Polypeptide sk56 and use thereof in the preparation of a medicament for preventing and / or treating diseases related to inflammatory disorders

Abstract

The application provides a polypeptide SK56 and application thereof in preparation of a medicine for preventing and / or treating a disease related to an inflammatory disorder, and belongs to the technical field of biological medicines.In the application, the amino acid sequence of the polypeptide SK56 is shown as SEQ ID NO.1.The polypeptide SK56 can be combined with a pore region of a GSDMD-N channel and inhibit the function of the channel to release inflammatory factors, so as to resist pyroptosis and inflammatory factor disorder.The test result shows that the polypeptide SK56 can effectively inhibit the release of inflammatory factors, and further inhibit the pyroptosis of cells;and it is found in the test that the polypeptide SK56 is basically non-toxic to cells.The polypeptide SK56 can be used as an inhibitor medicine for inhibiting the function of a GSDMD-N channel, and is used for resisting pyroptosis and inhibiting the release of inflammatory factors, and can be used for preparation of a medicine for treating or preventing sepsis or other inflammatory factor storm related diseases.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the peptide SK56 and its application in the preparation of drugs for the prevention and / or treatment of inflammatory dysregulation-related diseases. Background Technology

[0002] Pyroptosis, also known as inflammatory necrosis, is a type of programmed cell death characterized by the continuous swelling of cells until the cell membrane ruptures, leading to the release of cellular contents and activating a strong inflammatory response. Pyroptosis results in the massive release of inflammatory factors, subsequently causing dysregulation of the body's inflammatory response and resulting in physiological and organ dysfunction.

[0003] Shao Feng's team discovered GSDMD through genome-wide screening using CRISPR-Cas9 technology. This gene is closely related to pyroptosis; its presence triggers pyroptosis, while its absence prevents it. The terminal effector molecule of pyroptosis is the N-terminal protein of cleaved GSDMD, denoted as GSDMD-N. The study found that GSDMD-N translocates and anchors to the cell membrane, forming a channel for the release of inflammatory factors. This disrupts the osmotic pressure between the inside and outside of the cell, releasing inflammatory factors such as interleukin-1β, thus completing the pyroptosis process and triggering a cytokine storm. Therefore, blocking the binding of GSDMD-N to the cell membrane to form this inflammatory factor release channel and inhibiting the release of inflammatory factors can effectively reduce the negative harm of inflammatory factors and help restore balance to the inflammatory response.

[0004] However, there are currently no effective drugs that can treat inflammatory disorders by blocking the release of inflammatory factors from the GSDMD-N channel. Summary of the Invention

[0005] In view of this, the purpose of the present invention is to provide peptide SK56 and its use in the preparation of medicaments for the prevention and / or treatment of inflammatory dysregulation-related diseases, wherein peptide SK56 can block the function of GSDMD-N channel in releasing inflammatory factors and thereby treat inflammatory dysregulation-related diseases.

[0006] This invention provides a polypeptide SK56, comprising one or more of the polypeptides described in (1) to (3):

[0007] (1) A polypeptide with an amino acid sequence as shown in SEQ ID NO.1;

[0008] (2) A polypeptide that has the same function as the polypeptide defined in (1) after substitution, deletion or addition of one or more amino acids or chemical modification or cyclization of one or more amino acid residues based on the amino acid sequence of the polypeptide defined in (1).

[0009] (3) The salt form of the polypeptide described in item (1) and / or item (2).

[0010] The present invention also provides a medicament for the prevention and / or treatment of inflammatory dysregulation-related diseases, comprising the polypeptide SK56 described above and pharmaceutically acceptable excipients or carriers.

