A short peptide targeting ACLY and its application in bacterial infection

Abstract

This invention discloses a short peptide targeting ACLY and its application in bacterial infection, belonging to the field of biomedical technology. This invention obtained three candidate peptides through molecular docking and virtual screening. Experiments showed that the candidate peptide PVP can disrupt the ACLY-NLRP3 interaction and promote NLRP3 inflammasome activation, enhancing the host's defense against bacterial infection. This invention reveals a self-limiting circuit of immune-metabolic cross-talk, positioning the ACLY-NLRP3 axis as a novel therapeutic target for inflammasome-driven diseases, and also providing a new short peptide for the treatment of bacterial infections.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a short peptide targeting ACLY and its application in bacterial infection. Background Technology

[0002] Inflammatory bodies are crucial components of the innate immune system. They mediate caspase-1 activation and the maturation of pro-inflammatory cytokines IL-1β and IL-18 by recognizing pathogen-associated or damage-associated molecular patterns, and induce pyroptosis, playing a key role in host defense and various inflammatory diseases. Among these, the NLRP3 inflammatory body has become a research hotspot in recent years due to its broad responsiveness to various pathogens and metabolic signals, and its close association with chronic inflammation, metabolic diseases, and infectious diseases. Activation of the NLRP3 inflammatory body is tightly regulated; abnormal activation often leads to excessive inflammatory responses and tissue damage, while insufficient activity may result in decreased host resistance to infection. Therefore, finding molecular tools that can precisely regulate the activation state of the NLRP3 inflammatory body is of great significance for anti-infective and therapeutic purposes for inflammatory diseases.

[0003] ATP citrate lyase (ACLY) is a key enzyme linking mitochondrial metabolism and cytosolic acetyl-CoA synthesis. It not only participates in lipid synthesis but also regulates epigenetic processes such as histone acetylation. Recent studies have revealed that ACLY also plays a complex role in inflammatory responses, influencing macrophage polarization and inflammatory cytokine expression through metabolic remodeling. However, whether and how ACLY directly participates in the regulation of inflammasomes, particularly the NLRP3 inflammasome, remains unclear. Previous research by the inventors of this invention found that ACLY can directly bind to the Pyrin domain (PYD) of NLRP3 and inhibit its aggregation and activation, thereby negatively regulating NLRP3 inflammasome activation. This discovery reveals a non-classical function of the metabolic enzyme ACLY in innate immunity, providing a new perspective for understanding immune-metabolic interactions.

[0004] Based on the above mechanism, if a molecule capable of specifically interfering with the interaction between ACLY and NLRP3 could be developed, it would be possible to relieve ACLY's inhibition of NLRP3, thereby promoting inflammasome activation in the early stages of infection and enhancing the host's ability to clear pathogens. However, there are currently no reports on the development of short peptide intervention tools targeting the ACLY-NLRP3 interaction interface, nor are there any studies on using such short peptides to enhance the host's antibacterial infection function. Summary of the Invention

[0005] The purpose of this invention is to provide a short peptide targeting ACLY and its application in bacterial infection, in order to solve the problems existing in the prior art. The short peptide targeting ACLY obtained through screening can disrupt the ACLY-NLRP3 interaction and promote the activation of the NLRP3 inflammasome, thereby enhancing the host's defense against bacterial infection.

[0006] To achieve the above objectives, the present invention provides the following solution:

[0007] The present invention relates to the application of a short peptide in the preparation of a drug for treating bacterial infections, wherein the amino acid sequence of the short peptide is any one of those shown in SEQ ID NO.1, SEQ ID NO.3, and SEQ ID NO.5;

[0008] The bacteria in question is Klebsiella pneumoniae.

[0009] The present invention also provides the use of short peptides in the preparation of anti-inflammatory drugs, wherein the amino acid sequence of the short peptide is any one of those shown in SEQ ID NO.1, SEQ ID NO.3 and SEQ ID NO.5;

[0010] The anti-inflammatory drug is for inflammation caused by bacterial infection, and the bacteria is Klebsiella pneumoniae.

[0011] The present invention also provides a short peptide for antibacterial infection, said short peptide comprising any one of the peptides with an amino acid sequence as shown in SEQ ID NO.1, SEQ ID NO.3 and SEQ ID NO.5.

