CN111760026A

CN111760026A - Application of FGFR2b inhibitory molecule in preparation of medicine for treating PAF-mediated diseases

Info

Publication number: CN111760026A
Application number: CN202010783944.6A
Authority: CN
Inventors: 汪炬
Original assignee: Individual
Current assignee: Individual
Priority date: 2020-08-06
Filing date: 2020-08-06
Publication date: 2020-10-13
Also published as: WO2022027898A1

Abstract

The invention discloses application of FGFR2b inhibitory molecules in preparation of a medicament for treating PAF-mediated diseases. The present invention is based on the results of the present inventors' studies on the discovery that FGF-7 induces the production of platelet activating factor PAF. The inventor finds that after PAF is generated, the PAF can induce the level rise of proinflammatory factors, then the PAF can trigger inflammatory reaction, molecules which inhibit the combination of FGF-7 and FGFR2 are used as competitive antagonists of FGF7, the molecules are combined with FGF7 specific receptor FGFR2b, the FGFR2b is blocked from being activated into phosphorylated FGFR2b, excessive inflammatory reaction can be effectively inhibited, and therefore ARDS symptoms can be treated, and pulmonary fibrosis can be improved.

Description

Application of FGFR2b inhibitory molecule in preparation of medicine for treating PAF-mediated diseases

Technical Field

The invention belongs to the field of medical biology, and particularly relates to application of FGFR2b inhibitory molecules in preparation of a medicament for treating PAF-mediated diseases.

Background

Acute Respiratory Distress Syndrome (ARDS) is a clinical syndrome characterized by refractory hypoxemia, caused by intrapulmonary and/or extrapulmonary causes, that is of great interest for high mortality. Its pathogenesis involves an uncontrolled autoimmune defense response in the patient and leads to inflammation, endothelial damage, enhanced coagulation, reduced fibrinolysis and fibroproliferation, a clinical syndrome caused by a variety of pathogens. In 1994, the us-european consensus conference council for ARDS recommended a definition of the disease; the standards include: (1) acute episodes, (2) bilateral infiltration on chest X-ray patches, (3) pulmonary wedge pressure ≦ 18mm Hg or lack of clinical evidence of left atrial hypertension, and (4) Pao2/Fio2 ratio ≦ 300 (defined as ALI) or Pao2/Fio2 ratio ≦ 200 (defined ARDS as the more severe form of ALI) (Cepkova and Matthay, j. intensive Care med, 21: 119-143, 2006). Diffuse alveolar injury is a characteristic of ARDS. The disease has three overlapping stages: the exudation phase (first 4-7 days), the proliferation phase (. gtoreq.7-14 or 21 days) and the fibrosis phase (. gtoreq.14 or 21 days) (MacLaren and Stringge, Pharmacotherapy, 27: 860-873, 2007).

There is significant neutrophil accumulation during the acute phase. Neutrophils predominate in pulmonary edema fluid and alveolar lavage fluid obtained from infected persons. Alveolar macrophages secrete cytokines such as the interleukins IL-1, IL-6, IL-10, IL-8, and Tumor Necrosis Factor (TNF) - α, which stimulate neutrophil chemotaxis and activate neutrophils. Neutrophils then release oxidants, proteases (including neutrophil elastase), leukotrienes and other proinflammatory cellular mediators (Ware and Mattray, New England J. Med. 342: 1334-1349). These mediators interact in a complex manner to damage and inflame the alveolar-capillary interface, resulting in a series of inflammatory responses and other symptoms.

The existing ARDS treatment scheme mainly adopts methods of suppression immunity (glucocorticoid), supportive treatment (oxygen) and the like for relieving and treating. Glucocorticoids avoid further damage to the body by the immune system over-reaction, but have significant side effects including, but not limited to, exacerbation of infections, osteoporosis, complications of the cardiovascular system and digestive system. On the other hand, the supportive therapy is only to maintain the life of the patient, but not to treat the disease, and the improvement effect on the survival rate of the patient is very limited.

Since the origin of the disease is not clarified, a targeted therapeutic method for ARDS has not been developed, and thus the development of innovative therapeutic drugs is urgently needed.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provides application of FGFR2b inhibitory molecules in preparing medicines for treating PAF-mediated diseases.

