Peptides interacting with tlr2 and compositions comprising the same

Abstract

Peptide fragments of Amuc_1100* and uses thereof are disclosed. The peptides are shown to be ligands that activate TLR2 and are useful in the treatment of obesity and related diseases or disorders. The peptides of the invention are further useful in the treatment of small intestinal cancer, in the promotion of immune responses, and in intestinal epithelial barrier dysfunction.

Description

[0001] Cross-references

[0002] This application claims priority to Provisional U.S. Patent Application No. 62 / 959,519, filed January 10, 2020, pursuant to 35 USC §119(a), the entire contents of which are incorporated herein by reference. Technical Field

[0003] This invention relates to a peptide that interacts with Toll-like receptor 2 (TLR2), as well as peptides, compositions thereof, and uses for the treatment of obesity. Background Technology

[0004] The gut microbiome contains approximately 100 trillion microorganisms that are associated with the host's health. It has been reported that the gut microbiome influences and regulates the host's metabolism, nutrition, and immune response. Therefore, the gut microbiota is a potential target for diagnosis, prevention, treatment, and prognosis.

[0005] Akkermansia spp. is a family of bacteria isolated from the human gut microbiota. Reduced numbers of Akkermansia muciniphila in fecal samples from obese individuals and patients with inflammatory bowel disease have been previously reported. In particular, the Amuc_1100 protein, a specific protein isolated from the outer membrane of Akkermansia muciniphila, is considered an active agent for treating obesity and is therefore a potential drug. When Amuc_1100 interacts with steroid receptor 2, it improves the intestinal barrier and partially encapsulates the beneficial effects of this bacterium.

[0006] However, it has been reported that the pharmaceutically active proteins of *Ekkermansia myxophilus* are stable at the temperatures used for pasteurization, but become ineffective after autoclaving. There is a desire to find or develop new peptides that can be autoclaved but retain their activity. Summary of the Invention

[0007] Therefore, this invention provides some novel peptides that interact with TLR2 receptor 2.

[0008] This article provides, on the one hand, a synthetic peptide that interacts with TLR2, comprising an amino acid sequence consisting of SEQ ID NO:8; but with a maximum length of 50 amino acids.

[0009] In one specific embodiment, the synthetic peptide has a length of up to 30 amino acids.

[0010] In one specific embodiment, the synthetic peptide consists of the amino acid sequence of SEQ ID NO:8.

[0011] Another aspect provided herein is a composition for treating TLR2-related diseases, comprising a pharmaceutically acceptable carrier and at least one peptide comprising a fragment as described in SEQ ID NO:8.

[0012] Another aspect provided in this article is a treatment for TLR2-related diseases, comprising administering a therapeutically effective amount of a synthetic peptide to an individual in need, the synthetic peptide comprising an amino acid sequence consisting of SEQ ID NO:8.

[0013] In one specific embodiment, the synthetic peptide is administered orally or parenterally.

[0014] Another aspect provided in this article is a synthetic peptide with therapeutic effects on obesity, comprising an amino acid sequence selected from the group consisting of SEQ ID NO:10, SEQ ID NO:15, SEQ ID NO:19, and SEQ ID NO:20, but with a maximum length of 50 amino acids.

[0015] In one specific embodiment, the synthetic peptide has a length of up to 30 amino acids.

[0016] In one specific embodiment, the synthetic peptide consists of the amino acid sequence of SEQ ID NO:10.

[0017] In one specific embodiment, the synthetic peptide consists of the amino acid sequence of SEQ ID NO:8.

[0018] In one specific embodiment, the synthetic peptide consists of the amino acid sequence of SEQ ID NO:15.

[0019] In one specific embodiment, the synthetic peptide consists of the amino acid sequence of SEQ ID NO:19.

[0020] In one specific embodiment, the synthetic peptide consists of the amino acid sequence of SEQ ID NO:20.

[0021] Another aspect provided herein is a pharmaceutical composition for treating obesity, comprising a pharmaceutically acceptable carrier and at least one peptide comprising a fragment selected from the group consisting of SEQ ID NO:10, SEQ ID NO:15, SEQ ID NO:19, and SEQ ID NO:20.

[0022] Another aspect provided herein is a method for treating obesity, comprising administering to an individual in need a therapeutically effective amount of a synthetic peptide comprising an amino acid sequence consisting of SEQ ID NO:10, SEQ ID NO:15, SEQ ID NO:19, and SEQ ID NO:20.

[0023] In one specific embodiment, the synthetic peptide is administered orally or parenterally.

[0024] In one specific embodiment, the synthetic peptide provides the effect of promoting an immune response.

[0025] In one specific embodiment, the peptide is co-administered with a vaccine and serves as an adjuvant.

[0026] Another aspect provided herein is a method for preventing small bowel cancer and reducing the toxicity of small bowel carcinogens, comprising administering to an individual in need a therapeutically effective amount of an isolated peptide comprising the peptides disclosed herein.

[0027] In one specific embodiment, the synthetic peptide is administered orally or parenterally.

