CSF3R antagonistic peptides and their application in the preparation of drugs for treating allergic rhinitis

Abstract

This invention belongs to the field of biotechnology and pharmaceutical technology, and relates to a CSF3R antagonistic peptide and its application in the preparation of drugs for treating allergic rhinitis. This antagonistic peptide blocks the binding between neutrophil colony-stimulating factor receptor CSF3R and neutrophil colony-stimulating factor G-CSF, inhibits the STAT3 signaling pathway-mediated inflammatory response, reduces IgE levels and neutrophil infiltration, and decreases the expression levels of inflammatory factors, thereby exerting an anti-allergic effect. The antagonistic peptide of this invention can block inflammatory signal transduction upstream, inhibit abnormal neutrophil infiltration and excessive inflammatory activation, precisely improve pathological damage to the nasal mucosa, and has a clear mechanism of action and strong targeting.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and pharmaceutical technology, and more specifically, relates to CSF3R antagonistic peptides and their application in the preparation of drugs for treating allergic rhinitis. Background Technology

[0002] Allergic rhinitis (AR) is a common chronic inflammatory disease with main symptoms including sneezing, nasal itching, runny nose, and nasal congestion, which seriously affect patients' quality of life. Traditionally, allergic rhinitis was thought to be primarily mediated by a Th2 immune response; however, recent studies have found that neutrophils also play an important role in the inflammatory response of allergic rhinitis.

[0003] Currently, the conventional clinical treatments for allergic rhinitis mainly include nasal corticosteroids, antihistamines, leukotriene receptor antagonists, nasal decongestants, desensitization immunotherapy, and nasal physical irrigation, which can only relieve and control symptoms.

[0004] Existing treatment options have significant technical limitations: First, they are mostly symptomatic treatments and cannot target and block the core inflammatory pathways and immune cascade reactions that cause allergic rhinitis, nor can they mechanistically inhibit abnormal neutrophil activation, infiltration, and the release of inflammatory factors. Second, after stopping medication, patients are highly susceptible to relapse upon exposure to allergens, requiring long-term medication dependence. Third, long-term use of some medications can easily cause adverse reactions such as nasal dryness, nosebleeds, drowsiness, and drug-induced rhinitis, and desensitization treatment has a long cycle and is applicable to a limited population.

[0005] Therefore, existing drugs for treating allergic rhinitis have the technical problem of not being able to target and block the inflammatory pathways that cause allergic rhinitis. Summary of the Invention

[0006] In view of the above-mentioned defects or improvement needs of the prior art, the present invention provides a CSF3R antagonistic peptide and its application in the preparation of drugs for treating allergic rhinitis, with the aim of providing an antagonistic peptide that can target and block the inflammatory pathways that cause allergic rhinitis.

[0007] According to a first aspect of the present invention, an antagonistic polypeptide is provided, the amino acid sequence of which is shown in SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3.

[0008] According to another aspect of the present invention, the use of the antagonistic polypeptide in the preparation of a medicament for treating allergic rhinitis is provided.

[0009] Preferably, the antagonistic peptide is used to block the binding between neutrophil colony-stimulating factor receptor CSF3R and neutrophil colony-stimulating factor G-CSF.

[0010] Preferably, the antagonistic peptide is used to inhibit the STAT3 signaling pathway-mediated inflammatory response, reduce IgE levels and neutrophil infiltration, and decrease the expression levels of inflammatory factors.

[0011] Preferably, the inflammatory factor is Il4 Inflammatory factors.

[0012] Preferably, the IgE is specific immunoglobulin E.

[0013] Preferably, the antagonistic peptide is used to reduce mucosal thickening, inflammatory cell infiltration and / or goblet cell proliferation, thereby repairing pathological damage to the nasal mucosa and exerting an anti-allergic effect.

[0014] According to another aspect of the present invention, a pharmaceutical preparation for treating allergic rhinitis is provided, comprising the aforementioned antagonistic peptide.

[0015] In summary, compared with the prior art, the above-described technical solutions conceived by this invention can achieve the following beneficial effects: (1) Based on the PDB structure of the CSF3R complex, this invention utilizes computer-aided alanine scanning technology to resolve key hotspot residues at the CSF3R-G-CSF interaction interface. Furthermore, relying on the RFdiffusion protein design platform, de novo design was conducted, targeting the aforementioned key residues to screen and obtain antagonistic peptides P1, P2, and P3, shown in SEQ ID NO:1-3, which exhibit spatial conformational stability and high binding affinity. Molecular docking verification was performed using the AlphaFold3 protein structure and protein-protein complex prediction platform, confirming that antagonistic peptides P1, P2, and P3 all possess strong binding ability and targeting potential with CSF3R.

