Formyl peptide receptor 1 polypeptide blocker for resisting acute lung injury and pharmaceutical application of formyl peptide receptor 1 polypeptide blocker

Abstract

The invention discloses a formyl peptide receptor 1 polypeptide blocker for resisting acute lung injury and pharmaceutical application thereof. The invention provides a polypeptide with a sequence as shown in Sequence NO.1, and the polypeptide is reported for the first time. Pharmacological activity studies show that the polypeptide shown as Sequence NO.1 can significantly improve lung tissue structure damage of acute lung injury model mice, reduce the level of proinflammatory factors and reduce neutrophil infiltration, and shows an obvious acute lung injury resisting effect. Therefore, the polypeptide as shown in the Sequence NO.1 provided by the invention has a prospect of being developed into the acute lung injury resisting medicine. Specifically, the polypeptide shown as Sequence NO.1 can be used as a unique active ingredient for preparing the medicine for treating and resisting the acute lung injury, and can also be combined with other active ingredients to form a composition for preparing the medicine for treating and resisting the acute lung injury.

Description

Technical Field

[0001] The present invention belongs to the field of polypeptides and relates to novel polypeptides and uses thereof, and in particular to a formyl peptide receptor 1 polypeptide blocker for resisting acute lung injury and its pharmaceutical use. Background Art

[0002] Acute lung injury (ALI) is an acute, persistent inflammatory lung injury. Its main pathological features include damage to alveolar epithelial cells and capillary endothelial cells, increased permeability, and the resulting severe inflammatory response in lung tissue. Patients often experience symptoms such as dyspnea, pulmonary edema, and hypoxemia, which severely impact their quality of life and prognosis. In the absence of effective intervention, ALI can progress to acute respiratory distress syndrome, with a mortality rate as high as 40% to 56%. The pathogenesis of ALI is complex, involving the infiltration of multiple inflammatory cells and the mediation of inflammatory responses. Inflammatory factors such as interleukin-1β, interleukin-6, and tumor necrosis factor-α can damage pulmonary vascular endothelial cells, leading to changes in endothelial cell permeability and diffuse interstitial and alveolar edema. Treatment of ALI primarily focuses on symptomatic management, including mechanical ventilation and corticosteroids. However, these treatments lack specific drugs and are associated with adverse reactions. Therefore, there is an urgent need for new drugs to prevent and treat ALI.

[0003] Formyl Peptide Receptor 1 (FPR1) is a G protein-coupled receptor primarily expressed on the surface of immune cells. It activates immune responses by recognizing formyl peptides released by bacteria or damaged cells. When ALI occurs, activation of FPR1 on the surface of immune cells can lead to the recruitment and activation of immune cells such as neutrophils and macrophages, releasing large amounts of inflammatory mediators, amplifying the inflammatory response, exacerbating lung tissue damage, and causing pulmonary edema, ventilation / perfusion imbalance, and respiratory dysfunction. Studies have shown that targeted inhibition of FPR1 can block the binding of formyl peptides to FPR1, thereby inhibiting the overactivation and recruitment of immune cells such as neutrophils, reducing the release of inflammatory mediators, and alleviating the inflammatory response and damage to lung tissue. Therefore, targeted inhibition of FPR1 is an effective strategy to improve ALI.

[0004] The present invention is proposed to develop a polypeptide blocker of formyl peptide receptor 1 for the treatment of acute lung injury. Summary of the Invention

[0005] The first purpose of the present invention is to provide a polypeptide, the second purpose is to provide the use of the polypeptide for preparing a formyl peptide receptor 1 blocker drug, the third purpose is to provide the use of the polypeptide for preparing an anti-acute lung injury drug, the fourth purpose is to provide an anti-acute lung injury pharmaceutical composition, and the fifth purpose is to provide the use of the pharmaceutical composition for preparing an anti-acute lung injury drug.

[0006] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

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

[0008] The polypeptide is used for preparing a formyl peptide receptor 1 blocker drug.

[0009] The polypeptide is used for preparing an anti-acute lung injury drug.

[0010] Preferably, the drug uses the above-mentioned polypeptide as an active ingredient and is prepared into a pharmaceutically acceptable dosage form with pharmaceutically acceptable excipients or carriers.

[0011] More preferably, the excipient or carrier is solid, liquid or semisolid.

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

[0013] A pharmaceutical composition for preventing acute lung injury, wherein the active ingredient comprises a polypeptide having an amino acid sequence as shown in Sequence No. 1.

[0014] The pharmaceutical composition is used for preparing drugs for treating acute lung injury.