[0012] The present invention also provides a drug for antibacterial infection or anti-inflammation, the drug comprising the aforementioned short peptide.

[0013] The bacteria in question is Klebsiella pneumoniae.

[0014] The present invention discloses the following technical effects:

[0015] This invention develops a short peptide that disrupts the ACLY-NLRP3 interaction and promotes NLRP3 inflammasome activation, thereby enhancing the host's defense against bacterial infection. This invention reveals a self-limiting circuit of immune-metabolic crosstalk, positions the ACLY-NLRP3 axis as a novel therapeutic target for inflammasome-driven diseases, and also provides a novel short peptide for treating bacterial infections. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0017] Figure 1 To screen peptides targeting the ACLY region; (A) Flowchart of peptide screening targeting the ACLY region; (B) Molecular docking structures of short peptides GSQ, PSY and PVP (green) with ACLY-CCSα (blue);

[0018] Figure 2 The results of lactate dehydrogenase (LDH) release in primary peritoneal macrophages (PMs) and HEK293T cells treated with a short peptide (1 mM) for 72 h are shown; (A) PMs cells, (B) HEK293T cells; data are calculated as a percentage of the control group (not treated with the short peptide);

[0019] Figure 3 Immunofluorescence assay results of PMs and HEK293T cells after treatment with biotin-conjugated short peptide (5 μM) for 2 h; (A) PMs cells, (B) HEK293T cells; Scale bar: 10 μm;

[0020] Figure 4 To investigate the interaction between biotin-conjugated short peptides (5 μM) and HEK293T cells after 6 h of treatment with streptavidin magnetic beads, immunoprecipitation was performed. (A) PMs cells, (B) HEK293T cells; control group (Ctrl) was treated with PBS. Statistical significance was assessed by comparison with the control group. Data were from three independent experiments (n=3), and results are expressed as mean ± standard deviation. Statistical significance was calculated using one-way ANOVA; *p<0.05, **p<0.01, ***p<0.001, no significant difference in ns; β-actin was used as a control; Ctrl, control.

[0021] Figure 5 This study investigated the enzyme activity of ACLY after treatment with different concentrations of short peptides for 6 hours. The control group (Ctrl) was treated with PBS. Statistical significance was assessed by comparison with the control group. Data were obtained from three independent experiments (n=3), and results are expressed as mean ± standard deviation. Statistical significance was assessed using one-way ANOVA. *p<0.05, **p<0.01, ***p<0.001, no significant difference in ns. β-actin served as a control. Ctrl was the control.

[0022] Figure 6 The results of molecular docking and surface plasmon resonance (SPR) analysis of PVP short peptide with ACLY-CCSα are shown. (A) Molecular docking structure of PVP short peptide (green) and ACLY-CCSα (cyan); (B) Surface plasmon resonance (SPR) analysis results of ACLY-CCSα binding to PVP short peptide; The left figure shows the change in response units (RU) over time, and the right figure shows the relationship between equilibrium binding response and analyte concentration; the analyte concentration range is 2.1 × 10⁻⁶. -13 M to 1.1×10 -7 M;

[0023] Figure 7 Immunoprecipitation analysis was performed on primary peritoneal macrophages (PMs) treated with different short peptides or on HEK293T cells with exogenously expressed ACLY-Flag and NLRP3-Myc to detect the interaction between ACLY and NLRP3; (A) Immunoprecipitation analysis of the interaction between endogenous ACLY- and NLRP3 in primary peritoneal macrophages (PMs) after 12 h of treatment with PVP short peptide, BSA, and random peptide, followed by 6 h of LPS stimulation; (B) Immunoprecipitation analysis of the interaction between exogenously expressed ACLY-Flag and NLRP3-Myc in HEK293T cells treated with peptide PVP, BSA, and random controls for 12 h; Figure A uses anti-NLRP3. D4D8T Antibody detection of NLRP3 protein; β-actin as a loading control;

[0024] Figure 8 The secretion and expression of cytokines in PMs treated with PVP short peptide, BSA, and random peptide for 12 h, followed by LPS treatment for 6 h and ATP treatment for 1 h were detected by ELISA and quantitative PCR (qPCR); (A) IL-1β secretion; (B) IL-1β mRNA expression; (C) IL-6 secretion; (D) TNF-α secretion; data are expressed as mean ± standard deviation; statistical significance was assessed by comparison with the control group (Ctrl); differences were calculated using two-way ANOVA.

