Polypeptide DR57 and application thereof in preparation of medicine for treating sepsis immune disorder related diseases

Abstract

The invention discloses a polypeptide DR57 and application thereof in preparation of drugs for treating sepsis immune disorder related diseases, and belongs to the technical field of biological medicines. The amino acid sequence of the polypeptide DR57 is shown as SEQ ID NO.1, and the polypeptide DR57 can be specifically combined with GSDMD-NT protein and inhibit punching of the GSDMD-NT protein on cell membranes and mitochondrial intima, so that generation and development of pyroptosis are inhibited, release of inflammatory factors is reduced, the effect of treating inflammation disorder related diseases is achieved, and the polypeptide DR57 is a potential medicine for treating sepsis.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to a polypeptide DR57 and its application in the preparation of drugs for treating sepsis immune disorder-related diseases. Background Art

[0002] Pyroptosis is a programmed cell death mode triggered by inflammation-related signals. Its core feature is the formation of cell membrane pores depending on the Gasdermin family proteins (especially GSDMD), resulting in changes in cell permeability, swelling and rupture, and the release of a large amount of pro-inflammatory factors and cell contents, thus triggering a strong inflammatory response. Its molecular mechanism mainly involves the activation of inflammasomes. Through the classical pathway, caspase-1 or the non-classical pathway, caspase-4 / 5 / 11 cleaves GSDMD, making its N-terminal fragment form pores on 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 the dysregulation of the host's response to infection. When sepsis occurs, endotoxin can induce pyroptosis of the non-classical pathway, activate caspase-11 to cleave the GSDMD protein, resulting in the free GSDMD-NT terminus reaching the cell membrane, punching holes in the cell membrane, changing cell membrane permeability, releasing cell contents, triggering a systemic inflammatory response, endothelial damage and multiple organ failure, becoming an important driving factor for septic shock. Therefore, precisely inhibiting the specific activation of pyroptosis is a key target for balancing host defense and immunopathological damage. Currently, there is no effective drug for blocking the function of the GSDMD N-channel to release inflammatory factors and thus treating inflammation disorder-related diseases (sepsis). Summary of the Invention

[0004] Therefore, the main object of the present invention is to provide a polypeptide DR57 and its application in the preparation of drugs for treating sepsis immune disorder-related diseases. As a pyroptosis agent, the polypeptide DR57 can effectively treat sepsis. It can not only significantly inhibit the process of pyroptosis and reduce the release of inflammatory factors, but also has good safety.

[0005] The object of the present invention is achieved by the following technical solutions: A polypeptide DR57, whose amino acid sequence is as shown in SEQ ID NO.1, or a polypeptide obtained by substituting, deleting or adding one or several amino acid residues to the polypeptide as described in SEQ ID NO.1, which has the ability to inhibit the process of pyroptosis.

[0006] In some specific embodiments, its amino acid sequence is as shown in SEQ ID NO.1.

[0007] As the same concept of the present invention, the application of the polypeptide DR57 as a pyroptosis inhibitor in the preparation of a medicament for treating sepsis immune disorder-related diseases.

[0008] Wherein the sepsis is defined based on the international consensus diagnostic standard Sepsis-3, emphasizing organ dysfunction caused by infection, evaluated by the SOFA score, with a baseline score ≥ 2 points; In some specific embodiments, the polypeptide DR57 can specifically bind to the GSDMD-NT protein.

[0009] In some specific embodiments, the medicament can be delivered through a drug delivery system of hydrogel, or nanoion, or liposome.

[0010] Regarding the description of the drug: Preferably, the medicament further comprises a pharmaceutically acceptable carrier, and such carriers include (but are not limited to): diluents, buffers, suspensions, emulsions, granules, encapsulants, excipients, fillers, binders, sprays, transdermal absorbents, wetting agents, disintegrants, absorption promoters, surfactants, colorants, flavoring agents or adsorption carriers.

[0011] The medicament 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.