[0015] Preferably, the drug uses the above-mentioned pharmaceutical composition as an active ingredient and is prepared into a pharmaceutically acceptable dosage form with pharmaceutically acceptable excipients or carriers.

[0016] More preferably, the excipient or carrier is solid, liquid or semisolid, and the dosage form is tablet, capsule, injection or pill.

[0017] Beneficial effects:

[0018] The present invention provides a polypeptide having a sequence as shown in Sequence NO.1, which is reported for the first time. Pharmacological activity studies have shown that the polypeptide shown in Sequence NO.1 can significantly improve lung tissue structural damage in mice with acute lung injury models, reduce the level of pro-inflammatory factors, reduce neutrophil infiltration, and exhibit obvious anti-acute lung injury effects. Therefore, the polypeptide shown in Sequence NO.1 provided by the present invention has the prospect of being developed into an anti-acute lung injury drug. Specifically, the polypeptide shown in Sequence NO.1 can be used as the sole active ingredient in the preparation of drugs for treating acute lung injury, and can also be used with other active ingredients to form a composition for the preparation of drugs for treating acute lung injury. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is the HPLC chromatogram of the peptide blocker FPR1 Block Peptide-22;

[0020] Figure 2 This is the mass spectrometry detection diagram of the peptide blocker FPR1 Block Peptide-22;

[0021] Figure 3 Figure 2 shows the pathological changes of lung tissues in mice in each group; A shows the HE staining results of lung tissues in mice in each group, bar = 50 μm; B shows the alveolitis score in mice in each group; C shows the lung injury score in mice in each group; compared with the blank group: * P<0.05, ** P<0.01; compared with the model group: # P<0.05, ## P < 0.01; n = 6;

[0022] Figure 4 The changes of TNF-α, IL-6 and IL-1β in lung tissues of mice in each group compared with the blank group: ** P<0.01, ** P<0.01; compared with the model group: # P<0.05, ## P < 0.01; n = 6;

[0023] Figure 5 The changes of total protein content, total cell count and neutrophil ratio in bronchoalveolar lavage fluid of mice in each group; compared with the blank group: * P<0.05, ** P<0.01; compared with the model group: # P<0.05, ## P<0.01; n=3-6. DETAILED DESCRIPTION

[0024] The essential contents of the present invention are described in detail below with reference to the embodiments, but the protection scope of the present invention is not limited thereto.

[0025] Example 1: Peptide synthesis and detection

[0026] 1. Peptide Synthesis

[0027] Establishing the binding site and designing binders: Using the active region of the protein for substrate binding, we established an unbound region for binder design. After establishing the FPR1 docking site, we used RFdiffusion to design binding proteins for the docking region. The combined length of the binders was set to 15-25 amino acids. We generated a total of 100 binders with high specificity and affinity, and used molecular docking technology to identify the optimal binder for FPR1.

[0028] Hdock Precision Screening: We used the highly efficient protein interaction docking tool Hdock to accurately predict molecular interactions. We uploaded the PDB files for FPR1 and each candidate binding protein to the Hdock online server. We adjusted Hdock's Spacing and Angle parameters to 1.2 and 15, respectively. We extracted the Hdock score from the top 1 conformation of each binding protein and recorded it as the binding free energy. By calculating the binding score based on the bait protein FPR1 and the aligned proteins, we effectively identified interacting proteins with greater stability and affinity.

[0029] AlphaFold3 refined modeling analysis: AlphaFold 3 was used to predict the structures of the screened highly interactive binders to verify the stability and rationality of their complex structures with FPR1: the top 20 data screened by hdock were transferred to AlphaFold3 to calculate the pTM+ipTM value, and the value was subjected to Spearman rank correlation analysis with the docking score (ΔG) of Hdock to verify the consistency.

[0030] Finally, the peptide blocker FPR1 Block Peptide-22 (FBP-22) sequence was obtained, and the detailed information is shown in Table 1. It was commissioned to Qiangyao Biotechnology Co., Ltd. using conventional peptide solid phase synthesis method, and the purity was not less than 98%.

[0031] Table 1 Sequence details of peptide blocker FPR1 Block Peptide-22

[0032]

[0033] 2. HPLC detection

[0034] 1. Chromatographic conditions

[0035] Mobile phase A: 0.1% Trifluoroacetic in 100% Acetonitrile;

[0036] Mobile phase B: 0.1% Trifluoroacetic in 100% Water;

[0037] Flow rate: 1 mL / min;

[0038] Wavelength: 220 nm;

[0039] Chromatographic column: Kromasil 100 - 5C18, 4.6 mm * 250 mm, 5 micron;

[0040] Injection volume: 10 μL;

[0041] Elution gradient: 0.00 - 20.00 min, 63% - 38% B; 20.00 - 20.10 min, 38% - 0% B; 20.10 min, Stop.

