CN115073553A - Selective TNFR1 antagonistic peptide SN61-4G and application thereof in inflammatory bowel disease - Google Patents

Abstract

The invention relates to the field of biomedicine, and particularly provides a selective TNFR1 antagonistic peptide SN61-4G and application thereof in inflammatory bowel diseases. The selective TNFR1 antagonistic peptide SN61-4G is a highly selective linear chain polypeptide, contains 12 amino acid residues, has the molecular weight of 1210.32 daltons, the isoelectric point of 7.79 and the primary structure of the complete sequence of the polypeptide shown as SEQ ID NO. 1. The SN61-4G provided by the invention can directly interact with TNFR1, and the binding capacity of the SN61-4G to TNFR1 is about 446 nM; and only binds to TNFR1, not TNF-alpha and TNFR2, and can competitively inhibit the interaction between TNF-alpha and TNFR 1; has obvious anti-inflammatory activity on animal models of inflammatory bowel disease, and can be applied as a therapeutic drug for inflammatory bowel disease.

Description

Selective TNFR1 antagonistic peptide SN61-4G and application thereof in inflammatory bowel disease

Technical Field

The invention relates to the technical field of biomedicine, in particular to a selective TNFR1 antagonistic peptide SN61-4G and application thereof in inflammatory bowel diseases.

Background

Inflammatory Bowel Disease (IBD) is a nonspecific chronic inflammatory Disease of the intestinal tract, including Ulcerative Colitis (UC) and Crohn's Disease (CD), with the main clinical symptoms of abdominal pain, diarrhea and mucopurulent bloody stool, and long-term uncontrolled inflammation also leading to intestinal tumors and serious extra-intestinal complications in some patients. The morbidity and the prevalence rate of IBD are remarkably increased in the world in recent years, the incidence population is mostly young and old people of 20-40 years old, and a certain proportion of children and old people are difficult to cure, take medicine for a long time to a lifetime, even cause disability and death, and bring great influence to the patient, the national public health and social economy.

The pathogenesis of IBD is complex, and researches show that the pathogenesis of IBD is closely related to factors such as genetics, environment, hormones, immunity, microbial infection, physical and psychological stress caused by rapid development of modern society and the like. Clinically, salicylic acids, steroid hormones, immunosuppressive agents and targeted biological agents taking cytokines, receptors and signaling molecules as treatment targets are used for treating IBD of different degrees, but the problems of unsatisfactory curative effect, high price, serious side effect and poor prognosis exist, and no radical treatment is available at present. Therefore, new safe and effective therapeutic strategies and drugs are sought to solve the problems faced by current therapeutic regimens.

The anti-TNF-alpha biological agent can obviously improve the healing rate of intestinal mucosa of a patient as a standard intervention measure for treating moderate and severe IBD, however, the biological function of TNF-alpha is completely blocked, namely the signal transduction of TNF-TNFR1 and TNF-TNFR2 is blocked, so that the immune homeostasis and the immune monitoring function of an organism are influenced, and serious and potentially lethal infection risk and malignant tumor risk are induced. TNF-TNFR1 primarily transmits pro-inflammatory and apoptotic signals; TNF-TNFR2 is mainly used for transmitting repair and inflammation inhibition signals and regulating immune balance; therefore, selective blocking of TNF-TNFR1, opening of TNF-TNFR2 pathway, and modulation of Th17/Treg to maintain immune balance should be an effective strategy for treating IBD.

At present, no IBD treatment drug which specifically and selectively antagonizes TNFR1 is reported.

Disclosure of Invention

The invention aims to provide a selective TNFR1 antagonistic peptide SN61-4G and application thereof in inflammatory bowel diseases. The invention proves that SN61-4G has the application of treating inflammatory bowel disease through a mouse ulcerative colitis model induced by dextran sodium sulfate.

