when TIP1 treatment in the NLRP3-mediated inflamasome state induced by treatment with LPS and ATP. Figure 11 (b) is a diagram showing the results of performing the experiment in the mBMDM cells under the same conditions.
도 12는 TIP1의 전체 또는 부분적인 아미노산 서열을 갖는 펩타이드의 NF-κB 활성 및 NO 분비량을 확인한 결과를 나타낸 도이다. 도 12의 (a)는 SEAP 활성, 도 12의 (b)는 NO 분비량을 각각 나타낸 것이다. 12 is a diagram showing the results of confirming the NF-κB activity and the amount of NO secretion of the peptide having a full or partial amino acid sequence of TIP1. 12 (a) shows SEAP activity, and FIG. 12 (b) shows NO secretion amount, respectively.
도 13은 인간 단핵구세포인 THP1 세포에 FITC(fluorescein isothiocyanate)가 결합된 TIP1(TIP1-FITC) 또는 TIP1-FITC와 LPS를 함께 처리한 경우 TIP1(녹색)과 TLR4(빨간색) 및 MyD88(파란색) 간의 상호작용을 공초점 레이저 주사 현미경으로 관찰하여 TIP1이 TRL4와 결합하고 있음을 확인한 도이다.FIG. 13 shows TIP1 (TIP1-FITC) or TIP1-FITC combined with fluorescein isothiocyanate (FITC) or THS1 cells, which are human monocytes, between TIP1 (green) and TLR4 (red) and MyD88 (blue). Interactions were observed by confocal laser scanning microscopy to confirm that TIP1 binds to TRL4.
도 14는 C57BL/6J 쥐에 PBS(phosphate buffered saline)와 LPS 또는 TIP1과 LPS를 함께 처리한 후 1시간 또는 2시간 뒤 채취한 혈액을 통해 사이토카인 분비량을 확인한 결과를 나타낸 도이다. 도 14의 (a)는 TNF-α, 도 14의 (b)는 IL-12p40, 도 14의 (c)는 IL-6 분비량을 각각 나타낸 것이다.14 is a diagram showing the results of confirming the cytokine secretion through blood collected after 1 hour or 2 hours after treatment with PBS (phosphate buffered saline) and LPS or TIP1 and LPS in C57BL / 6J mice. FIG. 14A illustrates TNF-α, FIG. 14B shows IL-12p40, and FIG. 14C shows IL-6 secretion.
도 15는 C57BL/6J 쥐에 PBS와 LPS 또는 TIP1과 LPS를 함께 처리한 경우 사이토카인 분비량을 쥐의 간조직에서 확인한 결과를 나타낸 도이다. 도 15의 (a)는 TNF-α 및 IL-6의 분비량을 웨스턴 블롯팅을 통해 확인한 결과를 나타낸 것이고, 도 15의 (b)는 상기 웨스틴 블롯팅에 따른 밴드 강도를 정량화하여 그래프로 나타낸 것이다.15 is a diagram showing the results of confirming the cytokine secretion in liver tissue of rats when C57BL / 6J mice were treated with PBS and LPS or TIP1 and LPS. Figure 15 (a) shows the result of confirming the secretion amount of TNF-α and IL-6 through Western blotting, Figure 15 (b) is a graph showing the quantification of the band intensity according to the Westin blotting .
도 16은 C57BL/6J 쥐에 PBS와 LPS 또는 TIP1과 LPS를 함께 처리한 경우 TIP1이 신장 및 간 손상에 미치는 영향을 24시간 뒤 채취한 혈액을 통해 확인한 결과를 나타낸 도이다. 도 16의 (a) 및 (b)는 각각 TNF-α 및 IL-6 분비량을 나타낸 것이다. 도 16의 (c) 및 (d)는 각각 신장 손상 마커인 BUN(blood urea nitrogen) 및 크레아틴(creatinine, Cr)의 분비량을 나타낸 것이다. 도 16의 (e) 및 (f)는 간 손상 마커인 AST(aspartate aminotransferase) 및 ALT(alanine aminotransferase)의 분비량을 나타낸 도이다.16 is a diagram showing the results obtained through blood collected after 24 hours the effect of TIP1 on kidney and liver damage when PBS and LPS or TIP1 and LPS were treated together in C57BL / 6J mice. (A) and (b) of FIG. 16 show TNF-α and IL-6 secretion, respectively. (C) and (d) of FIG. 16 show secretion amounts of blood urea nitrogen (BUN) and creatin (creatinine, Cr), which are renal damage markers, respectively. Figure 16 (e) and (f) is a diagram showing the secretion amount of the liver damage markers AST (aspartate aminotransferase) and ALT (alanine aminotransferase).
도 17은 C57BL/6J 쥐에 PBS와 LPS 또는 TIP1과 LPS를 함께 처리한 경우 TIP1이 신장의 세포사멸(apoptosis)에 미치는 영향을 24시간 뒤 채취한 신장조직을 이용해 TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) 염색법으로 확인한 결과로 TIP1이 세포사멸을 감소시키고 있음을 나타낸 도이다.FIG. 17 shows TUNEL (terminal deoxynucleotidyl transferase dUTP nick end) using renal tissue harvested after 24 hours of treatment with PBS and LPS or TIP1 and LPS in C57BL / 6J mice. As a result of the labeling staining method, it is shown that TIP1 reduces apoptosis.
도 18은 BALB/c 쥐에 LPS 또는 TIP1과 LPS를 함께 처리한 경우 LPS에 의한 패혈증 모델 쥐의 생존율이 증가하고 있음을 보여주는 도이다.18 is a diagram showing that survival of LPS-induced sepsis model rats is increased when LPS or TIP1 and LPS are treated in BALB / c mice.
도 19의 (a)는 DAB-1J 쥐의 CIA(collagen-induced arthritis; 콜라겐에 의해 유도된 류마티스 관절염) 모델을 이용하여 류마티스 관절염에 대한 TIP1의 치료 효과를 확인하기 위한 실험 계획을 나타낸 도이다. 도 19의 (b)는 TIP1을 처리한 경우 치료효과를 시각적으로 관찰한 것으로 류마티스 관절염이 치료되고 있음을 나타낸 도이다.Figure 19 (a) is a diagram showing an experimental design for confirming the therapeutic effect of TIP1 on rheumatoid arthritis using a collagen-induced arthritis (collagen-induced rheumatoid arthritis) model of DAB-1J mice. Figure 19 (b) is a visual observation of the therapeutic effect when treated with TIP1 is a diagram showing that rheumatoid arthritis is being treated.
도 20은 DAB-1J 쥐의 CIA 류마티스 관절염 모델에 TIP1을 처리한 경우 치료효과를 확인한 결과를 나타낸 도이다. 도 20의 (a)는 체중, 도 20의 (b)는 squeaking (쥐가 찍찍거리는 소리), 도 20의 (c)는 발 부푼 정도, 도 20의 (d)는 관절염 인덱스를 관찰한 결과를 각각 나타낸 것이다.20 is a diagram showing the results of confirming the therapeutic effect when TIP1 treatment in the CIA rheumatoid arthritis model of DAB-1J mice. Figure 20 (a) is the weight, Figure 20 (b) is squeaking (rat rat squeaking), Figure 20 (c) is a foot swelling degree, Figure 20 (d) is a result of observing the arthritis index Respectively.
도 21은 DAB-1J 쥐의 CIA 모델에 TIP1을 처리한 후 Micro-CT(micro-computed tomography)를 이용해 3D 이미지 및 골밀도(born mineral density; BMD)를 측정한 것으로 관절염이 치료되고 있음을 나타낸 도이다.FIG. 21 shows that arthritis is being treated by measuring 3D image and bone mineral density (BMD) using Micro-CT (micro-computed tomography) after TIP1 treatment in a CIA model of DAB-1J mice. to be.
도 22는 DAB-1J 쥐의 CIA 모델에 TIP1을 처리한 후 무릎관절 부위를 헤마톡실린과 에오신으로 염색(Hematoxylin and eosin(H&E) staining)하여 기존의 치료제인 프레드니솔론과 치료 정도를 비교한 것으로 TIP의 치료 효과가 우수함을 나타낸 도이다.22 is a comparison of the degree of treatment with prednisolone, a conventional therapeutic agent, by treating the CIA model of DAB-1J mice with TIP1 and staining the knee joint with hematoxylin and eosin (H & E). Shows the excellent therapeutic effect.
도 23은 TLR4에 의해 유도되는 신호전달 과정과 TIP1이 제어하고 있는 부위를 모식적으로 나타낸 도이다.FIG. 23 is a diagram schematically showing a signaling process induced by TLR4 and a region controlled by TIP1. FIG.
이하, 본 발명에 대해 상세히 설명한다.Hereinafter, the present invention will be described in detail.
본 발명은 서열번호 1 의 아미노산 서열로 이루어진 펩타이드를 제공한다.The present invention provides a peptide consisting of the amino acid sequence of SEQ ID NO.
본 발명에서 용어, “펩타이드”는 펩타이드 결합에 의해 아미노산 잔기들이 서로 결합되어 형성된 선형의 분자를 의미한다. 상기 펩타이드는 당업계에 공지된 화학적 합성 방법에 따라 제조될 수 있으며, 바람직하게는 고체상 합성 기술에 따라 제조될 수 있으나, 이에 한정되지 않는다.As used herein, the term “peptide” refers to a linear molecule formed by binding amino acid residues to each other by peptide bonds. The peptide may be prepared according to chemical synthesis methods known in the art, and preferably may be prepared according to a solid phase synthesis technique, but is not limited thereto.
본 발명에서 용어, “TLR4”는 병원체 감염에 대한 감시자로서 기능하는 막관통(transmembrane) 단백질 패밀리인 톨-유사 수용체(Toll-like receptors, TLRs)에 속하는 단백질로서, TLR4 유전자에 의해 코딩되는 단백질을 말하며, CD284(cluster of differentiation 284)로 명명되기도 한다. 상기 TLR4는 그람-음성 박테리아의 LPS를 비롯한 다양한 PAMPs(pathogen-associated molecular patterns)를 인지하기 때문에 선천성 면역 시스템의 활성화에 매우 중요하다.As used herein, the term “TLR4” refers to a protein belonging to Toll-like receptors (TLRs), a family of transmembrane proteins that function as a monitor for pathogen infection. It is also called CD284 (cluster of differentiation 284). The TLR4 is very important for the activation of the innate immune system because it recognizes a variety of pathogen-associated molecular patterns (PAMPs), including LPS of Gram-negative bacteria.
본 발명에서 용어, “TLR3”은 병원체 감염에 대한 감시자로서 기능하는 막관통(transmembrane) 단백질 패밀리인 TLRs에 속하는 단백질로서, TLR3 유전자에 의해 코딩되는 단백질을 말하며, CD283(cluster of differentiation 283)으로 명명되기도 한다. 상기 TLR3은 바이러스의 double-strand RNA(dsRNA)를 비롯한 다양한 PAMPs를 인지하기 때문에 선천 면역 시스템의 활성화에 매우 중요하다.As used herein, the term “TLR3” refers to a protein that belongs to TLRs, a family of transmembrane proteins that function as a monitor for pathogen infection, and refers to a protein encoded by the TLR3 gene, and is named CD283 (cluster of differentiation 283). Sometimes. The TLR3 is very important for activation of the innate immune system because it recognizes a variety of PAMPs, including double-strand RNA (dsRNA) of the virus.
본 발명에서 용어, “NLRP3 인플라마좀”은 caspase-1의 활성화를 조절하는 내재 면역 체계의 수용체 및 센서이며, 미생물 감염 등에 반응하여 염증을 유발하는 인플라마좀의 한 종류로서, 다양한 자극들에 반응하여 활성화된다. NLRP3는 준비단계(priming)가 필요하며, 예시로 LPS의 TLR4로의 결합이 있다. LPS에 의한 TLR4 신호전달 경로는 ATP와 함께 NLRP3 인플라마좀 형성을 촉진한다. NLRP3 인플라마좀은 비정상적으로 활성화되면 다양한 염증성 질환의 발병을 유도하며, 특히 퇴행성 신경질환을 유발한다. 최근의 연구에 따르면, 알츠하이머 병의 주요 원인으로 꼽히는 베타아밀로이드(amyloid β)의 축적과 NLRP3 인플라마좀 사이에 직접적인 연관성이 있음이 밝혀졌고 파킨슨 병과의 관련성도 보고되었다.As used herein, the term “NLRP3 inflammasome” is a receptor and sensor of the intrinsic immune system that regulates the activation of caspase-1, and is a type of inflammasome that causes inflammation in response to microbial infections. Is activated in response. NLRP3 requires priming, for example the binding of LPS to TLR4. The TLR4 signaling pathway by LPS, along with ATP, promotes NLRP3 inflammasome formation. Abnormally activated NLRP3 inflammasomes lead to the development of a variety of inflammatory diseases, particularly neurodegenerative diseases. Recent studies have shown a direct link between the accumulation of beta amyloid (amyloid β), a major cause of Alzheimer's disease, and NLRP3 inflammasomes, and has also been linked to Parkinson's disease.
본 발명에서 용어, “TLR4에 의해 매개되는 신호전달 경로”는 TLR4를 통한 신호전달 경로를 말하며, 이는 TLR4 및 MD2에 의해 형성된 TLR4/MD2 복합체에 의존하는 LPS 반응일 수 있고, 이를 통해 신호가 전달된다. TLR4는 여러 가지 어댑터 단백질에 의해 신호를 전달하며, Mal(TIRAP로도 지칭됨)과 MyD88 및 트램(TRAM)과 트리프(TRIF) 등을 통해 작동한다. 활성화된 TLR4는 Myd88-의존적 신호전달 과정을 통해 NF-κB를 활성화시켜 핵으로 이동시키며, MAPK의 활성화를 유도한다. 상기 NF-κB와 MAPK의 활성화로 인해 TNF-α, IL-1β 및 IL-6와 같은 염증성 사이토카인이 분비되고, 대식세포에서 일산화질소(이하, NO라 한다)와 활성산소(이하, ROS라 한다)와 같은 산화 스트레스성 물질을 생성한다. 또한, 트램/트리프(TRAM/TRIF), 인터페론-조절자(IRF) 및 NF-κB의 활성화에 의해 MyD88-비의존적 신호전달 과정이 유도되어 타입 1 인터페론이 분비된다.As used herein, the term “TLR4 mediated signaling pathway” refers to a signaling pathway through TLR4, which may be an LPS response that depends on the TLR4 / MD2 complex formed by TLR4 and MD2, through which signals are transmitted. do. TLR4 carries signals by several adapter proteins and works through Mal (also called TIRAP), MyD88, and TRAM and TRIF. Activated TLR4 activates NF-κB to the nucleus through Myd88-dependent signaling, leading to the activation of MAPK. Due to the activation of NF-κB and MAPK, inflammatory cytokines such as TNF-α, IL-1β and IL-6 are secreted, and nitric oxide (hereinafter referred to as NO) and free radicals (hereinafter referred to as ROS) in macrophages To produce oxidative stressors. In addition, the activation of TRAM / TRIF, interferon-regulator (IRF), and NF-κB induces MyD88-independent signaling and secretes Type 1 interferon.
본 발명에서 용어, “TIR 도메인”은 세포 내 신호전달을 위한 도메인으로, 3개의 고도로 보존된 영역(highly conserved region)을 가지고 있으며 TLR과 다른 신호전달 분자 사이의 상호작용을 매개한다. 활성화된 TIR 도메인은 MyD88의 결합을 유도하고 TLR 신호전달 경로를 활성화 시킨다.In the present invention, the term “TIR domain” is a domain for intracellular signaling, which has three highly conserved regions and mediates the interaction between TLRs and other signaling molecules. Activated TIR domains induce binding of MyD88 and activate the TLR signaling pathway.
본 발명에서 용어, “억제”는 결핍, 부조화, 그 밖의 많은 원인에 의하여 생물 활동이나 신호활성이 저하되는 현상을 말하며, TLR의 활성을 부분적으로 또는 완전히 블로킹하거나, 감소시키거나, 방지하거나, 활성화를 지연시키거나, 불활성화 시키거나 또는 하향 조절하는 것일 수 있다. 본 발명의 일 실시예에 따르면, 본 발명에 따른 펩타이드 또는 융합 펩타이드는 TLR4 및 TLR3 신호전달 경로와 NLRP3 인플라마좀 억제용도를 제공한다. 또한 부분적으로 TLR1/2, TLR2/6, TLR7, TLR8 및 TLR9의 억제용도도 제공한다.As used herein, the term “inhibition” refers to a phenomenon in which biological activity or signaling activity is deteriorated due to deficiency, incompatibility, and many other causes, and partially, completely blocks, reduces, prevents, or activates TLR activity. May be delayed, inactivated or down regulated. According to one embodiment of the present invention, the peptide or fusion peptide according to the present invention provides TLR4 and TLR3 signaling pathways and NLRP3 inflamasome inhibition. It also provides partial inhibition of TLR1 / 2, TLR2 / 6, TLR7, TLR8 and TLR9.
본 발명에 있어서, 서열번호 1의 아미노산 서열로 이루어진 펩타이드의 S-H-C-R 서열(서열번호 2)은 TLR(Toll-like receptor)의 TIR(Toll/interleukin-1 receptor) 도메인에 특이적으로 결합하는 것을 특징으로 할 수 있다. In the present invention, the SHCR sequence (SEQ ID NO: 2) of the peptide consisting of the amino acid sequence of SEQ ID NO: 1 is characterized in that it specifically binds to the TIR (Toll / interleukin-1 receptor) domain of the TLR (Toll-like receptor) can do.
TLR4의 TIR 도메인과 결합함에 있어서, 본 발명에 따른 서열번호 1의 아미노산 서열로 이루어진 펩타이드는 서열 특이성을 가진다. 본 발명자들은 TLR4의 TIR 도메인과 결합하는 TIP1의 아미노산 최소부위를 탐색하고, 이 영역이 TLR4의 신호전달 경로를 효과적으로 억제할 수 있는지 알아보았다. 즉, 본 발명의 일실시예에 따르면, TIRAP의 TIR 도메인으로부터 decoy peptide를 선별하고 이의 아미노산 서열인 S-H-C-R-V-L-L-I를 이용하여 S-H-C-R(decoy peptide 1-2, 서열번호 2) 및 V-L-L-I(decoy peptide 1-3, 서열번호 7) 서열을 실시예 1-1에서 사용한 것과 동일한 CPP 서열의 N-말단에 각각 결합시켜 HEK-BlueTM-hTLR4 세포 및 hPBMC 세포에 LPS와 함께 처리한 후, 각각으로부터 NF-κB 활성 및 NO 분비량을 측정하였다. 그 결과, 상기 S-H-C-R(decoy peptide 1-2) 서열이 억제제 효과를 내는데 중요함을 확인하였다.In binding to the TIR domain of TLR4, the peptide consisting of the amino acid sequence of SEQ ID NO: 1 according to the invention has sequence specificity. The present inventors searched for the amino acid minimum of TIP1 that binds to the TIR domain of TLR4 and examined whether this region could effectively inhibit the signaling pathway of TLR4. That is, according to one embodiment of the present invention, decoy peptides are selected from the TIR domain of TIRAP and SHCR (decoy peptide 1-2, SEQ ID NO: 2) and VLLI (decoy peptide 1-3, VLLI) using the amino acid sequence SHCRVLLI. SEQ ID NO: 7) Binding the sequence to the N-terminus of the same CPP sequence as used in Example 1-1, respectively, to treat HEK-Blue TM -hTLR4 cells and hPBMC cells with LPS, followed by NF-κB activity and NO secretion was measured. As a result, it was confirmed that the SHCR (decoy peptide 1-2) sequence is important for producing an inhibitory effect.
