CN113730409B - Application of quinacrine in preparation of medicine for treating pulmonary fibrosis - Google Patents

Abstract

The invention relates to an application of quinacrine in preparing a medicament for treating pulmonary fibrosis, which has the structural formula shown as follows:

. The invention discovers for the first time that quinacrine can inhibit Smad2/3 phosphorylation by targeting CUL4B protein, further influences TGF-beta 1/Smads signal transduction pathway and realizes the effect of treating pulmonary fibrosis, and the discovery overcomes the defect that quinacrine is only used as a sensitizer to participate in the treatment process of cancers such as liver cancer and the like in the prior art, thereby remarkably widening the application range of the quinacrine.

Description

Application of quinacrine in preparation of medicine for treating pulmonary fibrosis

Technical Field

The invention relates to application of quinacrine in preparation of a medicament for treating pulmonary fibrosis, and belongs to the technical field of chemical medicament development.

Background

Pulmonary fibrosis is a group of interstitial lung diseases characterized by progressive dyspnea, cough, gas exchange failure and respiratory failure due to various etiologies, the pathogenesis of which has not been elucidated so far, and a specific treatment method is lacking. It is characterized by the proliferation and differentiation of fibroblasts residing in the pulmonary interstitium and perivascular spaces into myofibroblasts and the generation of excess extracellular matrix (ECM) deposited in the alveoli and pulmonary interstitium. It has been found that a key pathway for extracellular matrix (ECM) production is associated with TGF-. beta.1/Smads signaling.

During pulmonary fibrosis, the combined effects of many factors, especially the stimulation of a large number of cytokines, cause lung Fibroblasts (FBs) to constantly proliferate and transform into Myofibroblasts (MBs), which are capable of synthesizing and secreting large amounts of collagen. The most critical factor among these is transforming growth factor beta (TGF-. beta.). It is one of the cytokines currently considered to be the most potent fibrogenic, and it stimulates fibroblast proliferation and induces its transformation into MB by binding to the corresponding receptor, TGF- β also induces epithelial-mesenchymal transition differentiation (EMT), a biological process that transforms epithelial cells into cells with mesenchymal phenotype by a specific procedure. This process also plays a key role in pulmonary fibrosis.

The human transforming growth factor beta (TGF-. beta.) family includes 33 members, all dimeric, secreted polypeptides, including TGF-. beta.1, TGF-. beta.2, TGF-. beta.3, activin, nodal and bone morphogenic proteins. The family members participate in regulating and controlling cell differentiation, cell aging, cell apoptosis, cell adhesion and migration, extracellular matrix synthesis and remodeling, EMT, immunosuppressant, angiogenesis and other pathophysiological processes, and play a vital role in early embryonic development, tissue organ formation and adult homeostasis. Thus, the TGF- β family cytokines signal transduction pathway is tightly regulated spatio-temporally specifically, and dysregulation of signal transduction may lead not only to embryonic dysplasia, but also to a variety of human diseases, including tumors, tissue fibrosis, cardiovascular diseases, and autoimmune diseases. The Smads family of proteins are important TGF-. beta.signaling and regulatory molecules within the cell, and play a critical role in the transduction of TGF-. beta.signals into the nucleus. The research shows that the mechanism of TGF-beta 1 participating in the EMT process is mainly completed through Smads dependent pathways, and Smad2, 3, 4 and 7 are key proteins participating in various information transmission in the pathways. TGF-beta first binds to the extracellular domain located in TGF-beta RII, activating intracellular TGF-beta RI, which phosphorylates Smad2, Smad3 and binds to Smad4 to form a complex, which enters the nucleus, and Smad2/3/4 binds to specific DNA sequences, regulating transcription factors and promoting the development of EMT. The compound can also induce the expression of fibronectin and alpha-SMA, thereby inducing the generation of tissue fibrosis.

