CN114560817B - Small molecule drug for inhibiting fibrosis and application thereof - Google Patents

Abstract

By constructing a cell model and an animal model, the present invention takes PAX4 as a target for drug screening for the first time, and obtains a small molecule drug gfr-960v_5 that can effectively inhibit PAX4 to inhibit organ fibrosis. Experiments confirmed that the PAX4 inhibitor gfr‑960v_5 could reverse the increase of the transcription factor PAX4 caused by AngII and the content of fibronectin, αSMA and Col I, which are common markers in the development of cardiac fibrosis; in tissues with cardiac fibrosis, PAX4 inhibitor gfr‑960v_5 can reduce the area of cardiac fibrosis caused by AngII, and can improve the abnormal diastolic function (E/E' increase) caused by angiotensin II, the dose is much lower than the possible toxicological dose, there is a relatively wide Safe dose window. The above results show that the PAX4 inhibitor gfr‑960v_5 can alleviate the increase of extracellular matrix, inhibit the occurrence of fibrosis and improve cardiac function by inhibiting PAX4, which provides a new drug route and theoretical basis for the treatment of fibrotic diseases.

Description

A small molecule drug for inhibiting fibrosis and its application

technical field

The invention relates to the field of biomedicine, in particular to a small molecule PAX4 inhibitor and its application in preparing a medicament for inhibiting fibrosis.

Background technique

Cardiac fibrosis is an important pathological change in most heart diseases. Cardiac fibrosis is manifested by excessive deposition of extracellular matrix in cardiac tissue, resulting in the destruction of physiological cardiac tissue structure and ultimately heart failure, which seriously threatens human health and life. Altered gene expression in fibroblasts is central to the initiation and maintenance of the cardiac fibrotic response. Myofibroblasts are mainly differentiated from fibroblasts. Myofibroblasts have important contractile and secretory functions, and they express a large amount of α-smooth actin (αSMA), fibronectin (fibronectin), and type I collagen (Collagen I, Col I). its characteristics. Angiotensin II is a recognized polypeptide preparation that regulates vasoconstriction, affects cardiac function and induces cardiac fibrosis in current research. This preparation can usually be used to treat mice or fibroblasts to simulate the state of cardiac fibrosis. So far, cardiac fibrosis is still an important target and difficulty in clinical treatment of cardiac diseases. Since the treatment of cardiac fibrosis can delay the occurrence and development of heart failure, it is an important problem to find targets and drugs for the treatment of cardiac fibrosis.

The transcription factor PAX4 is a member of subfamily IV of the Paired box (PAX) family. Current research suggests that members of the PAX family perform important functions in many stages of embryonic development and organ formation, and also in all aspects of the body in adulthood. Members of the PAX family, from insects, amphibians, birds, and mammals, the evolution of their sequences is quite conserved. The protein structure of PAX4 includes a 128 amino acid bipartite paired domain (PD) and a homeodomain (HD), whose C-terminal not only has a transcriptional activation domain common to the PAX family, but also a unique negative regulatory structure. area. Current research suggests that members of the PAX family are important regulators of tissue development and cell differentiation. The research on PAX4 mainly focuses on pancreatic islet, cancer and retina related research. In our team's earlier research, it has been found that PAX4 is an important target of organ fibrosis (CN111214660B). On this basis, the development of drugs that can be used for fibrosis treatment has become the focus of further research.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide a small molecule drug that can be used to inhibit fibrosis and its application. More specifically, the present invention provides the following technical solutions:

A first aspect of the present invention provides a small molecule compound gfr-960v_5, the structure of which is as follows:

。

.

The second aspect of the present invention provides a pharmaceutical composition for inhibiting fibrosis, wherein the active ingredient of the pharmaceutical composition is the small molecule compound gfr-960v_5.

In one embodiment, the above-mentioned pharmaceutical composition further comprises pharmaceutically acceptable auxiliary ingredients.

In one embodiment, the above pharmaceutical composition is selected from solutions, suspensions, ointments, powders, granules, tablets or tinctures.