[0025] Figure 9 For patients who received peptide (5 mg / kg body weight) treatment for 3 consecutive days and were infected with Klebsiella pneumoniae (5 × 10⁻⁶) via intratracheal intubation. 4Analysis results of mouse survival rate (A) and bacterial load (B) at CFU / mouse; bacterial load in Figure B was measured on day 2 post-infection; data are expressed as mean ± standard deviation; in Figure A, n=10 per group; in Figure B, n=8 per group; statistical significance was assessed by comparison with the control group (Ctrl); log-rank test (Mantel-Cox) was used in Figure A; significance of differences in Figure B was calculated using two-way ANOVA.

[0026] Figure 10 For patients who received peptide (5 mg / kg body weight) treatment for 3 consecutive days and were infected with Klebsiella pneumoniae (5 × 10⁻⁶) via intratracheal intubation. 4 ELISA analysis results of IL-1β (A), IL-6 (B) and TNF-α (C) in mouse serum 24 h after CFU / mouse; data in the figure are expressed as mean ± standard deviation; mouse experimental groups: n=8 per group; statistical significance was assessed by comparison with the control group (Ctrl); the significance of differences was calculated by two-way ANOVA;

[0027] Figure 11 For patients who received peptide (5 mg / kg body weight) treatment for 3 consecutive days and were infected with Klebsiella pneumoniae (5 × 10⁻⁶) via intratracheal intubation. 4 ELISA analysis results of IL-1β (A), IL-6 (B) and TNF-α (C) in mouse lung tissue 24 h after CFU / mouse; data in the figure are expressed as mean ± standard deviation; mouse experimental group: n=8 per group; statistical significance was assessed by comparison with the control group (Ctrl); the significance of the difference was calculated by two-way ANOVA. Detailed Implementation

[0028] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0029] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0030] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0031] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0032] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0033] The inventors discovered that ACLY is a direct binding protein of NLRP3, specifically interacting with NLRP3-PYD independently of ACLY's enzymatic activity. This binding prevents NLRP3 aggregation and membrane localization, thereby inhibiting inflammasome activation. The results indicate that ACLY not only has metabolic functions but also plays a previously unknown role in directly controlling NLRP3 aggregation, thus defining a novel negative regulatory mechanism for inflammasome activation. Furthermore, the binding of NLRP3 to ACLY inhibited ACLY's enzymatic function and the production of ACLY-derived metabolites, which themselves have the ability to promote inflammasome activation. This interaction establishes NLRP3's function as a metabolic checkpoint to regulate ACLY-mediated metabolic processes, a function that transcends its traditional role in innate immunity. To translate this mechanism into applications, this invention developed a peptide that dissociates ACLY from NLRP3, promoting NLRP3 inflammasome activation and enhancing the host's defense against bacterial infection. Specific examples further illustrate the development and application of the peptide.

[0034] Example 1: Virtual screening of ACLY-targeting short peptides

[0035] (1) Molecular docking

[0036] Protein-protein docking was performed using GRAMM-X (https: / / gramm.compbio.ku.edu / ) and Rosetta software. First, an initial rigid-body docking search was conducted for NLRP3-PYD (PDB ID: 3QF2) and ACLY-CCSα (predicted by AlphaFold2). The highest-scoring model from the most frequent output clusters was selected for further optimization. Then, the selected complex structures were subjected to high-resolution flexible docking using the Rosetta docking protocol, generating 1000 substitute models. The final representative complex was selected based on the lowest interfacial energy score within the largest structure cluster.