TGF-. beta.1/Smads signals are also involved in the following physiological activities: 1. the TGF-beta 1/Smads signaling pathway and wound healing are divided into 3 processes, including (1) physiological inflammatory processes; (2) granulation tissue proliferation and collagen synthesis; (3) tissue remodeling and scarring; 2. ventricular remodeling is an important pathophysiological change of various heart diseases and can cause severe complications such as heart failure, arrhythmia, sudden cardiac death and the like. Ventricular remodeling from the cellular level includes the 3 major links of extracellular signal stimulation, intracellular signal transduction, and activation of nuclear gene transcription.

Therefore, the pulmonary fibrosis seriously threatens human health, but the exact pathogenesis of the pulmonary fibrosis is not completely understood so far, and a specific and effective treatment means is still lacked, so that the research on the pathogenesis of the pulmonary fibrosis has guiding significance on clinical treatment.

Quinacrine was discovered in the 1920 s and has historically been used as an antimalarial for both prophylaxis and therapy. Quinacrine is an acridine derivative that can be orally administered as quinacrine dihydrochloride, which has the following structural formula:

quinacrine is also used as an antibacterial against giardiasis, as an anti-inflammatory against systemic lupus erythematosus and rheumatoid arthritis, and as an intrapleural sclerosing agent for the prevention of recurrence of malignant pleural effusion and pneumothorax. In addition, it is still used for female sterilization in some countries and is being clinically evaluated for creutzfeldt-jakob disease. Because the side effect is small, the toxicity is low, most side effects can be reversed after the medicine is stopped, the compound is used as a potential anticancer medicine, and related researches show that the compound has anticancer potential in colon cancer, non-small cell lung cancer, prostate cancer, kidney cancer, head and neck cancer, leukemia, breast cancer, ovarian cancer and the like.

Disclosure of Invention

Aiming at the defects of the prior art, the invention provides the application of quinacrine in preparing a medicament for treating pulmonary fibrosis.

The application of quinacrine in preparing the medicine for treating pulmonary fibrosis diseases is that the structural formula of the quinacrine is as follows:

。

preferably, according to the invention, the pulmonary fibrotic disease is caused by an abnormality of the TGF-. beta.1/Smads signaling pathway.

Preferably, the quinacrine is used as a drug effect ingredient.

According to the invention, the medicament also comprises an accelerant, and the accelerant can improve the combination of the CUL4B protein and quinacrine.

A medicament suitable for pulmonary administration comprising quinacrine, an enantiomer or diastereomer of quinacrine, a pharmaceutically acceptable salt of quinacrine as a pharmaceutically effective ingredient.

Preferably, according to the invention, the medicament further comprises an enhancer for enhancing the binding of the CUL4B protein to quinacrine.

According to the invention, the medicament also contains a pharmaceutically acceptable carrier or auxiliary material.

According to the invention, the dosage form of the medicament comprises tablets, powder, injection, capsules or aerosol.

The CUL4B protein can be used as a target protein in screening drugs for treating pulmonary fibrosis diseases, and the drugs can increase the stability of the target protein CUL4B after being combined with the target protein CUL 4B.

Description of the principles

At present, researches prove that the quinacrine can be combined with other anticancer drugs so as to improve the treatment effect of the drugs on breast cancer, ovarian cancer, colorectal cancer and the like, and the research proves that the quinacrine can be in a sensitizer form to be synergistic with the chemotherapeutic drugs, so that the sensitivity of cancer cells to the chemotherapeutic drugs is increased, and the proliferation and the growth of tumors are inhibited. However, the pulmonary fibrosis is mainly regulated by TGF beta 1/Smads signals in the occurrence and development process, no research personnel in the field carry out research on quinacrine and TGF-beta 1/Smads signal pathways and pulmonary fibrosis, and the research of the inventor on the TGF-beta 1/Smads signal pathways and a lung disease model is pioneering.

