KR102236773B1 - TLR4 antagonizing peptide - Google Patents

Abstract

The present invention relates to a peptide that inhibits the TLR4 signaling pathway, and to a TLR4 antagonist comprising the peptide and a pharmaceutical composition for the treatment of autoimmune or inflammatory diseases. The peptide according to the present invention has excellent effects of inhibiting the secretion of inflammatory cytokines by inhibiting the TLR4 signaling pathway and inhibiting the activation of NF-κB and MAPKs. It is useful as a pharmaceutical composition for prophylaxis or treatment.

Description

TLR4 antagonizing peptide {TLR4 antagonizing peptide}

The present invention relates to a peptide that inhibits the TLR4 signaling pathway, and more particularly, to a peptide that binds to the TLR4/MD2 complex to inhibit inflammatory cytokine secretion and the activation of NF-κB and MAPKs, including the peptide. It relates to a TLR4 antagonist and a pharmaceutical composition for the treatment of autoimmune diseases or inflammatory diseases.

Toll-like receptors (TLRs) are capable of detecting pathogen-associated molecular patterns (PAMPs) or danger-associated molecular patterns (DAMPs), so the monitoring of the host It is very important in the system (Akira, S et al., Proceedings of the Japan Academy, Series B 85:143-156, 2009). Toll-like receptor 4 (TLR4) is the first receptor identified in the TLR family and has been extensively studied in immune-related pathology (Duffy, L. et al., ImmunoTargets and therapy 5:69, 2016; Achek, A et al., Arch. Pharmacal Res. 39:1032-1049, 2016). TLR4 is located on the cell surface and recognizes lipopolysaccharide (LPS) (Kawai, T. et al., Nat. Immunol. 11:373-384, 2010). LPS is a large glycolipid molecule located in the outer membrane of Gram-negative bacteria and is composed of lipid A, a key region, and a hydrophobic domain known as an O-antigen. Lipid A is responsible for the biological activity of LPS and is a major inducer of TLR4-mediated immune responses (Park, BS et al. Nature 458:1191-1195, 2009). When LPS (lipopolysaccharide)-binding protein (LBP) binds to LPS, it forms aggregates and is transferred to CD 14 (cluster of differentiation 14), and by binding to MD2 (myeloid differentiation factor 2), LPS-binding MD2 interacts with TLR4. This leads to the initiation of dimerization of the complex and subsequent signaling (Park, BS et al., Exp. Mol. Med. 45:e66, 2013). Unlike other TLRs, TLR4 can induce pathways dependent on Myd88 (myeloid differentiation primary response gene 88) and TRIF (Toll/interleukin-1 receptor (TIR)-domain-containing adapter-inducing interferon-β) (Nijland, R. et al., Mar. Drugs 12:4260-4273, 2014). Initiation of the MyD88-dependent pathway leads to early stage activation of nuclear factors NF-κB and MAPK, which leads to the production of pro-inflammatory cytokines such as IL-1β, IL-6 and TNF-α. TLR4 stimulation induces late-stage activation of NF-κB and interferon regulatory transcription factor 3 (IRF3) through a TRIF-dependent pathway. Both pathways induce maturation and activation of innate immune cells such as macrophages and dendritic cells (Akira, S et al., Proceedings of the Japan Academy, Series B 85:143-156, 2009; Kawai, T. et al. ., Nat. Immunol. 11:373-384, 2010; Kawai, T. et al., Immunity 34:637-650, 2011).

TLR4 activation in host defense mechanisms is due to the production of pro-inflammatory cytokines that help regulate immune responses to invading pathogens, but abnormal TLR4 signaling causes inflammatory responses and acute and chronic diseases (Yang, Y. et al. ., Cell Death Dis. 7:e2234, 2016; Lee, YS et al., EMBO Mol. Med. 7:577-592, 2015; Pierer, M. et al., PLoS One 6: e23539, 2011). In particular, TLR4 is known to play an important role in the pathogenesis of rheumatoid arthritis. The rheumatoid arthritis is a kind of autoimmune disease, mainly affecting synovial joints, causing chronic inflammation and destroying joint tissues. In addition, TLR4 is known to be associated with autoimmune diseases such as systemic lupus erythematosus, systemic sclerosis, Sjogren's syndrome, Myositis and inflammatory bowel disease (IBD) ( Laura Duffy et al., ImmunoTargets and Therapy. 2016:5 69-80, 2016; Zhang, PL, et al. J. Immunol. 177, 6880-6888, 2006).

Therefore, the development of a new TLR4 blocker that prevents inflammation and restores the immune response to pathogens is the development of a powerful therapeutic agent for TLR4-related autoimmune diseases such as rheumatoid arthritis, systemic lupus erythematosus, systemic sclerosis, Sjogren's syndrome, myositis and inflammatory bowel disease. Makes it possible.

TLR4-targeted therapeutics developed so far include polysaccharides, glycolipids, antibodies, small molecules and oligopeptides, some of which are drugs/adjuvants that can reduce the secretion of pro-inflammatory cytokines to prevent or alleviate inflammatory diseases. In clinical trials (Basith, S. et al., Expert Opin. Ther. Pat. 21:927-944, 2011; Patra, MC et al., Expert Opin. Ther. Pat. 26:719-730, 2016) . For example, TAK-242 (Resatorvid) (Matsunaga, N. et al., Mol. Pharmacol. 79:34-41, 2011) that binds to the intracellular TIR domain of TLR4 and Eritoran (E5564) that binds to the LPS binding pocket. ) (Shirey, KA et al., Nature 497:498-502, 2013) is known to block the lower signaling cascade. In addition, Mycobacterium w exhibits antagonistic action against TLR4, and phase 3 clinical trials for patients with severe sepsis are currently in progress (NCT02330432). NI-0101 (Novimmune's 15C1) is a monoclonal antibody that blocks TLR4 signaling and prevents the release of cytokines produced after LPS administration (Monnet, E. et al., Clin. Pharmacol. Ther. 101: 200-208, 2017), and phase 1 clinical trial has been completed, so safety and tolerability are excellent (NCT01808469 and NCT03241108).

Various approaches are being used to develop new drugs that can down-regulate TLR4-mediated inflammation, and one such strategy is phage display (PD) technology. PD is the most powerful research tool used to find new peptides from random phage libraries screened for specific target proteins in various protein interaction studies (Smith, GP et al., Methods Enzymol. 217:228-257, 1993 ). PD is based on the fact that the protein/peptide library is displayed on the surface of the M13 phage as a fusion product with one of the phage coat proteins, and is based on the binding affinity of the protein/peptide to the desired target in this random population of surface peptides. Peptides are selected (Azzazy, HM et al., Clin. Biochem. 35:425-445, 2002).

Accordingly, the present inventors identified a new peptide capable of inhibiting the TLR4 pathway using the above method, and as a result of earnest efforts to improve structural stability and efficacy, PIP2 (phage display-derived inhibitory peptide 2) in vitro LPS- It is possible to block induced TLR4 signaling and show that cPIP2 (cyclic PIP2) cyclized by a lactam bridge exhibits relatively stable protein kinetics and a strong inhibitory effect on the TLR4 signaling pathway induced by LPS. It was confirmed, and by confirming that cPIP2 relieves inflammatory symptoms in a rat model of rheumatoid arthritis (RA), the present invention was completed.

An object of the present invention is to provide a peptide capable of inhibiting the TLR4 signaling pathway and a TLR4 antagonist comprising the peptide.

Another object of the present invention is to provide a pharmaceutical composition for preventing or treating autoimmune diseases and inflammatory diseases caused by the TLR4 signaling pathway containing the peptide as an active ingredient.

In order to achieve the above object, the present invention provides a peptide represented by the amino acid sequence of SEQ ID NO: 1 or 2.

The present invention also provides a TLR4 (Toll-like receptor 4) antagonist comprising a peptide represented by the amino acid sequence of SEQ ID NO: 1 or 2.

The present invention also provides a pharmaceutical composition for preventing or treating autoimmune diseases containing a peptide represented by the amino acid sequence of SEQ ID NO: 1 or 2 as an active ingredient.

The present invention also provides a pharmaceutical composition for the prevention or treatment of inflammatory diseases containing the peptide represented by the amino acid sequence of SEQ ID NO: 1 or 2 as an active ingredient.

The peptide according to the present invention is excellent in inhibiting the secretion of inflammatory cytokines and the activation of NF-κB and MAPKs by inhibiting the TLR4 signaling pathway induced by LPS, so autoimmunity caused by the TLR4 signaling pathway It is useful as a pharmaceutical composition for preventing or treating diseases and inflammatory diseases.

