阅读说明：本技术 能够与炎性和代谢性疾病的生物标志物结合的新型肽及其用途 (Novel peptides capable of binding to biomarkers of inflammatory and metabolic diseases and uses thereof ) 是由 朴玧贞 郑钟平 李周娟 于 2019-08-29 设计创作，主要内容包括：本发明涉及：肽,其具有ATF3结合能力并由选自SEQ ID NOS:1至4的任一氨基酸序列表示；融合肽,其中细胞通透性肽与具有ATF3结合能力的肽键合；以及所述肽用于治疗炎性疾病、代谢性疾病、自身免疫性疾病和/或纤维化疾病的用途。根据本发明,提供了具有ATF3结合能力的新型肽,从而调节ATF3的细胞内浓度,并且因此可用于治疗炎性疾病、代谢性疾病、自身免疫性疾病和/或纤维化疾病,所述ATF3为在各种炎性疾病、代谢性疾病、自身免疫性疾病和/或纤维化疾病的发生中用作生物标志物和重要因子的蛋白质。此外,通过进一步将具有细胞内渗透性和炎症抑制性功能的肽与所述肽融合,可以增强疾病治疗效果。(The present invention relates to: a peptide having ATF3 binding ability and represented by any one amino acid sequence selected from the group consisting of SEQ ID NOS:1 to 4; a fusion peptide in which a cell-permeable peptide is bonded to a peptide having ATF 3-binding ability; and the use of said peptides for the treatment of inflammatory, metabolic, autoimmune and/or fibrotic diseases. According to the present invention, there is provided a novel peptide having ATF3 binding ability, thereby modulating the intracellular concentration of ATF3, and thus being useful for treating inflammatory diseases, metabolic diseases, autoimmune diseases, and/or fibrotic diseases, the ATF3 being a protein used as a biomarker and an important factor in the occurrence of various inflammatory diseases, metabolic diseases, autoimmune diseases, and/or fibrotic diseases. In addition, by further fusing a peptide having intracellular permeability and inflammation inhibitory functions to the peptide, the disease treatment effect can be enhanced.)

1. A peptide represented by an amino acid sequence selected from any one of SEQ ID NOS:1 to 4, the peptide having an ability to bind to ATF3 protein.

2. A fusion peptide comprising the peptide of claim 1 and a cell-permeable peptide bound to the N-terminus or C-terminus of the peptide of claim 1.

3. The fusion peptide according to claim 2, wherein the cell-permeable peptide is represented by the amino acid sequence of SEQ ID NO 5, SEQ ID NO 10 or SEQ ID NO 11.

4. The fusion peptide according to claim 3, wherein the fusion peptide is represented by any one of the amino acid sequences of SEQ ID NOS 6 to 9.

5. A pharmaceutical composition for treating inflammatory diseases, comprising the peptide according to any one of claims 1 to 4 as an active ingredient.

6. The pharmaceutical composition according to claim 5, wherein the inflammatory disease is at least one selected from the group consisting of arthritis, periodontitis, atopy, thyroiditis, uveitis, hashimoto's thyroiditis, gastritis, steatohepatitis, hepatitis, and enteritis.

7. A pharmaceutical composition for treating metabolic diseases, comprising the peptide of any one of claims 1 to 4 as an active ingredient.

8. The pharmaceutical composition according to claim 7, wherein the metabolic disease is at least one selected from the group consisting of obesity, weight loss, diabetes, atherosclerosis, arteriosclerosis, cardiovascular diseases, neurological diseases, Alzheimer's disease, cognitive disorders, oxidative stress, skin diseases, skin aging, damage caused by ultraviolet irradiation, hypertension, hypercholesterolemia, hyperlipidemia, immunodeficiency, cancer, and metabolic syndrome.

9. A pharmaceutical composition for treating autoimmune diseases, comprising the peptide of any one of claims 1 to 4 as an active ingredient.

10. The pharmaceutical composition of claim 9, wherein the autoimmune disease is at least one selected from the group consisting of insulin-dependent diabetes mellitus, multiple sclerosis, autoimmune encephalomyelitis, rheumatoid arthritis, osteoarthritis, myasthenia gravis, thyroiditis, uveitis, hashimoto's thyroiditis, thyrotoxicosis, pernicious anemia, autoimmune atrophic gastritis, autoimmune hemolytic anemia, idiopathic leukopenia, primary sclerosing cholangitis, alcoholic/non-alcoholic steatohepatitis, inflammatory bowel disease, crohn's disease, ulcerative enteropathy, psoriasis, Sjogren's syndrome, scleroderma, wegener's granulomatosis, polymyositis, dermatomyositis, discoid lupus erythematosus (discoid Le), and systemic lupus erythematosus.

11. A pharmaceutical composition for treating fibrotic diseases, comprising the peptide according to any one of claims 1 to 4 as an active ingredient.