[0037] (2) Virtual screening of peptides

[0038] This invention employs the Schrödinger software suite (version 12.8) for structure-based virtual screening of peptides. The receptor and peptide databases were constructed as follows: The NLRP3-ACLY complex obtained from the aforementioned flexible docking was first pretreated using the protein preparation wizard module under the OPLS4 force field. Subsequently, a receptor grid was generated using the receptor grid generation module by defining docking sites (Region 1) centered on the key residues Q505, E599, E669, and N668. This grid was specifically configured for peptide docking. Peptide libraries from the Top Science (https: / / www.tsbiochem.com / ) and MCE (https: / / www.medchemexpress.cn / ) databases were prepared using the LigPrep module to generate protonated states and low-energy three-dimensional conformations under physiological pH conditions.

[0039] A tiered docking strategy was implemented using a virtual screening workflow: high-throughput virtual screening (HTVS) was performed first, followed by standard precision (SP) and extra precision (XP) docking. The top 20% of compounds from each stage were retained for further optimization. Peptides selected from XP docking were evaluated for binding stability in Region 1 using Chai-1 (https: / / lab.chaidiscovery.com / ) (reflected by IpTM scores). Peptides exhibiting stable binding were advanced to flexible peptide docking. Molecular dynamics simulations were performed using the Desmond software package in an OPLS4 force field. Each protein-peptide system was solvated in an orthorhombic unit cell containing SPC water molecules and 0.15 M NaCl, neutralized by counterions. Simulations were run for 100 ns in an NPT system. The MM-GBSA binding free energy was calculated at 5-frame intervals during the production phase. The average value was used to assess binding affinity.

[0040] (3) Results and Analysis

[0041] This invention employed bioinformatics and computational investigations to screen peptides in the ACLY-Region 1 region, which has been shown to be crucial for NLRP3 binding. Initial screening was conducted through a multi-stage virtual screening process and cross-validation based on the Chai-1 model. Subsequently, flexible peptide docking, molecular dynamics simulations, and molecular mechanics-generalized Born surface area (MM-GBSA) combined with free energy calculations were performed. Figure 1 (A). The three highest-ranking peptide ligands were ultimately identified: PSYVYHQF (PSY, ΔG = -83.80 kcal / mol, SEQ ID NO.1), GSQKGAIIGLM (GSQ, ΔG = -74.54 kcal / mol, SEQ ID NO.3), and PVPQWAVGHFM (PVP, ΔG = -74.50 kcal / mol, SEQ ID NO.5). Figure 1 (See Table B and Table 1). Among all the screened candidate peptides, these peptides exhibited stable binding ability within ACLY-Region 1 and were therefore selected for further experimental validation. Figure 1 (See Table 1). This invention also synthesized two control peptides, one derived from BSA, and the other by fusing BSA with another randomly generated peptide (random peptide), and then linking these peptides to a TAT cell-penetrating peptide at the N-terminus (Table 1).

[0042] Table 1. Information on the three screened peptides and the two control peptides.

[0043]

[0044] Example 2: Performance determination of PVP short peptides

[0045] 1. The peptides were synthesized by Shanghai Sangon Biotech Co., Ltd. (Shanghai, China) with a purity >98%. The sequences of peptides with a cell membrane-penetrating TAT motif at the N-terminus are listed in Table 1. The peptides were dissolved in PBS and stored at -20°C.

[0046] The coding region of eukaryotic ACLY (i.e., amino acid design based on positions 487-810 of the full-length ACLY sequence (accession number NM_001096.3)) and CCSα were amplified under standard conditions and cloned into pEnCMV-3 × Flag-MCS or pCMV-MCS-3 × Myc-Neo vectors. Flag-tagged proteins were purified from HEK293T cells: Cells were collected 48 hours after transfection with the Flag-tagged plasmid. Cells were lysed on ice for 30 min in pre-chilled immunoprecipitation buffer. The lysate was then centrifuged at 15,000 × g for 15 min to clarify. The supernatant protein was collected at 4°C. The supernatant protein was incubated with pre-washed Flag-M2 agarose beads (Sigma-Aldrich) at 4°C with gentle rotation for 6 h. After washing the beads five times with 1 mL of immunoprecipitation buffer, elution was performed using 3×Flag peptide (Sigma-Aldrich) according to the manufacturer's instructions. The eluted protein components were analyzed by Western blotting and Coomassie Brilliant Blue staining to confirm purity and protein integrity. The purification procedures for His-tagged proteins in the prokaryotic system were strictly performed in accordance with the manufacturer's instructions (Beyotime Biotechnology, Shanghai, China).