[0016] (2) Neutrophil colony-stimulating factor (G-CSF) can bind to its receptor (CSF3R), activate the JAK / STAT3 signaling pathway, and promote neutrophil differentiation, migration, and inflammatory response. In the nasal mucosa tissue of patients with allergic rhinitis, the expression levels of G-CSF and CSF3R are significantly increased, accompanied by increased neutrophil infiltration. The application of the antagonistic peptides P1, P2, and P3 of this invention in the preparation of drugs for treating allergic rhinitis, in particular, can block the binding between the neutrophil colony-stimulating factor receptor CSF3R and neutrophil colony-stimulating factor G-CSF, inhibit the STAT3 signaling pathway-mediated inflammatory response, reduce IgE levels and neutrophil infiltration, and reduce the expression levels of inflammatory factors. It can effectively control abnormal neutrophil infiltration and persistent airway inflammation, thereby exerting an anti-allergic effect. It is especially suitable for moderate to severe, refractory allergic rhinitis with excessive neutrophil infiltration, and has the advantages of a clear mechanism of action and strong targeting. This addresses the technical problem that existing medications for allergic rhinitis cannot target and block the inflammatory pathways that cause allergic rhinitis, thus failing to intervene in the immune-inflammatory cascade response of allergic rhinitis, resulting in easy relapse after discontinuation of medication and the need for long-term continuous use.

[0017] (3) The application of the antagonistic peptides P1, P2 and P3 of the present invention in the preparation of drugs for treating allergic rhinitis can, in particular, reduce mucosal thickening, inflammatory cell infiltration and goblet cell proliferation, thereby repairing pathological damage to the nasal mucosa.

[0018] (4) Cell-level evaluation showed that the antagonistic peptides P1, P2 and P3 in this invention did not show obvious cytotoxicity after acting on human bronchial epithelial Beas-2B cells, and had good cell safety.

[0019] (5) Animal model experiments of allergic rhinitis confirmed that the antagonistic peptides P1, P2 and P3 in this invention can significantly alleviate the allergic phenotype of the model animals and reduce typical behavioral symptoms such as scratching the nose and sneezing; at the same time, they downregulate the serum OVA-sIgE content and inhibit the nasal mucosa Il4 Inflammatory factor expression. Attached Figure Description

[0020] Figure 1 This is a schematic diagram of the amino acid sequence of the antagonistic polypeptide described in this invention.

[0021] Figure 2 This is a diagram illustrating the binding pattern of the antagonistic peptides P1-P3 and CSF3R described in this invention.

[0022] Figure 3 This is the toxicity test result of the antagonistic peptides P1-P3 described in this invention at the cellular level.

[0023] Figure 4This invention relates to the behavioral observation and biochemical index detection of the antagonistic peptides P1-P3 in OVA-induced allergic rhinitis in mice at the animal level.

[0024] Figure 5 This invention relates to the pathological detection of the antagonistic peptides P1-P3 in OVA-induced allergic rhinitis in mice at the animal level. Detailed Implementation

[0025] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention. Furthermore, the technical features involved in the various embodiments of this invention described below can be combined with each other as long as they do not conflict with each other.

[0026] The first embodiment of the present invention provides an antagonistic polypeptide, the amino acid sequence of which is shown in SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3. The primary structure of the antagonistic polypeptide is as follows: Figure 1 As shown.

[0027] In some embodiments, the antagonistic peptide is synthesized according to standard solid-phase peptide synthesis procedures, with the synthesis direction from C-terminus to N-terminus. First, the resin is swollen, preferably using a chloroprene resin. Then, the first amino acid is attached to the resin; this attachment requires double coupling, meaning the amino acid reacts with the resin twice. Next, the Fmoc group at the amino terminus of the amino acid is removed (deprotection). Then, the peptide chain elongation process involves repeated condensation, washing, deprotection, washing, and condensation steps, linking amino acids one by one from the C-terminus to the N-terminus according to the peptide sequence until the desired peptide fragment is synthesized. After the resin deswells and dries, the peptide fragment is excised and the crude peptide is purified to obtain the synthesized peptide. Figure 1 The antagonistic peptides P1-P3 in the sample.

