CN111973578A

CN111973578A - Application of C188-9, Venetocalax and Bumetaside in medicament for treating fibrotic diseases

Info

Publication number: CN111973578A
Application number: CN202010767678.8A
Authority: CN
Inventors: 余鹰; 左胜锴; 申毓军; 崔辉
Original assignee: Tianjin Medical University
Current assignee: Tianjin Medical University
Priority date: 2020-08-03
Filing date: 2020-08-03
Publication date: 2020-11-24
Also published as: WO2022027953A1

Abstract

The application of C188-9, Venetocalax and Bumetanide in the medicament for treating the fibrotic diseases is based on clinical application, and takes an RNA recognition sequence (RRM) region of LARP6 protein as a docking target, virtual docking simulation screening is carried out in a computer, and finally cell and animal experiments show that in the process of the occurrence and development of the fibrosis induced by injury, C188-9, Venetocalax and Bumetanide (a powerful quick-acting diuretic which is commonly used clinically) are combined with the RRM region of LARP6 protein, so that the stability of collagen mRNA is inhibited to inhibit the generation of collagen, and further the diseases related to the fibrosis are inhibited. The research of the invention enriches the content of basic research on the pathogenesis of fibrosis related diseases and provides a new idea for further exploring the essential causes of the pathogenesis of fibrosis diseases. Meanwhile, C188-9, Venetocolx and Bumetanamide can be referred to as a new potential medicament for clinically treating the fibrotic diseases.

Description

Application of C188-9, Venetocalax and Bumetaside in medicament for treating fibrotic diseases

Technical Field

The invention belongs to the technical field of biology, and particularly relates to application of C188-9, Venetocalax and Bumetanamide in preparation of a medicament for preventing and treating a fibrotic disease.

Background

The major pathological changes of organ fibrosis are fibrous connective tissue increase in organ tissues, parenchymal cell reduction, and persistent fibrosis, which can lead to normal structural destruction and physiological function decline of organs, even failure, and serious threat to human health and life. Fibrosis plays an important role in diseases such as liver diseases, kidney diseases, idiopathic pulmonary fibrosis and heart failure. Fibrosis is also a major pathological feature of many chronic autoimmune diseases, such as scleroderma, rheumatoid arthritis, crohn's disease, ulcerative colitis, myelofibrosis, and systemic lupus erythematosus, among others. Fibrosis also plays an important role in the process of invasive metastasis of tumors, chronic rejection and many progressive myopathies. The common feature of these diseases is the excessive accumulation of collagen, especially type I collagen, but at present, few therapeutic targets are specifically aimed at the pathogenesis of fibrosis, and inhibition of collagen synthesis is one of the potential important targets for treating fibrosis-related diseases.

1. Collagen and tissue fibrosis

When a body organ is damaged or stressed, moderate fibrosis contributes to the repair of the damage, while excessive fibrosis exacerbates disease progression. TGF- β (transforming growth factor- β) and PDGF (platelet growth factor) signals play important roles in the process of fibrosis, primarily by mediating excessive secretion of extracellular matrix by fibroblasts. In these extracellular matrices, type I collagen, in proportions of up to 80% -90%, plays a dominant role in the process of fibrosis; type III collagen is also a component of extracellular matrix in fibrosis, but the proportion is small; other types of collagen, such as type II, type VI, etc., are not substantially detectable in fibrosis. Therefore, inhibition of collagen type i synthesis can effectively reduce fibrosis. Fibroblasts play an important role in the process of fibrosis. For example, after the injury of the intima of a blood vessel, the smooth muscle cells and the fibroblasts of the blood vessel excessively proliferate, and secrete a large amount of extracellular matrix including collagen, so that the blood vessel wall becomes thicker and the blood vessel cavity becomes narrower, and further various diseases such as atherosclerosis, hypertension, myocardial infarction and the like are caused. Elucidation of the molecular mechanism of collagen synthesis in fibroblasts not only deepens our understanding of fibrosis-related diseases, but also provides a new therapeutic target for anti-fibrosis.