[0011] Preferably, the excipients include salts; the salts include salts formed from polypeptides and organic acids; the organic acids include one or more of the following: tartaric acid, benzenesulfonic acid, formic acid, glycerophosphate, acetic acid, oxalic acid, camphoric acid, butyric acid, pectic acid, maleic acid, cyclopentanepropionic acid, succinic acid, nicotinic acid, adipic acid, alginic acid, citric acid, aspartic acid, camphorsulfonic acid, digluconic acid, dodecyl sulfate, ethanesulfonic acid, glucoheponic acid, 2-naphthalenesulfonic acid, heptanoic acid, neopentanoic acid, fumaric acid, 2-hydroxyethanesulfonic acid, 3-phenylpropionic acid, propionic acid, lactic acid, methanesulfonic acid, pyric acid, p-toluenesulfonic acid, and undecanoic acid.

[0012] Preferably, the excipients include excipients; the excipients include one or more of mannitol, lactose, sucrose and fructose.

[0013] Preferably, the dosage form of the drug includes an injection.

[0014] Preferably, the mass percentage of polypeptide SK56 in the drug is 5% to 99%.

[0015] The present invention also provides the use of the SK56 described above in the preparation of medicaments for the prevention and / or treatment of inflammatory dysregulation-related diseases.

[0016] Preferably, the inflammatory dysregulation-related disease includes sepsis.

[0017] The present invention also provides the use of the SK56 described above in the preparation of drugs that inhibit pyroptosis and / or suppress the release of inflammatory factors.

[0018] The present invention also provides the application of the SK56 described above in the preparation of a drug that binds to and blocks the release of inflammatory factors from the inflammatory factor release channel; the inflammatory factor release channel is formed by GSDMD-N binding to the cell membrane.

[0019] This invention provides polypeptide SK56, comprising one or more of the polypeptides described in (1) to (3): (1) a polypeptide with an amino acid sequence as shown in SEQ ID NO.1; (2) a polypeptide having the same function as the polypeptide described in (1) after substitution, deletion, or addition of one or more amino acids or chemical modification or cyclization of one or more amino acid residues based on the amino acid sequence of the polypeptide defined in (1); (3) a salt form of the polypeptide described in (1) and / or (2). The polypeptide SK56 of this invention can bind to the channel pore region formed by GSDMD-N binding to the cell membrane and inhibit the release of inflammatory factors from the channel to combat pyroptosis and dysregulation of inflammatory factors. Experimental results show that polypeptide SK56 can effectively inhibit the release of inflammatory factors, thereby inhibiting pyroptosis; and the experiment found that polypeptide SK56 is essentially non-toxic to cells. Polypeptide SK56 can be used as an inhibitory drug for the preparation of GSDMD-N channel function, for anti-pyroptosis, and for inhibiting the release of inflammatory factors, and can be used in the preparation of drugs for the treatment or prevention of sepsis or other cytokine storm-related diseases. Attached Figure Description

[0020] Figure 1 Images showing the binding of the screening peptide SK56 to the GSDMD-N channel provided for this invention; wherein, A: the screening workflow; B: showing the screening binding sites ( Figure 1 The top image of B (indicated by the arrow); the bottom image shows a schematic diagram of the binding of peptide SK56 to the GSDMD-N channel, as well as the sequence information of peptide SK56.

[0021] Figure 2 The polypeptide SK56 provided by this invention can bind to the GSDMD-N channel in vitro and has the activity of inhibiting the release of the inflammatory factor interleukin 1β and pyroptosis; wherein, A: enzyme-linked immunosorbent assay results; B: microscopic observation of pyroptosis; C: surface plasmon resonance assay to determine the binding constant of polypeptide SK56 to the GSDMD-N channel;

[0022] Figure 3 Image showing how the peptide SK56 provided by this invention reduces the mortality rate of mice with sepsis. Detailed Implementation

[0023] This invention provides a polypeptide SK56, comprising one or more of the polypeptides described in (1) to (3):

[0024] (1) A polypeptide with an amino acid sequence as shown in SEQ ID NO.1;

[0025] (2) A polypeptide that has the same function as the polypeptide defined in (1) after substitution, deletion or addition of one or more amino acids or chemical modification or cyclization of one or more amino acid residues based on the amino acid sequence of the polypeptide defined in (1).

[0026] (3) The salt form of the polypeptide described in item (1) and / or item (2).

