Fpr1 polypeptide blockers against lung inflammation and uses thereof

Abstract

The application discloses an FPR1 polypeptide blocker against lung inflammation and application thereof. The application provides a polypeptide shown in Sequence NO. 1, which can significantly improve lung tissue structure damage of a lung inflammation model mouse, reduce a proinflammatory factor level, reduce neutrophil infiltration, and show obvious anti-lung inflammation efficacy. Therefore, the polypeptide shown in Sequence NO. 1 has a prospect of development into an anti-lung inflammation drug.

Description

Technical Field

[0001] This invention belongs to the field of polypeptides, and relates to novel polypeptides and their applications, specifically to an FPR1 polypeptide blocker for anti-pulmonary inflammation and its applications. Background Technology

[0002] Pneumonia is an acute respiratory infection affecting the alveoli, pulmonary interstitium, and distal bronchi. It is usually caused by viruses or bacteria and is one of the major diseases threatening human health. If a patient's condition deteriorates rapidly, developing severe pneumonia symptoms such as hypothermia and hypoxemia, the mortality rate can exceed 50%. Pneumonia is broadly classified into community-acquired pneumonia (CAP) and hospital-acquired pneumonia. Currently, Western medicine treatment for pneumonia mainly relies on antibacterial and antiviral drugs, but due to drug resistance issues, some patients do not achieve ideal results during the acute phase. Therefore, strengthening effective prevention and treatment and clinical research on this disease is particularly urgent, in order to provide a scientific basis for the development of new drugs.

[0003] Neutrophil activation releases active substances to kill pathogens, while simultaneously amplifying the inflammatory response and inducing tissue damage. Neutrophils are key effector cells in the development and progression of pneumonia, responsible for tissue damage. Abnormal neutrophil function can significantly exacerbate lung inflammation and damage: pneumonia patients exhibit significantly increased neutrophil chemotaxis and lifespan, leading to excessive infiltration and the release of large amounts of IL-6 and TNF-α, triggering a cascade of inflammation and even causing acute respiratory distress syndrome and extrapulmonary organ damage. Inhibiting neutrophil infiltration can effectively improve adverse outcomes in pneumonia patients. Formyl peptide receptor 1 (FPR1) is mainly expressed on the surface of immune cells such as neutrophils and monocytes. It specifically recognizes formylated peptides released from pathogens or sites of injury, thereby activating G protein-mediated signal transduction. FPR1 participates in various key biological processes of neutrophils, including inducing neutrophil chemotactic migration and mediating the production of various pro-inflammatory cytokines and chemokines. Inhibiting FPR1 can effectively reduce neutrophil infiltration and improve pneumonia: Overexpression of FPR1 aggravates neutrophil infiltration and alveolar epithelial cell damage in mouse lung tissue, while inhibiting FPR1 expression can significantly reduce neutrophil infiltration, alveolar structural damage, and lung edema in mice. Therefore, inhibiting FPR1 to reduce inflammatory cell infiltration is an effective way to improve pneumonia.

[0004] This invention designs and prepares an FPR1 receptor peptide blocker, FPR1-PepBlok, which has the effects of reducing neutrophil infiltration in the lungs and reducing inflammatory response, and is suitable for the treatment of lung infections and inflammation. This invention is hereby proposed. Summary of the Invention

[0005] The first objective of this invention is to provide a polypeptide, the second objective is to provide the application of the polypeptide in the preparation of FPR1 receptor blocker drugs, and the third objective is to provide the application of the polypeptide in the preparation of anti-pulmonary inflammation drugs.

[0006] The above-mentioned objective of this invention is achieved through the following technical solution:

[0007] A polypeptide whose amino acid sequence is shown in Sequence NO.1.

[0008] The application of the polypeptide shown in Sequence NO.1 above in the preparation of FPR1 receptor blocker drugs.

[0009] The application of the polypeptide shown in Sequence NO.1 above in the preparation of drugs for treating lung inflammation.

[0010] Preferably, the drug uses the polypeptide shown in Sequence NO.1 as the active ingredient and is formulated into a pharmaceutically acceptable dosage form with pharmaceutically acceptable excipients.

[0011] More preferably, the excipient is a solid, liquid, or semi-solid.

[0012] More preferably, the dosage form is a tablet, capsule, injection, or pill.