[0012] The administration route of the medicament of the present invention is not limited as long as it can exert the desired therapeutic effect or prevention effect, including but not limited to oral administration, intravenous injection, intramuscular injection, subcutaneous injection, sublingual administration, rectal perfusion, nasal spray, oral spray, topical or systemic transdermal administration of the skin.

[0013] The medicament of the present invention can also be used in combination with other medicaments for treating sepsis immune disorder-related diseases, and the combined use of multiple medicaments can greatly improve the success rate of treatment.

[0014] Compared with the prior art, the present invention has at least the following advantages: DR57 provided by the present invention is designed according to the GSDMD-NT structure, and its sequence is DREEKEELSREANIPPEEAQFILHLFRLLERFGIPPEEVKKQIVRQLRVRNEELAR. It can specifically bind to GSDMD-NT, prevent GSDMD-NT protein from punching holes in the cell membrane, inhibit the process of pyroptosis, reduce the release of inflammatory factors, and thus treat sepsis. Moreover, it also has very good drug safety and is a very effective pyroptosis inhibitor. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] In order to more clearly illustrate the specific embodiments of the present invention, the drawings required for use in the specific embodiments or the description of the prior art will be briefly introduced below.

[0016] Figure 1 Design and synthesis of the polypeptide DR57 of the present invention; Figure 2 Mass spectrometry detection chart of the polypeptide DR57 of the present invention; Figure 3 Measurement of the binding rate of the polypeptide DR57 to GSDMD-NT by MST in the present invention (control group); Figure 4 Measurement of the binding rate of the polypeptide DR57 to GSDMD-NT by MST in the present invention (test group); Figure 5 In vitro cell experiments in the present invention to detect the inhibitory effect of the polypeptide DR57 on pyroptosis; Figure 6 Detection of the function of DR57 in inhibiting the release of cytokines (IL-1β) in the present invention; Figure 7 In vivo animal experiments in the present invention to detect the protective effect of DR57 on the survival rate of septic mice. SPECIFIC EMBODIMENTS

[0017] The present invention will be further described in detail below with reference to the drawings and embodiments. The following embodiments are only descriptive and not restrictive, and the protection scope of the present invention cannot be limited thereby.

[0018] When expressing a certain quantity, concentration or other value or parameter in the form of a range, a preferred range, or a preferred upper limit and lower limit of a value, it should be understood that any range formed by combining any upper limit of the range or a preferred value with any lower limit of the range or a preferred value is specifically disclosed, regardless of 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.

[0019] Unless otherwise specified, all percentages, parts, ratios, etc. in this article are by weight.

[0020] The materials, methods, and examples in this article are exemplary and should not be construed as restrictive unless otherwise specified.

[0021] In the following experiments, the LPS (lipopolysaccharide, brand: Sigma, catalog number: L2880); NIG (Nigericin sodium 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); GSDMD-NT (Recombinant Human GSDMD-N, brand: FineTest, catalog number: P9422) were used.

[0022] The experimental samples used were wild-type C57 mice and THP cell lines.

[0023] Based on the changes in the protein structure sites during the process of GSDMD being cleaved and transformed into GSDMD-NT, the polypeptide DR57 was designed. The amino acid sequence is: DREEKEELSREANIPPEEAQFILHLFRLLERFGIPPEEVKKQIVRQLRVRNEELAR. This polypeptide can specifically bind to the GSDMD-NT protein, inhibit its pore formation on the cell membrane and the inner mitochondrial membrane, thereby inhibiting the occurrence and development of pyroptosis, reducing the release of inflammatory factors, and then playing a role in treating diseases related to inflammatory disorders. Therefore, DR57 can be used as a pyroptosis inhibitor to inhibit the process of cell pyroptosis, reduce the release of inflammatory factors, and treat diseases related to sepsis immune disorders. It is a potential drug for treating sepsis. Specifically: Example 1 Materials and Experimental Methods: A plasmid was constructed based on the DR57 amino acid sequence: DREEKEELSREANIPPEEAQFILHLFRLLERFGIPPEEVKKQIVRQLRVRNEELAR (SEQ ID NO.1). Specifically, after transforming 2 μL of the plasmid into BL21(DE3) competent cells, it was spread on an LB resistant plate and cultured inverted at 37 °C for 17 h. For expression identification, a single colony was picked and inoculated into 4 mL of LB medium containing resistance, cultured at 37 °C and 220 rpm until OD600 = 0.6 - 0.8, then IPTG was added for induction (a blank control was also set). After centrifuging to collect the bacterial pellet, it was resuspended in buffer and sonicated on ice. The supernatant and precipitate were subjected to SDS-PAGE analysis (the sample needed to be mixed with reducing Loading buffer, boiled, and centrifuged).

