KR20190125197A - TLR4 antagonizing peptide - Google Patents

Abstract

The present invention relates to a peptide inhibiting the TLR4 signaling pathway, a TLR4 antagonist comprising the peptide, and a pharmaceutical composition, comprising the peptide, for treatment of autoimmune disease or inflammatory disease. The peptide according to the present invention exhibits an excellent effect of suppressing the secretion of inflammatory cytokines and the activation of NF-kB and MAPKs by inhibiting the TLR4 signaling pathway and thus is useful for a pharmaceutical composition for prevention or treatment of autoimmune diseases and inflammatory diseases mediated via the TLR4 signaling pathway.

Description

TLR4 antagonizing peptide

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

Toll-like receptors (TLRs) can detect pathogen-associated molecular patterns (PAMPs) or danger-associated molecular patterns (DAMPs), thus monitoring the host. 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; Toll-like receptor 4) is the first identified receptor in the TLR family and has been extensively studied in immunological 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 on the outer membrane of Gram-negative bacteria and consists of a hydrophobic domain known as lipid A, a key region and 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, LPS-binding MD2 interacts with TLR4 by forming aggregates, transferring to CD 14 (cluster of differentiation 14), and binding to myeloid differentiation factor 2 (MD2). To induce dimerization of the complex and initiation of subsequent signal transduction (Park, BS et al., Exp. Mol. Med. 45: e66, 2013). Unlike other TLRs, TLR4 can induce Myd88 (myeloid differentiation primary response gene 88) and TRIF (Toll / interleukin-1 receptor (TIR) -domain-containing adapter-inducing interferon-β) dependent pathways (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 results in the production of proinflammatory cytokines such as IL-1β, IL-6 and TNF-α. TLR4 stimulation induces late phase activation of NF-κB and interferon regulatory transcription factor 3 (IRF3) via TRIF dependent pathways. 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.Imunmun . 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 proinflammatory cytokines that help regulate the immune response to invading pathogens, but abnormal TLR4 signaling leads to inflammatory reactions 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 affects synovial joints, causes chronic inflammation and destroys joint tissue. TLR4 is also 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 new TLR4 blockers that prevent inflammation and restore immune responses to pathogens has led to the development of potent therapeutics for TLR4-related autoimmune diseases such as rheumatoid arthritis, systemic lupus erythematosus, systemic sclerosis, Sjogren's syndrome, myositis and inflammatory bowel disease To make it possible.

TLR4-target therapeutics developed to date include polysaccharides, glycolipids, antibodies, small molecules and oligopeptides, some of which are drugs / adjuvant that can reduce the secretion of proinflammatory 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 antagonizes TLR4 and is currently undergoing phase 3 clinical trials for patients with severe sepsis (NCT02330432). NI-0101 (15C1 from Novimmune) 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), phase 1 clinical trials have been completed, which is excellent in safety and tolerability (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 a protein / peptide library is expressed on the surface of M13 phage as a fusion product with one of the phage coat proteins, and based on the binding affinity of the protein / peptide to the desired target in this random surface peptide population Peptides are selected (Azzazy, HM et al., Clin. Biochem. 35: 425-445, 2002).

Accordingly, the present inventors have made efforts to identify novel peptides capable of inhibiting the TLR4 pathway and to improve structural stability and efficacy by using the above method. As a result, phage display-derived inhibitory peptide 2 (PIP2) was in vitro LPS- It is possible to block inducible TLR4 signaling and 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. The present invention was completed by confirming that cPIP2 alleviates inflammatory symptoms in a rheumatoid arthritis (RA) rat model.

It is an object of the present invention 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.

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 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 present invention also provides 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.

