Polypeptide for inhibiting TBK1 and inflammation and application thereof

Abstract

The invention relates to the field of biological medicine, in particular to polypeptide for inhibiting TBK1 and inflammation and application. The invention provides a series of polypeptides. A cell-penetrating peptide sequence TAT capable of penetrating a cell membrane and a connecting sequence (GSG) are added to the N end of the amino acid sequence of the polypeptides. The sequence can target TBK1 and influence the interaction of TBK1-STING-IRF3 so as to influence the activation of a pathway and achieve the purpose of inhibiting inflammation. The TBK1 targeting peptide LK shows remarkable anti-inflammatory activity in various established mouse inflammation or autoimmune disease models, including a lipopolysaccharide (LPS) induced systemic and acute inflammation model, a dextran sodium sulfate (DSS) induced ulcerative colitis model and a Trex1 defect AGS model. In addition, the polypeptide LK shows even a strong synergistic effect with a PD-1 antibody in cancer immunotherapy in various in vivo tumor models.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and particularly to polypeptides for inhibiting TBK1 and inflammation and their applications. Background Art

[0002] Inflammation is a physiological process by which the immune system responds to exogenous or endogenous stimuli, such as invading pathogens or injuries. However, when the response is uncontrolled or inappropriately activated due to various external or internal reasons, including infections, immunogenic exposures, and genetic abnormalities of certain immune components, it can lead to various inflammatory or autoimmune diseases, such as acute respiratory distress syndrome (ARDS), sepsis, inflammatory bowel disease (IBD), rheumatoid arthritis (RA), Aicardi-Goutières syndrome (AGS), systemic lupus erythematosus (SLE), etc. The symptoms of these diseases range from moderate to severe and are sometimes life-threatening. In addition, inflammation is associated with a variety of other diseases, including cancer and neurodegenerative diseases such as Alzheimer's disease. On the other hand, considering the diversity of the etiologies of inflammatory diseases, the development of novel anti-inflammatory drugs or treatment methods has attracted extensive attention and extensive efforts in the academic and industrial communities, because the expansion of the immunosuppressive repertoire will surely benefit the treatment of various inflammatory or autoimmune diseases. Summary of the Invention

[0003] In view of this, the present invention provides synthetic polypeptides and provides the applications of the above polypeptides in anti-inflammation.

[0004] In order to achieve the above-mentioned invention purposes, the present invention provides the following technical solutions:

[0005] In the first aspect, the present invention provides a polypeptide, which has:

[0006] (I) an amino acid sequence as shown in Formula I

[0007] X 1 -X 2 -K-K-X 5 -X 6 -X 7

[0008] Formula I

[0009] Wherein:

[0010] X 1 is selected from L;

[0011] X 2 is selected from R;

[0012] X 5 is selected from K;

[0013] X 6 is selected from A;

[0014] X 7 selected from C;

[0015] X 1 and X 2 and X 5 and X 6 and X 7 any one or more of which exist independently or are freely combined;

[0016] (II) An amino acid sequence obtained by substituting, deleting, or adding one or more amino acids to the amino acid sequence described in (I), and having the same function as the amino acid sequence described in (I); or

[0017] (III) An amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence homology with the amino acid sequence described in (I) or (II).

[0018] In some specific embodiments of the present invention, the polypeptide has:

[0019] (I) An amino acid sequence as shown in SEQ ID No. 4, 5, 7, 9, 15-19, 25-29;

[0020] (II) An amino acid sequence obtained by substituting, deleting, or adding one or more amino acids to the amino acid sequence described in (I), and having the same function as the amino acid sequence described in (I); or

[0021] (III) An amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence homology with the amino acid sequence described in (I) or (II).

[0022] In a second aspect, the present invention also provides a bioactive peptide, including the polypeptide described above, a transmembrane peptide, and a linker peptide.

[0023] In some specific embodiments of the present invention, in the bioactive peptide, the transmembrane peptide has:

[0024] (I) An amino acid sequence as shown in SEQ ID No. 3;

[0025] (II) An amino acid sequence obtained by substituting, deleting, or adding one or more amino acids to the amino acid sequence described in (I), and having the same function as the amino acid sequence described in (I); or

[0026] (III), an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence homology with the amino acid sequence described in (I) or (II);

[0027] and / or

[0028] The linker peptide has:

[0029] (I), an amino acid sequence as shown by GSG;

[0030] (II), an amino acid sequence obtained by substituting, deleting or adding one or more amino acids to the amino acid sequence described in (I), and having the same function as the amino acid sequence described in (I); or

[0031] (III), an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence homology with the amino acid sequence described in (I) or (II).

[0032] In some specific embodiments of the present invention, the bioactive peptide has:

[0033] (I), an amino acid sequence as shown by SEQ ID No.1, 2, 6, 8, 10-14, 20-24;

[0034] (II), an amino acid sequence obtained by substituting, deleting or adding one or more amino acids to the amino acid sequence described in (I), and having the same function as the amino acid sequence described in (I); or

[0035] (III), an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence homology with the amino acid sequence described in (I) or (II).

[0036] In the third aspect, the present invention also provides the use of the polypeptide or the bioactive peptide in the preparation of a drug for any of the following;

[0037] (I), targeting TBK1;

[0038] (II), affecting the interaction of TBK1, STING, IRF3 and thus affecting the activation of the pathway;

[0039] (III), inhibiting inflammation or autoimmune diseases.

[0040] In some specific embodiments of the present invention, the influencing of the interaction between TBK1-STING-IRF3 and thus the activation of the pathway includes:

[0041] (I) inhibiting the expression of downstream IFNβ, CXCL10, RANTES or ISG15 inflammatory factors induced by cGAS-STING;

[0042] (II) inhibiting the expression of downstream IFNβ, CXCL10 or RANTES inflammatory factors induced by TBK1;

[0043] (III) influencing the expression of downstream phosphorylated TBK1 or IRF3 proteins activated by Poly(dA:dT).

[0044] In some specific embodiments of the present invention, the inflammation includes lipopolysaccharide (LPS)-induced systemic and acute inflammation, dextran sulfate sodium (DSS)-induced ulcerative colitis or Trex1-deficient AGS.

[0045] In some specific embodiments of the present invention, the inhibition of inflammation includes:

[0046] (I) alleviating the pathological damage of the colon induced by DSS and / or inhibiting the expression of colonic inflammatory factors IL6 or RANTES;

[0047] (II) alleviating the expression of pulmonary inflammatory factors RANTES and CXCL10 induced by LPS;

[0048] (III) alleviating the pathological damage of the lungs caused by LPS;

[0049] (IV) inhibiting the expression of inflammatory factors RANTES or IL6 in the cardiac tissue of autoimmune Trex1-ko mice.

[0050] Fourthly, the present invention also provides a drug combination, including the polypeptide or the bioactive peptide as described above, and other active ingredients.

[0051] In some specific embodiments of the present invention, the active ingredients include but are not limited to PD-1 antibodies.

[0052] Fifthly, the present invention also provides the application of the drug combination as described above in the preparation of drugs for preventing and / or immunotherapizing cancer.

[0053] In some specific embodiments of the present invention, preventing and / or immunotherapizing cancer includes inhibiting tumors MC38 or B16F10.

