A selective TNFR1 antagonist peptide SN61-4G and its application in inflammatory bowel disease

Abstract

The present invention relates to the field of biomedicine, and specifically provides a selective TNFR1 antagonist peptide SN61‑4G and its application in inflammatory bowel disease. The selective TNFR1 antagonist peptide SN61‑4G of the present invention is a highly selective straight-chain polypeptide containing 12 amino acid residues, a molecular weight of 1210.32 Daltons, an isoelectric point of 7.79, and the primary structure of the full sequence of the polypeptide is shown in SEQ ID NO: 1. The SN61‑4G provided by the present invention can directly interact with TNFR1, and its binding ability with TNFR1 is about 446nM; and it only binds to TNFR1, not to TNF‑α and TNFR2, and can competitively inhibit the interaction between TNF‑α and TNFR1; it has obvious anti-inflammatory activity on animal models of inflammatory bowel disease, and can be used as a therapeutic drug for inflammatory bowel disease.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a selective TNFR1 antagonist peptide SN61-4G and its application in inflammatory bowel disease. Background Art

[0002] Inflammatory bowel disease (IBD) is a nonspecific, chronic intestinal inflammatory disease encompassing ulcerative colitis (UC) and Crohn's disease (CD). Key clinical symptoms include abdominal pain, diarrhea, and bloody stools containing mucus, pus, and pus. Long-term, uncontrolled inflammation can also lead to intestinal tumors and serious extraintestinal complications in some patients. The incidence and prevalence of IBD have increased significantly worldwide in recent years. IBD primarily affects young adults aged 20 to 40, but also affects a significant proportion of children and the elderly. Its incurable nature, requiring long-term or lifelong medication, and even resulting in disability and death, significantly impacts patients, national public health, and the socioeconomic landscape.

[0003] The pathogenesis of IBD is complex. Studies have shown that its onset is closely linked to genetic, environmental, hormonal, immune, microbial infections, and the physical and psychological stress associated with the rapid development of modern society. Clinically, salicylates, steroid hormones, immunosuppressants, and targeted biologics targeting cytokines, receptors, and signaling molecules are used to treat varying degrees of IBD. However, these treatments are associated with suboptimal efficacy, high costs, severe side effects, and poor prognosis. Currently, no curative treatment exists. Therefore, new, safe, and effective treatment strategies and drugs are needed to address the challenges faced by current treatment options.

[0004] Anti-TNF-α biologics, standard interventions for the treatment of moderate to severe IBD, can significantly improve intestinal mucosal healing rates. However, because they completely block the biological function of TNF-α, effectively blocking signaling from both TNF-TNFR1 and TNF-TNFR2, they can compromise the body's immune homeostasis and immune surveillance, leading to the risk of serious, potentially fatal infections and malignancies. TNF-TNFR1 primarily transmits pro-inflammatory and apoptotic signals, while TNF-TNFR2 primarily transmits repair and anti-inflammatory signals, regulating immune homeostasis. Therefore, selectively blocking TNF-TNFR1, opening the TNF-TNFR2 pathway, and regulating the Th17 / Treg immune system to maintain immune homeostasis should be an effective strategy for treating IBD.

[0005] Currently, there are no reports on IBD therapeutic drugs that specifically and selectively antagonize TNFR1. Summary of the Invention

[0006] The present invention aims to provide a selective TNFR1 antagonist peptide SN61-4G and its use in inflammatory bowel disease. The present invention demonstrates the use of SN61-4G in treating inflammatory bowel disease using a dextran sodium sulfate-induced mouse ulcerative colitis model.

[0007] The inventor's team conducted in-depth research on the biological structure of the interaction between the anti-inflammatory active peptide Hydrostatin-SN61 (SDPRASHLTGIR) from the sea snake Blue Ring Sea and TNF-α and TNFRs. Through homology modeling, molecular design and other technical means, SN61-4G (SDPGASHLTGIR) was obtained, that is, the 4th arginine (R) of Hydrostatin-SN61 was replaced with glycine (G). Using surface plasmon resonance technology (SPR), it was found that it can bind to TNFR1 with a binding capacity of approximately K D =446nM, significantly higher than the binding ability of Hydrostatin-SN61 to TNFR1 (K D =77.4 μM). Further investigation of whether SN61-4G has target specificity for TNFR1 and its application in the dextran sodium sulfate-induced mouse ulcerative colitis model will be of great significance for the development of therapeutic drugs for inflammatory bowel disease.

