Polypeptide DR57 and its application in the preparation of drugs for treating sepsis-related immune disorders

Abstract

The present invention discloses a polypeptide DR57 and its use in the preparation of a drug for treating sepsis-related immune disorders, belonging to the field of biomedicine technology. The polypeptide DR57 of the present invention, whose amino acid sequence is shown in SEQ ID NO. 1, can specifically bind to the GSDMD-NT protein, inhibiting it from forming pores in the cell membrane and the inner mitochondrial membrane, thereby inhibiting the occurrence and development of pyroptosis and reducing the release of inflammatory factors, thereby exerting a therapeutic effect on diseases related to inflammatory disorders. It is a potential drug for the treatment of sepsis.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to a polypeptide DR57 and its application in preparing a drug for treating sepsis-related immune disorders. Background Art

[0002] Pyroptosis is a type of programmed cell death triggered by inflammatory signals. Its core characteristic is the reliance on gasdermin family proteins (particularly GSDMD) to form cell membrane pores, leading to changes in cell permeability, swelling and rupture, and the release of a large number of proinflammatory cytokines and cellular contents, triggering a strong inflammatory response. Its molecular mechanism primarily involves the activation of the inflammasome, which cleaves GSDMD through either the canonical pathway (caspase-1) or the non-canonical pathway (caspase-4 / 5 / 11), causing its N-terminal fragment to form pores in the cell membrane, ultimately leading to cell death. Therefore, pyroptosis is also known as inflammatory necrosis.

[0003] Sepsis is a life-threatening organ dysfunction caused by a dysregulated host response to infection. During sepsis, endotoxins induce pyroptosis via the non-canonical pathway, activating caspase-11 to cleave the GSDMD protein. This releases the GSDMD-NT end into the cell membrane, where it perforates the cell membrane, altering cell permeability and releasing cellular contents, triggering systemic inflammation, endothelial damage, and multiple organ failure, becoming a key driver of septic shock. Therefore, precisely inhibiting the specific activation of pyroptosis is a key target for balancing host defenses with immune pathology. Currently, there are no effective drugs that block the GSDMD N channel's ability to release inflammatory factors and thereby treat diseases associated with inflammatory disorders, such as sepsis. Summary of the Invention

[0004] To this end, the main purpose of the present invention is to provide a polypeptide DR57 and its use in the preparation of a drug for treating sepsis-related immune disorders. As a pyroptotic agent, the polypeptide DR57 is effective in treating sepsis by not only significantly inhibiting the pyroptosis process and reducing the release of inflammatory factors, but also exhibiting a good safety profile.

[0005] The purpose of the present invention is achieved through the following technical solutions:

[0006] A polypeptide DR57, whose amino acid sequence is shown in SEQ ID NO.1, or a polypeptide having the ability to inhibit cell pyroptosis by substitution, deletion or addition of one or more amino acid residues of the polypeptide as shown in SEQ ID NO.1.

[0007] In certain specific embodiments, the amino acid sequence is shown as SEQ ID NO.1.

[0008] As the same concept of the present invention, the polypeptide DR57 is used as a cell pyroptosis inhibitor in the preparation of a drug for treating sepsis-related immune disorders.

[0009] Sepsis is defined based on the international consensus diagnostic criteria Sepsis-3, which emphasizes organ dysfunction caused by infection and is assessed by SOFA score, with a baseline score of ≥2 points;

[0010] In certain specific embodiments, the polypeptide DR57 can specifically bind to the GSDMD-NT protein.

[0011] In certain specific embodiments, the drug can be delivered via a hydrogel, nanoparticle, or liposome drug delivery system.

[0012] About the medicine:

[0013] Preferably, the drug further comprises a pharmaceutically acceptable carrier, such carriers including (but not limited to): diluents, buffers, suspensions, emulsions, granules, encapsulations, excipients, fillers, adhesives, sprays, transdermal absorbents, wetting agents, disintegrants, absorption enhancers, surfactants, colorants, flavoring agents or adsorption carriers.

[0014] The drug of the present invention can be prepared into various dosage forms as needed, including but not limited to tablets, solutions, granules, patches, ointments, capsules, aerosols or suppositories.