[0042] 2. Detection results

[0043] HPLC chromatogram and chromatographic peak integration area and other information are shown in Table 2 and Figure 1 as follows.

[0044] Table 2 HPLC Analysis Results

[0045]

[0046] III. Mass spectrometry detection

[0047] The mass spectrometry detection chart is as Figure 2 shown, and the measured molecular weight is 1915.53 (Calculation method: 639.51 × 3 - 3 = 1915.53).

[0048] Example 2: Anti - acute lung injury test

[0049] I. Experimental materials

[0050] Forty SPF - level C57BL / 6J male mice, weighing 20 - 22 g, were purchased from Beijing Speyford Biotechnology Co., Ltd., with the license number SCXK(Yu)2019 - 0010. The mouse rearing environment was: temperature 21 - 25 °C, humidity 45% - 65%, 12 - hour alternating light and darkness, and free access to food and water.

[0051] Dexamethasone: Dexamethasone sodium phosphate injection (1 mg / vial, approval number:国药准字H37021968, Chenxin Pharmaceutical Co., Ltd.), diluted with PBS before use.

[0052] FPR1 Block Peptide-22: the polypeptide shown in Sequence No. 1 obtained in Example 1, diluted with PBS to the working concentration.

[0053] Lipopolysaccharide (LPS), derived from Escherichia coli 055:B5, was purchased from Zhengzhou Dianqing Technology Co., Ltd. (Sigma-Aldrich, L2880-100MG). LPS powder was prepared at 10 mg / mL using sterile PBS buffer and stored at -80°C until use. Waste from the preparation process was sterilized by autoclaving at 121°C for 30 min and then disposed of uniformly.

[0054] Cell counter (JIMBIO Biological Co., Ltd.); high-resolution pathology scanning slide scanning system (Pannoramic MlDlⅡ, 3DHISTECH Company, Hungary); automatic slicer (Leica Biological Co., Ltd.).

[0055] 2. Experimental Methods

[0056] 1. Modeling method

[0057] After acclimating the mice for 7 days, a mouse model of acute lung injury was established using noninvasive endotracheal intubation. Scissors tied with string were secured to a foam board and inhaled with isoflurane. After the mice breathed deeply and rapidly, a string was passed through the base of the upper incisors to secure the upper jaw, suspending them vertically. A cold light was then applied to the throat. The tongue was lifted outward with a cotton swab in the right hand and pulled outward with the left hand to expose the pharynx. A bright spot opening and closing with each breath was visible, indicating the tracheal opening. LPS was injected into the tubing of an indwelling needle. The needle was then inserted through the throat into the endotracheal cannula. The needle core was removed. The fluid in the tubing moved up and down with the mouse's breathing, indicating successful intubation. 0.2 mL of gas was then injected into the trachea. The mice were then suspended for 1 minute and gently rotated to ensure even distribution of the bacterial solution throughout the lungs. A control group was instilled with sterile saline. Successful modeling was indicated by the development of pathological findings such as rapid breathing, lethargy, sustained weight loss, and significant lung inflammation.

[0058] 2. Grouping and Dosing

[0059] A total of 40 SPF-grade C57BL / 6J mice were randomly divided into five groups after 7 days of adaptive feeding, namely, control group, model group, dexamethasone (DXMS) group, low-dose peptide blocker (FPR1 Block Peptide-22 Low dose, FBP-22-L) group and high-dose peptide blocker (FPR1 Block Peptide-22 High dose, FBP-22-H) group, with 8 mice in each group. After the adaptive feeding phase, preventive medication was initiated: mice in the DXMS group received intraperitoneal injections of dexamethasone (10 mg / kg / d), while mice in the control, model, FBP-22-L, and FBP-22-H groups received intraperitoneal injections of PBS twice daily for 3 days. At the final dose, mice in the DXMS group received intraperitoneal injections of dexamethasone (10 mg / kg / d), mice in the control and model groups received intraperitoneal injections of PBS, and mice in the FBP-22-L and FBP-22-H groups received intraperitoneal injections of different concentrations of FBP-22 (3 mg / kg and 6 mg / kg). Two hours after the final dose, mice in the control group received intratracheal instillation of PBS, while mice in the other groups received intratracheal instillation of lipopolysaccharide (LPS, 10 mg / kg) to induce a mouse model of acute lung inflammation. Two hours after LPS induction, mice in the FBP-22-L and FBP-22-H groups were again intraperitoneally injected with different concentrations of FBP-22 (3 mg / kg and 6 mg / kg), while mice in the Control, Model, and DXMS groups were intraperitoneally injected with PBS. Samples were collected 24 hours after LPS induction.