[0008] To achieve the above objectives, the present invention provides the following technical solutions: Surface plasmon resonance (SPR) and microthermophoresis (MST) techniques demonstrated that SN61-4G has specific target sites and can directly interact with TNFR1, with a binding affinity of approximately 446 nM. It also binds exclusively to TNFR1 and not to TNF-α or TNFR2, demonstrating selectivity. Furthermore, it competitively inhibits the interaction between TNF-α and TNFR1, making it a selective TNFR1 antagonist peptide. Furthermore, using a dextran sodium sulfate (DSS)-induced mouse ulcerative colitis model, SN61-4G demonstrated significant anti-inflammatory activity in an animal model of inflammatory bowel disease. This demonstrates the therapeutic utility of the selective TNFR1 antagonist peptide SN61-4G in inflammatory bowel disease.

[0009] In a first aspect, the present invention provides a selective TNFR1 antagonist peptide SN61-4G, the amino acid sequence of which is shown in SEQ ID NO: 1.

[0010] The selective TNFR1 antagonist peptide SN61-4G is synthesized by solid-phase synthesis technology, and its purity and molecular weight are analyzed by HPLC and MS. The molecular weight of the selective TNFR1 antagonist peptide SN61-4G is 1210.32 Daltons and the isoelectric point is 7.79.

[0011] The selective TNFR1 antagonist peptide SN61-4G can directly interact with TNFR1, with a binding ability to TNFR1 of approximately 446nM. It only binds to TNFR1 and does not bind to TNF-α or TNFR2. It is selective and can competitively inhibit the interaction between TNFR1 and TNF-α.

[0012] The second aspect of the present invention provides a gene encoding the selective TNFR1 antagonist peptide SN61-4G, characterized in that the encoding gene is a DNA molecule as shown in SEQ ID NO: 2.

[0013] The third aspect of the present invention provides the use of the selective TNFR1 antagonist peptide SN61-4G in the preparation of a drug for treating inflammatory bowel disease.

[0014] Furthermore, the drug for treating inflammatory bowel disease is: a drug composition containing the selective TNFR1 antagonist peptide SN61-4G as the sole active ingredient, or a drug composition containing the selective TNFR1 antagonist peptide SN61-4G.

[0015] Furthermore, the inflammatory bowel disease includes Crohn's disease and ulcerative colitis; the drug for treating inflammatory bowel disease is the selective TNFR1 antagonist peptide SN61-4G.

[0016] Furthermore, the pharmaceutical composition is prepared into a pharmaceutical preparation with conventional pharmaceutical excipients in pharmacy.

[0017] Furthermore, the pharmaceutical preparation is a tablet, granule, dispersant, capsule, soft capsule, pill, injection, powder injection or aerosol.

[0018] The selective TNFR1 antagonist peptide SN61-4G provided by the present invention is a highly selective straight-chain polypeptide containing twelve amino acid residues. The primary structure of the entire polypeptide sequence is different from that of Hydrostatin-SN61, and its binding ability to TNFR1, TNFR2, and TNF-α is also different from that of Hydrostatin-SN61. It is a new selective TNFR1 antagonist peptide.

[0019] The present invention provides the use of a selective TNFR1 antagonist peptide SN61-4G in the treatment of inflammatory bowel disease, which has obvious anti-inflammatory activity in animal models of inflammatory bowel disease and can be used as a therapeutic drug for inflammatory bowel disease, providing an effective therapeutic drug for inflammatory bowel disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is the HPLC analysis result of SN61-4G.

[0021] Figure 2 This is the MS analysis result of SN61-4G.

[0022] Figure 3 The SPR technology was used to analyze the binding ability of SN61-4G to TNFR1; among them, the interaction between A, SN61-4G and TNFR1, the binding and dissociation of K D The value is about 446nM; B, interaction between SN61-4G and TNF-α, no binding; C, interaction between SN61-4G and TNFR2, no binding.

[0023] Figure 4 The MST technique was used to analyze the binding ability of SN61-4G to TNFR1; among them, the interaction between A, SN61-4G and TNFR1, the binding and dissociation of K D The value is about 453nM; B, interaction between SN61-4G and TNF-α, no binding; C, interaction between SN61-4G and TNFR2, no binding.

[0024] Figure 5 The competitive inhibition of SN61-4G on TNFR1-TNF-α binding was analyzed by MST technology; A, the interaction between TNFR1 and TNF-α (TNF-α fluorescent labeling), the binding and dissociation of K D The value is about 14nM; B, SN61-4G:TNFR1=10:1 (n:n) interacts with TNF-α (TNF-α fluorescently labeled), and the binding and dissociation constant K D The value is about 86nM; C, SN61-4G:TNFR1=20:1 (n:n) interacts with TNF-α (TNF-α fluorescent labeling), and the binding and dissociation constant K D The value is about 190nM; D, SN61-4G:TNFR1=40:1 (n:n) interacts with TNF-α (TNF-α fluorescent labeling), and the binding and dissociation constant K D The value is about 314nM; E, SN61-4G:TNFR1=80:1 (n:n) interacts with TNF-α (TNF-α fluorescent labeling), and the binding and dissociation constant K D The value is approximately 1040nM.