The team of the inventor deeply researches the biological structure of the interaction of Hydrostatin-SN61(SDPRASHLTGIR) as an anti-inflammatory peptide of the krait, TNF-alpha and TNFRs, obtains SN61-4G (SDPGASHLTGIR) by technical means such as homologous modeling, molecular design and the like, namely, replacing arginine (R) at the 4 th position of Hydrostatin-SN61 with glycine (G), and finds that the Hydrostatin-SN can be combined with TNFR1 by using the Surface Plasmon Resonance (SPR) technology and the combination capacity is about K _D 446nM, significantly higher than the ability of Hydrostatin-SN61 to bind TNFR1 (K) _D 77.4 μ M). Further research on whether SN61-4G has target specificity to TNFR1 or not, and application of the SN61-4G to a mouse ulcerative colitis model induced by dextran sodium sulfate has important significance for developing a therapeutic drug for inflammatory bowel diseases.

In order to achieve the purpose, the invention provides the following technical scheme: by Surface Plasmon Resonance (SPR), microcalorimetric electrophoresis (MST) techniques, it was shown that: SN61-4G target is specific, can directly interact with TNFR1, and has the binding capacity of about 446nM with TNFR 1; and only binds to TNFR1, not TNF-alpha and TNFR2, and has selectivity; capable of competitively inhibiting the interaction between TNF-alpha and TNFR1, is a selective TNFR1 antagonist peptide. Further, the mouse model of ulcerative colitis induced by Dextran Sodium Sulfate (DSS) showed that: SN61-4G has significant anti-inflammatory activity against animal models of inflammatory bowel disease. The selective TNFR1 antagonistic peptide SN61-4G was demonstrated to have utility in the treatment of inflammatory bowel disease.

In a first aspect of the invention, a selective TNFR1 antagonistic peptide SN61-4G is provided, wherein the amino acid sequence of the selective TNFR1 antagonistic peptide SN61-4G is shown in SEQ ID NO:1 is shown.

The selective TNFR1 antagonistic peptide SN61-4G is synthesized by the following steps: SN61-4G is synthesized by solid phase synthesis technology, and purity and molecular weight are analyzed by HPLC and MS, the molecular weight of the selective TNFR1 antagonistic peptide SN61-4G is 1210.32 daltons, and isoelectric point is 7.79.

The selective TNFR1 antagonist peptide SN61-4G was able to interact directly with TNFR1, had a binding capacity of about 446nM to TNFR1, and bound only to TNFR1, but not to TNF- α and TNFR2, was selective and able to competitively inhibit the interaction of TNFR1 and TNF- α.

In a second aspect of the present invention, there is provided a gene encoding the selective TNFR1 antagonist peptide SN61-4G, wherein the encoding gene is a DNA molecule represented by SEQ ID NO. 2.

In a third aspect of the invention, the selective TNFR1 antagonistic peptide SN61-4G is provided for use in the preparation of a medicament for treating inflammatory bowel disease.

Further, the medicine for treating inflammatory bowel disease is as follows: takes the selective TNFR1 antagonistic peptide SN61-4G as the only active ingredient, or comprises the selective TNFR1 antagonistic peptide SN 61-4G.

Further, the inflammatory bowel disease includes crohn's disease and ulcerative colitis; the drug for treating inflammatory bowel disease is selective TNFR1 antagonistic peptide SN 61-4G.

Furthermore, the pharmaceutical composition and the conventional pharmaceutic adjuvants in pharmaceutics are prepared into a pharmaceutical preparation.

Furthermore, the medicinal preparation is tablets, granules, dispersing agents, capsules, soft capsules, dripping pills, injections, powder injections or aerosols.

The selective TNFR1 antagonistic peptide SN61-4G provided by the invention is a highly selective straight-chain polypeptide, contains twelve amino acid residues, has a polypeptide complete sequence primary structure different from that of Hydrostatin-SN61, has different binding capacities with TNFR1, TNFR2 and TNF-alpha from that of Hydrostatin-SN61, and is a novel selective TNFR1 antagonistic peptide.