The purpose of the invention is realized by the following technical scheme: the application of FGFR2b inhibitory molecules in preparing drugs for treating PAF-mediated diseases is based on the research results of the inventor of the invention that FGF-7 can induce the generation of platelet activating factor PAF.

The PAF mediated diseases comprise Acute Respiratory Distress Syndrome (ARDS), acute inflammatory diseases such as pulmonary edema and the like, pulmonary fibrosis diseases, cardiovascular diseases such as arteriosclerosis and the like, various tumors related to PAF, PAF mediated tumor metastasis and relapse caused by radiotherapy and chemotherapy, and neurodegenerative diseases such as Alzheimer's Disease (AD) and Parkinson's Disease (PD).

The acute respiratory distress syndrome is preferably lung injury, pulmonary edema and acute respiratory distress syndrome caused by bacterial infection and/or viral infection including new coronary pneumonia.

The virus is preferably a novel coronavirus; more preferably SARS-CoV, MERS-CoV or 2019-nCoV.

The pulmonary fibrosis is idiopathic pulmonary fibrosis and pulmonary fibrosis caused by inflammation.

The FGFR2b inhibiting molecules comprise molecules for inhibiting FGF-7 expression, molecules for inhibiting FGFR2b from being activated into phosphorylated FGFR2b and molecules for competitively inhibiting the combination of FGF-7 and FGFR2 b.

Such molecules include, but are not limited to, siRNA, oligopeptides, polypeptides, and the like.

The molecule competitively inhibiting the binding of FGF-7 to FGFR2b is preferably selected from the group consisting of short peptides described in the national invention patent application having the application number 2019102390870 and the title of "short peptides, drugs and uses thereof for treating alopecia"; preferably, the short peptide is obtained by respectively adding, deleting or mutating 0-2 amino acids at two ends by taking EWVRTD, ELSGRA, QDVDS or VFSTTGV as a center; most preferred are EWVRTD, WVRTD, RHEWSRTD, ELSGRA, HTVELSGGRAK, QDVDS, VFSTTGV, FGSVFSTTGV.

The molecule for inhibiting FGFR2b from being activated to phosphorylate FGFR2b is preferably selected from short peptides described in the national invention patent with the application number of 201310714120.3 and the invention name of "gene sequence of extracellular domain of FGFR2b, polypeptide and application thereof".

Compared with the prior art, the invention has the following advantages and effects:

(1) the inventor of the invention firstly discovers that FGF7 can induce the generation of platelet activating factor PAF.

(2) The inventor also finds that after PAF is produced, the PAF can induce the level rise of proinflammatory factors, then the PAF can trigger inflammatory reaction, molecules for inhibiting the combination of FGF-7 and FGFR2 are used as competitive antagonists of FGF7, the molecules are combined with FGF7 specific receptor FGFR2b, the FGFR2b is blocked from being activated into phosphorylated FGFR2b, excessive inflammatory reaction can be effectively inhibited, and therefore the ARDS symptom can be treated, and lung fibrosis can be improved.

(3) The growth factor antagonist PI9(EWVRTD) used in the invention is a short peptide with 6 amino acids, plays a role by preventing FGF7 from being combined with a receptor on a membrane at the outside of a cell, and has the characteristics of no need of entering the cell and short half-life period; meanwhile, FGF7 has the unique binding receptor FGFR2b, so that the target is clear, the pharmacological effect is very specific, and the toxic and side effects are easy to evaluate. Therefore, the invention has wide application prospect in preparing ARDS medicines.

Drawings

FIG. 1 is a graph showing the results of detection and analysis of the group-differential lipid PAF by lipidomics; each column group represents group a, group B, group C and group D from left to right, respectively, group a being blank group, group B being PI9 group, group C being FGF7 group, group D being FGF7+ PI9 group.

FIG. 2 is a graph showing the results of the relative expression levels of IL-6 and IL-8 induced by PAF in sebaceous gland cells.

FIG. 3 is a graph showing the results of cytokine detection in an ARDS mouse model established by LPS induction.

Fig. 4 is a photograph of lung tissue of bleomycin-induced SD rat pulmonary fibrosis model.

Detailed Description

The present invention will be described in further detail with reference to examples and drawings, but the present invention is not limited thereto.