[0025] Figure 6 Effects of SN61-4G on body weight in a dextran sodium sulfate (DSS)-induced mouse ulcerative colitis model

[0026] Figure 7 The effect of SN61-4G on the disease activity index in the dextran sodium sulfate (DSS)-induced mouse ulcerative colitis model.

[0027] Figure 8 The effect of SN61-4G on the spleen index of mice in the dextran sulfate sodium (DSS)-induced mouse ulcerative colitis model.

[0028] Figure 9 The effect of SN61-4G on the colon length of mice in a dextran sodium sulfate (DSS)-induced mouse ulcerative colitis model.

[0029] Figure 10 shows the effect of SN61-4G on the pathological damage of the mouse colon in a dextran sulfate sodium (DSS)-induced mouse ulcerative colitis model. The image is a light micrograph of HE staining of tissue sections (200x).

[0030] Figure 11 This is the effect of SN61-4G on Foxp3+Treg cells in the mouse colon tissue in the dextran sulfate sodium (DSS)-induced mouse ulcerative colitis model. Light microscopic image of tissue section (200x).

[0031] Figure 12 The effect of SN61-4G on the MAPK pathway in the colon tissue of mice in the dextran sodium sulfate (DSS)-induced mouse ulcerative colitis model.

[0032] Figure 13 SN61-4G can inhibit the expression of CD4+IL-17A+ in the spleen of mice in the dextran sulfate sodium (DSS)-induced mouse ulcerative colitis model. Effect of Th17 cell ratio.

[0033] Figure 14 SN61-4G can inhibit the expression of CD4+CD25+Foxp3+ in the spleen of mice in the dextran sulfate sodium (DSS)-induced mouse ulcerative colitis model. Effect of Treg cell ratio.

[0034] Figure 15 SN61-4G has an effect on the expression of CD4+IL-17A+ in the mesenteric lymph nodes of mice in the dextran sulfate sodium (DSS)-induced mouse ulcerative colitis model. Effect of Th17 cell ratio.

[0035] Figure 16 SN61-4G inhibits CD4+CD25+Foxp3+ in mesenteric lymph nodes of mice in the dextran sulfate sodium (DSS)-induced mouse ulcerative colitis model. Effect of Treg cell ratio. DETAILED DESCRIPTION

[0036] The present invention will be described in detail below with reference to specific embodiments. The following examples will help those skilled in the art further understand the present invention, but the following examples should not be construed as limiting the present invention. It should be noted that those skilled in the art may make various adjustments and improvements without departing from the scope of the present invention. These modifications and improvements fall within the scope of protection of the present invention.

[0037] The experimental methods in the following examples are conventional methods unless otherwise specified.

[0038] Example 1: Synthesis and detection of selective TNFR1 antagonist peptide SN61-4G.

[0039] SN61-4G was synthesized by solid phase peptide synthesis and analyzed by HPLC ( Figure 1 ) and MS ( Figure 2 ) were used to analyze its purity and molecular weight. The results showed that its purity was >98% and its molecular weight was 1210.32 g / mol.

[0040] Hereinafter, the experiments of Examples 2-4 were carried out using SN61-4G prepared in Example 1.

[0041] Example 2: The interaction between SN61-4G and TNF-α, TNFR1 and TNFR2 coupled to the chip was detected using the biomacromolecule interaction analyzer BIACORE T200 based on surface plasmon resonance (SPR).

[0042] 1. Flow 1× PBS running buffer through the two channels set in the CM-5 sensor chip at a flow rate of 10 µl / min until the baseline level is reached.

[0043] 2. Activate the surface reactive groups of each channel of the chip with 1×PBS buffer and pre-enrich the ligands. Dissolve the lyophilized powders of TNF-α, TNFR1, and TNFR2 with 1×PBS buffer and determine the optimal pH value for the coating protein.

[0044] 3. Dilute each protein to a concentration of 50 μg / ml according to the optimal pH and inject it. Covalently couple TNF-α, TNFR1 and TNFR2 proteins to the surface of the sensor chip using the amino coupling method.

[0045] 4. When the 1× PBS running buffer reaches the baseline level, inject a series of SN61-4G concentrations and record the response value for each concentration.