12. The pharmaceutical composition according to claim 11, wherein the fibrotic disease is at least one selected from the group consisting of liver cirrhosis caused by fibrosis, pulmonary fibrosis, obstructive pulmonary disease, heart failure, arteriosclerosis, chronic renal failure, diabetes, and keloid caused by postoperative sequelae.

13. The pharmaceutical composition according to any one of claims 5 to 12, wherein the pharmaceutical composition is prepared in any one formulation selected from the group consisting of injection, oral formulation, patch, solution, capsule, granule, tablet, powder, spray, ointment, gel, mucosal formulation and suppository.

14. The pharmaceutical composition of claim 13, further comprising a pharmaceutically acceptable adjuvant.

15. The pharmaceutical composition according to claim 14, wherein the pharmaceutically acceptable adjuvant is at least one selected from the group consisting of an excipient, a diluent, a buffer, an antimicrobial preservative, a surfactant, an antioxidant, a thickener, and a viscosity modifier.

16. The peptide according to any one of claims 1 to 4, wherein the daily dose of the peptide is from 1 to 100mg/kg and the peptide is administered once daily or from 2 to 3 times weekly.

Technical Field

The present invention relates to novel peptides capable of binding to biomarkers of inflammatory and metabolic diseases and uses thereof, and more particularly, to peptides having ATF3 binding ability represented by amino acid sequences selected from SEQ ID NOS:1 to 4, fusion peptides in which the peptides having cell permeability are bound to the peptides having ATF3 binding ability, and uses of these peptides for treating inflammatory diseases, metabolic diseases, and/or autoimmune diseases.

Background

The ATF3 (activating transcription factor 3) protein is a component of the CAMP Response Element Binding (CREB) protein of mammalian activating transcription factors/transcription factors. The ATF3 gene is known to be expressed by various signals generated by various factors involved in cancer development and to be involved in a complex process of intracellular stress response.

ATF3 protein is used as activator or repressor of known target gene, more than 20 potential target genes of ATF3 are known in literature, and known potential target genes of ATF3 comprise AdipoR1, AdipoR2, bNIP3, Cdc25A, CCL2, CCL4, Cyclin D1, FN-1, GLUT4, HIF-2 alpha, IFN-gamma, IL-1 beta, IL-6, IL-12b, IRS2, MMP1, MMP13, Noxa, p15PAF, Slug, Snail, STAT1, TNF-alpha, TWIST1, p53, p73, PDX-1 and adiponectin.

In addition, in most cells including fibroblasts and epithelial cells, and in immune cells such as macrophages, mast cells, T cells, and dendritic cells, various pathways such as NF- κ B, JNK Erk, p38, and PKC induce ATF3, and the induced ATF3 is involved in apoptosis, cell proliferation, cell motility, and DNA repair and metabolism by regulating the transcription of various genes. Among them, NF-. kappa.B is known to be involved in the induction of the relevant inflammatory response.

At present, a number of studies are underway on the relationship of the expression of ATF3 protein to inflammatory diseases (Lai PF et al, Evi-Based comparative. Alternate. Med.2013; 2013:716481.), metabolic diseases such as obesity (Jang MK et al, biochem Biophys. Res. Commun.2013, 2.15.s.; 431(3):421-7.), diabetes (KR 10-1652957) and cardiovascular diseases (Zhou H. et al, Basic Res. Cardiol.2018, 8.9.s.2018; 113(5):37.) as well as autoimmune diseases (Smith C.K. et al, Ann. Rheum. Dis.76(2017)602- -611, Zheng S. et al, mol. cell biol 57, 33.2013), 48487 1). Substances that modulate the expression or function of ATF3 protein can inhibit the inflammatory response induced by ATF3, but no effective inhibitor of ATF3 protein has been developed so far.

Accordingly, as a result of extensive efforts to solve the problems of the prior art, the present inventors have developed peptides that bind to ATF3, inhibit the expression of ATF3, and inhibit the signaling pathway of ATF3, and pharmaceutical compositions comprising the peptides, and found that the peptides and compositions can reduce the production of inflammatory cytokines. Based on this finding, the present invention has been completed.

BRIEF SUMMARY OF THE PRESENT DISCLOSURE

Accordingly, the present invention has been made in view of the above problems, and it is an object of the present invention to provide a novel peptide capable of binding to ATF3 protein, a biomarker of inflammatory diseases and metabolic diseases, and uses thereof.

In accordance with one aspect of the present invention, the above and other objects can be accomplished by the provision of a peptide represented by any one amino acid sequence selected from the group consisting of SEQ ID NOS:1 to 4, the peptide having the ability to bind to ATF3 protein.

According to another aspect, there is provided a fusion peptide comprising the peptide and a peptide represented by the amino acid sequence of SEQ ID NOS:5 bound to the N-terminus or C-terminus of the peptide.

According to another aspect, there is provided a pharmaceutical composition for treating inflammatory diseases, metabolic diseases, autoimmune diseases and/or fibrotic diseases, comprising the peptide or fusion peptide as an active ingredient.