또한, 본 발명은 세포 투과성 펩타이드의 N-말단에 서열번호 1 또는 서열번호 2의 아미노산 서열로 이루어진 펩타이드가 연결된 융합 펩타이드를 제공한다.The present invention also provides a fusion peptide wherein a peptide consisting of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2 is linked to the N-terminus of the cell permeable peptide.
본 발명에 있어서, 상기 융합 펩타이드는 TLR(Toll-like receptor)에 의해 매개되는 신호전달 경로를 억제할 수 있고, 상기 TLR(Toll-like receptor)은 TLR1/2(Toll-like receptor 1/2), TLR2/6(Toll-like receptor 2/6), TLR3(Toll-like receptor 3), TLR4(Toll-like receptor 4), TLR7(Toll-like receptor 7), TLR8(Toll-like receptor 8) 및 TLR9(Toll-like receptor 9)로 이루어진 군으로부터 선택되는 어느 하나일 수 있고, 바람직하게는 TLR4(Toll-like receptor 4) 또는 TLR3(Toll-like receptor 3)일 수 있고, 보다 바람직하게는 TLR4(Toll-like receptor 4)인 것을 특징으로 할 수 있다.In the present invention, the fusion peptide may inhibit the signaling pathway mediated by TLR (Toll-like receptor), the TLR (Toll-like receptor) is TLR1 / 2 (Toll-like receptor 1/2) , Toll-like receptor 2/6 (TLR2 / 6), toll-like receptor 3 (TLR3), toll-like receptor 4 (TLR4), toll-like receptor 7 (TLR7), toll-like receptor 8 (TLR8), and It may be any one selected from the group consisting of TLR9 (Toll-like receptor 9), preferably TLR4 (Toll-like receptor 4) or TLR3 (Toll-like receptor 3), more preferably TLR4 ( Toll-like receptor 4) can be characterized.
또한, 본 발명에 있어서, 상기 융합 펩타이드의 TLR 신호전달경로 차단에 의해 TNF-α, IL-6 또는 IFN-β의 발현 억제; NO 또는 ROS의 분비 억제; 또는 NF-κB, MAPK 또는 NLRP3 인플라마좀의 활성이 억제될 수 있으며, 상기 융합 펩타이드는 MyD88-의존적 및 MyD88-비의존적 TLR4 신호전달 경로를 모두 억제하는 것을 특징으로 할 수 있다.In the present invention, the expression of TNF-α, IL-6 or IFN-β by blocking the TLR signaling pathway of the fusion peptide; Inhibition of secretion of NO or ROS; Alternatively, the activity of NF-κB, MAPK or NLRP3 inflamasomes may be inhibited, and the fusion peptide may be characterized by inhibiting both MyD88-dependent and MyD88-independent TLR4 signaling pathways.
본 발명에서 용어, “세포 투과성 펩타이드(cell penetrating peptide, CPP)”는 일종의 신호 펩타이드(Signal Peptide) 로서 단백질, DNA, RNA 등과 같은 고분자 물질을 세포 내로 전달하고자 하는 목적으로 사용되는 일종의 특정 아미노산 서열의 조합인 펩타이드이다. 현재까지 다양한 저분자 화합물, 단백질, 펩타이드, RNA, DNA 등 고분자 물질의 세포 내 전달을 위해 이용되고 있다.In the present invention, the term "cell penetrating peptide (CPP)" is a kind of signal peptide (Signal Peptide) of a kind of specific amino acid sequence used for the purpose of delivering a polymer material such as protein, DNA, RNA, etc. into the cell Peptides in combination. To date, various low molecular compounds, proteins, peptides, RNA, DNA, etc. have been used for intracellular delivery of high molecular materials.
본 발명의 융합 펩타이드는 세포 투과성 펩타이드를 이용하고 있으며, 상기 세포 투과성 펩타이드는 세포 내재화(endocytosis) 기전에 의해 세포 내로 들어가는 특징을 가지고 있는 것이라면 특별히 제한되지 않으나, 바람직하게는 하기 표 1에 나열된 세포 투과성 펩타이드 또는 이의 변이체 중에서 선택하여 사용할 수 있다.The fusion peptide of the present invention uses a cell-penetrating peptide, and the cell-penetrating peptide is not particularly limited as long as it has a characteristic of entering into a cell by a mechanism of cell internalization, but preferably the cell permeability listed in Table 1 below. It can be selected and used among the peptides or variants thereof.
펩타이드Peptide 유래origin 서열order 서열번호SEQ ID NO:
PenetratinPenetratin Drosophila Antennapedia homeodomainDrosophila Antennapedia homeodomain RQIKIWFQNRRMKWKKRQIKIWFQNRRMKWKK 99
TAT(48-60) TAT (48-60) Human immunodeficiency virus type 1 (HIV-1) TATHuman immunodeficiency virus type 1 (HIV-1) TAT GRKKRRQRRRPPQGRKKRRQRRRPPQ 1010
pVECpVEC VE-Cadherin(615-632)VE-Cadherin (615-632) LLIILRRRIRKQAHAHSKLLIILRRRIRKQAHAHSK 1111
Transportan 10/TP10Transportan 10 / TP10 Galanin-Lys-mastoparanGalanin-lys-mastoparan GWTLNSAGYLLGKINLKALAALAKKILGWTLNSAGYLLGKINLKALAALAKKIL 1212
MPGMPG A hydrophobic domain from the fusion sequence of HIV gp41 and NLS of SV40 T-antigenA hydrophobic domain from the fusion sequence of HIV gp41 and NLS of SV40 T-antigen GALFLGFLGAAGSTMGAWSQPKKKRKVGALFLGFLGAAGSTMGAWSQPKKKRKV 1919
Pep-1Pep-1 NLS from Simian Virus 40 large T antigen and reverse transcriptase of HIV-1NLS from Simian Virus 40 large T antigen and reverse transcriptase of HIV-1 KETWWETWWTEWSQPKKKRKVKETWWETWWTEWSQPKKKRKV 2020
MAPMAP Amphipathic model peptideAmphipathic model peptide KLALKLALKALKAALKLAKLALKLALKALKAALKLA 2121
R6/W3 R 6 / W 3 Based on penetratinBased on penetratin RRWWRRWRRRRWWRRWRR 2222
Polyarginine(R9,R8)Polyarginine (R 9 , R 8 ) Positively charged sequencePositively charged sequence Rn, n = 8 or 9R n , n = 8 or 9 23,2423,24
VP22VP22 Herpes simplex virusHerpes simplex virus NAKTRRHERRRKLAIERNAKTRRHERRRKLAIER 2525
YTA2YTA2 MMP cleavage site as seeding sequenceMMP cleavage site as seeding sequence YTAIAWVKAFIRKLRKYTAIAWVKAFIRKLRK 2626
YTA4YTA4 MMP cleavage site as seeding sequenceMMP cleavage site as seeding sequence IAWVKAFIRKLRKGPLGIAWVKAFIRKLRKGPLG 2727
M918M918 The tumor suppressor protein p14ARFThe tumor suppressor protein p14ARF MVTVLFRRLRIRRACGPPRVRVMVTVLFRRLRIRRACGPPRVRV 2828
CADYCADY Derived from PPTG1 peptide, W and charged amino acidsDerived from PPTG1 peptide, W and charged amino acids GLWRALWRLLRSLWRLLWRAGLWRALWRLLRSLWRLLWRA 2929
SAPSAP Designed based on a natural protein of maize, γ-zein VHL (PPP)8 Designed based on a natural protein of maize, γ-zein VHL (PPP) 8 (VRLLPPP)3 (VRLLPPP) 3 3030
SAP(E)SAP (E) Design inspired by SAP; Arg residue replaced by GluDesign inspired by SAP; Arg residue replaced by Glu Ac-CGGW(VELPPP)3Ac-CGGW (VELPPP) 3 3131
CyLoP-1CyLoP-1 Derived from crotamine toxin found in snake venom, crot(27-39)Derived from crotamine toxin found in snake venom, crot (27-39) CRWRWKCCKKCRWRWKCCKK 3232
gH 625gH 625 Based on the 625-644 residues of the glycoprotein HSV 1Based on the 625-644 residues of the glycoprotein HSV 1 HGLASTLTRWAHYNALIRAFHGLASTLTRWAHYNALIRAF 3333
GALAGALA Glu-rich an containing His (imidazole group) in order to be pH responsive (endosomes) (Designed to efficiently escape endosomes)Glu-rich an containing His (imidazole group) in order to be pH responsive (endosomes) (Designed to efficiently escape endosomes) WEAALAEALAEALAEHLAEALAEALEALAAWEAALAEALAEALAEHLAEALAEALEALAA 3434
CADYCADY Designed; based on chimeric peptide carrier PPTG1 derived from the fusion peptide JTS1Designed; based on chimeric peptide carrier PPTG1 derived from the fusion peptide JTS1 Ac-GLWRALWRLLRSLWRLLWRA-cysteamideAc-GLWRALWRLLRSLWRLLWRA-cysteamide 3535
L17EL17E Inspired by the spider venom M-lycotoxinInspired by the spider venom M-lycotoxin IWLTALKFLGKHAAKHEAKQQLSKLIWLTALKFLGKHAAKHEAKQQLSKL 3636
MPPsMPPs Designed to contain un-natural, cyclohexylalanine (Fx) residues and to have differential intracellular localizationDesigned to contain un-natural, cyclohexylalanine (F x ) residues and to have differential intracellular localization Mitochondria-penetrating peptides (example: FXrFXKFXrFXK)Mitochondria-penetrating peptides (example: F X rF X KF X rF X K) 3737
RR5-APPRR4-APPRR3-APPRR 5 -APPRR 4 -APPRR 3 -APP Small proteins (36-residue polypeptides)Small proteins (36-residue polypeptides) RPRRPRRPRRPGRRAPVEDLIRFYNDLQQYLNVVTRHRYCRPRRPRRPRRPGRRAPVEDLIRFYNDLQQYLNVVTRHRYC 3838
RRPRRPRRPGRRAPVEDLIRFYNDLQQYLNVVTRHRYCRRPRRPRRPGRRAPVEDLIRFYNDLQQYLNVVTRHRYC 3939
GPRRPRRPGRRAPVEDLIRFYNDLQQYLNVVTRHRYCGPRRPRRPGRRAPVEDLIRFYNDLQQYLNVVTRHRYC 4040
TATp-DTATp-D Analogue of TATAnalogue of TAT Ac-GRKKRRQRRRPPQ-K-KAc-GRKKRRQRRRPPQ- K -K 4141
Ac-GRKKRRQRRRPPQ Ac-GRKKRRQRRRPP Q 4242
Cyclic TatCyclic tat Lys- and Glu- amino acids added to the linear Tat sequence to obtain a ring with the same overall charge as the native formLys- 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]cc [K-rRrQrRkKrG-E] c 4343
K10(QW)6 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 nanostructuresThe design was based on combining W and K at the primary structure level to obtain self-assembly into a variety of nanostructures KKKKKKKKKKQWQWQWQWQWQWKKKKKKKKKKQWQWQWQWQWQW 4444
TAT (49-57) TAT (49-57) HIV-1 TAT proteinHIV-1 TAT protein RKKRRQRRRRKKRRQRRR 4545
DPV1047DPV1047 Chemically synthesizedChemically synthesized VKRGLKLRHVRPRVTRMDVVKRGLKLRHVRPRVTRMDV 4646
ARF(1-22) ARF (1-22) p14ARF proteinp14ARF protein MVRRFLVTLRIRRACGPPRVRVMVRRFLVTLRIRRACGPPRVRV 4747
BPrPr(1-28) BPrPr (1-28) N terminus of unprocessed bovine prion proteinN terminus of unprocessed bovine prion protein MVKSKIGSWILVLFVAMWSDVGLCKKRPMVKSKIGSWILVLFVAMWSDVGLCKKRP 4848
p28p28 AzurinAzurin LSTAADMQGVVTDGMASGLDKDYLKPDDLSTAADMQGVVTDGMASGLDKDYLKPDD 4949
VT5VT5 Chemically synthesizedChemically synthesized DPKGDPKGVTVTVTVTVTGKGDPKPDDPKGDPKGVTVTVTVTVTGKGDPKPD 5050
C105YC105Y α1-Antitrypsinα1-Antitrypsin CSIPPEVKFNKPFVYLICSIPPEVKFNKPFVYLI 5151
PFVYLIPFVYLI Derived from synthetic C105YDerived from synthetic C105Y PFVYLIPFVYLI 5252
한편, 상기 표 1의 세포 투과성 펩타이드 중 Transportan은 다음와 같은 변이체의 형태로 사용되는 것을 포함한다: AGYLLGKINLKALAALAKKIL-NH2(TP10, PepFect 3, 서열번호 13), AGYLLGKINLKALAALAKKIL-NH2(TP10, PepFect 6, 서열번호 14), AGYLLGKLLOOLAAAALOOLL-NH2(TP10, PepFect 14, 서열번호 15), AGYLLGKTNLKALAALAKKIL-NH2(NickFect 1, 서열번호 16), AGYLLGKTNLKALAALAKKIL-NH2(NickFect 2, 서열번호 17) 및 AGYLLGKTNLKALAALAKKIL-NH2(Nickfect 3, 서열번호 18). Meanwhile, the transportan of the cell permeable peptides of Table 1 includes those used in the form of the following variants: AGYLLGKINLKALAALAKKIL-NH 2 (TP10, PepFect 3, SEQ ID NO: 13), AGYLLGKINLKALAALAKKIL-NH 2 (TP10, PepFect 6, sequence No. 14), AGYLLGKLLOOLAAAALOOLL-NH 2 ( TP10, PepFect 14, SEQ ID NO: 15), AGYLLGKTNLKALAALAKKIL-NH 2 ( NickFect 1, SEQ ID NO: 16), AGYLLGKTNLKALAALAKKIL-NH 2 ( NickFect 2, SEQ ID NO: 17) and AGYLLGKTNLKALAALAKKIL-NH 2 ( Nickfect 3, SEQ ID NO: 18).
또한, 상기 표 1의 서열번호 37로 이루어진 아미노산 서열(MPPs)에 있어서 r은 d-Arginine을 의미하며, 서열번호 37의 아미노산 서열 내 2, 6번째 아미노산이 이에 해당한다. 아울러, 상기 표 1의 서열번호 41 및 42는 서로 측쇄를 이루는 하나의 펩타이드(TATp-D)를 구성하며, 서열번호 41의 아미노산 서열 내 14번째 Lys 잔기(K)가 서열번호 42의 아미노산 서열 내 13번째 Gln 잔기(Q)와 연결되어 측쇄를 이루는 것을 특징으로 한다. 나아가, 상기 표 1의 서열번호 43으로 이루어진 아미노산 서열(Cyclic Tat)에 있어서, r은 d-Arginine을 의미하며, 서열번호 43의 아미노산 서열 내 2, 4, 6, 10번째 아미노산이 이에 해당한다. 마찬가지로, k는 d-Lysine을 의미하며, 서열번호 43의 아미노산 서열 내 8번째 아미노산이 이에 해당한다. 상기 서열번호 43은 고리형 펩타이드인 것을 특징으로 한다.In addition, in the amino acid sequence (MPPs) consisting of SEQ ID NO: 37 of Table 1, r means d -Arginine, the second and sixth amino acid in the amino acid sequence of SEQ ID NO: 37 corresponds to this. In addition, SEQ ID NO: 41 and 42 of Table 1 constitutes one peptide (TATp-D) which is branched to each other, the 14th Lys residue (K) in the amino acid sequence of SEQ ID NO: 41 in the amino acid sequence of SEQ ID NO: 42 It is characterized in that the side chain is linked to the 13th Gln residue (Q). Furthermore, in the amino acid sequence (Cyclic Tat) consisting of SEQ ID NO: 43 of Table 1, r means d -Arginine, the 2nd, 4, 6, 10th amino acid in the amino acid sequence of SEQ ID NO: 43 corresponds to this. Likewise, k means d- Lysine, which corresponds to the eighth amino acid in the amino acid sequence of SEQ ID NO. SEQ ID NO: 43 is characterized in that the cyclic peptide.
본 발명의 일 실시예에서는 상기 표 1의 세포 투과성 펩타이드 중 Penetratin 서열(RQIKIWFQNRRMKWKK, 서열번호 9)을 선택하여 실험을 수행하였으며, 상기 실제로 사용한 세포 투과성 펩타이드 외에 다른 세포 투과성 펩타이드를 본 발명의 펩타이드와 융합시킨 경우에도 본 발명과 유사한 효과가 나타남은 당업자에게 자명할 것이다.In an embodiment of the present invention, the experiment was performed by selecting the Penetratin sequence (RQIKIWFQNRRMKWKK, SEQ ID NO: 9) among the cell permeable peptides of Table 1, and other cell permeable peptides in addition to the actually used cell permeable peptides were fused with the peptides of the present invention. In this case, it will be apparent to those skilled in the art that similar effects to the present invention can be obtained.
본 발명에 있어서, 세포 투과성 펩타이드의 N-말단에 서열번호 1 또는 서열번호 2의 아미노산 서열로 이루어진 펩타이드가 연결된 상기 융합 펩타이드는 바람직하게는 서열번호 3 또는 서열번호 4의 아미노산 서열로 이루어진 것을 특징으로 할 수 있다. 또한, 상기 아미노산 서열의 변이체도 본 발명의 범위 내에 포함될 수 있으며, 구체적으로 상기 변이체는 서열번호 3 또는 4와 각각 70% 이상, 바람직하게는 80% 이상, 더욱 바람직하게는 90% 이상, 보다 더욱 바람직하게는 95% 이상, 더욱더 바람직하게는 98% 이상, 가장 바람직하게는 99% 이상의 서열 상동성을 가지는 펩타이드를 모두 포함할 수 있다. 상기 용어 "상동성" 이란 야생형(wild type) 아미노산 서열 및 야생형 핵산 서열과의 유사한 정도를 나타내기 위한 것을 말한다.In the present invention, the fusion peptide is linked to the peptide consisting of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2 at the N-terminal end of the cell permeable peptide, characterized in that consisting of the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 4 can do. In addition, variants of the amino acid sequence may also be included within the scope of the present invention, and specifically, the variants may be 70% or more, preferably 80% or more, more preferably 90% or more, and even more, each of SEQ ID NO: 3 or 4, respectively. Preferably at least 95%, even more preferably at least 98%, most preferably at least 99% of all peptides having sequence homology. The term "homology" refers to a degree of similarity with a wild type amino acid sequence and a wild type nucleic acid sequence.