[0024] For Western Blot detection, the protein was transferred to a PVDF membrane by wet transfer method, developed after incubation with a primary antibody of His-tag polyclonal antibody (AP0032) and a secondary antibody labeled with HRP (BS13278); during scale-up culture, the seed solution was inoculated into 500 mL of LB medium at a ratio of 1:100, and the bacterial pellet was collected by centrifugation after induction; at the protein purification stage, it was sonicated at a ratio of 1:20 (bacterial solution / buffer), and eluted successively with equilibrium buffer, 50 mM and 350 mM imidazole buffer by Ni column chromatography. Finally, the eluate was dialyzed (1:1000) and then cleaved at 4 °C for 16 h by SUMO enzyme (10 μL of enzyme solution was added to 1 mg / mL sample), and the target protein was obtained by collecting the flow-through of the Ni column; finally, protein mass spectrometry detection was performed.

[0025] For the design and synthesis process of DR57, see Figure 1 , the protein structure of GSDMD-NT is shown in the left figure of 1A, and the molecular docking of DR57 and GSDMD-NT is as Figure 1 shown, the green part is the polypeptide DR57, and the yellow part is GSDMD-NT; the protein mass spectrometry detection is as Figure 2 shown, and the results show that the synthetic amino acid sequence of DR57 is consistent with the protein after extraction and purification, indicating the successful design and synthesis of DR57.

[0026] Example 2 The polypeptide DR57 in this application has excellent specific interaction with GSDMD-NT. Specifically: Materials and Experimental Methods: Based on the DR57 amino acid sequence, it was expressed prokaryotically in Escherichia coli, and the DR57 polypeptide protein with a FITC fluorescent label was extracted. 16 two-fold decreasing dilution gradients were set, and it was reacted with GSDMD-NT protein on ice for 15 minutes; in the control group, LPS was used to replace GSDMD-NT; then, a microscale thermophoresis instrument was used to detect the fluorescence intensity and data analysis was performed.

[0027] Results and Discussion: The binding rate results of DR57 and GSDMD-NT are shown in Figure 3 and Figure 4 , Figure 3 which is the control group, Figure 4 and this is the DR57 detection group. The MST detection results show that the binding constant of the control group is 0 KD, while that of the DR57 group is 7.6594x10 -6 KD. The experimental results suggest that there is a very strong specific interaction between DR57 and GSDMD-NT.

[0028] Example 3 The polypeptide DR57 in this application can inhibit pyroptosis and maintain the integrity of macrophages; Materials and Experimental Methods: After inducing THP-1 cells with 1 ng / ml PMA for 24 hours, wash them 3 times with PBS and divide them into NC control group, LPS-NIG group and LPS-NIG-DR57 group. Among them, the NC group is cultured in normal medium; the LPS-NIG group is added with 1 μg / ml LPS and 10 μM nigericin (NIG); the LPS-NIG-DR57 group is added with 1 μg / ml LPS, 10 μM nigericin (NIG) and 15 uM DR57. After stimulating for 2 hours, collect images under a microscope.