[0013] Beneficial effects:

[0014] This invention provides a polypeptide as shown in Sequence No. 1, which significantly improves lung tissue structural damage in a mouse model of lung inflammation, reduces pro-inflammatory factor levels, and decreases neutrophil infiltration, exhibiting significant anti-inflammatory efficacy. Therefore, the polypeptide shown in Sequence No. 1 provided by this invention has the potential to be developed into an anti-inflammatory drug. Attached Figure Description

[0015] Figure 1 The HPLC chromatogram of the peptide blocker FPR1-PepBlok is shown.

[0016] Figure 2 This is a mass spectrometry result of the peptide blocker FPR1-PepBlok.

[0017] Figure 3 The images show the pathological changes in the lung tissue of mice in each group; where a represents the HE staining results of the lung tissue of mice in each group (bar = 50 μm); b represents the alveolitis score of mice in each group; and c represents the lung injury score of mice in each group. Comparison with the control group is also provided. * P<0.05, ** P<0.01; Compared with the model group: # P<0.05, ##P<0.01; n=6;

[0018] Figure 4 Changes in TNF-α, IL-6, and IL-1β in lung tissue of mice in each group; compared with the control group: ** P<0.01, ** P<0.01; Compared with the model group: # P<0.05, ## P<0.01; n=6;

[0019] Figure 5 Changes in total protein content, total cell count, and neutrophil ratio in bronchoalveolar lavage fluid of mice in each group; compared with the control group: * P<0.05, ** P<0.01; Compared with the model group: # P<0.05, ## P<0.01; n=3~6. Detailed Implementation

[0020] The following describes the substantive content of the present invention in detail with reference to embodiments, but this does not limit the scope of protection of the present invention.

[0021] Example 1: Peptide Synthesis and Detection

[0022] I. Polypeptide Synthesis

[0023] Establishing binding sites and designing conjugates: Unbound sites were identified using the active regions of the protein that bind to the substrate for conjugate design. After establishing the docking site for FPR1, RFdiffusion was used to design binding proteins for the docking site regions. The length of the conjugates was set to 15-25 amino acids, resulting in 100 conjugates with high specificity and affinity. The optimal conjugate for FPR1 was then found using molecular docking technology.

[0024] Hdock Fine Screening: Utilizing the efficient protein interaction docking tool Hdock, this method accurately predicts intermolecular interactions. The PDB files of FPR1 and each candidate binding protein are uploaded to the Hdock online server. The Spacing and Angle parameters of Hdock are adjusted to 1.2 and 15, respectively. Hdock scores are extracted from the Top 1 conformation of each binding protein and recorded as binding free energy. By considering the binding scores of the decoy protein FPR1 and the structure-aligned proteins, binding scores are calculated, effectively identifying interacting proteins with better stability and stronger affinity.

[0025] AlphaFold3 Fine Modeling Analysis: AlphaFold3 was used to predict the structure of the selected high-interaction complexes and verify the stability and rationality of their complex structure with FPR1. The top 20 data selected by hdock were transferred into AlphaFold3 to calculate the pTM+ipTM values, and Spearman rank correlation analysis was performed on them with the docking score (ΔG) of hdock to verify consistency.

[0026] The final obtained peptide blocker FPR1-PepBlok sequence is shown in Table 1. It was prepared by Qiangyao Biotechnology Co., Ltd. using conventional solid-phase peptide synthesis methods, achieving a purity of no less than 98%.

[0027] Table 1. Detailed sequence information of the peptide inhibitor FPR1-PepBlok.

[0028]

[0029] II. HPLC Detection

[0030] 1. Chromatographic conditions

[0031] Mobile phase A: 0.1% Trifluoroacetic in 100% Acetonitrile;

[0032] Mobile phase B: 0.1% Trifluoroacetic in 100% Water;

[0033] Flow rate: 1 mL / min;

[0034] Wavelength: 220nm;

[0035] Column: Kromasil 100-5C18, 4.6mm*250mm, 5microns;

[0036] Injection volume: 10 μL;

[0037] Elution gradient: 0.00-20.00 min, 65%-40% B; 25.00-25.01 min, 40%-0% B; 25.01 min, Stop.

[0038] 2. Test Results

[0039] Information such as HPLC chromatograms and peak integral areas is shown in Table 2 and Figure 1 As shown.

