KR20200050715A - Composition for preventing or treating lupus comprising tlr-inhibiting peptides - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating lupus containing a peptide which inhibits a Toll-like receptor (TLR) signaling pathway as an active component. The pharmaceutical composition according to the present invention effectively modulates factors related to lupus symptoms in a lupus mouse model, thereby being useful for preventing or treating lupus.

Description

Composition for preventing or treating lupus comprising a TLR inhibitory peptide {COMPOSITION FOR PREVENTING OR TREATING LUPUS COMPRISING TLR-INHIBITING PEPTIDES}

The present invention relates to a composition for preventing or treating lupus, comprising a peptide that inhibits a TLR (Toll-like receptor) signaling pathway.

Innate immunity is the first defense against bacterial infections in the mammalian immune system. Pattern recognition receptors, such as toll-like receptors, are pathogen-associated molecular patterns (PAMPs) or risk-associated molecular patterns (dangers). -associated molecular pattern (DAMP).

TLRs play an important role in the innate immune response, and extracellular TLRs acting on the plasma membrane, including TLR1, TLR2, TLR4, TLR5, TLR6 and TLR11, and cells such as endosomes including TLR3, TLR7, TLR8 and TLR9 It can be divided into intracellular TLRs that function within. Structurally, the TLR has a leucine-rich repeat (RRR) site recognized by a ligand or accessory molecule at the N-terminus of the extracellular domain, and signals to the C-terminus of the intracellular part. It has a Toll / interleukin 1 receptor (TIR) domain.

In particular, TLR4 is the first identified receptor in the TLR family, which activates innate immune signals that are amplified through the myDoid (myeloid differentiation 88) -dependent signaling pathway and MyD88-independent signaling pathway. In contrast, TLR3 only activates the MyD88-independent signaling pathway. Due to this role of TLR, interest in research to utilize TLR as a target for treating various immune diseases is increasing.

The MyD88-dependent signaling of TLR4 is initiated by LPS recognition through accessory molecules such as clusters of differentiation 14 (CDA14) 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 together and form a complex with MyD88 Thereby activating the signaling pathway. Activated TLR4 induces the initial activation of NF-κB, transfer to the nucleus, and activation of MAPK through the Myd88-dependent signaling process. Activation of the 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 binding between the TIR domain of each TLR and the TIR domain of the TIR domain-containing adapter-inducing interferon-β (TRIF) -related adaptor molecule (TRAM) complex of TRAM / TRIF. And secretes type 1 interferon by activation of an interferon-regulatory factor (IRF). In addition, TLR4 activity produces oxidative stress substances such as NO and ROS in macrophages.

As such, TLRs may be targets for treating various diseases such as autoimmune diseases, inflammatory diseases, and cancers, and thus, research on substances targeting TLRs and medical compositions for treating diseases related to TLRs has been conducted. In particular, a large number of TLR4 accelerators and antagonists have been obtained by modifying the major skeletal structure of lipid A, eritoran, lipid A and Rhodobacter It has been found that sphaeroids lipid A (RsLA) can inhibit the interaction of LPS with MD2 and prevent shock induced by LPS 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 by participating in NLRP3 inflammasome formation.

On the other hand, studies have been actively conducted on peptides that act similar or opposite to pathogen-associated molecular patterns (PAMPs) using decoy peptides that control signaling by instead binding to proteins that interact originally using some of the protein's binding domains. Is going on. Peptides are known to have fewer side effects and easier modification and quality control than general small molecule therapeutics. Low cell permeability is also improved due to the development of cell penetrating peptide (CPP).

Lupus is a systemic autoimmune disease involving several organs, characterized by tissue damage caused by the formation of various autoantibodies and immune complexes. Skeletal system and skin symptoms are the most common, but a wide variety of clinical findings can occur in various organs of the body, such as the skin, joints, blood, and kidneys, during the early or course of the disease. : Vol. 78, No. 4, 2010). There are several types of lupus. The most common type is Systemic lupus erythematosus (SLE), which affects many parts of the body. Other types include discoid lupus erythematosus, which causes skin rash that does not go away, subacute skin erythema lupus, which causes skin ulcers on body parts exposed to the sun, drug-induced drug-induced lupus, and newborns with rare but newborn babies Lupus and others.

The present inventors are SLE mice treated with a novel peptide consisting of the amino acid sequence of SEQ ID NO: 1 (decoy peptide 1) and a fusion peptide (Toll-like receptor inhibitory peptide 1, TIP1) in which the new peptide is linked to the N-terminus of the cell permeable peptide. Compared to the SLE mouse model in which these peptides are not treated in the model, lymphocyte proliferation is suppressed, albumin content in urine is reduced, and anti-dsDNA antibody and anti-nuclear antibody (ANA) are produced less. Was confirmed.

Accordingly, an object of the present invention is to provide a pharmaceutical composition for preventing or treating lupus comprising a peptide consisting of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2 as an active ingredient.

In addition, another object of the present invention to provide a pharmaceutical composition for preventing or treating lupus comprising an 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 a cell permeable peptide as an active ingredient. will be.

In order to achieve the above object, the present invention provides a pharmaceutical composition for preventing or treating lupus comprising a peptide consisting of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2 as an active ingredient.

In addition, the present invention provides a pharmaceutical composition for preventing or treating lupus comprising an fusion peptide comprising a peptide consisting of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2 connected to the N-terminus of a cell permeable peptide as an active ingredient.

The pharmaceutical composition according to the present invention effectively regulates SLE symptom-related factors, such as inhibiting lymphocyte proliferation (lymphoproliferation) in the SLE mouse model, reducing albumin content in urine, and producing less anti-dsDNA antibodies and anti-nuclear antibodies. Therefore, it is useful for preventing or treating lupus.