[0027] In this invention, the amino acid sequence shown in SEQ ID NO.1 is specifically as follows:

[0028] SLEEFAKRVVEELVKEFNLDKRQESYLEMSALIQAQMGISERIIEIVLR HAAQTLK.

[0029] This invention utilizes computer artificial intelligence and virtual screening methods to screen peptides from a self-built peptide library that can bind to the GSDMD-N channel and inhibit the release of inflammatory factors. Combined with in vitro molecular experiments and activity verification, the peptide SK56, which inhibits the GSDMD-N channel, was ultimately screened out.

[0030] The present invention also provides a medicament for the prevention and / or treatment of inflammatory dysregulation-related diseases, comprising the polypeptide SK56 described above and pharmaceutically acceptable excipients or carriers.

[0031] In this invention, the pharmaceutically acceptable excipient or carrier is preferably a pharmaceutically acceptable pharmaceutical excipient or carrier that is non-toxic to humans and animals.

[0032] In this invention, the excipients preferably include salts; the salts preferably include salts formed from polypeptides and organic acids; the organic acids include one or more selected from tartaric acid, benzenesulfonic acid, formic acid, glycerophosphate, acetic acid, oxalic acid, camphoric acid, butyric acid, pectic acid, maleic acid, cyclopentanepropionic acid, succinic acid, nicotinic acid, adipic acid, alginic acid, citric acid, aspartic acid, camphorsulfonic acid, digluconic acid, dodecyl sulfate, ethanesulfonic acid, glucoheponic acid, 2-naphthalenesulfonic acid, heptanoic acid, neopentanoic acid, fumaric acid, 2-hydroxyethanesulfonic acid, 3-phenylpropionic acid, propionic acid, lactic acid, methanesulfonic acid, pyruvic acid, p-toluenesulfonic acid, and undecanoic acid. This invention does not have a specific limitation on the source of the above-mentioned organic acids; conventional commercially available products of the above-mentioned organic acids well known to those skilled in the art can be used.

[0033] In this invention, the excipients preferably include excipients; the excipients preferably include one or more of mannitol, lactose, sucrose, and fructose. This invention does not specifically limit the source of the excipients; conventional commercially available products of the above-mentioned excipients, well-known to those skilled in the art, are acceptable.

[0034] In this invention, the dosage form of the drug preferably includes an injection.

[0035] In this invention, the mass percentage of polypeptide SK56 in the drug is preferably 5% to 99%, more preferably 10% to 80%, more preferably 20% to 60%, and most preferably 30% to 50%.

[0036] The present invention also provides the use of the SK56 described above in the preparation of medicaments for the prevention and / or treatment of inflammatory dysregulation-related diseases.

[0037] In this invention, the inflammatory dysregulation-related diseases include sepsis.

[0038] In this invention, the prevention and / or treatment of inflammatory dysregulation-related diseases is preferably achieved by the peptide SK56 binding to the inflammatory factor release channel and blocking the inflammatory factor release function of the channel; the inflammatory factor release channel is formed by GSDMD-N binding to the cell membrane.

[0039] The present invention also provides the use of the SK56 described above in the preparation of drugs that inhibit pyroptosis and / or suppress the release of inflammatory factors.

[0040] In this invention, the anti-pyroptosis and / or inhibition of inflammatory factor release is preferably achieved by the peptide SK56 binding to the inflammatory factor release channel and blocking the inflammatory factor release function of the channel; the inflammatory factor release channel is formed by GSDMD-N binding to the cell membrane.

[0041] The present invention also provides the application of the SK56 described above in the preparation of a drug that binds to and blocks the release of inflammatory factors from the inflammatory factor release channel; the inflammatory factor release channel is formed by GSDMD-N binding to the cell membrane.

[0042] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments thereof.