[0054] Provided is an artificially synthesized polypeptide US, and the polypeptide is YGRKKRRQRRRGSGPPWLRKKKACALTRRS

[0055] The object of the present invention is to provide the application of the above polypeptide in anti-inflammation.

[0056] In order to achieve the above object, the present invention takes the following technical measures:

[0057] The present invention provides a series of polypeptides. At the same time, a cell-penetrating peptide sequence TAT and a linker sequence (GSG) that can penetrate the cell membrane are added to the N-terminus of the amino acid sequences of these polypeptides. Finally, a polypeptide LK with better antiviral effect is screened out from this series of polypeptides. This sequence can target TBK1 and affect the interaction of TBK1-STING-IRF3, thereby affecting the activation of the pathway to achieve the purpose of inhibiting inflammation. The polypeptide is YGRKKRRQRRRGSGLRKK. The TBK1-targeting peptide LK shows significant anti-inflammatory activity in a variety of established mouse inflammation or autoimmune disease models, including lipopolysaccharide (LPS)-induced systemic and acute inflammation models, dextran sulfate sodium (DSS)-induced ulcerative colitis models, and Trex1-deficient AGS models. In addition, in a variety of in vivo tumor models, the polypeptide LK even shows a strong synergistic effect with PD-1 antibody in cancer immunotherapy. Description of the Drawings

[0058] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art.

[0059] Figure 1 Showing the effect of US on the cGAS-STING signaling pathway;

[0060] Figure 2 Showing the cytotoxicity of polypeptide US on 293T cells detected by CCK-8;

[0061] Figure 3 Showing that polypeptide US has an inhibitory effect on the interaction of TBK1-STING-IRF3 in 293T cells;

[0062] Figure 4 Showing the inhibitory effect on the cGAS-STING signaling pathway detected by truncating polypeptide US in 293T cells;

[0063] Figure 5 Showing the inhibitory effect on the cGAS-STING signaling pathway by further truncating the polypeptide in 293T;

[0064] Figure 6 Showing the cytotoxicity of the further truncated polypeptide in 293T cells;

[0065] Figure 7 Show the inhibition of the cGAS-STING signaling pathway after the modification of polypeptide US;

[0066] Figure 8 Show that polypeptide LK inhibits the signaling pathway induced by TBK1;

[0067] Figure 9 Show the cytotoxicity of polypeptide LK on 293T cells detected by CCK-8;

[0068] Figure 10 Show that polypeptide LK has an inhibitory effect on the interaction of TBK1-STING-IRF3 in 293T cells;

[0069] Figure 11 Show that polypeptide LK inhibits the expression of downstream inflammatory factors induced by Poly(dA:dT);

[0070] Figure 12 Show that polypeptide LK inhibits the downstream protein phosphorylation induced by Poly(dA:dT);

[0071] Figure 13 Show the cytotoxicity of polypeptide LK on A549 cells detected by CCK-8;

[0072] Figure 14 Show the evaluation of the liver and kidney toxicity of LK in mice;

[0073] Figure 15 Show the distribution of LK in mice;

[0074] Figure 16 Show the anti-inflammatory effect of polypeptide LK on the DSS-induced mouse colitis model;

[0075] Figure 17 Show the HE staining results of the DSS-induced mouse colitis model treated with polypeptide LK;

[0076] Figure 18 Show the survival curve of polypeptide LK in the LPS-induced systemic inflammation in mice;

[0077] Figure 19 Show the anti-inflammatory effect of polypeptide LK in the LPS-induced systemic inflammation in mice;

[0078] Figure 20 Show the HE staining of the LPS-induced systemic inflammation in mice treated with polypeptide LK;

[0079] Figure 21 Show the anti-inflammatory effect of polypeptide LK in the Trex1-ko mouse model;

[0080] Figure 22Shown is that polypeptide LK combined with Anti-PD-1 inhibits MC38;

[0081] Figure 23 Shown is that polypeptide LK combined with Anti-PD-1 inhibits B16F10. Specific embodiments

[0082] The present invention discloses polypeptides for inhibiting TBK1 and inflammation and their applications. Those skilled in the art can draw on the content of this article and appropriately improve process parameters to achieve. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are all regarded as included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously make changes or appropriate alterations and combinations to the methods and applications described herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0083] Tank-binding kinase 1 (TBK1) is a member of the IKK kinase-related serine / threonine kinases, which can activate interferon regulatory factor 3 (IRF3) to induce type I interferon genes and interferon-stimulated genes (ISGs). It is a hub for downstream signal integration of multiple innate immune pathways and is related to various inflammatory or autoimmune diseases. TBK1 can also activate the NF-κB signaling pathway, promote NF-κB to enter the nucleus, bind to the promoter regions of multiple immune-related genes, and promote the expression of inflammatory factors (such as TNF-α, IL-6, etc.). These inflammatory factors play important roles in immune defense and regulation.

[0084] In addition, previous studies have reported the link between TBK1 and tumorigenesis and metabolic disorders. The study found that targeting TBK1 can overcome the resistance of cancer to PD-1 blockade immunotherapy by making tumors sensitive to RIPK1-dependent inflammatory cell death. Therefore, considering the association between dysregulation of TBK1 activity and inflammatory or autoimmune diseases as well as other diseases such as cancer and metabolic disorders, TBK1 is considered an attractive therapeutic target.

[0085] The applicant's research found that the UL37 exon-1 protein (UL37x1) encoded by human cytomegalovirus (HCMV) can directly target TBK1, block the TBK1-STING-IRF3 interaction, thereby inhibiting the TBK1-mediated immune pathway, and further determined the region of UL37x1 responsible for the interaction with TBK1. The immune escape mechanism of the virus is the optimal or relatively optimal immune intervention method evolved and retained by the virus during the long-term symbiosis and game with the host. Therefore, we developed a new strategy for immune intervention by drawing on the antagonistic mechanism of HCMV UL37x1 against TBK1.

[0086] The object of the present invention is to provide a synthetic polypeptide US, and the polypeptide is

[0087] YGRKKRRQRRRGSGPPWLRKKKACALTRRS, and the sequence is shown in SEQ ID No.1.

[0088] The object of the present invention is to provide the application of the above polypeptide in anti-inflammation.

[0089] In order to achieve the above object, the present invention takes the following technical measures:

[0090] The applicant designed a series of polypeptides based on the 35-50 key domains of the HCMV virus protein UL37, and added a cell-penetrating peptide sequence TAT and a linker sequence (GSG) that can penetrate the cell membrane to the N-terminus of the amino acid sequences of these polypeptides. Finally, a polypeptide LK with better antiviral effect was screened out from this series of polypeptides. This sequence can target TBK1 and affect the interaction of TBK1-STING-IRF3, thereby affecting the activation of the pathway to achieve the purpose of inhibiting inflammation. The polypeptide is YGRKKRRQRRRGSGLRKK, as shown in SEQ ID No.2. The TBK1-targeting peptide LK showed significant anti-inflammatory activity in a variety of established mouse inflammation or autoimmune disease models, including lipopolysaccharide (LPS)-induced systemic and acute inflammation models, dextran sulfate sodium (DSS)-induced ulcerative colitis models, and Trex1-deficient AGS models. In addition, in a variety of in vivo tumor models, the polypeptide LK even showed a strong synergistic effect with PD-1 antibodies in cancer immunotherapy. Therefore, the peptide mimicking the HCMV UL37x1 targeting TBK1 strategy has the potential for further development.