1 (a) shows that HEK-Blue ^TM hTLR4 (human TLR4) cells have different concentrations of TIPs (TIP1, TIP2; TIP2 is used for control), and when treated with LPS, secreted alkaline phosphatase (SEAP) activity is obtained. It is a diagram showing the decrease 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.
2 (a) is a diagram showing the results of confirming NF-κB (p65) and IRF3 activity through Western blotting when TIP1 is treated with LPS on RAW 264.7 cells, which are rat macrophages. Figure 2 (b) is a diagram showing the results of confirming the MAPK activity through the Western blotting under the same conditions.
FIG. 3 shows the results of confirming the activity (p-p65, green) of NF-κB by confocal microscopy when TIP1 was treated with LPS on RAW 264.7 cells, rat macrophages. It is a diagram showing that p65 nucleation is blocked.
4 is a diagram showing the result of confirming the cytokine secretion amount when TIP1 having different concentrations in rat macrophage RAW 264.7 cells was treated with LPS. FIG. 4 (a) shows TNF-α, FIG. 4 (b) IL-6, and FIG. 4 (c) shows IFN-β secretion, respectively.
5 is a diagram showing the results of confirming the amount of NO and ROS generation when TIP1 is treated with LPS on RAW 264.7 cells, which are rat macrophages. FIG. 5 (a) shows the amount of NO generated in the cytoplasm, FIG. 5 (b) shows the amount of ROS in the cytoplasm, and FIG. 5 (c) shows the amount of NO released outside the cell.
FIG. 6 is a diagram showing the results of confirming cytokine and NO generation when TIP1 was treated with LPS on mouse bone marrow-derived macrophage mBMDM (mouse bone marrow derived macrophage) cells. FIG. 6 (a) shows TNF-α, FIG. 6 (b) IL-6, FIG. 6 (c) NO, and FIG. 6 (d) shows IFN-β generation.
FIG. 7 shows p-ERK, ERK, p-JNK, JNK, pp38, p38, p-p65 and Iκ in cells when TIP1 is treated with LPS on human peripheral blood mononuclear cells (hPBMCs). It is a diagram showing that the expression level of -Bα is inhibited by TIP1 as a result of Western blotting.
8 is a mouse macrophage RAW 264.7 cells TIP1 to each TLR ligand corresponding to PAM ₃ CSK ₄ (TLR1 / 2), FSL-1 (TLR2 / 6), Poly (I: C) (TLR3), When treated with R848 (TLR7 / 8) or CpG-ODN (TLR9), this is a measure of the amount of TNF-α secretion in cells, showing that it is inhibited by TIP1.
FIG. 9 is a diagram showing that TIP1 is reduced by TIP1 as a result of confirming the secretion of IFN-β through ELISA when TIP1 is treated with Poly (I: C) in RAW 264.7 cells, which are rat macrophages.
10 (a) shows that the molecules involved in the formation of NLRP3 (NACHT, LRR and PYD domains-containing protein 3) -mediated inflamasome (inflammasome, inflammatory regulatory complex) induced by treatment with LPS and ATP are inhibited by TIP1. Is a diagram confirmed in THP1 cells. FIG. 10 (b) is a diagram showing the results of the experiments under the same conditions in mBMDM cells.
FIG. 11 (a) is a diagram confirming in THP1 cells that IL-1β secretion is inhibited when TIP1 is treated in the NLRP3-mediated inflammasome state induced by treatment of LPS and ATP. FIG. 11 (b) is a diagram showing the results of the experiments performed under the same conditions in mBMDM cells.
12 is a diagram showing the results of confirming the NF-κB activity and NO secretion of the peptide having the amino acid sequence of all or part of TIP1. FIG. 12 (a) shows SEAP activity, and FIG. 12 (b) shows NO secretion.
FIG. 13 shows the treatment between TIP1 (green) and TLR4 (red) and MyD88 (blue) when treated with TIP1 (TIP1-FITC) or TIP1-FITC combined with fluorescein isothiocyanate (FITC) in human monocyte THP1 cells. This is a diagram confirming that TIP1 is binding to TRL4 by observing the interaction with a confocal laser scanning microscope.
14 is a view showing the results of confirming the cytokine secretion amount through blood collected 1 hour or 2 hours after treatment with PBS (phosphate buffered saline) and LPS or TIP1 and LPS in C57BL / 6J mice. 14 (a) shows TNF-α, FIG. 14 (b) shows IL-12p40, and FIG. 14 (c) shows IL-6 secretion, respectively.
15 is a diagram showing the results of confirming the amount of cytokine secretion in liver tissue of rats when PBS and LPS or TIP1 and LPS were treated together in C57BL / 6J mice. FIG. 15 (a) shows the result of confirming the secretion amount of TNF-α and IL-6 through Western blotting, and FIG. 15 (b) shows the graph by quantifying the band intensity according to the Westin blotting. .
FIG. 16 is a diagram showing the results of confirming the effect of TIP1 on kidney and liver damage through blood collected after 24 hours when PBS and LPS or TIP1 and LPS were treated together in C57BL / 6J mice. 16 (a) and (b) show the secretion amounts of TNF-α and IL-6, respectively. 16 (c) and (d) show the secretion amounts of kidney damage markers BUN (blood urea nitrogen) and creatinine (Cr), respectively. 16 (e) and (f) are diagrams showing secretion amounts of liver damage markers AST (aspartate aminotransferase) and ALT (alanine aminotransferase).
FIG. 17 shows TUNEL (terminal deoxynucleotidyl transferase dUTP nick end) using kidney tissue collected after 24 hours of the effect of TIP1 on kidney apoptosis when C57BL / 6J mice were treated with PBS and LPS or TIP1 and LPS. This is a diagram showing that TIP1 is reducing apoptosis as a result of checking by labeling) staining.
FIG. 18 is a diagram showing that the survival rate of LPS-induced sepsis model rats is increased when LPS or TIP1 and LPS are treated in BALB / c rats.
FIG. 19 (a) is a diagram showing an experimental plan for confirming the therapeutic effect of TIP1 on rheumatoid arthritis using a CIA (collagen-induced arthritis) model of DAB-1J mice. FIG. 19 (b) is a diagram visually observing the treatment effect when TIP1 was treated and showing that rheumatoid arthritis is being treated.
20 is a diagram showing the results of confirming the treatment effect when treated with TIP1 in the CIA rheumatoid arthritis model of DAB-1J rats. 20 (a) shows weight, FIG. 20 (b) shows squeaking (rat squeaking), FIG. 20 (c) shows foot swelling, and FIG. 20 (d) shows the results of observing the arthritis index. Each is shown.
FIG. 21 shows the treatment of arthritis by measuring 3D image and bone mineral density (BMD) using micro-computed tomography (Micro-CT) after TIP1 treatment on the CIA model of DAB-1J rats. to be.
FIG. 22 is a comparison of the treatment level with prednisololone, a conventional therapeutic agent, by staining the knee joint area with hematoxylin and eosin (H & E) after TIP1 treatment on the CIA model of DAB-1J mice. It is a diagram showing that the therapeutic effect of is excellent.
23 is a diagram schematically showing a signal transduction process induced by TLR4 and a region controlled by TIP1.
24 is a photograph comparing the size of the spleen, kidney, and lymph nodes in wild-type mice (C57BL / 6), vehicle-treated SLE mouse model, and TIP1-treated SLE mouse model, respectively.
25A is a graph measuring the concentration of anti-dsDNA antibody in serum isolated from the SLE mouse model.
25B is a graph measuring the concentration of antinuclear antibody (ANA) in serum isolated from wild-type mouse and SLE mouse models.
25C is a graph measuring the concentration of C3 complement in serum isolated from wild-type mice and SLE mouse models.
25D is a graph measuring the concentration of albumin in urine isolated from wild-type mice and SLE mouse models.

Hereinafter, the present invention will be described in detail.

The present invention provides a peptide consisting of the amino acid sequence of SEQ ID NO: 1.

In the present invention, the term “peptide” refers to a linear molecule formed by combining amino acid residues by peptide bonds. The peptide may be prepared according to a chemical synthesis method known in the art, and preferably may be prepared according to a solid phase synthesis technique, but is not limited thereto.

In the present invention, the term, “TLR4” is a protein belonging to the toll-like receptors (TLLs), a family of transmembrane protein that functions as a monitor for pathogen infection, and a protein encoded by the TLR4 gene. It is also called CD284 (cluster of differentiation 284). The TLR4 recognizes various pathogen-associated molecular patterns (PAMPs), including LPS of Gram-negative bacteria, which is very important for the activation of the innate immune system.

In the present invention, the term “TLR3” is a protein belonging to the TLRs, a family of transmembrane proteins that functions as a monitor for pathogen infection, and refers to a protein encoded by the TLR3 gene, named CD283 (cluster of differentiation 283) It also becomes. The TLR3 recognizes a variety of PAMPs, including viral double-strand RNA (dsRNA), which is very important for the activation of the innate immune system.

In the present invention, 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 induces inflammation in response to microbial infections. It is activated by reaction. NLRP3 requires priming, for example, the binding of LPS to TLR4. The TLR4 signaling pathway by LPS promotes NLRP3 inflammasome formation along with ATP. NLRP3 inflammasome, when activated abnormally, induces the development of various inflammatory diseases, particularly degenerative neurological diseases. According to a recent study, it was found that there is a direct link between accumulation of beta amyloid (amyloid β), which is one of the main causes of Alzheimer's disease, and NLRP3 inflammasomes, and a relationship with Parkinson's disease was also reported.

In the present invention, the term, “TLR4 mediated signaling pathway” refers to a signaling pathway through TLR4, which may be an LPS response dependent on the TLR4 / MD2 complex formed by TLR4 and MD2, through which the signal is transmitted do. TLR4 is signaled by several adapter proteins, and works through Mal (also referred to as TIRAP) and MyD88 and TRAM and TRIF. Activated TLR4 activates NF-κB through the Myd88-dependent signaling process, moves to the nucleus, and induces the activation of MAPK. Due to the activation of the NF-κB and MAPK, inflammatory cytokines such as TNF-α, IL-1β and IL-6 are secreted, and nitrogen monoxide (hereinafter referred to as NO) and free radicals (hereinafter referred to as ROS) in macrophages. Oxidative stress). In addition, the MyD88-independent signaling process is induced by the activation of tram / TRIF, interferon-regulator (IRF) and NF-κB to secrete type 1 interferon.

In the present invention, the term “TIR domain” is a domain for intracellular signaling, having three highly conserved regions, and mediating the interaction between TLR and other signaling molecules. The activated TIR domain induces the binding of MyD88 and activates the TLR signaling pathway.

In the present invention, the term, "inhibition" refers to a phenomenon in which biological activity or signal activity decreases due to deficiency, mismatch, and many other causes, partially or completely blocking, reducing, preventing, or activating TLR activity. It 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 inflammasome inhibition. It also provides for partial inhibition of TLR1 / 2, TLR2 / 6, TLR7, TLR8 and TLR9.

In the present invention, the SHCR sequence of the peptide consisting of the amino acid sequence of SEQ ID NO: 1 (SEQ ID NO: 2) is characterized by specifically binding to the TIR (Toll / interleukin-1 receptor) domain of the Toll-like receptor (TLR). can do.

In binding to the TIR domain of TLR4, the peptide consisting of the amino acid sequence of SEQ ID NO: 1 according to the present invention has sequence specificity. The present inventors searched the minimum amino acid region of TIP1 that binds the TIR domain of TLR4 and investigated whether this region can effectively inhibit the signaling pathway of TLR4. That is, according to one embodiment of the present invention, a decoy peptide is selected from the TIR domain of TIRAP and SHCR (decoy peptide 1-2, SEQ ID NO: 2) and VLLI (decoy peptide 1-3, using amino acid sequence SHCRVLLI) SEQ ID NO: 7) The sequence was linked to the N-terminus of the same CPP sequence as used in Example 1-1, and then treated with LPS to HEK-Blue ^TM -hTLR4 cells and hPBMC cells, and 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.

In addition, the present invention provides a fusion peptide in which 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.

In the present invention, the fusion peptide is capable of inhibiting a signaling pathway mediated by a toll-like receptor (TLR), and the toll-like receptor (TLL) is a toll-like receptor 1/2 (TLR1 / 2). , TLR2 / 6 (Toll-like receptor 2), TLR3 (Toll-like receptor 3), TLR4 (Toll-like receptor 4), TLR7 (Toll-like receptor 7), TLR8 (Toll-like receptor 8) 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).

In addition, in the present invention, the expression of TNF-α, IL-6 or IFN-β is inhibited by blocking the TLR signaling pathway of the fusion peptide; Suppression of NO or ROS secretion; Alternatively, the activity of NF-κB, MAPK or NLRP3 inflammasomes may be inhibited, and the fusion peptide may be characterized by inhibiting both MyD88-dependent and MyD88-independent TLR4 signaling pathways.

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

The fusion peptide of the present invention uses a cell permeable peptide, and the cell permeable peptide is not particularly limited as long as it has a feature that enters into the cell by a mechanism of cell internalization (endocytosis), but is preferably cell permeability listed in Table 1 below. Peptides or variants thereof can be used.