Unless otherwise specified, all experimental techniques used in the following examples are conventional molecular biology techniques, and scientific researchers in the field can implement the results according to the conventional molecular biology techniques; the materials, reagents and the like used are, unless otherwise specified, commercially available reagents and materials.

The PBS used in the present invention was 0.1mol/L, pH 7.4.4 PBS, consisting of: NaCl 16.00g, KCl 0.40g, KH₂PO₄0.40g、Na₂HPO₄·12H₂O5.80 g, and the volume is adjusted to 1L by water.

Example 1

This example 1 provides a short peptide and a pharmaceutical preparation thereof, which is a sequence obtained by intercepting 5-12 amino acids from the extracellular domain of FGFR (SEQ NO id.52) and performing 0-2 point mutation. The 5-12 amino acids are intercepted by the FGFR extracellular segment and are spatially adjacent amino acids intercepted according to the three-dimensional structure of the FGFR extracellular segment. Specifically, the designed amino acids take EWVRTD (PI9, SEQ NO ID.9), ELSGRA (PI15, SEQ NO ID.15), QDVDS (PI29, SEQ NO ID.29) and VFSTTGV (PI47, SEQ NO ID.47) as centers, amino acid sequences are added or deleted at two ends respectively, and the short peptides are named as PI1-PI 51.

Wherein, the amino acid sequence (from amino terminal to carboxyl terminal) of the FGFR extracellular segment is as follows (SEQ NO ID.52):

APYWTNTEKMEKRLHAVPAANTVKFRCPAGGNPMPTMRWLKNGKEFKQEHRIGGYKVRNQHWSLIMESVVPSDKGNYTCVVENEYGSINHTYHLDVVERSPHRPILQAGLPANASTVVGGDVEFVCKVYSDAQPHIQWIKHVEKNGSKYGPDGLPYLKVLKHSGINSSNAEVLALFNVTEADAGEYICKVSNYIGQANQSAWLTVLPKQQAPGREKEITASPDYLEIAIYCIGVFLIACMVVTVIL。

the nomenclature of the short peptides, the amino acid sequences and the sequence numbers are shown in Table 1.

TABLE 1

The short peptide is added with certain Excipients (Excipients) and stored in the form of freeze-dried powder. Specifically, a short peptide solution with a certain concentration is prepared, for example, the concentration of the short peptide in the short peptide solution can be 10-1000 mu g/ml, preferably 100 mu g/ml, 3% (m/v) sucrose, 1% (m/v) lactose and 6% (m/v) sorbitol are added into the prepared short peptide solution to be used as excipients, the mixture is stored at 4 ℃ for 4-5h, so that the protective agent is fully dissolved and uniformly distributed in the short peptide solution, and the mixture is pre-frozen for 12-14h at the pre-freezing temperature of-20 ℃ and is pre-frozen.

Example 2

In this example 2, FGF7 was found to regulate the expression of Platlet Activating Factor (PAF) through lipidomics studies.

1.1 preparation of treated samples

Culturing SZ95 human sebaceous gland cells, digesting the cells with pancreatin for 5min when the cells grow to be heptagonal, discarding pancreatin, adding SEB-1 complete culture medium (SEB-1 sebaceous gland cell culture medium + 10% fetal bovine serum, Zenbio, USA) to terminate the digestion of pancreatin, blowing down the cells, mixing well to form cell suspension, counting, grouping the cells as shown in the following table, repeating 3 times per group, plating the primary sebaceous gland cells into 10cm culture dishes, adding 3.7 × 10 per well⁵Adding 10mL of SEB-1 complete culture medium into each cell, carrying out adherent culture, changing the culture solution once in 48h, and allowing the cells to grow to the length ofAt medium, starving with serum-free medium (SEB-1 medium) for 12h, discarding medium, treating cells as shown in Table 2, group A without any drug, group B with 4. mu.g/mL PI9 (solvent as medium), and group C with 10ng/mL FGF-7(FGF-7 is available from R.USA)&Company D; solvent as culture medium), group D was added with 4. mu.g/mL PI9+10ng/mL FGF7 (solvent as culture medium), 10mL of drug-containing culture medium was added to each well, and cultured for 48 h. The medium was aspirated off, cells were collected by trypsinization and processed for the next step.