본 발명의 일 실시예에 따르면, 본 발명에 따른 융합 펩타이드는 리포폴리사카라이드(LPS)에 의해 유도되는 TLR4 신호전달 경로를 억제함으로써 사이토카인(IL-6, TNF-α, IFN-β), NO 및 ROS의 분비와 NF-κB와 MAPK의 활성화를 억제함은 물론 동물에서 TLR4의 과활성에 의한 신장과 간 손상, 패혈증 및 류마티스 관절염 완화와 Poly(I:C)에 의해 유도된 TLR3 신호전달 경로 및 LPS/ATP에 의해 유도된 NLRP3 인플라마좀 형성을 억제하는 효과가 우수한바, 상기 신호전달 경로에 의해 발생하는 자가면역 질환, 염증성 질환 또는 NLRP3 인플라마좀에 의한 퇴행성 신경질환을 예방 및 치료하는데 유용하게 활용할 수 있다.According to one embodiment of the invention, the fusion peptide according to the present invention by inhibiting the TLR4 signaling pathway induced by lipopolysaccharide (LPS) cytokines (IL-6, TNF-α, IFN-β), TLR3 signaling induced by Poly (I: C), which inhibits NO and ROS secretion and activation of NF-κB and MAPK, as well as relieving kidney and liver damage, sepsis and rheumatoid arthritis due to overactivation of TLR4 in animals It is excellent in inhibiting the pathway and LPS / ATP-induced NLRP3 inflamasome formation, preventing and treating autoimmune diseases, inflammatory diseases or neurodegenerative diseases caused by NLRP3 inflamasome caused by the signaling pathway It can be useful to
또한, 본 발명에 있어서, 상기 본 발명에 따른 융합 펩타이드는 TLR4, TLR3 및 NLRP3 인플라마좀 억제제로서 사용될 수 있다.In addition, in the present invention, the fusion peptide according to the present invention can be used as TLR4, TLR3 and NLRP3 inflammasome inhibitor.
본 발명에서 용어, “억제제”는 임의의 메커니즘에 의하여, 수용체 또는 세포 내 매개체와 같은 다른 분자의 영향을 부분적으로 또는 완전히 저해하는 분자를 의미한다.As used herein, the term “inhibitor” refers to a molecule that, by any mechanism, partially or completely inhibits the effects of other molecules, such as receptors or intracellular mediators.
본 발명에서 용어, “TLR4, TLR3 및 NLRP3 인플라마좀 억제제”는 TLR4, TLR3 및 NLRP3 인플라마좀의 생물학적 활성을 직간접적으로, 또는 실질적으로 방해, 감소 또는 저해할 수 있는 물질을 말하며, 바람직하게는 TLR4, TLR3와 반응성인 펩타이드는 TLR4, TLR3의 TIR 도메인에 직접 결합하고, TLR4, TLR3의 활성을 중화시킴으로써 TLR4, TLR3 신호전달 경로를 차단하여, NF-κB 및 MAPK 그리고 NLRP3 인플라마좀 활성화의 감소를 유발해 염증성 사이토카인, NO 및 ROS의 분비를 감소시킬 수 있는 물질을 말한다.As used herein, the term “TLR4, TLR3 and NLRP3 inflammasome inhibitor” refers to a substance that can directly, indirectly, or substantially interfere with, reduce or inhibit the biological activity of TLR4, TLR3 and NLRP3 inflammasomes, preferably Peptides reactive with TLR4, TLR3 bind directly to the TIR domains of TLR4, TLR3 and neutralize the activity of TLR4, TLR3, thereby blocking TLR4, TLR3 signaling pathways, thereby inhibiting NF-κB and MAPK and NLRP3 inflammasome activation. A substance that can cause a decrease in the secretion of inflammatory cytokines, NO and ROS.
또한, 본 발명은 서열번호 1 또는 서열번호 2의 아미노산 서열로 이루어진 펩타이드를 유효성분으로 포함하는 자가면역질환, 염증성 질환 및 퇴행성 신경질환으로 이루어진 군으로부터 선택되는 1종 이상의 TLR 경로 매개성 질환의 예방 또는 치료용 약학적 조성물을 제공한다.The present invention also provides for the prevention of one or more TLR pathway mediated diseases selected from the group consisting of autoimmune diseases, inflammatory diseases and neurodegenerative diseases comprising peptides consisting of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2 as an active ingredient Or it provides a pharmaceutical composition for treatment.
또한, 본 발명은 세포 투과성 펩타이드의 N-말단에 서열번호 1 또는 서열번호 2의 아미노산 서열로 이루어진 펩타이드가 연결된 융합 펩타이드를 유효성분으로 포함하는 자가면역질환, 염증성 질환 및 퇴행성 신경질환으로 이루어진 군으로부터 선택되는 1종 이상의 TLR 경로 매개성 질환의 예방 또는 치료용 약학적 조성물을 제공한다. 상기 융합 펩타이드는 바람직하게는 서열번호 3 또는 서열번호 4의 아미노산 서열로 이루어진 것을 특징으로 할 수 있으며, 상기 아미노산 서열의 변이체도 본 발명의 범위 내에 포함될 수 있다.In addition, the present invention from the group consisting of autoimmune diseases, inflammatory diseases and neurodegenerative diseases comprising a fusion peptide linked to the peptide consisting of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2 at the N-terminal end of the cell permeable peptide as an active ingredient Provided are pharmaceutical compositions for the prophylaxis or treatment of one or more selected TLR pathway mediated diseases. The fusion peptide is preferably characterized in that consisting of the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 4, variants of the amino acid sequence may also be included within the scope of the present invention.
본 발명에서 용어, “경로 매개성 질환”은 하나 이상의 TLR의 활성화 및 TLR 매개 신호전달 경로가 기여 요인인 임의의 병리학적 상태를 의미한다. 상기 상태는 이로써 제한되는 것은 아니지만, 바람직하게는 자가면역 질환, 염증성 질환 및 퇴행성 질환으로 이루어진 군으로부터 선택되는 1종 이상인 것을 특징으로 할 수 있다.As used herein, the term "pathogenic disease" refers to any pathological condition in which the activation of one or more TLRs and the TLR mediated signaling pathway are contributing factors. The condition is not limited thereto, but may be preferably one or more selected from the group consisting of autoimmune diseases, inflammatory diseases and degenerative diseases.
본 발명에서 용어, “자가면역 질환”은 자기관용을 유도하거나 계속 유지하는데 있어서 문제가 발생하여 자기항원에 대한 면역반응이 일어나, 이로 인해 자신의 조직을 공격하는 현상이 발생하는 과정에 의해 발병되는 질환을 의미한다. 상기 자기관용이란 자기(self)를 구성하고 있는 항원물질에 대해서는 해롭게 반응하지 않는 면역학적 무반응성(immunologic unresponsiveness)을 말한다. 본 발명의 자기면역 질환은 인슐린-의존성 당뇨병, 다발 경화증, 실험적 자가면역 뇌척수염, 류마티스성 관절염, 실험적 자가면역 관절염, 중증 근무력증, 갑상선염, 실험적 형태의 포도막염, 하시모토 갑상선염, 원발성 점액수종, 갑상샘 중독증, 악성 빈혈, 자가면역 위축 위염, 애디슨 질환, 조기 폐경, 남성 불임증, 소아 당뇨병, 굿파스처 증후군, 보통 천포창, 유천포창, 교감성 안염, 수정체성 포도막염, 자가면역 용혈성 빈혈, 특발성 백혈구 감소, 원발성 담관 경화증, 만성 활동성 간염 Hbs-ve, 잠재성 간경변증, 궤양성 대장염, 쇼그렌 증후군, 경피증, 베게너 육아종증, 다발근육염/피부근육염, 원판상 LE 및 전신 홍반 루푸스를 포함하나, 이에 한정되는 것은 아니다.In the present invention, the term "autoimmune disease" is caused by a process in which a problem occurs in inducing or maintaining self-tolerance to cause an immune response to an autoantigen, thereby attacking its own tissue. Means disease. The self-tolerance refers to immunologic unresponsiveness that does not deleteriously react with the antigenic material constituting self. Autoimmune diseases of the present invention include insulin-dependent diabetes mellitus, multiple sclerosis, experimental autoimmune encephalomyelitis, rheumatoid arthritis, experimental autoimmune arthritis, myasthenia gravis, thyroiditis, experimental form of uveitis, Hashimoto thyroiditis, primary myxedema, thyroid poisoning, malignant Anemia, autoimmune atrophy gastritis, Addison's disease, premature menopause, male infertility, childhood diabetes, Goodpasture syndrome, common pemphigus, leiomyelitis, sympathetic ophthalmitis, lens uveitis, autoimmune hemolytic anemia, idiopathic leukocyte reduction, primary cholangiovascular sclerosis , Chronic active hepatitis Hbs-ve, latent cirrhosis, ulcerative colitis, Sjogren's syndrome, scleroderma, Wegener's granulomatosis, polymyositis / skin myositis, discoid LE and systemic lupus erythematosus.
또한, 본 발명에서 용어, “염증성 질환”은 염증유발인자 또는 방사선조사 등 유해한 자극으로 인해 면역체계를 과도하게 항진시켜 대식세포와 같은 면역세포에서 분비되는 TNF-α, IL-1, IL-6, 프로스타글란딘(prostaglandin), 루코트리엔(leukotriene) 또는 NO와 같은 염증 유발물질(염증성 사이토카인)에 의해 유발되는 질환을 의미하며, 본 발명의 염증성 질환은 천식, 습진, 건선, 알러지, 류마티스 관절염, 건선 관절염, 아토피성 피부염, 여드름, 아토피성 비염, 폐염증, 알레르기성 피부염, 만성 부비동염, 접촉성 피부염(contact dermatitis), 지루성 피부염(seborrheic dermatitis), 위염, 통풍, 통풍 관절염, 궤양, 만성 기관지염, 크론병, 궤양성 대장염, 강직성 척추염(ankylosing spondylitis), 패혈증, 맥관염, 활액낭염, 루프스, 류마티스 다발성 근육통, 측두 동맥염, 다발성 경화증, 고형암, 알츠하이머병, 동맥경화증, 비만 및 바이러스 감염을 포함하나, 이에 한정되는 것은 아니다.In addition, in the present invention, the term "inflammatory disease" refers to TNF-α, IL-1, IL-6, which are secreted by immune cells such as macrophages by excessively promoting the immune system due to harmful stimuli such as inflammation-inducing factors or irradiation. , Prostaglandin, leukotriene, or means a disease caused by inflammatory agents (inflammatory cytokines), such as NO, the inflammatory diseases of the present invention are asthma, eczema, psoriasis, allergy, rheumatoid arthritis , Psoriatic arthritis, atopic dermatitis, acne, atopic rhinitis, pneumonia, allergic dermatitis, chronic sinusitis, contact dermatitis, seborrheic dermatitis, gastritis, gout, gouty arthritis, ulcer, chronic bronchitis , Crohn's disease, ulcerative colitis, ankylosing spondylitis, sepsis, vasculitis, bursitis, lupus, rheumatoid polymyalgia, temporal arteritis, multiple sclerosis One including solid tumors, Alzheimer's disease, atherosclerosis, obesity and viral infection, but is not limited thereto.
본 발명에서 용어, “퇴행성 신경질환” 은 신경세포의 기능 감소 또는 소실에 의해 운동조절능력, 인지기능, 지각기능, 감각기능 및 자율신경의 기능 이상을 말하며, 주로 임상적 특징으로 분류되는데 주요 증상과 침범 부위를 기준으로 나누어진다. 본 발명의 퇴행성 신경질환은 알츠하이머병, 전두측두치매, 루이치매, 피질기저퇴행증, 파킨슨병, 다계통위축병, 헌팅턴병, 진핵성핵상마비, 루게릭병, 원발성측삭경화증, 척수근육위축병을 포함하나, 이에 한정되는 것은 아니다.In the present invention, the term "degenerative neuropathy" refers to a motor dysfunction, cognitive function, perceptual function, sensory function, and autonomic nerve abnormalities due to a decrease or loss of neuronal function, mainly classified as a clinical feature. And involvement sites. Neurodegenerative diseases of the present invention include Alzheimer's disease, frontal temporal dementia, Louis dementia, cortical hypodegeneration, Parkinson's disease, multiple system atrophy, Huntington's disease, eukaryotic nucleus palsy, Lou Gehrig's disease, primary lateral sclerosis, spinal muscular atrophy However, the present invention is not limited thereto.
본 발명의 약학적 조성물은 약학적으로 유효한 양의 상기 펩타이드를 단독으로 포함하거나 하나 이상의 약학적으로 허용되는 담체, 부형제 또는 희석제를 포함할 수 있다. 상기에서 약학적으로 유효한 양이란 자가면역 질환의 증상을 예방, 개선 및 치료하기에 충분한 양을 말한다.The pharmaceutical composition of the present invention may include a pharmaceutically effective amount of the peptide alone or may include one or more pharmaceutically acceptable carriers, excipients or diluents. The pharmaceutically effective amount herein refers to an amount sufficient to prevent, ameliorate and treat the symptoms of the autoimmune disease.
상기 “약학적으로 허용되는” 이란 생리학적으로 허용되고 인간에게 투여될 때, 통상적으로 위장 장애, 현기증과 같은 알레르기 반응 또는 이와 유사한 반응을 일으키지 않는 조성물을 말한다. 상기 담체, 부형제 및 희석제의 예로는, 락토즈, 덱스트로즈, 수크로즈, 솔비톨, 만니톨, 자일리톨, 에리스리톨, 말티톨, 전분, 아카시아 고무, 알지네이트, 젤라틴, 칼슘 포스페이트, 칼슘 실리케이트, 셀룰로즈, 메틸 셀룰로즈, 폴리비닐피롤리돈, 물, 메틸하이드록시벤조에이트, 프로필하이드록시벤조에이트, 탈크, 마그네슘 스테아레이트 및 광물유를 들 수 있다. 또한, 충진제, 항응집제, 윤활제, 습윤제, 향료, 유화제 및 방부제 등을 추가로 포함할 수 있다.The term “pharmaceutically acceptable” refers to a composition that is physiologically acceptable and, when administered to a human, typically does not cause gastrointestinal disorders, allergic reactions such as dizziness or the like. Examples of such carriers, 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.
또한, 본 발명의 약학적 조성물은 상기 펩타이드와 함께 자가면역 질환, 염증성 질환 또는 퇴행성 신경질환 치료 효과를 갖는 공지의 유효성분을 1종 이상 포함할 수 있다.In addition, the pharmaceutical composition of the present invention may include at least one known active ingredient having an effect of treating autoimmune diseases, inflammatory diseases or neurodegenerative diseases with the peptide.
본 발명의 약학적 조성물은 포유동물에 투여된 후 활성 성분의 신속, 지속 또는 지연된 방출을 제공할 수 있도록 당업계에 공지된 방법을 사용하여 제형화될 수 있다. 제형은 분말, 과립, 정제, 에멀젼, 시럽, 에어로졸, 연질 또는 경질 젤라틴 캅셀, 멸균 주사용액, 멸균 분말의 형태일 수 있다.The pharmaceutical compositions of the 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. The formulations may be in the form of powders, granules, tablets, emulsions, syrups, aerosols, soft or hard gelatin capsules, sterile injectable solutions, sterile powders.
본 발명의 약학적 조성물은 경구, 경피, 피하, 정맥 또는 근육을 포함한 여러 경로를 통해 투여될 수 있으며, 활성 성분의 투여량은 투여 경로, 환자의 연령, 성별, 체중 및 환자의 중증도 등의 여러 인자에 따라 적절히 선택될 수 있고, 자가면역 질환, 염증성 질환 또는 퇴행성 신경질환의 증상을 예방, 개선 또는 치료하는 효과를 가지는 공지의 화합물과 병행하여 투여할 수 있다.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 vary depending on the route of administration, the age, sex, weight and severity of the patient. It may be appropriately selected depending on factors, and may be administered in combination with known compounds having the effect of preventing, ameliorating or treating symptoms of autoimmune disease, inflammatory disease or neurodegenerative disease.
또한, 본 발명은 서열번호 1 또는 서열번호 2의 아미노산 서열로 이루어진 펩타이드를 이를 필요로 하는 개체에 투여하는 단계; 를 포함하는 TLR 경로 매개성 질환의 예방 또는 치료 방법을 제공한다.In addition, the present invention comprises the steps of administering a peptide consisting of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2 to a subject in need thereof; It provides a method for preventing or treating a TLR pathway mediated disease comprising a.
또한, 본 발명은 세포 투과성 펩타이드의 N-말단에 서열번호 1 또는 서열번호 2의 아미노산 서열로 이루어진 펩타이드가 연결된 융합 펩타이드를 이를 필요로 하는 개체에 투여하는 단계; 를 포함하는 TLR 경로 매개성 질환의 예방 또는 치료 방법을 제공한다.In addition, the present invention comprises the steps of administering to a subject in need thereof a fusion peptide linked to a peptide consisting of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2 at the N-terminal end of the cell permeable peptide; It provides a method for preventing or treating a TLR pathway mediated disease comprising a.
상기 개체는 인간을 포함한 포유류인 것이 바람직하며, TLR 경로 매개성 질환 치료를 필요로 하는 환자로 TLR 경로 매개성 질환 치료 중인 환자, TLR 경로 매개성 질환 치료를 받은 적이 있는 환자, TLR 경로 매개성 질환 치료를 받을 필요가 있는 환자를 모두 포함하며, TLR 경로 매개성 질환 치료를 위하여 외과적 수술을 시행한 환자 또한 포함될 수 있다.Preferably, the subject is a mammal including a human, a patient in need of treatment for a TLR pathway mediated disease, a patient being treated for a TLR pathway mediated disease, a patient who has been treated for a TLR pathway mediated disease, a TLR pathway mediated disease This includes all patients who need to be treated and may also include patients who have undergone surgical operations to treat TLR pathway mediated diseases.
또한 본 발명의 서열번호 1 또는 서열번호 2의 아미노산 서열로 이루어진 펩타이드; 또는 세포 투과성 펩타이드의 N-말단에 서열번호 1 또는 서열번호 2의 아미노산 서열로 이루어진 펩타이드가 연결된 융합 펩타이드는 이 외 기존의 TLR 경로 매개성 질환 치료를 위한 약물 또는 치료방법과 병용하여 동시에/순차적으로 처리될 수 있다.  In addition, the peptide consisting of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2 of the present invention; Alternatively, a fusion peptide in which the peptide consisting of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2 is linked to the N-terminus of the cell permeable peptide may be simultaneously / sequentially used in combination with other existing drugs or methods for treating TLR pathway mediated diseases. Can be processed.
이하, 본 발명의 내용을 실시예 및 실험예를 통하여 보다 구체적으로 설명한다. 하기 실시예 및 실험예는 본 발명을 예시하는 것일 뿐, 본 발명의 내용이 하기 실시예 및 실험예에 의해 한정되는 것은 아니다.Hereinafter, the content of the present invention will be described in more detail through Examples and Experimental Examples. The following Examples and Experimental Examples are only illustrative of the present invention, and the content of the present invention is not limited by the following Examples and Experimental Examples.