Advantageous effects

The invention discovers for the first time that quinacrine can inhibit Smad2/3 phosphorylation by targeting CUL4B protein, further influences TGF-beta 1/Smads signal transduction pathway and realizes the effect of treating pulmonary fibrosis, and the discovery overcomes the defect that quinacrine is only used as a sensitizer to participate in the treatment process of cancers such as liver cancer and the like in the prior art, thereby remarkably widening the application range of the quinacrine.

Drawings

FIG. 1 is a photograph showing the results of the experiment of the surface plasmon resonance technique (SPR experiment) in example 1;

FIG. 2 is a photograph showing the results of the computer-simulated molecular docking experiment (MOE-Dock experiment) of example 2;

FIG. 3 is a photograph of the DARTS experiment results demonstrating the binding of quinacrine to different CUL4B protein domains after transfection of different CUL4B protein domain plasmids in example 3;

FIG. 4 is a histogram of soluble collagen expression in mouse lung tissue extracted in example 4;

FIG. 5 is a histogram of RNA extracted from lung tissue of mouse in example 4 and detected by RT-PCR;

FIG. 6 shows the HE staining, MASSON trichrome staining and immunohistochemical staining results of lung tissue of mice in example 4;

FIG. 7 is a photograph showing the results of western blot analysis for extracting proteins from lung tissues of mice in example 4 to detect the protein level expression of P-SMAD 2/3;

FIG. 8 is a photograph showing the expression of P-SMAD2/3 in a mouse lung tissue in an immunohistochemical assay in example 4;

FIG. 9 is a bar graph of the results of RT-PCR assay of human fiber line HKF in example 5;

FIG. 10 is a photograph showing the results of western blot assay conducted after the cell nucleus and cell plasma proteins were extracted in example 6;

FIG. 11 is a photograph showing the results of western blot assay conducted on a stable transgenic cell line in which the CUL4B gene was knocked out in example 7;

FIG. 12 is a photograph showing the results of detecting the expression of the protein level of P-Smad2/3 in a western blot experiment after TGF- β stimulation is performed on the stable transgenic cell line in which the CUL4B gene was knocked out in example 7;

FIG. 13 is a photograph showing the results of detecting the expression of the protein level of α -SMA in a western blot experiment after TGF- β stimulation was performed on the stable transgenic cell line in which the CUL4B gene was knocked out in example 7.

Detailed Description

The technical solution of the present invention is further described with reference to the following examples, but the scope of the present invention is not limited thereto.

Example 1

Surface Plasmon Resonance experiment (Surface Plasmon Resonance SPR experiment)

1) Sample information

Stationary phase:

name (R)	Molecular weight	Buffer	Concentration of
				CUL4B	130.4 KD	Tris		30 μg/mL

Mobile phase:

name (R)	Molecular weight	Buffer (content of quinacrine hydrochloride)
			Quinacrine hydrochloride	472.88	65 mg

2) Parameters of the instrument

Protein fixation: the instrument comprises the following steps: biacore S200; chip type: S-CM 5; running buffer: PBS-P; the Ligand concentration is 30 mug/mL; flow rate: 10 μ L/min. Regeneration conditions are as follows: regeneration liquid: 10 mM glycine-HCl, pH 2.0; sample introduction time: 30 s; flow rate: 30 μ L/min. And (3) kinetic analysis: control channel: flow cell 1; running buffer: PBSP, 2% DMSO; contact time: 120 s; presentation time: 300 s; flow rate: 30 mu L/min; kinetic Analysis Wizard mode fitting was used.