The third aspect of the present invention provides an application of the above-mentioned small molecule compound gfr-960v_5 in the preparation of a medicament for inhibiting fibrosis.

In a preferred embodiment, the fibrosis refers to pulmonary fibrosis, cardiac fibrosis or pancreatic fibrosis.

The present invention has obtained the following technical effects with respect to the prior art :

By constructing a cell model and an animal model, the present invention takes PAX4 as a target for drug screening for the first time, and obtains a small molecule drug gfr-960v_5 that can effectively inhibit PAX4 to inhibit organ fibrosis. Experiments confirmed that the PAX4 inhibitor gfr-960v_5 (100 μg/kg) could reverse the increase of the transcription factor PAX4 caused by AngII and the content of fibronectin, αSMA and Col I, which are common markers of cardiac fibrosis; Among them, the PAX4 inhibitor gfr-960v_5 can reduce the area of cardiac fibrosis caused by AngII, and can improve the diastolic function (elevated E/E') caused by angiotensin II. This dose of gfr-960v_5 has no obvious damage to the heart, liver and kidney function in mice, and it is far lower than the possible toxicological dose. The above results show that the PAX4 inhibitor gfr-960v_5 can alleviate the increase of extracellular matrix, inhibit the occurrence of fibrosis, and improve cardiac function by inhibiting PAX4, which provides a new drug route and theoretical basis for the treatment of fibrotic diseases.

Description of drawings

The accompanying drawings are used to provide a further understanding of the present invention, and constitute a part of the specification, and are used to explain the present invention together with the embodiments of the present invention, and do not constitute a limitation to the present invention. In the attached image:

Figure 1A: Brightness of transcription factor PAX4, and fibrosis markers fibronectin, αSMA, Col I after stimulation with gfr-960v_5.

Figure 1B: Results of relative quantification and statistical analysis of brightness.

Figure 2: Real-time fluorescence quantitative PCR detection of cardiac fibroblasts pretreated with PAX4 inhibitor gfr-960v_5 for half an hour, and then stimulated with 1 μM angiotensin II for 2 days, the transcription factor PAX4, and fibrosis markers fibronectin, αSMA, Col I Transcription level changes.

Figure 3: After intraperitoneal injection of PAX4 inhibitor gfr-960v_5 into mice, the detection results of blood biochemical indexes AST, CR, GLU and CKMB in mice.

Figure 4A: Sirius red staining of the heart after administration of the PAX4 inhibitor gfr-960v_5, bar: 500 μm.

Figure 4B: Results of statistical analysis of cardiac fibrosis area.

Figure 5: After constructing a fibrosis model by injecting AngII into a micro-osmotic pump, and then intraperitoneally injecting the PAX4 inhibitor gfr-960v_5, echocardiography found that the use of the PAX4 inhibitor gfr-960v_5 could improve the cardiac relaxation caused by angiotensin II Dysfunction (elevated E/E').

Figure 6: The mice were injected with AngII to build a fibrosis model, and then the PAX4 inhibitor gfr-960v_5 was injected intraperitoneally. Real-time fluorescence quantitative PCR was used to detect the transcription factor PAX4 in cardiac tissue, as well as the fibrosis markers fibronectin, αSMA, and Col I. Transcription level changes.

Detailed ways

The present invention will be further described below with reference to specific embodiments. It should be understood that the specific embodiments described herein are only used to illustrate the present invention, but not to limit the scope of the present invention.

If no specific technique or condition is indicated in the examples, the technique or condition described in the literature in the field or the product specification is used. The reagents or instruments used without the manufacturer's indication are conventional products that can be purchased through formal channels.

The experimental methods in the following examples are conventional methods unless otherwise specified. The test materials used in the following examples are all commercially available products unless otherwise specified.