[0047] 2. Cell Culture

[0048] Human HEK293T and THP-1 cells, as well as mouse RAW264.7 cells, were purchased from the American Type Culture Collection (ATCC). THP-1 cells were cultured in RPMI-1640 medium supplemented with 10% (v / v) fetal bovine serum (FBS, LONSERA, Shanghai Shuangru Biotechnology Co., Ltd.), 100 U / mL penicillin, and 100 mg / mL streptomycin. All other cell lines were maintained in DMEM medium supplemented with 10% FBS, 100 U / mL penicillin, and 100 mg / mL streptomycin. Primary peritoneal macrophages (PMs) were collected from mice 3 days after starch injection and cultured in DMEM medium supplemented with 10% FBS. To induce THP-1 cell differentiation, adherent differentiation was induced using propylene glycol methyl ether acetate (PMA, Sigma-Aldrich). In summary, THP-1 cells were seeded in RPMI-1640 medium containing 10% FBS, and then treated with 20 nM PMA at 37 °C and 5% CO2 for 48 h. After incubation, the medium was replaced with fresh RPMI-1640 medium containing 10% FBS to promote recovery and further differentiation for subsequent experimental procedures. All cells were cultured in a humidified incubator at 37 °C and 5% CO2. The culture material was obtained from SORFA (Beijing, China).

[0049] 3. Immunoprecipitation and immunoblotting analysis

[0050] Cells were collected at designated time points following transfection or stimulation. Lysis was performed on ice for 30 min using pre-chilled lysis buffer [containing 1.0% (v / v) NP-40, 50 mM Tris-HCl (pH 7.4), 50 mM EDTA, 150 mM NaCl, and a mixture of protease inhibitors (Merck)]. The lysis buffer was centrifuged at 15,000 × g for 15 min at 4 °C to precipitate cell debris, and the supernatant was collected. For each immunoprecipitation reaction, 500 mL of clarified lysis buffer (containing approximately 500 mg of total protein) was incubated with the corresponding antibody (Mouse anti-ATP-citrate synthase (5F8D11) or Rabbit anti-NLRP3 (D4D8T)) and 40 mL of pre-washed Protein A / G agarose beads (Santa Cruz). The mixture was slowly rotated at 4°C for 8 h, then washed five times with 1 mL IP buffer, each wash including centrifugation at 1,000 × g for 5 min at 4°C to precipitate the beads. After the final wash, the immunoprecipitation complex was eluted by boiling the beads in 40 mL SDS sample buffer [containing 60 mM Tris-HCl (pH 6.8), 1% (w / v) SDS, 5% (v / v) glycerol, 0.005% (w / v) bromophenol blue, and 1% (v / v) 2-mercaptoethanol] for 10 min. For endogenous NLRP3 protein-related immunoprecipitation experiments in primary peritoneal macrophages (PMs), cells were first treated with LPS (200 ng / mL) for 6 h, followed by cell lysis and subsequent procedures as described above.

[0051] For immunoblotting analysis, samples were prepared by mixing eluted immunoprecipitate or whole-cell lysate with 2×SDS sample buffer. Proteins were separated on 8–15% Bis-Tris gels under reducing conditions and transferred to Immobilon-P PVDF membranes (Millipore). The membranes were blocked for 1 h with TBS containing 5% bovine serum albumin (BSA, DINGGUO CHANGSHENG) [50 mM Tris-HCl (pH 7.4), 150 mM NaCl], and then incubated overnight at 4°C with primary antibodies [mouse anti-ATP-citrate synthase (5F8D11), rabbit anti-NLRP3 (D4D8T)] diluted in TBST containing 3% BSA (TBS containing 0.1% Tween 20). After washing three times with TBST, the membranes were incubated for 1 h at room temperature with HRP-labeled secondary antibodies [horseradish peroxidase-labeled goat anti-mouse IgG (H+L), horseradish peroxidase-labeled goat anti-rabbit IgG (H+L)]. Chemiluminescence signals were detected using Clarity Western ECL substrate (Bio-Rad).