3) Experimental procedure

Sample dissolution and dilution:

3.1) exchange and dilution of the receptor protein CUL 4B:

the concentration of CUL4B protein is as follows: 30 μ g/mL, molecular weight of CUL4B protein: 130.4 KD

I.e. protein concentration C = 230.0613 nM

Treating the CUL4B protein solution with a desalting column, and replacing the solution with 10mM NaAc at pH 4.0;

3.2) dissolving and diluting ligand small-molecule quinacrine hydrochloride:

taking 65mg of quinacrine hydrochloride dry powder, wherein the molecular weight is as follows: 472.88, 6.8728 mL of DMSO was added

Fully and uniformly mixing to obtain: quinacrine hydrochloride solution at concentration C =20 mM, volume V =6.8728 mL;

adding 980. mu.L of PBSP into 20. mu.L of quinacrine hydrochloride solution with the concentration of C =20 mM, and fully and uniformly mixing to obtain: quinacrine hydrochloride solution at a concentration of C =400 μ M and a volume of V =1 mL;

taking 300 mu L of quinacrine hydrochloride solution with the concentration of C =400 mu M, adding 300 mu L of PBSP and 2% DMSO, and fully mixing to obtain: quinacrine hydrochloride solution at concentration C =200 μ M, volume V =600 μ L;

taking 300 mu L of quinacrine hydrochloride solution with the concentration of C =200 mu M, adding 300 mu L of PBSP and 2% DMSO, and fully mixing to obtain: quinacrine hydrochloride solution at concentration C =100 μ M, volume V =600 μ L;

taking 300 mu L of quinacrine hydrochloride solution with the concentration of C =100 mu M, adding 300 mu L of PBSP and 2% DMSO, and fully mixing to obtain: quinacrine hydrochloride solution at a concentration of C =50 μ M and a volume of V =600 μ L;

Taking 300 mu L of quinacrine hydrochloride solution with the concentration of C =50 mu M, adding 300 mu L of PBSP and 2% DMSO, and fully mixing to obtain: quinacrine hydrochloride solution at a concentration of C =25 μ M and a volume of V =600 μ L;

taking 300 mu L of quinacrine hydrochloride solution with the concentration of C =25 mu M, adding 300 mu L of PBSP and 2% DMSO, and fully mixing to obtain: quinacrine hydrochloride solution at a concentration of C =12.5 μ M and a volume of V =600 μ L;

taking 300 mu L of quinacrine hydrochloride solution with the concentration of C =12.5 mu M, adding 300 mu L of PBSP and 2% DMSO, and fully mixing to obtain: quinacrine hydrochloride solution at a concentration of C =6.25 μ M and a volume of V =600 μ L;

taking 300 mu L of quinacrine hydrochloride solution with the concentration of C =6.25 mu M, adding 300 mu L of PBSP and 2% DMSO, and fully mixing to obtain: quinacrine hydrochloride solution at a concentration of C =3.125 μ M and a volume of V =600 μ L;

taking 300 mu L of quinacrine hydrochloride solution with the concentration of C =3.125 mu M, adding 300 mu L of PBSP and 2% DMSO, and fully and uniformly mixing to obtain: quinacrine hydrochloride solution at a concentration of C =1.563 μ M and a volume of V =600 μ L;

taking 300 mu L of quinacrine hydrochloride solution with the concentration of C =1.563 mu M, adding 300 mu L of PBSP and 2% DMSO, and fully mixing to obtain: quinacrine hydrochloride solution at concentration C =0.781 μ M and volume V =600 μ L.

3.3) protein fixation:

The receptor protein CUL4B was coupled to a CM5 chip using 10 mM NaAc as Buffer at pH 4.0, with preset coupling values: 5000 RU (pg/mm 2);

3.4) affinity detection:

gradient concentration of ligand protein is used to flow through the surface of the chip, and response data and response curves are obtained. The resulting response data were fitted using a Kinetic Analysis Wizard fitting model.

Results of the experiment

SPR is to fix a biomolecule on the surface of a sensor chip, dissolve the molecule interacting with the biomolecule in a solution and flow the solution through the surface of the chip to cause the mass of the molecule bound on the surface of the chip to change the surface refractive index (the change of the refractive index is proportional to the mass of the biomolecule bound on the metal surface), and finally obtain a specific signal of the interaction between the biomolecules by detecting the dynamic change of an SPR angle in the biological reaction process (the SPR angle changes along with the change of the surface refractive index). SPR detection of the binding between the CUL4B protein and quinacrine was performed by using Biacore S200, and the binding between the CUL4B protein and quinacrine was found with Kd = 4.848E-4M, and the result is shown in FIG. 1.