Example 1. Detection of the expression levels of various markers in mouse cardiac fibroblasts

1. Isolation and culture of mouse cardiac fibroblasts:

About 8-week-old male C57/BL6 mice were killed by cervical dislocation, quickly immersed in 75% alcohol for about half a minute, and the heart was immediately removed from the chest on the ultra-clean workbench, and washed twice in PBS buffer at 4 degrees Celsius. , cut off the blood vessels at the atrium and the bottom of the heart, then cut the ventricle into small pieces, and wash it with PBS to wash away some residual blood. Digestion was performed by adding 0.1% collagenase type II in PBS balanced salt solution (330U, Worthington, Columbia, NJ, USA/Sigma, St. Louis, MO, USA). The whole digestion process was carried out under constant temperature stirring at 36-37 degrees Celsius. After every 8 minutes of digestion, the supernatant digestion solution was taken, added to an equal amount of DMEM medium containing 10% FBS, and mixed evenly. Repeat this process about 7-8 times until the tissue block is completely digested. Centrifuge the collected tubes of cells at 1000 rpm for 5 minutes at room temperature, discard the supernatant, resuspend the cells in DMEM medium containing 10% FBS, and combine the cardiomyocytes obtained each time. The suspension was seeded in a 100 mm diameter petri dish and placed in an incubator at 37 degrees Celsius and 5% CO ₂ for 2 hours to make the fibroblasts basically adhere to the wall. Aspirate and discard the culture medium in the petri dish, and add new DMEM medium containing 10% FBS to continue culturing. After 3 days the cells were confluent, passaged and subjected to subsequent experiments.

2. Experimental method:

2.1 Immunofluorescence staining experiment:

The mouse cardiac fibroblasts obtained in step 1 were fixed at 37 degrees Celsius with 4% paraformaldehyde at 37 degrees Celsius for 15 minutes, washed three times with warm PBS, and then ruptured with 0.2% Triton X-100 20 -30 minutes. After washing 3 times with warm PBS, blocking solution (5% BSA) was added to block for 30 minutes. This was followed by overnight incubation with primary antibodies αSMA (ab32575, abcam, Cambridge, MA, USA), fibronectin (ab2413, abcam, Cambridge, MA, USA), PAX4 (ab101721, abcam, Cambridge, MA, USA) at 4°C. After recovery and storage of the primary antibody, the cells were washed 3 times with PBS, and then incubated with the secondary antibody Alexa Fluor 488 for 1 hour at room temperature. Nuclei were stained using Hoechst (Invitrogen, Carlsbad, CA, USA) for 8 minutes at room temperature. Fluorescence intensities were counted and analyzed using the Morphology Explorer BioApplication module of the High Content Screening Imaging System CellomicsArrayScan VTI HCS Reader (Thermo Fisher Scientific, Rockford, IL, USA).

2.2 Extraction of cellular RNA:

RNA from cells was extracted with Trizol reagent, reverse transcribed with promega kit, and detected by qPCR (1 μg RNA) using Bio-Rad CFX96Touch.

3. Detection of markers in mouse cardiac fibroblast fibrosis model:

The P1 generation of mouse cardiac fibroblasts obtained in step 1 was cultured in a 96-well plate, and the cells were stimulated with 1 μM angiotensin II for 2 days to establish a cell fibrosis model. Cell samples were collected 2 days later, and the protein levels of endogenous PAX4 and fibrosis markers fibronectin, αSMA and Col I were detected by immunofluorescence after fixation. The brightness in Figure 1A indicates the localization and content of PAX4, fibronectin, αSMA and Col I recognized by specific antibodies, respectively. The experimental results showed that the transcription factor PAX4 was mainly expressed in the nucleus. The experimental results showed that the fluorescence intensity of PAX4, fibronectin, αSMA and Col I were enhanced to varying degrees under the stimulation of angiotensin II, and the fluorescence intensity of PAX4, fibronectin, αSMA and Col I in the cells after using the PAX4 inhibitor gfr-960v_5 significantly reduced. The quantitative results in Figure 1B and Figure 2 show that the transcription factor PAX4, as well as fibronectin, αSMA, and Col I, markers commonly found in cardiac fibrosis, increased significantly after 2 days of stimulation with angiotensin II, whereas the PAX4 inhibitor gfr- Half an hour after 960v_5 pretreatment, angiotensin II stimulation for 2 days could reverse the increase of transcription factor PAX4 caused by AngII and the content of fibronectin, αSMA and Col I, which are common markers of cardiac fibrosis.