[0040] Table 2 HPLC Analysis Results

[0041]

[0042]

[0043] III. Mass Spectrometry Detection

[0044] The mass spectrometry detection graph is as Figure 2 shown, and the measured molecular weight is 2232.45 (Calculation method: 447.49×5 - 5 = 2232.45).

[0045] Example 2: Anti - pulmonary inflammation test

[0046] I. Experimental materials

[0047] Forty SPF - level male C57BL / 6J mice, weighing 20 - 22 g, were purchased from Beijing Speyford Biotechnology Co., Ltd., with the license number SCXK(Yu)2019 - 0010. The mice were raised in an environment with a temperature of 21 - 25°C, a humidity of 45% - 65%, 12 - hour alternating light and dark, and free access to food and water.

[0048] Dexamethasone: Dexamethasone sodium phosphate injection (1 mg / vial, approval number:国药准字H37021968, Chenxin Pharmaceutical Co., Ltd.), which was diluted with PBS before use.

[0049] FPR1 blocker: the polypeptide shown in Sequence NO.1 obtained in Example 1, which was diluted with PBS to the working concentration.

[0050] Lipopolysaccharide (LPS), derived from Escherichia coli 055:B5, was purchased from Merck (Sigma - Aldrich, L2880 - 100MG). The LPS dry powder was configured into 10 mg / mL with sterile PBS buffer and stored at - 80°C for later use.

[0051] Cell counter (JIMBIO Co., Ltd.); High - resolution pathological scanning slide scanning system (3DHISTECH Co., Ltd., Hungary, Pannoramic MlDlⅡ); Automatic slicer (Leica Co., Ltd.).

[0052] II. Experimental methods

[0053] 1. Modeling method

[0054] After 7 days of acclimatization, a mouse model of lung inflammation was established using non-invasive endotracheal intubation. Scissors with a thin thread were fixed to a foam board, and the mouse was anesthetized with isoflurane via inhalation. The thread was threaded through the base of the upper incisors to fix the palate vertically. The throat was illuminated with a cold light source. The mouse's tongue was pulled out with a cotton swab in the right hand, and the tongue was pulled out with the left hand to expose the pharynx. A bright spot that kept opening and closing with the mouse's breathing was observed; this was the tracheal opening. LPS was injected into the indwelling needle catheter, and the catheter was inserted into the endotracheal tube through the pharynx. The needle core was removed, and the fluid in the catheter moved up and down with the mouse's breathing, indicating successful intubation. 0.2 mL of gas was then injected into the trachea. The mouse was then suspended for 1 minute and gently turned to ensure even distribution of the bacterial solution in the lungs. The control group received sterile PBS. Successful modeling was characterized by rapid breathing, lethargy, continuous weight loss, and significant inflammatory response in the lung tissue.

[0055] 2. Grouping and Dosing

[0056] Forty SPF-grade C57BL / 6J mice were randomly divided into five groups after seven days of acclimatization: a control group, a model group, a dexamethasone (DXMS) group, a low-dose peptide inhibitor (FPR1-PepBlok Low dose, PepBlok-L) group, and a high-dose peptide inhibitor (FPR1-PepBlok Highdose, PepBlok-H) group, with eight mice in each group. After the acclimatization phase, prophylactic treatment was initiated: mice in the DXMS group were intraperitoneally injected with dexamethasone (10 mg / kg / day), while mice in the Control, Model, PepBlok-L, and PepBlok-H groups were intraperitoneally injected with PBS, twice daily for 3 days. At the last administration, mice in the DXMS group were intraperitoneally injected with dexamethasone (10 mg / kg / day), mice in the Control and Model groups were intraperitoneally injected with PBS, and mice in the PepBlok-L and PepBlok-H groups were intraperitoneally injected with different concentrations of FPR1-PepBlok diluted solution (3 mg / kg and 6 mg / kg, respectively). Two hours after the last administration, mice in the Control group were intratracheally infused with PBS, and mice in the other groups were intratracheally infused with lipopolysaccharide (LPS, 10 mg / kg) to induce a mouse model of acute lung inflammation. Two hours after LPS induction, mice in the PepBlok-L and PepBlok-H groups were intraperitoneally injected with different concentrations of FPR1-PepBlok dilution (3 mg / kg and 6 mg / kg), while mice in the Control, Model, and DXMS groups were intraperitoneally injected with PBS. Twenty-four hours after LPS induction, samples were collected.