Three groups of 12 cell samples are divided into three groups, the grouping conditions are shown in the following table, LC-MS detection is carried out by adopting a positive ion mode and a negative ion mode, comparison among groups is carried out according to the detection result, and 3 times of comparison data are provided in the report.

Table 212 examples of cell sample grouping

1.2 reagents and consumables

TABLE 3 reagents used in LC-MS experiments

Acetonitrile (Acetonitrile)	Thermo Fisher
		Methanol (Methanol)	Thermo Fisher
Formic acid (Formic acid)	Sigma
		Ammonium formate	CNW
Ultrapure water (Ultrapure water)	Millipore
		Dichloromethane (Chloroform)	Merck(Darmstadt,Germany)

1.3 Experimental methods

1.3.1 sample pretreatment

1. Adding 800 μ L methanol, performing ultrasonic treatment for 30min, centrifuging to obtain supernatant, and blow-drying with N2;

2. adding 1.5mL of dichloromethane/methanol (2: 1, V/V) solution and 500. mu.L of water, vortexing and shaking for 1min, standing and centrifuging (3000rpm, 15min)

3. Taking down the organic phase of the lower layer to a new glass tube;

4. evaporating to dryness in a high-speed vacuum concentration centrifuge, re-dissolving with isopropanol/methanol (1: 1, V/V), transferring into a sampling bottle, and storing at-20 deg.C for use.

1.3.2 LC-MS analysis

1. An instrument analysis platform: LC-MS (Thermo, Ultimate 3000LC, Orbitrap Elite)

2. A chromatographic column: c18 column (Kinetex C18 (100X 2.1mm,1.9 μm))

3. The chromatographic separation conditions are as follows: the column temperature was 45 ℃; the flow rate is 0.4 mL/min;

mobile phase composition A: acetonitrile: water (60:40, V/V), the solution containing 10mmol/L ammonium formate; b: acetonitrile: isopropanol (10:90, V/V), the solution containing 10mmol/L ammonium formate and 0.1% formic acid;

the sample size was 4. mu.L, and the autosampler temperature was 4 ℃.

4. The mobile phase gradient elution procedure is shown in table 4.

Table 4 mobile phase elution procedure

1.3.3 data analysis

LC/MS detection data are extracted and preprocessed by using Lipid Search software (Thermo company), normalized and post-edited in Excel 2010, and the obtained data matrix is imported into SIMCA-P13.0 (Umetrics AB, Umea, Sweden) software for multivariate statistical analysis.

1.3.4 analysis of results

The differential lipid PAF comprises multiple forms, and the results of the relative content of the differential lipid PAF are shown in fig. 1 by testing different forms of PAF. It can be seen that FGF-7 can induce the expression of PAF, and that the combined use of PI9 and FGF-7 can inhibit the expression of PAF induced by FGF-7. In the research of FGF7 regulated lipid anabolism, PAF is a group of differential lipids, and the PAF is used as a strong proinflammatory mediator and is related to inflammatory reaction.

Example 3

20 mu.M PAF (Shanghai Yubo Biotech Co., Ltd.) was used to induce SZ95 human sebaceous gland cells, and then the effect of PAF on the induction of proinflammatory cytokine expression at different times was examined by ELISA. The detection steps are as follows:

collecting samples: sucking cell culture fluid, transferring into EP tube, centrifuging at 12000rpm for 5min at 4 deg.C in precooled refrigerated centrifuge, collecting supernatant, subpackaging in EP tube, storing in-20 deg.C refrigerator with each EP tube of 400 μ L, taking out when it is used, and thawing at room temperature;

holding the kit and the experimental sample stored at 4 ℃ to room temperature for 30min according to the specification of an IL-6ELISA detection kit and an IL-8ELISA detection kit of Beijing Si Zheng Bai Biotech Co., Ltd, and balancing to room temperature;

thirdly, adding a sample to be detected into corresponding enzyme-labeled holes, sealing the reaction holes by using sealing plate adhesive paper, and incubating for 90min in a constant temperature box at 37 ℃, wherein each hole is 100 mu L;

washing the plate for 4 times: taking out the enzyme label plate, throwing away the sample, adding 350 mu L of washing liquid, standing for 30s, throwing away the washing liquid, repeatedly washing for 4 times, and patting dry on absorbent paper;