[0028] Another aspect provided in this article is a method for treating a disease caused by intestinal epithelial barrier dysfunction, comprising administering a therapeutically effective amount of a peptide as disclosed herein to an individual in need.

[0029] In one specific embodiment, the disease caused by intestinal epithelial barrier dysfunction is inflammatory bowel disease, milk diarrhea, diarrheal disease, type II diabetes, obesity, or non-alcoholic fatty liver disease (NAFLD). Attached Figure Description

[0030] The foregoing description of the invention and the following detailed description of the invention will be better understood when read in conjunction with the accompanying drawings. Preferred embodiments are shown in the drawings to illustrate the invention.

[0031] As shown in the attached diagram:

[0032] Figure 1 shows the induction of TLR2 in recombinant HEK-293 by the peptides disclosed in this paper: (A) Recombinant HEK-293 functionally overexpressing human TLR and a reporter gene (SEAP); (B) HEK-293 blank as a negative control. Only AMS5 showed TLR-inducing activity, while other peptides did not. PC: positive control; NC: negative control; GST: glutamyl thiotransferase (blank).

[0033] Figure 2The image shows the reduction of zebrafish weight through peptide treatment as disclosed in Example 3 of this document. Zebrafish weight was measured on days 0, 1, 3, 6, 9, 12, and 13.

[0034] Figure 3 The figures show the body weight (top) and blood glucose levels (bottom) of zebrafish treated with the peptides disclosed herein on day 13 of Example 3. #: P < 0.05; ##: P < 0.01; ***: P < 0.001. (The statistical marker *** refers to the comparison between body weight on day 13 and day 0.)

[0035] Figure 4 The gene expression levels of MPX, IL10, CD86, and IL4 in AMS5-treated zebrafish were detected by qPCR on day 13. The Y-axis (-ΔΔCT) represents the difference in amplification threshold cycle. **: P < 0.01; ****: P < 0.0001.

[0036] Figure 5 The image shows the reduction of zebrafish body weight through peptide treatment as disclosed in Example 4 of this document. Zebrafish body weight was measured on days 0, 1, 3, 6, 9, 12, and 13.

[0037] Figure 6 The figures show the body weight (top) and blood glucose levels (bottom) of zebrafish treated with the peptides disclosed herein on day 12 of Example 4. ****: P < 0.0001.

[0038] Figure 7 The image shows the reduction of zebrafish weight through peptide treatment as disclosed in Example 5 of this document. Zebrafish weight was measured on days 0, 1, 3, 6, 9, 12, and 13.

[0039] Figure 8 The figures show the body weight (top and middle) and blood glucose levels (bottom) of zebrafish treated with the peptides described herein on day 13 of Example 5. *: P < 0.05; **: P < 0.01. Detailed Implementation

[0040] When read in conjunction with the accompanying drawings, the following embodiments are provided to clearly illustrate the above and other technical contents, features, and effects of the present invention. Since the contents disclosed in this invention should be readily understood and implemented by those skilled in the art, all equivalent changes or modifications that do not depart from the concept of the invention should be covered by the appended claims.

[0041] Unless otherwise stated, the following terms used in this application, including those in the specification and claims, have the definitions given below.

[0042] As used in the specification and appended claims, the singular forms “a,” “an,” and “the” include the plural objects unless the context clearly indicates otherwise. Unless otherwise stated, conventional methods of protein chemistry, biochemistry, recombinant DNA technology, and pharmacology are employed. In this application, the use of “or” or “and” means “and / or” unless otherwise stated. Furthermore, the use of the term “comprising (gerund)” and other forms such as “comprising (verb),” “comprising (singular verb),” and “comprising (passive)” is not restrictive. Section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described.

[0043] The term "amino acid" as used in this article is used in its broadest sense, including, but not limited to, naturally occurring L-type amino acids. -Amino acids or residues.

[0044] The terms “polypeptide,” “peptide,” and “protein,” used interchangeably herein, refer to polymeric forms of amino acids of any length, including naturally occurring amino acids, encoded or non-coding amino acids, chemically or biochemically modified, derived, or engineered amino acids, amino acid analogs, peptide mimics, and ester peptides, as well as peptides having modified, cyclic, bicyclic, ester-cyclic, or ester-bicyclic peptide backbones. This term includes both single-chain proteins and polymers.

[0045] The term "ligand" refers to a molecule that binds to another molecule, including receptors.

[0046] As used herein, terms such as “effective amount” or “therapeutic effective amount” refer to an adequate quantity of a drug or compound administered that will, to some extent, reduce one or more symptoms of the disease or condition being treated. The result may be a reduction and / or alleviation of the signs, symptoms, or cause of the disease, or any other desired alteration of the biological system. For example, an “effective amount” for therapeutic use is the amount of a composition containing the peptides or proteins disclosed herein required to provide a clinically significant reduction in the symptoms of a disease. In any case, the appropriate “effective” amount can be determined using techniques such as dose escalation studies.