[0091] In the present invention, YGRKKRRQRRR (TAT) in the polypeptide sequence is a cell-penetrating peptide, and the sequence is shown in SEQ ID No.3; GSG is a linker peptide, and the amino acid sequence of each polypeptide without the cell-penetrating peptide and the linker peptide sequence is the core sequence. For example, the core sequence is LRKK, as shown in SEQ ID No.4; or the core sequence is PPWLRKKKACALTRRS, as shown in SEQ ID No.5.

[0092] A negative control was set for the polypeptides in each embodiment of the present invention to prove that the core sequence of the polypeptides provided by the present invention has the corresponding anti-inflammatory effect.

[0093] The raw materials and reagents used in the polypeptides and applications for inhibiting TBK1 and inflammation provided by the present invention can all be purchased from the market.

[0094] The following further elaborates the present invention in conjunction with embodiments:

[0095] Example 1: Detection of the effect of polypeptide US on the cGAS-STING signaling pathway

[0096] 1. Experimental materials

[0097] 293T cells, 24-well plates. Polypeptide US was synthesized by GenScript (Nanjing). Total RNA extraction reagent (FORGENE) was purchased from Furi, and Two-step QPCRyeason was used.

[0098] 2. Experimental procedure

[0099] (1) Seed 293T cells in 24-well plates.

[0100] (2) When the cells reach 80%-90% confluence, transfect with cGAS-STING.

[0101] (3) After 4 h, change the medium and add 10 μM of polypeptide US. Use the group with TAT (YGRKKRRQRRR, as shown in SEQ ID No. 3) as the control group.

[0102] (4) Harvest the samples 24 h after transfection and extract RNA using a total RNA extraction kit.

[0103] (5) Discard the supernatant, add 250 μl of cRL1 lysis buffer to the wells, and lyse on a shaker for 15 min.

[0104] (6) First add the lysate to the DNA column, centrifuge at 12,000 g for 2 min, and collect the centrifuged liquid.

[0105] (7) Add 400 μl of cRL2 solution to the centrifuged liquid and pipette to mix well.

[0106] (8) Transfer the solution to the RNA extraction column and centrifuge at 12,000 g for 2 min.

[0107] (9) Add RNAwashingbuffer1 solution to the RNA column and centrifuge at 12,000 g for 30 s.

[0108] (10) Add RNA washingbuffer2 solution and centrifuge at 12,000 g for 1 min.

[0109] (11) Centrifuge the RNA column without sample at 12,000 g for 2 min to completely remove the residual RNA washingbuffer.

[0110] (12) Add 50 μl of DEPC water to the RNA column and centrifuge at 12,000 g for 2 min.

[0111] (13) Take 2 μl of RNA sample and perform fluorescence quantitative experiment using one step qRT-PCR kit.

[0112] The results are shown in Figure 1 Tables 1 - 3, and the addition of 10 μM polypeptide US inhibits the expression of downstream IFNβ, CXCL10, and RANTES inflammatory factors induced by cGAS-STING.

[0113] Table 1

[0114]

[0115] Table 2

[0116]

[0117] Table 3

[0118]

[0119] Example 2: Toxicity assay of polypeptide US on 293T cells

[0120] 1. Experimental materials

[0121] CCK-8 reagent (MCE) was purchased from Promoter Company.

[0122] 2. Experimental procedure

[0123] This study evaluated the cytotoxicity of polypeptide US, mainly using CCK-8 reagent to detect this index. Cells without any treatment were used as the control group.

[0124] The steps are as follows:

[0125] (1) Seed HEK293T cells in a 96-well cell culture plate, 100 μl per well.

[0126] (2) When the cells reach 80%-90% confluence, discard the DMEM medium containing 10% serum, wash the cells with PBS, and then add DMEM medium containing 2% serum (0.5 ml per well). Add polypeptide US at a certain concentration gradient so that the final concentration of polypeptide US in the wells is 0, 1.56 μM, 3.12 μM, 6.25 μM, 12.5 μM, 25 μM, 50 μM, 100 μM, and 200 μM respectively. The group without adding polypeptide US was used as the control group, and each group had 3 replicates.

[0127] (3) After adding the polypeptide and incubating for 24 h, collect the samples. Add 10 μl of the live cell detection reagent CCK-8 to each well and mix well.

[0128] (4) Incubate at 37 °C for 2 h.

[0129] (5) Detect the absorbance value of OD450 using a microplate reader.

[0130] The results are as Figure 2 shown in Table 4. When 25 μM of polypeptide US was added to the cells, there was still no difference in cell viability compared to the control group, indicating that the polypeptide US screened in this study has no toxicity to 293T cells within 25 μM.

[0131] Calculation method of cell survival rate:

[0132] Absorbance value of OD450 in the experimental group / Average absorbance value of OD450 in the control group * 100 (the survival rates in the following examples are all calculated according to this formula).

[0133] Table 4

[0134]

[0135] Example 3: Effect of polypeptide US on the interaction between TBK1-STING-IRF3 proteins

[0136] 1. Materials

[0137] HEK293T cells, polypeptide US, and TAT (YGRKKRRQRRR, as shown in SEQ ID No. 3) were synthesized by GenScript Corporation (Nanjing), and Protein A / G magnetic beads (MCE)

[0138] 2. Experimental procedure

[0139] (1) Seed HEK293T cells in a 6-cm dish.

[0140] (2) When the cell confluence reaches 70%-80%, co-transfect TBK1 (GenBank: KAL1288138.1) and STING (GenBank: AVQ94753.1); or TBK1 and IRF3 (GenBank: AAH71721.1) into 293T cells

[0141] (3) After 4 h of transfection, change the cell culture medium to DMEM medium containing 2% fetal bovine serum, and add 10 μM of polypeptide US to the medium. Use the group with 10 μM of TAT polypeptide as the control group.

[0142] (4) After 24 h, discard the supernatant, add 1 ml of PBS to the cells, collect the cells into an EP tube, and centrifuge at 800 x g for 10 min.

[0143] (5) Discard the supernatant, add 1 ml of cell lysis buffer, and lyse the cells at 4 °C for 30 min.

[0144] (6) Centrifuge the sample at 4°C, 12,000 g for 15 min.

[0145] (7) Subsequently, collect the supernatant, take 100 μl as lysate, and add the remaining to Protein A / G magnetic beads, incubate at 4°C for 2 hours. (8) After 2 hours, wash the magnetic beads 3 - 4 times with pre-cooled lysis buffer, 5 min each time.

[0146] (9) Treat the collected corresponding samples with 5×SDS loading buffer and boil.

[0147] (10) Detect the corresponding proteins in the samples by western blot.

[0148] The results are as Figure 3 shown, the polypeptide US affects the interaction between TBK1 and STING, and between TBK1 and IRF3.

[0149] Example 4: Determination of the anti-inflammatory efficiency of US truncated polypeptide in 293T cells

[0150] 1. Materials

[0151] 293T cells, 24-well plates, polypeptides UH, LS, UT, PS, PR were synthesized by GenScript (Nanjing), Total RNA extraction reagent (FORGENE) was purchased from Foji, Two-step QPCR yeason.

[0152] Table 5

[0153]

[0154] 2. Determination of the inhibitory effect of truncated polypeptide on cGAS-STING activation in 293T cells

[0155] (1) Seed 293T cells in 24-well plates.

