CN110672855B - Use of actin-binding protein 2 in screening of medicaments for treating smooth muscle dysfunction diseases - Google Patents

Abstract

The invention relates to the field of drug research and development, and in particular discloses an application of actin-binding protein 2 in screening of smooth muscle dysfunction disease treatment drugs. According to the invention, the Transgelin 2 is used as a target protein for drug action, 22 possible small molecular compounds are designed and primarily screened, and then 2 small molecular drugs truly having the function of dilating airway smooth muscle are screened out through further cell experiments, so that the method can be used for preparing airway smooth muscle dilating drugs; provides guidance and reference for the treatment of various diseases which may be caused by smooth muscle dysfunction, including vascular smooth muscle spasticity, intestinal canal smooth muscle spasticity, tracheal smooth muscle spasticity and endocrine diseases.

Description

Use of actin-binding protein 2 in screening of medicaments for treating smooth muscle dysfunction diseases

The application is a divisional application of the original application, and the application date of the original application is as follows: 2014-01-10; the application number is: 201410012512X; the invention is named as follows: use of actin-binding protein 2 for screening a medicament for the treatment of smooth muscle dysfunction disease.

Technical Field

The invention relates to the field of drug research and development, and in particular discloses a novel application of actin-binding protein 2 in screening of a smooth muscle dysfunction disease treatment drug.

Background

Actin-binding protein 2 (Transgelin 2, SM22-Alpha Homolog) was first discovered in 1998, whose gene was located on chromosome 1q21-q25, but so far the function and mechanism of action of this protein was unknown. The Transgelin 2 protein is expressed and positioned at the positions of cell membranes, actin microfilaments, cytoplasm and the like, contains 1 calmodulin homologous structural domain (calponin homolog domain) and can be combined with cytoskeletal proteins and the like. The existing literature suggests that the protein is related to smooth muscle cell morphology and B cell immune function and is probably a tumor marker of colorectal cancer and the like.

MT-2 (MT) is one of important members in the family of Metallothionein, and has main physiological characteristics of metal ion binding property and reducibility, can be reversibly bound with divalent ions such as zinc, copper, iron, cadmium and the like to maintain the balance of metal ions in a body, and can also be bound with oxygen free radicals of the body through mercapto (-SH) groups, so that the MT-2 has stronger reducibility. MT-2 is also involved in biological processes such as cell proliferation, apoptosis, differentiation, etc., and is closely related to diseases such as cancer, respiratory system, etc.

However, there has been no study on the relationship between MT-2 protein and Transgelin 2 protein, and on the involvement of Transgelin 2 in the development of drugs for asthma treatment.

Disclosure of Invention

The invention aims to disclose a novel method for screening a smooth muscle dysfunction disease treatment drug, which provides a convenient way for treating smooth muscle dysfunction disease and screening drugs by taking actin-binding protein 2 (Transgelin 2, SM22-Alpha Homolog) as a target spot of drug screening.

In a first aspect, the invention discloses the use of actin-binding protein 2 (Transgelin 2) in the screening of a medicament for the treatment of smooth muscle dysfunction disease.

Smooth muscle function as described herein includes smooth muscle relaxation and contraction.

The smooth muscle dysfunction disease treatment drug comprises: nucleic acids, carbohydrates, lipids, small molecules, polypeptides or proteins.

The nucleic acid comprises: antisense oligonucleotides, double-stranded RNAs (dsRNA), ribozymes, small interfering RNAs (esirnas) prepared by endoribonuclease III, or short hairpin RNAs (shrnas).

Further, the double stranded RNA is small interfering RNA (siRNA).

Preferably, the actin-binding protein 2 acts as a drug acting on the surface receptor of smooth muscle cells, for screening for therapeutic drugs for smooth muscle dysfunction diseases.