The above-mentioned operations are conventional in the art, and reference is made to Advances in Surface plasma research Imaging and Microcopy and therapy Biological Applications (Mark ta Bockov. et. Annual Review of Analytical Chemistry 2019.12: 10.1-10.26), etc.

Example 2

Computer simulation molecular docking experiment (MOE-Dock experiment)

MOE Dock was used for molecular docking of quinacrine hydrochloride with CUL 4B. Two-dimensional structures of quinacrine were downloaded from PubChem and converted to three-dimensional structures in MOE by energy minimization as ligands. The crystal structure of CUL4B was downloaded from the RCSB protein database (http:// www.rcsb.org /), and the PDB ID was 4A0C 2. The C chain of 4A0C was used as docking acceptor. Before docking, AMBER10, an implicit solvation model of the EHT force field and reaction field (r-field) was chosen. An "induction fit" scheme is chosen that allows the receptor binding site side chains to move according to the ligand conformation and impose constraints on their position. The weight used to bring the side chain atoms to the original position was 10. Firstly, scoring all the parking poses by adopting a London dG scoring function, then refining the force field of the front 30 poses, and then scoring by adopting a GBVI/WSA dG scoring function. The lowest binding free energy conformation is the best possible binding mode. PyMOL (www.pymol.org) shows a binding mode.

Results of the experiment

The binding pattern of quinacrine to CUL4B protein is shown in FIG. 2, and quinacrine is located deep in the binding pocket of CUL4B and shows a binding effect. Within the binding pocket, the pyridine and benzene rings on the quinacridan ring form a Pi-H interaction with the carbon atom of Glu834 in CUL 4B. VDW interactions are also formed between quinacrine and surrounding residues. These interactions contribute to the binding energy between quinacrine and CUL 4B. The MOE-Dock simulation study determined the binding affinity of quinacrine to CUL4B with a docking score of-7.9926 kcal/mol.

The above-mentioned procedures are conventional in The art, and can be referred to The Molecular basis of CRL4DDB2/CSA ubiquitin restriction, targeting, and activation (Fischer ES, etc.. cell.2011 Nov 23;147(5): 1024-39), and The software "Molecular Operating Environment (MOE) 2018.01", and (Chemical Computing Group Inc., 1010 Shooooke St. West, Suite #910, Montreal, QC, Canada, H3A 2R 7.2018).

Example 3

Stability assay (DARTS assay) of drug (quinacrine) binding to target protein (CUL 4B)

1) Quality of foodParticle construction and transfection: plasmid construction is the most common experimental technique used in molecular biology research. The principle depends on the action of restriction endonuclease, DNA ligase and other modifying enzyme, after the target gene and vector DNA are cut and modified respectively, they are connected together, then introduced into host cell to implement correct expression of target gene in host cell. Respectively constructing plasmids with FLAG labels and containing different CUL4B protein structural domains, respectively transfecting the constructed plasmids into tool cell 293T cells at 37 ℃ and 5% CO₂And culturing for 48h in an incubator with saturated humidity.

2) DARTS cell lysates were prepared (1 mL): 730 muL of M-PER mammalian protein extraction reagent, 10 muL of protease inhibitor, 10 muL of 200 mM phosphatase inhibitor sodium orthovanadate, 50 muL of 1M sodium fluoride solution, 100 muL of 100mM beta-sodium glycerophosphate solution and 100 muL of 50mM sodium pyrophosphate solution; formulation 10 × TNC (1 mL): 1M Tris-HCl buffer, pH 8.0500 μ L, 5M sodium chloride solution 100 μ L, 1M calcium chloride solution 100 μ L, sterile deionized water 300 μ L. When in use, 10 times of TNC is diluted by sterile deionized water by 10 times; the concentration of Pronase when used was 10 mg/mL.