The invention provides application of a selective TNFR1 antagonistic peptide SN61-4G in treating inflammatory bowel disease, has obvious anti-inflammatory activity on an inflammatory bowel disease animal model, can be applied as a therapeutic drug for inflammatory bowel disease, and provides an effective therapeutic drug for inflammatory bowel disease.

Drawings

FIG. 1 shows the results of HPLC analysis of SN 61-4G.

FIG. 2 shows the MS analysis results of SN 61-4G.

FIG. 3 is a graph showing the ability of SN61-4G to bind to TNFR1 using SPR techniques; wherein, the interaction between A, SN61-4G and TNFR1, the binding and dissociation of the mature K _D A value of about 446 nM; B. interaction of SN61-4G with TNF- α, no binding; C. SN61-4G interacts with TNFR2 without binding.

FIG. 4 is a MST assay for SN61-4G binding to TNFR 1; wherein, the interaction between A, SN61-4G and TNFR1, the binding and dissociation of the mature K _D A value of about 453 nM; B. interaction of SN61-4G with TNF- α, no binding; C. SN61-4G interacts with TNFR2 without binding.

FIG. 5 is a graph showing the competitive inhibition of TNFR1-TNF- α binding by SN61-4G analyzed using MST techniques; wherein A, TNFR1 interaction with TNF-alpha (TNF-alpha fluorescence labeling) binds to dissociation mature K _D A value of about 14 nM; B. SN 61-4G: TNFR 1-10: 1(n: n) interacts with TNF-a (TNF-a fluorescent label) and binds to dissociation constant K _D A value of about 86 nM; C. SN 61-4G: TNFR 1-20: 1(n: n) interacts with TNF-a (TNF-a fluorescent label) and binds to dissociation constant K _D A value of about 190 nM; D. SN 61-4G: TNFR 1-40: 1(n: n) interacts with TNF-a (TNF-a fluorescent label) and binds to dissociation constant K _D A value of about 314 nM; E. SN 61-4G: TNFR 1-80: 1(n: n) interacts with TNF-a (TNF-a fluorescent label) and binds to the dissociation constant K _D The value was approximately 1040 nM.

FIG. 6 is a graph of the effect of SN61-4G on mouse body weight in a Dextran Sodium Sulfate (DSS) -induced mouse ulcerative colitis model

FIG. 7 is a graph of the effect of SN61-4G on the disease Activity index in a mouse model of Dextran Sodium Sulfate (DSS) -induced ulcerative colitis.

FIG. 8 is a graph of the effect of SN61-4G on mouse spleen index in a Dextran Sodium Sulfate (DSS) -induced mouse model of ulcerative colitis.

FIG. 9 is an effect of SN61-4G on colon length in mice model of sodium dextran sulfate (DSS) -induced ulcerative colitis.

FIG. 10 is a graph of SN61-4G on the effects of histopathological lesions of the colon of mice in the Dextran Sodium Sulfate (DSS) -induced mouse ulcerative colitis model, histological sections HE stained by light microscopy (200-fold)

FIG. 11 is a photomicrograph (200-fold) of tissue sections showing the effect of SN61-4G on Foxp3+ Treg cells from mouse colon tissue in a Dextran Sodium Sulfate (DSS) -induced mouse ulcerative colitis model.

FIG. 12 is a graph of the effect of SN61-4G on MAPK pathway in mouse colon tissue in a Dextran Sodium Sulfate (DSS) -induced mouse model of ulcerative colitis.

FIG. 13 is a graph of the effect of SN61-4G on the proportion of CD4+ IL-17A + Th17 cells in the mouse spleen in a Dextran Sodium Sulfate (DSS) -induced model of mouse ulcerative colitis.

FIG. 14 is a graph of the effect of SN61-4G on the ratio of CD4+ CD25+ Foxp3+ Treg cells in the mouse spleen in a Dextran Sodium Sulfate (DSS) -induced mouse ulcerative colitis model.