More preferably, the smooth muscle dysfunction disease treatment drug comprises a cardiovascular disease treatment drug, a cerebral apoplexy, angina pectoris, myocardial infarction, heart failure, renal failure and other complications treatment drug caused by hypertension, a cerebral ischemic disease treatment drug, a myocardial ischemic disease treatment drug, a renal hypofunction and peripheral vasospastic disease treatment drug, a gastrointestinal spasmodic angina treatment drug, an irritable bowel syndrome treatment drug and gastrointestinal dysfunction treatment drug caused by irritable bowel syndrome, an airway smooth muscle relaxation drug, an airway remodeling treatment drug, a urogenital tract renal colic treatment drug, a bladder stimulation treatment drug and the like.

Further, the smooth muscle is airway smooth muscle.

Preferably, the use is the use of actin-binding protein 2 in the screening of a medicament for the treatment of diseases that relax airway smooth muscle. For example, it can be used for screening drugs for treating asthma based on the diastolic airway smooth muscle.

The invention designs 22 possible small molecular compounds by taking actin-binding protein 2 as a medicine to act on the receptor of airway smooth muscle cells and referring to the tertiary structure of actin-binding protein 2, and finally screens out two substances of isoquercetin and apigenin-7-glucoside from the 22 designed possible compounds, thereby having practical significance of dilating smooth muscle cells and being applicable to the treatment of asthma.

The actin-binding protein 2 gene is located on chromosome 1q21-q25, a known protein, but its function and mechanism of action are not known. The Transgelin 2 protein is expressed and positioned at the positions of cell membranes, actin microfilaments, cytoplasm and the like, contains 1 calmodulin homologous structural domain (calponin homolog domain) and can be combined with cytoskeletal proteins and the like. The existing literature suggests that the protein is related to smooth muscle cell morphology and B cell immune function and is probably a tumor marker of colorectal cancer and the like.

Smooth muscle is widely distributed in the digestive tract, respiratory tract, and vascular and urinary and reproductive systems of the human body, and smooth muscle dysfunction can cause a variety of diseases including vascular smooth muscle spasticity, intestinal canal smooth muscle spasticity, tracheal smooth muscle spasticity, and endocrine diseases. The invention discovers the new application of the Transgelin 2 in screening the asthma therapeutic drugs through researches: actin-binding protein 2 can act as a drug on the receptor of airway smooth muscle cells, and can screen out drugs capable of dilating airway smooth muscle, reducing airway resistance of asthma model rats, improving lung compliance, and/or treating airway remodeling; on one hand, the discovery provides a new way for the research and development of asthma therapeutic drugs, and on the other hand, the discovery has guiding significance and reference value for the treatment of cardiovascular diseases, hypertension and complications such as cerebral apoplexy, angina pectoris, myocardial infarction, heart failure, renal failure and the like caused by hypertension, cerebral ischemic diseases caused by vasospasm, myocardial ischemic diseases, renal hypofunction and peripheral vasospastic diseases caused by renal vasospasm, gastrointestinal spastic angina, irritable bowel syndrome and gastrointestinal dysfunction caused by irritable bowel syndrome, asthma, urogenital tract renal colic, bladder irritation and other smooth muscle dysfunction diseases.

In a second aspect, the invention discloses a method for screening a drug for treating smooth muscle dysfunction diseases, which comprises screening the drug by taking actin-binding protein 2 (Transgelin 2) as a drug action target.

Preferably, the smooth muscle dysfunction disease treatment drug is a drug for dilating airway smooth muscle.

More preferably, the smooth muscle dysfunction disease treatment drug is an asthma disease treatment drug.

The smooth muscle dysfunction disease treatment drug comprises: nucleic acid drugs, carbohydrate drugs, lipid drugs, small molecule drugs, polypeptide drugs or protein drugs.

Preferably, the smooth muscle dysfunction disease treatment agent is capable of competitively binding to actin-binding protein 2 with isoquercitrin or apigenin-7-glucoside.