[0047] As used herein, the term "composition" refers to a product obtained by mixing or combining more than one active ingredient, including both fixed and non-fixed combinations of that active ingredient. The term "fixed combination" refers to the simultaneous administration of the active ingredient and an adjuvant to the patient as a single entity or dose. The term "non-fixed combination" refers to the simultaneous, concurrent, or sequential administration of the active ingredient and an adjuvant as separate entities to the patient without a specific time limit for intervention, wherein such administration provides the efficacy of both compounds in the patient. The latter also applies to cocktail therapy, such as the administration of three or more active ingredients.

[0048] As used herein, the term "composition" also refers to a mixture that typically contains a pharmaceutically acceptable carrier, such as a pharmaceutically acceptable carrier or excipient conventional in the art.

[0049] As used in this article, the term "co-administration" is intended to cover the administration of selected therapeutic agents to a single patient, and is intended to include treatment regimens in which agents are administered sequentially or simultaneously via the same or different routes.

[0050] As used in this article, the term "carrier" refers to a relatively non-toxic compound or reagent that promotes the incorporation of a compound into cells or tissues.

[0051] Pharmaceutically acceptable carriers may be selected from, for example, excipients, adjuvants, diluents, fillers or bulking agents, granulators, coating agents, release control agents, binders, disintegrants, lubricants, preservatives, antioxidants, buffers, suspending agents, thickeners, flavoring agents, sweeteners, masking agents, stabilizers, or any other excipients commonly used in pharmaceutical compositions.

[0052] Examples of suitable excipients include, but are not limited to, lactose, glucose, sucrose, sorbitol, mannose, starch, gum arabic, calcium phosphate, alginate, astragalus gum, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone (PVP), cellulose, ddH2O, syrup, or methylcellulose.

[0053] "Pharmaceutical-acceptable carriers" refer to any conventional type of non-toxic solid, semi-solid, or liquid filler, diluent, encapsulation material, formulation adjuvant, or any conventional type of excipient. A pharmaceutically acceptable carrier is non-toxic to the receptor at the dose and concentration used and is compatible with other components of the formulation.

[0054] As used in this article, the terms "treatment" include reducing, alleviating, or improving at least one symptom of a disease or condition, preventing other symptoms, suppressing a disease or condition, for example, preventing the development of a disease or condition, relieving a disease or condition, causing a disease or condition to subside, alleviating symptoms caused by a disease or condition, or preventing and / or therapeutically stopping the symptoms of a disease or condition.

[0055] As used in this article, the term "disease" refers to any symptom, infection, condition, or syndrome that requires or necessitates medical intervention. Such medical intervention may include treatment, diagnosis, and / or prevention.

[0056] *Ekkermansia myxophilus* is a type of mucin-degrading bacterium in the human gut, first discovered in 2004. It is a Gram-negative, strictly anaerobic, non-motile, non-spore-forming oval bacterium. *Ekkermansia myxophilus* is believed to have anti-inflammatory effects in humans, and studies have shown an inverse relationship between its colonization and inflammatory conditions such as appendicitis or inflammatory bowel disease (IBD).

[0057] It is believed that a decrease in *Ekkermansia myxophilus* in the human gut is highly correlated with certain pathological conditions, thus *Ekkermansia myxophilus* is a potential beneficial microorganism because of its potential use as a medicine or nutritional food.

[0058] Amuc_1100* is an outer membrane protein of *Akermansia* that has been identified and found to activate intracellular signaling regulated by TLR2 (TLR2) in intestinal epithelial cells, thereby contributing to the enhancement of the intestinal barrier. Amuc_1100* has also been shown to participate in immune responses, particularly in inducing the production of interleukin-10 (IL-10), an anti-inflammatory cytokine.

[0059] TLRs are transmembrane proteins, comprising 13 different types, and are expressed in a wide range of cells and tissues, including immune-active cells such as macrophages and neutrophils, vascular endothelial cells, and intrinsic renal cells such as renal tubular epithelial cells. Activation of TLRs induces the expression of inflammatory cytokines, such as tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6), and induces inflammatory responses through, for example, activation of nuclear factor-kappa B (NF-κB), a transcription factor. The activation of various cells regulated by these TLRs is known to be involved in immune-inflammatory diseases such as sepsis, acute renal failure, chronic kidney disease, acute respiratory distress syndrome, scleroderma, acute pancreatitis, and chronic obstructive pulmonary disease.

[0060] TLR2, also known as TLR2 receptor 2, is a protein encoded by the TLR2 gene in humans. TLR2 is also referred to as CD282 (differentiation cluster 282). TLR2 is a type of TLR2 receptor and plays a role in the immune system. It is a membrane protein, a receptor, that appears on the surface of certain cells and recognizes foreign substances, transmitting appropriate signals to cells of the immune system. In the gut, TLR2 regulates the expression of CYP1A1, a key enzyme in the removal of carcinogenic polycyclic aromatic hydrocarbons (PAHs) such as benzopyrene.

[0061] As used in this article, "TLR2-related disease," "TLR2-related condition or disease," etc., refer to any disease state related to TLR2 receptor 2. These diseases or conditions include, but are not limited to, infectious diseases, inflammatory diseases, respiratory diseases, immune diseases, and gastrointestinal and colorectal diseases.