1 is a screening for a TLR4 binding peptide, (a) is an evaluation of the relative concentration of phage that specifically binds to the recombinant hTLR4 protein by the calculation/input ratio of the phage collected after each panning round, (b) Is the cell viability measured by MTT analysis by treating RAW264.7 cells with various concentrations of PIP for 24 hours, (c) is the TNF-α secretion evaluated by ELISA in RAW264.7 cells with different PIP concentrations. will be. All experiments were independently conducted three times, and the mean ±SEM of independent experiments was calculated. two-tailed paired Student t-test (*P <0.05, **P <0.01).
Figure 2 confirms the inhibitory effect of PIP2 on the TLR signaling pathway, (a) is an initial evaluation by monitoring the activity level of NF-κB through SEAP analysis in HEK-BlueTM hTLR4 cells, (b) The expression level of NF-κB (IκBα cytosolic degradation) and the expression levels of p-IRF3 and ATF3 were measured by Western blot in the total protein extract of RAW264.7 cells, and (c) is p-ERK, p-JNK and p The expression level of MAPK containing -p38 was measured in PIP2-treated cells, and inactive MAPK, ERK, JNK and p38 were used as controls. (d) is the expression level of p-p65 measured by immunofluorescence staining and detected by confocal microscopy. The part stained in red indicates phosphorylated NF-κB (p-p65), and Hoechst stained in blue indicates nuclear staining ( scale bar = 20μm). (e) and (f) are the levels of IL-6 and IFN-β secretion measured in RAW264.7 cells by ELISA, and (g) is the expression level of iNOS and COX2 in RAW264.7 cells by Western blot. It was measured, (h) is an evaluation of NO production using a NO secretion kit, (i) is a measurement of the cytoplasmic secretion level of NO by FACS using DAF-DA staining, (j) is DCF-DA ROS production was measured in the cytoplasm by FACS using staining, and (k) is a different TLR ligand [(TLR2/1 (PAM3CSK4), TLR2/6 (FSL-1), TLR9 (CpG-ODN), or TLR7/8 (R848)] to compare the secretion level of TNF-α in RAW264.7 cells treated with PIP2 under the influence of. All experiments were independently conducted three times, and the mean ±SEM of independent experiments was calculated. tailed paired Student t-test (* P <0.05, ** P <0.01).
Figure 3 shows that PIP2 inhibits cytokine secretion and activation of NF-κB and MAPKs in human peripheral blood mononuclear cells (hPBMCs), (a) and (b) TLR4 ligand LPS, TLR2/1 ligand PAM3CSK4 and TLR2/ 6 The secretion levels of TNF-α and IL-6 under the influence of the ligand FSL-1 were evaluated in PIP2-treated hPBMC, (c) NF-κB activation, IκBα degradation, and the expression level of MAPK by Western blot. As an evaluation, inactive MAPK was used as a control and β-actin was used as a loading control. All experiments were independently conducted three times, and the mean ±SEM of independent experiments was calculated. two-tailed paired Student t-test (*P <0.05, **P <0.01).
4 shows that PIP2 inhibits TLR4-mediated cytokine secretion in mouse bone marrow-derived macrophages (mBMDM) and alleviates TLR4-mediated inflammatory responses in vivo, (a) to (c) are TNF-α, IL- 6 and IFN-β showed that the levels of secretion were reduced by PIP2 treatment, and (d) evaluated NO secretion using a NO secretion kit. (e) to (g) are C57BL/6J mice intraperitoneally administered with 70 nmol/g of PIP2 (ip), followed by intraperitoneal administration of 5 μg/g of LPS (ip), and TNF-α, IL-6 and IL in plasma It monitored the level of -12p40. All experiments were independently conducted three times, and the mean ±SEM of independent experiments was calculated. two-tailed paired Student t-test (*P <0.05, **P <0.01).
Figure 5 shows the image of the PIP2-TLR4 complex, biophysical analysis, and the structure of PIP2.(a) The expression level of TLR4 (red staining) and the location of FITC-PIP2 (green staining) were evaluated by immunofluorescence and blank. As analyzed by focal microscopy, Hoechst staining (blue) is the nucleus (scale bar = 20 μm), and the confocal image shows the co-localization of FITC-PIP2 and TLR4. (b) is an analyte using chip-fixed hTLR4 and SPR as ligands, and PIP2 was used to evaluate the TLR4 binding affinity of PIP2.Sensorgram indicates that PIP2 is the extracellular domain of TLR4 (ECD; Extra Cellular Domain). ) In a concentration-dependent manner. (c) is a molecular dynamics simulation (MDS) for PIP2, where the N-terminus of PIP2 maintains a helical structure at the MDS end, but the C-terminus half acquires a loop structure. During MDS, the root-mean-square deviation (RMSD) for the initial conformation of the total protein (blue) and backbone atoms (purple) is presented using the least squares sum. The helical structure of PIP2 was stabilized through the formation of a lactam bridge between Asp6 and Lys10 (cPIP2).
Figure 6 shows the structural kinetics of TLR4 binding PIP2 and comparative interfacial analysis of TLR4/MD2 and PIP2-TLR4, where (a) is the root mean square deviation (RMSD) of the backbone atoms bound to TLR4 or MD2 and the entire PIP2 peptide is minimal. Calculated using the sum of squares, the hydrogen bond distance was calculated as a function of time between the interaction of the residues of PIP2 and the patch A and B residues of the ECD of TLR4. The top right panel of'A' shows the time-dependent formation of hydrogen bonding interactions between PIP2 and patch B of monomeric TLR4 (first simulation), the two panels below validate the initial simulation and PIP2 is PIP2-TLR4 ( When simulating docking with TLR4/MD2) composites, it is suggested that PIP2 establishes strong electrostatic interactions with Patch A and Patch B of TLR4 (second simulation). (b) describes amino acid identity/similarity between human and mouse TLR4 interacting with MD2 and PIP2, showing that PIP2 blocks LPS-induced TLR4 activation by competitively interacting with MD2-binding patches A and B of TLR4. Indicates that.
7 shows that cyclic PIP2 (cPIP2) inhibits inflammatory cytokine secretion, reduces oxidative stress, and interferes with MAPK expression in macrophages. And (b) to (e) were measured by ELISA for TNF-α, IL-6, IFN-β and IFN-α. (f) is the measurement of NO secretion using a NO secretion kit, (g) is the measurement of the cytoplasmic secretion of NO by FACS by DAF-DA staining, (h) is the ROS production in the cytoplasm by DCF-DA It was measured by FACS by staining. (i) Human monocyte (THP-1)-derived macrophages were treated with cPIP2 and multiple TLR ligands [TLR2/1 (PAM3CSK4), TLR2/6 (FSL-1), TLR9 (CpG-ODN) or TLR7/8 (R848)] was used to monitor the inhibitory effect of the peptide, showing that cPIP2 greatly inhibited TLR4 but insufficient inhibition of other TLRs. (j) is an evaluation of the expression level of MAPKs as well as NF-κB activation and IκBα decomposition by Western blot. Inactive MAPK was used as a control and β-actin was used as a loading control. All experiments were independently conducted three times, and the mean ±SEM of the independent experiments was calculated. two-tailed paired Student t-test (*P <0.05, **P <0.01).
Figure 8 confirms the biophysical interaction between PIP2 and TLR2, and the sensorgram shows that PIP2 binds to the extracellular domain (ECD) of TLR2 in a concentration-dependent manner.
9 is for PLIF (protein ligand interaction fingerprints) analysis of PIP2-binding TLR4 in the presence and absence of MD2, (a) is PIP2 docked to TLR4 without MD2, and PIP2 is mutually inconsistent with the ECD of TLR4. It shows an action and shows that about 43% of PIP2 peptides hydrophobicly interact with the N-terminus of TLR4. (b) is PIP2 docked with the TLR4/MD2 complex, where most of them are regulated by the hydrophobicity of MD2, and 80% of the docked PIP2 shows a hydrophobic interaction with Phe121 of MD2.
FIG. 10 shows the reduction of inflammation in the RA rat model of cyclic PIP2 (cPIP2), (a) visually monitoring the state of the paws at weeks 1, 3 and 6 in the normal group, the control group and the cPIP2 treatment group (scale bar = 10mm), (b) and (c) are measurements of the ankle diameter and joint index, respectively, and (d) to (f) are respectively hematoxylin and eosin (H&E) staining (d), safranin- O (SO) staining (e) and TNF-α staining (f) results (magnification = 200 times) are shown.

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

Overactivation of TLR4 is known to be a major cause of the pathophysiology of a wide variety of neurodegenerative, autoimmune and inflammatory diseases. For example, LPS-mediated activation of TLR4 signaling has been reported to affect the inflammatory process in cerebral ischemia through activation of microglia and secretion of inflammatory factors (Samson, Y et al., Rev. Neurol. (Paris) 161:1177-1182, 2005). The expression level of TLR4 was increased in tubular epithelial cells of the ischemia/reperfusion injury mouse model, whereas the TLR4-/- and MyD88-/- mice showed renal dysfunction, coronary injury, and accumulation of proinflammatory cytokines. It has been reported that TLR4 may play an important role in mediating kidney damage by being protected from secretion (Wu, H. et al., J. Clin. Invest. 117:2847-2859, 2007). In addition, LPS and HMGB1 (high-mobility group protein 1) induce memory abnormalities in the brain, thereby inducing activation of microglia and the onset of inflammatory stroke, leading to ischemic brain injury (Mazarati, A et al., Exp. Neurol). . 232:.... 143-148 , 2011; Shih, RH et al, Front Mol Neurosci 8:77, 2015). In this regard, Kaempferol, a natural flavonoid known for its antioxidant and anti-inflammatory properties, reduces HMGB1 levels, down-regulates TLR4-mediated inflammatory responses, and induces the destruction of the blood-brain barrier in mice and Protects against nerve inflammation (Cheng, X et al., Int. Immunopharmacol. 56:29-35, 2018). In addition, the production of ROS and NO is known to contribute to the pathogenesis of neurodegenerative diseases and cancer (Liou, GY et al., Free Radic. Res. 44:479-496, 2010; Bhattacharyya, A et al., Physiol Rev. 94:329-354, 2014; Uttara, B et al., Curr. Neuropharmacol. 7:65-74, 2009). Inhibition of ROS production is known to alleviate the TLR4-mediated inflammatory and proliferative phenotype of vascular smooth muscle cells (Pi, Y. et al., Lab. Invest. 93:880-887, 2013). Decreased TLR4 expression and decreased ROS production have been shown to delay the progression of acute lung injury (Asehnoune, K et al., J. Immunol. 172:2522-2529, 2004).

TLR4 appears with the onset of several diseases and is an important therapeutic target because it affects the activation of excessive secretion of inflammatory cytokines (Achek, A et al., Arch. Pharmacal Res. 39:1032-1049, 2016; Patra , MC et al., Expert Opin.Ther. Pat. 26:719-730, 2016). For this reason, the development of new TLR4 antagonists is very active, and so far, there are various proteins, small peptides, chemical agents and antibodies, and the like. Among them, TLR4 peptide inhibitor (VIPER) is TLR4 and its adapter protein Toll/interleukin-1 receptor (TIR) domain-containing adapter protein/MyD88 adapter-like (TIRAP/Mal) and TIR domain-containing adapter protein-inducing IFN-β. Blocking the interaction between the (TRIF)-related adapter molecule (TRAM) inhibited the LPS-induced secretion of IL-6 in RAW264.7 cells. In addition, Eritoran (E5564), a synthetic lipid A analog, prevents activation of TLR4 by blocking the binding of LPS and MD2 pockets (Opal, SM et al., AMA 309:1154-1162, 2013). E5564 progressed to phase 3 clinical trial (NCT00334828), but was discontinued due to failure to improve the patient's severe sepsis status (Opal, SM et al., AMA 309:1154-1162, 2013).