[0052] 4. Surface Plasmon Resonance (SPR) Detection Method

[0053] SPR-based purification and recombinant protein binding assays were performed using a Biacore T200 instrument (Cytiva) to assess interaction affinity. The SPR experiments utilized the S-series sensor chip CM5 (Cytiva). This chip utilizes Ni... 2+ Activation and fixation to form Ni 2+ -NTA (N-acetylated propionic acid) layer to capture proteins. The running buffer used is HBS-EP. + (Cytiva), containing 10 mM HEPES (pH 7.4), 150 mM NaCl, 3 mM EDTA, and 0.05% surfactant P20, ensures stable protein-protein interactions and minimizes non-specific binding. Analyte proteins were serially diluted to predetermined concentrations in the running buffer. Each analyte was injected into the sensor chip at a flow rate of 30 mL / min at 25°C, with real-time monitoring of binding and dissociation phases. Between each injection, the sensor chip surface was regenerated using 1 M imidazole regeneration buffer to dissociate the bound protein without damaging the immobilized ligands. The resulting sensor spectra were analyzed using Biacore T200 evaluation software version 3.1 to calculate the equilibrium dissociation constant (K0). D ).

[0054] 5. Inflammatory stimulation

[0055] Cells were treated with inflammatory stimuli according to a typical two-step procedure. First, cells were induced for a specified time with LPS (200 ng / mL). Then, inflammatory stimulants such as ATP, Nig (nigrin), aluminum hydroxide, poly(dA:dT), or flagellin were added to the cell culture medium and incubated for a specified time. The corresponding concentrations were as follows: ATP, 2 mM; Nig, 10 mM; aluminum hydroxide, 500 mg / mL; poly(dA:dT), 200 ng / mL; flagellin, 200 ng / mL.

[0056] 6. ACLY enzyme activity assay

[0057] The enzyme activity of ACLY was determined using the ATP citrate lyase activity assay kit (Solarbio, Beijing, China) according to the manufacturer's instructions. For fresh lung tissue samples, they were rinsed with pre-chilled PBS (pH 7.4) to remove residual blood, blotted dry, weighed, and then chopped on a cold plate. 0.1 g of lung tissue sample was homogenized in 1 mL of extraction buffer using a magnetic bead tissue homogenizer (3 mm stainless steel beads, 60 Hz, 30 pulses × 1 min, 4°C). The homogenate was centrifuged at 8,000 × g for 10 min at 4°C, and the supernatant was collected and stored on ice for immediate analysis. Results were standardized to tissue weight. For cell samples, 5 × 10⁵ cells were collected by centrifugation at 600 × g for 5 min at 4°C. 6 Cells were collected. The cell pellet was homogenized in 1 mL extraction buffer (60 Hz, 30 pulses × 1 min, 4 °C). The homogenate was centrifuged at 8,000 × g for 10 min at 4 °C, and the supernatant was collected and stored on ice for analysis. Results were normalized to a baseline of 10 cells. For purified ACLY, the reaction product of ACLY was used for detection, and results were normalized to a baseline of protein quality.

[0058] 7. Results and Analysis

[0059] First, the cytotoxicity of these peptides was tested, and it was found that even increasing the peptide concentration to 1 mM and incubating for 72 hours had no effect on the survival rate of PMs and HEK293T cells. Figure 2 Subsequently, the peptides were conjugated with biotin and immunofluorescence assays were performed, confirming that these peptides could effectively diffuse into the cells after 2 hours of pre-incubation with PMs and HEK293T cells. Figure 3 This invention further investigated the binding affinity of these peptides to PMs and ACLY derived from HEK293T cells using a biotin-streptavidin immunoprecipitation assay. Figure 4As shown, peptide-PVP exhibits the strongest binding affinity for ACLY. After incubation, these peptides retain most of the enzymatic activity of ACLY. Figure 5 Based on the above results, the PVP short peptide (i.e., the short peptide with the amino acid sequence shown in Table 1 as SEQ ID NO.5) was finally selected for further research.