Peptide origin order Sequence number 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 RRWWRRWRR 22 Polyarginine (R ₉ , R ₈ ) Positively charged sequence R _n , n = 8 or 9 23,24 VP22 Herpes simplex virus NAKTRRHERRRKLAIER 25 YTA2 MMP cleavage site as seeding sequence YTAIAWVKAFIRKLRK 26 YTA4 MMP cleavage site as seeding sequence IAWVKAFIRKLRKGPLG 27 M918 The tumor suppressor protein p14ARF MVTVLFRRLRIRRACGPPRVRV 28 CADY Derived from PPTG1 peptide, W and charged amino acids GLWRALWRLLRSLWRLLWRA 29 SAP Designed based on a natural protein of maize, γ-zein VHL (PPP) ₈ (VRLLPPP) ₃ 30 SAP (E) Design inspired by SAP; Arg residue replaced by Glu Ac-CGGW (VELPPP) 3 31 CyLoP-1 Derived from crotamine toxin found in snake venom, crot (27-39) CRWRWKCCKK 32 gH 625 Based on the 625-644 residues of the glycoprotein HSV 1 HGLASTLTRWAHYNALIRAF 33 GALA Glu-rich an containing His (imidazole group) in order to be pH responsive (endosomes) (Designed to efficiently escape endosomes) WEAALAEALAEALAEHLAEALAEALEALAA 34 CADY Designed; based on chimeric peptide carrier PPTG1 derived from the fusion peptide JTS1 Ac-GLWRALWRLLRSLWRLLWRA-cysteamide 35 L17E Inspired by the spider venom M-lycotoxin IWLTALKFLGKHAAKHEAKQQLSKL 36 MPPs Designed to contain un-natural, cyclohexylalanine (F _x ) residues and to have differential intracellular localization Mitochondria-penetrating peptides (example: F _X rF _X KF _X rF _X K) 37 RR ₅ -APPRR ₄ -APP
RR ₃ -APP Small proteins (36-residue polypeptides) RPRRPRRPRRPGRRAPVEDLIRFYNDLQQYLNVVTRHRYC
RRPRRPRRPGRRAPVEDLIRFYNDLQQYLNVVTRHRYC
GPRRPRRPGRRAPVEDLIRFYNDLQQYLNVVTRHRYC 38,39,40 TATp-D Analogue of TAT

41,42 Cyclic Tat Lys- and Glu- amino acids added to the linear Tat sequence to obtain a ring with the same overall charge as the native form c [K-rRrQrRkKrG-E] c 43 K ₁₀ (QW) ₆ The design was based on combining W and K at the primary structure level to obtain self-assembly into a variety of nanostructures KKKKKKKKKKQWQWQWQWQWQW 44 TAT _(49-57) HIV-1 TAT protein RKKRRQRRR 45 DPV1047 Chemically synthesized VKRGLKLRHVRPRVTRMDV 46 ARF _(1-22) p14ARF protein MVRRFLVTLRIRRACGPPRVRV 47 BPrPr _(1-28) N terminus of unprocessed bovine prion protein MVKSKIGSWILVLFVAMWSDVGLCKKRP 48 p28 Azurin LSTAADMQGVVTDGMASGLDKDYLKPDD 49 VT5 Chemically synthesized DPKGDPKGVTVTVTVTVTGKGDPKPD 50 C105Y α1-Antitrypsin CSIPPEVKFNKPFVYLI 51 PFVYLI Derived from synthetic C105Y PFVYLI 52 Pep-7 CHL8 peptide phage clone SDLWEMMMVSLACQY 53

Meanwhile, among the cell permeable peptides of Table 1, Transportan includes those used in the form of the following variants: AGYLLGKINLKALAALAKKIL-NH ₂ (TP10, PepFect 3, SEQ ID NO: 13), AGYLLGKINLKALAALAKKIL-NH ₂ (TP10, PepFect 6, SEQ ID NO: 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).

In addition, in the amino acid sequence (MPPs) consisting of SEQ ID NO: 37 in Table 1, r means d -Arginine, and the 2nd and 6th amino acids in the amino acid sequence of SEQ ID NO: 37 correspond to this. In addition, SEQ ID NO: 41 and 42 of Table 1 constitute one peptide (TATp-D) forming a side chain to each other, and the 14th Lys residue (K) in the amino acid sequence of SEQ ID NO: 41 is within the amino acid sequence of SEQ ID NO: 42. It is characterized by forming a side chain in connection with the 13th Gln residue (Q). Furthermore, in the amino acid sequence consisting of SEQ ID NO: 43 of Table 1 (Cyclic Tat), r means d -Arginine, and the 2, 4, 6, 10th amino acid in the amino acid sequence of SEQ ID NO: 43 corresponds to this. Similarly, k means d- Lysine, and the 8th amino acid in the amino acid sequence of SEQ ID NO: 43 corresponds to this. The SEQ ID NO: 43 is characterized by being a cyclic peptide.

In one embodiment of the present invention, an experiment was performed by selecting the Penetratin sequence (RQIKIWFQNRRMKWKK, SEQ ID NO: 9) among the cell permeable peptides in Table 1, and other cell permeable peptides other than the cell permeable peptides actually used were fused with the peptides of the present invention. It will be apparent to those skilled in the art that similar effects to the present invention are exhibited even in the case of prescribing.

In the present invention, the fusion peptide in which the 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 is preferably composed 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, specifically, the variants are SEQ ID NO: 3 or 4 and 70% or more, preferably 80% or more, more preferably 90% or more, and more Preferably, all peptides having a sequence homology of 95% or more, even more preferably 98% or more, and most preferably 99% or more may be included. The term “homology” refers to the degree of similarity between wild type amino acid sequences and wild type nucleic acid sequences.

According to one embodiment of the present invention, the fusion peptide according to the present invention is a cytokine (IL-6, TNF-α, IFN-β) by inhibiting the TLR4 signaling pathway induced by lipopolysaccharide (LPS), Inhibition of NO and ROS secretion and activation of NF-κB and MAPK, as well as renal and liver damage caused by TLR4 hyperactivity in animals, relief of sepsis and rheumatoid arthritis and TLR3 signaling induced by Poly (I: C) The effect of inhibiting the formation of NLRP3 inflammasomes induced by pathways and LPS / ATP is excellent. Preventing and treating autoimmune diseases, inflammatory diseases or degenerative neurological diseases caused by NLRP3 inflammasomes caused by the signaling pathway It can be useful to do.

In addition, in the present invention, the fusion peptide according to the present invention can be used as TLR4, TLR3 and NLRP3 inflammasome inhibitors.

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

In the present invention, the term, "TLR4, TLR3 and NLRP3 inflammasome inhibitor" refers to a substance capable of directly, indirectly or substantially interfering, reducing or inhibiting the biological activity of TLR4, TLR3 and NLRP3 inflammasomes, preferably Peptide reactive with TLR4 and TLR3 binds directly to the TIR domains of TLR4 and TLR3 and blocks the TLR4 and TLR3 signaling pathways by neutralizing the activity of TLR4 and TLR3, thereby NF-κB and MAPK and NLRP3 inflammasome activation It refers to substances that can reduce the secretion of inflammatory cytokines, NO and ROS by causing a decrease.

In addition, the present invention provides a pharmaceutical composition for the prevention or treatment of lupus comprising a peptide consisting of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2 as an active ingredient.

In addition, the present invention provides a pharmaceutical composition for the prevention or treatment of lupus comprising a fusion peptide linked to a peptide consisting of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2 to the N-terminus of a cell permeable peptide as an active ingredient. The fusion peptide may be preferably made of the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 4, and variants of the amino acid sequence may also be included within the scope of the present invention.

The lupus may be systemic lupus erythematosus (SLE), discoid erythema lupus, subacute cutaneous lupus, drug-induced lupus, neonatal lupus.

The pharmaceutical composition of the present invention may contain a pharmaceutically effective amount of the peptide alone or may include one or more pharmaceutically acceptable carriers, excipients or diluents. The pharmaceutically effective amount in the above refers to an amount sufficient to prevent, ameliorate and treat lupus symptoms.

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

The pharmaceutical composition of the present invention can be formulated using methods known in the art to provide rapid, sustained or delayed release of the active ingredient after administration to a mammal. 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 be administered in various ways, such as the route of administration, patient's age, gender, weight, and severity of the patient. It may be appropriately selected according to factors, and may be administered in combination with a known compound having an effect of preventing, improving or treating lupus symptoms.

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 an individual in need thereof; It provides a method of preventing or treating lupus comprising.

In addition, the present invention comprises the steps of administering a fusion peptide linked to a peptide consisting of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2 to the N-terminal of the cell permeable peptide to an individual in need thereof; It provides a method of preventing or treating lupus comprising. Preferably, the individual is a mammal, including a human.

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, the 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 can be simultaneously / sequentially treated in combination with other drugs or treatments for the treatment of lupus. .

Hereinafter, the contents 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 contents of the present invention are not limited by the following examples and experimental examples.

Example  1. Preparation of test sample

1-1. TLR4 of TIR  Domain-specific binding Peptide  Selection

The present inventors 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 Drosophila at the N-terminus of the sequence. New peptides TIP1 and TIP2 were synthesized by linking antennapedia homeodomain-derived cell permeable peptides (Cell-Penetrating Peptides, CPP). The sequences of all the peptides used in this example are shown in Table 2 below.

name order Sequence number Decoy peptide 1 (TIP1 W / O CPP) SHCRVLLI One TIP1 RQIKIWFQNRRMKWKK SHCRVLLI 3 Decoy peptide 2 (TIP2 W / O CPP) TIPLLS 5 TIP2 RQIKIWFQNRRMKWKK TIPLLS 6 CPP RQIKIWFQNRRMKWKK 9

* The sequence of Decoy peptide is underlined.