[0156] (2) When the cells reach 80% - 90% confluence, transfect cGAS-STING.

[0157] (3) After 4 h, change the medium and add 10 μM of polypeptides UH, LS, UT, PS, PR, using the TAT group as the control.

[0158] (4) Harvest the samples 24 h after transfection and extract RNA using a total RNA extraction kit.

[0159] (5) Discard the supernatant, add 250 μl of cRL1 lysis buffer to the wells, and lyse on a shaker for 15 min.

[0160] (6) Add the lysate to the DNA column first, centrifuge at 12,000 g for 2 min, and collect the centrifuged liquid.

[0161] (7) Add 400 μl of cRL2 solution to the centrifuged liquid and pipette to mix well.

[0162] (8) Transfer the solution to the RNA extraction column and centrifuge at 12,000 g for 2 min.

[0163] (9) Add RNA washing buffer 1 solution to the RNA column and centrifuge at 12,000 g for 30 s.

[0164] (10) Add RNA washing buffer 2 solution and centrifuge at 12,000 g for 1 min.

[0165] (11) Centrifuge the RNA column without sample at 12,000 g for 2 min to completely remove the residual RNA washing buffer.

[0166] (12) Add 50 μl of DEPC water to the RNA column and centrifuge at 12,000 g for 2 min.

[0167] (13) Take 2 μl of the RNA sample and perform fluorescence quantitative experiment using the one step qRT-PCR kit.

[0168] The results are as Figure 4 shown in Tables 6 - 8, and the polypeptides UH, LS, UT, PS, and PR all inhibit the expression of downstream IFNβ, CXCL10, and RANTES inflammatory factors induced by cGAS - STING.

[0169] The significance analysis was performed by comparing with the TAT group as the control.

[0170] Table 6

[0171]

[0172] Table 7

[0173]

[0174] Table 8

[0175]

[0176] Example 5: Determination of the anti - inflammatory efficiency of further truncated polypeptides in 293T cells

[0177] 1. Materials

[0178] 293T cells, 24-well plates, polypeptides LT, RT, KKA, KC, and RK were synthesized by GenScript (Nanjing). The Total RNA extraction reagent (FORGENE) was purchased from Furi. Two-step QPCR yeason.

[0179] Table 9

[0180]

[0181] 2. Determination of the inhibitory effect of truncated polypeptides on cGAS-STING activation in 293T cells

[0182] (1) Seed 293T cells in 24-well plates.

[0183] (2) When the cells reach 80%-90% confluence, transfect with cGAS-STING

[0184] (3) After 4 h, change the medium and add 10 μM of polypeptides LT, RT, KKA, KC, and RK. Use the TAT group as the control.

[0185] (4) Harvest the samples 24 h after transfection and extract RNA using a total RNA extraction kit.

[0186] (5) Discard the supernatant, add 250 μl of cRL1 lysis buffer to the wells, and lyse on a shaker for 15 min.

[0187] (6) First add the lysate to the DNA column, centrifuge at 12,000 g for 2 min, and collect the centrifuged liquid.

[0188] (7) Add 400 μl of cRL2 solution to the centrifuged liquid and pipette to mix well.

[0189] (8) Transfer the solution to the RNA extraction column and centrifuge at 12,000 g for 2 min.

[0190] (9) Add RNAwashingbuffer1 solution to the RNA column and centrifuge at 12,000 g for 30 s.

[0191] (10) Add RNA washingbuffer2 solution and centrifuge at 12,000 g for 1 min.

[0192] (11) Centrifuge the RNA column without sample at 12,000 g for 2 min to completely remove residual RNA washingbuffer.

[0193] (12) Add 50 μl of DEPC water to the RNA column and centrifuge at 12,000 g for 2 min.

[0194] (13) Take 2 μl of RNA sample and perform fluorescence quantitative experiment using one step qRT-PCR kit.

[0195] The results are as Figure 5 shown in Tables 10 and 11. The polypeptides LT, RT, KKA, KC, and RK all inhibited the expression of downstream IFNβ and RANTES inflammatory factors induced by cGAS-STING.

[0196] Table 10

[0197]

[0198] Table 11

[0199]

[0200] Example 6 Toxicity Detection of Truncated Polypeptides in 293T Cells

[0201] 1. Experimental Materials

[0202] CCK-8 reagent (MCE) was purchased from Promoter Company.

[0203] 2. Experimental Procedure

[0204] This study evaluated the cytotoxicity of polypeptides LT, RT, KKA, KC, and RK on 293T cells, mainly using CCK-8 reagent to detect this index. Cells without any treatment were used as the control group.

[0205] The steps are as follows:

[0206] (1) Seed 293T cells in a 96-well cell culture plate, 100 μl per well.

[0207] (2) When the cells reach 80%-90% confluence, discard the DMEM medium containing 10% serum, wash the cells with PBS, and then add DMEM medium containing 2% serum (0.5 ml per well). Add polypeptide UH at a certain concentration gradient so that the final concentrations in the wells are 0, 1.56 μM, 3.12 μM, 6.25 μM, 12.5 μM, 25 μM, 50 μM, 100 μM, and 200 μM respectively. The group without adding polypeptide was used as the control group, with 3 replicates in each group.

[0208] (3) After adding the polypeptide and incubating for 24 h, collect the samples. Add 10 μl of CCK-8, a live cell detection reagent, to each well and mix well.

[0209] (4) Incubate at 37 °C for 2 h.

[0210] (5) Measure the absorbance at OD450 using an enzyme-linked immunosorbent assay (ELISA) reader.

[0211] The results are asFigure 6 As shown in Table 12, when 200 μM of the truncated polypeptides LT, RT, KKA, KC, and RK were added to the cells, the cell viabilities of LT, RT, KC, and RK, except for KKA, were still not different from those of the control group, suggesting that the polypeptides screened in this study had no toxicity to 293T cells within 200 μM.

[0212] Calculation method of cell survival rate:

[0213] Absorbance value of OD450 in the experimental group / Average absorbance value of OD450 in the control group * 100 (the survival rates in the following examples were all calculated according to this formula).

[0214] Table 12

[0215]

[0216]

[0217] Example 7: Determination of the anti-inflammatory efficiency of US-modified polypeptides in 293T cells

[0218] 1. Materials

[0219] 293T cells, 24-well plates, polypeptides LA (YGRKKRRQRRRGSGLRKKKA, the sequence is shown in SEQ ID No. 6; among them, the core sequence LRKKKA is shown in SEQ ID No. 7), LK, KA

[0220] (YGRKKRRQRRRGSGKKKA, the sequence is shown in SEQ ID No. 8; among them, the core sequence KKKA is shown in SEQ ID No. 9) were synthesized by GenScript (Nanjing), and the Total RNA extraction reagent (FORGENE) was purchased from Fujian, and Two-step QPCRyeason.

[0221] 2. Determination of the inhibitory effect of truncated polypeptides on the activation of cGAS-STING in 293T cells

[0222] (1) Seed 293T cells in 24-well plates.

[0223] (2) When the cells reached 80%-90% confluence, transfect cGAS-STING

[0224] (3) After 4 h, change the medium and add 10 μM of polypeptides LA, LK, and KA respectively. The group with TAT (YGRKKRRQRRR, as shown in SEQ ID No. 3) was used as the control group.