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

1 shows screening of TLR4 binding peptides, (a) assessing the relative concentration of phage that specifically binds to recombinant hTLR4 protein by the yield / dosage ratio of phage collected after each panning round, (b) Cell viability was measured by MTT assay by treating RAW264.7 cells with various concentrations of PIP for 24 hours, and (c) TNF-α secretion was evaluated by ELISA in RAW264.7 cells with different PIP concentrations. will be. All experiments were performed three times independently and the mean ± SEM of the 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) was initially evaluated 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 in whole protein extracts of RAW264.7 cells were measured by Western blot, and (c) is p-ERK, p-JNK and p Expression levels of MAPK including -p38 were measured in PIP2 treated cells, 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, where the red stained portion represents phosphorylated NF-κB (p-p65) and the blue stained Hoechst is nuclear stained ( scale bar = 20 μm). (e) and (f) measured the secretion levels of IL-6 and IFN-β in RAW264.7 cells by ELISA, and (g) measured the expression levels of iNOS and COX2 in RAW264.7 cells by Western blot. (H) is to evaluate NO production using the NO secretion kit, (iii) is to measure the cellular secretion level of NO by FACS using DAF-DA staining, (j) to DCF-DA The production of ROS in the cytoplasm by FACS using staining is measured, and (k) represents a different TLR ligand [(TLR2 / 1 (PAM3CSK4), TLR2 / 6 (FSL-1), TLR9 (CpG-ODN), or TLR7 / 8 (R848)] TNF-α secretion levels were compared in PIP2-treated RAW264.7 cells, all experiments were performed three times independently and the mean ± SEM of the independent experiments was calculated. tailed paired Student t-test (* P <0.05, ** P <0.01).
Figure 3 confirms PIP2 cytokine secretion and inhibition of NF-κB and MAPKs activation in human peripheral blood mononuclear cells (hPBMCs), (a) and (b) are TLR4 ligand LPS, TLR2 / 1 ligand PAM3CSK4 and TLR2 / The secretion levels of TNF-α and IL-6 under the influence of 6 ligand FSL-1 were evaluated in PIP2-treated hPBMCs, and (c) shows NF-κB activation, degradation of IκBα, and expression levels of MAPK in Western blots. As an evaluation, inactive MAPK was used as a control and β-actin was used as a loading control. All experiments were performed three times independently and the mean ± SEM of the independent experiments was calculated. two-tailed paired Student t-test (* P <0.05, ** P <0.01).
Figure 4 confirms that PIP2 inhibits TLR4-mediated cytokine secretion and mitigates TLR4-mediated inflammatory responses in vivo in mouse bone marrow-derived macrophages (mBMDM), and (a) to (c) show TNF-α, IL- The secretion levels of 6 and IFN-β are shown to be decreased by PIP2 treatment, and (d) is the NO secretion kit assessed NO secretion. (e)-(g) shows C57BL / 6J mice intraperitoneally administered with 70 nmol / g PIP2 (ip) followed by intraperitoneal administration of 5 μg / g LPS (ip), resulting in TNF-α, IL-6 and IL in plasma. Monitored the level of -12p40. All experiments were performed three times independently and the mean ± SEM of the independent experiments was calculated. two-tailed paired Student t-test (* P <0.05, ** P <0.01).
FIG. 5 shows images of PIP2-TLR4 complexes and biophysical analysis and the structure of PIP2. (A) shows the expression level of TLR4 (red staining) and the location of FITC-PIP2 (green staining) by immunofluorescence. As analyzed by focus 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 assay using chip-fixed hTLR4 and SPR as ligands to evaluate the TLR4 binding affinity of PIP2 using PIP2. The sensorgram shows that PIP2 is an extracellular domain (ECD; Extra Cellular Domain) of TLR4. Concentration-dependent binding). (c) is Molecular Dynamics Simulation (MDS) for PIP2, where the N-terminus of PIP2 maintains a helical structure at the MDS terminus, while the C-terminal half obtains a loop structure. During MDS, the root-mean-square deviation (RMSD) for the initial conformation of the whole protein (blue) and backbone atoms (purple) is presented using the minimum sum of squares. The spiral structure of PIP2 was stabilized through the formation of a lactam bridge between Asp6 and Lys10 (cPIP2).
6 shows the structural kinetics of TLR4 binding PIP2 and comparative interfacial analysis of TLR4 / MD2 and PIP2-TLR4, wherein (a) shows that the mean square root deviation (RMSD) of the backbone atoms and total PIP2 peptides bound to TLR4 or MD2 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 upper right panel of 'A' shows the time-dependent formation of hydrogen bond interactions between PIP2 and Patch B of monomeric TLR4 (first simulation), the two panels below verify the initial simulation and PIP2 is PIP2-TLR4 ( When docking simulations with TLR4 / MD2) complexes, we propose 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, wherein PIP2 competitively interacts with MD2-binding patches A and B of TLR4 to block LPS-induced TLR4 activation Indicates.
Figure 7 confirms that cyclic PIP2 (cPIP2) inhibits inflammatory cytokine secretion, reduces oxidative stress and interferes with MAPK expression in macrophages, (a) measuring cell viability by MTT assay to determine that cPIP2 is nontoxic. (B) to (e) are measured by ELISA of TNF-α, IL-6, IFN-β and IFN-α. (f) is the measurement of NO secretion using the NO secretion kit, (g) is the measurement of cytoplasmic secretion of NO by FACS with DAF-DA staining, (h) is DCF-DA for ROS production in the cytoplasm Measured by FACS by staining. (i) Treated human monocyte (THP-1) derived macrophages 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 peptides, showing that cPIP2 significantly inhibits TLR4 but insufficient inhibition of other TLRs. (j) evaluated the expression level of MAPKs as well as NF-κB activation and degradation of IκBα by Western blot, using inactive MAPK as a control and β-actin as a loading control. All experiments were performed three times independently and the mean ± SEM of the independent experiments was calculated. two-tailed paired Student t-test (* P <0.05, ** P <0.01).
8 confirms the biophysical interaction of PIP2 with TLR2. Sensorgrams show that PIP2 binds to the extracellular domain (ECD) of TLR2 in a concentration dependent manner.
FIG. 9 is for the analysis of protein ligand interaction fingerprints (PLIF) of PIP2-binding TLR4 in the presence and absence of MD2, where (a) is PIP2 docked in TLR4 without MD2, and PIP2 is a mutually inconsistent ECD of TLR4 It demonstrates that about 43% of PIP2 peptides hydrophobicly interact with the N-terminus of TLR4. (b) is PIP2 docked with TLR4 / MD2 complex, most of which is controlled by hydrophobicity of MD2, with 80% of docked PIP2 exhibiting hydrophobic interaction with Phe121 of MD2.
10 confirms the reduction in inflammation in the RA rat model of cyclic PIP2 (cPIP2), (a) visually monitoring the status of paws 1, 3 and 6 in normal, control and cPIP2 treated groups (scalebar = 10 mm), (b) and (c) are measured ankle diameters and joint indices, respectively, and (d) to (f) are hematoxylin and eosin (H & E) staining (d) and safranin-, respectively. O (SO) staining (e) and TNF-α staining (f) result (magnification = 200-fold) are shown.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In general, the nomenclature used herein 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 neurodegeneration, autoimmune and inflammatory diseases. For example, LPS mediated activation of TLR4 signaling has been reported to affect the inflammatory process in cerebral ischemia through the activation of microglial cells and the secretion of inflammation-inducing factors (Samson, Y et al., Rev. Neurol. (Paris) 161: 1177-1182, 2005). The expression levels of TLR4 are increased in tubular epithelial cells of an ischemia / reperfusion injury mouse model, whereas TLR4-/-and MyD88-/-mice have increased renal dysfunction, coronary damage and accumulation of proinflammatory cytokines. It has been reported that TLR4 may play an important role in mediating renal injury, protected from secretion (Wu, H. et al., J. Clin. Invest. 117: 2847-2859, 2007). In addition, LPS and high-mobility group protein 1 (HMGB1) induce memory abnormalities in the brain, leading to the activation of microglial cells and the development of inflammatory strokes (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, well known for its antioxidant and anti-inflammatory effects, reduces HMGB1 levels, down-regulates TLR4-mediated inflammatory responses, and destroys the blood-brain barrier in mice. Protect from 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 cancers (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 mitigate 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 to date there are various proteins, small peptides, chemical agents and antibodies. Of these, the 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 (TRIF) -related adapter molecule (TRAM) inhibited LPS-induced secretion of IL-6 in RAW264.7 cells. In addition, Eritoran (E5564), a synthetic lipid A analog, prevents the activation of TLR4 by blocking the binding of LPS and MD2 pockets (Opal, SM et al., AMA 309: 1154-1162, 2013). E5564 advanced to phase 3 clinical trials (NCT00334828), but was discontinued because it failed to improve the patient's severe sepsis status (Opal, SM et al., AMA 309: 1154-1162, 2013).

In the present invention, peptide libraries designed through the PD system were screened for TLR4-binding and specificity to inhibit TLR4. The peptide selected from the PD library was named PIP2 using hTLR4, and PIP2 was a slightly polar hydrophobic dodecapeptide due to two serines and one lysine, resulting in 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 signal transduction. That is, PIP2 significantly inhibited LPS-induced inflammatory responses, disrupting the proinflammatory cytokine and type I IFNs secretion, 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 inhibited (FIG. 4).