1-2. Cell culture and preparation

HEK-Blue ^TM -hTLR4 cells (InvivoGen, San Diego, CA, USA) with 1% penicillin / streptomycin, 10% fetal bovine serum (FBS) (Thermo Fisher Scientific Inc., Waltham, MA, USA) and 0.2% Normocin (InvivoGen) was added and cultured in DMEM (Dulbecco's modified Eagle's medium) (Thermo Fisher Scientific Inc.) medium. Rat macrophage RAW 264.7 cells (Korean Cell Line Bank, Seoul, Korea) were added to low-glucose DMEM (DMEM) with 1% penicillin / streptomycin and 10% FBS (Thermo Fisher Scientific, Inc.). And cultured. Human monocyte cells, THP1 cells, were cultured in RPMI 1640 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 differentiate into macrophages for 24 hours. Human peripheral blood mononuclear cells, hPBMC cells (PromoCell, Heidelberg, Germany) were cultured in RPMI 1640 (Thermo Fisher Scientific Inc.) with 2.05 mM L-glutamine, 1% penicillin / streptomycin and 10% FBS. Rat marrow-derived macrophages, mBMDM cells, were cultured in Thermo Fisher Scientific Inc. (DMEM) with 1% penicillin / streptomycin and 10% FBS. All cells were cultured in a humidified culture system (Thermo Fisher Scientific Inc.) at 5% CO ₂ , 37 ° C., and the medium was changed every 16 hours. PAM ₃ CSK ₄ , Poly (I: C), R848 and CpG-ODN from Thermo Fisher Scientific, Inc., FSL-1 from InvivoGen, LPS ( Escherichia coli 0111: B4) and ATP from Sigma-Aldrich Co. ( St. Louis, MO, USA), and all peptides used in the experiment were synthesized and purchased from Peptron, Inc. (Daejeon, Korea).

Example  2. Analysis method

2-1. MTT  analysis

HEK-Blue ^TM -hTLR4 cells are dispensed into 96-well plates (BD Biosciences, San Jose, CA, USA) to a cell number of 5 x 10 ⁴ cells / well, and RAW264.7 cells are 2 x 10 ⁵ cells / well The cells were dispensed to the number of cells and cultured overnight. Thereafter, MTT analysis was performed using a 1- (4,5-dimethylthiazol-2-yl) -3,5-diphenylformazan (MTT) solution (Sigma-Aldrich Co. LLC).

2-2. SEAP  Active analysis

HEK-Blue ^TM -hTLR4 cells were dispensed into a 24-well plate (BD Biosciences) to a cell number of 2 x 10 ⁵ cells / well and incubated overnight. The next day, after removing the culture medium and replacing 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 culture was then transferred to a 96-well plate (BD Biosciences) and the 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 was analyzed.

2-3. Western Blot

To perform western blotting, a pre-protein extraction solution (M-PER, Thermo Fisher Scientific Inc.) was mixed with a protease and phosphatase inhibitory mixture and added to RAW 264.7 cells or hPBMC cell pellets. After cooling the pellets for 10 minutes, the lysate was centrifuged at 16000 X g for 10 minutes. Then, the NE-PER nuclear and cytoplasmic extraction reagent (Thermo Fisher Scientific Inc.) was used to extract the cytoplasmic and nuclear proteins, respectively, and the concentration of the protein was measured using a BCA kit (Sigma-Alderich Co. LLC). Did. Thereafter, the same amount of protein was developed on an SDS-polyacrylamide gel, and electrophoresis was performed using a Mini-PROTEAN Tetra Cell two-dimensional electrophoresis system (Bio-Rad Laboratories, Hercules, CA, USA). The membrane was immunoblotted by gently shaking overnight with a primary antibody 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) Antibody). Then, after thoroughly shaking with PBST, the membrane was incubated for 2 hours with an anti-rat / rabbit HRP-conjugated secondary antibody (Thermo Fisher Scientific Inc.), and SuperSignal West Pico ECL solution (Thermo Fisher Scientific Inc. .) And the detected protein was visualized using Fuji LAS-3000 system (Fujifilm, Tokyo, Japan).

2-4. Confocal  Microscopic analysis (Confocal microscopy)

RAW 264.7 cells and THP1 cells were aliquoted into 24-well plates to a cell count of 2 x 10 ⁵ cells / well and grown in an incubator overnight. After 24 hours after treatment with TIP1 and LPS, 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 3 times with PBS and blocked with 2% BSA solution. The blocked cells were TIP1-FITC (25 μM; Peptron, Inc., Daejeon, Korea), p-p65, TLR4, Myd88, TOM20 (1: 1000; Santa Cruz Biotechnology Inc.) and NLRP3 (Adipogen) antibodies and 2 After incubation for an hour, washed three times with PBS. The cells were then incubated for 1 hour with AlexaFluor 408 and / or 488 and / or 546-conjugated secondary antibody (Invitrogen, Carlsbad, CA, USA) and washed 3 times with PBS. Then, using a 5 μM Hoechst 33258 (Sigma-Aldrich Co.) for 15 minutes at room temperature, using a confocal laser scanning microscope (LSM-700, Carl Zeiss MicroImaging GmbH) to count the number of the fluorescently stained cells Images were analyzed using Zen 2009 software.

2-5. TNF -α, IL-6, IFN -β and IL-1β analysis

RAW 264.7 cells, THP1 cells, and mBMDM cells were dispensed to a cell number of 2 x 10 ⁵ cells / well in a 96-well plate or to a cell number of 5 x 10 ⁵ cells / well in a 24-well plate and cultured overnight. . Then, 24 hours after treatment with TIP1 and LPS, the amount of IFN-β, IL-6 and TNF-α secretion was 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! It was measured using a kit (eBiosciences). Absorbance at 450 nm was measured using a microplate reader spectrophotometer (Molecular Devices) and the results were analyzed using Softmax Pro 5.3 software (Molecular Devices Inc.).

2-6. NO in the cytoplasm and ROS  analysis

* RAW 264.7 cells were dispensed in a 6-cm dish (SPL Life Sciences., Pochun, Korea) to a cell number of 1 x 10 ⁶ cells / well and cultured overnight. Thereafter, TIP1 was treated and stained with DAF-FM and DCF-DA (Thermo Fisher Scientific, Inc.), respectively, and then incubated for 1 hour. Thereafter, centrifugation was performed at 200 X g / min for 5 minutes, and the mixture was transferred to a brown tube and stored in PBS at a temperature of 4 ° C. The intensity of DAF-FM and DCF-DA fluorescent materials was measured and quantified using FACSAria III with Diva software (BD Biosciences).

2-7. NO secretion analysis

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 value of the culture supernatant was detected in 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. surface Plasmon  Resonance analysis (surface plasmon  resonance; SPR )

Surface plasmon resonance analysis was performed using a ProteOn XPR36 instrument (Bio-Rad Laboratories, Inc.) with a ProteOn NLC sensor chip. Specifically, the synthetic peptide encoding the BB-loop (RDFIPGVAIAA) and C-terminal region (EGTVGTGCNWQEATSI) of the TIR domain is fixed to the surface of the NLC sensor chip using NeutrAvidin bound to the GLC polymer layer, and the running buffer is used as a control. PBS supplemented with phosphorus 0.05% Tween 20 was flowed. Various types of TIP1, TIP1-1, TIP1-2 and Tip1-3 (50 μM) and different concentrations of TIP1 (12.5, 25 and 50 μM) were flowed on the chip to confirm the binding affinity with the fixed peptide. After measurement, the surface of the sensor chip was regenerated using 0.85% phosphoric acid or PBST. Dissociation constants were calculated using ProteOn manager ^TM software (version 2.0), and data from various doses of TIP1 were grouped to fit the kinetic rate constants (K _a and K _d ), equilibrium dissociation constant K _D Was calculated using the equation K _D = K _d / K _a .

Experimental Example  One. TIPs of TLR4 -Check binding affinity

To confirm the TLR4-binding affinity of the TIPs (TIP1, TIP2) prepared in Example 1-1, the activity of NF-κB in HEK-Blue ^TM -hTLR4 cells cultured in Example 1-2 was measured. To this end, the IL-12 p40 minimal promoter (IL-12 p40 is a TLR4 stimulator) that activates NF-κB and AP-1 after the NF-κB and AP-1 (activator protein 1) DNA binding sites are included. Inducible secreted embryonic alkaline phosphatase (SEAP) reporter gene. After that, the concentration of TIP or CPP-unconnected TIP (TIP W / O CPP) was varied to 12.5, 25, 50 μM and 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.

As shown in Fig. 1 (a), when only TIP was added, the activity of SEAP did not change significantly, but when TLR4 was stimulated with LPS after TIP treatment, TIP1 was SEAP induced by LPS unlike TIP2. It was confirmed that the activity was decreased 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 TIP1 (TIP1 W / O CPP) and TIP2 (TIP2 W / O CPP) had no change in SEAP activity. Through this, the TIP1 of the present invention, once translocated into the cell as it is connected to the CPP, interferes with downstream adapter molecules of the signaling pathway and blocks the activation of the TLR4-mediated signaling pathway induced by LPS, thereby effective TLR4. It was confirmed that it can be used as an inhibitor.