According to another aspect, there is provided the use of a peptide, fusion peptide or composition for the treatment of an inflammatory disease, a metabolic disease, an autoimmune disease and/or a fibrotic disease.

According to another aspect, there is provided the use of a peptide, fusion peptide or composition in the manufacture of a medicament for the treatment of an inflammatory disease, a metabolic disease, an autoimmune disease and/or a fibrotic disease.

According to another aspect, there is provided a method for preventing and/or treating an inflammatory disease, a metabolic disease, an autoimmune disease and/or a fibrotic disease, comprising administering the peptide, fusion peptide or composition to a subject in need of treatment of the inflammatory disease, metabolic disease, autoimmune disease and/or fibrotic disease.

Drawings

FIG. 1 shows the binding of the peptides of SEQ ID NOS:1 to 4 to the ATF3 protein in a concentration-dependent manner.

FIG. 2 is the result of Western blotting for detecting the amount of ATF3 protein when the peptides of SEQ ID NOS:1 to 4 were used for the treatment.

FIG. 3 is a confocal microscopy image showing cell permeability of peptides of SEQ ID NO 1, SEQ ID NO 5, SEQ ID NO 6.

FIG. 4 shows the results of ELISA for detecting the expression level of IL-6 in the treatment with the peptides of SEQ ID NOS:1 to 6.

FIG. 5a shows the results of H & E staining for detecting reduction of liver tissue inflammation and fat in a non-alcoholic steatohepatitis animal model when treated with the peptide of SEQ ID NO: 1.

FIG. 5b shows the results of Sirius red (Sirius red) staining for testing the effect of reducing fibrosis in liver tissue in a non-alcoholic steatohepatitis animal model, when treated with the peptide of SEQ ID NO: 1.

FIG. 5c shows the results of Masson trichrome staining (Masson trichrome staining) to test the effect of reducing fibrosis in liver tissue in an animal model of non-alcoholic steatohepatitis when treated with the peptide of SEQ ID NO: 1.

FIG. 5d shows the measurement of NAS score for each group in non-alcoholic steatohepatitis animal models when the peptide of SEQ ID NO:1 was administered.

FIG. 6 shows the results of H & E staining for observation of intestinal villi after treatment of an animal model of inflammatory bowel disease with the peptide of SEQ ID NO: 1.

Best mode

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 terminology used herein is well known in the art and is commonly used.

In the present invention, a peptide sequence having ATF3 binding ability as a biomarker of inflammatory and metabolic diseases was found using a phage display method, and the peptide was found to have ATF3 binding ability in a concentration-dependent manner.

Thus, in one aspect, the invention relates to peptides capable of binding to the ATF3 protein.

The peptide may be represented by any one of amino acid sequences selected from SEQ ID NOS:1 to 4.

SEQ ID NO:1(ABP1):AESPLTNRGWNP

SEQ ID NO:2(ABP2):MLDTNIQSRPNL

SEQ ID NO:3(ABP3):TLGLRPVPVATT

SEQ ID NO:4(ABP4):VLNIPEHFTAQN

In the present invention, a peptide having the ability to bind to an ATF protein may be represented by any one of amino acid sequences selected from SEQ ID NOS:1 to 4, and may have 60% or more, 70% or more, 80% or more, 90% or more or 95% or more homology with the amino acid sequence. It is obvious to those skilled in the art that any one of the amino acid sequences selected from SEQ ID NOS:1 to 4 may be appropriately substituted to achieve the object of the present invention; for example, some amino acids (e.g., 1 to 5 amino acids) in the amino acid sequence of the peptide of SEEQ ID NOS:1 to 4 may be substituted with other amino acids, or some amino acids (e.g., 1 to 5 amino acids) in the amino acid sequence of the peptide of SEQ ID NOS:1 to 4 may be added or deleted if a beneficial effect on peptide stability is provided without affecting the desired structure or function of the peptide.

The present invention is based on the finding that peptides of SEQ ID NOS:1 to 4 have low cell permeability, whereas peptides of EQ ID NOS:6 to 9 (having cell permeability) obtained by binding the peptides of EQ ID NOS:1 to 4 to the peptides of SEQ ID NO:5 have improved cell permeability.

Thus, in another aspect, the invention relates to fusion peptides having cell permeability and ATF3 binding capacity.

The fusion peptide may have a structure in which a peptide having cell permeability is bound to a peptide of each of SEQ ID NOS:1 to 4, and the peptide having cell permeability may be a peptide having an amino acid sequence of SEQ ID NO:5, SEQ ID NO:10, or SEQ ID NO: 11.

In the present invention, the peptide having cell permeability may be represented by the amino acid sequence of SEQ ID NO. 5, SEQ ID NO. 10 or SEQ ID NO. 11, or may have 60% or more, 70% or more, 80% or more, 90% or more or 95% or more homology with the amino acid sequence. It is obvious to those skilled in the art that the amino acid sequences of SEQ ID NO 5, 10 or 11 may be appropriately substituted for the purpose of the present invention; for example, some amino acids (e.g., 1 to 5 amino acids) in the amino acid sequence of SEQ ID NO 5, 10 or 11 may be substituted with other amino acids, or some amino acids (e.g., 1 to 5 amino acids) in the amino acid sequence of the peptide of SEQ ID NO 5, 10 or 11 may be added or deleted, if a beneficial effect on peptide stability is provided without affecting the desired structure or function of the peptide.