[0060] 3. Detection indicators

[0061] 3.1 Daily Situation

[0062] Three days before modeling, mice were weighed and recorded at the same time every morning. After modeling, the mice's mental state, activity level, respiratory status, body weight, food intake, coat color, and death were closely observed.

[0063] 3.2 Total cell count of bronchoalveolar lavage fluid

[0064] When sampling, the mouse trachea was fully exposed, an incision was made on the trachea, the tracheal intubation needle was inserted into the trachea, and it was fixed with surgical thread. 1 mL of pre-cooled PBS was drawn with a 1 mL syringe and injected into the whole lung through the trachea, and pumped back and forth 3 times. The lavage fluid was collected into a centrifuge tube, centrifuged at 3500 rpm / min for 15 minutes, and the supernatant was extracted and stored at -80°C; 1 mL of red blood cell lysis buffer was added to the precipitate, and the lysis was carried out at room temperature in the dark for 15 minutes, after which 5 mL of PBS was added to stop the lysis. The supernatant was centrifuged and discarded, and 1 mL of staining buffer was added to resuspend the cells. After mixing with a pipette, 10 μL of cell suspension was extracted and dropped into a cell counter for automatic counting.

[0065] 3.3 Detection of inflammatory factors

[0066] Enzyme-linked immunosorbent assay (ELISA) was used to measure the levels of tumor necrosis factor-α, interleukin-6, and interleukin-1β in lung tissue. The experimental steps were followed: antibody coating, adding standard and sample, enzyme-labeled antibody, substrate development solution, and stop solution. Finally, absorbance was measured at 450 nm and 570 nm, respectively. Inflammatory factor levels in BALF were calculated based on the standard curve.

[0067] 3.4 Lung tissue pathology

[0068] The left lung of mice was perfused and filled with 4% paraformaldehyde and then fixed in formaldehyde for 72 hours, with the formaldehyde fixative changed every 24 hours. Tissue blocks approximately 3 mm thick were cut transversely near the hilum of the lung, dehydrated in an automatic dehydrator, and then fixed and embedded in paraffin to create paraffin blocks. The paraffin blocks were cut into 4-μm thin sections using an automatic microtome and stained with hematoxylin and eosin using the following procedure: dewaxing with a reversed alcohol gradient, staining with hematoxylin, differentiation with 1% hydrochloric acid and alcohol, counterstaining with eosin, dehydration with a continuous alcohol gradient, and mounting with neutral gum. Organ pathology was observed. Six sections were randomly selected from each group and photographed under a microscope at ×400 magnification. At least 20 randomly selected fields (50% of the alveolar area) were scored independently and blindly by at least three individuals. The degree of alveolitis and lung injury was scored according to the method of Szapie and Matute-Bello.

[0069] 3.5 Flow cytometry

[0070] Centrifuge the alveolar lavage fluid and remove the precipitate, resuspend it in PBS, and add TruStainFcX TM Anti-mouse CD16 / 32 antibodies were blocked at room temperature for 30 minutes. After grouping, corresponding surface antibodies such as CD11b and Ly6G were added and incubated at room temperature. After subsequent treatment, the cells were tested on a microscope. FlowJo software was used to analyze CD11b. + Ly6G +The percentage of neutrophils in total cells.

[0071] 4. Statistical processing

[0072] The experimental data were analyzed using IBM SPSS 26.0 statistical software. One-way ANOVA was used for comparison between groups, and the least significant difference (LSD) method was used for homogeneous variance, and the Dunnett method was used for unequal variance. ' The T3 method was used, the significance level was α = 0.05, and the data were expressed as mean ± standard deviation for statistical description.

[0073] 3. Experimental Results

[0074] 1. Effects of FBP-22 on the daily conditions of ALI mice

[0075] Compared with the Control group, the mice in the Model group showed rapid breathing, curled up, decreased activity, poor mental state, and significantly reduced autonomous activity after modeling; compared with the Model group, the FBP-22 group and dexamethasone intervention can effectively improve the daily condition of the mice. After treatment, the conditions of the mice in the FBP-22-L group, FBP-22-H group, and DXMS group improved, and their autonomous activity increased.