In the present invention, in order to inhibit TLR4, a peptide library designed through the PD system was screened for TLR4-binding and specificity. Using hTLR4, the peptide selected from the PD library was named PIP2, and PIP2 was a hydrophobic dodecapeptide with a slightly strong polarity due to two serine and one lysine, and of SEQ ID NO: 1 (NH ₂ -LGVFSSWWIKLV-COOH). It is represented by the amino acid sequence (Fig. 5c). The PIP2 showed an antagonist effect on TLR4 signaling. That is, PIP2 significantly inhibited the LPS-induced inflammatory response, interfering with the secretion of pro-inflammatory cytokines and type I IFNs, lowering the expression level of MAPK, and reducing the production of oxidative stress markers in other cell lines (Figs. 2 and 3). In addition, TLR4-mediated inflammation observed in vivo after challenge with LPS was also suppressed (FIG. 4).

Cyclization of peptides has been widely practiced to improve the half-life of peptide drugs and also to increase cellular uptake (Taylor, JW et al., Biopolymers 66:49-75, 2002; Davies, JS et al., J. Pept. Sci. 9:471-501, 2003). Thus, in silico , the stability and efficacy of PIP2 were improved by improving the structural information of the TLR4-PIP2 complex. That is, by connecting Asp6 and Lys10 through a lactam bridge, the helical structure of PIP2 was stabilized (FIG. 5C), and the amino acid sequence of SEQ ID NO: 2 (LGVFSDWWIKLV) was shown. cPIP2 not only maintains the TLR4 inhibitory effect, but also significantly reduces the inhibitory concentration (FIG. 7).

Accordingly, the present invention relates to a peptide represented by the amino acid sequence of SEQ ID NO: 1 or 2 in a single point of view.

In the present invention, the peptide may be characterized by inhibiting the TLR4 (Toll-like receptor 4) signaling pathway, and the TLR4 signaling pathway may be characterized by being induced by LPS (lipopolysaccharide).

In the present invention, the peptide may be characterized in that it binds to the TLR4/MD2 complex.

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

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

In the present invention, the term "TLR4 signaling pathway" refers to a signaling pathway through TLR4, and may be an LPS reaction dependent on the TLR4/MD2 complex, which is a transmembrane complex formed by TLR4 and MD2, through which signals are transmitted. do. TLR4 is signaled by several adapter proteins, and the signaling pathways act as Mal (also referred to as TIRAP) and MyD88, as well as TRAM and TRIF. The TLR4 mediated signaling pathway induces activation of MyD88-dependent and MyD88-independent signaling through a variety of sub-signaling molecules. Initiation of the MyD88-dependent pathway induces early stage activation of NF-κB and secretion of pro-inflammatory cytokines such as TNF-α and IL-6, and initiation of the MyD88-independent pathway leads to the activation of IRF3 and 7, the activation of IFNs. Induces secretion and late stage activation of NF-κB (Park, BS et al., Exp. Mol. Med. 45:e66, 2013; Nijland, R et al., Mar. Drugs 12:4260-4273, 2014) . In addition, TLR4 induces the activation of MAPK, including ERK, JNK and p38, and secretes inflammatory cytokines and IFN (Achek, A et al., Arch. Pharmacal Res. 39:1032-1049, 2016; Kawai, T et al., Nat. Immunol. 11:373-384, 2010; Kawai, T et al., Semin. Immunol. 19:24-32, 2007). Stimulation of TLR4 by related ligands in macrophages induces COX2 and iNOS, induces NO production, and produces mitochondria and intracellular ROS (Kim, JY et al., Eur. J. Pharmacol. 584:175- 184, 2008; Liu, K.-L. et al. J. Agric. Food Chem. 54:3472-3478, 2006; West, AP et al. Nature 472:476, 2011; Mancek-Keber, M. et al. .. Sci Signal 8:. ra60 -ra60, 2015).

In the present invention, the term "inhibition" refers to a phenomenon in which biological activity or activity is decreased due to deficiency, incongruity, and many other causes, and partially or completely blocks, reduces, prevents, or activates the activity of TLR4. It may be delaying, inactivating or down-regulating.

In the present invention, the term "TLR4/MD2 complex" refers to a transmembrane complex formed by TLR4 and MD2, and the peptide represented by SEQ ID NO: 1 or 2 of the present invention binds to the TLR4/MD2 complex, thereby making the TLR4 signaling pathway Can be suppressed.

In silico analysis confirmed that PIP2 can block TLR4 signaling by two possible mechanisms. Because of its hydrophobicity, PIP2 preferably binds MD2 in the TLR4/MD2 complex, and because TLR4 requires MD2, co-receptor and LPS binding pockets (Kim, HM et al., Cell 130:906-917, 2007; Ohnishi, T et al., Clin. Diagn. Lab. Immunol. 10:405-410, 2003), blocking the influx of LPS into MD2 may be one of the TLR4-antagonistic mechanisms of PIP2 (Park, S. et al. al., Biomaterials 126:49-60, 2017). Since PIP2 was first selected through PD using the ECD of hTLR4, PIP2 docks with TLR4 in the absence of MD2. PIP2 is located at four different locations in the extracellular region of TLR4 (Figure 9). In addition, MD2 interacts with ECD during TLR4 signaling, and patches A and B of TLR4 form electrostatic contacts in mice and humans (Park, BS et al., Nature 458:1191-1195, 2009; Kim, HM. et al., Cell 130:906-917, 2007). As a result of comparative structural analysis, residues of this patch are conserved and are critically important for TLR4-MD2 interactions. Small peptides derived from MD2 are known to bind to these patches and interfere with TLR4-MD2 interactions (Slivka, PF et al., ChemBioChem 10:645-649, 2009). In the present invention, it was found that PIP2 can also bind to these residues and make similar contact, and PIP2 specifically recognizes Val108 and Asp155 and stabilizes itself through C-terminal electrostatic interaction with patch B of TLR4. In conclusion, in the present invention, it was found that it is important to block TLR4-MD2 interaction in addition to MD2 in order to block TLR4 signaling.

Accordingly, in another aspect, the present invention relates to a TLR4 (Toll-like receptor 4) antagonist comprising a peptide represented by the amino acid sequence of SEQ ID NO: 1 or 2.

As long as the "peptide of SEQ ID NO: 1 or 2" of the present invention has the ability to effectively bind to the TLR4/MD2 complex, not only the same peptide as the sequence of SEQ ID NO: 1 or 2, but also the amino acid thereof is substituted by conservative substitution. , It may include all peptides having a sequence homology of 70% or more, preferably 80% or more, and more preferably 90% or more. The term "homology" refers to a wild type amino acid sequence and a wild type nucleic acid sequence. It is meant to indicate the degree of similarity.

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

In the present invention, the term "TLR4 antagonist" refers to a substance capable of directly or indirectly or substantially interfering, reducing or inhibiting the biological activity of TLR4, and preferably, a peptide reactive with TLR4 is a TLR4 or TLR4/MD2 complex. By direct binding and neutralizing the activity of TLR4, it is possible to block the TLR4 signaling pathway, causing a decrease in the activation of NF-κB and MAPKs, thereby reducing the secretion of inflammatory cytokines, NO and ROS.

According to an embodiment of the present invention, the peptide represented by SEQ ID NO: 1 or 2 of the present invention inhibits the TLR4 signaling pathway induced by lipopolysaccharide (LPS), thereby inhibiting interleukin-6 (IL-6), NO And it is excellent in the effect of inhibiting the secretion of ROS and the activation of NF-κB and MAPKs, it can be usefully used as a pharmaceutical composition for the prevention or treatment of autoimmune diseases and inflammatory diseases caused by the TLR4 signaling pathway.

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

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

Peptide stapling is used to limit short peptides in the alpha-helical form, and during stapling, the sidechains of the two amino acids are covalently bonded through lactam bridges to form macrocyclic peptides. Lactam ligation strategies are being studied to improve the efficacy of therapeutic peptides, and it has been reported that peptides restricted using lactam ligation strategies are superior to linear analogues (Harrison, RS et al., Proc. Natl. Acad. Sci. USA 107:11686-11691, 2010).

Accordingly, in the present invention, a protein design technique was implemented to stabilize the helical structure of PIP2 by connecting Asp6 and Lys10 through a lactam bridge (FIG. 5C and Formula 1). Structural dynamics confirmed that the lactam linkage stabilized the helical structure in cPIP2 and limited the free moment at the N- and C-terminals compared to the linear structure of PIP2. In addition, the backbone of cPIP2 and the total protein RMSD were very stable compared to PIP2 (Fig. 5c). As a result of confirming the TLR4 inhibitory effect of cPIP2 in vitro, it was found that cPIP2 not only maintains the TLR4 inhibitory effect, but also significantly reduces the inhibitory concentration (PIP2 IC50 = 40 μM, cPIP2 IC50 = 25 μM) (FIG. 7 ).

Accordingly, in another aspect, the present invention relates to a pharmaceutical composition for the prevention or treatment of autoimmune diseases containing the peptide represented by the amino acid sequence of SEQ ID NO: 1 or 2 as an active ingredient.