실시예 1. 시험 시료의 준비Example 1. Preparation of Test Samples
1-1. TLR4의 TIR 도메인에 특이적으로 결합하는 펩타이드 선별1-1. Peptide selection that specifically binds to the TIR domain of TLR4
본 발명자들은 TLR4의 TIR 도메인에 특이적으로 결합하는 펩타이드를 선별하기 위해 TIRAP의 TIR 도메인으로부터 2종의 서열(Decoy peptide 1, 2)을 선별하였고 상기 서열의 N 말단에 Drosophila antennapedia 호메오도메인 유래의 세포 투과성 펩타이드(Cell-Penetrating Peptides, CPP)를 연결하여 신규펩타이드 TIP1 및 TIP2를 합성하였다. 본 실시예에서 사용한 모든 펩타이드의 서열을 하기 표 2에 나타내었다.We selected two sequences (Decoy peptide 1, 2) from the TIR domain of TIRAP to select peptides that specifically bind to the TIR domain of TLR4, and at the N terminus of the sequence, Drosophila New peptides TIP1 and TIP2 were synthesized by linking cell-penetrating peptides (CPP) derived from antennapedia homeodomain. The sequences of all peptides used in this example are shown in Table 2 below.
이름name 서열order 서열번호SEQ ID NO:
Decoy peptide 1(TIP1 W/O CPP)Decoy peptide 1 (TIP1 W / O CPP) SHCRVLLISHCRVLLI 1One
TIP1 TIP1 RQIKIWFQNRRMKWKKSHCRVLLI RQIKIWFQNRRMKWKK SHCRVLLI 33
Decoy peptide 2(TIP2 W/O CPP)Decoy peptide 2 (TIP2 W / O CPP) TIPLLS TIPLLS 55
TIP2 TIP2 RQIKIWFQNRRMKWKKTIPLLS RQIKIWFQNRRMKWKK TIPLLS 66
CPP CPP RQIKIWFQNRRMKWKKRQIKIWFQNRRMKWKK 99
* Decoy peptide의 서열을 밑줄로 표시하였다.* The sequence of the Decoy peptide is underlined.
1-2. 세포 배양 및 준비1-2. Cell Culture and Preparation
HEK-BlueTM-hTLR4 세포(InvivoGen, San Diego, CA, USA)를 1%의 페니실린/스트렙토마이신, 10%의 소 태아 혈청(FBS; fetal bovine serum)(Thermo Fisher Scientific Inc., Waltham, MA, USA) 및 0.2%의 노모신(normocin)(InvivoGen)이 첨가된 DMEM(Dulbecco's modified Eagle's medium)(Thermo Fisher Scientific Inc.) 배지에 넣어 배양하였다. 쥐 대식세포인 RAW 264.7 세포(Korean Cell Line Bank, Seoul, Korea)는 1%의 페니실린/스트렙토마이신, 10%의 FBS(Thermo Fisher Scientific, Inc.)가 첨가된 저당 DMEM(Low-Glucose DMEM)에 넣어 배양하였다. 인간 단핵구 세포인 THP1 세포는 1%의 페니실린/스트렙토마이신, 10%의 FBS(Thermo Fisher Scientific, Inc.)가 첨가된 RPMI 1640에서 배양 후, 10 nM의 phorbol 12-myristate 13-acetate(PMA)(Sigma-Aldrich Co. LLC., St. Louis, MO, USA)를 이용하여 24시간 동안 대식세포로 분화 유도하였다. 인간 말초혈액 단핵구 세포인 hPBMC 세포(PromoCell, Heidelberg, Germany)는 2.05mM L-글루타민, 1% 페니실린/스트렙토마이신 및 10%의 FBS가 첨가된 RPMI 1640(Thermo Fisher Scientific Inc.)에서 배양하였다. 쥐 골수유래 대식세포인 mBMDM 세포는 1%의 페니실린/스트렙토마이신과 10%의 FBS가 첨가된 DMEM(Thermo Fisher Scientific Inc.)에서 배양하였다. 모든 세포들은 5% CO2, 37 ℃의 습한 조건의 배양 시스템(Thermo Fisher Scientific Inc.)에서 배양하였고, 배지는 16시간마다 교체하였다. PAM3CSK4, Poly(I:C), R848 및 CpG-ODN은 Thermo Fisher Scientific, Inc.에서, FSL-1은 InvivoGen에서, LPS(Escherichia coli 0111:B4)와 ATP는 Sigma-Aldrich Co.(St. Louis, MO, USA)에서 구입하였고, 실험에 사용된 모든 펩타이드들은 Peptron, Inc.(Daejeon, Korea)에서 합성 및 구입하였다. HEK-Blue ™ -hTLR4 cells (InvivoGen, San Diego, CA, USA) were treated with 1% penicillin / streptomycin, 10% fetal bovine serum (FBS) (Thermo Fisher Scientific Inc., Waltham, MA, USA) and 0.2% normocin (InvivoGen) added to DMEM (Dulbecco's modified Eagle's medium) (Thermo Fisher Scientific Inc.) medium. Rat macrophages, RAW 264.7 cells (Korean Cell Line Bank, Seoul, Korea) were added to low-glucose DMEM (DMEM) supplemented with 1% penicillin / streptomycin and 10% FBS (Thermo Fisher Scientific, Inc.). Put incubated. THP1 cells, which are human monocytes, were cultured in RPMI 1640 supplemented with 1% penicillin / streptomycin and 10% FBS (Thermo Fisher Scientific, Inc.), followed by 10 nM of phorbol 12-myristate 13-acetate (PMA) ( Sigma-Aldrich Co. LLC., St. Louis, MO, USA) was used to induce differentiation into macrophages for 24 hours. HPBMC cells (PromoCell, Heidelberg, Germany), which are human peripheral blood monocytes, were cultured in RPMI 1640 (Thermo Fisher Scientific Inc.) with 2.05 mM L-glutamine, 1% penicillin / streptomycin and 10% FBS. MBMDM cells, which are mouse bone marrow-derived macrophages, were cultured in DMEM (Thermo Fisher Scientific Inc.) with 1% penicillin / streptomycin and 10% FBS. All cells were incubated in a humidified culture system (Thermo Fisher Scientific Inc.) at 5 ° C. CO 2 , 37 ° C., and the medium was changed every 16 hours. PAM 3 CSK 4 , Poly (I: C), R848 and CpG-ODN are from Thermo Fisher Scientific, Inc., FSL-1 is from InvivoGen, LPS ( Escherichia coli 0111: B4) and ATP are from Sigma-Aldrich Co. St. Louis, MO, USA) and all peptides used in the experiment were synthesized and purchased from Peptron, Inc. (Daejeon, Korea).
실시예 2. 분석 방법Example 2. Analytical Methods
2-1. MTT 분석2-1. MTT analysis
HEK-BlueTM-hTLR4 세포를 96-웰 플레이트(BD Biosciences, San Jose, CA, USA)에 5 x 104 cells/웰의 세포수가 되도록 분주하고, RAW264.7 세포는 2 x 105 cells/웰의 세포수가 되도록 분주하여 하룻밤 동안 배양하였다. 이후 1-(4,5-dimethylthiazol-2-yl)-3,5-diphenylformazan(MTT) 용액(Sigma-Aldrich Co. LLC)을 사용하여 MTT 분석을 수행하였다.HEK-Blue TM- hTLR4 cells are dispensed in 96-well plates (BD Biosciences, San Jose, CA, USA) to a cell number of 5 x 10 4 cells / well, RAW264.7 cells are 2 x 10 5 cells / well The cells were aliquoted and cultured overnight. Thereafter, MTT analysis was performed using 1- (4,5-dimethylthiazol-2-yl) -3,5-diphenylformazan (MTT) solution (Sigma-Aldrich Co. LLC).
2-2. SEAP 활성 분석2-2. SEAP Activity Assay
HEK-BlueTM-hTLR4 세포를 24-웰 플레이트(BD Biosciences)에 2 x 105 cells/웰의 세포수가 되도록 분주하고 하룻밤 동안 배양하였다. 다음날, 배양액을 제거하고 배지를 교체한 후, 배양액 일부(200㎕)를 미세원심분리(microcentrifuge) 튜브에 옮겨 heating block(FINEPCR. Co., Seoul, Korea)을 사용하여 65℃에서 10분간 가열시켰다. 이후 배양액을 96-웰 플레이트(BD Biosciences)에 옮기고 HEK-BlueTM detection 키트(InvivoGen) 및 마이크로플레이트 리더 분광 광도계(Molecular Devices Inc., Silicon Valley, CA, USA)를 사용하여 405nm에서 흡광도를 측정하여 SEAP(Secreted Alkaline phosphatase) 발현량을 분석하였다. HEK-Blue ™ -hTLR4 cells were aliquoted into 24-well plates (BD Biosciences) to a cell number of 2 × 10 5 cells / well and incubated overnight. The next day, after removing the culture medium and changing the medium, a portion of the culture medium (200 μl) was transferred to a microcentrifuge tube and heated at 65 ° C. for 10 minutes using a heating block (FINEPCR. Co., Seoul, Korea). . The cultures were then transferred to 96-well plates (BD Biosciences) and absorbance was measured at 405 nm using a HEK-Blue TM detection kit (InvivoGen) and a microplate reader spectrophotometer (Molecular Devices Inc., Silicon Valley, CA, USA). SEAP (Secreted Alkaline phosphatase) expression levels were analyzed.
2-3. 웨스턴 블롯2-3. Western Blot
웨스턴 블롯팅을 수행하기 위해 전-단백질 추출 용액(M-PER, Thermo Fisher Scientific Inc.)을 프로테아제 및 포스파타아제 억제 혼합물과 섞어 RAW 264.7 세포 또는 hPBMC 세포 펠릿(pellet)에 첨가하였다. 상기 펠릿을 10분간 냉각시킨 후 10분 동안 16000 X g에서 상기 용해물을 원심 분리하였다. 그런 다음, NE-PER 핵 및 세포질 추출 시약(Thermo Fisher Scientific Inc.)을 사용하여 세포질과 핵의 단백질을 각각 추출하고, BCA 키트(Sigma-Alderich Co. LLC)를 사용하여 상기 단백질의 농도를 측정하였다. 이후, 동일한 양의 단백질을 SDS-폴리아크릴아마이드 겔에 전개하고, Mini-PROTEAN Tetra Cell 이차원 전기영동 시스템(Bio-Rad Laboratories, Hercules, CA, USA)을 이용하여 전기영동을 수행하였다. 상기 막을 1차 항체로 4℃의 온도에서 하룻밤 동안 가볍게 쉐이킹하여 면역블롯팅하였다(상기 1차 항체는 p-p65, p-JNK, p-IRF3, ERK, p38 및 인간 IL-1β(Cell Signaling Technology Inc., Danvers, MA, USA); p-ERK, p-p38, Iκ-Bα, JNK, ATF3, COX2, caspase-1 및 β-액틴(Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA); iNOS(BD Biosciences); IL-6 및 마우스 IL-1β(R&D Systems Inc., Minneapolis, MN, USA); TNF-α(Thermo Fisher Scientific, Inc.); NLRP3(Adipogen, San Diego, CA, USA)에 대한 항체이다). 그런 다음, PBST로 철저히 쉐이킹한 후 상기 막을 항-쥐/-토끼 HRP-컨쥬게이티드 2차 항체(Thermo Fisher Scientific Inc.)와 함께 2시간 동안 배양하고, SuperSignal West Pico ECL 용액(Thermo Fisher Scientific Inc.)으로 단백질을 검출하고 Fuji LAS-3000 시스템(Fujifilm, Tokyo, Japan)을 사용하여 검출된 단백질을 시각화하였다.Pre-protein extraction solutions (M-PER, Thermo Fisher Scientific Inc.) were mixed with protease and phosphatase inhibition mixtures and added to RAW 264.7 cells or hPBMC cell pellets to perform western blotting. The pellet was cooled for 10 minutes and the lysate was centrifuged at 16000 X g for 10 minutes. Then, the proteins of the cytoplasm and the nucleus were extracted using NE-PER nuclear and cytoplasmic extraction reagents (Thermo Fisher Scientific Inc.), and the concentration of the protein was measured using a BCA kit (Sigma-Alderich Co. LLC). It was. The same amount of protein was then developed on SDS-polyacrylamide gels, and electrophoresis was performed using a Mini-PROTEAN Tetra Cell two-dimensional electrophoresis system (Bio-Rad Laboratories, Hercules, CA, USA). The membranes were immunoblotted by light shaking with primary antibody overnight at a temperature of 4 ° C. (The primary antibodies were p-p65, p-JNK, p-IRF3, ERK, p38 and human IL-1β (Cell Signaling Technology). Inc., Danvers, MA, USA) p-ERK, p-p38, Iκ-Bα, JNK, ATF3, COX2, caspase-1 and β-actin (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA); iNOS (BD Biosciences); IL-6 and Mouse IL-1β (R & D Systems Inc., Minneapolis, MN, USA); TNF-α (Thermo Fisher Scientific, Inc.); NLRP3 (Adipogen, San Diego, CA, USA) Is an antibody against). Then, after thorough shaking with PBST, the membrane was incubated with anti-rat / rabbit HRP-conjugated secondary antibody (Thermo Fisher Scientific Inc.) for 2 hours and the SuperSignal West Pico ECL solution (Thermo Fisher Scientific Inc.) Protein was detected and the detected protein was visualized using the Fuji LAS-3000 system (Fujifilm, Tokyo, Japan).
2-4. 공초점 현미경 분석(Confocal microscopy)2-4. Confocal microscopy
RAW 264.7 세포 및 THP1 세포를 24-웰 플레이트에 2 x 105 cells/웰의 세포수가 되도록 분주한 후 하룻밤 동안 인큐베이터에서 키웠다. 이후 TIP1 및 LPS를 함께 처리하고 24시간 뒤, 상기 RAW 264.7 세포 및 THP1 세포를 15분 동안 3.7% 포름알데히드(Sigma-Aldrich Co. LLC)로 고정하고, 15분 동안 0.2% 트리톤 X-100(AMRESCO, Solon, OH, USA)에 침지한 후 PBS로 3회 세척하고 2% BSA 용액으로 블로킹하였다. 상기 블로킹된 세포들을 TIP1-FITC(25μM; Peptron, Inc., Daejeon, Korea), p-p65, TLR4, Myd88, TOM20(1:1000; Santa Cruz Biotechnology Inc.) 및 NLRP3(Adipogen) 항체들과 2시간 동안 배양한 다음, PBS로 3회 세척하였다. 이후, 상기 세포들을 AlexaFluor 408 및/또는 488 및/또는 546-컨쥬게이션된 2차 항체(Invitrogen, Carlsbad, CA, USA)와 함께 1시간 동안 배양하고 PBS로 3회 세척하였다. 이후, 5μM의 Hoechst 33258(Sigma-Aldrich Co.)을 이용하여 상온에서 15분간 염색하고, 공초점 레이저 주사 현미경(LSM-700, Carl Zeiss MicroImaging GmbH)을 사용하여 상기 형광 염색된 세포의 수를 세었으며, Zen 2009 소프트웨어를 사용하여 이미지를 분석하였다.RAW 264.7 cells and THP1 cells were seeded in 24-well plates to a cell number of 2 × 10 5 cells / well and grown in incubator overnight. After 24 hours of treatment with TIP1 and LPS together, the RAW 264.7 cells and THP1 cells were fixed with 3.7% formaldehyde (Sigma-Aldrich Co. LLC) for 15 minutes and 0.2% Triton X-100 (AMRESCO) for 15 minutes. , Solon, OH, USA), washed three times with PBS and blocked with 2% BSA solution. The blocked cells were treated with TIP1-FITC (25 μM; Peptron, Inc., Daejeon, Korea), p-p65, TLR4, Myd88, TOM20 (1: 1000; Santa Cruz Biotechnology Inc.) and NLRP3 (Adipogen) antibodies. Incubated for hours, then washed three times with PBS. The cells were then incubated with AlexaFluor 408 and / or 488 and / or 546-conjugated secondary antibodies (Invitrogen, Carlsbad, CA, USA) for 1 hour and washed three times with PBS. Thereafter, the cells were stained at room temperature for 15 minutes using 5 μM of Hoechst 33258 (Sigma-Aldrich Co.), and the number of the fluorescently stained cells was counted using a confocal laser scanning microscope (LSM-700, Carl Zeiss MicroImaging GmbH). The image was analyzed using Zen 2009 software.
2-5. TNF-α, IL-6, IFN-β 및 IL-1β 분석2-5. TNF-α, IL-6, IFN-β and IL-1β Assays
RAW 264.7 세포, THP1 세포 및 mBMDM 세포를 96-웰 플레이트에 2 x 105 cells/웰의 세포수가 되도록 분주 또는 24-웰 플레이트에 5 x 105 cells/웰의 세포수가 되도록 분주하고 하룻밤 동안 배양하였다. 이후 TIP1 및 LPS를 함께 처리하고 24시간 뒤, IFN-β, IL-6 및 TNF-α 분비량을 LEGEND MAXTM Mouse IL-6 pre-coated ELISA 키트(BIoLegend), Mouse IL-6 Platinum ELISA(eBiosciences) 및 Mouse TNF alpha ELISA Ready SET-Go! 키트(eBiosciences)를 사용하여 측정하였다. 마이크로플레이트 리더 분광 광도계(Molecular Devices)를 사용하여 450nm에서 흡광도를 측정하고, 그 결과를 소프트맥스 프로 5.3 소프트웨어(Molecular Devices Inc.)를 사용하여 분석하였다.RAW 264.7 cells, THP1 cells and mBMDM cells were aliquoted to 2 x 10 5 cells / well in 96-well plates or 5 x 10 5 cells / well to 24-well plates and incubated overnight. . After treatment with the TIP1 and LPS and 24 hours later, IFN-β, IL-6 and TNF-α secretion by LEGEND MAX TM Mouse IL-6 pre-coated ELISA kit (BIoLegend), Mouse IL-6 Platinum ELISA (eBiosciences) And Mouse TNF alpha ELISA Ready SET-Go! Measurement was made using the kit (eBiosciences). Absorbance was measured at 450 nm using a microplate reader spectrophotometer (Molecular Devices) and the results were analyzed using SoftMax Pro 5.3 software (Molecular Devices Inc.).
2-6. 세포질 내 NO 및 ROS 분석2-6. Intracellular NO and ROS Analysis
RAW 264.7 세포를 6-cm 디쉬(SPL Life Sciences., Pochun, Korea)에 1 x 106 cells/웰의 세포수가 되도록 분주하고 하룻밤 동안 배양하였다. 이후 TIP1을 처리하고 각각 DAF-FM 및 DCF-DA(Thermo Fisher Scientific, Inc.)로 염색한 후, 1시간 동안 배양하였다. 그 후, 5분당 200 X g으로 원심 분리하여 수거하고 갈색 튜브에 옮겨 4℃의 온도에서 PBS에 보관하였다. 디바 소프트웨어(BD Biosciences)로 FACSAria Ⅲ를 사용하여 DAF-FM, DCF-DA 형광물질의 강도를 측정하여 정량화하였다.RAW 264.7 cells were aliquoted into 6-cm dishes (SPL Life Sciences., Pochun, Korea) to a cell number of 1 × 10 6 cells / well and incubated overnight. TIP1 was then treated and stained with DAF-FM and DCF-DA (Thermo Fisher Scientific, Inc.), respectively, and incubated for 1 hour. Thereafter, the cells were collected by centrifugation at 200 X g per 5 minutes, transferred to a brown tube, and stored in PBS at a temperature of 4 ° C. The intensity of DAF-FM, DCF-DA phosphors was measured and quantified using FACSAria III with Diva software (BD Biosciences).