[0062] In some specific embodiments of the present invention, TLR2-related diseases are intestinal epithelial barrier dysfunction, such as inflammatory bowel disease, diarrhea, diarrheal diseases, type II diabetes, obesity, or non-alcoholic fatty liver disease (NAFLD). Therefore, as intestinal epithelial barrier dysfunction is reduced, the absorption of carcinogens by the intestine decreases, further reducing the toxicity of intestinal carcinogens and preventing small bowel cancer. Furthermore, due to intestinal epithelial barrier failure, TLR2-related diseases are further associated with obesity. Therefore, TLR2-related diseases include obesity.

[0063] The term "obesity" as used in this article refers to the excessive accumulation of body fat.

[0064] As used herein, the terms “immune response” or “immunological response” to an antigen or composition refer to the development of a humoral and / or cellular immune response in an individual to that antigen or composition.

[0065] According to the present invention, compositions or pharmaceutical compositions comprising the peptide can be prepared in any form suitable for oral, parenteral, topical, intranasal, intrabronchial, sublingual, rectal, or transdermal administration. When the compositions are intended for parenteral administration, they can be formulated for intravenous, intramuscular, intraperitoneal, subcutaneous administration or direct delivery to the target organ or tissue by injection, infusion, or other delivery methods.

[0066] Pharmaceutical dosage forms suitable for oral administration include tablets, capsules (hard or soft shell), capsule tablets, pills, lozenges, syrups, solutions, powders, granules, elixirs, suspensions, sublingual tablets, thin sheets, or patches, such as buccal patches.

[0067] For oral administration, fine powders or granules may contain diluents, dispersants, and / or surfactants, and may be present in aqueous or syrup form, in a dry state as capsules or sachets, or in non-aqueous or suspension form, which may contain suspending agents, tablets that may contain binders and lubricants, or suspensions in water or syrup. Flavoring agents, preservatives, suspending agents, thickeners, or emulsifiers may be included when needed or necessary. Tablets and granules are preferred forms for oral administration and may be coated.

[0068] The pharmaceutical compositions disclosed herein can be preserved by lyophilization and can be reconstituted with a suitable carrier prior to use. Lyophilization and reconstitution can be performed according to techniques common to the art, and those skilled in the art will understand that lyophilization and reconstitution result in a certain degree of loss of activity, and the pharmaceutical dosage should be adjusted upwards to compensate for this. The pharmaceutical compositions disclosed herein can also be manufactured by, for example, spray drying, drum drying, or vacuum drying, but are not limited thereto.

[0069] As defined herein, Amuc_1100 refers to the full length of the protein Amuc_1100, which is available from a general database; Amuc_1100* as defined herein refers to the protein Amuc_1100 that does not contain its transmembrane domain, and is represented herein as SEQ ID NO:1.

[0070] According to the present invention, a number of synthetic peptides have been designed based on amino acid fragments derived from Amuc_1100. Specifically, the synthetic peptides comprise the group consisting of SEQ ID NO:10, SEQ ID NO:15, SEQ ID NO:19, and SEQ ID NO:20.

[0071] In some embodiments, the peptide has a length of up to 50 amino acids. In some embodiments, the peptide has a length of up to 40 amino acids. In some embodiments, the peptide has a length of up to 30 amino acids. In some embodiments, the peptide has a length of up to 20 amino acids.

[0072] In some specific embodiments, the peptides used herein serve as ligands that initiate downstream signaling of TLR receptors (TLRs), particularly TLR2.

[0073] This article provides, on the one hand, a synthetic peptide that interacts with TLR2, which contains an amino acid sequence consisting of SEQ ID NO:8; but the length is at most 50 amino acids.

[0074] In one specific embodiment, the synthetic peptide has a length of up to 30 amino acids.

[0075] In one specific embodiment, the synthetic peptide consists of the amino acid sequence of SEQ ID NO:8.

[0076] Another aspect provided herein is a composition for treating TLR2-related diseases, comprising a pharmaceutically acceptable carrier and at least one peptide comprising the fragment shown in SEQ ID NO:8.

[0077] Another aspect of this article provides a method for treating TLR2-related diseases, comprising administering a therapeutically effective amount of a synthetic peptide to an individual in need, the synthetic peptide comprising an amino acid sequence consisting of SEQ ID NO:8.

[0078] In one specific embodiment, the synthetic peptide is administered orally or parenterally.

[0079] Another aspect provided in this article is a synthetic peptide with therapeutic effects on obesity, comprising an amino acid sequence selected from the group consisting of SEQ ID NO:10, SEQ ID NO:15, SEQ ID NO:19, and SEQ ID NO:20, but with a maximum length of 50 amino acids.

[0080] In one specific embodiment, the synthetic peptide has a length of up to 30 amino acids.

[0081] In one specific embodiment, the synthetic peptide consists of the amino acid sequence of SEQ ID NO:10.

[0082] In one specific embodiment, the synthetic peptide consists of the amino acid sequence of SEQ ID NO:8.

[0083] In one specific embodiment, the synthetic peptide consists of the amino acid sequence of SEQ ID NO:15.

[0084] In one specific embodiment, the synthetic peptide consists of the amino acid sequence of SEQ ID NO:19.