For example, most drugs are effective by interfering with the function of enzymes or receptors in cells, which are proteins, which have many sites on their surface that are occupied by small molecules, which are called ligands when they bind to the sites on the enzyme or receptor as a key to a lock. When a drug has the same or similar shape as the ligand, the drug can bind to the enzyme or receptor and the binding drug enzyme or receptor will send a signal to the cell causing the cell to undergo a series of physiological reactions. The enzyme or receptor that binds to the drug is the direct target (target protein) for the drug.

The third aspect of the invention discloses an application of isoquercetin in preparing medicaments for dilating airway smooth muscle; and application of apigenin-7-glucoside in preparing medicaments for dilating airway smooth muscle.

The invention also discloses a drug for dilating airway smooth muscle, which contains isoquercetin or apigenin-7-glucoside.

Preferably, the isoquercetin or apigenin-7-glucoside is the only effective amount of the drug for dilating airway smooth muscle.

Preferably, the pharmaceutical formulation further comprises a pharmaceutically acceptable carrier.

Pharmaceutically acceptable carriers are various pharmaceutically commonly used adjuvants and/or excipients, including, but not limited to, sugars (such as lactose, dextrose, and sucrose), starches (such as corn starch and potato starch), celluloses and derivatives thereof (such as sodium carboxymethyl cellulose, ethyl cellulose, and methyl cellulose), tragacanth powder, malt, gelatin, talc, solid lubricants (such as stearic acid and magnesium stearate), calcium sulfate, vegetable oils, such as peanut oil, cottonseed oil, sesame oil, olive oil, corn oil, and cocoa butter, polyols (such as propylene glycol, glycerol, sorbitol, mannitol, and polyethylene glycol), alginic acid, emulsifying agents (such as Tween, polyoxyethylated castor oil), wetting agents (such as sodium lauryl sulfate), colorants, flavoring agents, tableting agents, stabilizers, antioxidants, preservatives, pyrogen-free water, isotonic saline and phosphate buffers and the like; the carrier can improve the stability, activity, biological effectiveness and the like of the formula according to the requirement.

The airway smooth muscle dilating medicine of the present invention may be prepared into any conventional preparation form according to the general pharmaceutical method.

Preferably, the airway smooth muscle dilating medicine is injection or aerosol.

The airway smooth muscle-dilating drug containing isoquercetin or apigenin-7-glucoside of the present invention may be administered by inhalation, nasal, intravenous, oral, subcutaneous, intramuscular or transdermal routes.

Preferably, the airway smooth muscle dilating drug is an asthma disease treating drug.

In a final aspect the invention discloses a method of dilating airway smooth muscle comprising administering isoquercitrin or apigenin-7-glucoside to a patient.

Preferably, isoquercitrin or apigenin-7-glucoside acts on airway smooth muscle of the patient.

Preferably, the mode of administration includes inhalation, oral administration or injection.

Preferably, the mode of administration is by aerosol delivery of isoquercetin or apigenin-7-glucoside to the patient.

The invention discloses a novel function of a Transgelin 2 protein, and discovers that the Transgelin 2 can act on receptors of airway smooth muscle cells as medicines through ligand metallothionein MT-2 of the Transgelin 2, and screens the novel function of the medicines for treating smooth muscle dysfunction diseases. According to the invention, the Transgelin 2 is used as a target protein for drug action, 22 possible small molecular compounds are designed and primarily screened, and then 2 small molecular drugs truly having airway smooth muscle dilating effect are screened out through further cell experiments, so that the method can be used for treating tracheal smooth muscle spasmodic diseases such as asthma and preparing asthma therapeutic drugs. Therefore, the method for screening the smooth muscle dysfunction disease treatment medicine by using the Transgelin 2 protein has the advantages of practical and effective screening and high screening efficiency; provides guidance and reference for the treatment of various diseases which may be caused by smooth muscle dysfunction, including vascular smooth muscle spasticity, intestinal canal smooth muscle spasticity, tracheal smooth muscle spasticity and endocrine diseases.