3) Washing the transfected 293T cells with precooled Phosphate Buffered Saline (PBS) for 2 times, sucking up the PBS, adding 500 mu L M-PER lysate, scraping the cells, transferring the cells into 1.5mL EP tubes, incubating for 1.5 hours at 4 ℃ in a shaking table, centrifuging for 15 minutes at 13000rpm and 4 ℃, then sucking the supernatant, respectively adding 225 mu L of supernatant protein into two groups of EP tubes, respectively adding 5 mu L of DMSO (drug solvent, control group) into one group, adding 5 mu L of quinacrine hydrochloride (drug final concentration is 200 mu M, drug group) into the other group, incubating for 1.5 hours at a dark room temperature in a shaking table, dividing each group of proteins into 4 tubes, (50 mu L PER tube), and totally 8 tubes of drug solution. Using 1 XTNC to dilute the Pronase, and preparing two groups of protease solutions with four concentrations according to the mass of the Pronase/the supernatant protein of 0, 1:1600, 1:800 and 1:400, wherein the total amount of the protease solutions is 8 tubes. Then, the prepared 8 tubes of protease solution are respectively added into the tubes of the corresponding 8 tubes of drug solution (-0, +0, -1:1600, +1:1600, -1:800, +1:800, -1:400, +1:400, -as a control group and + as a drug group), and incubated for 10min at room temperature, 1 μ L of cocktail is added into each tube, and incubated for 5min on ice. According to the protein: the loading buffer is added into the water bath kettle in a ratio of 4:1, and the water bath kettle is boiled for 10 min. Loading the sample in the above order, separating by sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) electrophoresis, and transferring to membrane. After 5% skim milk was blocked for 1.5 hours, color developed after 12 hours of incubation with 4 degrees shaker FLAG antibody primary antibody.

4) As shown in FIG. 3, in the grouping transfected with the full-length domain 1-913 of CUL4B, the target band is shown at about 130kDa, and it can be clearly seen that quinacrine protects the CUL4B protein from proteolysis, whereas in the grouping transfected with the domain plasmid of CUL4B protein 1-813, quinacrine loses its protective effect on the protein, but in the grouping transfected with the domain of 813-913 protein, the target band at about 40kDa clearly shows that the target protein is obviously protected by quinacrine. It was demonstrated that the binding of quinacrine to the target protein CUL4B was mainly concentrated in the 813-913 segment domain. Subsequently, in the structural domain plasmid grouping after GAA glutamic acid point at the 834 site is mutated into AAG lysine in the 1-913 and 813-913 segment domains respectively, the original protective effect of quinacrine on the target protein is lost, the target protein can be inactivated by protease hydrolysis, and the experiment combined with the MOE-Dock experiment in example 2 can prove that quinacrine can be combined with the CUL4B protein, and the combining site of the two is positioned at the 834 amino acid of the CUL4B protein. It was shown that quinacrine could bind to CUL4B and probably act to inhibit the TGF-. beta.1/Smads signaling pathway.

The above-mentioned procedures are routine technical procedures in the art, for example, refer to the development of target stability of Drug Affinity Reaction (DARTS) technology, a method for targeting drug targets (Lideljun et al, Chinese veterinary medicine impurities, vol.40, No. 2, 2021), etc.

Example 4

Pulmonary fibrosis in vivo experiment

Experimental materials: c57BL/6J mice, purchased from Choneman animal Breeding Co., Ltd; bleomycin, available from MedChemExpress; quinacrine hydrochloride, available from MedChemExpress.