adding 100 mu L/hole of biotinylated antibody working solution, sealing the reaction hole with sealing plate paper, and incubating in a constant temperature oven at 37 ℃ for 60 min; the plate washing is repeated;

sixthly, adding 100 mu L/hole of the working solution of the enzyme conjugate, sealing the reaction hole by using sealing paper, incubating for 30min in a constant temperature box at 37 ℃, and repeating the step IV;

adding 100 mu L/hole of color development liquid, incubating for 15min at 37 ℃ in a dark place, taking out the enzyme-labeled plate, adding 100 mu L/hole of stop solution, slightly oscillating and uniformly mixing, and measuring the OD450 value within 5 min.

The result analysis shows that the expression result of the proinflammatory cytokines is shown in figure 2, PAF induces sebaceous gland cells to express IL-6 and IL-8 to increase along with the increase of time, IL-6 and IL-8 reach the maximum value after 16h induction, and the IL-6 and IL-8 are increased by 4.1 times and 3.4 times compared with the control group IL-6. After 24h and 48h of induction, the expression of the two is reduced.

Example 4

Mouse lung lavage fluid cell count and lung lavage fluid cytokine detection were performed by establishing an LPS-induced Acute Respiratory Distress Syndrome (ARDS) mouse model.

4.1 experiments were performed using BALB/c mice from Shanghai Ling laboratory animals Co., Ltd, with the grouping information shown in Table 5.

Table 5 grouping and dosing regimens

4.1.1 LPS solution preparation: LPS was dissolved in PBS to a final concentration of 1.0mg/mL of LPS solution.

Dexamethasone was first administered orally at 4.1.2-0.5 hours.

Acute lung injury was induced by LPS in groups G2-G5 at 4.1.30 hours and in groups G6-G7 at 6 hours, and 50. mu.l of LPS was administered as a model agent through the trachea using a special atomized aerosol needle. G1 received vehicle treatment of an equivalent volume of LPS. Prior to tracheal injection, animals received 2-5% isoflurane inhalation anesthesia.

4.1.4 dosing was performed according to Table 5.

4.1.5 animals were anesthetized 24 hours after LPS molding, all animals received 25mg/kg of Suitai intraperitoneal injection, tracheal intubation was performed, and the lungs were first lavaged with 0.5mL of PBS (containing 0.04M EDTA-K2). Another 0.5mL PBS (containing 0.04M EDTA-K2) was used to perform a second lavage of the lungs. Two lavages (BALF) were placed on ice.

Cells were obtained by centrifugation and resuspended in PBS. Diluting the cell sap to a concentration of about 10⁷Cells/ml, 100. mu.L of cell fluid was removed to a blood cell separator and centrifuged at 800rpm for 5 minutes. Slides were air dried, fixed in methanol solution as appropriate, and then stained with Wright-Giemsa stain to differentiate eosinophils, neutrophils, macrophages, and lymphocytes. Counting under an optical microscope.

4.1.6 and all supernatants were stored in ultra-low temperature refrigerator and transferred to an in vitro laboratory for total protein determination and detection of TNF- α, IL-1 β, IL-6, FGF7 and PAF, all in duplicate wells.

Experimental mice were divided into G1-7 groups, in which G1 was a normal group, G2 was a model-vehicle group, G3 was a model-dexamethasone group, G4 was a model-0 hour administration group (100 μ G/mouse), G5 was a model-0 hour administration group (150 μ G/mouse), G6 was a model-6 hour administration group (100 μ G/mouse), and G7 was a model-6 hour administration group (150 μ G/mouse), and the results of detection of cytokines in lung lavage fluid were as shown in fig. 3, and it was found that the contents of TNF- α, IL-6, IL-1 β, FGF7, and BALF proteins in the model-vehicle group were significantly higher than in the normal group. The model-dexamethasone group can obviously inhibit TNF-alpha, IL-6, IL-1 beta and FGF7 in BALF supernatant, the model-administration group is administered once in 6 hours and once in 12 hours after the model is constructed by 100 mu g/mouse dose, PAF in BALF supernatant can be obviously inhibited 24 hours after the model is constructed, and the degree of inhibition of cytokine expression by FGF-7 short peptide inhibitor is time-effect.