Meanwhile, the fusion peptide may be represented by any one of amino acid sequences selected from SEQ ID NOS:6 to 9.

SEQ ID NO:5(hBD-3-3):GKCSTRGRKCCRRKK

SEQ ID NO:6(ABP1-hBD3-3):AESPLTNRGWNPGKCSTRGRKCCRRKK

SEQ ID NO:7(ABP2-hBD3-3):MLDTNIQSRPNLGKCSTRGRKCCRRKK

SEQ ID NO:8(ABP3-hBD3-3):TLGLRPVPVATTGKCSTRGRKCCRRKK

SEQ ID NO:9(ABP4-hBD3-3):VLNIPEHFTAQNGKCSTRGRKCCRRKK

SEQ ID NO:10(LMWP):VSRRRRRRGGRRRR

SEQ ID NO:11(H4):HRRCNKNNKKR

The fusion peptide can be synthesized by linking the amino acid sequence of SEQ ID NO. 5, 10 or 11 to the C-terminus of the peptide represented by the amino acid sequence of each of SEQ ID NO. 1 to SEQ ID NO. 4, or can also be synthesized by linking the peptide represented by the amino acid sequence of SEQ ID NO. 5, 10 or 11 to the N-terminus of the peptide represented by the amino acid sequence of each of SEQ ID NO. 1 to 4.

In addition, a peptide represented by the amino acid sequence of each of SEQ ID NOS:1 to 4 may be linked to a peptide represented by the amino acid sequence of SEQ ID NO:5, SEQ ID NO:10, or SEQ ID NO:11 through a linker. Any linker may be used as the linker as long as it can provide a region in which each peptide can form a functional structure. For example, the linker may be a naturally derived and/or synthetic peptide linker. A naturally derived and/or synthetic peptide linker may have an amino acid chain consisting of 1 to 50 amino acids, and may include a sequence of repeating amino acids of a naturally occurring polypeptide (such as a polypeptide having a hinge function). In another embodiment, the peptide linker amino acid sequence may be a synthetic linker amino acid sequence designed to be rich in glycine, glutamine and/or serine residues. For example, these residues can be arranged in small repeat units of 5 or fewer amino acids, and the small repeat units can be arranged repeatedly to form a multimeric unit. 6 or fewer additional optional naturally occurring amino acids can be added at the amino and/or carboxyl terminus of the multimer unit. Other synthetic peptide linkers can have a single configuration of amino acids repeated 10 to 20 times, and can have 6 or fewer additional optional naturally occurring amino acids at the amino and/or carboxy termini. Meanwhile, the linker may be in the form of chemically modified amino acid. For example, Fmoc-6-aminocaproic acid (Fmoc- ε -Acp-OH) bound to Fmoc- (9-fluorenylmethyloxycarbonyl) can be used as a blocking group, but is not limited thereto.

A part of the peptides of SEQ ID NOS:1 to 4 was found to pass through cells, but its cell permeability was not excellent. However, when the peptide of SEQ ID NO. 5 having cell permeability is linked to each of the peptides of SEQ ID NOS:1 to 4, the cell permeability of the peptides of SEQ ID NOS:1 to 4 is increased, and since the ATF3 protein exists in the cell, the peptides can be more easily bound to the ATF3 protein in the cell. Therefore, the effect on ATF3 can be enhanced.

ATF3 is known to be an important factor in inflammatory, metabolic, autoimmune and fibrotic diseases.

Thus, in another aspect, the present invention relates to a pharmaceutical composition for treating inflammatory diseases, comprising the peptide or fusion peptide as an active ingredient.

In the present invention, the inflammatory disease may include at least one selected from the group consisting of arthritis, periodontitis, atopy, thyroiditis, uveitis, hashimoto's thyroiditis, gastritis, steatohepatitis, hepatitis, and enteritis, but is not limited thereto.

In another aspect, the present invention relates to a pharmaceutical composition for treating metabolic diseases, comprising the peptide or the fusion peptide as an active ingredient.

In the present invention, the metabolic disease includes at least one selected from the group consisting of obesity, weight loss, diabetes, atherosclerosis, arteriosclerosis, cardiovascular diseases, neurological diseases, alzheimer's disease, cognitive disorders, oxidative stress, skin diseases, skin aging, damage caused by ultraviolet irradiation, hypertension, hypercholesterolemia, hyperlipidemia, immunodeficiency, cancer and metabolic syndrome, but is not limited thereto.

In the present invention, the term "hypercholesterolemia" refers to a condition characterized by a concentration of LDL, HDL, and/or VLDL in the blood higher than a normal level, and the term "hyperlipidemia" refers to a disease characterized by a concentration of triglycerides higher than a normal level.