[0043] Example 1: Virtual Drug Screening

[0044] A three-dimensional model of the GSDMD-N channel protein was constructed based on its publicly available structure, and molecular dynamics simulations were used to optimize the 3D structure. Subsequently, ligand peptides were screened for the region of the GSDMD-N channel facing the pore center to identify drugs that could interfere with its binding. The screening database consisted of a self-built peptide library containing clinically approved peptide drugs, naturally occurring peptides, peptide fragments derived from the structural dissection of proteins with existing structures, and novel peptides generated by artificial intelligence. The top 12 candidate peptides were directly expressed and purified in prokaryotes, and their activity was further screened using in vitro experimental methods to obtain the active peptide SK56 (see...). Figure 1 , Figure 1 Images show the screening process for GSDMD-N channel inhibitory peptides. A: The screening workflow; after screening, each high-scoring candidate peptide was validated in vitro to ensure that the candidate peptide, while binding to the GSDMD-N channel, also exhibits pyroptosis-inhibiting activity; B: Images show the selected binding sites. Figure 1 The top image of B (indicated by the arrow); the bottom image shows a schematic diagram of the binding of peptide SK56 to the GSDMD-N channel, as well as the sequence information of peptide SK56.

[0045] Example 2

[0046] Figure 2 This diagram illustrates the activity of peptide SK56 in in vitro binding to GSDMD-N channels and its inhibitory activity against pyroptosis. A: Enzyme-linked immunosorbent assay (ELISA) results show that peptide SK56 exhibits the highest inhibitory activity against IL-1β release compared to other candidate peptides; B: Direct microscopic observation of cells shows that no pyroptosis occurred in cells treated with peptide SK56 within the same treatment time (3 h); C: Surface plasmon resonance (SPR) assays determined that the binding constant of peptide SK56 to GSDMD-N channels is approximately 3.39e⁻⁹ mol.

[0047] Pyroptosis assay. THP-1 cells were first cultured in 200 ng / ml PMA (phorbol ester) for 24 hours, after which the culture medium was changed. The next day, 1 μg / ml LPS (lipopolysaccharide) and 10 μM Nigericin were added. The treatment group received approximately 15 μM of peptide SK56. Microscopic observation was performed every 10 molecules to determine the inhibitory effect of peptide SK56 on pyroptosis. Results showed that, compared to the control group, peptide SK56 significantly inhibited pyroptosis activity, and no significant pyroptosis was observed after 6 hours of treatment. (See...) Figure 2 (A) in the middle.

[0048] Example 3

[0049] Enzyme-linked immunosorbent assay (ELISA). The solutions used in the experiment were formulated as follows: Coating buffer: 0.05M carbonate buffer (pH 9.6); 1.59g sodium carbonate + 2.93g sodium bicarbonate dissolved in 1L deionized water. Washing buffer: Cell-grade phosphate-buffered saline (PBS) + 0.1% Tween-20. Note: Cell-grade PBS: 0.2g potassium chloride, 0.2g potassium dihydrogen phosphate, 8g sodium chloride, 2.16g sodium dihydrogen phosphate heptahydrate, 1L deionized water. Blocking buffer (antibody dilution): 1g bovine serum albumin (BSA) per 100ml of washing buffer. Chromogenic solution: 3,3',5,5'-tetramethylbenzidine (TMB) single-component chromogenic solution. Stop solution: 2mol / L sulfuric acid, 178.3ml water + 21.7ml concentrated sulfuric acid, slowly stirred until well mixed.

[0050] The specific steps are as follows: First, add the cell supernatant treated in Example 2 to the test plate coated with interleukin-1β, incubate at room temperature for 0.5 h, wash three times, add 100 μL of horseradish peroxidase (HRP)-labeled secondary antibody (diluted according to the instructions) to each well, and incubate at 37°C for 1 h. After washing three times, add 100 μL of chromogenic solution to each well, incubate at 37°C in the dark for 20 min, then add 100 μL of stop solution, and measure the dual-wavelength readings at 450 / 630 nm using a microplate reader.

[0051] The results showed that peptide SK56 could also significantly inhibit the release of the inflammatory cytokine interleukin-1β. (See...) Figure 2 (B in the middle).