[0029] Results and Discussion: The specific results of in vitro cell detection of the inhibitory effect of DR57 on pyroptosis are shown in Figure 5 , the left side is the NC group, the middle is the LPS-NIG group, and the right side is the LPS-NIG-DR57 group. Microscopic photography shows that: oval macrophages with clear boundaries and good three-dimensional sense can be observed in the NC group; while the cell morphology of the LPS-NIG group is enlarged and round, showing the typical balloon shape of pyroptosis; the LPS-NIG-DR57 group can observe clear boundaries, good three-dimensional sense, no enlargement, and no pyroptotic balloon shape; the experimental results suggest that the DR57 polypeptide can inhibit pyroptosis and maintain the integrity of macrophages.

[0030] Example 4 The polypeptide DR57 in this application has a significant effect of inhibiting the release of inflammatory factors. Specifically: Materials and Experimental Methods: After inducing THP-1 cells with PMA at a concentration of 1 ng / ml for 24 hours, wash them 3 times with PBS and divide them into an NC control group, an LPS-NIG group, and an LPS-NIG-DR57 group. Among them, the NC group is cultured in normal medium; the LPS-NIG group is added with 1 μg / ml LPS and 10 μM nigericin (NIG); the LPS-NIG-DR57 group is added with 1 μg / ml LPS, 10 μM nigericin (NIG), and 15 uM DR57. After stimulating for 2 hours, collect the cell supernatant, centrifuge at 3000 RPM for 20 minutes, remove the precipitate, and keep the cell supernatant on ice for later use; according to the experimental steps of the BBI brand IL-1β ELISA kit, detect the IL-1β protein and perform data analysis.

[0031] Results and Discussion: Detection of the inhibition of cytokine (IL-1β) release by DR57, and the results are as Figure 6 . The experimental results show that the expression level of IL-1β in the NC group is 147.4 ± 27.8 pg / ml; while the expression level of IL-1β in the LPS-NIG group is 768.6 ± 102.9 pg / ml; the expression level of IL-1β in the LPS-NIG-DR57 group is 345 ± 49.6 pg / ml; suggesting that a large amount of inflammatory factor IL-1β is released during pyroptosis, and DR57 has a significant effect on inhibiting the release of inflammatory factors.

[0032] Example 5 The polypeptide DR57 in this application can effectively increase the mortality rate of LPS-induced systemic inflammation mice and has a preventive and therapeutic effect on systemic inflammatory sepsis, specifically as follows: Materials and Experimental Methods: Use 6-8 week-old C57 mice to establish a systemic inflammation model by intraperitoneal injection of LPS at a dose of 25 mg / kg according to the mouse body weight. A total of 2 groups are divided into a control group and a DR57 intervention group. Among them, in the DR57 intervention group, 6 hours after modeling, inject DR57 polypeptide via the tail vein at a dose of 5 mg / kg; the control group is injected with an equal volume of normal saline; observe the mortality rate of mice within 72 hours and perform data statistics.

[0033] Results and Discussion: Verify the protective effect of DR57 on the survival rate of septic mice in vivo, and the results are as Figure 7 . The experimental results show that: the mortality rate of mice in the control group within 72 hours is 90%; while the mortality rate of mice in the DR57 intervention group within 72 hours is 40%; suggesting that DR57 polypeptide can effectively increase the mortality rate of LPS-induced systemic inflammation mice and has a preventive and therapeutic effect on systemic inflammatory sepsis.

[0034] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that: they can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements on some or all of the technical features; and these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the various embodiments of the present invention, and they should all be covered within the scope of the claims and the 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 pyroptosis inhibitor in the preparation of a medicament for treating sepsis immune disorder-related diseases.

3. The application according to claim 2, wherein The polypeptide DR57 can specifically bind to the GSDMD-NT protein.

4. The application according to claim 2, characterized in that The medicament can be delivered through a drug delivery system of hydrogel, or nanoion, or liposome.

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