Experimental Example  2. In vitro in TLR  For signaling pathways TIP1 Inhibitory effect of

The TLR4-mediated response induced by LPS induces a direct interaction between the TIR domain of TIRAP and MyD88 to activate the MyD88-dependent signaling pathway. In addition, it induces the interaction between the TIR domain of TRAM and TRIF to activate 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, the MyD88-independent signaling pathway induces late secretion 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 experiment after treating LPS to RAW 264.7 cells to investigate the effect of TIP1 on the TLR4 signaling pathway.

2-1. NF -κB and MAPK of Western Blotting

Western blotting was performed according to the method described in Example 2-3 to confirm the effect of TIP1 prepared in Example 1-1 on the activity of NF-κB and MAPK. The results are shown in FIG. 2.

As shown in FIG. 2 (a), when only LPS was treated with RAW 264.7 cells, which are the control rat macrophages, the activity of NF-κB was increased to degrade Iκ-Bα and the activity of IRF3 and ATF3 increased. , When TIP1 was treated together, it was confirmed that the activity of NF-κB was suppressed and the degree of Iκ-Bα decomposition and the activity of IRF3 and ATF3 decreased. In addition, as shown in Fig. 2 (b), when only LPS was treated, the activity of MAPK increased, and ERK, JNK, and p38 were phosphorylated, but when treated with TIP1, the activity of MAPK was inhibited and the enzyme was inhibited. It was confirmed that the phosphorylation degree of the was reduced. Through this, it was confirmed that TIP1 according to the present invention inhibits MyD88-dependent signaling pathway as well as MyD88-dependent signaling pathway induced by LPS by binding to TIR domain of TLR4.

2-2. NF -kB activation confirmation

To confirm the effect of TIP1 prepared in Example 1 on the activity of NF-κB, analysis was performed with a confocal laser microscope according to the method described in Example 2-4. The results are shown in FIG. 3.

As shown in FIG. 3, when LPS alone was treated, activated NF-κB (p-p65) was expressed in the nucleus, but when TIP1 was treated together, the phosphorylation level of p65 induced by LPS in the nucleus was decreased. It was confirmed that the activity of NF-κB decreased.

Experimental Example  3. TIP1 This cytokine and NO and ROS  Effect on secretion

Cytokines (TNF-α, IL-6, IFN-β) and NO (Nitric) when TIP1 prepared in Example 1-1 was treated with RAW 264.7 cells, rat macrophages cultured in Example 1-2. The following experiment was performed to confirm whether the secretion of Oxide) and the generation of reactive oxygen species (ROS) in the cytoplasm are suppressed.

3-1. TIP1 Effects on this cytokine secretion

TNF-α, IL-6, and IFN-β levels of the culture supernatant of RAW 264.7 cells according to the method described in Example 2-5 are rat TNF-α, IL-6, and IFN-β ELISA kit Ready-SET-Go ! It measured using, and the results are shown in Figure 4.

As shown in FIG. 4, when TIP1 and LPS were treated together, it was confirmed that secretion of TNF-α, IL-6, and IFN-β was decreased in a concentration-dependent manner. Through this, it was confirmed that TIP1 according to the present invention inhibits the secretion of cytokines induced by LPS.

3-2. TIP1 This NO and ROS  Effect on secretion

According to the method described in Example 2-6, TIP1 was treated with RAW 264.7 cells to determine the effect of TIP1 on the generation of NO and reactive oxygen species (ROS) in the cytoplasm, and DAF-FM (Invitrogen Corp., CA, respectively) , USA) and DCF-DA (Invitrogen Corp.) to quantify cytoplasmic NO and ROS, respectively. Further, 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.

As shown in (a) to (c) of FIG. 5, when TIP1 and LPS were treated together, it was confirmed that TIP1 effectively reduces the amount of NO and ROS in the cytoplasm as well as the secretion of extracellular NO. Through this, it was confirmed that TIP1 according to the present invention inhibits oxidative stress induced by LPS.

Experimental Example  4. In primary cells TIP1 The impact of this

Cytokines (TNF-α, IL-6, IFN-β) when TIP1 prepared in Example 1 was treated with mBMDM, a mouse bone marrow-derived macrophage cultured in Example 2, and hPBMC cells, which are human peripheral blood mononuclear cells. ) And NO (Nitric Oxide) secretion and TLR signaling proteins to confirm whether the inhibition was performed as follows.

4-1. TIP1 this mBMDM  Cell impact

TNF-α, IL-6, and IFN-β levels of the culture supernatant of mBMDM cells according to the method described in Example 2-5 were rat TNF-α, IL-6, IFN-β ELISA kit Ready-SET-Go! It was measured using a kit, and 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.

As shown in Fig. 6 (a) to (d), TIP1 reduces the secretion 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. Through this, 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 mouse bone marrow-derived macrophages.

4-2. TIP1 this hPBMC  Cell impact

To determine 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 proteins were visualized, and the results are shown in FIG. 7.

As shown in FIG. 7, when only LPS was treated with hPBMC cells, a control group of human peripheral blood mononuclear cells, the activity of NF-κB was increased to decompose Iκ-Bα, whereas when treated with TIP1, NF- The activity of κB was suppressed and the degree of decomposition of Iκ-Bα decreased. In addition, when only LPS was treated, ERK, JNK, and p38 were phosphorylated due to increased activity of MAPK, but when TIP1 was treated together, it was confirmed that the activity of MAPK was inhibited to decrease phosphorylation of the enzymes.

Through this, it was confirmed that TIP1 according to the present invention inhibits the TLR4 signaling pathway not only in immortalized cell lines, but also in primary cells extracted directly from animals.

Experimental Example  5. TIP1 This other TLR  Effect on signal transmission

In addition to TLR4, in order to confirm the effect of TIP1 on the signaling pathways of other TLR families, the TNF-α levels induced by different TLR ligands in the culture supernatant of RAW 264.7 cells were measured in Examples 2-5. It measured using the method of, and the results are shown in Figure 8. In addition, the IFN-β level of the culture supernatant of RAW 264.7 cells was measured using the ELISA kit Ready-SET-Go! It measured using, and the results are shown in Figure 9.

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, TNF-α secretion was decreased, and TIP1 was confirmed to have a high inhibitory effect on TLR3 signaling (92% reduction at 50 μM). In addition, as shown in FIG. 9, when TIP1 and Poly (I: C) were treated together, it was confirmed that the secretion amount of IFN-β decreased. Through this, it was confirmed that TIP1 according to the present invention has an inhibitory effect on cytokine secretion in TLR4 as well as TLR1 / 2, TLR2 / 6, TLR3, TLR7 / 8, and TLR9.

Experimental Example  6. TIP1 Effects on degenerative neurological diseases

Activation of the TLR4 signaling pathway induced by LPS results in the activity of NF-κB, which in turn is a 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 agents reduce the concentration of potassium inside the cell and induce the production of mature IL-1β by the NLRP3 inflamasome.

By paying attention to the correlation between the activity of the TLR4 signaling pathway and the formation of NLRP3 inflammasomes, the present inventors show that LPS / ATP or TIP1 and LPS / ATP are present in human monocyte cells THP1 cells and mouse marrow-derived macrophages mBMDM THP1 cells. After treatment with NLRP3, pro-caspase-1 (45 kDa), active capase-1 (10 kDa), pro-IL-1β (35 kDa) and mature IL according to the method described in Example 2-3. Protein expression of -1β (17 kDa) was visualized through Western blotting. The results are shown in FIG. 10.

In addition, after confirming the effect of TIP1 on cytokine secretion when NLRP3 inflammasome was induced, after treating LPS / ATP or TIP1 with LPS / ATP on THP1 cells and mBMDM cells, IL-1β in culture supernatant The values were measured according to the method described in Example 2-5. Human and Rat IL-1β ELISA Kit Ready-SET-Go! It was measured using. The results are shown in FIG. 11.

10 (a) to (b), it was confirmed that the expression of NLRP3, Caspase-1, and 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 level of NLRP3, active capase-1 (10 kDa) and mature IL-1β (17 kDa), and ultimately inhibited IL-1β secretion. In addition, as shown in FIG. 11, when TIP1 and LPS / ATP were treated together, it was confirmed that the amount of IL-1β secretion decreased. Through this, it was confirmed that TIP1 according to the present invention blocks the TLR4 signaling pathway, thereby inhibiting NLRP3 inflammasome activation.

NLRP3 inflammasomes, when activated abnormally, cause various inflammatory diseases, especially degenerative neurological diseases.If it can effectively inhibit the activity of NLRP3 inflammasomes, it can be used as a therapeutic agent for degenerative neurological diseases through suppression of an inflammatory response. I can expect. Therefore, the present inventors confirmed through the above results that TIP1 according to the present invention can have a prophylactic or therapeutic effect against degenerative neurological diseases.

Experimental Example  7. TIP1  Confirmation of sequence specificity

The present inventors used the amino acid sequence of the decoy peptide prepared in Example 1-1, SHCRVLLI (SEQ ID NO: 1), to determine the minimum amino acid region of TIP1 binding 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 used in Example 1-1. All peptides used in this example are listed in Table 3 below.

name order Sequence number 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 cultured in Example 1-2 with TIP1 (CPP-SHCRVLLI), TIP1-1 (CPP), TIP1-2 (CPP-SHCR) and TIP1-3 (CPP-VLLI) of Table 3 above. 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) with LPS. Then, the amount of NO secretion was measured using the method of Example 2-7. The results are shown in FIG. 12.