[0057] 3. Detection indicators

[0058] 3.1 Daily Situation

[0059] Three days before modeling, the mice were weighed and their weight recorded at the same time every morning. After modeling, the mice were closely observed for their mental state, activity level, respiratory status, weight, food intake, coat color, and mortality.

[0060] 3.2 Total cell count in bronchoalveolar lavage fluid

[0061] During sample collection, the mouse trachea was fully exposed, an incision was made in the trachea, and the endotracheal cannula was inserted into the trachea and secured with surgical sutures. 1 mL of pre-cooled PBS was drawn into the lungs using a 1 mL syringe and injected into the lungs via the trachea. This was pumped back and forth 3 times, and the lavage fluid was collected into a centrifuge tube. The tubes were centrifuged at 3500 rpm for 15 min, and the supernatant was collected and stored at -80℃. 1 mL of erythrocyte lysis buffer was added to the pellet, and the cells were lysed at room temperature in the dark for 15 min. Then, 5 mL of PBS was added to stop the lysis, and the supernatant was discarded after centrifugation. 1 mL of staining buffer was added to resuspend the cells, and the cells were mixed by pipetting. 10 μL of the cell suspension was then added to a cell counter for automatic counting.

[0062] 3.3 Detection of inflammatory factors

[0063] The levels of tumor necrosis factor-α, interleukin-6, and interleukin-1β in lung tissue were detected using an enzyme-linked immunosorbent assay (ELISA). The experimental procedure involved antibody coating, addition of standards and samples, enzyme-labeled antibody, substrate chromogenic solution, and stop solution. Absorbance was measured at 450 nm and 570 nm, and the levels of inflammatory factors in BALF were calculated based on the standard curve.

[0064] 3.4 Lung tissue pathology

[0065] The left lung of mice was perfused with 4% paraformaldehyde and fixed in formaldehyde for 72 hours, with the formaldehyde fixative changed every 24 hours. A tissue block approximately 3 mm thick was transversely excised near the hilum, dehydrated using an automated dehydrator, and then fixed and embedded in paraffin to form a paraffin block. The paraffin block was then cut into 4 μm thick sections using an automated microtome for HE staining: reverse gradient alcohol dewaxing → hematoxylin staining → 1% hydrochloric acid alcohol differentiation → eosin counterstaining → gradient alcohol dehydration → neutral resin mounting. Organ pathological morphology was observed. Six sections were randomly selected from each group and photographed under a microscope at 400x magnification. At least 20 fields of view (50% alveolar area) were randomly selected, and at least three individuals independently scored the sections in a blinded manner, using the Szapie and Matte-Bello method to assess the degree of alveolitis and lung injury.

[0066] 3.5 Flow cytometry

[0067] Centrifuge the bronchoalveolar lavage fluid and collect the lower precipitate, resuspend in PBS, and add TruStainFcX. TM (anti-mouse CD16 / 32) antibody blocking was performed at room temperature for 30 min. After grouping, corresponding surface antibodies such as CD11b and Ly6G were added, and the mixture was incubated at room temperature. After further processing, the samples were analyzed using FlowJo software. CD11b was analyzed using FlowJo software. + Ly6G + Percentage of neutrophils in total cells.

[0068] 4. Statistical processing

[0069] Experimental data were analyzed using IBM SPSS 26.0 statistical software. One-way ANOVA was used for comparisons between groups; the Least Significant Difference (LSD) method was used for groups with homogeneous variances, and the Dunnett method was used for groups with unequal variances. ' The s-T3 method was used, with a significance level of α = 0.05. Data were expressed as mean ± standard deviation for statistical description.

[0070] III. Experimental Results

[0071] 1. Effects of FPR1-PepBlok on the daily condition of mice with lung inflammation model

[0072] Compared with the control group, the mice in the model group showed rapid breathing, curling up, decreased activity, poor mental state, and significantly reduced spontaneous activity. Compared with the model group, the FPR1-PepBlok group and dexamethasone intervention can effectively improve the daily condition of the mice. After treatment, the mice in the PepBlok-L group, PepBlok-H group and DXMS group showed improved condition and increased spontaneous activity.