Drawings

Fig. 1: western blot verification result diagram of Transgelin 2 protein after RNAi silencing

Fig. 2: western blot results of Pull Down experiments

Fig. 3: transgelin 2 protein band mass spectrum

Fig. 4: enolase 1 protein band mass spectrum

Fig. 5: specific binding curves for MT-2 proteins at different concentrations and Scatchard receptor radioassay (n=3)

Fig. 6: specific binding curves of different concentrations of MT-2 protein to smooth muscle cells 4 days after RNAi transfection

Fig. 7: specific binding percentage graph of MT-2 iodine labeled protein after incubation of different antibodies and proteins

Fig. 8: results of cell axial length change

Fig. 9: change in rat tracheal smooth muscle cell index (cell index)

Detailed Description

Before the embodiments of the invention are explained in further detail, it is to be understood that the invention is not limited in its scope to the particular embodiments described below; it is also to be understood that the terminology used in the examples of the invention is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the invention.

Example 1 detection of Transgelin 2 protein and its specific ligand MT-2 protein interactions

1. Experimental materials

1.1 His-tagged MT-2 proteins

According to NCBI tissue distribution information, selecting normal rat lung tissue, extracting total RNA by guanidine isothiocyanate by a one-step method, synthesizing cDNA by AMV reverse transcriptase, and performing PCR amplification under the action of DNA taq enzyme (the amplification primer is shown in Table 1, the amplification product sequence is shown as SEQ ID NO: 2) as a template for PCR amplification of MT-2 gene, wherein the PCR amplification procedure is as follows: after pre-denaturation at 94℃for 5min, denaturation at 94℃for 30s, annealing at 58℃for 30s, extension at 72℃for 60s, a total of 35 cycles were performed, and extension at 72℃for 5min.

After the amplified product is digested by EcoR I enzyme and Xho I enzyme, the T4 DNA ligase is connected to the corresponding digestion site of the pET32a vector to obtain a recombinant vector, and the recombinant vector which is detected to be correct by sequencing is converted into BL21 (DE 3) competent cells by heat shock at 42 ℃. MT-2 recombinant protein was obtained by induction of expression at 37℃using 1mM IPTG (final concentration). The MT-2 fusion protein is treated by enterokinase, and the MT-2 protein with His TAG is obtained by S-TAG affinity chromatography and anion exchange resin purification method.

TABLE 1 primer sequences

1.2 Transgelin 2 low expression tracheal smooth muscle cells

Reducing the expression of the Transgelin 2 by using an RNA interference technology to obtain the low-expression tracheal smooth muscle cells of the Transgelin 2; the specific method for RNA interference is as follows:

1) One day prior to transfection, 6-well plates were plated at the appropriate tracheal smooth muscle cell density. When in transfection, the cells are required to achieve 80% fusion;

2) Transfection reagents according to siRNA (shown in table 2) and fugene (purchased from plamcger company, usa) were used at 3: mixing in proportion 1, incubating for 15min at room temperature, and adding into cells to be transfected;

3) After 6h of transfection, the cells are changed into a complete culture medium, protein is extracted 4 days after transfection to detect the expression of the transgelin 2 protein, the expression level of the transgelin 2 protein is reduced to 5% of the original expression level, and the western blot result after RNA interference is shown in figure 1.

TABLE 2 RNA interference sequences

2. Experimental methods and analysis

2.1 His pull-Down experiment

After mixing and incubating MT-2 recombinant protein containing His tag and rat tracheal smooth muscle cell lysate for 24 hours (4 ℃ C., on ice), adding Ni-NTA agarose gel, centrifuging at 5000rpm for 10min after full incubation, and discarding the supernatant. Imidazole solutions with different concentrations are added, and after full incubation, centrifugation is carried out at 5000rpm for 10min, and the supernatant is reserved. Taking the supernatants of each group, and simultaneously carrying out non-denaturing gel electrophoresis and western blot identification, wherein the result of the western blot is shown in figure 2; according to the result of western blot, the bands on the corresponding gel are cut for mass spectrum identification, and the result shows that two bands of pulldown are found, through mass spectrum identification, one is transgelin 2 (actin binding protein 2, protein coding sequence reference genebank number Gene ID: 304983), the other is Enolase 1 (phosphopyruvate hydratase-1), and the mass spectrum result is shown in figures 3-4.