In the present invention, the term "autoimmune disease" is caused by a process in which a problem occurs in inducing or maintaining self-tolerance and an immune response to an autoantigen occurs, thereby attacking the own tissue. The self-tolerance refers to immunologic unresponsiveness that does not adversely respond to antigenic substances constituting the self. The autoimmune diseases of the present invention are insulin-dependent diabetes mellitus, multiple sclerosis, and experimental. Autoimmune encephalomyelitis, rheumatoid arthritis, experimental autoimmune arthritis, myasthenia gravis, thyroiditis, experimental form of uveitis, Hashimoto's thyroiditis, primary myxedema, thyroid poisoning, pernicious anemia, autoimmune atrophy gastritis, Addison's disease, early menopause, male infertility, Childhood diabetes, Goodpasture syndrome, common pemphigus, pemphigus, sympathetic ophthalmitis, lens uveitis, autoimmune hemolytic anemia, idiopathic leukocytosis, primary cholangiosclerosis, latent cirrhosis, ulcerative colitis, Sjogren's syndrome, scleroderma, Wegener's parenting Including, but not limited to, polymyositis/dermatomyositis and systemic lupus erythematosus.

The composition for preventing or treating autoimmune diseases of the present invention may contain a pharmaceutically effective amount of the peptide alone or may contain one or more pharmaceutically acceptable carriers, excipients, or diluents. Iran refers to an amount sufficient to prevent, ameliorate, and treat symptoms of autoimmune diseases.

The term "pharmaceutically acceptable" refers to a composition that is physiologically acceptable and does not usually cause allergic reactions such as gastrointestinal disorders, dizziness or similar reactions when administered to humans. Examples of the carrier, excipient and diluent include, Lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, polyvinylpyrrolidone, water, methylhydride Oxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oils. In addition, fillers, anti-aggregating agents, lubricants, wetting agents, fragrances, emulsifiers and preservatives, etc. may be further included.

Accordingly, in the present invention, the protective effect of cPIP2 was confirmed in rheumatoid arthritis (RA), which is a kind of autoimmune disease. The rheumatoid arthritis is a multifactorial autoimmune disease mainly characterized by erosive inflammation of joint cartilage, leading to swelling of synovial fluid such as a tumor and gradual destruction of adjacent joint cartilage. The exact cause of rheumatoid arthritis is not yet known, but immunological dysregulation observed during rheumatoid arthritis is known to be involved in promoting inflammation and synovial cell proliferation (Isaacs, JD Nature reviews. 605-611, 2010 and Smolen, JS et al. Nature reviews. Disease primers , 18001, 2018).

In the present invention, as a result of confirming the effect of cPIP2 on rheumatoid arthritis in the RA rat model, it was found that cPIP2 significantly reduces inflammation and promotes regeneration of cartilage tissue (FIG. 10). In conclusion, PIP2, especially cPIP2, is a promising inhibitor of TLR4 signaling, and can be usefully used in treatment groups for autoimmune diseases or inflammatory diseases.

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

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

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

In another aspect, the present invention relates to a pharmaceutical composition for the prevention or treatment of inflammatory diseases containing a peptide represented by the amino acid sequence of SEQ ID NO: 1 or 2 as an active ingredient.

In the present invention, the term "inflammatory disease" refers to TNF-α, IL-1, IL-6, and prostaglandins secreted by immune cells such as macrophages by excessively promoting the immune system due to harmful stimulation such as inflammation inducing factors or irradiation. (prostaglandin), leukotriene (leukotriene) or NO refers to a disease caused by an inflammatory substance (inflammatory cytokine) such as, the inflammatory disease of the present invention is asthma, eczema, psoriasis, allergies, rheumatoid arthritis, Psoriatic arthritis, contact dermatitis, atopic dermatitis, acne, atopic rhinitis, allergic dermatitis, chronic sinusitis, seborrheic dermatitis, gastritis, gout, gouty arthritis, ulcers, chronic bronchitis , Pulmonary inflammation, Crohn's disease, ulcerative colitis, ankylosing spondylitis, sepsis, vasculitis, bursitis, lupus, rheumatoid multiple myalgia, temporal arteritis, multiple sclerosis, solid cancer, Alzheimer's disease, arteriosclerosis, obesity and viral infections. Including, but not limited to.

Since the pharmaceutical composition for preventing or treating inflammatory diseases includes a pharmaceutical preparation containing the above-described peptide as an active ingredient, descriptions of the contents overlapping with the above-described composition of the present invention will be omitted.

[Example]

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

Example 1: Construction and screening method of TLR4 binding peptide library

1-1: PD peptide library

The PD (phage display) peptide library was constructed and screened by the previously described method (Park, S. et al. Biomaterials 126:49-60, 2017).

12mer peptide library is forward primer 5'-GCC CAG CCG GCC ATG GCC (NNK) ₁₂ TCG AGT GGT GGA GGC GGT TCA G-3' (SEQ ID NO: 3) and reverse primer 5'-GCC AGC ATT GAC AGG AGG TTG AG- It was synthesized as 3'(SEQ ID NO: 4). In the case of the NNK random codon (N=A/C/G/T, K=G/T), all amino acids were selected in order to minimize the stop codon in a state capable of encoding. The DNA product was cloned into pHEN2 phagemid and transfected into DH10B (E. coli ) cells, and then the phage library was transformed into XL1-Blue (E. coli ) cells and amplified. Hyperphage, M13K07ΔpIII (PROGEN Biotechnik GmbH, Heidelberg, Germany) was added to the culture solution (final concentration, 1 x 10 ¹² PFU / mL), and incubated at 37°C for 30 minutes. Thereafter, the cell pellet was centrifuged (3300×g, 10 min, 4°C), resuspended in 30 mL of fresh 2×YT medium (100 μg/ml ampicillin and 25 μg/mL kanamycin), and cultured at 30° C. overnight, The phage particles were recovered.

1-2: biopanning

The biopanning process was also performed by the previously described method (Park, S. et al. Biomaterials 126:49-60, 2017).

Recombinant hTLR4 protein (2.5 μg/mL) was coated on a Nunc MaxiSorp 96-well plate (Thermo Fisher Scientific Inc., Waltham, MA, USA), incubated overnight at 4°C, and blocked with 5% skim milk at room temperature the next day. Was exposed to the phage library. The bound phage was eluted, recovered, and subjected to phage titration, and the phage titer was calculated as a colony forming unit (cfu) on a plate containing ampicillin (10 μg/mL). Thereafter, the phage was amplified and purified and used for panning, and the concentration efficiency was estimated by calculating the input/output ratio in each round (6 rounds in total). Selection and isolation of transformed clones was performed after 6th biopanning through phage ELISA, as previously described (Park, S. et al. Biomaterials 126:49-60, 2017).

1-3: DNA sequencing and peptide synthesis

Phage DNA was isolated from the phage selected through ELISA of Example 1-2 using the Miniprep Kit (GeneAll Biotechnology, Seoul, Korea). The isolated DNA was sequenced using a primer of 5'-TTG TGA GCG GAT AAC AAT TTC-3' (SEQ ID NO: 5) (Macrogen, Inc. Korea, Seoul). The degree of diversity was evaluated by translating the DNA sequence into an amino acid sequence using BioEdit software and aligning to evaluate the modification (Hall, TA et al., Nucleic Acids Symposium Series, 95-98, 1999). All peptides used are Peptron, Inc. It was synthesized and purchased from (Daejeon, Korea).

Example 2: Screening of PD (phage-display) peptide library for TLR4

2-1: TLR4 specific binding peptide

A solid-phase screening method was used to identify the TLR4 specific binding peptide. As in Example 1, a pHEN2 (12-mer) PD library was prepared and exposed to a 96-well plate coated with recombinant human TLR4 (hTLR4) protein. Biopanning was performed 6 times to enrich the PD library for hTLR4, and the concentration ratio after each round was evaluated through the titer of the eluted phage (Fig. 1A). Then, phage clones capable of binding hTLR4 were selected by phage-ELISA and sequenced to estimate their nucleotide variations using BioEdit software.

As a result, five 12-mer peptides named PIP1-PIP5 having a unique sequence were synthesized. PIP1 and PIP4 were identical except for the two residues at the N-terminus, but the other peptides had unique sequences and mainly contained hydrophobic residues.

2-2: Cytotoxicity of TLR4 binding peptide

Using the PIP1-PIP5 peptide synthesized in Example 2-1 at a concentration of 20-100 μM, cytotoxicity to RAW264.7 (ATCC, Manassas, VA, USA) cells was measured by MTT (1-(4,5-dimethylthiazol-2- yl)-3,5-diphenylformazan; Sigma-Aldrich Co.) was used to investigate.

RAW264.7 cells were inoculated at a density of 2 x 10 ⁵ cells/well and treated with different concentrations of peptides for 24 hours, the next day the medium was removed and the diluted MTT solution (final concentration: 500 μg/mL) (100 μl/well ), and then incubated at 37°C for 3 hours. The MTT solution was removed and a dimethyl sulfoxide solution (100 μL/well, Sigma-Aldrich Co.) was added, and the absorbance at 540 nm was measured after 30 minutes with a spectrophotometer (Molecular Devices Inc.).

As a result, at a concentration of less than 80 μM, the peptide did not show cytotoxicity, but the viability of the cells treated with the 100 μM peptide decreased slightly (FIG. 1B ).

2-3: Inhibition of TNF-α secretion by TLR4 binding peptide

According to the results of Example 2-2, this time, TNF-α production was further investigated at a concentration of 20 to 80 μM. Microplate spectrophotometer system at specific absorbance using Mouse TNF alpha ELISA Ready-SET-Go kit (eBioscience, San Diego, CA, USA) or Human TNF alpha ELISA MAX™ Deluxe (BioLegend, San Diego, CA, USA) (Molecular Devices Inc.) and all data were analyzed using SoftMax Pro 5.3 software (Molecular Devices Inc.).

As a result, it was found that among PIP1-PIP5, PIP2 (SEQ ID NO: 1: LGVFSSWWIKLV) significantly reduced TNF-α (tumor necrosis factor α) secretion in a concentration-dependent manner (FIG. 1C).

Therefore, TNF-α production was once again confirmed through ELISA in RAW264.7 cells treated with PIP in the presence of LPS. In addition, the physicochemical properties of PIP2 were further investigated to evaluate the TLR4-specific trend, and the antagonistic effect of PIP2 on TLR4 signaling was investigated as a follow-up experiment.