2-7. NO 분비량 분석2-7. NO secretion analysis
RAW 264.7 세포 및 mBMDM 세포를 96-웰 플레이트(BD Biosciences)에 2 x 105 cells/웰의 세포수가 되도록 분주하고 하룻밤 동안 배양한 뒤, 배양 상층액의 NO 수치를 NO 검출 키트(iNtRON Biotechnology Inc., Seongnam, Korea)를 사용하여 측정하였다. 마이크로플레이트 리더 분광 광도계(Molecular Devices In.)를 사용하여 550nm에서 흡광도를 측정하고, 그 결과를 소프트맥스 프로 5.3 소프트웨어(Molecular Devices Inc.)를 사용하여 분석하였다.After dispensing RAW 264.7 cells and mBMDM cells in a 96-well plate (BD Biosciences) to a cell number of 2 x 10 5 cells / well and incubating overnight, the NO level of the culture supernatant was determined using the NO detection kit (iNtRON Biotechnology Inc.). , Seongnam, Korea). Absorbance was measured at 550 nm using a microplate reader spectrophotometer (Molecular Devices In.) And the results were analyzed using Softmax Pro 5.3 software (Molecular Devices Inc.).
2-8. 표면 플라즈몬 공명 분석(surface plasmon resonance; SPR)2-8. Surface plasmon resonance (SPR)
ProteOn NLC 센서 칩과 함께 ProteOn XPR36 instrument(Bio-Rad Laboratories, Inc.)를 사용하여 표면 플라즈몬 공명 분석을 수행하였다. 구체적으로, GLC polymer layer에 결합된 NeutrAvidin을 이용하여 TIR 도메인의 BB-loop(RDFIPGVAIAA) 및 C-말단 부위(EGTVGTGCNWQEATSI)를 코딩하는 합성 펩타이드를 NLC 센서 칩의 표면에 고정시키고, 대조군으로는 러닝 버퍼인 0.05% Tween 20으로 보충된 PBS를 흘려주었다. 다양한 종류의 TIP1, TIP1-1, TIP1-2 및 Tip1-3(50μM) 및 서로 다른 농도의 TIP1(12.5, 25 및 50 μM)를 칩에 흘려 고정된 펩타이드와의 결합 친화도를 확인하였다. 측정 후에 센서 칩의 표면은 0.85% 인산(phosphoric acid) 또는 PBST를 사용하여 재생되었다. 해리(dissociation) 상수는 ProteOn managerTM 소프트웨어(버전 2.0)를 사용하여 계산하였고, 다양한 투여량의 TIP1로부터 얻은 데이터를 운동 속도 상수(Ka 및 Kd)에 맞추기 위해 그룹화 하였으며, 평형 해리상수 KD는 KD=Kd/Ka 식을 사용하여 계산하였다. Surface plasmon resonance analysis was performed using a ProteOn XPR36 instrument (Bio-Rad Laboratories, Inc.) with a ProteOn NLC sensor chip. Specifically, the synthetic peptides encoding the BB-loop (RDFIPGVAIAA) and the C-terminal region (EGTVGTGCNWQEATSI) of the TIR domain are fixed to the surface of the NLC sensor chip using NeutrAvidin bound to the GLC polymer layer. PBS supplemented with 0.05% Tween 20 was flowed. Various kinds of TIP1, TIP1-1, TIP1-2 and Tip1-3 (50 μM) and different concentrations of TIP1 (12.5, 25 and 50 μM) were flowed into the chip to confirm binding affinity with the immobilized peptide. After the measurement, the surface of the sensor chip was regenerated using 0.85% phosphoric acid or PBST. Dissociation constants were calculated using ProteOn manager ™ software (version 2.0), and the data from the various doses of TIP1 were grouped to fit the kinetic rate constants (K a and K d ), and the equilibrium dissociation constant K D Was calculated using the equation K D = K d / K a .
실험예 1. TIPs의 TLR4-결합 친화도 확인Experimental Example 1. Confirmation of TLR4-binding affinity of TIPs
실시예 1-1에서 제조한 TIPs(TIP1, TIP2)의 TLR4-결합 친화도를 확인하기 위해 상기 실시예 1-2에서 배양한 HEK-BlueTM-hTLR4 세포에서 NF-κB의 활성을 측정하였다. 이를 위해, NF-κB와 AP-1(activator protein 1)의 DNA가 결합하는 부위를 포함하는 IL-12 p40 미니멀 프로모터(IL-12 p40은 TLR4의 자극 후 NF-κB와 AP-1의 활성화에 의해 생성된다)의 조절 부분 아래에 유도성 SEAP(secreted embryonic alkaline phosphatase) 리포터 유전자를 위치시켰다. 그 후, TIP 또는 CPP가 연결되지 않은 TIP(TIP W/O CPP)의 농도를 12.5, 25, 50 μM로 다양하게 하여 HEK-BlueTM-hTLR4 세포에 처리한 다음, SEAP 활성의 평균값을 계산하여 TLR4의 활성을 측정하고, 그 결과를 도 1에 나타내었다.In order to confirm the TLR4-binding affinity of the TIPs (TIP1, TIP2) prepared in Example 1-1, the activity of NF-κB was measured in HEK-Blue TM -hTLR4 cells cultured in Example 1-2. To this end, the IL-12 p40 minimal promoter (IL-12 p40 containing a site where DNA of NF-κB and AP-1 (activator protein 1) binds to the activation of NF-κB and AP-1 after stimulation of TLR4). Underneath the regulatory portion of the inducible secreted embryonic alkaline phosphatase (SEAP) reporter gene. Thereafter, the concentration of TIP or CPP-linked TIP (TIP W / O CPP) was varied to 12.5, 25, and 50 μM, and then treated to HEK-Blue TM -hTLR4 cells, and then the average value of SEAP activity was calculated. The activity of TLR4 was measured and the results are shown in FIG. 1.
도 1의 (a)에 나타낸 바와 같이, 오직 TIP만을 첨가한 경우 SEAP의 활성은 큰 변화가 없으나, TIP를 처리한 후 LPS로 TLR4를 자극하였을 때, TIP1은 TIP2과 달리 LPS에 의해 유도된 SEAP 활성을 농도 의존적으로 감소시킴을 확인하였다. 또한, 도 1의 (b)에 나타낸 바와 같이, CPP 서열이 연결되지 않은 경우에는 TIP1(TIP1 W/O CPP) 및 TIP2(TIP2 W/O CPP) 모두 SEAP 활성에 변화가 없음을 확인하였다. 이를 통해 본 발명의 TIP1은 CPP와 연결됨에 따라 일단 세포 내부로 전위되면(translocated) 신호전달 경로의 하류 어댑터 분자들을 방해하고, LPS에 의해 유도되는 TLR4-매개 신호전달 경로의 활성화를 차단하여 효과적인 TLR4 억제제로 사용될 수 있음을 확인하였다.As shown in (a) of FIG. 1, when only TIP is added, the SEAP activity does not change significantly, but when TLR4 is stimulated with LPS after TIP treatment, TIP1 is induced by LPS unlike TIP2. It was found that the activity was reduced in a concentration dependent manner. In addition, as shown in (b) of FIG. 1, when the CPP sequence was not linked, it was confirmed that there was no change in SEAP activity in both TIP1 (TIP1 W / O CPP) and TIP2 (TIP2 W / O CPP). Through this, TIP1 of the present invention, once associated with CPP, interferes with adapter molecules downstream of the signaling pathway once translocated into the cell, and blocks the activation of the TLR4-mediated signaling pathway induced by LPS, thus providing an effective TLR4. It was confirmed that it can be used as an inhibitor.
실험예 2. Experimental Example 2. In vitroIn vitro 에서 TLR 신호전달 경로에 대한 TIP1의 억제적 효과Inhibitory Effect of TIP1 on TLR Signaling Pathway in Rats
LPS에 의해 유도된 TLR4 매개 반응은 TIRAP의 TIR 도메인과 MyD88 간의 직접적인 상호작용을 유도하여 MyD88-의존적 신호전달경로를 활성화한다. 또한, TRAM의 TIR 도메인과 TRIF 간의 상호작용을 유도하여 MyD88-비의존적 신호전달경로를 활성화하기도 한다. 상기 MyD88-의존적 신호전달 경로에서, NF-κB의 초기 활성은 TNF-α(tumor necrosis factor alpha) 및 IL-6(interleukin 6)와 같은 염증 유발(pro-inflammatory) 사이토카인의 분비를 유도한다. 한편, MyD88-비의존적 신호전달 경로에서는 IRF3 및 7의 활성을 비롯하여 IFN-α 및 IFN-β와 같은 type Ⅰ 인터페론(IFNs)의 후기 분비 반응이 유도된다. 본 발명자들은 TLR4 신호전달 경로에 대한 TIP1의 영향을 알아보기 위하여 RAW 264.7 세포에 LPS를 처리한 후, 하기와 같은 실험을 수행하였다.TLR4-mediated responses induced by LPS induce direct interactions between the TIR domain of TIRAP and MyD88 to activate MyD88-dependent signaling pathways. It also induces interactions between the TIR domain of the TRAM and TRIF, activating the MyD88-independent signaling pathway. In the MyD88-dependent signaling pathway, the initial activity of NF-κB induces the secretion of pro-inflammatory cytokines such as tumor necrosis factor alpha (TNF-α) and interleukin 6 (IL-6). On the other hand, MyD88-independent signaling pathways induce late secretory responses of type I interferons (IFNs) such as IFN-α and IFN-β, as well as the activity of IRF3 and 7. The present inventors performed the following experiments after LPS treatment of RAW 264.7 cells to determine the effect of TIP1 on the TLR4 signaling pathway.
2-1. NF-κB 및 MAPK의 웨스턴 블롯팅2-1. Western blotting of NF-κB and MAPK
상기 실시예 1-1에서 제조한 TIP1이 NF-κB 및 MAPK의 활성에 미치는 영향을 확인하기 위해 실시예 2-3에 기재된 방법에 따라 웨스턴 블롯팅을 수행하였다. 그 결과를 도 2에 나타내었다.Western blotting was performed according to the method described in Example 2-3 to determine the effect of TIP1 prepared in Example 1-1 on the activity of NF-κB and MAPK. The results are shown in FIG.
도 2의 (a)에 나타낸 바와 같이, 대조군인 쥐 대식세포인 RAW 264.7 세포에 오직 LPS만을 처리한 경우, NF-κB의 활성이 증가하여 Iκ-Bα가 분해되고 IRF3와 ATF3의 활성이 증가한 반면, TIP1을 함께 처리한 경우, NF-κB의 활성이 억제되어 Iκ-Bα가 분해되는 정도 및 IRF3와 ATF3의 활성이 감소함을 확인하였다. 또한, 도 2의 (b)에 나타낸 바와 같이, 오직 LPS만을 처리한 경우, MAPK의 활성이 증가하여 ERK, JNK, p38이 인산화되지만, TIP1을 함께 처리한 경우, MAPK의 활성이 억제되어 상기 효소들의 인산화된 정도가 감소함을 확인하였다. 이를 통해 본 발명에 따른 TIP1은 TLR4의 TIR 도메인에 결합하여 LPS에 의해 유도된 MyD88-의존적 신호전달 경로는 물론 MyD88-비의존적 신호전달 경로 역시 억제함을 확인하였다.As shown in (a) of FIG. 2, when only LPS was treated to control macrophage RAW 264.7 cells, NF-κB activity was increased to degrade Iκ-Bα and to increase IRF3 and ATF3 activities. When treated with TIP1, it was confirmed that NF-κB activity was inhibited to decrease the degree of Iκ-Bα degradation and the activities of IRF3 and ATF3. In addition, as shown in (b) of FIG. 2, when only LPS was treated, the activity of MAPK was increased to phosphorylate ERK, JNK, and p38. However, when TIP1 was treated together, the activity of MAPK was inhibited, resulting in the enzyme. It was confirmed that their phosphorylation degree is reduced. Through this, it was confirmed that TIP1 according to the present invention binds to the TIR domain of TLR4 and inhibits MyD88-dependent signaling pathway as well as MyD88-dependent signaling pathway induced by LPS.
2-2. NF-κB의 활성화 확인2-2. Confirmation of NF-κB Activation
상기 실시예 1에서 제조한 TIP1이 NF-κB의 활성에 미치는 영향을 확인하기 위해 실시예 2-4에 기재된 방법에 따라 공초점 레이저 현미경으로 분석을 수행하였다. 그 결과를 도 3에 나타내었다.In order to confirm the effect of TIP1 prepared in Example 1 on the activity of NF-κB was analyzed by confocal laser microscope according to the method described in Example 2-4. The results are shown in FIG.
도 3에 나타낸 바와 같이, LPS만을 처리한 경우 활성화된 NF-κB(p-p65)가 핵 안에서 발현되고 있으나, TIP1을 함께 처리한 경우 핵에서 LPS에 의해 유도된 p65의 인산화 수준이 감소함을 통해 NF-κB의 활성도가 감소함을 확인하였다. As shown in FIG. 3, the activated NF-κB (p-p65) is expressed in the nucleus when only LPS is treated, but the phosphorylation level of p65 induced by LPS in the nucleus is reduced when TIP1 is treated together. It was confirmed that the activity of NF-κB decreases through.
실험예 3. TIP1이 사이토카인과 NO 및 ROS 분비에 미치는 영향Experimental Example 3. Effect of TIP1 on cytokine, NO and ROS secretion
상기 실시예 1-1에서 제조한 TIP1을 상기 실시예 1-2에서 배양한 쥐 대식세포인 RAW 264.7 세포에 처리하였을 때 사이토카인(TNF-α, IL-6, IFN-β)과 NO(Nitric Oxide)의 분비 및 세포질 내의 ROS(reactive oxygen species)의 발생이 억제되는지를 확인하기 위해 하기와 같은 실험을 수행하였다.Cytokines (TNF-α, IL-6, IFN-β) and NO (Nitric) when TIP1 prepared in Example 1-1 were treated to RAW 264.7 cells, which are mouse macrophages cultured in Example 1-2 Oxygen secretion and the experiment to determine whether the generation of reactive oxygen species (ROS) in the cytoplasm is inhibited.
3-1. TIP1이 사이토카인 분비에 미치는 영향3-1. Effect of TIP1 on Cytokine Secretion
실시예 2-5에 기재된 방법에 따라 RAW 264.7 세포의 배양 상층액의 TNF-α, IL-6, IFN-β 수치를 쥐 TNF-α, IL-6, IFN-β ELISA 키트 Ready-SET-Go! 를 사용하여 측정하고, 그 결과를 도 4에 나타내었다.TNF-α, IL-6, IFN-β levels of the culture supernatant of RAW 264.7 cells were determined according to the method described in Example 2-5. The rat TNF-α, IL-6, IFN-β ELISA kit Ready-SET-Go ! It measured using, and the result is shown in FIG.
도 4에 나타낸 바와 같이, TIP1과 LPS를 함께 처리하였을 경우 TNF-α, IL-6, IFN-β의 분비가 농도 의존적으로 감소함을 확인하였다. 이를 통해 본 발명에 따른 TIP1이 LPS에 의해 유도된 사이토카인의 분비를 억제함을 확인하였다.As shown in FIG. 4, when TIP1 and LPS were treated together, it was confirmed that the secretion of TNF-α, IL-6, IFN-β decreased in a concentration-dependent manner. This confirmed that TIP1 according to the present invention inhibits the secretion of cytokines induced by LPS.
3-2. TIP1이 NO 및 ROS 분비에 미치는 영향3-2. Effect of TIP1 on NO and ROS Secretion
실시예 2-6에 기재된 방법에 따라 TIP1이 세포질에서의 NO 및 ROS(reactive oxygen species) 발생에 미치는 영향을 확인하기 위해 RAW 264.7 세포에 TIP1을 처리하고, 각각 DAF-FM(Invitrogen Corp., CA, USA) 및 DCF-DA(Invitrogen Corp.)로 염색하여 세포질의 NO, ROS를 각각 정량화하였다. 또한, 실시예 2-7에 기재된 방법에 따라 RAW 264.7 세포의 배양 상층액의 NO 수치를 NO 검출 키트를 사용하여 측정하였다. 상기 결과를 도 5에 나타내었다.To determine the effect of TIP1 on NO and reactive oxygen species (ROS) development in the cytoplasm according to the method described in Examples 2-6, RAW 264.7 cells were treated with TIP1 and DAF-FM (Invitrogen Corp., CA, respectively) , USA) and DCF-DA (Invitrogen Corp.) to quantify cytosolic NO and ROS, respectively. In addition, according to the method described in Example 2-7, the NO value of the culture supernatant of RAW 264.7 cells was measured using the NO detection kit. The results are shown in FIG. 5.
도 5의 (a) 내지 (c)에 나타낸 바와 같이, TIP1과 LPS를 함께 처리하였을 경우 TIP1은 세포 외 NO 분비뿐만 아니라 세포질에서의 NO 및 ROS 발생량 역시 효과적으로 감소시킴을 확인하였다. 이를 통해 본 발명에 따른 TIP1은 LPS에 의해 유도된 산화적 스트레스를 억제함을 확인하였다.As shown in (a) to (c) of FIG. 5, when TIP1 and LPS were treated together, it was confirmed that TIP1 effectively reduced NO and ROS generation in the cytoplasm as well as extracellular NO secretion. Through this it was confirmed that TIP1 according to the present invention inhibits the oxidative stress induced by LPS.
실험예 4. 일차세포(primary cells)에서 TIP1이 미치는 영향 확인Experimental Example 4 Confirmation of the Effect of TIP1 on Primary Cells
상기 실시예 1에서 제조한 TIP1을 상기 실시예 2에서 배양한 쥐 골수유래 대식세포인 mBMDM과 인간 말초혈액 단핵구 세포인 hPBMC 세포에 처리하였을 때 사이토카인(TNF-α, IL-6, IFN-β)과 NO(Nitric Oxide)의 분비 및 TLR 신호전달 단백질들의 활성화가 억제되는지를 확인하기 위해 하기와 같은 실험을 수행하였다.Cytokines (TNF-α, IL-6, IFN-β) when TIP1 prepared in Example 1 were treated with mBMDM, a mouse bone marrow-derived macrophage, and hPBMC cells, which are human peripheral blood mononuclear cells, ) And to confirm whether NO (Nitric Oxide) secretion and activation of TLR signaling proteins are inhibited.
4-1. TIP1이 mBMDM 세포에 미치는 영향4-1. Effect of TIP1 on mBMDM Cells
실시예 2-5에 기재된 방법에 따라 mBMDM 세포의 배양 상층액의 TNF-α, IL-6, IFN-β 수치를 쥐 TNF-α, IL-6, IFN-β ELISA 키트 Ready-SET-Go! 키트를 사용하여 측정하고, 실시예 2-7에 기재된 방법에 따라 상층액의 NO 수치를 측정하여 그 결과를 도 6에 나타내었다.TNF-α, IL-6, IFN-β levels of the culture supernatant of mBMDM cells were measured according to the method described in Example 2-5 in the rat TNF-α, IL-6, IFN-β ELISA kit Ready-SET-Go! Measured using the kit, the NO value of the supernatant was measured according to the method described in Example 2-7, and the results are shown in FIG. 6.