The results of the lung lavage fluid cell counts are shown in Table 6, and the mean number of neutrophils (neu) in the administered group is greater than that in the dexamethasone group, which indicates that the inhibition of the neutrophils by PI9 is weaker than that by dexamethasone, and that the inhibition of the inflammatory response by dexamethasone is too strong, which may affect the body to resist the foreign pathogens.

TABLE 6 Lung lavage fluid cell count

Example 5

A study of the effect of PI1-PI51 on PAF expression in lung lavage fluid of ARDS (performed as in example 4) was performed by establishing a mouse model of LPS-induced Acute Respiratory Distress Syndrome (ARDS) (see example 4) using the short peptide PI1-PI51 described in example 1 (at a concentration of 100. mu.g/ml).

TABLE 7

Note: relative value, i.e. inhibition rate, based on normal control group

As can be seen from Table 7, compared with the negative control group, the short peptides with large similarity to EWVRTD (PI9, SEQ NO ID.9), ELSGRA (PI15, SEQ NO ID.15), QDVDS (PI29, SEQ NO ID.29), VFSTTGV (PI47, SEQ NO ID.47) have better effect of inhibiting PAF expression.

Example 6

Study of inhibition of pulmonary fibrosis by PI9 conducted by establishing a bleomycin-induced pulmonary fibrosis rat (purchased from the animal experimental center in guangdong province) model.

I. Construction of bleomycin-induced SD rat pulmonary fibrosis model

All rats are fasted before operation and are forbidden to be watered for 12 hours, the rats are weighed, 10% chloral hydrate is injected into the abdominal cavity to anaesthetize the rats, and the dosage of the anaesthetics is 3 ml/Kg; preparing skin at neck, fixing four feet, head and incisor teeth of the rat after skin preparation on an operation table, locally sterilizing with iodophor, and wiping with alcohol; using an ophthalmologic scissors to make a vertical incision about 0.5cm long on the upper part of the bronchus; separating muscles layer by layer with ophthalmological forceps and straight forceps, exposing the air outlet pipe, and confirming the position of the thyroid cartilage; raising one end of the mouse head to form an included angle of more than 30 degrees with a desktop, then pricking the trachea cricoid cartilage with a syringe needle under direct vision, and slowly injecting the bleomycin solution when the bleomycin solution is pricked to have a falling feeling; standing the rat board, keeping the rat upright, and rotating the rat board for 2min to make the medicinal liquid uniformly distributed in the two lungs; after the muscle and skin layers are sutured, the iodophor is disinfected locally, and the penicillin solution with the dosage of 80000 IU/body is injected into the abdominal cavity, so that postoperative infection is prevented.

Experimental grouping and dosing regimens

Grouping:

normal control group: 250 mul of physiological saline is injected into the trachea during modeling;

group BLM: 5mg/Kg bleomycin solution is injected into the trachea during modeling;

BLM + PI9 group: 5mg/Kg bleomycin solution is injected into the trachea during modeling, and the drug is administered 7 days after modeling.

The administration mode comprises the following steps:

dosing was initiated on day 7 of the model and continued for 14 days.

Normal control group: injecting 400 mul normal saline into the abdominal cavity;

group BLM: injecting 400 mul normal saline into the abdominal cavity;

BLM + PI9 dosing group: p-8 polypeptide was injected intraperitoneally at 100. mu.g/day.

The results of various rat lung tissues are shown in fig. 4, and by observing the bleomycin-induced SD rat pulmonary fibrosis model, compared with the model group, the treatment group rat lung tissue Hematoxylin-eosin staining (HE) has a relatively complete lung structure, and collagen and FGF-2 are obviously reduced, and the results show that PI9 has an obvious inhibitory effect on pulmonary fibrosis.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

Sequence listing

<110> river-south university

Application of <120> FGFR2b inhibitory molecule in preparation of drugs for treating PAF-mediated diseases

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<213> Artificial Sequence (Artificial Sequence)

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His Thr Lys Glu Leu Ser Gly Arg Ala Lys Leu Ile

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<213> Artificial Sequence (Artificial Sequence)