[0028] The second embodiment of the present invention provides the application of the antagonistic peptide in the first embodiment in the preparation of a drug for treating allergic rhinitis.

[0029] In some embodiments, the antagonistic peptide is used to block the binding between neutrophil colony-stimulating factor receptor CSF3R and neutrophil colony-stimulating factor G-CSF, reduce IgE levels and neutrophil infiltration, and reduce the expression levels of inflammatory factors, thereby effectively controlling abnormal neutrophil infiltration and persistent airway inflammation, and thus exerting an anti-allergic effect.

[0030] In some embodiments, the antagonistic peptide is used to reduce mucosal thickening, inflammatory cell infiltration and goblet cell proliferation, thereby repairing pathological damage to the nasal mucosa and exerting an anti-allergic effect.

[0031] The third embodiment of the present invention provides a pharmaceutical composition for treating allergic rhinitis, comprising the antagonistic peptide of the first embodiment.

[0032] Example 1: Screening of antagonistic peptides The antagonistic peptides P1, P2 and P3 of the present invention have strong binding energies to CSF3R (Table 1).

[0033] Based on the publicly available PDB structure file of the CSF3R complex (PDB: 2D9Q), key hotspot residues at the CSF3R-G-CSF interaction interface were identified using computer-aided alanine scanning. Six hotspot residues on CSF3R (R166, L170, L172, H237, R287, L290) were determined. De novo protein design was performed using the RFdiffusion protein design platform. de novo Based on the design, candidate peptide sequences with stable spatial conformation and high affinity potential were obtained by targeting the hotspot binding region of CSF3R. Using the AlphaFold3 protein structure and protein-protein complex structure prediction platform, antagonistic peptides P1, P2, and P3 all formed stable binding interfaces with both human and mouse CSF3R. The antagonistic peptides P1 (human cumulative binding energy: -4.4 kcal / mol; mouse cumulative binding energy: -3.0 kcal / mol), P2 (human cumulative binding energy: -7.5 kcal / mol; mouse cumulative binding energy: -6.0 kcal / mol), and P3 (human cumulative binding energy: -7.0 kcal / mol; mouse cumulative binding energy: -6.4 kcal / mol) showed strong binding affinity for CSF3R, especially P2 and P3 (Table 1 & Figure 2 ).

[0034] Table 1. Statistical table of prediction results for antagonistic peptides and CSF3R AlphaFold3.

[0035] Example 2: Cytotoxicity Investigation Human bronchial epithelial cells (Beas-2B) were seeded in 96-well plates. After cell adhesion, they were treated with antagonistic peptides P1, P2, and P3, while a blank control was administered for 24 hours. MTT solution (10 μL / well) was added, and DMSO was added after 4 hours. The absorbance was measured at 490 nm. MTT cytotoxicity assays confirmed that, compared to the blank control, there was no significant difference in cell viability after intervention with antagonistic peptides P1, P2, and P3, and no significant cell death was observed, indicating that antagonistic peptides P1, P2, and P3 had no significant cytotoxic effect on human bronchial epithelial cells. Figure 3 In the figures, A, B, and C represent the MTT assay results of human bronchial epithelial cells after intervention with antagonistic peptide P1, antagonistic peptide P2, and antagonistic peptide P3, respectively. It can be seen that after treating cells with different concentration gradients of antagonistic peptides P1, P2, and P3, the cell survival rates in each group were not different from those in the blank control group. Therefore, antagonistic peptides P1, P2, and P3 have no significant cytotoxic side effects on human bronchial epithelial cells and exhibit good in vitro safety.

[0036] Example 3: Investigation of drug efficacy in animals Six- to eight-week-old female C57BL / 6 mice (SPF grade) were selected and sensitized for one week. On days 0, 7, and 14, they were sensitized by intraperitoneal injection of a sensitization solution containing 50 μg OVA and 100 μL of 2% aluminum hydroxide vaccine adjuvant (dissolved in 200 μL PBS). The control group received an equal volume of PBS buffer intraperitoneally. Sensitized mice were challenged nasally from days 21 to 27 by intranasal administration of a challenge solution containing 1 mg OVA (dissolved in 20 μL PBS, 10 μL / nostril). The control group received an equal volume of PBS buffer intranasally. Ten minutes after the final OVA challenge and acclimatization, the number of times each mouse scratched its nose, sneezed, and produced clear, watery nasal discharge was recorded within 10 minutes. For antagonistic peptide therapy, prophylactic treatment was administered via nasal instillation of 1 nmol of the antagonistic peptide (dissolved in 20 μL PBS, 10 μL / nostril) or intraperitoneal injection of 50 nmol of the antagonistic peptide (dissolved in 200 μL PBS) on days 18 to 27. The model group and control group received an equal volume of PBS buffer. One day later, mice were sacrificed, and serum was collected for IgE and OVA-sIgE measurements; nasal bone tissue was collected, embedded, and stained into tissue sections; and nasal mucosa tissue was collected for inflammatory factor measurement.