[0046] like Figure 3As shown, SN61-4G can directly interact with TNFR1, and the binding ability to TNFR1 is about 446nM; SN61-4G does not bind to TNFR2 and TNF-α.

[0047] Comparative Example: The interaction between SN61 and TNF-α, TNFR1 and TNFR2 coupled to the chip was detected using the biomacromolecule interaction analyzer BIACORE T200 based on surface plasmon resonance (SPR).

[0048] The inventors' previously filed Chinese patent application CN111763244B discloses an anti-inflammatory peptide, Hydrostatin-SN61, from the sea snake (Hydrostatin cyanobacteria). Its gene sequence is: 5'-TCGGATCCCC GAGCATCACA CCTGACTGGA ATACGA-3'; the primary structure of the full polypeptide sequence is: Ser-Asp-Pro-Arg-Ala-Ser-His-Leu-Thr-Gly-Ile-Arg (SDPRASHLTGIR). It can directly bind to TNFR1 (see Example 2).

[0049] However, the target of Hydrostatin-SN61 disclosed in this patent is not specific. It binds to TNF-α, TNFR1 and TNFR2 to varying degrees, with binding abilities of approximately 100µM, 77.4µM and 200µM, respectively. It can competitively inhibit the binding ability of TNF-α to TNFR1 and TNF-α to TNFR2 to varying degrees.

[0050] SN61-4G of the present invention is target-specific and selective, binding only to TNFR1, but not to TNF-α or TNFR2. Its binding capacity to TNFR1 is approximately 446 nM, and it competitively inhibits the binding of TNFR1 to TNF-α. Therefore, SN61-4G of the present invention exhibits superior target specificity and target binding capacity, significantly outperforming Hydrostatin-SN61.

[0051] Example 3: Microthermophoresis (MST) was used to detect the interaction between SN61-4G and TNF-α, TNFR1 and TNFR2.

[0052] 1. Interaction of SN61-4G with TNF-α, TNFR1, and TNFR2

[0053] A series of gradient concentrations of SN61-4G were prepared at a dilution ratio of 1:3. Equal volumes of fluorescently labeled TNF-α / TNFR1 / TNFR2 200 nM were mixed with SN61-4G, and incubated in the dark for 15 min. An appropriate amount of sample was drawn with a capillary for MST detection. The time trajectory of the relative fluorescence value and the dose-response curve of thermophoresis were observed, and the affinity K was calculated by fitting using the software NTAffinityAnalysis v2.0.2. D value, to determine whether SN61-4G has target selectivity.

[0054] like Figure 4 As shown, SN61-4G can directly interact with TNFR1, and the binding ability to TNFR1 is about 453nM; SN61-4G does not bind to TNFR2 and TNF-α.

[0055] 2. Competitive inhibition of SN61-4G on the binding of TNF-α to TNFR1

[0056] A series of gradient concentrations of TNFR1 were prepared at a 1:1 dilution ratio. Equal volumes of fluorescently labeled TNF-α (200 nM) were evenly mixed with TNFR1 and incubated in the dark for 15 min. An appropriate amount of sample was drawn with a capillary tube for MST detection. The time trajectory of the relative fluorescence value and the dose-response curve of thermophoresis were observed, and the affinity K was calculated using the software NTAffinityAnalysis v2.0.2. D value.

[0057] SN61-4G and TNFR1 were mixed evenly at ratios of 10:1, 20:1, 40:1, and 80:1 (n:n). A series of gradient concentrations of TNFR1 and SN61-4G mixed solutions were prepared at a 1:1 dilution ratio. Equal volumes of fluorescently labeled TNF-α 200 nM were evenly mixed with the TNFR1 and SN61-4G mixed solutions and incubated in the dark for 15 minutes. An appropriate amount of sample was drawn with a capillary for MST detection. The time trajectory of the relative fluorescence value and the dose-response curve of thermophoresis were observed, and the affinity K was calculated by fitting using the software NTAffinityAnalysis v2.0.2. D value.

[0058] like Figure 5 As shown in Figure 2, with the increase of SN61-4G concentration, the K between TNFR1 and TNF-α D The value increased, indicating that SN61-4G can competitively inhibit the TNFR1-TNF-α interaction.

[0059] Example 4: Effect of SN61-4G on dextran sodium sulfate (DSS)-induced ulcerative colitis in mice.