2.2 specific binding experiments

The change of intracellular ligand MT-2 protein binding to the smooth muscle cells of the trachea with normal expression and low expression of Transgelin 2 is observed by using an isotope method and a scatchard analysis method, and the specific method is as follows:

1) Normal tracheal smooth muscle cells

After marking MT-2 protein with iodine 125, MT-2 protein marked with a final concentration of 2nM-160nM is sequentially added to a six-well plate paved with normal tracheal smooth muscle cells, and incubated for 15min, and washed twice with PBS, 1ml each time. Then, the cells were digested with 600. Mu.l of pancreatin, neutralized with 600. Mu.l of DMED after 15min, and 1ml of the cell suspension was taken to measure the gamma-ray intensity. Equilibrium dissociation constants and maximum binding amounts were calculated using conventional scatchard analysis methods.

The experimental results are shown in FIG. 5, which proves that MT-2 protein has single receptor on the surface of rat tracheal smooth muscle cells, the equilibrium dissociation constant Kd value is 33.4nM, the maximum binding value Bmax is 110pM, and the number of binding sites on each cell surface is 6.6X10 ⁵ 。

2) Low expression tracheal smooth muscle cells

The MT-2 radioreceptor assay (reduced expression of the Transgelin 2 protein to 5% of the original) was performed in the same manner as described above using the transgenic lin 2-expressing smooth muscle cells transfected for 4 days with RNAi (SEQ ID NO: 5-6), and the binding curve was shown in FIG. 6, 9 points in total, and fitted with curve R ² = 0.9239.Scatchard receptor radioanalysis MT-2 transfected RNAi for 4 days smooth muscle cell equilibrium dissociation constant Kd value was still 33.4nM, but Bmax was reduced to 23.3nM (6 points total, scatchard equation R) ² ＝0.8078)。

However, when the cells with reduced expression of enolase 1 were also detected by the siRNA method, it was found that the Kd value and the maximum binding amount were not changed.

Based on the results of the pre-mass spectrometry, the specific binding of MT-2 iodine 125 labeled protein and cell surface receptor was detected after antagonism using different antibodies (including unlabeled MT-2 protein, transgelin 2 antibody, enolase 1 antibody, D2DR antibody, and BSA protein, wherein the Transgelin 2 antibody was purchased from Proteintech Group, the Enolase 1 antibody was purchased from cell signaling technology, and the D2DR antibody was purchased from santa cruz), thereby preliminarily verifying the potential receptor. The specific method comprises the following steps: selecting unlabeled MT-2 protein, a Transgelin 2 antibody, an Enolase 1 antibody and a D2DR antibody, adding the factors with the concentrations of 10nM,20nM and 50nM respectively into culture dish supernatants paved with rat tracheal smooth muscle cells, incubating for 5min, adding the MT-2 protein marked by iodine 125, and detecting the specific binding condition of the MT-2 iodine 125 marked protein and a cell surface receptor according to the marked receptor radioassay. As shown in FIG. 7, after the MT-2 protein without iodine 125 and the transgelin 2 antibody are respectively incubated and antagonized, the specific binding CPM value of the MT-2 iodine 125 labeled protein is obviously reduced, and the maximum reduction is 72.8% and 69.9% of the blank control group respectively in the experimental system. After incubation antagonism of the D2DR antibody, the specific binding CPM values of the MT-2 iodo 125 marker protein were not significantly altered.