As shown in Fig. 12 (a), the effect of inhibiting NF-κB activity of TIP1 was the highest, and TIP1-2 also reduced NF-κB activity, whereas when treated with TIP1-1 and TIP1-3, there was no or little change. Was confirmed. In addition, as shown in (b) of FIG. 12, the effect of reducing the amount of NO secretion of TIP1 was the highest, and TIP1-2 also inhibited NO secretion, whereas when treated with TIP1-1 and TIP1-3, there was no change or insignificance. Confirmed. Through this, it was confirmed that the S-H-C-R sequence of the sequence of TIP1 according to the present invention is important in generating an inhibitory effect.

Experimental Example  8. TIP1 and TLR4  And MyD88  The interaction between people

Protein-protein docking was performed to analyze the binding interface between the TIR4 TIR domain and TIP1. As a result, TIP1 was expected to bind to the BB loop, DD loop, and C-terminal tail in the TIR domain of TLR4, and to confirm this, surface plasmon resonance was performed according to the method described in Example 2-8. SPR) analysis was performed.

In addition, to confirm the binding of TIP1 to TLR4, TIP1-FITC was prepared by binding fluorescein isothiocyanate (FITC) to the N-terminus of TIP1. After treating TIP1 (TIP1-FITC) or LPS with TIP1 in THP1 cells of Example 1-2, fluorescently stained using immunofluorescence staining and confocal microscopy according to the method described in Example 2-4 The interaction between proteins was analyzed by counting the number of cells. The results are shown in FIG. 13.

As shown in FIG. 13, when TIP1-FITC and LPS were treated together, TIP1-FITC (green) and TLR4 (red) bind to form a complex, whereas MyD88 (blue) cannot bind to the complex site. Confirmed. On the other hand, in the absence of LPS, TLR4 is expressed in the plasma membrane, while MyD88 is spread in the cell.When LPS is treated on the cell, both TLR4 and TIP1-FITC enter the cell through the process of endocytosis, and MyD88 is the cell. It was confirmed that it was not spread inside but aggregated on the plasma membrane. In addition, it was confirmed that while TIP1-FITC-TLR4 strongly binds, MyD88 forms a weak binding to TIP1-FITC. Through this, the inhibitory effect of TIP1 on the TLR4 signaling pathway is due to binding to the C-terminal tail and BB loop among the TIR domains of TLR4, and at the same time, the interaction between TIP1 and MyD88 is inhibited, ultimately leading to the TLR4 signaling process. It was confirmed that it was inhibited.

Experimental Example  9. In vivo in TIP1 Effects of this mouse on cytokine secretion

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 (20-25g, n = 8) mice were purchased from Orient Bio, Inc. (Seoul, Korea) and used in the experiment. The mice were intraperitoneally injected with TIP1 (10 nmol per gram of animal body weight) and after 1 hour, LPS (5 µg per gram of animal body weight) was injected for 2 hours. The control group was injected with the same volume of PBS. Thereafter, plasma was separated from the rat blood through centrifugation and stored at -80 ° C until analysis of secretion. Plasma TNF-α, IL-12p40 (diluted at 1: 100) and IL-6 (diluted at 1: 100) levels in plasma Rat 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. 14.

14 (a) to (c), when TIP1 and LPS were treated together, it was confirmed that the secretion of TNF-α, IL-12p40 and IL-6 was significantly reduced. This was confirmed as well as the TIP1 to inhibit TLR4 signaling pathway in vitro according to the present invention inhibits cytokine secretion in in vivo.

Experimental Example  10. TIP1 The effect on sepsis

To confirm the effect of TIP1 on sepsis, C57BL / 6J and BALB / c mice were treated with PBS and LPS or TIP1 and LPS together, and then the following experiment was performed to confirm kidney and liver damage and changes in survival rate. .

10-1. TIP1 The effect of this on the secretion of cytokines from the liver

After treatment with PBS and LPS or TIP1 and LPS in C57BL / 6J mice (2 hours), liver tissue was extracted to include M-PER mammalian protein extraction reagent with Protease inhibitor cocktail (Thermo Fisher Scientific, Inc.). After homogenization using a Kontes Pellet Pestle Cordless Motor (Thermo Fisher Scientific, Inc.), proteins were extracted according to the manufacturer's protocol. Thereafter, according to the method described in Example 2-3, IL-6, TNF-α, and β-actin protein expression was visualized through Western blotting, and the intensity of the bands was graphed and the results are shown in FIG. 15. Shown.

15 (a) and (b), when LPS and TIP1 were treated together, it was confirmed that the expression level of TNF-α and IL-6 induced by LPS in rat liver tissue was significantly reduced.

10-2. TIP1 Effects on kidney and liver damage

In order to more accurately confirm the anti-inflammatory effect of TIP1 in the mouse sepsis model, experiments were performed in the same manner as above. Specifically, PBS and LPS or TIP1 and LPS were treated together in C57BL / 6J mice (24 hours), and then plasma was separated from the 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 kidney damage markers were blood urea nitrogen (BUN) and creatinine (Cr) and liver damage markers. Changes in the secretion amounts of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were analyzed through VETTEST-8008-system (IDEXX). The results are shown in FIG. 16.

As shown in (a) to (f) of FIG. 16, when TIP1 and LPS were treated together, it was confirmed that the secreted amounts 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 can suppress TLR4 signaling pathway, thereby alleviating cytokine secretion as well as kidney and liver damage.

10-3. TIP1 Apoptosis of this kidney ( apoptosis ) Impact

After 8 weeks of age, C56BL / 6 (20-25g, n = 8) mice were treated with TIP1 and LPS or PBS and LPS together (24 hours), kidney tissue was separated and electronic balance (ER-180A, A & D Company, Tokyo, Japan), and then cut into sagittal slices. This was placed in a 10% formalin solution overnight, and then paraffin wax was poured to solidify it and made into a thin section of 4 μm using a microtome to fix it. To determine the effect of TIP1 on apoptosis in fixed kidney tissue, stained with terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) (Merck Millipore, Billerica, MA, USA) and confocal laser scanning microscope (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. 17.

As shown in FIG. 17, when LPS alone was treated, TUNEL-positive kidney cells significantly increased, that is, apoptosis (green dot) in kidney tissue increased, whereas when treated with TIP1 and LPS, kidney increased by LPS It was confirmed that apoptosis (green spot) in the tissue was reduced.

10-4. TIP1 Impact of this survival rate

To determine the effect of TIP1 on LPS-induced sepsis, LPS (5 or 10 μg / g animal weight) and TIP1 (g / g animal weight) in 8-week-old BALB / c (20-25g, n = 8) male mice 10nmol) and LPS were treated together, and survival was measured for 5 days. The control group was injected with the same volume of PBS. The results are shown in FIG. 18.

As shown in FIG. 18, when TIP1 and LPS were treated together, it was confirmed that the survival rate increased compared to the case where only LPS was treated. Specifically, in the case of the mild sepsis model treated with LPS at 5 μg / g, the survival rate was reduced by 100% in 3 days, and when treated with TIP1, the survival rate of 30% was maintained for 5 days. In addition, in the case of a severe sepsis model treated with 10 μg / g LPS, the survival rate was reduced by 70% in one day, and after two days, all mice died, and when treated with TIP1, the survival rate was maintained for one day despite causing severe sepsis. It was maintained at 100% and the survival rate was reduced by 90% after two days. Through this, it was confirmed that TIP1 according to the present invention suppresses the TLR4 signaling pathway to relieve the initial stage of inflammation, thereby reducing cytokine secretion and kidney and liver damage and eventually septicemia.

Experimental Example  11. TIP1 this Rheumatism  Effects on arthritis

To confirm the effect of TIP1 on rheumatoid arthritis, arthritis was induced by applying a collagen-induced arthritis (CIA) model to 6-7 week old DBA / 1J (20-23g) male mice. Specifically, chicken collagen type II (Sigma-Aldrich Co. LLC) and complete Freund's adjuvant (Sigma-Aldrich Co. LLC.) Were mixed in 1: 1 to emulsify Then, 100 μg of the emulsified collagen solution was injected intradermally into the tail of the mouse to induce primary immunity and designated as day 0. After 14 days of primary immunization, the emulsified collagen solution was again injected intradermally into the tail of the mouse to induce secondary boosting.

The following two types of TIP1 treatment schemes were designed: ① After the second immunization (day 15), TIP1 (2.5, 5 and 10 nmol / g) for 30 days or positive control prednisolone 5 mg, which is well known as a conventional arthritis treatment. plan to inject / kg daily; And ② After completely inducing arthritis (day 35), a plan to inject 10 nmol / g of TIP1 for 10 days. This was a total of 7 experimental groups (n = 8) and was named as follows: (1) Untreated normal group (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 (pre-existing arthritis treatment) treatment group (CIA-prednisolone (5 μg / g); (7) 10 nmol / g TIP1 treatment group (PAP CIA-TIP1 in post-arthritis phase (PAP)) (10 nmol / g) TIP1 and prednisolone were dissolved in saline and injected intraperitoneally, and along with this, changes in behavioral development and disability such as apparent symptoms and weight, rat crying, foot volume, and arthritis index were analyzed. 19 and 20 are shown.

As shown in FIG. 19, it was visually confirmed that when treated with TIP1, the paws swollen by the CIA model were alleviated similarly to normal mice. In addition, as shown in FIG. 20, it was confirmed that TIP1 suppresses weight loss caused by arthritis induction, crying and foot volume, and an increase in arthritis index to a level similar to that of prednisolone, an existing arthritis treatment.