Cyclization of peptides is widely practiced to enhance 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, the stability and efficacy of PIP2 were improved by improving the structural information of the TLR4-PIP2 complex in In silico . That is, Asp6 and Lys10 were linked through a lactam bridge to stabilize the helical structure of PIP2 (FIG. 5C), and the amino acid sequence of SEQ ID NO: 2 (LGVFSDWWIKLV) was expressed. cPIP2 not only retained the TLR4 inhibitory effect but also significantly reduced the inhibitory concentration (FIG. 7).

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

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

In the present invention, the peptide may be characterized by binding to the TLR4 / MD2 complex.

As used herein, the term "peptide" refers to a linear molecule formed by binding amino acid residues to each other by a peptide bond. The peptide may be prepared according to chemical synthesis methods known in the art, and preferably, a solid phase synthesis technique. It may be prepared according to, but is not limited thereto.

As used herein, the term "TLR4" refers to a protein encoded by the TLR4 gene, which belongs to TLRs, a family of transmembrane proteins that function as a monitor for pathogen infection, and refers to a protein encoded by the TLR4 gene, referred to as CD284 (cluster of differentiation 284). It may be named. The TLR4 is very important for the activation of the innate immune system because it recognizes a variety of pathogen-associated molecular patterns (PAMPs), including LPS of Gram-negative bacteria.

As used herein, the term "TLR4 signaling pathway" refers to a signaling pathway through TLR4, and may be an LPS response that depends on the TLR4 / MD2 complex, a membrane-crossing complex formed by TLR4 and MD2, through which signal is transmitted. do. TLR4 carries signals by several adapter proteins, and these signaling pathways work with Mal (also referred to as TIRAP) and MyD88, and with trams (TRAM) and trippes (TRIF). TLR4-mediated signal transduction pathways induce activation of MyD88-dependent and MyD88-independent signal transduction through various sub signaling molecules. Initiation of the MyD88-dependent pathway leads to early stage activation of NF-κB and secretion of proinflammatory 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 secretory 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 causes activation of MAPK including ERK, JNK and p38 and secretes inflammatory cytokines and IFNs (Achek, A et al., Arch. Pharmacal Res. 39: 1032-1049, 2016; Kawai, T et al., Nat. Immunol. 11: 373-384, 2010; 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).

As used herein, the term "inhibition" refers to a phenomenon in which biological activity or activity is lowered due to deficiency, incompatibility, and many other causes, and partially or completely block, reduce, prevent, or prevent activation of TLR4. Delaying, inactivating or down-regulating.

As used herein, the term "TLR4 / MD2 complex" refers to a membrane-transmembrane complex formed by TLR4 and MD2, wherein the peptide represented by SEQ ID NO: 1 or 2 of the present invention binds to the TLR4 / MD2 complex, thereby binding the TLR4 signaling pathway. Can be suppressed.

In silico analysis confirmed that PIP2 can block TLR4 signaling by two possible mechanisms. Because of the hydrophobicity, PIP2 preferably binds MD2 in the TLR4 / MD2 complex, since TLR4 requires MD2, a coceptor and LPS binding pocket (Kim, HM et al., Cell 130: 906-917, 2007; Ohnishi, T et al., Clin. Diagn. Lab. Immunol. 10: 405-410, 2003), blocking LPS entry into MD2 may be one of the TLR4-antagonist mechanisms of PIP2 (Park, S. et. al., Biomaterials 126: 49-60, 2017). Since PIP2 was initially selected via PD using the ECD of hTLR4, PIP2 docks with TLR4 in the absence of MD2. PIP2 is at four different positions in the extracellular region of TLR4 (FIG. 9). In addition, MD2 interacts with the 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, the residues of this patch are conserved and are critically important for TLR4-MD2 interaction. Small peptides from MD2 are known to bind to these patches and interfere with TLR4-MD2 interaction (Slivka, PF et al., Chem BioChem 10: 645-649, 2009). In the present invention, it was found that PIP2 was able to bind to these residues and make similar contact, and PIP2 specifically recognized Val108 and Asp155 and stabilized 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 to block TLR4 signal transduction.

Thus, 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.

"Peptide of SEQ ID NO: 1 or 2" of the present invention, as long as it has the ability to effectively bind to the TLR4 / MD2 complex, as well as peptides identical to the sequence of SEQ ID NO: 1 or 2, peptides whose amino acids are substituted by conservative substitution And peptides having at least 70%, preferably at least 80%, and more preferably at least 90% sequence homology therewith. The term “homologous” refers to a wild type amino acid sequence and a wild type nucleic acid sequence. To indicate a similar degree of.

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

As used herein, the term "TLR4 antagonist" refers to a substance that can directly, indirectly, or substantially interfere with, reduce or inhibit the biological activity of TLR4. Preferably, the peptide that is reactive with TLR4 is directed to a TLR4 or TLR4 / MD2 complex. Blocking the TLR4 signaling pathway by directly binding and neutralizing the activity of TLR4 can lead to a decrease in the activation of NF-κB and MAPKs, thereby reducing the secretion of inflammatory cytokines, NO and ROS.

According to one embodiment of the present invention, the peptide represented by SEQ ID NO: 1 or 2 of the present invention by inhibiting the TLR4 signaling pathway induced by lipopolysaccharide (LPS) interleukin-6 (IL-6), NO And excellent in the effect of inhibiting the secretion of ROS and 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 the organism is represented by the size of the protein-protein interaction (PPI) interaction (Stumpf, MP et al., Proc. Natl. Acad. Sci. USA 105: 6959-6964, 2008). Approximately half of the drug 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 crucial for finding powerful regulators of deregulate PPI. In addition, inhibiting the PPI interface with small molecules is a difficult problem.

Thus, peptide-based therapeutics can overcome the effects of protein on PPI treatment due to protein properties and similar binding mode. The development of peptides or peptide-like drugs is a key way to expand the 'druggable genome', particularly with the aim of PPI interfaces that are less suitable for small molecule-based therapies. Peptide drugs have lower toxicity and improved target selectivity than small molecules. Despite the promising effects as therapeutics, the development of peptide-based drugs has low stability against host proteolytic enzymes, resulting in low bioactivity and low bioavailability.