In another aspect, the present invention relates to a pharmaceutical composition for treating autoimmune diseases, comprising the peptide or the fusion peptide as an active ingredient.

In the present invention, the autoimmune disease includes at least one disease selected from the following diseases: insulin-dependent diabetes mellitus, multiple sclerosis, autoimmune encephalomyelitis, rheumatoid arthritis, osteoarthritis, myasthenia gravis, thyroiditis, uveitis, hashimoto's thyroiditis, thyrotoxicosis, pernicious anemia, autoimmune atrophic gastritis, autoimmune hemolytic anemia, idiopathic leukopenia, primary sclerosing cholangitis, alcoholic/non-alcoholic steatohepatitis, inflammatory bowel disease, crohn's disease, ulcerative enteropathy, psoriasis, Sjogren's syndrome, scleroderma, wegener's granulomatosis, polymyositis, dermatomyositis, discoid lupus erythematosus (discoid LE), and systemic lupus erythematosus, but are not limited thereto.

In another aspect, the present invention relates to a pharmaceutical composition for treating fibrotic diseases, comprising the peptide or the fusion peptide as an active ingredient.

In the present invention, the fibrotic disease may include at least one selected from the group consisting of fibrosis-induced liver cirrhosis, pulmonary fibrosis, obstructive pulmonary disease, heart failure, arteriosclerosis, chronic renal failure, diabetes, and keloid caused by postoperative sequelae, but is not limited thereto.

In another aspect, the invention relates to the use of a peptide, fusion peptide or composition for the treatment of an inflammatory disease.

In another aspect, the invention relates to the use of a peptide, fusion peptide or composition for the treatment of a metabolic disease.

In another aspect, the invention relates to the use of a peptide, fusion peptide or composition for the treatment of an autoimmune disease.

In another aspect, the invention relates to the use of a peptide, fusion peptide or composition for the treatment of fibrotic diseases.

In another aspect, the invention relates to the use of a peptide, fusion peptide or composition in the manufacture of a medicament for the treatment of an inflammatory disease.

In another aspect, the invention relates to the use of a peptide, fusion peptide or composition for the manufacture of a medicament for the treatment of a metabolic disease.

In another aspect, the invention relates to the use of a peptide, fusion peptide or composition for the manufacture of a medicament for the treatment of an autoimmune disease.

In another aspect, the invention relates to the use of a peptide, fusion peptide or composition for the manufacture of a medicament for the treatment of a fibrotic disease.

In another aspect, the present invention relates to a method for preventing and treating inflammatory diseases comprising administering the peptide, fusion peptide or composition to a subject in need of treatment for inflammatory diseases.

In another aspect, the present invention relates to a method for preventing and treating a metabolic disease, comprising administering the peptide, fusion peptide or composition to a subject in need of treatment of a metabolic disease.

In another aspect, the present invention relates to a method for preventing and treating an autoimmune disease comprising administering the peptide, fusion peptide or composition to a subject in need of treatment of the autoimmune disease.

In another aspect, the present invention relates to a method for preventing and treating a fibrotic disease, comprising administering the peptide, fusion peptide or composition to a subject in need of treatment of the fibrotic disease.

In addition to the peptide, the pharmaceutical composition of the present invention may further comprise at least one pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier may be saline, sterile water, ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, and combinations of one or more of these components. Other conventional additives such as antioxidants, buffers and bacteriostats may be added if desired. In addition, diluents, dispersants, surfactants, binders and lubricants may also be added to prepare injectable formulations such as aqueous solutions, suspensions and emulsions, and pills, capsules, granules or tablets.

Therefore, the composition of the present invention is prepared into any one formulation selected from the group consisting of injection, formulation for oral administration, patch, solution, capsule, granule, tablet, powder, spray, ointment, gel, mucosal formulation and suppository, but is not limited thereto. These preparations can be prepared by a conventional method used for preparations in the art, or by a method disclosed in Remington's Pharmaceutical Science (latest edition), Mack Publishing Company, Easton PA, and can be prepared into various preparations according to each disease or component.

The therapeutic pharmaceutical composition may be formulated such that it further comprises a pharmaceutically acceptable adjuvant, and the pharmaceutically acceptable adjuvant may include at least one selected from the group consisting of an excipient, a diluent, a buffer, an antimicrobial preservative, a surfactant, an antioxidant, a thickener, and a viscosity modifier, but is not limited thereto.

The composition of the present invention may be administered orally or parenterally (e.g., intravenous, subcutaneous, intraperitoneal, or topical application) according to a desired method, and the dosage may vary according to the body weight, age, sex, health condition, diet, administration time, administration method, excretion rate, and severity of disease of a patient. When the peptides of SEQ ID NOS:1 to 9 are used for treating inflammatory diseases, metabolic diseases, autoimmune diseases and/or fibrotic diseases, the daily dose of the peptides is about 1 to about 100mg/kg, preferably 5 to 50mg/kg, and may be administered once per day or2 to 3 times per week, but is not limited thereto.