[0225] (5) Harvest the samples 24 h after transfection and extract RNA using a total RNA extraction kit.

[0226] (5) Discard the supernatant, add 250 μl of cRL1 lysis solution to the wells, and lyse for 15 min on a shaker.

[0227] (6) First add the lysis solution to the DNA column, centrifuge at 12,000 g for 2 min, and collect the centrifuged liquid.

[0228] (7) Add 400 μl of cRL2 solution to the centrifuged liquid and pipette to mix well.

[0229] (8) Transfer the solution to the RNA extraction column and centrifuge at 12,000 g for 2 min.

[0230] (9) Add RNA washing buffer1 solution to the RNA column and centrifuge at 12,000 g for 30 s.

[0231] (10) Add RNA washing buffer2 solution and centrifuge at 12,000 g for 1 min.

[0232] (11) Centrifuge the RNA column empty at 12,000 g for 2 min to completely remove the residual RNA washing buffer.

[0233] (12) Add 50 μl of DEPC water to the RNA column and centrifuge at 12,000 g for 2 min.

[0234] (13) Take 2 μl of the RNA sample and perform a fluorescence quantitative experiment using a one step qRT-PCR kit.

[0235] The results are as Figure 7 , shown in Table 13 and Table 14. The polypeptides LA, LK, and KA all inhibit the expression of downstream CXCL10 and ISG15 inflammatory factors induced by cGAS-STING.

[0236] Table 13

[0237]

[0238] Table 14

[0239]

[0240] Example 8: Determination of the anti-inflammatory effect of polypeptide LK targeting TBK1 in 293T cells

[0241] 1. Materials

[0242] 293T cells, 24-well plates, polypeptide LK was synthesized by GenScript (Nanjing), and the Total RNA extraction reagent (FORGENE) was purchased from Furi, Two-step QPCRyeason.

[0243] 2. Determination of the inhibitory effect of polypeptide LK on TBK1 activation in 293T cells

[0244] (1) Seed 293T cells in 24-well plates.

[0245] (2) When the cells reach 80%-90% confluence, transfect TBK1

[0246] (3) After 4 h, change the medium and add 10 μM of polypeptide LK. The group adding TAT polypeptide was used as the control group.

[0247] (4) Harvest the samples 24 h after transfection and extract RNA using a total RNA extraction kit.

[0248] (5) Discard the supernatant, add 250 μl of cRL1 lysis buffer to the wells, and lyse on a shaker for 15 min.

[0249] (6) First add the lysate to the DNA column, centrifuge at 12,000 g for 2 min, and collect the centrifuged liquid.

[0250] (7) Add 400 μl of cRL2 solution to the centrifuged liquid and pipette to mix well.

[0251] (8) Transfer the solution to the RNA extraction column and centrifuge at 12,000 g for 2 min.

[0252] (9) Add RNAwashingbuffer1 solution to the RNA column and centrifuge at 12,000 g for 30 s.

[0253] (10) Add RNA washingbuffer2 solution and centrifuge at 12,000 g for 1 min.

[0254] (11) Centrifuge the RNA column empty at 12,000 g for 2 min to completely remove the residual RNA washingbuffer.

[0255] (12) Add 50 μl of DEPC water to the RNA column and centrifuge at 12,000 g for 2 min.

[0256] (13) Take 2 μl of the RNA sample and perform fluorescence quantitative experiments using a one step qRT-PCR kit.

[0257] The results are as Figure 8, as shown in Tables 15 to 17, the polypeptide LK inhibits the expression of downstream IFNβ, CXCL10, and RANTES inflammatory factors induced by TBK1.

[0258] Table 15

[0259]

[0260] Table 16

[0261]

[0262] Table 17

[0263]

[0264] Example 9: Toxicity of Polypeptide LK in 293T Cells

[0265] 1. Experimental Materials

[0266] The CCK-8 reagent (MCE) was purchased from the promoter company.

[0267] 2. Experimental Procedure

[0268] This study evaluated the cytotoxicity of polypeptide LK, mainly using the CCK-8 reagent to detect this index. Cells without any treatment were used as the control group.

[0269] The steps are as follows:

[0270] (1) Seed HEK293T cells in a 96-well cell culture plate, 100 μl per well.

[0271] (2) When the cells reach 80%-90% confluence, discard the DMEM medium containing 10% serum, wash the cells with PBS, and then add DMEM medium containing 2% serum (0.5 ml per well). Add polypeptide LK at a certain concentration gradient so that the final concentrations of polypeptide LK in the wells are 0, 1.56 μM, 3.12 μM, 6.25 μM, 12.5 μM, 25 μM, 50 μM, 100 μM, and 200 μM, respectively. The group without adding LK polypeptide was used as the control group, with 3 replicates in each group.

[0272] (3) After adding the polypeptide and incubating for 24 h, collect the samples. Add 10 μl of the live cell detection reagent CCK-8 to each well and mix well.

[0273] (4) Incubate at 37 °C for 2 h.

[0274] (5) Measure the absorbance value at OD450 using an enzyme-linked immunosorbent assay (ELISA) reader.

[0275] The results are as Figure 9, as shown in Table 18, when 200 μM of polypeptide LK was added to the cells, there was still no difference in cell viability compared to the control group, indicating that the polypeptide LK screened in this study has no toxicity to 293T cells within 200 μM.

[0276] Calculation method of cell survival rate:

[0277] Absorbance value of OD450 in the experimental group / Average absorbance value of OD450 in the control group * 100 (the survival rates in the following examples are all calculated according to this formula).

[0278] Table 18

[0279]

[0280] Example 10: Effect of polypeptide LK on the interaction of TBK1-STING-IRF3 in 293T cells

[0281] 1 Materials

[0282] HEK293T cells, polypeptide LK, and TAT were synthesized by GenScript Corporation (Nanjing).

[0283] 2. Experimental procedure

[0284] (1) Seed HEK293T cells in a 6-cm dish.

[0285] (2) When the cell confluence reaches 70%-80%, co-transfect TBK1 and STING or TBK1 and IRF3 into 293T cells.

[0286] (3) 4 hours after transfection, change the cell culture medium to DMEM medium containing 2% fetal bovine serum, and add 10 μM of polypeptide LK to the medium. The group with 10 μM of TAT polypeptide added is used as the control group.

[0287] (4) After 24 hours, discard the supernatant, add 1 ml of PBS to the cells, collect the cells into an EP tube, and centrifuge at 800 x g for 10 minutes. (5) Discard the supernatant, add 1 ml of cell lysis buffer, and lyse the cells at 4°C for 30 minutes.

[0288] (6) Centrifuge the samples at 4°C, 12000 x g for 15 minutes.

[0289] (7) Then collect the supernatant, take 100 μl as lysate, and add the remaining to Protein A / G magnetic beads, and incubate at 4°C for 2 hours. (10) After 2 hours, wash the magnetic beads 3-4 times with pre-cooled lysis buffer, 5 minutes each time.

[0290] (11) Treat the collected corresponding samples with 5×SDS loading buffer and boil.

[0291] (10) Detect the corresponding protein in the sample by Western blot.

[0292] The results are as Figure 10 shown, and the polypeptide LK affects the interaction between TBK1 and STING, and between TBK1 and IRF3.