In addition, arthritis-induced DAB-1J rats were injected once daily for 30 days with TIP1 and prednisolone through the CIA model, and then at day 45, the mice were sacrificed to sample the joint tissue. Thereafter, 3D images and bone mineral density (BMD) of the joint tissue were analyzed through micro-CT (micro-computed tomography). In addition, hematoxylin and eosin (H & E) staining was performed using the knee joint region of the rat, and the synovial membrane was excessive in cartilage, subchondral bone, femur, tibia, and meniscus. Changes in proliferative (synovial hyperplasia) were observed. The results are shown in FIGS. 21 and 22.

As shown in FIGS. 21 and 22, when treated with TIP1, bones damaged by the CIA model are alleviated similarly to normal mice, and cartilage (C), subchondral bone (S), femur (F), tibia (T) , It was confirmed that histopathologic transformation of meniscus (M) also decreased. Through this, it was confirmed that TIP1 according to the present invention can be effectively used as a therapeutic agent for various acute or chronic inflammatory diseases, including rheumatoid arthritis, by effectively suppressing the TLR4 signaling pathway.

Experimental Example  12. On TIP1  Treatment effect in lupus mouse model by

Systemic lupus erythematosus (SLE) mouse model was used to evaluate the therapeutic efficacy of TIP1 in systemic autoimmunity.

Three female wild-type mice (C57BL / 6) weighing an initial weight of 18-20 g and three lupus-prone mice (MRL / lpr) weighing an initial weight of 38-40 g were tested in Jackson Laboratory (Bar Harbor, ME, USA). All animal experiments were conducted with the approval of the Ajou University Medical Center Animal Ethics Committee (Approval No. 2017-0022). According to the guidelines published in the animal laboratory at Ajou University School of Medicine, the mice were acclimatized for a week before the experiment while rearing them under pathogen-free conditions. Mice from the vehicle group were injected intraperitoneally with 1% DMSO, and mice from the TIP1 treated group were injected with TIP1 dissolved in 1% DMSO daily for 10 days at 10 nmol / g body weight for 20 days. During the treatment period, the body weight of the mice was measured. At the end of the experiment, mice were sacrificed and blood, urine and tissues were collected. After incubation for 1 hour, blood samples were collected in serum separation tubes and centrifuged at 20 ° C and 3000 rpm for 10 minutes. Then, serum samples were stored at -80 ° C. The collected urine samples were immediately stored at -80 ° C. The tissues were quickly removed and washed thoroughly with PBS, then immersed in RNA stabilization solution (QIAGEN Sciences, Maryland, MD, USA). The concentrations of anti dsDNA antibody, antinuclear antibody (ANA) and C3 complement in mouse serum were analyzed by ELISA. Specifically, urine albumin levels of mice were analyzed according to the manufacturer's protocol with a mouse albumin ELISA kit (41-ALBMS-E01, Alpco Diagnostics, Salem, NH, USA). Measured 3 times per sample.

As seen in FIG. 24, the size of the spleen, kidney and lymph nodes was smaller in the SLE mouse model treated with TIP1 compared to the vehicle-treated SLE mouse model. In addition, lymphocyte proliferation (lymphoproliferation) was significantly inhibited in SLE mice treated with TIP1 compared to mice treated with vehicle.

As a result of analyzing the concentrations of the anti-dsDNA antibody and the antinuclear antibody (ANA), the anti-dsDNA antibody and the anti-nuclear antibody were present in a lower concentration in the SLE mouse model treated with TIP1 than in the SLE mouse model not treated with TIP1 (vehicle group). (See Figures 25A and 25B).

The low concentration of complement components in serum is a common marker of SLE, and the SLE mouse model treated with TIP1 recovered the concentration of C3 complement components in serum (see FIG. 25C).

The albumin content in the urine acts as a marker of kidney pathology called glomerulonephritis. As shown in FIG. 25D, the SLE mouse model treated with TIP1 significantly reduced the albumin content in urine than the SLE mouse model without TIP1 treatment.

As described above, TIP1 effectively regulates the factors related to lupus symptoms, and thus can be usefully used to prevent or treat lupus.

