WO2018203613A1 - Peptide pour l'inhibition du récepteur de type toll (tlr) et composition pharmaceutique le comprenant - Google Patents

Peptide pour l'inhibition du récepteur de type toll (tlr) et composition pharmaceutique le comprenant Download PDF

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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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peptide
tip1
seq
disease
diseases
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PCT/KR2018/004803
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English (en)
Korean (ko)
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최상돈
권혁권
신현준
계향애
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아주대학교산학협력단
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Priority claimed from KR1020180046715A external-priority patent/KR102024186B1/ko
Application filed by 아주대학교산학협력단 filed Critical 아주대학교산학협력단
Priority to US16/610,278 priority Critical patent/US11352399B2/en
Priority to CN201880045288.0A priority patent/CN111094321B/zh
Priority to EP18793867.5A priority patent/EP3647321A4/fr
Priority to JP2020512344A priority patent/JP6906692B2/ja
Publication of WO2018203613A1 publication Critical patent/WO2018203613A1/fr

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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

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  • 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

La présente invention concerne un polypeptide de fusion qui inhibe les voies de signalisation TLR1/2, TLR2/6, TLR7, TLR8 et TLR9 ainsi que le récepteur 4 de type Toll (TLR4) et TLR3 et une composition pharmaceutique destinée à prévenir ou à traiter des maladies médiées par la voie TLR. Le peptide de fusion de la présente invention présente un excellent effet d'inhibition de TLR4 et de diverses voies TLR et peut être utilisé efficacement dans la prévention et le traitement de diverses maladies, médiées par la voie TLR, provoquées par les voies de signalisation, telles que des maladies auto-immunes, des maladies inflammatoires et des maladies neurologiques dégénératives, par inhibition des réponses immunitaires médiées par TLR.
PCT/KR2018/004803 2017-05-04 2018-04-25 Peptide pour l'inhibition du récepteur de type toll (tlr) et composition pharmaceutique le comprenant WO2018203613A1 (fr)

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US16/610,278 US11352399B2 (en) 2017-05-04 2018-04-25 Peptide for toll-like receptor (TLR) inhibition and pharmaceutical composition comprising same
CN201880045288.0A CN111094321B (zh) 2017-05-04 2018-04-25 用于抑制Toll样受体(TLR)的肽和包含该多肽的药物组合物
EP18793867.5A EP3647321A4 (fr) 2017-05-04 2018-04-25 Peptide pour l'inhibition du récepteur de type toll (tlr) et composition pharmaceutique le comprenant
JP2020512344A JP6906692B2 (ja) 2017-05-04 2018-04-25 トール様受容体（ｔｌｒ）阻害のためのペプチドおよびそれを含む医薬組成物

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