[0060] Figure 6 Figure A illustrates the binding mode of the PVP short peptide to ACLY-Region 1. This invention uses SPR experiments to determine the binding affinity of ACLY-CCSα to the PVP short peptide, and the results show that the K1 of the PVP short peptide binding to ACLY-CCSα is... D The value is 3.74 × 10 -8 M ( Figure 6 (B) Subsequently, the effect of PVP short peptides on regulating the interaction between ACLY and NLRP3 was investigated. LPS-treated PMs were prepared, and co-immunoprecipitation experiments were performed using anti-NLRP3 antibody with or without the addition of PVP short peptides. Figure 7 As shown in Figure A, the addition of PVP peptides significantly reduced the amount of ACLY immunoprecipitated by NLRP3. Similarly, in HEK293T cells, PVP peptides also reduced the amount of ACLY immunoprecipitated by exogenously expressed Myc-labeled NLRP3 (NLRP3-Myc) (ACLY-Flag). Figure 7 (Middle B). These results indicate that PVP short peptides can inhibit the interaction between ACLY and NLRP3. To further investigate the effect of PVP short peptides on NLRP3 inflammasome activation, PMs were treated with PVP short peptides and stimulated with LPS and ATP. The results showed that PVP short peptides could increase IL-1β secretion but not its expression, and had no significant effect on TNF-α and IL-6 secretion. Figure 8 This indicates that the short PVP peptide promotes the activation of the NLRP3 inflammasome.

[0061] Example 3: Application of PVP short peptides in bacterial infections

[0062] This example uses Klebsiella pneumoniae (K. pneumoniae 43816) as an example. Klebsiella pneumoniae was purchased from ATCC and cultured according to the manufacturer's instructions.

[0063] Eight-week-old pathogen-free male C57BL / 6J mice were anesthetized and placed supine on a 45° inclined board. The tongue was gently everted, and the glottis was observed using a cold light source. A sterile plastic catheter was inserted 5 mm into the trachea. A single dose of 5.0 × 10⁻⁶ ppm of [unspecified substance] was administered. 4100 mL of CFU bacterial culture was administered, followed by the injection of 200 mL of air to purge the catheter. Mice were held vertically for 5 min to ensure bilateral distribution. Control mice received an equal volume of sterile PBS. Bacterial administration was prepared with a small amount of sterile PBS to ensure accurate dosing. Mice were sacrificed on day 2 post-infection, and lung tissue was collected for bacterial load determination and hematoxylin and eosin (H&E) staining. Serum was collected for ELISA kit analysis of IL-1β, IL-6, and TNF-α.

[0064] Mice were intraperitoneally injected with these peptides at a dose of 5 mg / kg for three consecutive days, followed by intratracheal infection with Klebsiella pneumoniae. Results showed that administration of the short PVP peptide effectively reduced mortality caused by Klebsiella pneumoniae, and this treatment regimen significantly reduced the bacterial load in infected tissues. Figure 9 This confirmed its crucial role in defending against bacterial infections. Furthermore, when mice were infected with Klebsiella pneumoniae, the serum and lung tissue levels of IL-1β were significantly increased in the PVP short peptide treatment group (…). Figures 10-11 Based on the above findings, it can be concluded that PVP short peptides exert anti-infective protective effects by blocking the binding of ACLY to NLRP3 and promoting the activation of NLRP3 inflammasomes, which provides important evidence for in vivo treatment.

[0065] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. The application of short peptides in the preparation of drugs for antibacterial infection, characterized in that, The amino acid sequence of the short peptide is shown in SEQ ID NO. 5, and the bacteria is Klebsiella pneumoniae (Klebsiella pneumoniae). Klebsiella pneumoniae ).

2. The application of short peptides in the preparation of anti-inflammatory drugs, characterized in that, The amino acid sequence of the short peptide is shown in SEQ ID NO.5; The anti-inflammatory drug is for inflammation caused by bacterial infection, and the bacteria is Klebsiella pneumoniae (Klebsiella pneumoniae). Klebsiella pneumoniae ).

3. A short peptide for antibacterial infection, characterized in that, The amino acid sequence of the short peptide is shown in SEQ ID NO.5, and the bacteria is Klebsiella pneumoniae (Klebsiella pneumoniae). Klebsiella pneumoniae ).

4. A drug for treating bacterial infection or inflammation, characterized in that, The drug comprises the short peptide of claim 3, and the bacteria is Klebsiella pneumoniae (…). Klebsiella pneumoniae ).

Images