[0085] In one specific embodiment, the synthetic peptide consists of the amino acid sequence of SEQ ID NO:20.

[0086] Another aspect provided herein is a pharmaceutical composition for treating obesity, comprising a pharmaceutically acceptable carrier and at least one peptide comprising a fragment selected from the group consisting of SEQ ID NO:10, SEQ ID NO:15, SEQ ID NO:19, and SEQ ID NO:20.

[0087] Another aspect provided herein is a method for treating obesity, comprising administering a therapeutically effective amount of a synthetic peptide to an individual in need, the synthetic peptide comprising an amino acid sequence consisting of SEQ ID NO:10, SEQ ID NO:15, SEQ ID NO:19, and SEQ ID NO:20.

[0088] In one specific embodiment, the synthetic peptide is administered orally or parenterally.

[0089] In one specific embodiment, the synthetic peptide provides the effect of promoting an immune response.

[0090] In one specific embodiment, the peptide is co-administered with a vaccine and serves as an adjuvant.

[0091] Another aspect of the method provided herein for preventing small bowel cancer and reducing the toxicity of intestinal carcinogens includes administering a therapeutically effective amount of a separated peptide containing the peptide disclosed herein to an individual in need.

[0092] In one specific embodiment, the synthetic peptide is administered orally or parenterally.

[0093] Another aspect provided in this article is a method for treating a disease caused by intestinal epithelial barrier dysfunction, which involves administering a therapeutically effective amount of a peptide as disclosed herein to an individual in need.

[0094] In one specific embodiment, the disease caused by intestinal epithelial barrier dysfunction is inflammatory bowel disease, milk diarrhea, diarrheal disease, type II diabetes, obesity, or non-alcoholic fatty liver disease (NAFLD).

[0095] Example

[0096] Example 1. Preparation of Amuc_1100* cleavage peptide

[0097] The full length of Amuc_1100* consists of approximately 317 amino acids, totaling about 17,384 kDa (SEQ ID NO:1).

[0098] Amuc_1100* can also be divided into the segments shown in Table 1 below.

[0099] Table 1

[0100] name Serial Number sequence AMS1 SEQ ID NO:2 IVNSKRSELDKKISIAAKEIKSANAAEITPSR AMS2 SEQ ID NO:3 SSNEELEKELNRYAKAVGSLETAYKPFLASSA AMS3 SEQ ID NO:4 LVPTTPTAFQNELKTFRDSLISSCKKK AMS4 SEQ ID NO:5 NILITDTSSWLGFQVYSTQAPSVQAASTLGFELK AMS4-1 SEQ ID NO:6 NILITDTSSWLGFQVY AMS4-2 SEQ ID NO:7 STQAPSVQAASTLGFELK AMS5 SEQ ID NO:8 AINSLVNKLAECGLSKFIKVYRPQL AMS5-1 SEQ ID NO:9 AINSLVNKLAEC AMS5-2 SEQ ID NO:10 GLSKFIKVYRPQL AMS6 SEQ ID NO:11 PIETPANNPEESDEADQAPWTPMP AMS7 SEQ ID NO:12 LEIAFQGDRESVLKAMNAITGMQDYLF AMS7-1 SEQ ID NO:13 LEIAFQGDRESVLK AMS7-2 SEQ ID NO:14 AMNAITGMQDYLF AMS8 SEQ ID NO:15 TVNSIRIRNER AMS9 SEQ ID NO:16 MMPPPIANPAAAKPAAAQPATGAASL AMS10 SEQ ID NO:17 TPADEAAAPAAPAIQQVIKPYMGK AMS11 SEQ ID NO:18 EQVFVQVSLNLVHFNQPKAQEPSED AMS11-1 SEQ ID NO:19 EQVFVQVSLNL AMS11-2 SEQ ID NO:20 VHFNQPKAQEPSED

[0101] The generation and purification of the peptides listed in Table 1 include the following steps.

[0102] The effector plastids were derived from pET28a containing the Amuc_1100* fragment. For expression in *E. coli*, the fragment was selected and implanted into the C-terminus of glutamyl-1100* S-transferase (GST) to form a fusion protein. Bacterial cultures were cultured in LB medium, and then induced in the mid-exponential phase by adding 1 mM IPTG to the growth medium. After 3 hours of IPTG induction, the bacteria were centrifuged and stored at -20°C.

[0103] The bacteria were resuspended and lysed with lysozyme and by ultrasound. After centrifugation, the supernatant was collected and the desired peptide fragments were purified by metal affinity purification using ABT glutamic acid thioresin under natural conditions. After buffer exchange in 0.1x PBS using a 10K snakeskin dialysis column, the washed peptides were confirmed by BCA analysis, and the peptide samples were stored at -20°C.

[0104] After purification, use Biorad's CHT TM Ceramic hydroxyapatite microbeads were used to remove endotoxins from washed peptides. Endotoxin removal was confirmed by LAL (Limulis Amebocyte Lysate) assay kit. Endotoxin levels in all peptide samples were measured at 10 EU / mL.