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

Thus, the present invention implements a protein design technique for stabilizing the helical structure of PIP2 by connecting Asp6 and Lys10 through a lactam bridge (FIG. 5c and Formula 1). Structural kinetics confirmed that lactam linkages stabilize the helical structure in cPIP2 and limit the free moment at the N- and C-terminus as compared to PIP2, a linear structure. In addition, the backbone and total protein RMSD of cPIP2 were significantly stabilized compared to PIP2 (FIG. 5C). In vitro confirmation of the TLR4 inhibitory potency of cPIP2 found that cPIP2 not only retained the TLR4 inhibitory effect but also significantly reduced inhibitory concentrations (PIP2 IC50 = 40 μM, cPIP2 IC50 = 25 μM) (FIG. 7).

Therefore, in another aspect, the present invention relates to 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.

In the present invention, the term "autoimmune disease" is caused by a process in which a problem occurs in inducing or maintaining self-tolerance to cause an immune response to an autoantigen, thereby attacking its own tissue. The term "self-tolerance" refers to immunologic unresponsiveness, which does not deleteriously react with antigens constituting self. The autoimmune diseases of the present invention are insulin-dependent diabetes mellitus, multiple sclerosis, and experimental results. 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 disease, early menopause, male infertility, Pediatric Diabetes, Goodpasture Syndrome, Common Cheonpoeum, Yucheonpoe, Sympathetic It includes, but is not limited to, ophthalmitis, crystalline uveitis, autoimmune hemolytic anemia, idiopathic leukocyte reduction, primary cholangiovascular sclerosis, latent cirrhosis, ulcerative colitis, Sjogren's syndrome, scleroderma, Wegener's granulomatosis, polymyositis / dermatitis, and systemic lupus erythematosus It is not limited to this.

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

The term "pharmaceutically acceptable" refers to a composition which, when administered physiologically and is physiologically acceptable and does not normally cause an allergic reaction such as gastrointestinal disorders, dizziness, or the like, examples of the carrier, excipient and diluent include: Lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, polyvinylpyrrolidone, water, methyl hydride And oxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. Further, fillers, anti-coagulants, lubricants, wetting agents, fragrances, emulsifiers and preservatives may be further included.

Accordingly, the present invention confirmed the protective effect of cPIP2 in rheumatoid arthritis (RA), which is a type of autoimmune disease. Rheumatoid arthritis is a multifactorial autoimmune disease characterized primarily by erosive inflammation of articular cartilage, leading to the expansion of synovial fluid such as tumors and the gradual destruction of adjacent articular cartilage. Although the exact cause of rheumatoid arthritis is not yet known, immunological dysregulations observed during rheumatoid arthritis are known to be involved in promoting inflammation and synovial cell proliferation (Isaacs, JD Nature reviews. Immunology , 605-611, 2010 and Smolen, JS et al. Nature reviews. Disease primers , 18001, 2018).

In the present invention, the effect of cPIP2 on rheumatoid arthritis in the RA rat model was confirmed, and cPIP2 was found to significantly reduce inflammation and promote the regeneration of cartilage tissue (FIG. 10). In conclusion, PIP2, particularly cPIP2, is a promising inhibitor of TLR4 signal transduction and may be usefully used in the treatment of autoimmune 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 with the peptide.

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

The pharmaceutical composition of the present invention may be administered through various routes including oral, transdermal, subcutaneous, intravenous or intramuscular, and the dosage of the active ingredient is determined by various factors such as the route of administration, the age, sex, weight and severity of the patient The pharmaceutical composition for the prevention or treatment of autoimmune diseases according to the present invention may be administered in parallel with known compounds having the effect of preventing, ameliorating or treating the symptoms of autoimmune diseases.

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

As used herein, the term "inflammatory disease" refers to TNF-α, IL-1, IL-6, and prostaglandin, which are secreted from immune cells such as macrophages by excessively promoting the immune system due to harmful stimuli such as inflammation-inducing factors or irradiation. (prostaglandin), means a disease caused by inflammatory agents (inflammatory cytokines), such as leukotriene or NO, the inflammatory diseases of the present invention are asthma, eczema, psoriasis, allergic, rheumatoid arthritis, Psoriatic arthritis, contact dermatitis, atopic dermatitis, acne, atopic rhinitis, allergic dermatitis, chronic sinusitis, seborrheic dermatitis, gastritis, gout, gouty arthritis, ulcers, chronic bronchitis , Pneumonia, Crohn's disease, ulcerative colitis, ankylosing spondylitis, sepsis, vasculitis, bursitis, lupus, rheumatic polymyalgia, temporal arteritis, St. sclerosis, solid tumors, Alzheimer's disease, atherosclerosis, obesity, and one containing the virus, but is not limited thereto.

Since the pharmaceutical composition for preventing or treating the inflammatory disease includes the pharmaceutical preparation including the above-mentioned peptide as an active ingredient, the description overlapping with the above-described composition of the present invention will be omitted.

EXAMPLE

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention, and it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as being limited by these examples.

Example 1 Construction and Screening Method of TLR4 Binding Peptide Library

1-1: PD Peptide Library

PD (phage display) peptide libraries were constructed and screened by the methods previously described (Park, S. et al. Biomaterials 126: 49-60, 2017).

12mer peptide library consists of 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- 3 '(SEQ ID NO: 4). In the case of NNK random codons (N = A / C / G / T, K = G / T), all amino acids were selected to minimize stop codons in an encoding state. The DNA product was cloned into pHEN2 phagemid and transfected into DH10B ( E. coli ) cells, and then phage libraries were transformed into XL1-Blue ( E. coli ) cells and amplified. Hyperphage, M13K07ΔpIII (PROGEN Biotechnik GmbH, Heidelberg, Germany) was added to the culture (final concentration, 1 × 10 ¹² PFU / mL) and incubated at 37 ° C. for 30 minutes. Cell pellets are then centrifuged (3300 × g, 10 min, 4 ° C.) and resuspended in 30 mL of fresh 2 × YT medium (100 μg / ml ampicillin and 25 μg / mL kanamycin) and incubated at 30 ° C. overnight. Phage particles were recovered.

1-2: Bio Panning

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) and incubated overnight at 4 ° C. and the next day blocked with 5% skim milk at room temperature Was exposed to phage library. Bound phages were eluted and recovered for phage titration, and phage titers were calculated as colony forming units (cfu) on plates containing ampicillin (10 μg / mL). Thereafter, phages were amplified and purified and used for panning. The concentration efficiency was estimated by calculating the input / output ratio in each round (6 rounds in total). Selection and isolation of the transformed clones was performed after 6th biopanning via phage ELISA (Park, S. et al. Biomaterials 126: 49-60, 2017), as previously described.