[0060] The specific implementation steps are as follows: 6-8 week old male C57BL / 6 mice were selected and randomly divided into 5 groups, with 8 mice in each group. The blank group (Control) did not receive any treatment; 3% (w / v) DSS (commercially available, purchased from MP, MW 36,000-50,000) was added to the drinking water of the model group (Model) mice to induce colitis for 7 days; the other groups were given drugs at the same time as the modeling for 7 consecutive days. Positive control group: infliximab (IFX, 4 mg / kg / d, ip) and sulfasalazine (SASP, 400 mg / kg / d, ig); SN61-4G administration group: 400 μg / kg / d, ip. The weight changes of mice in each group were recorded daily ( Figure 6 ), SN61-4G can effectively alleviate the weight loss caused by colitis. The disease activity index (DAI) score was calculated according to Table 1. The results are as follows Figure 7 As shown, SN61-4G can effectively alleviate the symptoms of diarrhea and bloody stools in mice with colitis. After 7 days of treatment, the mice were killed by cervical dislocation, and the spleens were removed. The spleen weight was measured and the spleen index was calculated. The results showed that SN61-4G can effectively inhibit the spleen swelling caused by colitis ( Figure 8 ); The whole colon of the mouse was removed and it was found that SN61-4G could significantly improve the length of the mouse colon ( Figure 9 The terminal colon tissue was fixed with 10% formalin, sliced, and stained with HE to observe the changes in the colon. It was found that SN61-4G could effectively inhibit the colonic lesions in mice with ulcerative colitis ( Figure 10 ); Foxp3+ staining was performed on tissue sections, and it was found that SN61-4G could significantly increase the number of Foxp3+Treg cells in colon tissue ( Figure 11 Total protein of colon tissue was extracted and Western blot was performed to detect changes in MAPK signaling pathway. It was found that SN61-4G could inhibit the phosphorylation of ERK, JNK, and P38 ( Figure 12 The mouse spleen was ground and single cell suspension was prepared, which was then stained with fluorescent antibodies. The proportion of CD4+IL-17A+ Th17 cells in the spleen of the model mice was detected by flow cytometry ( Figure 13 ), and the proportion of CD4+CD25+Foxp3+ Treg cells ( Figure 14 The mesenteric lymph nodes of mice were removed, ground to prepare single cell suspension, and stained with fluorescent antibodies. The proportion of CD4+IL-17A+ Th17 cells in the mesenteric lymph nodes of model mice was detected by flow cytometry ( Figure 15 ), and the proportion of CD4+CD25+Foxp3+ Treg cells ( Figure 16 The results showed that SN61-4G can reduce the proportion of Th17 cells, increase the proportion of Treg cells, and shift the Th17 / Treg balance towards Treg.

[0061]

[0062] The above results show that SN61-4G can effectively treat DSS-induced colitis in mice.

[0063] The preferred embodiments of the present invention have been specifically described above, but the present invention is not limited to the described embodiments. Those skilled in the art may make various equivalent modifications or substitutions without departing from the spirit of the present invention. These equivalent modifications or substitutions are all included in the scope defined by the claims of this application. Sequence Listing <110> Shanghai University <120> A selective TNFR1 antagonist peptide SN61-4G and its application in inflammatory bowel disease <130> Description, claims <160> 2 <170> SIPOSequenceListing 1.0 <210> 1 <211> 12 <212> PRT <213> Artificial Sequence <400> 1 Ser Asp Pro Gly Ala Ser His Leu Thr Gly Ile Arg 1 5 10 <210> 2 <211> 36 <212> DNA <213> Artificial Sequence <400> 2 tcggatcccg gtgcatcaca cctgactgga atacga 36

Claims

1. Use of a selective TNFR1 antagonist peptide SN61-4G in the preparation of a drug for treating inflammatory bowel disease, wherein: The amino acid sequence of the selective TNFR1 antagonist peptide SN61-4G is shown in SEQ ID NO:

1.

2. The use according to claim 1, characterized in that: The drug for treating inflammatory bowel disease is: a drug composition with the selective TNFR1 antagonist peptide SN61-4G as the only active ingredient, or containing the selective TNFR1 antagonist peptide SN61-4G.

3. The use according to claim 1, characterized in that: The inflammatory bowel diseases include Crohn's disease and ulcerative colitis.

4. The use according to claim 1, characterized in that: The drug is prepared into a pharmaceutical preparation with conventional pharmaceutical excipients in pharmacy.

5. The use according to claim 4, characterized in that: The pharmaceutical preparation is tablet, granule, dispersant, capsule, dripping pill, injection, powder injection or aerosol.

6. The use according to claim 1, characterized in that: The selective TNFR1 antagonist peptide SN61-4G has a molecular weight of 1210.32 Daltons and an isoelectric point of 7.

79.

7. The use according to claim 1, characterized in that: The coding gene of the selective TNFR1 antagonist peptide SN61-4G is a DNA molecule as shown in SEQ ID NO:2.

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