[0015] The administration route of the drug of the present invention is not limited as long as it can exert the desired therapeutic effect or preventive effect.

[0016] The administration method may include, but is not limited to, oral administration, intravenous injection, intramuscular injection, subcutaneous injection, sublingual dissolution, rectal irrigation, nasal spray, oral spray, topical or systemic transdermal administration.

[0017] The drug of the present invention can also be used in combination with other drugs for treating sepsis-related immune disorders. The combined use of multiple drugs can greatly improve the success rate of treatment.

[0018] Compared with the prior art, the present invention has at least the following advantages:

[0019] The DR57 provided by this invention is designed based on the GSDMD-NT structure and has the sequence DREEKEELSREANIPPEEAQFILHLFRLLERFGIPPEEVKKQIVRQLRVRNEELAR. It specifically binds to GSDMD-NT, hindering the formation of pores in the cell membrane, inhibiting the pyroptosis process and reducing the release of inflammatory factors, thereby treating sepsis. It also has excellent drug safety and is a highly effective pyroptosis inhibitor. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the specific embodiments of the present invention, the following briefly introduces the drawings required for the specific embodiments or the description of the prior art.

[0021] Figure 1 Design and synthesis of the polypeptide DR57 of the present invention;

[0022] Figure 2 This is a mass spectrometry detection diagram of the polypeptide DR57 of the present invention;

[0023] Figure 3 The binding rate between the polypeptide DR57 and GSDMD-NT detected by MST in the present invention (control group);

[0024] Figure 4 The binding rate between the polypeptide DR57 and GSDMD-NT was detected by MST in the present invention (detection group);

[0025] Figure 5 The in vitro cell experiment in the present invention is used to detect the inhibitory effect of polypeptide DR57 on cell pyroptosis;

[0026] Figure 6 This is the detection of the function of DR57 in inhibiting the release of cytokines (IL-1β) in the present invention;

[0027] Figure 7 The in vivo animal experiment in the present invention detected the protective effect of DR57 on the survival rate of septic mice. DETAILED DESCRIPTION

[0028] The present invention will be further described in detail below with reference to the accompanying drawings and examples. The following examples are merely illustrative and non-restrictive, and should not be used to limit the scope of protection of the present invention.

[0029] When expressing a certain amount, concentration or other value or parameter in the form of a range, preferred range, or preferred upper and lower numerical limits, it should be understood that it is equivalent to specifically disclosing any range by combining any pair of upper range limits or preferred numerical values ​​with any lower range limit or preferred numerical value, without considering whether the range is specifically disclosed. Unless otherwise indicated, the numerical range values ​​listed herein include the endpoints of the range and all integers and fractions within the range.

[0030] Unless otherwise indicated, all percentages, parts, ratios, etc. herein are by weight.

[0031] The materials, methods, and examples herein are illustrative and, unless otherwise indicated, are not to be construed as limiting.

[0032] In the following examples, the following reagents were used: LPS (lipopolysaccharide, brand: Sigma, catalog number: L2880); NIG (Nigericinsodium salt, brand: MCE, catalog number: HY-100381); PMA (Phorbol 12-myristate 13-acetate, brand: MCE, catalog number: HY-18739); IL-1β ELISA kit (mouse interleukin-1β, brand: BBI, catalog number: D721017-0096); and GSDMD-NT (Recombinant Human GSDMD-N, brand: FineTest, catalog number: P9422).

[0033] The experimental samples used were wild-type C57 mice and THP cell lines.