[0073] 2. Effects of FPR1-PepBlok on lung pathology in a mouse model of lung inflammation

[0074] HE staining of lung tissue showed that the lung tissue structure of mice in the Control group was normal, the alveoli were of regular size and arrangement, and no obvious inflammatory cell infiltration was observed; the inflammatory cell infiltration of mice in the Model group was obvious, the alveolar structure was broken up more, and the alveolar wall was edematous and thickened; the lung tissue structure of mice in the FPR1-PepBlok group and DXMS group was relatively intact, and the inflammatory cell infiltration was less.

[0075] Pathological scoring results showed that the Model group mice had significant structural damage and significantly higher pathological scores (P < 0.05, P < 0.01), while the PepBlok-L, PepBlok-H, and DXMS groups all had significantly lower pathological scores (P < 0.05, P < 0.01). Results are shown in Table 3 and... Figure 3 .

[0076] Table 3. Changes in lung tissue pathological scores of mice in each group (mean ± s)

[0077]

[0078] Note: n = 6. Compared with the Control group, *P < 0.05, **P < 0.01; compared with the Model group, # P<0.05, ## P<0.01.

[0079] 3. Effects of FPR1-PepBlok on inflammatory factors in lung tissue of mice with lung inflammation model

[0080] ELISA results showed that, compared with the Control group, the levels of inflammatory factors such as TNF-α, IL-1β, and IL-6 in the lung tissue of mice in the Model group were significantly increased (P < 0.05, P < 0.01); compared with the Model group, the levels of inflammatory factors such as TNF-α, IL-1β, and IL-6 in the lung tissue of mice in the PepBlok-L group, PepBlok-H group, and DXMS group were significantly decreased (P < 0.05, P < 0.01). The results are shown in Table 4 and... Figure 4 .

[0081] Table 4. Changes in IL-6, IL-1β, and TNF-α levels in lung tissue of mice in each group (pg / mg, mean ± s)

[0082]

[0083] Note: n = 6. Compared with the Control group, * P<0.05, ** P<0.01; compared with the Model group, # P<0.05, ## P<0.01.

[0084] 4. Effects of FPR1-PepBlok on neutrophil infiltration in bronchoalveolar lavage fluid of mice with lung inflammation model

[0085] Compared with the Control group, the total cell count, total protein content, and MPO content in the lung tissue of mice in the model group were significantly increased (P < 0.05, P < 0.01); compared with the Model group, the total cell count, total protein content, and MPO content in the lung tissue of mice in the PepBlok-L group, PepBlok-H group, and DXMS group were significantly decreased (P < 0.05, P < 0.01). Results are shown in Table 5 and... Figure 5 .

[0086] Table 5. Changes in total protein content, total cell number, and neutrophil ratio in bronchoalveolar lavage fluid of mice in each group (mean ± s)

[0087]

[0088] Note: n = 6. Compared with the Control group, *P < 0.05, **P < 0.01; compared with the Model group, # P<0.05, ## P<0.01.

[0089] The above experimental results show that the polypeptide represented by Sequence NO.1 provided by this invention can significantly improve lung tissue structural damage in mice with pulmonary inflammation, reduce the level of pro-inflammatory factors, and decrease neutrophil infiltration, exhibiting significant anti-pulmonary inflammation efficacy and can be used for the prevention and treatment of pulmonary inflammation. Therefore, the polypeptide represented by Sequence NO.1 provided by this invention has the potential to be developed into an anti-pulmonary inflammation drug.

[0090] The purpose of the above embodiments is to specifically illustrate the substantive content of the present invention, but those skilled in the art should know that the scope of protection of the present invention should not be limited to the specific embodiments.

Claims

1. A polypeptide, characterized in that: Its amino acid sequence is shown in Sequence NO.

1.

2. The use of the polypeptide of claim 1 in the preparation of a medicament for treating lung inflammation.

3. The application according to claim 2, characterized in that: The drug uses the polypeptide described in claim 1 as the active ingredient and is formulated into a pharmaceutically acceptable dosage form with pharmaceutically acceptable excipients.

4. The application according to claim 3, characterized in that: The auxiliary material is a solid, liquid, or semi-solid.

5. The application according to claim 3, characterized in that: The dosage form is tablet, capsule, injection, or pill.

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