[0105] Example 2

[0106] Human embryonic kidney cells 293 (HEK-293) were treated with the peptide fragments revealed in this paper.

[0107] Samples and controls were tested on recombinant HEK-293 cell lines, with duplicate replicates. These cell lines functionally overexpress human TLRs and a reporter gene, a secreted alkaline phosphatase (SEAP). The generation of this reporter gene is driven by an NF-κB inducible promoter. Activation levels are expressed as optical density (OD).

[0108] The 100 μg / ml sample was diluted to a final concentration of 10 μg / ml in the reaction volume. Samples and controls were tested in duplicate wells. BSA was used as a negative control.

[0109] SEAP reporters were detected by OD values. The mean non-inducible (NI) value was subtracted from the OD value. The average of the duplicates after subtracting NI was taken and represented by a histogram. The reagents and materials used in this example were provided by Invivogen.

[0110] Figure 1 shows the screening of peptides for TLR-induced TLR as revealed in this paper. Figure 1A As shown, the human TLR receptor (TLR) responds strongly and specifically to AMS5 activation. In contrast, the blank control group, HEK-293, a reporter gene with an inactive TLR, showed no response to treatment with any of the peptides described herein. Figure 1B ).

[0111] Therefore, it can be concluded that AMS5 can activate TLR2, while other fragments of Amuc_1100 cannot. Thus, AMS5 is a potential drug for TLR2-related diseases.

[0112] Example 3: Treatment of Obesity

[0113] Zebrafish have long been used as a model of obesity in numerous reports (Hasumura et al. Nutrition & Metabolism (2012); Tainaka et al. Nutrition & Metabolism (2011); Oka et al. BMCPhysiology (2010); Shimada et al. Nutrition & Metabolism (2015); Meguro et al. Scientific Reports (2019); Montalbano et al. Endocrine (2018); Yang et al. Cancer (2019)). In this paper, zebrafish are also used as a subject for the treatment of obesity with the peptides disclosed in this paper.

[0114] All peptides used in this example were purified using CHT beads to remove endotoxins. Further LAL assays confirmed that the residual endotoxin level was less than 0.25 EU / ml.

[0115] Zebrafish were placed in water at 25°C (pH 6.5-7.5, conductivity 250-750 S·m). -1 Zebrafish were raised in environments with NH3 < 0.25 ppm, NO2 < 0.25 ppm, and NO3 < 0.5 ppm, and fed a normal diet (brine shrimp). Each experimental group included 10 zebrafish (n = 10).

[0116] Zebrafish were tube-fed according to the method described in Collymore et al. (Journal of Visualized Experiment (2013)). For 13 days of the experiment, zebrafish were fed the peptide fragment described herein at a dose of 0.03 μg / day / fish, double the usual dose. Zebrafish weights were measured on days 0, 1, 3, 6, 9, 12, and 13. In this embodiment, five groups of zebrafish were administered the peptide described herein in combination with:

[0117] Group 1: AMS4-1 + AMS4-2

[0118] Group 2: GST (blank)

[0119] Group 3: AMS1 + AMS2

[0120] Group 4: AMS3 + AMS9

[0121] Group 5: AMS5

[0122] Figure 2 The image shows the results of weight changes in zebrafish. Figure 2 As shown, zebrafish administered AMS5 in group 5 experienced a significant decrease in body weight. After 13 weeks of the experiment, zebrafish administered AMS5 showed an average weight loss of approximately 20 mg. In contrast, zebrafish in the other groups did not show any reduction in body weight.

[0123] Figure 3 The figure shows the body weight distribution of zebrafish at the end of the experiment (day 13), where the weight loss of zebrafish treated with AMS5 was statistically significant. However, glucose levels in the zebrafish serum did not decrease. Figure 3 (See the image below).

[0124] In addition, reverse transcription (RT)-qPCR was performed to detect the gene expression levels of MPX, IL10, CD86, and IL4 in zebrafish treated with AMS5 according to this embodiment. At the end of the experiment (day 13), the gene expression of IL10 and IL4 was significantly reduced, while the gene expression of MPX and CD86 was not reduced.

[0125] The study concluded that the peptide fragment AMS5 significantly reduced the body weight of zebrafish, and the reduction in body weight was associated with a decrease in the expression of IL10 and IL4, indicating that the inflammatory response was suppressed.

[0126] Example 4

[0127] The materials and methods used in this embodiment are similar to those in Embodiment 3, except for the following differences, which will not be repeated here.

[0128] In this embodiment, five groups of zebrafish were administered the peptides described herein:

[0129] Group 1: AMS11-2

[0130] Group 2: AMS11-1

[0131] Group 3: AMS8

[0132] Group 4: AMS5-2

[0133] Group 5: GST (blank)

[0134] Except for the GST (blank) treatment, the body weight of zebrafish in all groups was significantly reduced. Figure 5 Furthermore, compared to the control group (GST), the zebrafish maintained their original body weight, and their glucose levels remained unchanged after administration of the peptides used in this study. Figure 6 ).

[0135] Example 5

[0136] The materials and methods used in this embodiment are similar to those in Embodiment 3, except for the following differences, which will not be repeated here.