[0034] Based on the changes in the protein structure site during the cleavage and conversion of GSDMD into GSDMD-NT, the present invention designed the polypeptide DR57, with the amino acid sequence: DREEKEELSREANIPPEEAQFILHLFRLLERFGIPPEEVKKQIVRQLRVRNEELAR. This polypeptide can specifically bind to the GSDMD-NT protein, inhibiting it from forming pores in the cell membrane and the inner mitochondrial membrane, thereby inhibiting the occurrence and development of pyroptosis, reducing the release of inflammatory factors, and thus playing a role in treating diseases related to inflammatory disorders. Therefore, DR57 can act as a pyroptosis inhibitor, inhibiting the process of cell pyroptosis, reducing the release of inflammatory factors, and treating diseases related to sepsis and immune disorders. It is a potential drug for the treatment of sepsis. Specifically:

[0035] Example 1

[0036] Materials and experimental methods: A plasmid was constructed based on the DR57 amino acid sequence: DREEKEELSREANIPPEEAQFILHLFRLLERFGIPPEEVKKQIVRQLRVRNEELAR (SEQ ID NO. 1). Specifically, 2 μL of the plasmid was transformed into BL21 (DE3) competent cells, spread on LB resistance plates and cultured inverted at 37°C for 17 h. For expression identification, a single clone was picked and inoculated into 4 mL of resistance-containing LB medium. The cells were cultured at 37°C and 220 rpm until OD600 = 0.6-0.8, and then IPTG was added for induction (while keeping a blank control). The bacterial sludge was collected by centrifugation, resuspended in buffer, and ultrasonically disrupted in an ice bath. The supernatant and precipitate were taken for SDS-PAGE analysis (the sample needed to be mixed with reducing loading buffer, boiled, and centrifuged).

[0037] For Western Blot analysis, proteins were transferred to PVDF membranes using the wet transfer method and developed after incubation with a His-tag polyclonal antibody (AP0032) as the primary antibody and an HRP-labeled secondary antibody (BS13278). During expansion, the seed solution was inoculated into 500 mL of LB medium at a ratio of 1:100, and the bacterial sludge was collected by centrifugation after induction. During protein purification, the cells were ultrasonically disrupted at a ratio of 1:20 (bacterial solution / buffer), and eluted by Ni column chromatography using a gradient of equilibration buffer, 50 mM, and 350 mM imidazole buffer. The final eluate was dialyzed (1:1000) and then cleaved by SUMO enzyme (1 mg / mL sample plus 10 μL enzyme solution) at 4°C for 16 h. The flow-through was collected by Ni column to obtain the target protein. Protein spectrum analysis was finally performed.

[0038] The design and synthesis process of DR57 can be found in Figure 1 The GSDMD-NT protein structure is shown in the left figure of Figure 1A, and the DR57 and GSDMD-NT molecules are docked as shown in Figure 1 As shown, the green part is the peptide DR57, and the yellow part is GSDMD-NT; protein spectrum detection is as follows Figure 2 As shown, the results showed that the synthetic amino acid sequence of DR57 was consistent with the extracted and purified protein, indicating that the design and synthesis of DR57 was successful.

[0039] Example 2

[0040] The polypeptide DR57 in this application has excellent specific interaction with GSDMD-NT, specifically:

[0041] Materials and experimental methods:

[0042] Based on the DR57 amino acid sequence, the DR57 polypeptide protein was expressed in Escherichia coli and fluorescently labeled with FITC. Sixteen 2-fold decreasing dilution gradients were set and reacted with GSDMD-NT protein on ice for 15 minutes. In the control group, LPS was used instead of GSDMD-NT. Then, the fluorescence intensity was detected by microthermophoresis, and the data were analyzed.

[0043] Results and Discussion

[0044] The results of the binding rate between DR57 and GSDMD-NT are shown in Figure 3 and Figure 4 , Figure 3 As the control group, Figure 4 The MST test results showed that the binding constant of the control group was 0 KD, while the binding constant of the DR57 group was 7.6594x10 -6 KD. The experimental results suggest that DR57 and GSDMD-NT have a very strong and specific interaction.

[0045] Example 3

[0046] The peptide DR57 in this application can inhibit cell pyroptosis and maintain the integrity of macrophages;

[0047] Materials and experimental methods:

[0048] THP-1 cells were induced with PMA at a concentration of 1 ng / ml for 24 hours, washed three times with PBS, and divided into NC control group, LPS-NIG group, and LPS-NIG-DR57 group. The NC group was cultured in ordinary culture medium; the LPS-NIG group was added with 1 μg / ml LPS and 10 μM nigericin (NIG); and the LPS-NIG-DR57 group was added with 1 μg / ml LPS, 10 μM nigericin (NIG), and 15uM DR57. After 2 hours of stimulation, images were collected under a microscope.