In such cases, the administration regimen and dosage will vary according to the age, weight and response of the individual patient. In view of these factors, the skilled person can easily select the appropriate regimen and dosage.

Examples

Hereinafter, the present invention will be described in more detail with reference to examples. However, it will be apparent to those skilled in the art that these examples are provided only for illustrating the present invention and should not be construed as limiting the scope of the present invention.

Example 1: discovery of peptide having ATF3 protein binding ability

Example 1-1: searching peptide sequence with ATF3 binding ability by using phage display technology

Phage display technology was used to find specific peptide sequences that bind to ATF 3. After M13 phage labeled with a random peptide library having high diversity and complexity was attached to a well of a polystyrene plate coated with ATF3 protein, the suspended phage were washed out to collect only the attached phage. This process was repeated several times. By sequencing the DNA encoding the peptide labeled on the finally obtained phage using subtractive panning, a peptide sequence having ATF3 binding ability was found. Subtractive panning is a novel phage display method that can be used to find sequences that bind to a particular substance but not to other substances.

The Phage Display kit used herein was the ph.d. -12Phage Display kit (New England Biolabs (USA)), and the final 17 clones in the form of e.coli cultures were sequenced by Cosmo Genetech co., Ltd using Phage discovery tagged with a random peptide library consisting of 12 amino acids provided in the kit.

As shown in table 1, as a result, four sequences were identified, two of which clones had the same DNA sequence, and by translating the same sequence, the sequence of the final peptide consisting of 12 amino acids could be obtained: SEQ ID NO 1(ABP1, AESPLTNRGWNP), SEQ ID NO 2(ABP2(MLDTNIQSRPNL), SEQ ID NO 3(ABP3, TLGLRPVPVATT) and SEQ ID NO 4(ABP3, VLNIPEHFTAQN). peptide sequences are likely to be specific sequences that bind to ATF3 and are therefore selected as candidates for specific peptide sequences that bind to ATF 3.

[ Table 1]

Examples 1 to 2: synthesis of peptide having ATF3 binding ability

The peptides of SEQ ID NOS:1 to 4 were synthesized using F-moc solid phase peptide synthesis in the order from the N-terminus. The synthesized peptide sequence was cleaved from the resin, washed, lyophilized, and then isolated and purified by liquid chromatography. The molecular weight of the purified peptide was determined by MALDI-TOF analysis.

Examples 1 to 3: determination of binding affinity of peptides having ATF 3-binding ability to ATF3 protein

Biotin was labeled on the peptides of examples 1-2 to determine the binding affinity of the peptides to the ATF3 protein. Each peptide was biotinylated using EZ-link sulfo-NHS-biotin (Pierce Biotechnology, USA) according to the manufacturer's instructions and unbound byproducts were removed using ultrafiltration, a membrane separation method using differential pressure as the driving force. Then, the molecular weight of the synthesized product was detected by measuring the molecular weight using mass spectrometry. Analytical reverse phase liquid chromatography was used for analysis and purification. By using C with a diameter of 4.6mm₁₈Column, flow rate of 1 ml/min, 0.1% TFA/H₂O and 0.092% TFA/acetonitrile (gradient 0% to 60%) were flowed for 30 minutes for analysis. At this time, the wavelength of the ultraviolet detector was 220 nm. Purification was performed using a column having a diameter of 2.2cm, under the same conditions of solvent and detection wavelength as described above, at a flow rate of 20 ml/min. The solvent was removed from a portion of the biotin-conjugated peptide using a rotary evaporator, followed by lyophilization.

The binding affinity of peptides with the binding capacity of ATF3 to ATF3 protein was measured using an avidin-biotin complex binding assay. Each well of a 96-well polystyrene plate was coated with ATF3 protein at a concentration of 0.02. mu.g/. mu.l for 16 hours, followed by blocking solution (0.1M NaHCO)₃(pH 8.6),5mg/ml BSA,0.02％NaN₃(optional), filter-sterilized) for at least 1 hour, discarding the blocking solution, and washing with washing solution (TBS + 0.1% [ v/v ]]Tween-20) was washed strongly 6 times or more. Then, the peptide of SEQ ID NOS:1 to 4(ABP 1-4) which had been labeled with biotin and obtained by performing synthesis, isolation and purification, i.e., the peptide having ATF3 binding ability, was added in an amount of 150. mu.l at various concentrations of 10. mu.M to 100. mu.MSeeded on the surface of wells coated with ATF3 protein. After 2 hours of reaction at room temperature, the resulting product was washed vigorously at least 6 times with a wash solution suitable for each case, and 150. mu.l of a dilution of Extravidin-Peroxidase (Cat. No. E2886, Sigma-Aldrich, USA) diluted 1:500 in blocking solution was inoculated into each well. After 1 hour of reaction at room temperature, the resulting product was washed vigorously 6 or more times with appropriate washes, 150. mu.l of a substrate solution (2, 2' -azino-BIS, Cat. No. A3219, Sigma-Aldrich, USA) was inoculated into the wells, and color development was carried out at room temperature for 20 minutes. After 20 minutes, the resulting product was treated with 50. mu.l of a 1% SDS solution to stop the reaction, and the absorbance was measured at 405 nm.