[0037] In vivo pharmacodynamic studies in animals showed that the antagonistic peptides P1, P2, and P3 could effectively improve the typical clinical symptoms of OVA-induced allergic rhinitis in mice, significantly reduce the frequency of nose scratching and sneezing in model mice, and effectively reduce the abnormal expression level of OVA-specific immunoglobulin E (OVA-sIgE) in mouse serum. Figure 4 The values ​​of A and C in the table represent the statistics of the number of times the model mice scratched their noses, the number of times they sneezed, and the serum OVA-sIgE content, respectively. It can be seen that, compared with the model control group, the mice in each of the P1, P2, and P3 antagonistic peptide intervention groups showed a significant reduction in nose scratching and sneezing behaviors, and a significant decrease in serum OVA-sIgE levels. This suggests that the antagonistic peptides can effectively alleviate the clinical symptoms of allergic rhinitis and inhibit IgE-mediated allergic reactions.

[0038] Figure 5 Histopathological staining results showed: HE staining ( Figure 5 (A) allows for direct observation of the thickness of the mouse nasal mucosa and the infiltration of eosinophils in the lamina propria. PAS staining can assess abnormal goblet cell proliferation and mucus secretion levels. In OVA-induced allergic rhinitis model mice, the nasal mucosa showed significant epithelial thickening, lamina propria edema with abundant inflammatory cell infiltration, and marked goblet cell proliferation. After intervention with antagonistic peptides P1, P2, and P3, these pathological changes were significantly improved: the nasal mucosa thickness was significantly reduced (…). Figure 5 In B), the density of inflammatory cell infiltration in the lamina propria was significantly decreased ( Figure 5 In C), the number of goblet cells and the level of mucus secretion decreased significantly. Figure 5 (D in the text). The above results indicate that the antagonistic peptides can comprehensively reduce the degree of inflammatory damage and pathological lesions in nasal mucosa tissue, and effectively alleviate the pathological damage to the nasal mucosa caused by allergic rhinitis.

[0039] In vivo pharmacological studies in animals have confirmed that the antagonistic peptides P1, P2, and P3 can effectively alleviate the clinical symptoms of OVA-induced allergic rhinitis in mice, improve the pathological damage of nasal mucosal inflammation, and downregulate key indicators related to allergies and inflammation. They have excellent application value in the prevention and treatment of allergic rhinitis and can be used as candidate active peptides for the treatment of allergic rhinitis.

[0040] Those skilled in the art will readily understand that the above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. An antagonistic polypeptide, characterized in that, The amino acid sequence of the antagonistic polypeptide is shown in SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:

3.

2. The use of the antagonistic peptide as described in claim 1 in the preparation of a drug for treating allergic rhinitis.

3. The application as described in claim 2, characterized in that, The antagonistic peptide is used to block the binding between neutrophil colony-stimulating factor receptor CSF3R and neutrophil colony-stimulating factor G-CSF.

4. The application as described in claim 3, characterized in that, The antagonistic peptide is used to inhibit the STAT3 signaling pathway-mediated inflammatory response, reduce IgE levels and neutrophil infiltration, and decrease the expression levels of inflammatory factors.

5. The application as described in claim 4, characterized in that, The inflammatory factors are Il4 Inflammatory factors.

6. The application as described in claim 4, characterized in that, The IgE mentioned is specific immunoglobulin E.

7. The application as described in any one of claims 2-6, characterized in that, The antagonistic peptides are used to reduce mucosal thickening, inflammatory cell infiltration and / or goblet cell proliferation, thereby repairing pathological damage to the nasal mucosa and exerting an anti-allergic effect.

8. A pharmaceutical preparation for treating allergic rhinitis, characterized in that, Includes the antagonistic polypeptide of claim 1.

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