[0137] In this embodiment, zebrafish were fed a normal diet (brine shrimp) six times a day and the peptides disclosed herein were fed once a day to induce a spontaneous animal model.

[0138] In this embodiment, five groups of zebrafish were administered the peptides described herein:

[0139] Group 1: AMS5-2

[0140] Group 2: Amuc_1100*

[0141] Group 3: GST (blank)

[0142] Except for the GST (blank) treatment, the body weight of zebrafish in all groups was significantly reduced. Figure 7 ).

[0143] In addition, such as Figure 8 As shown, at the end of the experiment (day 13), compared to the original group and the GST group, the zebrafish treated with AMS5-2 and Amuc_1100* showed a further decrease in body weight. Furthermore, a decrease in glucose content was observed in the groups treated with AMS5-2 and Amuc_1100*. Figure 8 (See the image below).

[0144] Although preferred embodiments of the invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments have been provided by way of example only. Many variations, modifications, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the specific embodiments of the invention described herein can be used to practice the invention. It is intended that the following claims define the scope of the invention and thereby cover the methods and structures within the scope of these claims and their equivalents. sequence list <110> Huang Zhihong <120> TLR2-interacting peptides and compositions thereof <130> HJH0001CN <160> 20 <170> PatentIn version 3.5 <210> 1 <211> 287 <212> PRT <213> Akkermansia muciniphila <400> 1 Ile Val Asn Ser Lys Arg Ser Glu Leu Asp Lys Lys Ile Ser Ile Ala 1 5 10 15 Ala Lys Glu Ile Lys Ser Ala Asn Ala Ala Glu Ile Thr Pro Ser Arg 20 25 30 Ser Ser Asn Glu Glu Leu Glu Lys Glu Leu Asn Arg Tyr Ala Lys Ala 35 40 45 Val Gly Ser Leu Glu Thr Ala Tyr Lys Pro Phe Leu Ala Ser Ser Ala 50 55 60 Leu Val Pro Thr Thr Pro Thr Ala Phe Gln Asn Glu Leu Lys Thr Phe 65 70 75 80 Arg Asp Ser Leu Ile Ser Ser Cys Lys Lys Lys Asn Ile Leu Ile Thr 85 90 95 Asp Thr Ser Ser Trp Leu Gly Phe Gln Val Tyr Ser Thr Gln Ala Pro 100 105 110 Ser Val Gln Ala Ala Ser Thr Leu Gly Phe Glu Leu Lys Ala Ile Asn 115 120 125 Ser Leu Val Asn Lys Leu Ala Glu Cys Gly Leu Ser Lys Phe Ile Lys 130 135 140 Val Tyr Arg Pro Gln Leu Pro Ile Glu Thr Pro Ala Asn Asn Pro Glu 145 150 155 160 Glu Ser Asp Glu Ala Asp Gln Ala Pro Trp Thr Pro Met Pro Leu Glu 165 170 175 Ile Ala Phe Gln Gly Asp Arg Glu Ser Val Leu Lys Ala Met Asn Ala 180 185 190 Ile Thr Gly Met Gln Asp Tyr Leu Phe Thr Val Asn Ser Ile Arg Ile 195 200 205 Arg Asn Glu Arg Met Met Pro Pro Pro Ile Ala Asn Pro Ala Ala Ala 210 215 220 Lys Pro Ala Ala Ala Gln Pro Ala Thr Gly Ala Ala Ser Leu Thr Pro 225 230 235 240 Ala Asp Glu Ala Ala Ala Pro Ala Ala Pro Ala Ile Gln Gln Val Ile 245 250 255 Lys Pro Tyr Met Gly Lys Glu Gln Val Phe Val Gln Val Ser Leu Asn 260 265 270 Leu Val His Phe Asn Gln Pro Lys Ala Gln Glu Pro Ser Glu Asp 275 280 285 <210> 2 <211> 32 <212> PRT <213> Akkermansia muciniphila <400> 2 Ile Val Asn Ser Lys Arg Ser Glu Leu Asp Lys Lys Ile Ser Ile Ala 1 5 10 15 Ala Lys Glu Ile Lys Ser Ala Asn Ala Ala Glu Ile Thr Pro Ser Arg 20 25 30 <210> 3 <211> 32 <212> PRT <213> Akkermansia muciniphila <400> 3 Ser Ser Asn Glu Glu Leu Glu Lys Glu Leu Asn Arg Tyr Ala Lys Ala 1 5 10 15 Val Gly Ser Leu Glu Thr Ala Tyr Lys Pro Phe Leu Ala Ser Ser Ala 20 25 30 <210> 4 <211> 27 <212> PRT <213> Akkermansia muciniphila <400> 4 Leu Val Pro Thr Thr Pro Thr Ala Phe Gln Asn Glu Leu Lys Thr Phe 1 5 10 15 Arg Asp Ser Leu Ile Ser Ser Cys Lys Lys Lys 20 25 <210> 5 <211> 34 <212> PRT <213> Akkermansia