As a result, as can be seen from fig. 1, when the ATF3 protein was treated with the peptide having ATF3 binding ability of example 1-2, the tendency of the peptide to bind to the ATF3 protein in a concentration-dependent manner was determined.

Example 2: peptides with cell permeability and ATF3 binding capacity

The peptide of each of SEQ ID NOS:6 to 9 was synthesized by F-moc solid phase peptide synthesis by linking the peptide of SEQ ID NOS:5 to the C-terminal of the peptide of each of SEQ ID NOS:1 to 4 having the ability to bind to ATF3 protein. The synthesized peptide sequence was cleaved from the resin, washed, lyophilized, and isolated and purified by liquid chromatography. The molecular weight of the purified peptide was determined by MALDI-TOF analysis.

Example 3: determination of peptide action in non-alcoholic steatohepatitis animal models

Methionine-choline deficiency (MCD) diet was fed to C57BL/6J mice for 8 weeks to induce a non-alcoholic fatty liver model, to which the peptide of SEQ ID NO:1 was administered at each dose for 6 weeks, and then the effect was evaluated.

Example 4: determination of peptide action in animal models of inflammatory bowel disease

The therapeutic effect was determined by inducing inflammatory bowel disease in ICR mice using drinking water solution in 5% DSS for 10 days while administering the peptide to the mice.

Experimental example 1: determination of the Effect of ATF 3-binding peptides on the reduction of expression of the liver disease marker ATF3 protein

Western blotting was performed to determine the effect of the peptides of SEQ ID NOS:1 to 4 on the change in the expression of ATF3 protein. HepG2 cells were plated at 70% density on 6-well plates. After 16 hours, the cells were starved for 2 hours with DMEM medium containing 0.5% FBS. Cells were treated with a medium containing each peptide of SEQ ID NOS:1 to 4 at a concentration of 200. mu.M for 2 hours, and then with palmitic acid at a concentration of 200. mu.M for 24 hours to induce an inflammatory response. Cells were lysed using RIPA lysis buffer (25mM Tris HCl pH 7.6,150mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS) containing protease inhibitors and phosphatase inhibitors. The protein was determined by BCA protein assay and the expression levels of ATF3 and tubulin were detected by western blot. Western blots were performed by loading equal amounts of each sample and size marker on an 8% SDS PAGE gel, followed by electrophoresis for about 2 hours and transfer to nitrocellulose membrane. The transferred membrane was blocked with 5% skim milk for 1 hour and reacted with primary antibody at a ratio of 1:1000 overnight. Then, the membrane was washed with TBST containing 0.1% tween-20 and reacted with a secondary HRP-attached antibody for 1 hour, and then chemiluminescence was detected with an ECL substrate.

As a result, as shown in FIG. 2, the expression of ATF3 protein, which is a marker of liver disease, was increased when treated with palmitic acid alone, but the expression of ATF3 protein was found to be decreased when treated with palmitic acid in combination with the peptides of SEQ ID NOS:1 to 4. This indicates that the peptides of SEQ ID NOS:1 to 4 have an effect of inhibiting the expression of ATF3 protein as a marker of liver diseases, and the peptides of SEQ ID NOS:1-4 have an effect of treating liver diseases.

Experimental example 2: determination of the Effect of SEQ ID NO 5 on the increase in cell Permeability of peptides having the ability to bind to ATF3 protein

RAW 264.7 cells were plated at 2X 10⁴Density of individual/well was seeded on 6-well plates. After 24 hours, replacement was performed with DMEM medium containing 0.5% FBS. Cells were treated with 50 μ M of each fluorescent (rhodamine) labeled peptide of SEQ ID NOS:1, 5 and 6. After 20 minutes, the cells were fixed and observed with a confocal microscope.

As a result, as can be seen from FIG. 3, the peptides of SEQ ID NO:1 fluoresce reddish in cells, indicating that some cells are permeated, and the peptides of SEQ ID NO:5 and SEQ ID NO:6 fluoresce more in cells than the peptides of SEQ ID NO:1, indicating that the peptides of SEQ ID NO:5 and SEQ ID NO:6 have excellent cell permeability.

This means that, by binding the peptide of SEQ ID NO:5 having high cell permeability to the peptide of SEQ ID NO:1 having low cell permeability, the intracellular permeability of the peptide capable of binding to the ATF3 protein (i.e., SEQ ID NO:6) can be further enhanced and the effect in cells can be improved.