From the above results, MT-2 is a Transgelin 2 protein as a receptor on the surface of rat tracheal smooth muscle cells

Example 2 Effect of intracellular ligand MT-2 protein on the systolic Effect of Transgelin 2-underexpressed tracheal smooth muscle cells

1. Experimental method

The trans-gelin 2 low-expression tracheal smooth muscle cell suspension is added into a culture dish with a special glass bottom, MT-2 proteins (0, 100, 200 and 400 ng/ml) with different concentrations are added into culture supernatant after cell adhesion, 30 cell axial length changes (axial length change = axial length after intervention-axial length before intervention in mu m) in the visual field before and after 10min are observed by using an Olympic live cell workstation polarized light stereoscopic imaging method, and the experimental result is shown in figure 8.

The trans-gelin 2 low-expression tracheal smooth muscle cell suspension was added to an xcelligent real-time cell analyzer, and after cell attachment, different concentrations of MT-2 protein (0, 100, 200, 400 ng/ml) were added to the culture supernatant, and different observation reagents: 400ng/ml Terbutaline (TB), 400ng/ml Hydrocortisone (HC), 30min incubation, 20 μM Ach stimulation were added, and changes in rat tracheal smooth muscle cell index (cell index) before and after 10min were recorded, as shown in FIG. 9.

Rat tracheal smooth muscle cell suspension was added to 3% rat tail collagen, after cell-collagen complex formation, MT-2 protein (0, 100, 200 and 400 ng/ml) was added at different concentrations for incubation, imageJ software observed changes in cell-collagen complex area at different time points of 4h,8h,24h etc. after shrink initiation, and the ratio of shrinkage ratio of experimental group (MT-2 protein addition 100, 200, 400 ng/ml) to shrinkage ratio of control group (MT-2 protein addition 0) (ratio of shrinkage ratio of experimental group to control group = shrinkage ratio of experimental group/shrinkage ratio of control group) was calculated, and experimental results are shown in table 3.

TABLE 3 results of ratio of the shrinkage ratio of the experimental group to the control group

2. Analysis of experimental results:

(1) Comparing normal tracheal smooth muscle cells with the Transgelin 2 low-expression tracheal smooth muscle cells, the MT-2 protein has reduced diastole effect on the Transgelin 2 low-expression cells, no dose dependency is presented, only 100ng/ml group has diastole effect, the maximum difference of diastole is reduced to 1/4 times of the same concentration of the normal cell group (1.6 microns of 100ng/ml group is compared with 6.2 microns of the normal cell group), and the result is shown in figure 8.

(2) For the Transgelin 2-low expressing cells, the difference over 10min after addition of acetylcholine to each group was as follows. It was suggested that 30min incubation of 100, 200, 400ng/ml MT-2 protein, 400ng/ml TB,400ng/ml HC did not significantly reduce the contractile effect caused by Ach. Comparing with normal ASM cells, the data suggest that the inhibition effect of MT-2 protein on the cells with low expression of Transgelin 2 is obviously reduced. The specific results are shown in FIG. 9.

(3) After incubation of normal ASM with 100, 200, 400ng/ml MT-2 protein for 30min, there was a significant tendency for the cell-collagen complex to slow down, with a statistical difference, P <0.05, between the control group and 200, 400ng/ml MT-2 at the 4h time point; at the 8h time point, there was a statistical difference between the control group and 400ng/ml MT-2, P <0.05; at 24h time point, there was a statistical difference between the control group and 400ng/ml MT-2, P <0.05. In contrast, after 200 ng/ml MT-2 was added to the airway smooth muscle cells under the condition of Transgelin 2, the contraction ratio at each time point was compared with that of the control group, and no significant change was observed. The specific data are shown in Table 3.

EXAMPLE 3 study of the use of Transgelin 2 to screen asthma therapeutic drugs

The aim of the embodiment is to design a small molecular medicine aiming at the target protein by taking the Transgelin 2 protein as a medicine to act on a receptor on the surface of airway smooth muscle cells and screen effective asthma therapeutic medicines from the small molecular medicine.