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Glu His Ser Gly Arg

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<213> Artificial Sequence (Artificial Sequence)

<220>

<223>PI29

<400>29

Gln Asp Val Asp Ser

1 5

<210>30

<211>12

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223>PI30

<400>30

Pro Leu Arg Gln Asp Val Asp Ser Arg Ser Thr His

1 5 10

<210>31

<211>11

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223>PI31

<400>31

Pro Leu Arg Gln His Val Asp Ser Arg Ser Thr

1 5 10

<210>32

<211>7

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223>PI32

<400>32

Arg Gln Asp Val Asp Ser Arg

1 5

<210>33

<211>7

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223>PI33

<400>33

Leu Gly Gln Asp Val Asp Ser

1 5

<210>34

<211>6

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223>PI34

<400>34

Pro Leu Arg Gln Asp Val

1 5

<210>35

<211>6

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223>PI35

<400>35

Arg Gln Asp Val Asp Ser

1 5

<210>36

<211>6

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223>PI36

<400>36

Leu Gly Gln Asp Val Asp

1 5

<210>37

<211>10

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223>PI37

<400>37

Pro Leu Arg Gln Asp Val Asp Ser Arg Ser

1 5 10

<210>38

<211>8

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223>PI38

<400>38

Leu Gly Gln Asp Val Asp Ser Arg

1 5

<210>39

<211>9

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223>PI39

<400>39

Pro Leu Arg Gln Asp Val Asp Ser Arg

1 5

<210>40

<211>9

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223>PI40

<400>40

Phe Gly Ser Val Phe Ser Thr Thr Gly

1 5

<210>41

<211>9

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223>PI41

<400>41

Gly Ser Val Phe Ser Thr Thr Gly Val

1 5

<210>42

<211>8

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223>PI42

<400>42

Ser Val Phe Ser Thr Thr Gly Val

1 5

<210>43

<211>7

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223>PI43

<400>43

Phe Gly Ser Val Phe Ser Thr

1 5

<210>44

<211>13

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223>PI44

<400>44

Phe Gly Ser Val Phe Ser Thr Thr Gly Val Ile Ser Arg

1 5 10

<210>45

<211>7

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223>PI45

<400>45

Ser Val Phe Ser Thr Thr Gly

1 5

<210>46

<211>6

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223>PI46

<400>46

Val Phe Ser Thr Thr Ile

1 5

<210>47

<211>7

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223>PI47

<400>47

Val Phe Ser Thr Thr Gly Val

1 5

<210>48

<211>12

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223>PI48

<400>48

Phe Gly Ser Val Phe Ser Thr Thr Gly Val Ile Ser

1 5 10

<210>49

<211>8

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223>PI49

<400>49

Phe Gly Ser Val Phe Ser Thr Thr

1 5

<210>50

<211>10

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223>PI50

<400>50

Phe Gly Ser Val Phe Ser Thr Thr Gly Val

1 5 10

<210>51

<211>11

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223>PI51

<400>51

Phe Gly Ser Val Phe Ser Thr Cys Gly Val Ile

1 5 10

<211>246

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> FGFR extracellular domain amino acid sequence