<110> GENESEN CO., LTD.          AJOU UNIVERSITY INDUSTRY-ACADEMIC COOPERATION FOUNDATION <120> COMPOSITION FOR PREVENTING OR TREATING LUPUS COMPRISING          TLR-INHIBITING PEPTIDES <130> FPD / 201810-0044 / C <160> 53 <170> KoPatentIn 3.0 <210> 1 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Decoy peptide 1 <400> 1 Ser His Cys Arg Val Leu Leu Ile   1 5 <210> 2 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> Decoy peptide 1-2 <400> 2 Ser His Cys Arg   One <210> 3 <211> 24 <212> PRT <213> Artificial Sequence <220> <223> TIP1 <400> 3 Arg Gln Ile Lys Ile Trp Phe Gln Asn Arg Arg Met Lys Trp Lys Lys   1 5 10 15 Ser His Cys Arg Val Leu Leu Ile              20 <210> 4 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> TIP1-2 <400> 4 Arg Gln Ile Lys Ile Trp Phe Gln Asn Arg Arg Met Lys Trp Lys Lys   1 5 10 15 Ser His Cys Arg              20 <210> 5 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Decoy peptide 2 <400> 5 Thr Ile Pro Leu Leu Ser   1 5 <210> 6 <211> 22 <212> PRT <213> Artificial Sequence <220> <223> TIP2 <400> 6 Arg Gln Ile Lys Ile Trp Phe Gln Asn Arg Arg Met Lys Trp Lys Lys   1 5 10 15 Thr Ile Pro Leu Leu Ser              20 <210> 7 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> Decoy peptide 1-3 <400> 7 Val Leu Leu Ile   One <210> 8 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> TIP1-3 <400> 8 Arg Gln Ile Lys Ile Trp Phe Gln Asn Arg Arg Met Lys Trp Lys Lys   1 5 10 15 Val Leu Leu Ile              20 <210> 9 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> CPP (Penetratin) <400> 9 Arg Gln Ile Lys Ile Trp Phe Gln Asn Arg Arg Met Lys Trp Lys Lys   1 5 10 15 <210> 10 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> TAT (48-60) <400> 10 Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Pro Pro Gln   1 5 10 <210> 11 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> pVEC <400> 11 Leu Leu Ile Ile Leu Arg Arg Arg Ile Arg Lys Gln Ala His Ala His   1 5 10 15 Ser Lys         <210> 12 <211> 27 <212> PRT <213> Artificial Sequence <220> <223> Transportan10 / TP10 <400> 12 Gly Trp Thr Leu Asn Ser Ala Gly Tyr Leu Leu Gly Lys Ile Asn Leu   1 5 10 15 Lys Ala Leu Ala Ala Leu Ala Lys Lys Ile Leu              20 25 <210> 13 <211> 21 <212> PRT <213> Artificial Sequence <220> <223> TP10, PepFect 3 <220> <221> MOD_RES <222> (21) <223> Amidation on the C-terminal end <400> 13 Ala Gly Tyr Leu Leu Gly Lys Ile Asn Leu Lys Ala Leu Ala Ala Leu   1 5 10 15 Ala Lys Lys Ile Leu              20 <210> 14 <211> 21 <212> PRT <213> Artificial Sequence <220> <223> TP10, PepFect 6 <220> <221> MOD_RES <222> (21) <223> Amidation on the C-terminal end <400> 14 Ala Gly Tyr Leu Leu Gly Lys Ile Asn Leu Lys Ala Leu Ala Ala Leu   1 5 10 15 Ala Lys Lys Ile Leu              20 <210> 15 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> TP10, PepFect 14 <220> <221> MOD_RES <222> (18) <223> Amidation on the C-terminal end <400> 15 Ala Gly Tyr Leu Leu Gly Lys Leu Leu Leu Ala Ala Ala Ala Leu Ala   1 5 10 15 Leu Leu         <210> 16 <211> 21 <212> PRT <213> Artificial Sequence <220> <223> NickFect 1 <220> <221> MOD_RES <222> (21) <223> Amidation on the C-terminal end <400> 16 Ala Gly Tyr Leu Leu Gly Lys Thr Asn Leu Lys Ala Leu Ala Ala Leu   1 5 10 15 Ala Lys Lys Ile Leu              20 <210> 17 <211> 21 <212> PRT <213> Artificial Sequence <220> <223> NickFect 2 <220> <221> MOD_RES <222> (21) <223> Amidation on the C-terminal end <400> 17 Ala Gly Tyr Leu Leu Gly Lys Thr Asn Leu Lys Ala Leu Ala Ala Leu   1 5 10 15 Ala Lys Lys Ile Leu              20 <210> 18 <211> 21 <212> PRT <213> Artificial Sequence <220> <223> NickFect 3 <220> <221> MOD_RES <222> (21) <223> Amidation on the C-terminal end <400> 18 Ala Gly Tyr Leu Leu Gly Lys Thr Asn Leu Lys Ala Leu Ala Ala Leu   1 5 10 15 Ala Lys Lys Ile Leu              20 <210> 19 <211> 27 <212> PRT <213> Artificial Sequence <220> <223> MPG <400> 19 Gly Ala Leu Phe Leu Gly Phe Leu Gly Ala Ala Gly Ser Thr Met Gly   1 5 10 15 Ala Trp Ser Gln Pro Lys Lys Lys Arg Lys Val              20 25 <210> 20 <211> 21 <212> PRT <213> Artificial Sequence <220> <223> Pep-1 <400> 20 Lys Glu Thr Trp Trp Glu Thr Trp Trp Thr Glu Trp Ser Gln Pro Lys   1 5 10 15 Lys Lys Arg Lys Val              20 <210> 21 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> MAP <400> 21 Lys Leu Ala Leu Lys Leu Ala Leu Lys Ala Leu Lys Ala Ala Leu Lys   1 5 10 15 Leu Ala         <210> 22 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> R6 / W3 <400> 22 Arg Arg Trp Trp Arg Arg Trp Arg Arg   1 5 <210> 23 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Polyarginine (R8) <400> 23 Arg Arg Arg Arg Arg Arg Arg Arg   1 5 <210> 24 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Polyarginine (R9) <400> 24 Arg Arg Arg Arg Arg Arg Arg Arg Arg   1 5 <210> 25 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> VP22 <400> 25 Asn Ala Lys Thr Arg Arg His Glu Arg Arg Arg Lys Leu Ala Ile Glu   1 5 10 15 Arg     <210> 26 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> YTA2 <400> 26 Tyr Thr Ala Ile Ala Trp Val Lys Ala Phe Ile Arg Lys Leu Arg Lys   1 5 10 15 <210> 27 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> YTA4 <400> 27 Ile Ala Trp Val Lys Ala Phe Ile Arg Lys Leu Arg Lys Gly Pro Leu   1 5 10 15 Gly     <210> 28 <211> 22 <212> PRT <213> Artificial Sequence <220> <223> M918 <400> 28 Met Val Thr Val Leu Phe Arg Arg Leu Arg Ile Arg Arg Ala Cys Gly   1 5 10 15 Pro Pro Arg Val Arg Val              20 <210> 29 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> CADY <400> 29 Gly Leu Trp Arg Ala Leu Trp Arg Leu Leu Arg Ser Leu Trp Arg Leu   1 5 10 15 Leu Trp Arg Ala              20 <210> 30 <211> 21 <212> PRT <213> Artificial Sequence <220> <223> SAP <400> 30 Val Arg Leu Leu Pro Pro Pro Val Arg Leu Leu Pro Pro Pro Val Arg   1 5 10 15 Leu Leu Pro Pro Pro              20 <210> 31 <211> 22 <212> PRT <213> Artificial Sequence <220> <223> SAP (E) <220> <221> MOD_RES <222> (1) <223> Acetylation on the N-terminal end <400> 31 Cys Gly Gly Trp Val Glu Leu Pro Pro Pro Val Glu Leu Pro Pro Pro   1 5 10 15 Val Glu Leu Pro Pro Pro              20 <210> 32 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> CyLoP-1 <400> 32 Cys Arg Trp Arg Trp Lys Cys Cys Lys Lys   1 5 10 <210> 33 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> gH 625 <400> 33 His Gly Leu Ala Ser Thr Leu Thr Arg Trp Ala His Tyr Asn Ala Leu   1 5 10 15 Ile Arg Ala Phe              20 <210> 34 <211> 30 <212> PRT <213> Artificial Sequence <220> <223> GALA <400> 34 Trp Glu Ala Ala Leu Ala Glu Ala Leu Ala Glu Ala Leu Ala Glu His   1 5 10 15 Leu Ala Glu Ala Leu Ala Glu Ala Leu Glu Ala Leu Ala Ala              20 25 30 <210> 35 <211> 21 <212> PRT <213> Artificial Sequence <220> <223> CADY <220> <221> MOD_RES <222> (1) <223> Acetylation on the N-terminal end <220> <221> MISC_FEATURE <222> (21) <223> X = cysteamide <400> 35 Gly Leu Trp Arg Ala Leu Trp Arg Leu Leu Arg Ser Leu Trp Arg Leu   1 5 10 15 Leu Trp Arg Ala Xaa              20 <210> 36 <211> 25 <212> PRT <213> Artificial Sequence <220> <223> L17E <400> 36 Ile Trp Leu Thr Ala Leu Lys Phe Leu Gly Lys His Ala Ala Lys His   1 5 10 15 Glu Ala Lys Gln Gln Leu Ser Lys Leu              20 25 <210> 37 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> MPPs <220> <221> MISC_FEATURE <222> (1) <223> X = cyclohexylalanine <220> <221> MISC_FEATURE <222> (3) <223> X = cyclohexylalanine <220> <221> MISC_FEATURE <222> (5) <223> X = cyclohexylalanine <220> <221> MISC_FEATURE <222> (7) <223> X = cyclohexylalanine <400> 37 Xaa Arg Xaa Lys Xaa Arg Xaa Lys   1 5 <210> 38 <211> 40 <212> PRT <213> Artificial Sequence <220> <223> RR5-APP <400> 38 Arg Pro Arg Arg Pro Arg Arg Pro Arg Arg Pro Gly Arg Arg Ala Pro   1 5 10 15 Val Glu Asp Leu Ile Arg Phe Tyr Asn Asp Leu Gln Gln Tyr Leu Asn              20 25 30 Val Val Thr Arg His Arg Tyr Cys          35 40 <210> 39 <211> 38 <212> PRT <213> Artificial Sequence <220> <223> RR4-APP <400> 39 Arg Arg Pro Arg Arg Pro Arg Arg Pro Gly Arg Arg Ala Pro Val Glu   1 5 10 15 Asp Leu Ile Arg Phe Tyr Asn Asp Leu Gln Gln Tyr Leu Asn Val Val              20 25 30 Thr Arg His Arg Tyr Cys          35 <210> 40 <211> 37 <212> PRT <213> Artificial Sequence <220> <223> RR3-APP <400> 40 Gly Pro Arg Arg Pro Arg Arg Pro Gly Arg Arg Ala Pro Val Glu Asp   1 5 10 15 Leu Ile Arg Phe Tyr Asn Asp Leu Gln Gln Tyr Leu Asn Val Val Thr              20 25 30 Arg His Arg Tyr Cys          35 <210> 41 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> TATp-D-1 <220> <221> MOD_RES <222> (1) <223> Acetylation on the N-terminal end <400> 41 Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Pro Pro Gln Lys Lys   1 5 10 15 <210> 42 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> TATp-D-2 <220> <221> MOD_RES <222> (1) <223> Acetylation on the N-terminal end <400> 42 Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Pro Pro Gln   1 5 10 <210> 43 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Cyclic Tat <400> 43 Lys Arg Arg Arg Gln Arg Arg Lys Lys Arg Gly Glu   1 5 10 <210> 44 <211> 22 <212> PRT <213> Artificial Sequence <220> <223> K10 (QW) 6 <400> 44 Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Gln Trp Gln Trp Gln Trp   1 5 10 15 Gln Trp Gln Trp Gln Trp              20 <210> 45 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> TAT (49-57) <400> 45 Arg Lys Lys Arg Arg Gln Arg Arg Arg   1 5 <210> 46 <211> 19 <212> PRT <213> Artificial Sequence <220> <223> DPV1047 <400> 46 Val Lys Arg Gly Leu Lys Leu Arg His Val Arg Pro Arg Val Thr Arg   1 5 10 15 Met Asp Val             <210> 47 <211> 22 <212> PRT <213> Artificial Sequence <220> <223> ARF (1-22) <400> 47 Met Val Arg Arg Phe Leu Val Thr Leu Arg Ile Arg Arg Ala Cys Gly   1 5 10 15 Pro Pro Arg Val Arg Val              20 <210> 48 <211> 28 <212> PRT <213> Artificial Sequence <220> <223> BPrPr (1-28) <400> 48 Met Val Lys Ser Lys Ile Gly Ser Trp Ile Leu Val Leu Phe Val Ala   1 5 10 15 Met Trp Ser Asp Val Gly Leu Cys Lys Lys Arg Pro              20 25 <210> 49 <211> 28 <212> PRT <213> Artificial Sequence <220> <223> p28 <400> 49 Leu Ser Thr Ala Ala Asp Met Gln Gly Val Val Thr Asp Gly Met Ala   1 5 10 15 Ser Gly Leu Asp Lys Asp Tyr Leu Lys Pro Asp Asp              20 25 <210> 50 <211> 26 <212> PRT <213> Artificial Sequence <220> <223> VT5 <400> 50 Asp Pro Lys Gly Asp Pro Lys Gly Val Thr Val Thr Val Thr Val Thr   1 5 10 15 Val Thr Gly Lys Gly Asp Pro Lys Pro Asp              20 25 <210> 51 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> C105Y <400> 51 Cys Ser Ile Pro Pro Glu Val Lys Phe Asn Lys Pro Phe Val Tyr Leu   1 5 10 15 Ile     <210> 52 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> PFVYLI <400> 52 Pro Phe Val Tyr Leu Ile   1 5 <210> 53 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Pep-7 <400> 53 Ser Asp Leu Trp Glu Met Met Met Val Ser Leu Ala Cys Gln Tyr   1 5 10 15

Claims (4)

A pharmaceutical composition for preventing or treating lupus, comprising a peptide consisting of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2 as an active ingredient.
A pharmaceutical composition for preventing or treating lupus, comprising a fusion peptide in which 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 as an active ingredient.
The pharmaceutical composition for preventing or treating lupus according to claim 2, wherein the fusion peptide consists of the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 4.
The system according to any one of claims 1 to 3, wherein the lupus is Systemic lupus erythematosus (SLE), discoid erythema lupus, subacute cutaneous lupus, drug Induced lupus or neonatal lupus, pharmaceutical composition for preventing or treating lupus.

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