Example 2. Therapeutic effect of PAX4 inhibitor on the pathological model of cardiac fibrosis in mice

In animal pathological model experiments, a mouse cardiac fibrosis model was constructed, and cardiac tissue was obtained, and the size of cardiac fibrosis was detected by the method of Sirius red staining experiment.

1. Preparation of angiotensin II-induced mouse cardiac fibrosis model:

10-week-old male C57BL/6 mice were randomly divided into two groups, model group and treatment group:

Model group: Mice were constructed with angiotensin (3 mg·kg-1·day-1) micro-osmotic pump (Alzet MODEL1007D, DURECT, Cupertino, CA) for 7 days to establish a fibrosis model. Preparation of the micro-osmotic pump: 1 day before surgery, inject angiotensin II (dissolved in sterile PBS buffer) into the micro-osmotic pump with a 1 mL syringe, soak the micro-osmotic pressure pump in sterile PBS buffer, and equilibrate at 37 degrees Celsius overnight. During the operation, the mice were anesthetized with 2% to 3% isoflurane, a transverse incision of about 0.7 cm was cut in the back of the mouse's neck, and the subcutaneous tissue was bluntly separated with tweezers. The pump is buried, the wound is sutured, and neomycin ointment is applied to prevent infection. The operation group received continuous infusion of angiotensin II at a concentration of 3 mg/kg/d for 7 days.

Treatment group: One day after the angiotensin II micro-osmotic pump was used to embed the pump, the PAX4 inhibitor gfr-960v_5 (50 μg/kg/d and 100 μg/kg/d for seven consecutive days) was intraperitoneally injected, and 10-week-old male C57BL was selected. /6 mice established a mouse cardiac fibrosis model to detect the degree of fibrosis in the heart.

Seven days after the mice were intraperitoneally injected with the PAX4 inhibitor gfr-960v_5, the blood biochemical indicators AST, CR, GLU and CKMB were detected (Figure 3), suggesting that gfr-960v_5 did not damage the heart, liver and kidneys of the mice.

2. Tissue sectioning and staining:

The above-mentioned two groups of mice were respectively taken and sacrificed, and the cross-sections at the level of the cardiac papillary muscle were fixed in 4% paraformaldehyde solution (W/V%, prepared in PBS) for 6-8 hours. Dehydrated in 20% sucrose solution (w/v% in PBS). Then put it into 70% (3 hours), 80% (3 hours) ethanol solution for gradient dehydration, and finally put it in 90% ethanol plus n-butanol solution (volume ratio 1:1) overnight. The next day, 95% ethanol plus n-butanol solution (twice for 45 minutes), n-butanol (30 minutes), and butanol (20 minutes) were placed in sequence, the surface liquid was blotted with filter paper, and the tissue blocks were embedded in paraffin. Afterwards, the heart was sectioned with a microtome, and the thickness of the paraffin section was 5 μm, which was cross-sectioned at the level of the papillary muscle, and then stained with Sirius red to detect the collagen deposition. First, dewaxing was performed with xylene 3 times for 10 min, 100% ethanol twice for 3 min, 95% ethanol twice for 3 min, 80% ethanol once for 3 min, and 70% ethanol once for 3 min. Finally wash with distilled water 3 times. After that, the Sirius red staining was carried out, first dipped in water, put it into the Sirius red solution for 1 minute, washed with distilled water to remove the floating color (3 times), quickly washed once with 95% ethanol, and then put it in 100% ethanol for 1 2 times per minute (be careful not to wash off the yellow stained color), and finally clear with 80% xylene (2 times in 10 minutes), cover the surface of the section with neutral resin, and seal it with a cover slip. Finally, the tissue sections were observed and quantitatively analyzed. Quantitative collagen fiber area (Sirius red staining) was analyzed using the NanoZoomer-SQ (Hamamatsu, Japan) image analysis system. The Sirius red-stained sections were observed, the collagen fibrosis area (red-stained part) was measured for each specimen, the cross-sectional area of the entire heart was measured, and the fibrosis area was divided by the total heart area, that is, the percentage of fibrosis.