<400>52

Ala Pro Tyr Trp Thr Asn Thr Glu Lys Met Glu Lys Arg Leu His Ala

1 5 10 15

Val Pro Ala Ala Asn Thr Val Lys Phe Arg Cys Pro Ala Gly Gly Asn

2025 30

Pro Met Pro Thr Met Arg Trp Leu Lys Asn Gly Lys Glu Phe Lys Gln

35 40 45

Glu His Arg Ile Gly Gly Tyr Lys Val Arg Asn Gln His Trp Ser Leu

50 55 60

Ile Met Glu Ser Val Val Pro Ser Asp Lys Gly Asn Tyr Thr Cys Val

65 70 75 80

Val Glu Asn Glu Tyr Gly Ser Ile Asn His Thr Tyr His Leu Asp Val

85 90 95

Val Glu Arg Ser Pro His Arg Pro Ile Leu Gln Ala Gly Leu Pro Ala

100 105 110

Asn Ala Ser Thr Val Val Gly Gly Asp Val Glu Phe Val Cys Lys Val

115 120 125

Tyr Ser Asp Ala Gln Pro His Ile Gln Trp Ile Lys His Val Glu Lys

130 135 140

Asn Gly Ser Lys Tyr Gly Pro Asp Gly Leu Pro Tyr Leu Lys Val Leu

145 150 155 160

Lys His Ser Gly Ile Asn Ser Ser Asn Ala Glu Val Leu Ala Leu Phe

165 170 175

Asn Val Thr Glu Ala Asp Ala Gly Glu Tyr Ile Cys Lys Val Ser Asn

180 185 190

Tyr Ile Gly Gln Ala Asn Gln Ser Ala Trp Leu Thr Val Leu Pro Lys

195 200 205

Gln Gln Ala Pro Gly Arg Glu Lys Glu Ile Thr Ala Ser Pro Asp Tyr

210 215 220

Leu Glu Ile Ala Ile Tyr Cys Ile Gly Val Phe Leu Ile Ala Cys Met

225 230 235 240

Val Val Thr Val Ile Leu

245

Claims

Use of an FGFR2b inhibitory molecule in the preparation of a medicament for the treatment of a PAF-mediated disease.
2. The use of an FGFR2b inhibitory molecule according to claim 1 for the preparation of a medicament for the treatment of a PAF-mediated disease, wherein: the PAF mediated diseases comprise acute respiratory distress syndrome, pulmonary edema, pulmonary fibrosis diseases, arteriosclerosis cardiovascular diseases, various tumors related to PAF, PAF mediated tumor metastasis and recurrence caused by radiotherapy and chemotherapy, and neurodegenerative diseases such as Alzheimer disease and Parkinson disease.
3. The use of an FGFR2b inhibitory molecule according to claim 1 for the preparation of a medicament for the treatment of a PAF-mediated disease, wherein: the acute respiratory distress syndrome is caused by viral infection and bacterial infection.
4. The use of an FGFR2b inhibitory molecule according to claim 3 in the preparation of a medicament for the treatment of a PAF-mediated disease, wherein: the virus is coronavirus.
5. The use of an FGFR2b inhibitory molecule according to claim 4, in the preparation of a medicament for the treatment of PAF-mediated diseases, wherein: the coronavirus is SARS-CoV, MERS-CoV or 2019-n CoV.
6. The use of an FGFR2b inhibitory molecule according to claim 2 in the preparation of a medicament for the treatment of a PAF-mediated disease, wherein: the pulmonary fibrosis is idiopathic pulmonary fibrosis and pulmonary fibrosis caused by inflammation.
7. The use of an FGFR2b inhibitory molecule according to any one of claims 1 to 6, in the preparation of a medicament for the treatment of PAF-mediated diseases, wherein: the FGFR2b inhibiting molecules comprise molecules for inhibiting FGF-7 expression, molecules for inhibiting FGFR2b from being activated into phosphorylated FGFR2b and molecules for competitively inhibiting the combination of FGF-7 and FGFR2 b.
8. The use of an FGFR2b inhibitory molecule according to claim 7 in the preparation of a medicament for the treatment of a PAF-mediated disease, wherein: the molecules include, but are not limited to, siRNA, oligopeptide, polypeptide.
9. The use of an FGFR2b inhibitory molecule according to claim 7 in the preparation of a medicament for the treatment of a PAF-mediated disease, wherein: the molecule which competitively inhibits the combination of FGF-7 and FGFR2b is selected from the short peptides described in the national invention patent application with the application number of 2019102390870 and the invention name of 'short peptides, medicaments and application thereof for treating alopecia'; further, the peptide is a short peptide which is obtained by respectively adding, deleting or mutating 0-2 amino acids at two ends by taking EWVRTD, ELSGRA, QDVDS or VFSTTGV as a center; further EWVRTD, WVRTD, RHEWSRTD, ELSGRA, HTVELSGGRAK, QDVDS, VFSTTGV or FGSVFSTTGV.
10. The use of an FGFR2b inhibitory molecule according to claim 7 in the preparation of a medicament for the treatment of a PAF-mediated disease, wherein: the molecule for inhibiting FGFR2b from being activated into phosphorylated FGFR2b is selected from short peptides described in the national invention patent with the application number of 201310714120.3 and the invention name of 'gene sequence of FGFR2b extracellular domain, polypeptide and application thereof'.