Example 3: Inhibition of the TLR4 signaling pathway of PIP2 in macrophages

3-1: SEAP (secreted embryonic alkaline phosphatas) analysis

In order to confirm the regulatory effect of PIP2 on the TLR signaling pathway, SEAP (secreted embryonic alkaline phosphatas) analysis was performed. The SEAP reporter gene is under the control of the IL-12p40 minimal promoter containing NF-κB (nuclear factor-κB) and AP-1 (activator protein 1) binding sites, and is induced upon TLR4 activation (Fujioka, S. et al. Mol. Cell. Biol. 24:7806-7819, 2004). Therefore, TLR4 inhibitory activity was measured using different concentrations of PIP2 (20, 40 or 80 μM) in the presence of LPS.

HEK-Blue™ hTLR4 cells (InvivoGen, San Diego, CA, USA) were inoculated into a 96-well plate at ^{a density of 2 x 10 4} cells/well and cultured for 2 days. The supernatant was removed and treated with different concentrations of PIP2 (20, 40 or 80 μM) for 1 hour in HEK-Blue detection medium (IvivoGen, San Diego, CA, USA), followed by LPS (100 ng/mL) for 24 hours. Processed. Culture supernatant was collected and placed in a new 96-well plate, and SEAP activity was evaluated by measuring absorbance at 630 nm with a VersaMax Microplate Reader (Molecular Devices Inc., Sunnyvale, CA, USA).

As a result, activation of NF-κB was induced in LPS-treated cells, but in cells stimulated with LPS for 24 hours after pretreatment with PIP2 for 1 hour, SEAP activity was decreased in a concentration-dependent manner (FIG. 2A ). On the other hand, in cells treated with PIP2 alone, there was no effect on SEAP activity.

3-2: Inhibition of activation of N F-κB and MAPK

In LPS-stimulated RAW264.7 cells, the activation of NF-κB and mitogen-activated protein kinases (MAPK) was investigated by Western blot to analyze the inhibitory effect of PIP2.

Primary antibodies to p-JNK (c-Jun N-terminal kinase), p-IRF3 (interferon regulatory transcription factor 3), ERK (extracellular signal-regulated kinase), and p38 (p38 mitogen-activated protein kinase) (Cell Signaling) Technology Inc., Danvers, MA, USA); primary antibodies to p-ERK, p-p38, Iκ-Bα, JNK, ATF3, COX2 (cyclooxygenase 2), β-actin (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA); And a primary antibody against iNOS (inducible nitric oxide synthase) (BD Biosciences) was diluted in a ratio of 1:500-1000. The secondary antibody, anti-mouse/-rabbit peroxidase-conjugated immunoglobulin G antibody (Thermo Fisher Scientific Inc.) was used in a ratio of 1:1,000, and the protein was detected with SuperSignal West Pico ECL solution (Thermo Fisher Scientific Inc.). Visualized with the Fusion solo S system (Vilber Lourmat, France).

As a result, PIP2 inhibited the LPS-induced NF-κB and IκBα degrading activity (FIG. 2B). In addition, PIP2 inhibited the activation of ATF3 (activating transcription factor 3) and phosphorylation of IRF3, and successfully inhibited the activation of MAPKs including phosphorylation of ERK, JNK and p38 compared to the activation level of LPS-treated cells. (Fig. 2c).

Then, RAW264.7 cells treated with PIP2 (80 μM) for 1 hour were ^{grown overnight at a concentration of 2 x 10 5} cells/well in a 24-well plate, and then treated with LPS (100 ng/ml) for 30 minutes to p -p65 was analyzed. In the case of FICT-PIP2 (Peptron, Inc), cells were pretreated with 40 μM of FITC conjugated peptide for 1 hour, followed by LPS treatment (100 ng/mL) for 30 minutes, and the cells were subjected to 3.7% formaldehyde solution (Sigma-Aldrich Co. .) for 5 minutes and infiltrated with 0.2% Triton X-100 solution (AMRESCO, Solon, OH, USA) for 5 minutes. Cells were reacted with a primary antibody against p-p65 (Cell Signaling Technology 1:1000) or TLR4 (Abcam, Cambridge, UK, 1:1000) for 2 hours, and then Alexa Fluor 408 and/or 488 secondary antibody ( Invitrogen) and treated with Hoechst 33258 solution (5 μM, Sigma-Aldrich Co.) to stain the nuclei. Fluorescence intensity was measured using a confocal microscope (LSM-700, Carl Zeiss Microscopy GmbH), and images were analyzed using Zen 2009 software.

As a result, it was confirmed that the LPS-induced p-p65 expression level was inhibited by PIP2 (FIG. 2D).

3-3: inhibition of IL-6, IFN-β and TNF-α

RAW264.7 cells were ^{seeded in a 96-well plate (BD Biosciences) at a density of 2 x 10 5} cells/well to treat PIP2, and IL-6 secretion was performed by Mouse/Human IL-6 ELISA MAX™ Deluxe (BioLegend) or Mouse IL-6 Platinum ELISA (eBioscience, San Diego, CA, USA) was measured, and TNF-α production was measured by Mouse TNF alpha ELISA Ready-SET-Go kit (eBioscience, San Diego, CA, USA) or Human TNF alpha ELISA Measured with MAX™ Deluxe (BioLegend, San Diego, CA, USA). The specific absorbance was read with a microplate spectrophotometer system (Molecular Devices Inc.), and all data were analyzed using SoftMax Pro 5.3 software (Molecular Devices Inc.).

As a result, the production of LPS-induced IL-6 and IFN-β was also inhibited in a concentration-dependent manner by PIP2 (Fig. 2e-f).

In addition, in order to confirm the effect of PIP2 on other TLR and its subsignaling, cells were transferred to PAM ₃ CSK ₄ (TLR1/2), FSL-1 (TLR2/6), Resiquimod/R848 (TLR7/8) or CpG. Treatment with PIP2 with -ODN (TLR9), and then the level of secretion of TNF-α was monitored by ELISA. TNF-α production was measured with Mouse TNF alpha ELISA Ready-SET-Go kit (eBioscience, San Diego, CA, USA) or Human TNF alpha ELISA MAX™ Deluxe (BioLegend, San Diego, CA, USA). Similarly, the specific absorbance was read with a microplate spectrophotometer system (Molecular Devices Inc.), and all data were analyzed using SoftMax Pro 5.3 software (Molecular Devices Inc.).

As a result, PIP2 inhibited TLR2/1- and 2/6-, and inhibited TLR7/8- and 9-mediated TNF-α secretion (FIG. 2K ).

That is, because of the structural and sequence similarity of the TLRs external domain (ECD), PIP2 in TLR-1/2/6 may have a binding site of low affinity, indicating that sub-signal transduction may be non-specifically altered. This was partially supported by surface plasmon resonance (SPR) results (FIG. 8).

In conclusion, it can be seen that in RAW264.7 cells stimulated with LPS, PIP2 remarkably inhibits the TLR4 pathway (MyD88-dependent and TRIF-independent pathway).

3-4: check iNOS and COX2 levels

The production of ROS and NO is directly related to the cellular level and other factors of iNOS (Kim, JY et al., Eur. J. Pharmacol. 584:175-184, 2008; Liu, K.-L. et al. al. J. Agric. Food Chem. 54:3472-3478, 2006; West, AP et al. Nature 472:476, 2011; Mancek-Keber, M. et al. Sci. Signal. 8:ra60-ra60, 2015 )), iNOS and COX2 levels were monitored in LPS-stimulated cells pretreated with PPS2.

As a result, PIP2 effectively inhibited the expression of iNOS and COX2 induced by LPS (Fig. 2g).

Measurement of cytoplasmic NO and ROS was performed using DAF-FM and DCF-DA staining.

RAW264.7 cells were inoculated in a 6 cm dish (SPL Life Sciences, Pochun, Korea) at ^{a density of 1 x 10 6} to treat PIP2, and DAF-FM and DCF-DA dyes (Thermo Fisher Scientific, Inc.) were used to inoculate the cells. Each was analyzed. The fluorescence intensity detected in the cells was measured using a FACSAria III instrument equipped with Diva software (BD Biosciences).

In addition, NO secretion was performed using a nitric oxide detection kit (iNtRON Biotechnology, Gyeonggi, Korea).

RAW264.7 cells were ^{seeded at a density of 2 x 10 5} cells/well, cultured in 96-well plates (BD Biosciences) and treated with PIP2. Absorbance was measured at 540 nm using a microplate reader spectrophotometer (Molecular Devices Inc.), and data was analyzed using SoftMax Pro 5.3 software (Molecular Devices Inc.).

As a result, the extracellular secretion of NO as well as the intracellular production of NO and ROS were inhibited by PIP2 (Fig. 2h-j).

Example 4: Inhibition of the TLR4 signaling pathway of PIP2 in human peripheral blood mononuclear cells (hPBMCs)

The immunosuppressive effect of PIP2 was evaluated in hPBMCs through evaluation of cytokine secretion and protein expression through Western blot. Monocytes in the peripheral blood are important for a protective immune response against pathogen invasion. These cells secrete cytokines and chemokines, play an important role in detecting pathogens and danger signals, and are precursors of tissue-infiltrating dendritic cells (DC). DCs are potent antigen presenting cells and are important in coordinating the first immune response to risk (Pierer, M et al., PLoS One 6:e23539, 2011). In addition, high expression of TLRs in hPBMCs is associated with the pathogenesis of diseases such as systemic lupus erythematosus and chronic pain syndrome (Guo, Y. et al. nt. J. Clin. Exp. Med. 8:20472-20480 , 2015; Kwok, YH et al., PLoS One 7:e44232, 2012; Wong, CK et al., Clin. Exp. Immunol. 159:11-22, 2010).