1-3: DNA Sequencing and Peptide Synthesis

Phage DNA was isolated from the selected phage by ELISA of Example 1-2 using a Miniprep Kit (GeneAll Biotechnology, Seoul, Korea). The isolated DNA was sequenced using primers of 5′-TTG TGA GCG GAT AAC AAT TTC-3 ′ (SEQ ID NO: 5) (Macrogen, Inc., Seoul, Korea). BioEdit software was used to translate DNA sequences into amino acid sequences and align to assess modifications to assess the degree of diversity (Hall, TA et al., Nucleic Acids Symposium Series, 95-98, 1999). All peptides used were Peptron, Inc. It was synthesized and purchased from (Daejeon, Korea).

Example 2: Screening of PD (phage-display) Peptide Libraries for TLR4

2-1: TLR4 Specific Binding Peptides

Solid phase screening methods were used to identify TLR4 specific binding peptides. As in Example 1 above, a pHEN2 (12-mer) PD library was constructed and exposed to 96-well plates coated with recombinant human TLR4 (hTLR4) protein. Six biopannings were performed to enrich the PD library for hTLR4, and after each round the concentration ratio was assessed via the titer of the eluted phage (FIG. 1A). Phage clones capable of binding hTLR4 were then selected by phage-ELISA and sequenced to estimate their nucleotide variations using BioEdit software.

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

2-2: Cytotoxicity of TLR4 Binding Peptides

Cytotoxicity against RAW264.7 (ATCC, Manassas, VA, USA) cells was determined using MIP (1- (4,5-dimethylthiazol-2-) concentrations using 20-100 μM concentration of PIP1-PIP5 peptide synthesized in Example 2-1. yl) -3,5-diphenylformazan; Sigma-Aldrich Co.).

RAW264.7 cells were seeded at a density of 2 x 10 ⁵ cells / well and treated with different concentrations of peptides for 24 hours to remove the media the next day and 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 dimethylsulfoxide solution (100 μL / well, Sigma-Aldrich Co.) was added and the absorbance at 540 nm was measured spectrophotometrically after 30 minutes (Molecular Devices Inc.).

As a result, the peptide showed no cytotoxicity at concentrations below 80 μM, but the viability of cells treated with 100 μM peptide was slightly reduced (FIG. 1B).

2-3: TNF-α Secretion Inhibition of TLR4 Binding Peptides

According to the result of Example 2-2, this time the TNF-alpha production was further investigated about the density | concentration of 20-80 micrometers. 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.) were read and all data were analyzed using SoftMax Pro 5.3 software (Molecular Devices Inc.).

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

Therefore, TNF-α production was once again confirmed by ELISA in P26 treated RAW264.7 cells in the presence of LPS. In addition, the physicochemical properties of PIP2 were further investigated to assess TLR4 specific trends, and subsequent experiments examined the antagonistic effects of PIP2 on TLR4 signal transduction.

Example 3: Inhibition of TLR4 Signaling Pathway of PIP2 in Macrophages

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

In order to confirm the regulatory effect of PIP2 on the TLR signaling pathway, secreted embryonic alkaline phosphatas (SEAP) analysis was performed. The SEAP reporter gene is under the control of an IL-12p40 minimal promoter containing a nuclear factor-κB (NF-κB) and an activator protein 1 (AP-1) binding site and is induced upon TLR4 activation (Fujioka, S. et al. Mol. Cell. Biol. 24: 7806-7819, 2004). Thus, 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, Calif., USA) were seeded in 96-well plates at a density of 2 × 10 ⁴ cells / well and incubated for 2 days. The supernatant was removed and treated with HEK-Blue detection medium (IvivoGen, San Diego, CA, USA) with different concentrations of PIP2 (20, 40 or 80 μM) for 1 hour, followed by LPS (100 ng / mL) for 24 hours. Treated. Culture supernatants were collected and placed in new 96-well plates and SEAP activity was assessed by measuring absorbance at 630 nm with VersaMax Microplate Reader (Molecular Devices Inc., Sunnyvale, Calif., USA).

As a result, LPS-treated cells induced NF-κB activation, but cells stimulated with LPS for 24 hours after PIP2 pretreatment with PIP2 for 1 hour had a concentration-dependent decrease in SEAP activity (FIG. 2A). On the other hand, cells treated with PIP2 alone did not affect SEAP activity.

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

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

Cell Signaling for 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) Technology Inc., Danvers, MA, USA); p-ERK, p-p38, Iκ-Bα, JNK, ATF3, COX2 (cyclooxygenase 2), primary antibody against β-actin (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA); And primary antibodies against inducible nitric oxide synthase (iNOS) (BD Biosciences) were used at a dilution of 1: 500-1000. The secondary antibody, anti-mouse / -rabbit peroxidase-conjugated immunoglobulin G antibody (Thermo Fisher Scientific Inc.) was used at a ratio of 1: 1,000 and the protein was detected by SuperSignal West Pico ECL solution (Thermo Fisher Scientific Inc.). Visualized with Fusion solo S system (Vilber Lourmat, France).

As a result, PIP2 inhibited LPS-induced NF-κB and IκBα degradation 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).

Next, RAW264.7 cells treated for 1 hour with PIP2 (80 μM) were grown overnight at a concentration of 2 × 10 ⁵ cells / well in 24-well plates, followed by 30 minutes treatment with LPS (100 ng / ml). -p65 was analyzed. For FICT-PIP2 (Peptron, Inc), cells were pretreated with 40 μM of FITC conjugated peptides for 1 hour, followed by LPS treatment (100 ng / mL) for 30 minutes, and cells were 3.7% formaldehyde solution (Sigma-Aldrich Co.). 5 minutes and infiltrated with 0.2% Triton X-100 solution (AMRESCO, Solon, OH, USA) for 5 minutes. Cells were reacted with primary antibodies 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 antibodies ( Invitrogen) and stained by treatment with Hoechst 33258 solution (5 μM, Sigma-Aldrich Co.). Fluorescence intensity was measured using confocal microscopy (LSM-700, Carl Zeiss Microscopy GmbH) and images were analyzed using Zen 2009 software.