[0049] Results and Discussion

[0050] For the specific results of in vitro cell assays on the inhibitory effect of DR57 on cell pyroptosis, please refer to Figure 5 The NC group is on the left, the LPS-NIG group is in the middle, and the LPS-NIG-DR57 group is on the right. Microscopic images showed that in the NC group, oval macrophages with clear boundaries and good three-dimensionality were observed; in the LPS-NIG group, cells were swollen and round, typical of pyroptotic balloons; in the LPS-NIG-DR57 group, clear boundaries, good three-dimensionality, and no swelling were observed, showing pyroptotic balloons. These results suggest that the DR57 peptide can inhibit pyroptosis and maintain macrophage integrity.

[0051] Example 4

[0052] The polypeptide DR57 in this application has a significant effect of inhibiting the release of inflammatory factors, specifically:

[0053] Materials and experimental methods:

[0054] THP-1 cells were induced with 1 ng / ml PMA for 24 hours, washed three times with PBS, and divided into NC control group, LPS-NIG group, and LPS-NIG-DR57 group. The NC group was cultured in ordinary culture medium; the LPS-NIG group was added with 1 μg / ml LPS and 10 μM nigericin (NIG); and the LPS-NIG-DR57 group was added with 1 μg / ml LPS, 10 μM nigericin (NIG), and 15 μM DR57. After 2 hours of stimulation, the cell supernatant was collected and centrifuged at 3000 RPM for 20 minutes. The precipitate was removed and the cell supernatant was kept on ice for later use. IL-1β protein was detected according to the experimental procedures of the BBI brand IL-1β ELISA kit, and the data were analyzed.

[0055] Results and Discussion

[0056] DR57 inhibited the release of cytokines (IL-1β), and the results were as follows Figure 6 The experimental results showed that the expression level of IL-1β in the NC group was 147.4±27.8 pg / ml; while the expression level of IL-1β in the LPS-NIG group was 768.6±102.9 pg / ml; and the expression level of IL-1β in the LPS-NIG-DR57 group was 345±49.6 pg / ml, indicating that a large amount of inflammatory factor IL-1β was released during cell pyroptosis, and DR57 had a significant inhibitory effect on the release of inflammatory factors.

[0057] Example 5

[0058] The polypeptide DR57 in this application can effectively increase the mortality rate of mice with LPS systemic inflammation and has a preventive and therapeutic effect on systemic inflammation and sepsis, specifically:

[0059] Materials and experimental methods:

[0060] A systemic inflammation model was established in 6-8 week old C57 mice by intraperitoneal injection of LPS at a dose of 25 mg / kg based on their body weight. The mice were divided into two groups: a control group and a DR57 intervention group. The DR57 intervention group received a 5 mg / kg tail vein injection of DR57 peptide 6 hours after modeling; the control group received an equal volume of saline. The mice were observed for mortality within 72 hours, and statistical data were collected.

[0061] Results and Discussion

[0062] The protective effect of DR57 on the survival rate of septic mice was verified in vivo. Figure 7 The experimental results showed that the mortality rate of mice in the control group was 90% within 72 hours, while the mortality rate of mice in the DR57 intervention group was 40% within 72 hours, indicating that DR57 polypeptide can effectively increase the mortality rate of mice with LPS systemic inflammation and has a preventive and therapeutic effect on systemic inflammatory sepsis.

[0063] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some or all of the technical features therein. These modifications or replacements do not deviate the essence of the corresponding technical solutions from the scope of the technical solutions of the embodiments of the present invention, and they should all be included in the scope of the claims and description of the present invention.

Claims

1. A polypeptide DR57, characterized in that Its amino acid sequence is shown in SEQ ID NO.

1.

2. Use of the polypeptide DR57 according to claim 1 as a cell pyroptosis inhibitor in the preparation of a drug for treating sepsis.

3. The use according to claim 2, characterized in that The drug-carrying system of the drug is one of hydrogel, nanoparticles or liposomes.

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