Role of LARP6 in collagen synthesis

TGF- β is currently thought to be the most prominent pro-fibrotic cytokine in many organs and a key molecule in the progression of fibrosis. TGF- β signaling has been reported to be associated with fibrosis in the liver, lung, kidney, skin and heart, and TGF- β 1 signaling pathways can reduce the progression of many models of fibrosis at the animal level. In recent years, the signaling pathway of TGF-beta synthesis and activity regulation is a new hot target for developing anti-fibrosis drugs. However, TGF- β signaling also plays an important role in other life activities, such as the development of multiple autoimmune disorders and increased susceptibility to tumor development in TGF- β 1-deficient mice. Thus, the TGF- β signaling pathway is not an ideal target for the treatment of fibrosis-related diseases.

Few studies on the regulation of collagen after transcription have been reported, and only reports that LARP6 can be used as an RNA-binding protein to increase the stability of collagen mRNA and further influence the synthesis of collagen. The type I collagen is heterotrimer composed of two alpha 1 and one alpha 2 peptide chains, and is the protein with the highest content in human body. Normally, type i collagen is highly expressed in skin, bone and tendon, and is less expressed in parenchymal organs; once the organ is damaged, the level of the type I collagen is rapidly increased in transcription and after transcription, the protein expression amount of the type I collagen can reach 50-100 times of the original expression amount, and the type I collagen is involved in the repair process after the organ is damaged. Pathologically, increased type i collagen synthesis is more regulated at the level of transcription than at the level of posttranscriptional mRNA stability. mRNAs encoding type I collagen contain cis-acting elements in their 3 'and 5' non-coding regions (UTRs) to control the stability of the mRNAs. In all vertebrate type I mRNAs, in the 5 ' UTR region, there is a separate 5 ' neck loop (5 ' SL) structure, which is also present in the mRNA encoding type III collagen. LARP6(La ribosomal protein family 6) is a member of the La-domain containing ribonucleoprotein family, which has a conserved La domain and a highly variable RNA recognition sequence (RRM). LARP6 is the only one of the currently discovered approximately 800 RNA binding proteins involved in the stability of type I mRNAs and binds to type I and type III mRNAs 5' SL, thereby modulating the stability and translation of these mRNAs. Knock-down LARP6 greatly reduced the expression level of type i collagen in both mRNA and protein. Insulin-like growth factor-1 (IGF-1) increases collagen synthesis by increasing LARP6 expression, and overexpression of IGF-1 in Apoe-/-mice increases the degree of fibrosis in atherosclerotic plaques, a signal that also plays a role in regulating collagen type I synthesis during osteoblast differentiation. The regulation of collagen type i and iii synthesis by LARP6, including interaction with myosin filaments to regulate collagen type i secretion and with vimentin (vimentin) to increase collagen mRNA half-life, but it is unclear whether the specific regulatory proteins of LARP6 and the binding site of LARP6 to collagen can bind other molecules and needs to be explored.

Disclosure of Invention

The invention aims to provide application of C188-9, Venetocalax and Bumetanamide in preparing medicines for preventing and treating fibrotic diseases.

The invention relates to the application of at least one compound of C188-9, Venetocalax and Bumetanamide in the preparation of functional products for treating or preventing organism organ fibrosis diseases;

wherein, the structural formulas of C188-9 (formula I), Venetoclax (formula II) and Bumetanide (formula III) are respectively as follows:

wherein, the functional product is a product or potential substance which can generate beneficial effects of treating, relieving, inhibiting and regulating the occurrence and development of the fibrotic diseases of the body organs; the functional product is a single preparation or a composition containing effective dose of preparation components.

Wherein the functional product targets the RRM region of the LARP6 protein.

Wherein the functional product is used for playing one or more of the following functions:

i) reducing the expression of collagen in fibroblasts;

ii) inhibits the binding of LARP6 to collagen mRNA in fibroblasts.

Wherein the fibrotic disease comprises pulmonary fibrosis, hepatic fibrosis, renal fibrosis, cardiac fibrosis, endometrial fibrosis, ocular fibrosis, pancreatic fibrosis, a fibroproliferative disease of the spleen, a fibrotic disease of the bone marrow or a disease induced by fibrosis.