The staining results are shown in Figure 4A. In the tissue with cardiac fibrosis, the PAX4 inhibitor gfr-960v_5 can reduce the area of cardiac fibrosis caused by AngII. The statistical results are shown in Figure 4B and Figure 6.

3. Echocardiography:

Two groups of mice were placed in an anesthesia box and given isoflurane (2.5% isoflurane, 0.8 L/min) for anesthesia. After anesthesia, the mice were taken out of the anesthesia box and quickly placed in a supine position in a heated position. Put on the board and put on the nasal mask connected with anesthetics, fix the limbs of the mice with adhesive tape, and adjust the concentration of isoflurane to 1% to maintain anesthesia. Depilation cream (Nail, Canada) was used on the chest for hair removal. Mice echocardiographic examinations were performed using a Vevo 2100 ultrasound machine (Fujifilm Visual sonics, Canada).

The echocardiographic results are shown in Figure 5. The use of the PAX4 inhibitor gfr-960v_5 was able to improve angiotensin II-induced diastolic dysfunction (elevated E/E') (Figure 5). The above echocardiographic results suggest that inhibition of PAX4 has a protective effect on cardiac diastolic function in mice.

Based on the above experimental results, it is suggested that the PAX4 inhibitor gfr-960v_5 can alleviate the increase of extracellular matrix, inhibit the occurrence of fibrosis and improve cardiac function by inhibiting PAX4.

Example 3, PAX4 inhibitor drug safety test

In order to estimate the safety of the small molecule compounds involved in the present invention, drug toxicity prediction algorithms Lazar (https://lazar.in-silico.ch/predict), Toxicity Predictor (my-pharm.ac.jp), eMolTox ( http://xundrug.cn/moltox) estimated the safety indicators such as the highest dose, liver and kidney toxicity of the compound. The results are shown in Table 1 below. The table shows that the highest dose of PAX4 inhibitor gfr-960v_5 is more than 100 times higher than the effective dose of anti-fibrosis shown in animal experiments, and it is highly sensitive to the heart, respiratory, nervous system and other organs. No toxic side effects.

Table 1 gfr-960v_5 safe dose analysis

index result algorithm Maximum dose (human) ~ 2.0 mg/kg_bw/day Lazar Maximum dose (mice) ~ 18.0 mg/kg_bw/day Lazar Maximum dose used (rat) ~ 12.5 mg/kg_bw/day Lazar cardiotoxicity none eMolTox Pulmonary toxicity none Toxicity Predictor central nervous system toxicity none eMolTox hepatotoxicity Have eMolTox Nephrotoxicity none eMolTox carcinogenicity none eMolTox reproductive toxicity none eMolTox Cytotoxicity none eMolTox Skin allergies none eMolTox mitochondrial toxicity none eMolTox endocrine toxicity Have eMolTox Mutational genotoxicity none eMolTox acute oral toxicity none eMolTox P450 Drug Metabolism Toxicity none eMolTox

It will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the spirit and scope of the invention. Thus, provided that these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims (4)

1. the application of a small molecule compound gfr-960v-5 in the preparation of a pharmaceutical composition for suppressing cardiac fibrosis, is characterized in that, the structure of described small molecule compound gfr-960v-5 is as follows:

. 2. The use according to claim 1, wherein the medicament further comprises a pharmaceutically acceptable auxiliary ingredient. 3. The use according to claim 1 or 2, wherein the pharmaceutical composition is selected from solutions, suspensions, ointments, powders, granules, tablets or tinctures. 4. application as claimed in claim 1 is characterized in that, the dosage of described medicine is lower than 2.0 mg/kg_bw/day.

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