FIG. 15 is a graph of the effect of SN61-4G on the proportion of CD4+ IL-17A + Th17 cells in the mesenteric lymph nodes of mice in a Dextran Sodium Sulfate (DSS) -induced mouse model of ulcerative colitis.

FIG. 16 is a graph of the effect of SN61-4G on the proportion of CD4+ CD25+ Foxp3+ Treg cells in the mesenteric lymph nodes of mice in a Dextran Sodium Sulfate (DSS) -induced mouse model of ulcerative colitis.

Detailed Description

The present invention will be described in detail with reference to specific examples. The following examples will assist those skilled in the art in further understanding the present invention, but the following examples should not be construed as limiting the present invention. It should be noted that it would be apparent to those skilled in the art that several modifications and improvements can be made without departing from the inventive concept. All falling within the scope of the present invention.

The experimental procedures in the following examples are conventional unless otherwise specified.

Example 1: synthesis and detection of selective TNFR1 antagonistic peptide SN61-4G

SN61-4G was synthesized by solid phase peptide synthesis and analyzed for purity and molecular weight by HPLC (FIG. 1) and MS (FIG. 2), indicating that the purity was > 98% and the molecular weight was 1210.32G/mol.

Next, experiments of examples 2 to 4 were carried out using SN61-4G obtained in example 1.

Example 2: the biomacromolecule interaction analyzer BIACORE T200 based on Surface Plasmon Resonance (SPR) is adopted to detect the interaction between SN61-4G and TNF-alpha, TNFR1 and TNFR2 coupled on the chip.

1. The 1 XPBS running buffer was flowed through the channels set in the two CM-5 sensor chips at a flow rate of 10. mu.l/min, respectively, until a baseline level was reached.

2. Activating surface reaction groups of each channel of the chip by using 1 XPBS buffer, simultaneously performing pre-enrichment of ligands, dissolving TNF-alpha, TNFR1 and TNFR2 freeze-dried powder by using 1 XPBS buffer, and determining the optimal pH value of the coating protein.

3. Diluting each protein to the concentration of 50ug/ml according to the optimum pH, injecting samples, and covalently coupling TNF-alpha, TNFR1 and TNFR2 proteins on the surface of the sensor chip by an amino coupling method.

4. After running a 1 XPBS running buffer to baseline levels, serial concentrations of SN61-4G were injected and response values were recorded for each concentration.

As shown in FIG. 3, SN61-4G was able to interact directly with TNFR1 and had approximately 446nM binding capacity to TNFR 1; SN61-4G does not bind to TNFR2, TNF-alpha.

Comparative example: the biomacromolecule interaction analyzer BIACORE T200 based on Surface Plasmon Resonance (SPR) is used for detecting the interaction between SN61 and TNF-alpha, TNFR1 and TNFR2 coupled on the chip.

Chinese patent CN111763244B previously filed by the inventor discloses anti-inflammatory peptide Hydrostatin-SN61 of a Qinghuan sea snake, the gene sequence of which is as follows: 5'-TCGGATCCCC GAGCATCACA CCTGACTGGA ATACGA-3', respectively; the primary structure of the polypeptide complete sequence is as follows: Ser-Asp-Pro-Arg-Ala-Ser-His-Leu-Thr-Gly-Ile-Arg (SDPRASHLTGIR). It binds directly to TNFR1 (see example 2).

However, the Hydrostatin-SN61 disclosed in the patent is not specific in target, and has different degrees of binding with TNF-alpha, TNFR1 and TNFR2, the binding capacity is about 100 mu M, 77.4 mu M and 200 mu M respectively, and the binding capacity of TNF-alpha and TNFR1 and TNF-alpha and TNFR2 can be competitively inhibited in different degrees.