1. Experimental method

1) Design of small molecule compounds

Based on the three-dimensional structure of the Transgelin 2 protein (PDB number: 1 WYM), a virtual screening of small molecules based on molecular docking was performed using the sequence (a 46-a63, QPGRENFQNWLKDGTVLC, 18 amino acids total, SEQ ID NO: 1) of the Transgelin 2 antibody (Proteintech Group Co.) as the main target site. The software used for virtual screening is maestro2010 software (the main modules include glide modules, ligperp, protein preparation wizard, etc.), the preparation parameters (default parameters) of small molecules: the ph is controlled to be 5-9 during the protonation; using the epi-k standpoint; three-dimensional configuration: retain specified chiralities (from 3D structure) is selected; setting a box with a side length of 30A, adopting an SP method to dock, adopting an XP method to dock 1500 molecules at the top of the ranking, selecting molecules with scoring better than-6, and finally preliminarily obtaining 22 possible small molecules.

2) Screening of the compounds: testing the effect of the small molecule compound screened in step 1) on the resistance value of the tracheal smooth muscle, thereby deducing the systolic and diastolic effects of the compound on cells.

The effect of the screened 22 possible small molecule compounds on airway smooth muscle resistance was tested using the Roche real-time cell analysis system (xCELLigence RTCA DP, roche) to infer the systolic effect of the compounds on cells.

The specific experimental method is as follows:

1) 3000 smooth muscle cells were distributed into the electrode-containing test wells, and cells were allowed to adhere overnight;

2) Adding 0.1, 1, 10, 50 and 100um (final concentration) of each small molecule compound to the system cell index to 1.0, and incubating for 30min; PBS was used as baseline, DMSO was used as negative control, and terbutaline was used as positive control.

3) Then 20um acetylcholine (Ach) is added to stimulate for 10min, and the resistance change in the process is recorded in the whole course.

2. Experimental results

TABLE 4 systolic and diastolic effects of small molecule compounds on cells

As can be seen from the above data, isoquercetin (CAS number: 21637-25-2, molecular formula C) ₂₁ H ₂₀ O ₁₂ ) And apigenin-7-glucanGlucoside (CAS number 578-74-5, molecular formula C) ₂₁ H ₂₀ O ₁₀ ) The two compounds have practical dilating effect on cells, and can be used for treating asthma. Therefore, the 22 possible small molecular compounds screened by taking MT-2 receptor protein Transgelin 2 as a drug action target point have the effect of dilating airway smooth muscle, and can be used as asthma therapeutic drugs.

Sequence listing

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Shanghai Medicine Inst., Chinese Academy of Sciences

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Claims (6)

1. Use of an actin-binding protein 2 binding agent in the manufacture of a medicament for dilating airway smooth muscle;

the actin-binding protein 2 binding agent is selected from isoquercetin or apigenin-7-glucoside;

the airway smooth muscle dilating medicine is an asthma treating medicine.

2. The use according to claim 1, wherein the actin-binding protein 2 binding agent is a nucleic acid drug, a carbohydrate drug, a lipid drug, a small molecule drug, a polypeptide drug, or a protein drug.

3. A method for screening a drug for treating smooth muscle dysfunction disease, comprising screening a drug using actin-binding protein 2 as a drug action target.

4. A method according to claim 3, characterized in that it comprises, in particular, the following steps:

1) Design of small molecule compounds: based on the three-dimensional structure of actin-binding protein 2, taking actin-binding protein 2 antibody sequence as main target site to perform molecular docking-based small molecule virtual screening;

2) Screening of the compounds: testing the effect of the small molecule compound screened in step 1) on the resistance value of the tracheal smooth muscle, thereby deducing the systolic and diastolic effects of the compound on cells.

5. The method of claim 3, wherein the smooth muscle dysfunction disease treatment drug is a nucleic acid drug, a carbohydrate drug, a lipid drug, a small molecule drug, a polypeptide drug, or a protein drug.

6. The method of claim 5, wherein the smooth muscle dysfunction disease treatment agent is capable of competitively binding actin-binding protein 2 with isoquercetin or apigenin-7-glucoside.

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