[0052] Example 4: Surface Plasmon Resonance Experiment

[0053] This experiment was performed using a ForteBio Octet instrument with phosphate buffer (pH 7.4). The instrument was operated according to the instruction manual. First, an optical fiber was loaded. Then, N-hydroxysuccinimide (EDS / NHS) was used to activate the functional groups on the fiber, crosslinking the peptide SK56 onto the fiber through this functional group reaction, thus completing the immobilization. Next, 1M ethanolamine hydrochloride (pH 8.5) was used to block excess active carboxyl groups on the fiber. Subsequently, multichannel detection was performed, and the binding constant was calculated by adding different concentrations of the target GSDMD-N protein and extracted cell membranes.

[0054] The results showed that the binding constant between peptide SK56 and GSDMD-N was approximately 3.39e-9 moles. (See...) Figure 2 (C in the middle).

[0055] Example 5

[0056] Mortality experiment in septic mice. Ten 5-week-old C57 mice (half male and half female) were intraperitoneally injected with 15 mg / kg LPS to induce sepsis. Twenty hours later, or half an hour before LPS injection, the drug-treated group received a tail vein injection of 20 mg / kg peptide SK56. The survival of the mice was observed. LPS caused a severe inflammatory response in the mice, leading to death. The addition of peptide SK56 inhibited pyroptosis and the release of inflammatory factors, protecting the mice. The more mice that survived after the addition of the peptide, the better the drug's effect.

[0057] Experimental results showed that the mortality rate of mice treated with peptide SK56 was significantly reduced, demonstrating a protective effect. See results below. Figure 3 , Figure 3 Image showing how peptide SK56 reduced mortality in septic mice. The group treated with peptide SK56 showed a significantly higher survival rate, indicating that peptide SK56 played a protective role.

[0058] This invention employs a strategy of designing novel peptides based on target structure, utilizing computer artificial intelligence and virtual screening methods to screen inhibitors binding to the GSDMD-N channel from a proprietary peptide library, combined with in vitro molecular experiments and activity verification. The subsequent validation experiments significantly increase the reliability of the screening results. This invention provides a novel molecule for the treatment and prevention of diseases related to pyroptosis and dysinflammatory responses by inhibiting GSDMD-N channel function—peptide K56.

[0059] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. The polypeptide SK56, characterized in that, The polypeptide is one or both of the polypeptides described in (1) to (2): (1) A polypeptide with an amino acid sequence as shown in SEQ ID NO.1; (2) The salt form of the polypeptide described in item (1).

2. A drug for the prevention and / or treatment of sepsis, characterized in that, It includes the polypeptide SK56 as described in claim 1 and pharmaceutically acceptable excipients or carriers.

3. The drug according to claim 2, characterized in that, The excipients include salts; the salts include salts formed from polypeptides and organic acids; the organic acids include one or more of the following: tartaric acid, benzenesulfonic acid, formic acid, glycerophosphate, acetic acid, oxalic acid, camphoric acid, butyric acid, pectic acid, maleic acid, cyclopentanepropionic acid, succinic acid, nicotinic acid, adipic acid, alginic acid, citric acid, aspartic acid, camphorsulfonic acid, digluconic acid, dodecyl sulfate, ethanesulfonic acid, glucoheponic acid, 2-naphthalenesulfonic acid, heptanoic acid, neopentanoic acid, hexanoic acid, fumaric acid, 2-hydroxyethanesulfonic acid, 3-phenylpropionic acid, propionic acid, lactic acid, methanesulfonic acid, pyric acid, p-toluenesulfonic acid, and undecanoic acid.

4. The drug according to claim 2 or 3, characterized in that, The excipients include excipients; the excipients include one or more of mannitol, lactose, sucrose and fructose.

5. The drug according to claim 2, characterized in that, The dosage form of the drug includes injections.

6. The drug according to claim 2 or 5, characterized in that, The mass percentage of polypeptide SK56 in the drug is 5% to 99%.

7. The use of the SK56 of claim 1 in the preparation of a medicament for the prevention and / or treatment of sepsis.

8. The use of SK56 as described in claim 1 in the preparation of an anti-pyroptosis drug.

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