[0293] Example 11: Expression of downstream inflammatory factors activated by Poly(dA:dT) by polypeptide LK in A549 cells

[0294] 1. Materials

[0295] A549 cells, 24-well plates, polypeptide LK, and TAT were synthesized by GenScript (Nanjing). Total RNA extraction reagent (FORGENE) was purchased from Furi, and Poly(dA:dT) was purchased from InvivoGen, Two-step QPCRyeason.

[0296] 2. Determination of the inhibitory effect of polypeptide LK on the activation of Poly(dA:dT) in 293T cells

[0297] (1) Seed A549 cells in 24-well plates.

[0298] (2) When the cells reach 80%-90% confluence, add 10 μM of LK, and use the group adding 10 μM of TAT as the control group.

[0299] (3) After 12 h, change the medium and transfect Poly(dA:dT) with lipo6000.

[0300] (4) Harvest the samples 6 h after transfection and extract RNA using a total RNA extraction kit.

[0301] (5) Discard the supernatant, add 250 μl of cRL1 lysis buffer to the wells, and lyse on a shaker for 15 min.

[0302] (6) First add the lysate to the DNA column, centrifuge at 12000 g for 2 min, and collect the centrifuged liquid.

[0303] (7) Add 400 μl of cRL2 solution to the centrifuged liquid and pipette to mix well.

[0304] (8) Transfer the solution to the RNA extraction column and centrifuge at 12000 g for 2 min.

[0305] (9) Add RNAwashingbuffer1 solution to the RNA column and centrifuge at 12000 g for 30 s.

[0306] (10) Add RNA washing buffer 2 solution and centrifuge at 12,000 g for 1 min.

[0307] (11) Centrifuge the RNA column at 12,000 g for 2 min to completely remove the residual RNA washing buffer.

[0308] (12) Add 50 μl of DEPC water to the RNA column and centrifuge at 12,000 g for 2 min.

[0309] (13) Take 2 μl of the RNA sample and perform fluorescence quantitative experiment using the one step qRT-PCR kit.

[0310] The results are as Figure 11 shown in Tables 19 - 21, and the polypeptide LK inhibits the expression of downstream CXCL10, RANTES, and ISG15 inflammatory factors induced by Poly(dA:dT).

[0311] Table 19

[0312]

[0313] Table 20

[0314]

[0315] Table 21

[0316]

[0317] Example 12: Effect of polypeptide LK on the phosphorylation of downstream proteins by Poly(dA:dT) in A549 cells 1. Experimental materials

[0318] A549 cells, 6 cm dish, polypeptide LK, and TAT were synthesized by GenScript Corporation (Nanjing)

[0319] 2. Experimental procedure

[0320] (1) Seed A549 cells in a 24-well plate.

[0321] (2) When the cell confluence reaches 80% - 90%, add 10 μM of LK, and use the group with 10 μM of TAT added as the control group.

[0322] (3) After 12 h, change the medium and transfect Poly(dA:dT) using lipo6000.

[0323] (3) Harvest the samples 6 h after transfection, discard the supernatant, and add 1 ml of PBS to the cells.

[0324] (4) Collect the cells into an EP tube and centrifuge at 800 x g for 10 min.

[0325] (5) Discard the supernatant, add 1 ml of cell lysis buffer, and lyse at 4°C for 30 min.

[0326] (6) Centrifuge the samples at 4°C, 12,000 g for 15 min.

[0327] (7) Subsequently, collect the supernatant, treat it with 5×SDS loading buffer, and boil it.

[0328] (8) Detect the expression of the corresponding protein in the samples by western blot.

[0329] The results are as Figure 12 shown, and the polypeptide LK affects the expression of downstream phosphorylated proteins of TBK1 and IRF3 activated by Poly(dA:dT).

[0330] Example 13: Toxicity of polypeptide LK in A549 cells

[0331] 1. Experimental materials

[0332] CCK-8 reagent (MCE) was purchased from Qidong Company.

[0333] 2. Experimental procedure

[0334] This study evaluated the cytotoxicity of polypeptide LK on A549, mainly using the CCK-8 reagent to detect this index. Cells without any treatment were used as the control group.

[0335] The steps are as follows:

[0336] (1) Seed A549 cells in a 96-well cell culture plate, 100 μl per well.

[0337] (2) When the cells reach 80%-90% confluence, discard the DMEM medium containing 10% serum, wash the cells with PBS, then add DMEM medium containing 2% serum (0.5 ml per well), and add polypeptide LK at a certain concentration gradient so that the final LK concentrations in the wells are 0, 1.56 μM, 3.12 μM, 6.25 μM, 12.5 μM, 25 μM, 50 μM, 100 μM, and 200 μM respectively. The group without adding LK polypeptide was used as the control group, with 3 replicates in each group.

[0338] (3) After adding the polypeptide and incubating for 24 h, collect the samples, add 10 μl of the live cell detection reagent CCK-8 to each well, and mix well.

[0339] (4) Incubate at 37°C for 2 h.

[0340] (5) Measure the absorbance value at OD450 using an enzyme-linked immunosorbent assay reader.

[0341] The results are as Figure 13 shown in Table 22. When 200 μM of polypeptide LK was added to the cells, there was still no difference in cell viability compared to the control group, indicating that the polypeptide LK screened in this study has no toxicity to 293T cells within 200 μM.

[0342] Calculation method of cell survival rate:

[0343] Absorbance value of OD450 in the experimental group / Average absorbance value of OD450 in the control group * 100 (the survival rates in the following examples are all calculated according to this formula).

[0344] Table 22

[0345]

[0346] Example 14: Safety detection of LK in mice

[0347] 1. Materials

[0348] Polypeptide LK was synthesized by GenScript. C57BL / 6 mice, ELISA kit (mlbio)

[0349] 2. Experimental procedure

[0350] (1) The mice were randomly divided into three groups, with 6 mice in each group. Blood was collected from the eyes of the mice before injecting the polypeptide drug.

[0351] (2) Using a test tube without pyrogen and endotoxin, after collecting the blood, centrifuge at 1000 × g for 10 minutes to quickly and carefully separate the serum and red blood cells.

[0352] (3) Except for the mock group, the other two groups were intraperitoneally injected with 10 mg / kg and 25 mg / kg of polypeptide LK respectively.

[0353] (4) Blood was collected from the eyes of the mice again on the first day and the third day.

[0354] (5) Dilute the specimen 1:1 with the specimen diluent and add 50 μl to the reaction well.

[0355] (6) Add 50 μl of the diluted standard product to the reaction well and 50 μl of the sample to be tested to the reaction well. Immediately add 50 μl of the biotin-labeled antibody. Cover the membrane plate, gently shake and mix well, and incubate at 37 °C for 1 hour.

[0356] (7) Discard the liquid in the well, fill each well with the washing solution, shake for 30 seconds, discard the washing solution, and pat dry with absorbent paper. Repeat this operation 3 times. If using a plate washer for washing, increase the number of washing times by one.

[0357] (8) Add 80 μl of streptavidin-HRP to each well, gently mix by shaking, and incubate at 37 °C for 30 minutes.

[0358] (9) Discard the liquid in the wells, fill each well with washing solution, shake for 30 seconds, discard the washing solution, and pat dry with absorbent paper. Repeat this operation 3 times. If using a plate washer for washing, increase the number of washing times by one.

[0359] (10) Add 50 μl of each of substrate A and B to each well, gently mix by shaking, and incubate at 37 °C for 10 minutes. Avoid light.