도 6의 (a) 내지 (d)에 나타낸 바와 같이, TIP1은 LPS에 의해 유도된 TNF-α, IL-6 및 NO의 분비량을 감소시키고, Poly(I:C)에 의해 유도된 IFN-β의 생성을 억제시킴을 확인하였다. 이를 통해 본 발명에 따른 TIP1이 쥐 골수유래 대식세포에서 LPS 또는 Poly(I:C)에 의해 유도된 사이토카인과 NO의 분비를 억제함을 확인하였다.As shown in (a) to (d) of FIG. 6, TIP1 reduces the secretion amount of TNF-α, IL-6 and NO induced by LPS, and IFN-β induced by Poly (I: C). It was confirmed to inhibit the production of. It was confirmed that TIP1 according to the present invention inhibits the secretion of cytokines and NO induced by LPS or Poly (I: C) in rat bone marrow-derived macrophages.
4-2. TIP1이 hPBMC 세포에 미치는 영향4-2. Effect of TIP1 on hPBMC Cells
TIP1이 hPBMC 세포에서 NF-κB 및 MAPK의 활성에 미치는 영향을 확인하기 위해 실시예 2-3에 기재된 방법에 따라 웨스턴 블롯팅을 수행하였고, 단백질을 시각화하였으며, 그 결과를 도 7에 나타내었다.In order to confirm the effect of TIP1 on the activity of NF-κB and MAPK in hPBMC cells, Western blotting was performed according to the method described in Example 2-3, the protein was visualized, and the results are shown in FIG.
도 7에 나타낸 바와 같이, 대조군인 인간 말초혈액 단핵구 세포인 hPBMC 세포에 오직 LPS만을 처리한 경우, NF-κB의 활성이 증가하여 Iκ-Bα가 분해되는 반면, TIP1을 함께 처리한 경우, NF-κB의 활성이 억제되어 Iκ-Bα가 분해되는 정도가 감소하였다. 또한, 오직 LPS만을 처리한 경우, MAPK의 활성이 증가하여 ERK, JNK, p38이 인산화되지만, TIP1을 함께 처리한 경우, MAPK의 활성이 억제되어 상기 효소들의 인산화된 정도가 감소함을 확인하였다.As shown in FIG. 7, when only LPS was treated to hPBMC cells, which are control human peripheral blood mononuclear cells, NF-κB activity was increased to degrade Iκ-Bα, whereas when TIP1 was treated together, NF- Inhibition of κB activity reduced the level of Iκ-Bα degradation. In addition, when only LPS was treated, the activity of MAPK was increased to phosphorylate ERK, JNK, and p38, but when TIP1 was treated together, it was confirmed that the activity of MAPK was inhibited to decrease the phosphorylated degree of the enzymes.
이를 통해 본 발명에 따른 TIP1은 불멸화된 세포주(cell line) 뿐만 아니라 동물에서 직접 추출한 일차세포에서도 TLR4 신호전달경로를 억제함을 확인하였다.Through this, it was confirmed that TIP1 according to the present invention inhibits TLR4 signaling pathways not only in immortalized cell lines, but also in primary cells directly extracted from animals.
실험예 5. TIP1이 다른 TLR 신호전달에 미치는 영향Experimental Example 5. Effect of TIP1 on Other TLR Signaling
TLR4 외에, 다른 TLR 패밀리의 신호전달 경로에 대한 TIP1의 효과를 확인하기 위하여, RAW 264.7 세포의 배양 상층액의 각기 다른 TLR 리간드(ligand)에 의해 유도된 TNF-α 수치를 상기 실시예 2-5의 방법을 이용하여 측정하고, 그 결과를 도 8에 나타내었다. 또한, RAW 264.7 세포의 배양 상층액의 IFN-β 수치를 ELISA 키트 Ready-SET-Go! 를 사용하여 측정하고, 그 결과를 도 9에 나타내었다.In addition to TLR4, in order to confirm the effect of TIP1 on the signaling pathways of other TLR families, TNF-α levels induced by different TLR ligands in the culture supernatant of RAW 264.7 cells were measured in Example 2-5. It measured using the method of, and the result is shown in FIG. In addition, IFN-β levels in the culture supernatant of RAW 264.7 cells were determined by ELISA kit Ready-SET-Go! It measured using, and the result is shown in FIG.
도 8에 나타낸 바와 같이, TIP1과 PAM3CSK4(TLR1/2) 또는 FSL-1(TLR2/6) 또는 Poly(I:C)(TLR3) 또는 R848(TLR7/8) 또는 CpG-ODN(TLR9)을 함께 처리하였을 경우, TNF-α 분비량이 감소하였고, 특히 TIP1은 TLR3 신호전달에 대한 억제 효과가 높음을 확인하였다(50μM에서 92% 감소). 또한, 도 9에 나타낸 바와 같이, TIP1과 Poly(I:C)를 함께 처리하였을 경우, IFN-β의 분비량이 감소함을 확인하였다. 이를 통해 본 발명에 따른 TIP1이 TLR4뿐만 아니라 TLR1/2, TLR2/6, TLR3, TLR7/8, TLR9에서도 사이토카인 분비 억제 효과가 있음을 확인하였다.As shown in FIG. 8, TIP1 and PAM3CSK4 (TLR1 / 2) or FSL-1 (TLR2 / 6) or Poly (I: C) (TLR3) or R848 (TLR7 / 8) or CpG-ODN (TLR9) together When treated, it was confirmed that the amount of TNF-α secretion decreased, in particular TIP1 has a high inhibitory effect on TLR3 signaling (92% reduction at 50μM). In addition, as shown in Figure 9, when treated with TIP1 and Poly (I: C), it was confirmed that the secretion amount of IFN-β. Through this, it was confirmed that TIP1 according to the present invention has an inhibitory effect on cytokine secretion not only in TLR4 but also in TLR1 / 2, TLR2 / 6, TLR3, TLR7 / 8, and TLR9.
실험예 6. TIP1이 퇴행성 신경질환에 미치는 영향Experimental Example 6. Effect of TIP1 on Degenerative Neuropathy
LPS에 의해 유도되는 TLR4 신호전달 경로의 활성화는 NF-κB의 활성을 야기하고, 이는 곧 대식세포에서 NOD-유사 수용체(NOD-like receptor, NLR), NLRP3(NACHT, LRR 및 PYD-domains-containing protein 3) 및 IL-1β의 발현으로 이어진다. 이러한 조건 하에서, ATP와 칼륨 유출 물질(potassium efflux agent)은 세포 내부의 칼륨 농도를 감소시키고, NLRP3 인플라마좀(inflammasome)에 의한 성숙한 IL-1β의 생성을 유도한다. Activation of the TLR4 signaling pathway induced by LPS results in the activity of NF-κB, which is NOD-like receptor (NLR), NLRP3 (NACHT, LRR and PYD-domains-containing) in macrophages. protein 3) and IL-1β. Under these conditions, ATP and potassium efflux agent reduce potassium levels in cells and induce the production of mature IL-1β by NLRP3 inflammasomes.
이와 같은 TLR4 신호전달 경로의 활성과 NLRP3 인플라마좀 형성의 상관관계에 주목하여, 본 발명자들은 인간 단핵구 세포인 THP1 세포 및 쥐 골수유래 대식세포인 mBMDM THP1 세포에 LPS/ATP 또는 TIP1과 LPS/ATP를 함께 처리한 후, 실시예 2-3에 기재된 방법에 따라 NLRP3, pro-caspase-1 (45 kDa), 활성형 capase-1 (10 kDa), pro-IL-1β(35 kDa) 및 성숙한 IL-1β(17 kDa)의 단백질 발현을 웨스턴 블롯팅을 통해 시각화 하였다. 그 결과를 도 10에 나타내었다.Noting the correlation between the activity of the TLR4 signaling pathway and NLRP3 inflammasome formation, the present inventors have found that LPS / ATP or TIP1 and LPS / ATP are expressed in THP1 cells, which are human monocytes, and mBMDM THP1 cells, which are mouse bone marrow-derived macrophages. And NLRP3, pro-caspase-1 (45 kDa), active capase-1 (10 kDa), pro-IL-1β (35 kDa) and mature IL according to the methods described in Examples 2-3. Protein expression of -1β (17 kDa) was visualized by Western blotting. The results are shown in FIG.
또한, TIP1이 NLRP3 인플라마좀이 유도되었을 때 사이토카인 분비에 미치는 영향을 확인하기 위해 THP1 세포 및 mBMDM 세포에 LPS/ATP 또는 TIP1과 LPS/ATP를 함께 처리한 후, 배양 상층액의 IL-1β 수치를 실시예 2-5에 기재된 방법에 따라 인간 및 쥐 IL-1β ELISA 키트 Ready-SET-Go! 를 사용하여 측정하였다. 그 결과를 도 11에 나타내었다.In addition, to determine the effect of TIP1 on cytokine secretion when NLRP3 inflamasome was induced, THP1 cells and mBMDM cells were treated with LPS / ATP or TIP1 and LPS / ATP, and then IL-1β of the culture supernatant. The values were determined according to the method described in Example 2-5. The human and rat IL-1β ELISA kit Ready-SET-Go! Measured using. The results are shown in FIG.
도 10의 (a) 내지 (b)에 나타낸 바와 같이, THP1과 mBMDM 세포에서 LPS와 ATP에 의해 유도된 NLRP3, Caspase-1, IL-1β의 발현이 TIP1에 의해 억제됨을 확인하였다. 구체적으로, TIP1은 NLRP3, 활성형 capase-1 (10 kDa) 및 성숙한 IL-1β(17 kDa)의 세포 내 발현량을 현저히 감소시키고, 궁극적으로 IL-1β분비를 억제시킴을 확인하였다. 또한, 도 11에 나타낸 바와 같이, TIP1과 LPS/ATP를 함께 처리하였을 경우, IL-1β의 분비량이 감소함을 확인하였다. 이를 통해 본 발명에 따른 TIP1이 TLR4 신호전달 경로를 차단하여 NLRP3 인플라마좀 활성화를 억제함을 확인하였다.As shown in (a) to (b) of Figure 10, it was confirmed that the expression of NLRP3, Caspase-1, IL-1β induced by LPS and ATP in THP1 and mBMDM cells is inhibited by TIP1. Specifically, it was confirmed that TIP1 significantly reduced the intracellular expression levels of NLRP3, active capase-1 (10 kDa) and mature IL-1β (17 kDa) and ultimately inhibited IL-1β secretion. In addition, as shown in Figure 11, when treated with TIP1 and LPS / ATP, it was confirmed that the secretion amount of IL-1β decreases. Through this, it was confirmed that TIP1 according to the present invention inhibits NLRP3 inflammasome activation by blocking the TLR4 signaling pathway.
NLRP3 인플라마좀은 비정상적으로 활성화되면 다양한 염증성 질환, 그 중에서도 퇴행성 신경질환을 유발하여, NLRP3 인플라마좀의 활성을 효과적으로 억제할 수 있다면 염증 반응의 억제를 통해 퇴행성 신경질환의 치료제로 사용될 수 있음을 기대할 수 있다. 따라서, 본 발명자들은 상기 결과를 통해 본 발명에 따른 TIP1이 퇴행성 신경질환에 대하여 예방 또는 치료 효과를 나타낼 수 있음을 확인하였다.If NLRP3 inflamasome is abnormally activated, it can cause various inflammatory diseases, especially degenerative neurological diseases. If NLRP3 inflamasome can be effectively inhibited, it can be used as a therapeutic agent for neurodegenerative diseases through inhibition of inflammatory response. You can expect Accordingly, the present inventors confirmed that the TIP1 according to the present invention may have a prophylactic or therapeutic effect on neurodegenerative diseases.
실험예 7. TIP1 서열 특이성 확인Experimental Example 7. Confirmation of TIP1 Sequence Specificity
본 발명자들은 TLR4의 TIR 도메인과 결합하는 TIP1의 아미노산 최소부위를 확인하기 위해, 실시예 1-1에서 제조한 decoy peptide의 아미노산 서열인 S-H-C-R-V-L-L-I(서열번호 1)를 이용하여 S-H-C-R(decoy peptide 1-2, 서열번호 2) 및 V-L-L-I(decoy peptide 1-3, 서열번호 7) 서열을 실시예 1-1에서 사용한 것과 동일한 CPP 서열의 N-말단에 각각 결합하였다. 본 실험예에서 사용한 모든 펩타이드를 하기 표 3에 기재하였다.The present inventors used SHCR (decoy peptide 1-2) using SHCRVLLI (SEQ ID NO: 1), which is the amino acid sequence of the decoy peptide prepared in Example 1-1, to identify the amino acid minimum region of TIP1 that binds to the TIR domain of TLR4. , SEQ ID NO: 2) and VLLI (decoy peptide 1-3, SEQ ID NO: 7) sequences were respectively bound to the N-terminus of the same CPP sequence as used in Example 1-1. All peptides used in this Experimental Example are listed in Table 3 below.
이름name 서열order 서열번호SEQ ID NO:
Decoy peptide 1(TIP1 W/O CPP)Decoy peptide 1 (TIP1 W / O CPP) SHCRVLLISHCRVLLI 1One
Decoy peptide 1-2Decoy peptide 1-2 SHCR SHCR 22
TIP1 TIP1 RQIKIWFQNRRMKWKKSHCRVLLI RQIKIWFQNRRMKWKK SHCRVLLI 33
TIP1-2TIP1-2 RQIKIWFQNRRMKWKKSHCR RQIKIWFQNRRMKWKK SHCR 44
Decoy peptide 1-3Decoy peptide 1-3 VLLIVLLI 77
TIP1-3TIP1-3 RQIKIWFQNRRMKWKKVLLI RQIKIWFQNRRMKWKK VLLI 88
상기 표 3의 TIP1(CPP-SHCRVLLI), TIP1-1(CPP), TIP1-2(CPP-SHCR) 및 TIP1-3(CPP-VLLI)을 실시예 1-2에서 배양한 HEK-BlueTM-hTLR4 세포에 LPS와 함께 처리한 후, 상기 실험예 1의 방법을 이용하여 NF-κB 활성을 측정하였다. 또한, 상기 실시예 1-2에서 배양한 hPBMC 세포에 LPS와 함께 TIP1(CPP-SHCRVLLI), TIP1-1(CPP), TIP1-2(CPP-SHCR) 및 TIP1-3(CPP-VLLI)를 처리한 후, 상기 실시예 2-7의 방법을 사용하여 NO 분비량을 측정하였다. 그 결과를 도 12에 나타내었다.HEK-Blue TM -hTLR4 in which TIP1 (CPP-SHCRVLLI), TIP1-1 (CPP), TIP1-2 (CPP-SHCR) and TIP1-3 (CPP-VLLI) of Table 3 were cultured in Examples 1-2. After the cells were treated with LPS, NF-κB activity was measured using the method of Experimental Example 1 above. In addition, the hPBMC cells cultured in Example 1-2 were treated with TIP1 (CPP-SHCRVLLI), TIP1-1 (CPP), TIP1-2 (CPP-SHCR) and TIP1-3 (CPP-VLLI) together with LPS. Afterwards, the amount of NO secretion was measured using the method of Example 2-7. The results are shown in FIG.
도 12의 (a)에 나타낸 바와 같이, TIP1의 NF-κB 활성 억제 효과가 가장 높았고 TIP1-2 역시 NF-κB 활성을 감소시킨 반면, TIP1-1과 TIP1-3를 처리한 경우 변화가 없거나 미미함을 확인하였다. 또한, 도 12의 (b)에 나타낸 바와 같이, TIP1의 NO 분비량 감소 효과가 가장 높았고 TIP1-2 역시 NO 분비를 억제시킨 반면, TIP1-1과 TIP1-3를 처리한 경우 변화가 없거나 미미함을 확인하였다. 이를 통해 본 발명에 따른 TIP1의 서열 중 S-H-C-R 서열이 억제제 효과를 내는데 중요함을 확인하였다.As shown in (a) of FIG. 12, TIP1 had the highest inhibitory effect on NF-κB activity and TIP1-2 also decreased NF-κB activity, whereas TIP1-1 and TIP1-3 were not changed or insignificant. It was confirmed. In addition, as shown in (b) of FIG. 12, TIP1 had the highest NO secretion reducing effect and TIP1-2 also suppressed NO secretion, whereas TIP1-1 and TIP1-3 were not changed or were insignificant. Confirmed. This confirmed that the S-H-C-R sequence of the sequence of TIP1 according to the present invention is important for producing an inhibitory effect.
실험예 8. TIP1과 TLR4 및 MyD88 간의 상호작용 확인Experimental Example 8. Confirmation of the interaction between TIP1 and TLR4 and MyD88
단백질-단백질 도킹(protein-protein docking)을 수행하여 TLR4의 TIR 도메인과 TIP1 간의 결합 인터페이스(binding interface)를 분석하였다. 그 결과, TIP1이 TLR4의 TIR 도메인에서 BB loop, DD loop 및 C-말단 꼬리에 결합하는 것으로 예상되었으며, 이를 확인하기 위하여 실시예 2-8에 기재한 방법에 따라 표면 플라즈몬 공명(surface plasmon resonance; SPR) 분석을 수행하였다.Protein-protein docking was performed to analyze the binding interface between the TIR domain of TLR4 and TIP1. As a result, TIP1 was expected to bind to the BB loop, the DD loop and the C-terminal tail in the TIR domain of TLR4, to confirm this by surface plasmon resonance according to the method described in Examples 2-8; SPR) analysis was performed.
또한, TLR4에 대한 TIP1의 결합을 확인하기 위해 TIP1의 N-말단에 FITC(fluorescein isothiocyanate)를 결합시켜 TIP1-FITC를 제조하였다. 상기 실시예 1-2의 THP1 세포에 TIP1(TIP1-FITC) 또는 LPS와 TIP1을 함께 처리한 후, 실시예 2-4에 기재한 방법에 따라 면역형광 염색 및 공초점 현미경을 이용하여 형광 염색된 세포의 수를 세어 단백질 간의 상호작용을 분석하였다. 그 결과를 도 13에 나타내었다.In addition, TIP1-FITC was prepared by binding fluorescein isothiocyanate (FITC) to the N-terminus of TIP1 to confirm binding of TIP1 to TLR4. TIP1 (TIP1-FITC) or LPS and TIP1 were treated together with THP1 cells of Example 1-2, and then fluorescently stained using immunofluorescent staining and confocal microscopy according to the method described in Examples 2-4. The number of cells was counted to analyze the interactions between the proteins. The results are shown in FIG.