[0076] 2. Effect of FBP-22 on lung pathology in ALI mice

[0077] HE staining of lung tissue showed: Figure 3 As shown in Figure A, the lung tissue structure of mice in the Control group was normal, with regular alveolar size and arrangement, and no obvious inflammatory cell infiltration. The inflammatory cell infiltration of mice in the Model group was obvious, with increased alveolar structural fragmentation and edema and thickening of the alveolar wall. The lung tissue structure of mice in the FBP-22 and DXMS groups was relatively complete, with less inflammatory cell infiltration.

[0078] The pathological scoring results are shown in Table 3 and Figure 3 As shown in Figures B and C, the pathological structures of mice in the Model group were significantly damaged and the pathological scores were significantly increased (P < 0.05, P < 0.01). The pathological scores of mice in the FBP-22-L group, FBP-22-H group, and DXMS group were significantly decreased (P < 0.05, P < 0.01).

[0079] Table 3 Changes in lung tissue pathological scores of mice in each group (mean ± s)

[0080]

[0081]

[0082] 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.

[0083] 3. Effect of FBP-22 on inflammatory factors in lung tissue of ALI mice

[0084] 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 tissues 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 tissues of mice in the FBP-22-L group, FBP-22-H group, and DXMS group were significantly decreased (P < 0.05, P < 0.01). The results are shown in Tables 4 and Figure 4 .

[0085] Table 4 Changes of IL-6, IL-1β and TNF-α in lung tissues of mice in each group (pg / mg, mean ± s)

[0086]

[0087] 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.

[0088] 4. Effect of FBP-22 on Neutrophil Infiltration in Bronchoalveolar Lavage Fluid of ALI Mice

[0089] Compared with the Control group, the total cell count, total protein content in the bronchoalveolar lavage fluid and MPO content in the lung tissue of the 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 the mice in the FBP-22-L group, FBP-22-H group and DXMS group were significantly decreased (P < 0.05, P < 0.01). The results are shown in Tables 5 and Figure 5 .

[0090] Table 5 Changes in total protein content, total cell count and neutrophil ratio in bronchoalveolar lavage fluid of mice in each group (mean ± s)

[0091]

[0092]

[0093] 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.

[0094] In summary, the present invention provides a polypeptide having a sequence as shown in Sequence NO.1, which is reported for the first time. Pharmacological activity studies have shown that the polypeptide shown in Sequence NO.1 can significantly improve lung tissue structural damage in mice with acute lung injury models, reduce the level of pro-inflammatory factors, reduce neutrophil infiltration, and exhibit obvious anti-acute lung injury effects. Therefore, the polypeptide shown in Sequence NO.1 provided by the present invention has the prospect of being developed into an anti-acute lung injury drug. Specifically, the polypeptide shown in Sequence NO.1 can be used as the sole active ingredient for the preparation of drugs for treating acute lung injury, and can also be used with other active ingredients to form a composition for the preparation of drugs for treating acute lung injury.

[0095] The purpose of the above embodiments is to specifically introduce the essential content of the present invention, but those skilled in the art should know that the protection scope of the present invention should not be limited to this specific embodiment.

Claims

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

1.

2. Use of the polypeptide according to claim 1 for preparing a formyl peptide receptor 1 blocker drug.

3. Use of the polypeptide according to claim 1 for preparing an anti-acute lung injury drug.

4. The use according to claim 3, characterized in that: The drug uses the polypeptide according to claim 1 as an active ingredient and is prepared into a pharmaceutically acceptable dosage form with pharmaceutically acceptable excipients or carriers.

5. The use according to claim 4, characterized in that: The auxiliary material or carrier is solid, liquid or semi-solid.

6. The use according to claim 4, characterized in that: The dosage form is tablet, capsule, injection or pill.

7. A pharmaceutical composition for treating acute lung injury, characterized in that: The active ingredient includes a polypeptide having an amino acid sequence as shown in Sequence No.

1.

8. Use of the pharmaceutical composition according to claim 7 for preparing an anti-acute lung injury drug.

9. The use according to claim 8, characterized in that: The drug uses the pharmaceutical composition according to claim 7 as an active ingredient and is prepared into a pharmaceutically acceptable dosage form with pharmaceutically acceptable excipients or carriers.

10. The use according to claim 8, characterized in that: The auxiliary material or carrier is solid, liquid or semi-solid, and the dosage form is tablet, capsule, injection or pill.

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