HPBMC used for Western blot was purchased from PromoCell (Heidelberg, Germany), and hPBMC used for evaluation of cytokine secretion was Lonza Inc. (Allendale, NJ, USA). The secretion of TNF-α and IL-6 was analyzed by ELISA in cells treated with various concentrations of PIP2 in the presence of _{LPS, PAM 3} CSK _{4 or FSL-1 (InvivoGen, San Diego, CA, USA).}

As a result, PIP2 significantly inhibited the secretion of LPS-mediated TNF-α, and _{slightly inhibited the secretion in PAM 3} CSK ₄ or FSL-1 stimulated cells (FIG. 3A). In addition, LPS-induced IL-6 secretion was significantly suppressed after simultaneous treatment with PIP2, but _{had little effect in PAM 3} CSK ₄ or FSL-1 stimulating cells (Fig. 3B).

As a result of evaluating the activity of LPS-mediated MAPKs in hPBMCs, PIP2-treated cells significantly reduced the phosphorylation of NF-κB and inhibited the degradation of IκBα (FIG. 3C ). In addition, the expression levels of MAPKs including p-ERK, p-JNK and p-p38 were also significantly suppressed in PIP2 treated cells.

Example 5: Inhibition of TLR4 signaling pathway and LPS-induced systemic cytokine response in mouse bone marrow-derived macrophages (mBMDMs) by PIP2

5-1: Inhibition of TLR4 signaling pathway by PIP2 in mBMDMs

Similar to RAW264.7 cells and hPBMC, the TLR inhibitory effect of PIP2 on mBMDM was further investigated by evaluating the secretion of LPS-induced TNF-α, IL-6 and IFN-β.

As a result, PIP2 significantly inhibited the secretion of pro-inflammatory cytokines and IFN-β in these cells (Figs. 4a-c). Moreover, PIP2 inhibited LPS-induced NO secretion in mBMDMs in a concentration-dependent manner (Fig. 4D).

5-2: Inhibition of LPS-induced systemic cytokine response in vivo

Next, the immunosuppressive effect of PIP2 was evaluated in vivo by measuring the level of systemic cytokines in mice.

Orient Bio, Inc. 8-week-old C57BL/6J mice (20-25g, n = 5) were obtained from (Seoul, Korea). After pretreatment by intraperitoneal administration (i.p.) of PIP2 (70 nmol/g body weight) to mice for 1 hour, LPS (5 μg/g animal weight) was antigen-treated for 2 hours. The same amount of PBS was injected into the control group. Plasma samples were collected and stored frozen for evaluation of proinflammatory cytokine secretion. TNF-α (1:100), IL-12p40 (1:50) and IL-6 (1:100) were analyzed using an ELISA kit (BioLegend, San Diego, CA, USA). All animal experiments were approved by the Institutional Animal Care and Use Committee (approval number: KHNMC AP 2016-006).

TNF-α, IL-12p40 and IL-6 were measured in plasma samples collected before and for 2 hours after stimulation, and as a result, systemic secretion of cytokines induced by LPS was significantly reduced due to PIP2 treatment. It could be seen that (Fig. 4e-g).

Example 6: Structure of PIP2 and TLR4 Inhibition Mechanism

6-1: TLR4 expression and FITC-PIP2 location confirmation by immunofluorescence and confocal microscopy

To confirm the binding of PIP2 and TLR4, FITC (fluorescein isothiocyanate) was conjugated to the N-terminus of the peptide. RAW264.7 cells were pretreated with FITC-PIP2 for 1 hour before LPS stimulation, and the fluorescence intensities of FITC-PIP2 and TLR4 were analyzed using immunofluorescence staining and confocal microscopy. The localization of TLR4 and FITC-PIP2 was monitored with respect to location.

As a result, TLR4 was located on the plasma membrane of LPS-treated cells, and FITC-PIP2 was also located on the plasma membrane, and the signal was almost identical to the fluorescence of TLR4. In the cells treated with both FITC-PIP2 and LPS, FITC-PIP2 and TLR4 completely overlapped the plasma membrane, resulting in a yellow signal (FIG. 5A).

6-2: Confirmation of binding kinetics of PIP2 and TLR4 using SPR

By studying the binding kinetics of PIP2 and TLR4 through surface plasmon resonance (SPR), it is possible to study the biophysical interactions of macromolecules, nucleic acids, and small molecules (Drescher, DG et al., Methods Mol. Biol. 493:323-343, 2009).

SPR was performed using a ProteOn XPR36 instrument (Bio-Rad Laboratories, Inc.) with a ProteOn GLR sensor chip. hTLR4 and hTLR2 (R&D system) were immobilized on the surface of the GLM sensor chip by amine coupling. Various concentrations of PIP2 molecules (0, 12.5, 25, 50, 100 and 200 μM) were injected into the chip to analyze binding to the immobilized protein. Data was analyzed using ProteOnTM Manager software (version 2.0), and the data on the protein surface were grouped according to the kinetic rate constants (ka and kd). The binding constant KD was calculated by Equation 1 below.

Recombinant hTLR4 protein was immobilized on a ProteOn GLM sensor chip (140 μM ligand) and PIP2 (analyte) was added to the sensor chip at various concentrations between 12.5 and 200 μM. _a ) and dissociation (k _d ) constants were 3.45 × 10 ⁴ M ^-1 S ^-1 and 1.13 × 10 ^-2 S ^-1 , respectively, and the sensorgram indicated that the binding of PIP2 to TLR4 increased in a concentration-dependent manner ( Fig. 5b).

In addition, the same conditions were applied to the PIP2 and TLR2 binding kinetics, but showed a concentration-independent interaction pattern between PIP2 and TLR2 (FIG. 8).

PIP2 was used as an analyte at concentrations of 12.5, 25 and 50 μM, which corresponds to a narrow range of reaction units (RU) of 8-9, which is further supported by overlapping RU values when 100 and 200 μM of PIP2 were used. Can be. These and insignificant in vitro results can partially explain that the interaction of PIP2 and TLR2 is non-specific.

6-3: protein modeling

The potential interaction between PIP2 and TLR4, identified through cell imaging and SPR analysis, was further investigated through in silico analysis. The PIP2 oligopeptide was modeled through a computer procedure used to predict the binding structure of TLR4, which was confirmed to be regulated in an in vitro system treated with PIP2.

A 3D model of the PIP2 oligopeptide was created through MOE (2016.08) and I-TASSER online server (Zhang, Y et al., BMC Bioinformatics 9:40, 2008). The structure was selected and verified with the same fold and TM score> 0.5 in I-TASSER (Bowie, JU et al., Science 253:164-170, 1991). Overall geometric and stereochemical properties of the final 3D model peptide were investigated through Ramachandran plots generated by MOE and PROCHECK68 (Laskowski, RA et al., J. Biomol. NMR 8:477-486, 1996).

As a result, in the secondary structure predicted by I-TASSER34 and PSIPRED, the N-terminus and C-terminus of the peptide were wound, whereas the central part was mainly spiral (Table 1). Table 1 shows the secondary structure prediction and homology modeling parameters of PIP2. In addition, the 3D structure proposed by I-TASSER34 and MOE did not show any strands and mainly showed a helical topology (Fig. 5c).

6-4: Molecular dynamics and structure stabilization of peptides

To standardize and stabilize the structure of the predicted PIP2, an established molecular dynamics simulation (MDS) system was utilized.

The best model of PIP2 selected from I-TASSER is cPIP2, a circulating form, MD simulation was performed on the AMBER99SB-ILDNP force field using GROMACS v5.6.2 (Lindorff-Larsen, K. et al., Proteins 78:1950 -1958, 2010). The system is a known method (Jorgensen, WL et al., The Journal of chemical physics 79:926-935, 1983; Bussi, G et al., J. Chem. Phys. 126:014101, 2007; Parrinello, M et al. al., J. Appl. Phys. 52:7182-7190, 1981; Hess, B et al., J. Comput. Chem. 18:1463-1472, 1997). Equilibrium structures were simulated in NPT conditions without positional restrictions for 1 μs (PIP2) and 150 ns (cPIP2), respectively (Parrinello, M et al., J. Appl. Phys. 52:7182-7190, 1981).

As a result, it was confirmed that the N-terminus of PIP2 maintained a helical structure and the C-terminus was transformed into a cyclic structure. The helical topology is maintained by the electrostatic interaction between the polar side chain of Ser6 and the backbone oxygen of Val3 (Figure 5c).

In addition, stapling small peptides to increase half-life and maintain secondary structure has been widely used (Dietrich, L. et al., Cell Chem Biol 24:958-968, 2017; Verdine, GL et al., Methods. Enzymol.503 :3-33, 2012; White, AM et al., Expert Opin Drug Discov 11:1151-1163, 2016). Taking into account the flexible C-terminus of PIP2, and assuming that this could reduce the TLR4 inhibitory efficacy, peptide stapling was performed to improve the stability of PIP2.

As a result, polar Ser6 was replaced by Asp, and a lactam bridge was established between Asp6 and Lys10 (Chemical Formula 1).

In order to adjust the stapling effect to the overall structure of cPIP2, the circulation form of PIP2; The MD trajectory was visualized and compared with the trajectory of PIP2.

As a result, it was found that cPIP2 maintains the helical shape, but PIP2 loses the C-terminal helix. This suggests that due to the stable properties of cPIP2, TLR4 signaling can be suppressed for a long time at low concentrations compared to PIP2.

Example 7: Docking simulation and interfacial analysis of PIP2-binding TLR4 complex

The in vitro results of Examples 3 and 4 suggest that PIP2 significantly inhibited LPS-induced TLR4 activation. Therefore, the possible binding patterns and TLR4 antagonism of PIP2 were investigated.

PIP2 selected through phage ELISA using recombinant ECD of human TLR4 was docked in the following three different states of TLR4 to determine specific binding sites. That is, in order to evaluate the binding affinity of the human TLR4/MD2 complex (3FXI) and PIP2, the related X-ray crystal structure was retrieved from the PDB. To assess binding affinity and to suggest a mechanism of TLR4 antagonism of PIP2, the moiety that PIP2 can bind to TLR4 is i) the extracellular domain of monomeric TLR4; ii) the extracellular domain of monomeric TLR4 bound to MD2; iii) It can be seen as a dimeric extracellular domain of TLR4 in which one monomer is attached to MD2 and the other monomer is empty.