Experimental example 3: determination of the Effect of ATF 3-binding peptides on the reduction of IL-6 expression

HepG2 cells were plated at 70% density on 6-well plates. After 16 hours, the cells were starved for 2 hours with DMEM medium containing 0.5% FBS. The cells were treated with a medium containing each of the peptides of SEQ ID NOS:1 to 6 at a concentration of 200. mu.M for 2 hours and further treated with palmitic acid at a concentration of 200. mu.M to induce an inflammatory response for 24 hours. Each medium was collected and the expression level of the inflammatory cytokine IL-6 was measured using an ELISA kit (R & D, Minneapolis, MN, USA).

As a result, as can be seen from FIG. 4, the expression level of IL-6 increased by palmitic acid was decreased by the ATF 3-binding peptide. This demonstrates that ATF 3-binding peptides are effective in treating liver disease by reducing the production of inflammatory-causing cytokines. In addition, SEQ ID NO. 6 has the lowest IL-6 expression level, indicating that the inhibition of cytokine expression is further enhanced by the cell permeability of SEQ ID NO. 5.

Experimental example 4: determination of peptide action in non-alcoholic steatohepatitis animal model

According to the present invention, an MCD mouse model is used to determine whether the peptides are effective at treating steatohepatitis at the animal level. Male C57BL/6J mice were used and fed a normal diet, MCD diet, low dose of the SEQ ID NO:1 peptide (20mg/kg) in combination with MCD diet, and high dose of the SEQ ID NO:1 peptide (80mg/kg) in combination with MCD diet. Mice were fed the MCD diet freely for 8 weeks and then administered the peptide once daily, or once every three days, for a total of 12 administrations. For all animals, body weights were measured shortly before the start of administration, twice weekly throughout the experiment. After completion of the last administration, blood was collected from the subject through the posterior vena cava under isoflurane respiratory anesthesia, and then the subject was subjected to painless death by exsanguination. After painless death, the appearance was visually observed and the characteristics of the organs in the abdominal and thoracic cavities were observed. For all animals at necropsy, tissues were fixed in neutral buffered formalin solution, paraffin blocks were prepared by tissue processing, and microtomes were cut. Tissue sections were H & E stained (fig. 5a), sirius red stained (fig. 5b) and masson trichrome stained (fig. 5c) to detect the extent of fat accumulation and fibrosis in liver tissue.

As a result, as can be seen from FIG. 5a, the number and size of inflammation and fat particles increased in the MCD diet-fed group, while the number and size of inflammation and fat particles decreased in the group injected with the peptide of SEQ ID NO: 1. As can be seen from fig. 5b and 5c, fibrosis was very severe in the MCD diet-fed group (red in fig. 5b, and blue in fig. 5c), and fibrosis was significantly reduced in the group injected with the SEQ ID NO:1 peptide.

NAS score is an index of the sum of balloon degeneration (ballooning degeneration), lobular inflammation and steatosis. NAS scores in the peptide-treated groups were reduced by 50% or more, indicating improvement in non-alcoholic fatty liver disease (fig. 5 d).

Experimental example 5: determination of peptide action in animal models of inflammatory bowel disease

Determining whether the peptide has a therapeutic effect in an animal level inflammatory bowel disease model according to the present invention. Using ICR mice, the peptides were injected by IP injection while inducing inflammation using 5% DSS as drinking water for 10 days to induce inflammation. Each of normal, defective, positive control (SAHA), immunoregulatory control (anti-TNF-a antibody), and SEQ ID NO:1 peptide treatments were performed daily for 10 days. After 10 days, by CO₂Mice were sacrificed by hyperventilation, colon and spleen were collected, and H was performed by colon length and production using paraffin&E staining to determine if intestinal villi are damaged (fig. 6).

As a result, as can be seen from fig. 6, more inflammatory cells were observed in the DSS-induced group, and the villi were severely damaged. In the group treated with the peptide of SEQ ID NO. 1, the number of inflammatory cells was reduced and villi were preserved without damage.

While specific configurations of the present invention have been described in detail, those skilled in the art will appreciate that the description is provided for purposes of illustration of preferred embodiments and should not be construed as limiting the scope of the invention. Accordingly, the substantial scope of the present invention is defined by the appended claims and equivalents thereof.

INDUSTRIAL APPLICABILITY

The present invention provides a novel peptide having ATF 3-binding ability, the ATF3 being a protein that plays an important role in the pathogenesis of various inflammatory, metabolic, autoimmune and/or fibrotic diseases in the prior art. Therefore, the peptide can be used for treating inflammatory diseases, metabolic diseases, autoimmune diseases, and/or fibrotic diseases by controlling the intracellular concentration of ATF3, and the effect of treating diseases can be increased by further fusing a peptide having intracellular permeability and inflammation inhibitory function to the peptide.

Non-sequence text

The electronic file is attached.

Sequence listing

<110> nanometer Intelligent biomedical engineering Co., Ltd

<120> novel peptides capable of binding to biomarkers of inflammatory and metabolic diseases and uses thereof

<130> 0165-PA-011CN

<150> KR 10-2018-0103614

<151> 2018-08-31

<150> KR 10-2019-0105916

<151> 2019-08-28

<160> 33

<170> PatentIn version 3.5

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