[0360] (11) Take out the enzyme-labeled plate, quickly add 50 μl of stop solution, and measure the results immediately after adding the stop solution.

[0361] (12) Measure the OD value of each well at a wavelength of 450 nm.

[0362] The results are as Figure 14 shown. The polypeptide LK has no effect on the liver and kidney functions of mice at both 10 mg / kg and 25 mg / kg.

[0363] Example 15: Detection of the Distribution of LK in Mice

[0364] 1. Materials

[0365] The polypeptide Cy5-LK was synthesized by GenScript Corporation. C57BL / 6 mice.

[0366] 2. Experimental Procedure

[0367] (1) Randomly divide 8-week-old C57BL / 6J mice into two groups, and intraperitoneally inject 10 mg / kg Cy5-LK or the control group (n = 3) (for background fluorescence measurement).

[0368] (2) One hour after injection, image the distribution of Cy5-LK in mice using an IVIS Lumina K Series III in vivo imaging system (Perkin Elmer).

[0369] (3) Sacrifice the mice by injecting sodium pentobarbital, obtain the corresponding tissues, and image and perform fluorescence evaluation on the brain, heart, lungs, liver, and kidneys of the mice.

[0370] (4) Use Living Image 4.4 software to calculate the relevant radiant efficiency (Ps -1 cm -2 sr -1 ) / (μW / cm 2 ).

[0371] As Figure 15 shown, Cy5-LK has an obvious distribution in the heart, liver, spleen, lungs, kidneys, brain, and intestine.

[0372] Example 16: Anti-inflammatory Effect of Polypeptide LK in DSS Colitis Model

[0373] 1. Experimental Materials

[0374] Polypeptide LK was synthesized by GenScript Corporation. C57BL / 6 mice and DSS (MPbio) were used.

[0375] 2. Experimental Procedure

[0376] (1) Dissolve DSS in drinking water to prepare a 2.5% DSS solution.

[0377] (2) Randomly divide C57BL / 6 mice into a control group, a model group, and a drug administration group, with 10 mice in each group. The model group and the drug administration group were allowed to freely drink the 2.5% DSS solution, while the control group drank ordinary water.

[0378] (3) Inject DSS colitis model mice intraperitoneally with polypeptide LK at a dose of 5 mg / kg once a day for 7 consecutive days.

[0379] (4) Record the body weight of the mice daily.

[0380] (5) After the experiment, euthanize the mice and take their colon tissues. After grinding and inactivating a part with Trizol, extract the total RNA in the tissues for inflammatory factor detection, and perform HE staining on another part.

[0381] (5) Add 200 μl of chloroform to the RNA lysis solution, mix well by shaking, let it stand, and centrifuge at 12000 g for 10 min. Collect the upper layer liquid after centrifugation.

[0382] (6) Add an equal volume of isopropanol to the collected liquid, mix well, precipitate at -20 °C for 10 min, and centrifuge at 12000 g for 15 min.

[0383] (7) Discard the supernatant, retain the precipitate, add 1 ml of 75% ethanol solution, and centrifuge at 12000 g for 5 min.

[0384] (8) Discard the supernatant, retain the precipitate, and dissolve the precipitate with 50 μl of DEPC water.

[0385] (9) Take 3 μl of the RNA sample and perform fluorescence quantitative experiments using a Two step qRT-PCR kit.

[0386] As Figure 16 、 17 shown in Table 23 and Table 24, adding polypeptide LK can reduce the pathological damage of the colon induced by DSS and inhibit the expression of colonic inflammatory factors IL6 and RANTES.

[0387] Table 23

[0388]

[0389] Table 24

[0390]

[0391] Example 17: Survival Curve of Polypeptide LK in Systemic Acute Inflammation Induced by LPS

[0392] 1. Materials

[0393] Polypeptide LK was synthesized by GenScript Corporation (Nanjing). C57BL / 6 mice, LPS

[0394] 2. Effect of Polypeptide LK on LPS in C57BL / 6 Mice

[0395] (1) Polypeptide LK was intraperitoneally injected at a dose of 5 mg / kg for 3 consecutive days

[0396] (2) Three days after administration, the mice were intraperitoneally injected with 8 mg / kg of LPS to induce systemic acute inflammation

[0397] (3) The survival of the mice was recorded every day

[0398] As Figure 18 shown, it can significantly rescue the mice from death caused by LPS

[0399] Example 18: Determination of Anti-inflammatory Effect of Polypeptide LK in Systemic Acute Inflammation Induced by LPS

[0400] 1. Materials

[0401] Polypeptide LK was synthesized by GenScript Corporation (Nanjing). C57BL / 6 mice, DSS, Two-step QPCRyeason

[0402] 2. Determination of the Inhibitory Effect of Polypeptide LK on LPS Activation in C57BL / 6 Mice

[0403] (1) Polypeptide LK was intraperitoneally injected at a dose of 5 mg / kg for 3 consecutive days

[0404] (2) Eighteen 6-week-old C57BL / 6 mice were divided into 3 groups of 6 mice each and injected intraperitoneally. One group was the polypeptide LK 10 mg / kg group, one group was given 10 mg / kg of TAT, and the other group was given an equal amount of PBS as a control

[0405] (3) Three days after administration, LPS, LPS+LK mice were treated with 8 mg / kg of LPS by intraperitoneal injection

[0406] (4) Twenty-four hours after LPS injection, the mice were euthanized and their lung tissues were taken. One part was ground and inactivated with Trizol to extract total RNA from the tissue, and the other part was used for HE staining.

[0407] (5) Add 200 μl of chloroform to the RNA lysis solution, mix well by shaking, let it stand, centrifuge at 12,000 g for 10 min, and collect the upper layer liquid after centrifugation.

[0408] (6) Add an equal volume of isopropanol to the collected liquid, mix well, precipitate at -20 °C for 10 min, and centrifuge at 12,000 g for 15 min.

[0409] (7) Discard the supernatant, retain the precipitate, add 1 ml of 75% ethanol solution, and centrifuge at 12,000 g for 5 min.

[0410] (8) Discard the supernatant, retain the precipitate, and dissolve the precipitate with 50 μl of DEPC water.

[0411] (9) Take 3 μl of the RNA sample and perform a fluorescence quantitative experiment using a Two step qRT-PCR kit.

[0412] As Figure 19 、 Figure 20 、Table 25, Table 26 show that for LPS-induced systemic acute inflammation, the polypeptide LK group can significantly reduce the expression of the pulmonary inflammatory factors RANTES and CXCL10; at the same time, HE staining shows that polypeptide LK significantly reduces the pathological damage of LPS to the lungs.

[0413] Table 25

[0414]

[0415] Table 26

[0416]

[0417] Example 19: Detection of the distribution of LK in mice

[0418] 1. Materials

[0419] The polypeptide LK was synthesized by GenScript. C57BL / 6 Trex1-ko mice.

[0420] 2. Experimental procedure

[0421] (1) Randomly divide 4- to 5-week-old C57BL / 6J Trex1-ko mice into two groups, intraperitoneally inject 10 mg / kg of polypeptide LK, and the control group injects the same volume of PBS

[0422] (2) Inject continuously for 21 days, euthanize the mice, take their heart tissues, after grinding and inactivating with Trizol, extract the total RNA in the tissues.