As a result, it was confirmed that 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 inoculated in 96-well plates (BD Biosciences) at a density of 2 × 10 ⁵ cells / well to treat PIP2, and IL-6 secretion was determined using Mouse / Human IL-6 ELISA MAX ™ Deluxe (BioLegend) or Measured by Mouse IL-6 Platinum ELISA (eBioscience, San Diego, CA, USA), TNF-α production was determined using 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 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 concentration dependently inhibited by PIP2 (FIGS. 2E-F).

In addition, to determine the effect of PIP2 on other TLRs and their subsignal signaling, cells were cultured with PAM ₃ CSK ₄ (TLR1 / 2), FSL-1 (TLR2 / 6), Resiquimod / R848 (TLR7 / 8) or CpG. Treatment with PIP2 with -ODN (TLR9) followed by monitoring the secretion level of TNF-α by ELISA. TNF-α production was measured with Mouse TNF alpha ELISA Ready-SET-Go kit (eBioscience, San Diego, Calif., USA) or Human TNF alpha ELISA MAX ™ Deluxe (BioLegend, San Diego, Calif., USA). Likewise read at a specific absorbance 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).

In other words, due to the structural and sequence similarity of the TLRs outer domains (ECD), it indicates that PIP2 may have a low affinity binding site in TLR-1 / 2/6 and may alter nonspecific signaling. This was supported in part by surface plasmon resonance (SPR) results (FIG. 8).

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

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 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 iNOS and COX2 expression 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 using DAF-FM and DCF-DA dyes (Thermo Fisher Scientific, Inc.) 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 used for nitric oxide detection kit (iNtRON Biotechnology, Gyeonggi, Korea).

RAW264.7 cells were seeded at a density of 2 × 10 ⁵ cells / well, incubated 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 were analyzed using SoftMax Pro 5.3 software (Molecular Devices Inc.).

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

Example 4: Inhibition of TLR4 Signaling Pathway of PIP2 in Human Peripheral Blood Mononuclear Cells (hPBMCs)

The immunosuppressive effect of PIP2 was assessed in hPBMCs through evaluation of cytokine secretion and protein expression via Western blot. Mononuclear cells in peripheral blood are important for the protective immune response against pathogen invasion. These cells secrete cytokines and chemokines and play an important role in detecting pathogens and danger signals and are precursors of tissue infiltrating dendritic cells (DCs). DC is a potent antigen presenting cell and is 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).

HPBMCs used for Western blot were purchased from PromoCell (Heidelberg, Germany), and hPBMCs used for evaluation of cytokine secretion were obtained from Lonza Inc. (Allendale, NJ, USA). 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, Calif., 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 stimulating cells (FIG. 3A). In addition, LPS-induced secretion of IL-6 was significantly inhibited after co-treatment with PIP2, but had little effect on PAM ₃ CSK ₄ or FSL-1 stimulating cells (FIG. 3B).

Activity of LPS mediated MAPKs evaluated in hPBMCs, PIP2-treated cells significantly reduced phosphorylation of NF-κB and inhibited degradation of IκBα (FIG. 3C). In addition, expression levels of MAPK including p-ERK, p-JNK and p-p38 were also significantly inhibited 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 hPBMCs, 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 proinflammatory cytokines and IFN-β in these cells (FIGS. 4A-C). Moreover, PIP2 inhibited LPS-induced NO secretion in concentration-dependent manner in mBMDMs (FIG. 4D).

5-2: Inhibition of LPS Induced Systemic Cytokine Responses in Vivo

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

Orient Bio, Inc. Eight-week old C57BL / 6J mice (20-25 g, n = 5) were obtained from (Seoul, Korea). Mice were pretreated by intraperitoneal administration (i.p.) of PIP2 (70 nmol / g body weight) for 1 hour, followed by antigen treatment with LPS (5 μg / g animal weight) for 2 hours. The control group was injected with the same amount of PBS. 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 (Approved Number: KHNMC AP 2016-006).

TNF-α, IL-12p40 and IL-6 were measured in plasma samples collected before and after stimulation for 2 hours, resulting in a significant decrease in systemic secretion of LPS-induced cytokines due to PIP2 treatment. It can be seen that (Fig. 4e-g).

Example 6: Structure of PIP2 and Mechanism of TLR4 Inhibition

6-1: TLR4 Expression and FITC-PIP2 Location by Immunofluorescence and Confocal Microscopy

To confirm the binding of PIP2 and TLR4, fluorescein isothiocyanate (FITC) was conjugated to the N terminus of the peptide. RAW264.7 cells were pretreated with FITC-PIP2 for 1 hour before LPS stimulation and fluorescence intensities of FITC-PIP2 and TLR4 were analyzed using immunofluorescence staining and confocal microscopy. 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, FITC-PIP2 was also located on the plasma membrane, and the signal was almost identical to the fluorescence of TLR4. Cells treated with both FITC-PIP2 and LPS showed a yellow signal with FITC-PIP2 and TLR4 perfectly overlapping the plasma membrane (FIG. 5A).

6-2: Confirmation of Binding Kinetics of PIP2 and TLR4 Using SPR

Surface plasmon resonance (SPR) studies the binding kinetics of PIP2 and TLR4 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 by amine coupling to the surface of the GLM sensor chip. 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 ProteOn ™ Manager software (version 2.0), and data on protein surfaces were grouped according to kinematic rate constants (ka and kd). The binding constant KD was calculated by the following equation.

Recombinant hTLR4 protein was immobilized on the 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, and the calculated binding (k) for PIP2-TLR4 interaction. _a ) and dissociation (k _d ) constants are 3.45 × 10 ⁴ M ^-1 S ^-1 and 1.13 × 10 ^-2 S ^-1 , respectively, and the sensorgram shows that the binding of PIP2 to TLR4 increases concentration-dependently ( 5b).

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

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

6-3: Protein Modeling

The potential interaction of PIP2 with TLR4 identified by cell imaging and SPR analysis was further investigated by in silico analysis. PIP2 oligopeptides were modeled through computer procedures used to predict the binding structure with TLR4 found to be regulated in a PIP2-treated in vitro system.

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

As a result, the secondary structure predicted by I-TASSER34 and PSIPRED was wound around the N-terminus and C-terminus of the peptide, while the central part was mainly helical (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 showed no strands and showed a predominantly helical topology (FIG. 5C).