도 13에 나타낸 바와 같이, TIP1-FITC와 LPS를 함께 처리하였을 경우, TIP1-FITC(녹색)와 TLR4(빨간색)가 결합하여 복합체를 형성하는 반면, 복합체 부위에 MyD88(파란색)이 결합하지 못함을 확인하였다. 한편, LPS의 부존재 시 TLR4는 원형질막에서 발현되는 반면 MyD88은 세포 내에 퍼져있는데, 세포에 LPS를 처리하는 경우 TLR4 및 TIP1-FITC 모두 세포 내재화(endocytosis) 과정을 통해 세포 내부로 유입되며, MyD88은 세포 내부에 퍼져있는 것이 아니라 원형질막 상에서 응집됨을 확인하였다. 또한, TIP1-FITC-TLR4가 강력하게 결합하는 반면에, MyD88은 TIP1-FITC에 대하여 약한 결합을 형성함을 확인하였다. 이를 통해 TLR4 신호전달 경로에 대한 TIP1의 억제적 효과는 TLR4의 TIR 도메인 중에서도 C-말단 꼬리와 BB loop에 결합함에 따른 것이며, 동시에 TIP1과 MyD88과의 상호작용은 억제되어 궁극적으로 TLR4 신호전달 과정이 저해됨을 확인하였다.As shown in FIG. 13, when TIP1-FITC and LPS were treated together, TIP1-FITC (green) and TLR4 (red) combined to form a complex, whereas MyD88 (blue) did not bind to the complex site. Confirmed. On the other hand, in the absence of LPS, TLR4 is expressed in the plasma membrane, whereas MyD88 is spread within the cell. When LPS is treated in the cell, both TLR4 and TIP1-FITC are introduced into the cell through the process of endocytosis, and MyD88 is in the cell. It was confirmed that agglomerates on the plasma membrane rather than spreading inside. In addition, while TIP1-FITC-TLR4 strongly binds, MyD88 was found to form a weak bond to TIP1-FITC. The inhibitory effect of TIP1 on the TLR4 signaling pathway is due to the binding of the C-terminal tail and the BB loop among TLR4's TIR domains. At the same time, the interaction between TIP1 and MyD88 is inhibited, ultimately leading to TLR4 signaling. It was confirmed to be inhibited.
실험예 9. Experimental Example 9. In vivoIn vivo 에서 TIP1이 쥐의 사이토카인 분비에 미치는 영향Effect of TIP1 on cytokine secretion in rats
모든 동물 실험은 실험동물 운영위원회(Institutional Animal Care and Use Committee)의 승인을 받아 진행하였다(KHNMC AP 2016-006). TIP1의 in vivo 효과를 확인하기 위해 8주령 C56BL/6 (20-25g, n=8) 마우스를 Orient Bio, Inc.(Seoul, Korea)로부터 구매하여 실험에 사용하였다. 상기 마우스에 TIP1(동물 체중 g당 10nmol)을 복강 내 주사하고 1시간 후 LPS(동물 체중 g당 5㎍)를 2시간 동안 주사하였다. 대조군에는 동일 부피의 PBS를 주사하였다. 이후 쥐 혈액으로부터 원심분리를 통해 혈장을 분리하여 분비량 분석 전까지 -80℃에서 보관하였다. 혈장 안의 TNF-α, IL-12p40(1:100으로 희석) 및 IL-6(1:100으로 희석) 수치를 쥐 TNF-α, IL-12p40, IL-6 ELISA 키트 ELISA MAX Deluxe (BioLegend, San Diego, CA, USA)를 사용하여 측정하였다. 마이크로플레이트 리더 분광 광도계(Molecular Devices In.)를 사용하여 450nm에서 흡광도를 측정하고, 그 결과를 소프트맥스 프로 5.3 소프트웨어(Molecular Devices Inc.)를 사용하여 분석하였다. 그 결과를 도 14에 나타내었다.All animal experiments were conducted with the approval of the Institutional Animal Care and Use Committee (KHNMC AP 2016-006). To confirm the in vivo effect of TIP1, 8-week-old C56BL / 6 (20-25 g, n = 8) mice were purchased from Orient Bio, Inc. (Seoul, Korea) and used for the experiment. The mice were intraperitoneally injected with TIP1 (10 mmol per g body weight of the animal) and 1 hour later LPS (5 μg per g body weight of the animal) was injected for 2 hours. The control group was injected with the same volume of PBS. The plasma was then separated from rat blood by centrifugation and stored at −80 ° C. until secretion analysis. TNF-α, IL-12p40 (diluted at 1: 100) and IL-6 (diluted at 1: 100) levels in plasma were determined using the mouse TNF-α, IL-12p40, IL-6 ELISA Kit ELISA MAX Deluxe (BioLegend, San Diego, CA, USA). Absorbance was measured at 450 nm using a microplate reader spectrophotometer (Molecular Devices In.) And the results were analyzed using Softmax Pro 5.3 software (Molecular Devices Inc.). The results are shown in FIG.
도 14의 (a) 내지 (c)에 나타낸 바와 같이, TIP1과 LPS를 함께 처리하였을 경우 TNF-α, IL-12p40 및 IL-6의 분비량이 현저히 감소함을 확인하였다. 이를 통해 본 발명에 따른 TIP1이 TLR4 신호전달 경로를 억제하여 in vitro 뿐만 아니라 in vivo에서도 사이토카인 분비를 억제함을 확인하였다. As shown in (a) to (c) of FIG. 14, when TIP1 and LPS were treated together, the secretion of TNF-α, IL-12p40, and IL-6 was significantly reduced. Through this, it was confirmed that TIP1 according to the present invention inhibits TLR4 signaling pathway and inhibits cytokine secretion not only in vitro but also in vivo .
실험예 10. TIP1이 패혈증에 미치는 영향Experimental Example 10 Effect of TIP1 on Sepsis
TIP1이 패혈증에 미치는 영향을 확인하기 위해 C57BL/6J 및 BALB/c 쥐에 PBS와 LPS 또는 TIP1과 LPS를 함께 처리한 후, 신장 및 간 손상과 생존율 변화를 확인하기 위해 하기와 같은 실험을 수행하였다.In order to determine the effect of TIP1 on sepsis, the C57BL / 6J and BALB / c mice were treated with PBS and LPS or TIP1 and LPS, and then the following experiments were performed to check for changes in kidney and liver damage and survival rate. .
10-1. TIP1이 간에서 사이토카인 분비에 미치는 영향10-1. Effect of TIP1 on Cytokine Secretion in the Liver
C57BL/6J 쥐에 PBS와 LPS 또는 TIP1과 LPS를 함께 처리한 후(2시간), 간 조직은 적출하여 Protease inhibitor cocktail(Thermo Fisher Scientific, Inc.)과 함께 M-PER 포유동물 단백질 추출 시약을 포함하는 Kontes Pellet Pestle Cordless Motor(Thermo Fisher Scientific, Inc.)를 이용하여 균질화시킨 후 제조사의 프로토콜에 따라 단백질을 추출하였다. 이후, 실시예 2-3에 기재한 방법에 따라 IL-6, TNF-α, β-actin의 단백질 발현을 웨스턴 블롯팅을 통해 시각화 하였으며, 밴드의 강도를 그래프로 나타내었고 그 결과를 도 15에 나타내었다.After treatment with PBS and LPS or TIP1 and LPS in C57BL / 6J mice (2 hours), liver tissues were extracted and included with the Protease inhibitor cocktail (Thermo Fisher Scientific, Inc.) and M-PER mammalian protein extraction reagent. After homogenization using Kontes Pellet Pestle Cordless Motor (Thermo Fisher Scientific, Inc.), the protein was extracted according to the manufacturer's protocol. Then, protein expression of IL-6, TNF-α and β-actin were visualized by Western blotting according to the method described in Example 2-3, and the intensity of the band was graphically shown and the results are shown in FIG. 15. Indicated.
도 15의 (a) 및 (b)에 나타낸 바와 같이, LPS와 TIP1을 함께 처리하였을 경우 쥐의 간 조직에서 LPS에 의해 유도된 TNF-α 및 IL-6의 발현량이 현저히 감소함을 확인하였다.As shown in (a) and (b) of FIG. 15, when LPS and TIP1 were treated together, it was confirmed that the expression levels of TNF-α and IL-6 induced by LPS in rat liver tissues were significantly reduced.
10-2. TIP1이 신장 및 간 손상에 미치는 영향10-2. Effect of TIP1 on Kidney and Liver Damage
마우스 패혈증 모델에서 TIP1의 항염증 효과를 보다 정확하게 확인하기 위하여, 상기와 동일한 방법으로 실험을 수행하였다. 구체적으로, C57BL/6J 쥐에 PBS와 LPS 또는 TIP1과 LPS를 함께 처리한 후 (24시간), 쥐 혈액으로부터 원심분리를 통해 혈장을 분리하였다. 분리한 혈장을 이용하여, 상기 실험예 9의 방법에 의해 TNF-α와 IL-6의 분비량을 측정하였고, 신장 손상 마커인 BUN(blood urea nitrogen) 및 크레아틴(creatinine; Cr)과 간 손상 마커인 AST(aspartate aminotransferase) 및 ALT(alanine aminotransferase)의 분비량 변화를 VETTEST-8008-system (IDEXX)을 통해 분석하였다. 그 결과를 도 16에 나타내었다.In order to more accurately identify the anti-inflammatory effect of TIP1 in the mouse sepsis model, the experiment was carried out in the same manner as above. Specifically, C57BL / 6J mice were treated with PBS and LPS or TIP1 and LPS together (24 hours), and then plasma was separated from rat blood by centrifugation. Using the separated plasma, the secretion amount of TNF-α and IL-6 was measured by the method of Experimental Example 9, and the blood damage markers (BUN (blood urea nitrogen) and creatinine (Cr) and liver damage markers), which are kidney damage markers, were measured. The secretion changes of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were analyzed by VETTEST-8008-system (IDEXX). The results are shown in FIG.
도 16의 (a) 내지 (f)에 나타낸 바와 같이, TIP1과 LPS를 함께 처리하였을 경우 TNF-α와 IL-6 및 BUN, Cr, AST, ALT의 분비량이 현저히 감소하였음을 확인하였다. 이를 통해 본 발명에 따른 TIP1이 TLR4 신호전달 경로를 억제하여 사이토카인 분비는 물론 신장 및 간 손상을 완화 시킬 수 있음을 확인하였다. As shown in (a) to (f) of FIG. 16, when TIP1 and LPS were treated together, it was confirmed that the secretions of TNF-α and IL-6 and BUN, Cr, AST, and ALT were significantly reduced. Through this, it was confirmed that TIP1 according to the present invention inhibits the TLR4 signaling pathway and can mitigate kidney and liver damage as well as cytokine secretion.
10-3. TIP1이 신장의 세포사멸(apoptosis)에 미치는 영향10-3. Effect of TIP1 on Apoptosis in Kidney
8주령 C56BL/6 (20-25g, n=8) 마우스에 TIP1과 LPS 또는 PBS와 LPS를 함께 처리한 후(24시간), 신장조직을 분리하고 electronic balance(ER-180A, A&D Company, Tokyo, Japan)를 사용하여 무게를 측정한 후, 시상면으로 절단하여 슬라이스로 만들었다. 이를 10% 포르말린(formalin) 용액에 하룻밤 동안 둔 뒤 파라핀 왁스를 부어 고체화시키고 미세박절기(microtome)를 이용하여 4㎛의 얇은 절편으로 만들어 고정(fixation)하였다. 고정한 신장조직에서 TIP1이 세포사멸에 미치는 영향을 확인하기 위해 TUNEL(terminal deoxynucleotidyl transferase dUTP nick end labeling) (Merck Millipore, Billerica, MA, USA) 염색을 하고 공초점 레이저 주사 현미경(LSM-700, Carl Zeiss MicroImaging GmbH, Jena, Germany)을 사용하여 상기 형광 염색된 세포의 수를 세었으며, Zen 2009 소프트웨어(Carl Zeiss MicroImaging GmbH.)를 사용하여 이미지를 분석하였다. 그 결과를 도 17에 나타내었다.After treatment with TIP1 and LPS or PBS and LPS (24 hours) in 8-week-old C56BL / 6 (20-25 g, n = 8) mice, kidney tissues were separated and electronic balance (ER-180A, A & D Company, Tokyo, Japan) was weighed, and then cut into sagittal planes and sliced. This was left overnight in a 10% formalin (formalin) solution, followed by solidification by pouring paraffin wax and fixing it by making a thin section of 4 μm using a microtome. To determine the effect of TIP1 on apoptosis in fixed kidney tissue, stain with TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) (Merck Millipore, Billerica, MA, USA) and confocal laser scanning microscopy (LSM-700, Carl Zeiss). The fluorescently stained cells were counted using MicroImaging GmbH, Jena, Germany) and images were analyzed using Zen 2009 software (Carl Zeiss MicroImaging GmbH.). The results are shown in FIG.
도 17에 나타낸 바와 같이, LPS만을 처리한 경우 TUNEL-양성 신장세포가 현저히 증가, 즉 신장조직에서의 세포사멸(녹색점)이 증가한 반면, TIP1과 LPS를 함께 처리하였을 경우, LPS에 의해 증가한 신장조직에서의 세포사멸(녹색점)이 감소함을 확인하였다.As shown in FIG. 17, TUNEL-positive kidney cells were significantly increased when LPS was treated only, ie, cell death (green dot) in kidney tissues was increased, whereas when TIP1 and LPS were treated together, kidneys increased by LPS. It was confirmed that apoptosis (green dot) in the tissue was reduced.
10-4. TIP1이 생존율에 미치는 영향10-4. Effect of TIP1 on Survival Rate
TIP1이 LPS에 의해 유도된 패혈증에 미치는 영향을 알아보기 위해 8주령 BALB/c (20-25g, n=8) 수컷 마우스에 LPS(동물 체중 g당 5 또는 10㎍)와 TIP1(동물 체중 g당 10nmol)과 LPS를 함께 처리한 후 5일 동안 생존율을 측정하였다. 대조군에는 동일 부피의 PBS를 주사하였다. 그 결과를 도 18에 나타내었다.To determine the effect of TIP1 on LPS-induced sepsis, 8-week-old BALB / c (20-25 g, n = 8) male mice had LPS (5 or 10 μg / g animal weight) and TIP1 (g / g animal weight). 10 nmol) and LPS were treated together and the survival rate was measured for 5 days. The control group was injected with the same volume of PBS. The results are shown in FIG.
도 18에 나타낸 바와 같이, TIP1과 LPS를 함께 처리하였을 경우, LPS만 처리하였을 경우에 비해 생존율이 증가함을 확인하였다. 구체적으로, LPS를 5㎍/g으로 처리한 온화한 패혈증 모델의 경우 3일만에 생존율이 100% 감소하였고, TIP1을 함께 처리하였을 경우 30%의 생존율을 5일 동안 유지하였다. 또한, LPS를 10㎍/g으로 처리한 중증 패혈증 모델의 경우 하루 만에 생존율이 70% 감소하였고, 이틀 후 모든 마우스가 사망하였으며, TIP1을 함께 처리하였을 경우 중증 패혈증을 유발시켰음에도 하루 동안 생존율이 100% 유지되었고, 이틀 후 생존율이 90% 감소하였다. 이를 통해 본 발명에 따른 TIP1이 TLR4 신호전달 경로를 억제하여 염증의 초기 단계를 완화시킴으로써 사이토카인 분비와 신장 및 간 손상을 감소시키고 결국 패혈증을 완화 시킴을 확인하였다.As shown in FIG. 18, when TIP1 and LPS were treated together, it was confirmed that the survival rate was increased compared with the case where only LPS was treated. Specifically, in the mild sepsis model treated with LPS at 5 μg / g, survival was reduced by 100% after 3 days, and when treated with TIP1, 30% survival was maintained for 5 days. In addition, in the severe sepsis model treated with 10 μg / g LPS, the survival rate decreased by 70% in one day, and all mice died two days later, and when treated with TIP1, the survival rate during the day was increased even though it induced severe sepsis. It remained 100% and after two days the survival rate decreased by 90%. Through this, it was confirmed that TIP1 according to the present invention inhibits the TLR4 signaling pathway to alleviate the early stage of inflammation, thereby reducing cytokine secretion and kidney and liver damage and eventually alleviating sepsis.
실험예 11. TIP1이 류마티스 관절염에 미치는 영향Experimental Example 11. Effect of TIP1 on Rheumatoid Arthritis
TIP1이 류마티스 관절염에 미치는 영향을 확인하기 위해 6 내지 7주령 DBA/1J (20-23g) 수컷 마우스에 콜라겐 유도 관절염(Collagen-induced arthritis, CIA) 모델을 적용시켜 관절염을 유도하였다. 구체적으로, 닭의 제2형 콜라겐(chicken collagen type Ⅱ, Sigma-Aldrich Co. LLC)과 완전 프로인트 보조제(complete Freund's adjuvant, Sigma-Aldrich Co. LLC.)를 1:1로 혼합하여 에멀젼화한 후, 상기 에멀젼화된 콜라겐 용액 100㎍을 마우스 꼬리에 피내 주사하여 1차 면역을 유도하고 이를 day 0으로 지정하였다. 1차 면역 14일 후에 상기 에멀젼화된 콜라겐 용액을 재차 마우스 꼬리에 피내 주사하여 2차 면역(boosting)을 유도하였다. To determine the effect of TIP1 on rheumatoid arthritis, arthritis was induced by applying collagen-induced arthritis (CIA) model to 6-7 week old DBA / 1J (20-23g) male mice. Specifically, emulsified by mixing chicken collagen type II (Sigma-Aldrich Co. LLC) and complete Freund's adjuvant (Sigma-Aldrich Co. LLC.) In a 1: 1 ratio. Thereafter, 100 μg of the emulsified collagen solution was injected intravenously into the tail of the mouse to induce primary immunity and designated day 0. After 14 days of primary immunization, the emulsified collagen solution was again injected intradermal into the mouse tail to induce secondary immunity.
이후 다음의 두 종류의 TIP1 처리 계획을 설계하였다: ① 2차 면역 후(day 15), 30일 동안 TIP1(2.5, 5 및 10nmol/g) 또는 기존 관절염 치료제로 잘 알려진 양성 대조군 프레드니솔론(prednisolone) 5mg/kg을 매일 주사하는 계획; 및 ② 관절염을 완전히 유발시킨 후(day 35), 10일 동안 TIP1 10nmol/g을 주사하는 계획. 이는 총 7개 실험군(n=8)으로, 다음과 같이 명명하였다: (1) 무처리 정상군(Normal); (2) 대조군(vehicle-treated)(CIA, n=8); (3) 2.5nmol/g TIP1 처리군(CIA-TIP1(2.5nmol/g)); (4) 5nmol/g TIP1 처리군(CIA-TIP1(5nmol/g)); (5) 10nmol/g TIP1 처리군(CIA-TIP1(10nmol/g)); (6) 5㎍/g 프레드니솔론(prednisolone, 기존 관절염 치료제) 처리군(CIA-prednisolone(5㎍/g); (7) post-arthritis phase(PAP)에서 10nmol/g TIP1 처리군(PAP CIA-TIP1(10nmol/g). TIP1과 프레드니솔론은 식염수에 용해시켜 복강 내 주사하였고, 이와 함께 외관상 보이는 증상과 체중, 쥐의 울음소리, 발의 부피, 관절염 인덱스 등 행동발달 및 장애의 변화를 분석하였다. 그 결과를 도 19 및 도 20에 나타내었다.Two TIP1 treatment plans were then designed: ① TIP1 (2.5, 5 and 10 nmol / g) or 30 mg of positive control prednisolone, a well known treatment for conventional arthritis, after secondary immunization (day 15) plans to inject / kg daily; And ② plan to inject 10 mmol / g of TIP1 for 10 days after fully inducing arthritis (day 35). This was a total of seven experimental groups (n = 8), named as follows: (1) no treatment normal (Normal); (2) vehicle-treated (CIA, n = 8); (3) 2.5 nmol / g TIP1 treated group (CIA-TIP1 (2.5 nmol / g)); (4) 5 nmol / g TIP1 treated group (CIA-TIP1 (5 nmol / g)); (5) 10 nmol / g TIP1 treated group (CIA-TIP1 (10 nmol / g)); (6) 5 µg / g prednisolone (preexisting arthritis treatment) treatment group (CIA-prednisolone (5 µg / g); (7) 10 nmol / g TIP1 treatment group in the post-arthritis phase (PAP) (PAP CIA-TIP1 (10nmol / g) TIP1 and prednisolone were dissolved in saline and injected intraperitoneally and analyzed for changes in behavioral development and disorders such as apparent symptoms and weight, rat crying, paw volume, and arthritis index. Are shown in FIGS. 19 and 20.