The experimental method comprises the following steps: 6-8w of C57BL/6J mice, separating trachea by operation, injecting bleomycin (2 mg/Kg) into the trachea by using a micro-syringe, feeding the mice with quinine hydrochloride starting on the first day after the operation, wherein the high-concentration group of the quinine hydrochloride is as follows: the 25mg/kg (abbreviated as high in the figure) and the low concentration group of quinacrine hydrochloride are as follows: 10mg/kg (abbreviated as "low" in the figure), and the mice were killed by spinal dislocation under 4w of postanesthesia, and lung tissues of the mice were taken out. RNA was extracted from lung tissue, and the expression levels of col1a1, FN1, and ACTA2 were measured by RT-PCR. Preparing a paraffin section from lung tissue, observing lung structures and fiber scar formation degree by using a HE staining method and a MASSON trichrome staining method, and detecting the expression amounts of col1a and alpha SMA by using an immunohistochemical method.

The experimental results are as follows: extraction of lung tissue the content of Soluble Collagen was detected using a Biocolor Soluble Collagen detection kit (Sircol Soluble Collagen Assay), and the results are shown in fig. 4, where it was found that the quinacrine treatment group significantly reduced the expression of the lung tissue Collagen content caused by bleomycin. The lung tissue RT-PCR result is shown in FIG. 5, and the mRNA expression level of the fibrosis related index in the quinacrine treatment group is obviously reduced compared with that in the control group; the results of HE staining, MASSON trichrome staining and immunohistochemistry of paraffin sections of lung tissues are shown in fig. 6, and compared with a quinacrine treatment group, HE staining and MASSON trichrome staining are seen to be more serious in collagen deposition of a control group, obvious in mononuclear cell infiltration, obvious in thickening of pulmonary parenchyma alveolar spaces, higher in positive expression of alpha SMA and col1a of the control group than a normal group, and significantly lower in positive ratio of two indexes by quinacrine.

Mouse lung tissues are extracted for western blot detection, and found that quinacrine inhibits the expression level of P-SMAD2/3 protein (figure 7), and in an immunohistochemical experiment (figure 8), quinacrine also significantly inhibits the expression of P-SMAD2/3, thereby fully proving that quinacrine can influence the generation and development of lung fibrosis by inhibiting TGF-beta 1/Smads signal pathways.

The above-mentioned operation is a conventional technique in the art, and for example, refer to Regulation OF transformation Growth Factor-b 1-drive Lung fibers by Galectin-3 (Alison C. Mackinon 1, etc.. AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE, VOL 185,2012), etc.

Example 5

In vitro assay of human fibroblast cell line HKF

Experimental materials: human fibroblast cell line HKF, purchased from Shanghai-Chui Biotech, Inc.; TGF-. beta.from PeproTech; quinacrine hydrochloride, available from MedChemExpress.

The experimental method comprises the following steps: inoculating human fibroblast cell line HKF in logarithmic growth phase to 6-well plate, placing at 37 deg.C and 5% CO₂And culturing for 24h in an incubator with saturated humidity. And when the cells grow to 70% of fusion degree, carrying out different treatments according to grouping conditions, wherein the final concentration of quinacrine is 1uM, the concentration of TGF beta is 10ng/ml, placing the cells in an incubator for continuous culture, extracting RNA after 24 hours, and detecting related indexes of fibrosis by using RT-PCR.

The experimental results are as follows: HKF cell line RT-PCR results are shown in figure 9, the index of fibrosis is increased after TGF beta stimulation is added, but the index is obviously reduced and is dose-dependent when quinacrine is added after TGF stimulation, which proves that quinacrine can treat pulmonary fibrosis.

Example 6

The experimental method comprises the following steps: taking human fibroblast cell line HKF in logarithmic growth phase, inoculating into 6-well plate, placing at 37 deg.C and 5% CO₂And in an incubator with saturated humidity, when cultured cells grow to 70% of fusion degree, a control group is fed with DMSO, a drug group is fed with quinine hydrochloride (the drug solvent is DMSO, the final concentration is 10 ng/mL), TGF-beta (10 ng/mL) is added into each hole to stimulate the cells, cell proteins are extracted according to 0min, 15min, 30min and 60min after the TGF-beta is added, cell nucleus proteins and cell plasma protein extraction kits (P0028) of Biyunnan company are used for extracting cell nucleus and cell plasma proteins respectively, and related indexes of a TGF-beta pathway are detected through Western Blot.