An online protein-protein docking server ZDOCK69 and a standalone MOE suite were used to predict and evaluate the interaction mechanism between PIP2 and TLR4 (Shah, M et al., Sci. Rep. 5:13446, 2015). TLR was considered a receptor and PIP2 was considered a ligand in each round of docking. ZDOCK and MOE were used separately to create 500 docking constructs for each TLR and PIP2.

The hydrophobic nature of PIP2 suggests a high potential for nonpolar contact with interacting proteins, but excess lysine can form salt bridges with residues exposed to negatively charged solvents. When docked with TLR4 alone in monomeric form, PIP2 exhibits inconsistent binding with ECD. Of the four docked clusters that PIP2 formed with the TLR4 ECD, one cluster at the N-terminus was slightly constant and non-dispersive. About 43% of the docked PIP2 is clustered at a specific location in TLR4, and the second cluster, which is very important in relation to MD2-dependent TLR4 activation, accounted for ~9% of the docked PIP2.

Therefore, the interaction database of the top 150 dockings of PIP2-TLR4 was constructed and expressed through protein ligand interaction fingerprints (PLIF) (FIG. 9). Most of the moieties that interact are non-polar or hydrophobic. In addition, the main clustering of PIP2 at the N-terminus of the TLR4 ECD was confirmed by PLIF analysis.

Next, to confirm the preferred binding affinity of PIP2 between the TLR4 and TLR4/MD2 heterodimers, the TLR4/MD2 dimeric and trimeric (one of the bound MD2s was removed and the second remained bound to the TLR4/MD2 tetramer). ) The complex and PIP2 were docked. Docked PIP2 almost all had the hydrophobic cavity of MD2 and showed no non-specific clustering with TLR4 ECD. In addition, PLIF analysis was performed on the top 150 PIP2-MD2 (TLR4) docking complexes to investigate key residues that consistently interact.

As a result, it was confirmed that the gating loop (Ser120-Lys128) of MD2, known to be involved in the activation of TLR4 (Kim, HM et al., Cell 130:906-917, 2007), is particularly involved in the PIP2 interaction. Ile52 and Phe121 of the gating loop interacted with more than 80% of the docked PIP2 (Fig. 9b), and the main residues of PIP2 involved in this interaction were generally similar to those observed in the PIP2-TLR4 docking.

From the above preliminary docking results, it was found that PIP2 inhibited TLR4 signaling by competitively interfering with MD2 binding with MD2 cavity or TLR4 (cluster 2). The binding of PIP2 in cluster 1 is the most reliable way to block TLR4 signaling, but inhibition of the N-terminus of TLR4 does not appear to inhibit its subsignals. Since these two combinations can be seen to inhibit TLR4 signaling, further investigations are needed for structural kinetics using MDS.

Accordingly, after initial evaluation of PIP2 as a potent TLR4 antagonist, peptide cyclization was performed to stabilize the proposed helical structure and improve inhibitory efficacy. The protein design algorithm performed in MOE was used to improve the TLR4 binding affinity by examining residues that can be mutated or modified to improve the stability of the peptide.

As a result, a Ser6/Asp mutation was selected among the proposed modifications, and a lactam bridge was created between NH3 + of Lys10 and COO- of the Asp6 side chain, and was named cPIP2 (SEQ ID NO: 2: LGVFSDWWIKLV). cPIP2 was further investigated for its structural stability through protein kinetics and in vitro efficacy analysis.

Example 8: Structural kinetics of PIP2 binding TLR4

8-1: Stability of TLR4-PIP2

Two PIP2 docking complexes were simulated for 100 ns because PIP2 has the potential to interfere with TLR4 signaling by binding to the cavity of MD2 or by competingly interfering with TLR4 and MD2 interactions. First, the stability of TLR4-PIP2 (PIP2 binds to the center of TLR4) was investigated.

The structural flexibility and limited movement provided by MDS show that the backbone oxygen of the C-terminal Val and Leu of PIP2 form strong hydrogen bonds with the positively charged Arg208, 238 and polar Ser291 of TLR4. This was further confirmed by tracking the minimum distance between these residues along the MD trajectory (FIG. 6A ). In addition, a similar site of TLR4 was found to be crucial for MD2 binding, and was indicated as Patch A and Patch B (Kim, HM et al. Cell 130:906-917, 2007).

Analysis of the PIP2-MD2(TLR4) MD trajectory shows a sustained rise in RMSD (FIG. 6A). This is a result of the translational motion of MD2 that affects the relative stability of PIP2 rather than the conformational change induced by PIP2 in the TLR4/MD2 complex.

8-2: binding affinity of PIP2 and TLR4

Next, in order to confirm the binding affinity of PIP2 in patches A and B of TLR4, a single complex including two TLR4 molecules, one MD2 and two PIP2 molecules was assembled and analyzed by MDS.

As a result, one of the PIP2 peptides bound to MD2 and the other did not bind to MD2 near patches A and B of adjacent TLR4 (Fig. 6B). Since the external translational movement of MD2 was not observed, it can be seen that the second TLR4 has a significant effect on the stability of MD2 binding PIP2. However, we did not observe the transition of Phe126 from an active state to an inactive state. Phe126, located in the gating loop of MD2, is known to be crucial for ligand-dependent TLR4 signaling (Shah, M et al., Sci. Rep. 6:39271, 2016).

In addition, in order to evaluate the binding affinity of PIP2 and TLR4 to the MD2-binding patch, the PIP2-TLR4 interface in the PIP2-TLR4 (TLR4/MD2) complex was analyzed over time.

As a result, in patch A of TLR4, Trp8 of PIP2 bonded to Val108 and C-H-π (arene), which was further strengthened by electrostatic contact between Asp155 and nitrogen atom in the indole group of Trp8. Trajectory analysis confirmed that these interactions were quite stable and maintained throughout the MD run.

In addition, Lys10 established salt bridges with Asp155 and Asp183 after 25 ns of MD, and it was found that the C-terminal residue of PIP2 not in contact with TLR4 had a strong hydrogen bond after 20 ns (FIG. 6A).

This is a similar trend with the PIP2-TLR4 system, this consistency was crucial for the stability of the PIP2-TLR4 interaction in both the dimer and tetrameric forms of the C-terminal residue, especially the carboxyl group of Val12 and the backbone oxygen of Leu11 [PIP2- TLR4, PIP2-TLR4 (TLR4/MD2)] (Fig. 6B).

Example 9: Inhibition of TLR-induced inflammatory response of Cyclic PIP2

9-1: The ability of cPIP2 to inhibit TLR4 in macrophages

The TLR4 inhibitory potential of cPIP2 was confirmed in the same macrophages used in the PIP2 assay of Example 3.

As a result of examining cytotoxicity to RAW264.7 cells at concentrations of 5, 25, and 50 μM of cPIP2, it was confirmed that there was no toxicity (Fig. 7a).

Subsequently, the TLR4 inhibitory effect of cPIP2 was evaluated by ELISA by monitoring the level of inflammatory cytokines secreted from RAW264.7 cells.

As a result, it was confirmed that the secretion of TNF-α and IL-6 was significantly inhibited in a concentration-dependent manner. (Figs. 7b and c). In addition, a decrease in type 1 IFNs (IFN-α and IFN-β) was observed in cells stimulated with LPS and treated with cPIP2- (Figs. 7d and e).

Next, the inhibitory effect of cPIP2 on oxidative stress markers (ie, NO and ROS) was investigated. As a result, it was found that the extracellular secretion of NO and the intracellular production of NO and ROS were inhibited by cPIP2, which is consistent with the results of PIP2 (Fig. 7f-h).

9-2: The ability of cPIP2 to inhibit TLRs in THP-1 cells

The effect of cPIP2 on TLR4 and other TLRs was investigated through the evaluation of IL-6 secretion in THP-1 cells.

THP-1 (Ajou University Medical Center) cells were differentiated into macrophages by treatment with 80 nM of PMA (phorbol 12-myristate 13-acetate; Sigma-Aldrich Co., St. Louis, MO, USA). Thereafter, cells were treated with different TLR ligands (LPS (TLR4), PAM3CSK4 (TLR1/2), FSL-1 (TLR2/6), resiquimod / R848 (TLR7 / 8) or CpG-ODN (TLR9)].

As a result, it was found that cPIP2 inhibits TLR4-mediated IL-6 secretion, and it was confirmed that it slightly inhibited other TLRs at 50 μM (Fig. 7i).

In addition, the activation of MAPK induced by LPS was evaluated through Western blot analysis in THP-1 cells. As a result, compared to the expression level after LPS treatment, cPIP2 successfully inhibited the activation of NF-κB (phosphorylation of p65 and degradation of IκBα) and inhibited the phosphorylation level of MAPK including ERK, JNK and p38. cPIP2 also inhibited LPS-induced activation of ATF3 and phosphorylation of IRF3 (Fig. 7j).

In conclusion, cPIP2 not only retained the inhibitory properties of PIP2, but also reduced the inhibitory concentration (IC50) from 40 μM to 25 μM. That is, it was confirmed that the efficiency of the peptide was improved.

Example 10: Effect of Cyclic PIP2 on the treatment of rheumatoid arthritis in vivo

After confirming the inhibitory activity of cPIP2 on the LPS-induced inflammatory response in vitro , the effect of cPIP2 in vivo was further investigated using a rheumatoid arthritis (RA) rat model.

10-1: Preparation of RA rat model

RA rat models were prepared as follows.

First, a 1:1 solution of bovine type 2 collagen (bovine collagen type II, Sigma-Aldrich Co. LLC) and complete Freund's adjuvant (Sigma-Aldrich Co. LLC.) was mixed for 30 minutes, and the above was mixed. 250 µl of the mixed solution (including 0.5 mg of bovine type 2 collagen) was injected into the tail of a 4-week-old male Lewis rat. After 3 weeks, evidence of swelling and erythema was observed in the ankle muscles, ankle joints, knee joints and feet of the Lewis rats, respectively.