도 19에 나타낸 바와 같이, TIP1을 처리하였을 경우 CIA 모델에 의해 심하게 부었던 발이 정상 쥐와 유사하게 완화됨을 육안으로 확인하였다. 또한, 도 20에 나타낸 바와 같이, TIP1가 관절염 유도에 의한 체중감소, 울음소리와 발의 부피 및 관절염 인덱스 증가를 기존 관절염 치료제인 프레드니솔론과 유사한 수준으로 억제함을 확인하였다. As shown in FIG. 19, it was confirmed visually that the foot severely swollen by the CIA model was relieved similarly to the normal rat when TIP1 was treated. In addition, as shown in Figure 20, it was confirmed that TIP1 inhibits weight loss, crying and foot volume and arthritis index increase by arthritis induction to a similar level to prednisolone, an existing arthritis treatment agent.
또한, CIA 모델을 통해 관절염이 유도된 DAB-1J 쥐에 30일간 TIP1 및 프레드니솔론을 매일 1회 주사한 후 day 45에 마우스를 희생시켜 관절 조직을 샘플링하였다. 이후, Micro-CT(micro-computed tomography)를 통해 관절 조직의 3D 이미지 및 골밀도(BMD; born mineral density)를 분석하였다. 또한, 쥐의 무릎관절 부위를 이용하여 Hematoxylin and eosin(H&E) 염색을 하고 연골(cartilage), 연골 하골(subchondral bone), 대퇴골(femur), 경골(tibia), 메니스커스(meniscus)에서 활막 과다 증식증(synovial hyperplasia)의 변화를 관찰하였다. 그 결과를 도 21 및 도 22에 나타내었다.In addition, arthritis-induced DAB-1J rats were injected once daily with TIP1 and prednisolone every 30 days through the CIA model, and then the mice were sacrificed on day 45 to sample the joint tissue. Thereafter, 3D image and bone mineral density (BMD) of joint tissue were analyzed by micro-computed tomography (Micro-CT). In addition, hematoxylin and eosin (H & E) staining was performed using the knee joints of rats, and the synovial excess in cartilage, subchondral bone, femur, tibia, and meniscus. Changes in synovial hyperplasia were observed. The results are shown in FIGS. 21 and 22.
도 21과 도 22에 나타낸 바와 같이, TIP1을 처리하였을 경우, CIA 모델에 의해 손상된 뼈가 정상 쥐와 유사하게 완화되고 연골(C), 연골 하골(S), 대퇴골(F), 경골(T), 메니스커스(M)의 조직병리학적 변형 역시 감소함을 확인하였다. 이를 통해 본 발명에 따른 TIP1은 TLR4 신호전달경로를 효과적으로 억제함에 따라 류마티스 관절염을 비롯한 다양한 급성 또는 만성 염증성 질환의 치료제로서 유용하게 사용될 수 있음을 확인하였다.21 and 22, when treated with TIP1, bones damaged by the CIA model were alleviated similarly to normal rats, and cartilage (C), cartilage lower bone (S), femur (F), and tibia (T). , Histopathological modification of meniscus (M) was also reduced. Through this, TIP1 according to the present invention effectively inhibits the TLR4 signaling pathway, it was confirmed that it can be usefully used as a therapeutic agent for various acute or chronic inflammatory diseases including rheumatoid arthritis.
전술한 본 발명의 설명은 예시를 위한 것이며, 본 발명이 속하는 기술 분야의 통상의 지식을 가진 자는 본 발명의 기술적 사상이나 필수적인 특징을 변경하지 않고서 다른 구체적인 형태로 쉽게 변형이 가능하다는 것을 이해할 수 있을 것이다. 그러므로 이상에서 기술한 실시예들은 모든 면에서 예시적인 것이며 한정적이 아닌 것으로 이해되어야 한다.The above description of the present invention is intended for illustration, and it will be understood by those skilled in the art that the present invention may be easily modified in other specific forms without changing the technical spirit or essential features of the present invention. will be. Therefore, the embodiments described above are to be understood in all respects as illustrative and not restrictive.

Claims (17)

  1. 서열번호 1의 아미노산 서열로 이루어진 펩타이드. Peptide consisting of the amino acid sequence of SEQ ID NO: 1.
  2. 제1항에 있어서, 상기 펩타이드의 S-H-C-R 서열(서열번호 2)은 TLR(Toll-like receptor)의 TIR(Toll/interleukin-1 receptor) 도메인에 특이적으로 결합하는 것을 특징으로 하는, 펩타이드.The peptide of claim 1, wherein the S-H-C-R sequence (SEQ ID NO: 2) of the peptide specifically binds to a Toll / interleukin-1 receptor (TIR) domain of a toll-like receptor (TLR).
  3. 세포 투과성 펩타이드의 N-말단에 서열번호 1 또는 서열번호 2의 아미노산 서열로 이루어진 펩타이드가 연결된 융합 펩타이드. A fusion peptide wherein a peptide consisting of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2 is linked to the N-terminus of the cell permeable peptide.
  4. 제3항에 있어서, 상기 융합 펩타이드는 TLR(Toll-like receptor)에 의해 매개되는 신호전달 경로를 억제하는 것을 특징으로 하는, 융합 펩타이드.The fusion peptide of claim 3, wherein the fusion peptide inhibits a signaling pathway mediated by a toll-like receptor (TLR).
  5. 제4항에 있어서, 상기 융합 펩타이드는 TLR4(Toll-like receptor 4)에 의해 매개되는 신호전달 경로를 억제하는 것을 특징으로 하는, 융합 펩타이드.The fusion peptide of claim 4, wherein the fusion peptide inhibits a signaling pathway mediated by TLR4 (Toll-like receptor 4).
  6. 제4항에 있어서, 상기 융합 펩타이드는 TLR1/2(Toll-like receptor 1/2), TLR2/6(Toll-like receptor 2/6), TLR3(Toll-like receptor 3), TLR7(Toll-like receptor 7), TLR8(Toll-like receptor 8) 및 TLR9(Toll-like receptor 9)로 이루어진 군으로부터 선택되는 어느 하나에 의해 매개되는 신호전달 경로를 억제하는 것을 특징으로 하는, 융합 펩타이드.The method of claim 4, wherein the fusion peptide is TLR1 / 2 (Toll-like receptor 1/2), TLR2 / 6 (Toll-like receptor 2/6), TLR3 (Toll-like receptor 3), TLR7 (Toll-like) A fusion peptide, characterized in that it inhibits signaling pathways mediated by any one selected from the group consisting of receptor 7), toll-like receptor 8 (TLL8) and toll-like receptor 9 (TLL9).
  7. 제3항에 있어서, 상기 융합 펩타이드의 TLR 신호전달경로 차단에 의해 TNF-α, IL-6 또는 IFN-β의 발현 억제; NO 또는 ROS의 분비 억제; 또는 NF-κB, MAPK 또는 NLRP3 인플라마좀의 활성이 억제되는 것을 특징으로 하는, 융합 펩타이드.The method of claim 3, further comprising: inhibiting the expression of TNF-α, IL-6 or IFN-β by blocking TLR signaling pathways of the fusion peptides; Inhibition of secretion of NO or ROS; Or fusion peptides, characterized in that the activity of NF-κB, MAPK or NLRP3 inflamasome is inhibited.
  8. 제3항에 있어서, 상기 융합 펩타이드는 MyD88-의존적 및 MyD88-비의존적 TLR4 신호전달 경로를 모두 억제하는 것을 특징으로 하는, 융합 펩타이드.The fusion peptide of claim 3, wherein the fusion peptide inhibits both MyD88-dependent and MyD88-independent TLR4 signaling pathways.
  9. 제3항에 있어서, 상기 융합 펩타이드는 서열번호 3 또는 서열번호 4의 아미노산 서열로 이루어진 것을 특징으로 하는, 융합 펩타이드.The fusion peptide of claim 3, wherein the fusion peptide comprises an amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 4. 5.
  10. 서열번호 1 또는 서열번호 2의 아미노산 서열로 이루어진 펩타이드를 유효성분으로 포함하는 자가면역질환, 염증성 질환 및 퇴행성 신경질환으로 이루어진 군으로부터 선택되는 1종 이상의 TLR 경로 매개성 질환의 예방 또는 치료용 약학적 조성물.Pharmaceutical for the prophylaxis or treatment of at least one TLR pathway mediated disease selected from the group consisting of autoimmune diseases, inflammatory diseases and neurodegenerative diseases comprising a peptide consisting of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2 as an active ingredient Composition.
  11. 세포 투과성 펩타이드의 N-말단에 서열번호 1 또는 서열번호 2의 아미노산 서열로 이루어진 펩타이드가 연결된 융합 펩타이드를 유효성분으로 포함하는 자가면역질환, 염증성 질환 및 퇴행성 신경질환으로 이루어진 군으로부터 선택되는 1종 이상의 TLR 경로 매개성 질환의 예방 또는 치료용 약학적 조성물.At least one selected from the group consisting of autoimmune diseases, inflammatory diseases and neurodegenerative diseases comprising a fusion peptide linked to a peptide consisting of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2 at the N-terminus of the cell permeable peptide as an active ingredient A pharmaceutical composition for preventing or treating a TLR pathway mediated disease.
  12. 제11항에 있어서, 상기 융합 펩타이드는 서열번호 3 또는 서열번호 4의 아미노산 서열로 이루어진 것을 특징으로 하는, 자가면역질환, 염증성 질환 및 퇴행성 신경질환으로 이루어진 군으로부터 선택되는 1종 이상의 TLR 경로 매개성 질환의 예방 또는 치료용 약학적 조성물.The method of claim 11, wherein the fusion peptide is one or more TLR pathway mediated mediation selected from the group consisting of autoimmune diseases, inflammatory diseases and neurodegenerative diseases, characterized in that consisting of the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 4. Pharmaceutical compositions for the prevention or treatment of diseases.
  13. 제10항 내지 제12항 중 어느 한 항에 있어서, 상기 자가면역질환은 인슐린-의존성 당뇨병, 다발 경화증, 실험적 자가면역 뇌척수염, 류마티스성 관절염, 실험적 자가면역 관절염, 중증 근무력증, 갑상선염, 실험적 형태의 포도막염, 하시모토 갑상선염, 원발성 점액수종, 갑상샘 중독증, 악성 빈혈, 자가면역 위축 위염, 애디슨 질환, 조기 폐경, 남성 불임증, 소아 당뇨병, 굿파스처 증후군, 보통 천포창, 유천포창, 교감성 안염, 수정체성 포도막염, 자가면역 용혈성 빈혈, 특발성 백혈구 감소, 원발성 담관 경화증, 만성 활동성 간염 Hbs-ve, 잠재성 간경변증, 궤양성 대장염, 쇼그렌 증후군, 경피증, 베게너 육아종증, 다발근육염/피부근육염, 원판상 LE 및 전신 홍반 루푸스로 이루어진 군으로부터 선택된 1종 이상인 것을 특징으로 하는, 자가면역질환, 염증성 질환 및 퇴행성 신경질환으로 이루어진 군으로부터 선택되는 1종 이상의 TLR 경로 매개성 질환의 예방 또는 치료용 약학적 조성물.The method of claim 10, wherein the autoimmune disease is insulin-dependent diabetes mellitus, multiple sclerosis, experimental autoimmune encephalomyelitis, rheumatoid arthritis, experimental autoimmune arthritis, myasthenia gravis, thyroiditis, experimental form of uveitis. , Hashimoto's thyroiditis, primary myxedema, thyroid poisoning, pernicious anemia, autoimmune atrophy, gastritis, Addison's disease, early menopause, male infertility, childhood diabetes, Goodpasture's syndrome, common pemphigus, ileal swelling, sympathetic ophthalmitis, lens uveitis, Autoimmune hemolytic anemia, idiopathic leukocyte reduction, primary cholangiovascular sclerosis, chronic active hepatitis Hbs-ve, latent cirrhosis, ulcerative colitis, Sjogren's syndrome, scleroderma, Wegener's granulomatosis, polymyositis / dermal myositis, discoid LE and systemic lupus erythematosus At least one member selected from the group consisting of, autoimmune disease, inflammatory vagina Pharmaceutical composition for the prevention or treatment of at least one TLR pathway mediated disease selected from the group consisting of ring and degenerative neurological diseases.
  14. 제10항 내지 제12항 중 어느 한 항에 있어서, 상기 염증성 질환은 천식, 습진, 건선, 알러지, 류마티스 관절염, 건선 관절염, 아토피성 피부염, 여드름, 아토피성 비염, 폐염증, 알레르기성 피부염, 만성 부비동염, 접촉성 피부염(contact dermatitis), 지루성 피부염(seborrheic dermatitis), 위염, 통풍, 통풍 관절염, 궤양, 만성 기관지염, 크론병, 궤양성 대장염, 강직성 척추염(ankylosing spondylitis), 패혈증, 맥관염, 활액낭염, 루프스, 류마티스 다발성 근육통, 측두 동맥염, 다발성 경화증, 고형암, 알츠하이머병, 동맥경화증, 비만 및 바이러스 감염으로 이루어진 군으로부터 선택된 1종 이상인 것을 특징으로 하는, 자가면역질환, 염증성 질환 및 퇴행성 신경질환으로 이루어진 군으로부터 선택되는 1종 이상의 TLR 경로 매개성 질환의 예방 또는 치료용 약학적 조성물.The method according to claim 10, wherein the inflammatory disease is asthma, eczema, psoriasis, allergy, rheumatoid arthritis, psoriatic arthritis, atopic dermatitis, acne, atopic rhinitis, pulmonary inflammation, allergic dermatitis, chronic Sinusitis, contact dermatitis, seborrheic dermatitis, gastritis, gout, gout arthritis, ulcers, chronic bronchitis, Crohn's disease, ulcerative colitis, ankylosing spondylitis, sepsis, vasculitis, bursitis, Group consisting of autoimmune disease, inflammatory disease and degenerative neuropathy, characterized by at least one member selected from the group consisting of lupus, rheumatoid polymyalgia, temporal arteritis, multiple sclerosis, solid cancer, Alzheimer's disease, arteriosclerosis, obesity and viral infection A pharmaceutical composition for the prophylaxis or treatment of at least one TLR pathway mediated disease selected from.
  15. 제10항 내지 제12항 중 어느 한 항에 있어서, 상기 퇴행성 신경질환은 알츠하이머병, 전두측두치매, 루이치매, 피질기저퇴행증, 파킨슨병, 다계통위축병, 헌팅턴병, 진핵성핵상마비, 루게릭병, 원발성측삭경화증, 척수근육위축병으로 이루어진 군으로부터 선택된 1종 이상인 것을 특징으로 하는, 자가면역질환, 염증성 질환 및 퇴행성 신경질환으로 이루어진 군으로부터 선택되는 1종 이상의 TLR 경로 매개성 질환의 예방 또는 치료용 약학적 조성물.The neurodegenerative disease according to any one of claims 10 to 12, wherein the neurodegenerative disease is Alzheimer's disease, frontal temporal dementia, Louis dementia, cortical basal degeneration, Parkinson's disease, multi-system atrophy, Huntington's disease, eukaryotic nucleus palsy, Lou Gehrig's disease. Prevention of at least one TLR pathway mediated disease selected from the group consisting of autoimmune diseases, inflammatory diseases and neurodegenerative diseases, characterized in that at least one selected from the group consisting of diseases, primary sclerosis, spinal muscular atrophy, or Therapeutic pharmaceutical composition.
  16. 서열번호 1 또는 서열번호 2의 아미노산 서열로 이루어진 펩타이드를 이를 필요로 하는 개체에 투여하는 단계; 를 포함하는 TLR 경로 매개성 질환의 예방 또는 치료 방법.Administering a peptide consisting of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2 to an individual in need thereof; Method for preventing or treating a TLR pathway mediated disease comprising a.
  17. 세포 투과성 펩타이드의 N-말단에 서열번호 1 또는 서열번호 2의 아미노산 서열로 이루어진 펩타이드가 연결된 융합 펩타이드를 이를 필요로 하는 개체에 투여하는 단계; 를 포함하는 TLR 경로 매개성 질환의 예방 또는 치료 방법.Administering to a subject in need thereof a fusion peptide linked to a peptide consisting of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2 at the N-terminus of the cell permeable peptide; Method for preventing or treating a TLR pathway mediated disease comprising a.
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2021517561A (en) * 2018-01-02 2021-07-26 ラッシュ・ユニバーシティ・メディカル・センター Compositions and Methods for Treating Neurological Disorders and Other Disorders
WO2024096261A1 (en) * 2022-10-31 2024-05-10 아주대학교산학협력단 Composition for preventing or treating autoinflammatory diseases, and uses thereof

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
DATABASE Protein [O] 15 August 2014 (2014-08-15), "Deformed [Echinococcus Granulosus", XP055561138, Database accession no. CDS15727.1 *
DATABASE Protein [O] 18 July 2016 (2016-07-18), "PREDICTED: Toll/interleukin-1 Receptor Domain-containing Adapter Protein Isoform X1 [Cebus Capucinus Imitator]", XP055561131, Database accession no. XP_017382104.1 *
DATABASE Protein [O] 8 December 2015 (2015-12-08), "Deformed [Hymenolepis Microstoma", XP055561141, Database accession no. CDS27878.1 *
LOIARRO, MARIA ET AL.: "Targeting TLR/IHR Signalling in Human Diseases", MEDIATORS OF INFLAMMATION, vol. 2010, 2010, pages 1 - 12, XP055401730 *
See also references of EP3647321A4 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2021517561A (en) * 2018-01-02 2021-07-26 ラッシュ・ユニバーシティ・メディカル・センター Compositions and Methods for Treating Neurological Disorders and Other Disorders
JP7212694B2 (en) 2018-01-02 2023-01-25 ラッシュ・ユニバーシティ・メディカル・センター Compositions and methods for treating neurological and other disorders
WO2024096261A1 (en) * 2022-10-31 2024-05-10 아주대학교산학협력단 Composition for preventing or treating autoinflammatory diseases, and uses thereof

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