The experimental results are as follows: as shown in figure 10, compared with a control group, Smad2/3 of a drug group is mainly accumulated in plasma protein, and quinacrine obviously inhibits the expression of Smad2/3 in nuclear protein, so that quinacrine can inhibit the phosphorylation of Smad2/3 into a core, and the TGF beta 1/Smads signaling pathway is inhibited.

Example 7

CUL4B knockout verification experiment

CRISPR-Cas9 system: the CRISPR/Cas9 is a gene precise editing technology, single gene or multiple gene knockout can be carried out at the cellular level by using the technology, and the principle is that the endonuclease Cas9 protein carries out PAM-dependent recognition on a target DNA sequence through guide RNA (guide RNA, gRNA) and initiates DNA cutting at a specific site 3bp upstream of a PAM region (5' -NGG). Double-stranded breaks generated by Cas9 endonuclease can be repaired by Homology-mediated repair (HDR) or Non-homologous end joining pathway (NHEJ). NHEJ repair randomly introduces base insertions or deletions (indels) at the DSB site, if the indels are not multiples of 3, the subsequent reading frame is shifted, the frame shifting often generates a Premature Termination Codon (PTC), the Function of the protein is lost (the premature translation termination polypeptide is generally degraded), and gene knockout (KO, Loss of Function) is realized.

HKF cell CUL4B encoding genes are knocked out by using a CRISPR-Cas9 system, Western Blot is used for verifying the knocking-out condition of the CUL4B encoding genes, and the result is shown in figure 11, HKF cells do not express CUL4B after CUL4B encoding genes are knocked out, which shows that CUL4B is knocked out successfully in a HKF cell line by using the CRISPR-Cas9 system.

Two stable transfer cell lines, CUL4B knock-out cell line (KO) and KONC cell line (KONC), were seeded in 6-well plates at 37 ℃ with 5% CO₂And when the cultured cells grow to 70% -80% in an incubator with saturated humidity, DMSO is given to the KONC group and the KO group, TGF-beta 1 (the drug solvent is DMSO, and the final drug concentration is 10 mu M) is given to the KONC + quinacrine group and the KO + quinacrine group, and the cells are placed in the incubator to be cultured for 6 hours continuously. Western Blot and RT-PCR verify whether the activation of Smad2/3 and the development of fibrosis can be inhibited after the target protein CUL4B coding gene is knocked out.

Western Blot results are shown in FIG. 12, and after CUL4B is knocked out, the addition of quinacrine hydrochloride cannot inhibit the phosphorylation of Smad2/3 in NIH HKF cells, which indicates that the CUL4B protein is knocked out, and the inhibition effect of quinacrine hydrochloride on TGF-beta 1/Smads signal transduction pathways disappears, as shown in FIG. 13, after CUL4B is knocked out, the addition of quinacrine hydrochloride cannot inhibit the high expression of the fibrosis-related protein alpha-SMA induced by TGF-beta 1, and indicates that the inhibition effect of quinacrine hydrochloride on fibrosis is also cancelled after the CUL4B target protein is knocked out.

The results all prove that the quinacrine hydrochloride has the function of treating the pulmonary fibrosis by combining with CUL4B protein and then inhibiting the activation of TGF-beta 1/Smads signaling pathway.

Claims (1)

1. The CUL4B protein is used as a target protein in screening drugs for treating pulmonary fibrosis diseases in vitro, and the drugs are combined with the Glu834 site of the target protein CUL4B to increase the stability of the target protein CUL 4B;

   the pulmonary fibrosis disease is caused by the abnormality of TGF-beta 1/Smads signal transduction pathway.

Images