10-2: Confirmation of the therapeutic effect of cPIP2 in RA rat model

The RA treatment experiment was conducted with the approval of the Animal Testing Committee of Ajou University School of Medicine (approval number 2013-0070).

The experiment was conducted in three experimental groups: normal group, untreated group, and cPIP2 (1 nmol/g body weight) treatment group.

In the case of the cPIP2 treatment group, 100 μl of cPIP2 (suspended in PBS at a final concentration of 1154.4 μg/mL) was individually injected into the knee joints of RA rat models. RA rat models were sacrificed at week 1, 3 or 6 (n=3), respectively, to evaluate the treatment effect.

First, the effect of cPIP2 on RA rats was evaluated through visual monitoring of the foot, measurement of ankle diameter, and articular index (AI) measurement for 6 weeks.

Specifically, the ankle diameter and AI score were measured at weeks 1, 2, 3, 4, 5 and 6 by observing and measuring the hind paws of the three experimental groups. The ankle diameter of 3 rats was measured using a Vernier caliper. AI was blind tested and scored as follows: no edema or erythema (score 0), mild edema and erythema (+1), moderate edema and shackles (+2), edema and metatarsals due to limited use of the joint. Signs of enlargement in (+3), and excessive swelling as severe symptoms associated with joint stiffness and total hind paws (+4).

All ankle diameters and AI scores were independently measured three times, and the results were expressed as mean ± standard deviation (SD).

As a result, as shown in Fig. 10a, in the case of the untreated group, swollen joints, stiff plantar, severe swelling and erythema were observed for 6 weeks, but in the case of cPIP2-treated group, at 3 weeks It was confirmed that swelling and swelling of the ankle were significantly reduced, and recovered to a level similar to that of the normal group at week 6 (see FIG. 10A).

In addition, as a result of monitoring the ankle diameter and AI score of RA rats during the treatment period for 6 weeks, as shown in FIGS. 10B and 10C, in the case of the untreated group, the ankle diameter and average AI value of RA rats were significantly higher. , In the case of the cPIP2 treatment group, the ankle diameter and AI values were significantly reduced (see FIGS. 10B and 10C).

In addition, hematoxylin and eosin (H&E) staining, safranin-O (SO) staining, and TNF-α staining were used to evaluate the degree of inflammation and cartilage degradation during RA onset.

Specifically, the experimental group was sacrificed at week 1, 3 or 6 and the knee joint was dissected (n=3 for each week). The removed joint was fixed with 10% formalin (Sigma-Aldrich, St. Louis, MO, USA) for 3 days, and calcined for 2 days using 6% nitric acid (Sigma-Aldrich). The fixed tissue was dehydrated with 100% ethanol and impregnated with paraffin. The impregnated specimen was cut into 8 μm slices. The cut samples were stained with hematoxylin and eosin (H&E), safranin-O (SO) and TNF-α. For SO staining, paraffin was removed from the specimen and then hydrated. Then, the slides were washed 3 times *?**?* and treated with Mayer's hematoxylin solution (Muto Pure Chemicals, Tokyo, Japan) for 5 minutes, and then the slides were washed again for 20 minutes. . The stained tissue was colored with a 0.002% fast green solution (Sigma-Aldrich) for 30 seconds and washed with 1% glacial acetic acid. The slide was placed in a 0.1% SO solution (Sigma-Aldrich) for 6 minutes and fixed with a mounting medium (Muto Pure Chemicals).

^{For TNF-α staining, the slide was 120 in a 10 × 10 -3} M sodium citrate buffer solution consisting of citric anhydride (Daejung Chemicals, Shiheung, Korea) and trisodium citrate anhydrous (Yakuri Pure Chemicals, Kyoto, Japan). To 130° C. for 10 minutes, and washed with PBS. After blocking the slide with PBS containing 10% BSA at 37° C. for 60 minutes, 4 with a primary antibody (rabbit anti-rat TNF-a (1:100); Novus Biologicals, Littleton, CO, USA) It was left overnight at °C. Thereafter, the sample was treated with a secondary antibody (goat anti-rabbit Alexa Fluor® 594; Invitrogen, Carlsbad, CA, USA) at a ratio of 1:200 for 2 hours. The slides were later washed with ProLong Gold Antifade Reagent (Thermo Fisher Scientific Inc.) and mounted. Immunofluorescence images were obtained using Axio Imager A1 (Carl Zeiss Microimaging GmbH, Gottingen, German) and analyzed with Axiovision software (Rel. 4.8; Carl Zeiss Microimaging GmbH). The TNF-α positive area was calculated as a percentage of the total cartilage area using ImageJ software (National Institute of Health, Bethesda, MD, USA) in three fields randomly selected for each sample.

As a result of H&E staining, as shown in FIG. 10D, in the case of the untreated group, no significant histological changes were observed in all the parking lots. On the other hand, in the cPIP2 treatment group, there was a remarkable increase in cartilage thickness after 1 week, and it was confirmed that numerous osteocytes were present in the lacunae after 4 and 6 weeks (see FIG. 10D).

As a result of SO staining, as shown in FIG. 10E, a clear SO positive reaction was observed in the synovial tissue portion of the cPIP2 treated group compared to the untreated group. The round-shaped mature chondrocytes were surrounded by the deposition of glycosaminoglycans (GAGs; dark red), and it was confirmed that GAGs were remarkably regenerated at the boundary between cartilage and bone at the 3rd week (see FIG. 10e). The stained area increased further at 6 weeks. The above results show that treatment of cPIP2 in RA rats successfully promotes regeneration of cartilage tissue in vivo and relieves the inflammatory state commonly observed in rheumatoid arthritis.

In addition, as a result of measuring the expression level of TNF-α in the synovial tissue obtained from the three experimental groups, as shown in Fig. 10f, TNF-α was not expressed in the synovial tissue of the normal group, and TNF- in the tissue of RA rats. The expression level of α was measured to be high. On the other hand, in the cPIP2 treatment group, the expression level of TNF-α was significantly decreased during the first week of treatment (see FIG. 10F).

As described above, specific parts of the present invention have been described in detail, and it will be apparent to those of ordinary skill in the art that these specific techniques are only preferred embodiments, and the scope of the present invention is not limited thereby. will be. Therefore, it will be said that the practical scope of the present invention is defined by the appended claims and their equivalents.

<110> GENESEN CO., LTD. <120> TLR4 antagonizing peptide <130> FPD/201809-0023/C <150> KR 10-2018-0049101 <151> 2018-04-27 <160> 5 <170> KoPatentIn 3.0 <210> 1 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> PIP2 <400> 1 Leu Gly Val Phe Ser Ser Trp Trp Ile Lys Leu Val 1 5 10 <210> 2 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> cPIP2 <400> 2 Leu Gly Val Phe Ser Asp Trp Trp Ile Lys Leu Val 1 5 10 <210> 3 <211> 76 <212> DNA <213> Artificial Sequence <220> <223> F-primer <400> 3 gcccagccgg ccatggccnn knnknnknnk nnknnknnkn nknnknnknn knnktcgagt 60 ggtggaggcg gttcag 76 <210> 4 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> R-primer <400> 4 gccagcattg acaggaggtt gag 23 <210> 5 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> S-primer <400> 5 ttgtgagcgg ataacaattt c 21

Claims (10)

A peptide represented by the amino acid sequence of SEQ ID NO: 1 or 2.
The peptide according to claim 1, wherein the peptide of SEQ ID NO: 2 is characterized in that Asp6 and Lys10 form a lactam bridge.
The peptide according to claim 1, wherein the peptide inhibits the TLR4 (Toll-like receptor 4) signaling pathway.
The peptide according to claim 3, wherein the TLR4 signaling pathway is induced by LPS (lipopolysaccharide).
The peptide according to claim 1, wherein the peptide binds to the TLR4/MD2 complex.
delete As a pharmaceutical composition for the prevention or treatment of autoimmune diseases containing a peptide represented by the amino acid sequence of SEQ ID NO: 1 or 2 as an active ingredient, the autoimmune disease is insulin-dependent diabetes, multiple sclerosis, autoimmune encephalomyelitis, rheumatoid arthritis , Autoimmune arthritis, myasthenia gravis, thyroiditis, uveitis, Hashimoto's thyroiditis, primary myxedema, thyroid poisoning, pernicious anemia, autoimmune atrophy gastritis, Addison's disease, early menopause, male infertility, childhood diabetes, Goodpasture's syndrome, pemphigus, breast As pemphigus, sympathetic blepharitis, lens uveitis, autoimmune hemolytic anemia, idiopathic leukocytosis, primary cholangiosclerosis, latent cirrhosis, ulcerative colitis, Sjogren's syndrome, scleroderma, Wegener's granulomatosis, polymyositis/dermatomyositis and systemic lupus erythematosus A pharmaceutical composition for the prevention or treatment of autoimmune diseases, characterized in that at least one selected from the group consisting of.
delete As a pharmaceutical composition for preventing or treating inflammatory diseases containing a peptide represented by the amino acid sequence of SEQ ID NO: 1 or 2 as an active ingredient, the inflammatory diseases include asthma, eczema, psoriasis, allergies, rheumatoid arthritis, psoriatic arthritis, atopic Dermatitis, acne, atopic rhinitis, pulmonary inflammation, allergic dermatitis, chronic sinusitis, contact dermatitis, seborrheic dermatitis, gastritis, gout, gout arthritis, ulcers, chronic bronchitis, Crohn's disease, ulcerative Colitis, ankylosing spondylitis, sepsis, vasculitis, bursitis, lupus, rheumatoid multiple myalgia, temporal arteritis, multiple sclerosis, solid cancer, Alzheimer's disease, arteriosclerosis, obesity, and viral infection. Pharmaceutical composition for the prevention or treatment of inflammatory diseases. delete

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