6-4: Molecular Dynamics and Structure Stabilization of Peptides

In order 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 the circulating form cPIP2, which performed MD simulations in the AMBER99SB-ILDNP force field using GROMACS v5.6.2 (Lindorff-Larsen, K. et al., Proteins 78: 1950). -1958, 2010). The system can be obtained from known methods (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., 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 position limitation 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 the PIP2 maintained the helical structure and the C terminus was modified into the cyclic structure. The helical topology is maintained by the electrostatic interaction between the polar side chain of Ser6 and the backbone oxygen of Val3 (FIG. 5C).

In addition, stapling small peptides to increase half-life and maintain secondary structure is 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). Considering the flexible C-terminus of PIP2, assuming that it can reduce TLR4 inhibitory efficacy, peptide stapling was performed to enhance the stability of PIP2.

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

Circulating forms of PIP2 to adjust the stapling effect to the overall structure of cPIP2; MD trajectories were visualized and compared with the trajectories of PIP2.

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

Example 7: Docking Simulation and Interfacial Analysis of PIP2-Binding TLR4 Complexes

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

PIP2 selected via 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, to evaluate the binding affinity of human TLR4 / MD2 complex (3FXI) with PIP2, the relevant X-ray crystal structure was retrieved from PDB. In order to assess binding affinity and suggest a TLR4 antagonistic mechanism of PIP2, the portion of PIP2 capable of binding to TLR4 includes: i) the extracellular domain of monomer TLR4; ii) the extracellular domain of monomer TLR4 bound to MD2; iii) a dimeric extracellular domain of TLR4 with one monomer attached to MD2 and the other monomer empty.

An online protein-protein docking server ZDOCK69 and a standalone MOE suit 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 the receptor and PIP2 was considered the ligand in each round of docking. Using ZDOCK and MOE separately, 500 docking constructions were created 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 alone with TLR4 in monomeric form, PIP2 exhibits a binding that is inconsistent with the ECD. Of the four docked clusters PIP2 formed with the TLR4 ECD, one cluster at the N terminus was slightly constant and undispersed. About 43% of the docked PIP2 is clustered at a specific location of TLR4, and the second cluster, which is very important with respect to MD2-dependent TLR4 activation, accounts for ˜9% of docked PIP2.

Thus, an interaction database of the top 150 docks of PIP2-TLR4 was constructed and shown through protein ligand interaction fingerprints (PLIF) (FIG. 9). The interacting residues are mostly nonpolar or hydrophobic. In addition, major clustering of PIP2 at the N-terminus of TLR4 ECD was confirmed by PLIF analysis.

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

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

From the preliminary docking results, it can be seen that PIP2 inhibits TLR4 signal transduction by competitively interfering with MD2 cavities or MD2 binding with TLR4 (cluster 2). Binding of PIP2 in cluster 1 is the most obvious way to block TLR4 signaling, but inhibition of the N-terminus of TLR4 does not appear to inhibit its subsignal. Since these two combinations can be seen to inhibit TLR4 signal transduction, further investigation of the structural kinetics using MDS is needed.

Thus, after initial evaluation of PIP2 as a potent TLR4 antagonist, peptide cyclization was performed to stabilize the proposed helix structure and enhance inhibition efficacy. Protein design algorithms performed in MOE were used to enhance TLR4 binding affinity by investigating residues that could be mutated or modified to enhance 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 named cPIP2 (SEQ ID NO: 2: LGVFSDWWIKLV). cPIP2 was further investigated for its structural stability through protein kinetic and in vitro potency assays.

Example 8: Structural Kinetics of PIP2 Binding TLR4

8-1: Stability of TLR4-PIP2

Two PIP2 docking complexes were simulated for 100ns because PIP2 is likely to interfere with TLR4 signal transduction by binding to the cavity of MD2 or competitively interfering with TLR4 and MD2 interactions. First, the stability of TLR4-PIP2 (PIP2 binds to the center of TLR4) was investigated.

Structural flexibility and limited movement provided by MDS show that the C-terminal Val and Leu backbone oxygen 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, analogous sites of TLR4 have been identified as critical for MD2 binding and have been labeled Patch A and Patch B (Kim, HM et al. Cell 130: 906-917, 2007).

Analysis of the PIP2-MD2 (TLR4) MD trajectory shows a steady rise in RMSD (FIG. 6A). This is a result of the translational motion of MD2 which 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 with TLR4

Next, 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 no external translational motion of MD2 was observed, the second TLR4 could be seen to have a significant effect on the stability of MD2 binding PIP2. However, no Phe126 transition was observed from active to inactive. Phe126, located in the gating loop of MD2, is known to be critical for ligand dependent TLR4 signal transduction (Shah, M et al., Sci. Rep. 6: 39271, 2016).

In addition, PIP2-TLR4 interface in the PIP2-TLR4 (TLR4 / MD2) complex was analyzed over time to assess the binding affinity of PIP2 with TLR4's MD2-binding patch.

As a result, patch A of TLR4, Trp8 of PIP2 bonds Val108 and C-H-π (arene), which was further enhanced by electrostatic contact between Asp155 and nitrogen atoms in the indole group of Trp8. Trajectory analysis confirmed that this interaction was quite stable and maintained throughout the MD run.

 Lys10 also established salt bridges with Asp155 and Asp183 after 25 ns of MD and found that the C-terminal residue of PIP2 not in contact with TLR4 had strong hydrogen bonds after 20 ns (FIG. 6A).

This is a similar trend with the PIP2-TLR4 system, with this consistency being the C-terminal residues, in particular the carboxyl groups of Val12 and the backbone oxygen of Leu11, were critical to the stability of the PIP2-TLR4 interaction in both dimeric and tetrameric forms [PIP2- TLR4, PIP2-TLR4 (TLR4 / MD2)] (FIG. 6B).

Example 9 Inhibition of TLR-Induced Inflammatory Response of Cyclic PIP2

9-1: TLR4 Inhibitory Activity of cPIP2 in Macrophages

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

Examination of cytotoxicity on RAW264.7 cells at concentrations of 5, 25 and 50 μM of cPIP2 confirmed no toxicity (FIG. 7A).

The TLR4 inhibitory effect of cPIP2 was then assessed by ELISA by monitoring the level of proinflammatory cytokines secreted in RAW264.7 cells.

As a result, it was confirmed that secretion of TNF-α and IL-6 was significantly inhibited in a concentration-dependent manner. (Figures 7b and c). In addition, reduction of type 1 IFNs (IFN-α and IFN-β) was observed in LPS stimulated and cPIP2-treated cells (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 are inhibited by cPIP2, which is consistent with the results of PIP2 (Fig. 7F-H).