The SN61-4G target of the invention is specific and selective, and only binds to TNFR1, but not to TNF-alpha and TNFR 2; the binding capacity of the TNFR1 is about 446nM, and the TNFR1 can competitively inhibit the binding of TNF-alpha. Therefore, the SN61-4G of the invention shows better target specificity and target binding capacity, which is obviously superior to that of Hydrostatin-SN 61.

Example 3: the interaction between SN61-4G and TNF-alpha, TNFR1 and TNFR2 was examined using microcalorimetry technology (MST).

1. Interaction of SN61-4G with TNF- α, TNFR1, TNFR2

Preparing SN61-4G with a series of gradient concentrations by a dilution ratio of 1:3, respectively and uniformly mixing fluorescence labeling TNF-alpha/TNFR 1/TNFR 2200 nM with SN61-4G in equal volume, incubating for 15min in a dark place, sucking a proper amount of sample by using a capillary tube for MST detection, observing a time track of a relative fluorescence value and a dose-response curve of Thermophoresis, and fitting and calculating affinity K by software NTAffinityanalysis v2.0.2 _D And (4) judging whether the SN61-4G has target selectivity or not.

As shown in FIG. 4, SN61-4G was able to interact directly with TNFR1 and bound TNFR1 at approximately 453 nM; SN61-4G does not bind to TNFR2, TNF-alpha.

2. Competitive inhibition of TNF-alpha binding to TNFR1 by SN61-4G

According to a 1:1 dilution ratioPreparing TNFR1 with a series of gradient concentrations, uniformly mixing isovolumetric fluorescently-labeled TNF-alpha 200nM with TNFR1, incubating for 15min in a dark place, sucking a proper amount of sample by using a capillary tube to perform MST detection, observing a time track of a relative fluorescence value and a dose-response curve of Thermophoresis, and fitting and calculating affinity K through software NTAffinityAnalysis v2.0.2 _D The value is obtained.

Uniformly mixing SN61-4G and TNFR1 according to a ratio of 10:1, 20:1, 40:1 and 80:1(n: n), preparing TNFR1 and SN61-4G mixed solution with a series of gradient concentrations according to a dilution ratio of 1:1, uniformly mixing fluorescence-labeled TNF-alpha 200nM with the TNFR1 and SN61-4G mixed solution with the same volume, incubating for 15min in a dark place, sucking a proper amount of sample by using a capillary tube for MST detection, observing a time track of a relative fluorescence value and a dose-response curve of Thermophoresis, and fitting and calculating affinity K by software NTAffinityAnalysis v2.0.2 _D The value is obtained.

As shown in FIG. 5, K between TNFR1 and TNF-alpha increases with the concentration of SN61-4G _D The value increased, indicating that SN61-4G is able to competitively inhibit the TNFR1-TNF- α interaction.

Example 4: effect of SN61-4G on Dextran Sodium Sulfate (DSS) -induced ulcerative colitis in mice