[0423] (5) Add 200 μl of chloroform to the RNA lysis solution, mix well by shaking, let it stand, centrifuge at 12000 g for 10 min, and collect the upper layer liquid after centrifugation.

[0424] (6) Add an equal volume of isopropanol to the collected liquid, mix well, precipitate at -20 °C for 10 min, and centrifuge at 12000 g for 15 min.

[0425] (7) Discard the supernatant, retain the precipitate, add 1 ml of 75% anhydrous ethanol solution, and centrifuge at 12000 g for 5 min.

[0426] (8) Discard the supernatant, retain the precipitate, and dissolve the precipitate with 50 μl of DEPC water.

[0427] (9) Take 3 μl of the RNA sample and perform a fluorescence quantitative experiment using a Two step qRT-PCR kit.

[0428] As Figure 21 shown in Table 27 and Table 28, the polypeptide LK can inhibit the expression of inflammatory factors RANTES and IL6 in the heart tissues of autoimmune Trex1-ko mice.

[0429] Table 27

[0430]

[0431] Table 28

[0432]

[0433] Example 20: Detection of the therapeutic effect of polypeptide LK combined with Anti PD-1 on tumors

[0434] 1. Experimental materials

[0435] The polypeptide LK was synthesized by GenScript Corporation, C57BL / 6 mice, B16F10 cells, MC38 cells

[0436] 2. Experimental procedure

[0437] (1) Ensure to obtain tumor cells B16F10 and MC38 in the logarithmic growth phase with a cell density of approximately 80-90%.

[0438] (2) Remove the old culture medium, wash the cells with PBS 1 - 2 times first to remove serum components. Then, digest the cells with trypsin and prepare a cell suspension. After that, centrifuge the cells and remove the supernatant. Finally, resuspend the cells with PBS or serum-free medium and count them to determine the required final cell concentration.

[0439] (3) Dilute according to the cell concentration to ensure that the number of cells inoculated into each mouse is 1x10 6 cells, and the inoculation volume is 100 μl.

[0440] (4) Inoculate 60 mice with each type of cell. After fixing the nude mouse with the left hand, perform subcutaneous injection at the right axilla. When injecting, insert the needle about 1 cm deep into the subcutaneous tissue and inject 100 μl of the cell suspension. To slow down cell apoptosis, keep the cell suspension on ice throughout the inoculation process.

[0441] (5) Measure the volume 5 - 7 days after inoculation, group the mice, with 10 mice in each group, and evenly divide them into 4 groups (Control, polypeptide LK, Anti PD-1, Anti PD-1 + LK groups).

[0442] (6) Inject polypeptide LK intraperitoneally at a dose of 10 mg / kg per day, and inject Anti PD-1 intraperitoneally at a dose of 150 mg / mouse (inject 3 times a week).

[0443] (7) Measure the volume every three days.

[0444] As Figure 22 、 Figure 23 shown, polypeptide LK can enhance the therapeutic effects on tumors MC38 and B16F10 in combination with Anti PD-1.

[0445] The above description is only the preferred embodiment of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and modifications can be made, and these improvements and modifications should also be regarded as the protection scope of the present invention.

Claims

1. A polypeptide, characterized in that It has: (I), the amino acid sequence shown in Formula I X1-X2-KK-X5-X6-X7 Formula I in: X1 is selected from L; X2 is selected from R; X5 is selected from K; X6 is selected from A; X7 is selected from C; Any one or more of X1, X2, X5, X6, X7 exist independently or in free combination; (II) an amino acid sequence obtained by substituting, deleting or adding one or more amino acids to the amino acid sequence described in (I), and having the same function as the amino acid sequence described in (I); or (III) an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence homology to the amino acid sequence described in (I) or (II).

2. The polypeptide according to claim 1, characterized in that It has: (I), the amino acid sequence shown in SEQ ID No. 4, 5, 7, 9, 15 to 19, 25 to 29; (II) an amino acid sequence obtained by substituting, deleting or adding one or more amino acids to the amino acid sequence described in (I), and having the same function as the amino acid sequence described in (I); or (III) an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence homology to the amino acid sequence described in (I) or (II).

3. A bioactive peptide, characterized in that It comprises the polypeptide as claimed in claim 1 or 2, as well as a membrane-penetrating peptide and a connecting peptide.

4. The bioactive peptide according to claim 3, characterized in that The cell-penetrating peptide has: (I), the amino acid sequence shown in SEQ ID No.3; (II) an amino acid sequence obtained by substituting, deleting or adding one or more amino acids to the amino acid sequence described in (I), and having the same function as the amino acid sequence described in (I); or (III), an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence homology to the amino acid sequence described in (I) or (II); and / or The connecting peptide has: (I) the amino acid sequence shown in GSG; (II) an amino acid sequence obtained by substituting, deleting or adding one or more amino acids to the amino acid sequence described in (I), and having the same function as the amino acid sequence described in (I); or (III) an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence homology to the amino acid sequence described in (I) or (II).

5. The bioactive peptide according to claim 3 or 4, characterized in that It has: (I), the amino acid sequence shown in SEQ ID No. 1, 2, 6, 8, 10-14, 20-24; (II) an amino acid sequence obtained by substituting, deleting or adding one or more amino acids to the amino acid sequence described in (I), and having the same function as the amino acid sequence described in (I); or (III) an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence homology to the amino acid sequence described in (I) or (II).

6. Use of the polypeptide according to claim 1 or 2 or the bioactive peptide according to any one of claims 3 to 5 in the preparation of any of the following drugs; (I), targeting TBK1; (II), affecting the interaction between TBK1, STING, and IRF3, thereby affecting the activation of the pathway; (III) Suppressing inflammatory or autoimmune diseases.

7. The use according to claim 6, characterized in that The effects on the interaction of TBK1-STING-IRF3 and thus on the activation of the pathway include: (I) inhibiting the expression of downstream IFNβ, CXCL10, RANTES or ISG15 inflammatory factors induced by cGAS-STING; (II) inhibiting the expression of downstream IFNβ, CXCL10 or RANTES inflammatory factors induced by TBK1; (III) affecting the expression of downstream TBK1 or IRF3 phosphorylated proteins activated by Poly(dA:dT); The inflammation includes systemic and acute inflammation induced by lipopolysaccharide (LPS), dextran sodium sulfate (DSS)-induced ulcerative colitis, or Trex1-deficient AGS; Preferably, the inhibition of inflammation comprises: (I) reducing the pathological damage of the colon induced by DSS and / or inhibiting the expression of colon inflammatory factors IL6 or RANTES; (II) Alleviate the expression of lung inflammatory factors RANTES and CXCL10 induced by LPS; (III) Alleviate the pathological damage of LPS to the lungs; (IV) Inhibit the expression of inflammatory factors RANTES or IL6 in the heart tissue of autoimmune Trex1-ko mice.

8. A drug combination, characterized in that The method comprises the polypeptide according to claim 1 or 2 or the bioactive peptide according to any one of claims 3 to 5, and other effective ingredients.

9. The pharmaceutical combination according to claim 8, characterized in that The active ingredients include but are not limited to PD-1 antibodies.

10. Use of the drug combination according to claim 8 or 9 in the preparation of a drug for preventing and / or immunotherapeutic cancer; Preferably, the prevention and / or immunotherapy of cancer comprises inhibiting the tumor MC38 or B16F10.

Images