9-2: Inhibition of TLRs of cPIP2 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 cells were treated with 80 nM of PMA (phorbol 12-myristate 13-acetate; Sigma-Aldrich Co., St. Louis, MO, USA) to differentiate into macrophages. The cells were then 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 slightly inhibited other TLRs at 50 μM (FIG. 7I).

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

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

10-1: Preparation of RA Rat Model

The RA rat model was prepared as follows.

First, a 1: 1 solution of bovine collagen type II (Sigma-Aldrich Co. LLC) and complete Freund's adjuvant (Sigma-Aldrich Co. LLC.) Is mixed for 30 minutes, and 250 μl of the mixed solution (containing 0.5 mg of bovine type 2 collagen) was injected into the tail of 4 week old male Lewis rats. Three weeks later, evidence of edema and erythema was observed in the ankle, ankle, knee and foot of the Lewis rats, respectively.

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

The RA treatment trial was approved by the Animal Experiment Committee of Ajou University School of Medicine (Approved Number 2013-0070).

Three experimental groups were tested: normal, untreated, and cPIP2 (1 nmol / g body weight) treated groups.

For 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 assess treatment effect.

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

Specifically, the hind paws of the three types of experimental groups were observed and measured, and ankle diameter and AI score were measured at 1, 2, 3, 4, 5, and 6 weeks. Ankle diameters of three rats were measured using Vernier calipers. AI was blind tested and scored as follows: with no edema or erythema (score 0), mild edema and erythema (+1), moderate edema and shackles (+2), edema and metatarsal bone due to limited use of the joint Signs of enlargement in (+3), and excessive swelling (+4) with serious symptoms associated with joint stiffness and full hind paws.

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

As a result, as shown in FIG. 10A, in the untreated group, swollen joints, stiff plantar swelling, severe edema and erythema were observed for 6 weeks, but in the cPIP2 treated group (cPIP2-treated), at 3 weeks Swelling and swelling of the ankles were considerably reduced, and at 6 weeks it was confirmed that they recovered to a level similar to normal (see FIG. 10A).

In addition, as a result of monitoring the ankle diameter and AI score of the RA rat during the treatment period of 6 weeks, as shown in Figures 10b and 10c, in the untreated group, the ankle diameter and mean AI value of the RA rat was significantly higher. , cPIP2 treatment group, ankle diameter and AI value was significantly reduced (see Fig. 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 the onset of RA.

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

For TNF-α staining, the slides were placed in 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). 10 minutes at 130 [deg.] C. and washed with PBS. The slides were blocked with PBS containing 10% BSA for 60 minutes at 37 ° C., followed by 4 with primary antibody (rabbit anti-rat TNF-a (1: 100); Novus Biologicals, Littleton, CO, USA). It was left overnight at ℃. Thereafter, the samples were treated with a second antibody (goat anti-rabbit Alexa Fluor® 594; Invitrogen, Carlsbad, Calif., USA) at a ratio of 1: 200 for 2 hours. The slides were later washed and mounted with ProLong Gold Antifade Reagent (Thermo Fisher Scientific Inc.). 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). TNF-α positive regions were calculated as percentage of total cartilage area using ImageJ software (National Institute of Health, Bethesda, MD, USA) in three randomly selected fields for each sample.

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

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

In addition, TNF-α expression level was measured in synovial tissues obtained from the three types of experimental groups. As shown in FIG. The expression level of a was measured high. In contrast, the expression level of TNF-α in the cPIP2 treatment group significantly decreased during the first week of treatment (see FIG. 10F).

As described above in detail specific parts of the present invention, it will be apparent to those skilled in the art that these specific descriptions are merely preferred embodiments, and thus the scope of the present invention is not limited thereto. will be. Therefore, the substantial scope of the present invention will be 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)

Peptide represented by the amino acid sequence of SEQ ID NO: 1 or 2.
The peptide of claim 1, wherein the peptide of SEQ ID NO: 2 is characterized in that Asp6 and Lys10 form a lactam bridge.
The peptide of claim 1, wherein the peptide inhibits Toll-like receptor 4 (TLR4) signaling pathway.
The peptide of claim 3, wherein the TLR4 signaling pathway is induced by lipopolysaccharide (LPS).
The peptide of claim 1, wherein the peptide binds to the TLR4 / MD2 complex.
A TLR4 (Toll-like receptor 4) antagonist comprising a peptide represented by the amino acid sequence of SEQ ID NO: 1 or 2.
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 method of claim 7, wherein the autoimmune disease is insulin-dependent diabetes mellitus, multiple sclerosis, experimental autoimmune encephalomyelitis, rheumatoid arthritis, experimental autoimmune arthritis, myasthenia gravis, thyroiditis, experimental form uveitis, Hashimoto thyroiditis, primary myxedema, Thyroid addiction, pernicious anemia, autoimmune atrophy gastritis, Addison disease, early menopause, male infertility, juvenile diabetes, Goodpasture syndrome, common swelling, vulgaris ulcer, sympathetic ophthalmic, crystalline uveitis, autoimmune hemolytic anemia, idiopathic leukocyte reduction Prevention or treatment of autoimmune diseases, characterized in that at least one selected from the group consisting of primary biliary sclerosis, latent cirrhosis, ulcerative colitis, Sjogren's syndrome, scleroderma, Wegener's granulomatosis, polymyositis / dermatitis, and systemic lupus erythematosus Pharmaceutical composition for.
A pharmaceutical composition for preventing or treating an inflammatory disease containing a peptide represented by the amino acid sequence of SEQ ID NO: 1 or 2 as an active ingredient.
The method of claim 9, wherein the inflammatory disease is asthma, eczema, psoriasis, allergy, rheumatoid arthritis, psoriatic arthritis, atopic dermatitis, acne, atopic rhinitis, pulmonary inflammation, allergic dermatitis, chronic sinusitis, contact dermatitis dermatitis, seborrheic dermatitis, gastritis, gout, gout, gout, ulcers, chronic bronchitis, Crohn's disease, ulcerative colitis, ankylosing spondylitis, sepsis, vasculitis, bursitis, lupus, rheumatic polymyalgia, temporal A pharmaceutical composition for preventing or treating inflammatory diseases, characterized in that at least one selected from the group consisting of arteritis, multiple sclerosis, solid cancer, Alzheimer's disease, arteriosclerosis, obesity and viral infection.

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