The specific implementation steps are as follows: male C57BL/6 mice, 6-8 weeks old, were selected and randomized into 5 groups of 8 mice each. Blank group (Control) does not do any processing; to the drinking water of Model group (Model) mice, 3% (w/v) DSS (commercially available from MP, MW 36,000-50,000) was added for 7 days to induce colitis; additional groups were given the drug simultaneously with molding for 7 consecutive days. Positive control group: infliximab (IFX, 4mg/kg/d, ip) and sulfasalazine (SASP, 400mg/kg/d, ig); SN61-4G group: 400. mu.g/kg/d, ip. The change in body weight was recorded daily for each group of mice (fig. 6), and SN61-4G was effective in reducing body weight loss due to colitis. Disease Activity Index (DAI) scores were performed according to Table 1, and the results are shown in FIG. 7, where SN61-4G was effective in relieving symptoms of diarrhea and hematochezia in mice with colitis. 7 days after the administration treatment, mice were sacrificed by cervical dislocation, spleens of the mice were removed, spleen weights were measured and spleen indices were calculated, and the results showed that SN61-4G could effectively inhibit spleen swelling caused by colitis (FIG. 8); taking out the whole colon of the mouse, and finding that SN61-4G can obviously improve the length of the colon of the mouse (figure 9); fixing the end tissues of the colon by 10% formalin, carrying out tissue sectioning, observing colon changes after HE staining, and finding that SN61-4G can effectively inhibit colon lesions in mouse ulcerative colitis (figure 10); foxp3+ staining of tissue sections revealed that SN61-4G significantly increased the number of Foxp3+ Treg cells in colon tissue (FIG. 11); extracting total protein of colon tissue, detecting change of MAPK signal channel by Western blot, and finding that SN61-4G can inhibit phosphorylation of ERK, JNK and P38 (figure 12); grinding mouse spleen, preparing single cell suspension, performing fluorescent antibody staining, and detecting the proportion of CD4+ IL-17A + Th17 cells (figure 13) and the proportion of CD4+ CD25+ Foxp3+ Treg cells (figure 14) in the model mouse spleen by flow cytometry; taking out mouse mesenteric lymph nodes, grinding to prepare single cell suspension, carrying out fluorescent antibody staining, and detecting the proportion of CD4+ IL-17A + Th17 cells (figure 15) and the proportion of CD4+ CD25+ Foxp3+ Treg cells (figure 16) in the model mouse mesenteric lymph nodes by flow cytometry; as a result, SN61-4G can reduce the proportion of Th17 cells and increase the proportion of Treg cells, and promote the balance of Th17/Treg to shift towards Treg.

TABLE 1 DAI Scoring criteria

The results show that SN61-4G can effectively treat a DSS-induced mouse colitis animal model.

While the preferred embodiments of the present invention have been described in detail, it will be understood by those skilled in the art that the invention is not limited thereto, and that various changes and modifications may be made without departing from the spirit of the invention, and the scope of the appended claims is to be accorded the full scope of the invention.

Sequence listing

<110> university at Shanghai

<120> a selective TNFR1 antagonistic peptide SN61-4G and application thereof in inflammatory bowel diseases

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Ser Asp Pro Gly Ala Ser His Leu Thr Gly Ile Arg

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tcggatcccg gtgcatcaca cctgactgga atacga 36

Claims (8)

1. A selective TNFR1 antagonist peptide SN61-4G, wherein the amino acid sequence of the selective TNFR1 antagonist peptide SN61-4G is as set forth in SEQ ID NO:1 is shown.

2. The selective TNFR1 antagonist peptide SN61-4G of claim 1, wherein: the selective TNFR1 antagonistic peptide SN61-4G has a molecular weight of 1210.32 daltons and an isoelectric point of 7.79.

3. A gene encoding the selective TNFR1 antagonistic peptide SN61-4G according to claim 1 or 2, wherein the coding gene is a DNA molecule as shown in SEQ ID No. 2.

4. Use of the selective TNFR1 antagonistic peptide SN61-4G of claim 1 or 2 in the preparation of a medicament for the treatment of inflammatory bowel disease.

5. The use of claim 4, wherein the agent for the treatment of inflammatory bowel disease is: takes the selective TNFR1 antagonistic peptide SN61-4G as the only active ingredient, or comprises the selective TNFR1 antagonistic peptide SN 61-4G.

6. The use of claim 4, wherein the inflammatory bowel disease comprises Crohn's disease and ulcerative colitis; the drug selective TNFR1 antagonistic peptide SN61-4G for treating inflammatory bowel disease is provided.

7. The use of claim 4, wherein the pharmaceutical composition is formulated with pharmaceutically acceptable conventional pharmaceutical excipients into a pharmaceutical formulation.

8. The use of claim 7, wherein the pharmaceutical preparation is a tablet, a granule, a dispersion, a capsule, a soft capsule, a drop pill, an injection, a powder injection or an aerosol.

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