muciniphila <400> 5 Asn Ile Leu Ile Thr Asp Thr Ser Ser Trp Leu Gly Phe Gln Val Tyr 1 5 10 15 Ser Thr Gln Ala Pro Ser Val Gln Ala Ala Ser Thr Leu Gly Phe Glu 20 25 30 Leu Lys <210> 6 <211> 16 <212> PRT <213> Akkermansia muciniphila <400> 6 Asn Ile Leu Ile Thr Asp Thr Ser Ser Trp Leu Gly Phe Gln Val Tyr 1 5 10 15 <210> 7 <211> 18 <212> PRT <213> Akkermansia muciniphila <400> 7 Ser Thr Gln Ala Pro Ser Val Gln Ala Ala Ser Thr Leu Gly Phe Glu 1 5 10 15 Leu Lys <210> 8 <211> 25 <212> PRT <213> Akkermansia muciniphila <400> 8 Ala Ile Asn Ser Leu Val Asn Lys Leu Ala Glu Cys Gly Leu Ser Lys 1 5 10 15 Phe Ile Lys Val Tyr Arg Pro Gln Leu 20 25 <210> 9 <211> 12 <212> PRT <213> Akkermansia muciniphila <400> 9 Ala Ile Asn Ser Leu Val Asn Lys Leu Ala Glu Cys 1 5 10 <210> 10 <211> 13 <212> PRT <213> Akkermansia muciniphila <400> 10 Gly Leu Ser Lys Phe Ile Lys Val Tyr Arg Pro Gln Leu 1 5 10 <210> 11 <211> 24 <212> PRT <213> Akkermansia muciniphila <400> 11 Pro Ile Glu Thr Pro Ala Asn Asn Pro Glu Glu Ser Asp Glu Ala Asp 1 5 10 15 Gln Ala Pro Trp Thr Pro Met Pro 20 <210> 12 <211> 27 <212> PRT <213> Akkermansia muciniphila <400> 12 Leu Glu Ile Ala Phe Gln Gly Asp Arg Glu Ser Val Leu Lys Ala Met 1 5 10 15 Asn Ala Ile Thr Gly Met Gln Asp Tyr Leu Phe 20 25 <210> 13 <211> 14 <212> PRT <213> Akkermansia muciniphila <400> 13 Leu Glu Ile Ala Phe Gln Gly Asp Arg Glu Ser Val Leu Lys 1 5 10 <210> 14 <211> 13 <212> PRT <213> Akkermansia muciniphila <400> 14 Ala Met Asn Ala Ile Thr Gly Met Gln Asp Tyr Leu Phe 1 5 10 <210> 15 <211> 11 <212> PRT <213> Akkermansia muciniphila <400> 15 Thr Val Asn Ser Ile Arg Ile Arg Asn Glu Arg 1 5 10 <210> 16 <211> 26 <212> PRT <213> Akkermansia muciniphila <400> 16 Met Met Pro Pro Pro Ile Ala Asn Pro Ala Ala Ala Lys Pro Ala Ala 1 5 10 15 Ala Gln Pro Ala Thr Gly Ala Ala Ser Leu 20 25 <210> 17 <211> 24 <212> PRT <213> Akkermansia muciniphila <400> 17 Thr Pro Ala Asp Glu Ala Ala Ala Pro Ala Ala Pro Ala Ile Gln Gln 1 5 10 15 Val Ile Lys Pro Tyr Met Gly Lys 20 <210> 18 <211> 25 <212> PRT <213> Akkermansia muciniphila <400> 18 Glu Gln Val Phe Val Gln Val Ser Leu Asn Leu Val His Phe Asn Gln 1 5 10 15 Pro Lys Ala Gln Glu Pro Ser Glu Asp 20 25 <210> 19 <211> 11 <212> PRT <213> Akkermansia muciniphila <400> 19 Glu Gln Val Phe Val Gln Val Ser Leu Asn Leu 1 5 10 <210> 20 <211> 14 <212> PRT <213> Akkermansia muciniphila <400> 20 Val His Phe Asn Gln Pro Lys Ala Gln Glu Pro Ser Glu Asp 1 5 10

Claims

1. A pharmaceutical composition for treating TLR2 (Toll-like receptor 2) related diseases, comprising: a synthetic peptide consisting of the amino acid sequence of SEQ ID NO: 8; and a pharmaceutically acceptable carrier.

2. A pharmaceutical composition for treating obesity, comprising a synthetic peptide consisting of the amino acid sequence of SEQ ID NO:8; and a pharmaceutically acceptable carrier.

3. The pharmaceutical composition of claim 1, further comprising a synthetic peptide consisting of the amino acid sequence of SEQ ID NO:

15.

4. The pharmaceutical composition of claim 2, further comprising a synthetic peptide consisting of the amino acid sequence of SEQ ID NO:

15.

5. Use of a synthetic peptide in the preparation of a medicament for treating obesity, the synthetic peptide comprising the amino acid sequence of SEQ ID NO:

8.

6. The use as described in claim 5, wherein the synthetic peptide is administered orally or parenterally.

7. The use as claimed in claim 5, wherein the medicament further comprises a synthetic peptide consisting of the amino acid sequence of SEQ ID NO: 15.

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