Further, the pulmonary fibrosis disease is drug-induced pulmonary fibrosis, the drug-induced pulmonary fibrosis is sarcoidosis, pneumoconiosis, hypersensitivity pneumonitis or radiation-induced pulmonary fibrosis, pulmonary fibrosis with unknown etiology, and diseases induced by pulmonary fibrosis; the hepatic fibrosis is viral hepatitis, alcoholic hepatitis, autoimmune diseases, fatty liver, malnutrition, chronic congestive heart failure or liver fibrosis caused by drugs, liver fibrosis of unknown etiology, and diseases induced by hepatic fibrosis; the renal fibrosis is renal fibrosis caused by hypertension, glomerulonephritis, systemic lupus erythematosus, scleroderma, renal transplant rejection, pyelonephritis, renal calculus, hyperlipidemia, diabetes, hyperuricemia and hypercalciuria, renal fibrosis with unknown etiology, and diseases induced by renal fibrosis; the cardiac fibrosis is cardiac fibrosis, cardiac remodeling and cardiac hypertrophy caused by ischemic heart disease, hypertension, viral myocarditis, metabolic cardiomyopathy, keshan disease, dilated cardiomyopathy, hypertrophic cardiomyopathy, restrictive cardiomyopathy or arrhythmia, cardiac fibrosis with unknown etiology, and diseases induced by cardiac fibrosis; the endometrial fibrosis is endometrial fibrosis lesion caused by different reasons, and diseases induced by the endometrial fibrosis; the ocular fibrosis is a retinal fibroproliferative disease of the eye caused by ocular trauma, ocular surgery or diabetes, and a disease induced by ocular fibrosis.

In addition, the three compounds C188-9, Venetocclax and Bumetanide related in the invention are preferably selected from:

c188-9: the goods number is: t4650, CAS, 432001-19-9, Targetmol (Wellesley Hills, MA, USA).

Venetocalax: the goods number is: t2119, CAS, 1257044-40-8, Targetmol (Wellesley Hills, MA, USA).

Bumetanide: the goods number is: t0108, CAS, 28395-03-1, Targetmol (Wellesley Hills, MA, USA).

The invention mainly adopts advanced biological information and molecular biology technical means such as a blood pressure remote measuring technology, a laser confocal microscopy technology, a Real-time PCR technology, a microcalorimetric electrophoresis (MST), an RNA co-immunoprecipitation, an immunoblotting technology and the like, and researches the effect of LARP6 in collagen stability from collagen regulation key protein LARP 6. Firstly, a small molecule library with the known activity of the ceramic biological is taken as a screening object, an RRM region of LARP6 protein is taken as a docking target, and a molecule with strong binding force with the RRM region of LARP6 is screened out through high-throughput virtual docking screening. Again, the first fifty small molecule compounds with high docking scores were selected and treated with NIH 3T3 fibroblast cell line, and the five small molecule compounds C188-9 (cat # T4650), Venetocalax (cat # T2119), AT7519 (cat # T1778) Bumetanide (cat # T0108) and Anidulafungin (cat # T6088) were found to reduce the mRNA level of collagen AT the cellular level by RT-PCR assay. Then, further by MST technology, the purified LARP6-RRM protein and five small molecules are subjected to direct interaction analysis, and three compounds of C188-9, Venetocalax and Bumetanide are found to be capable of directly interacting with LARP 6-RRM. Finally we chose Bumetanide for further cellular and animal fibrosis model validation. In both mouse fibroblast NIH 3T3 cells and human fibroblast MRC-5 cells, Bumetandide can effectively inhibit the combination of LARP6 and collagen mRNA, and reduce the expression of collagen. In bleomycin-induced pulmonary fibrosis model in mice, Bumetanide can effectively reduce pulmonary fibrosis and collagen content in mice.

The invention discovers that the three compounds of C188-9, Venetocalax and Bumetaside can effectively inhibit the synthesis of collagen by docking with LARP 6. Through the research, the invention can confirm that the three compounds of C188-9, Venetocalax and Bumetanide can be used as potential medicaments for preventing or treating fibrosis of body organs.

Drawings

FIG. 1 is a flow chart of compound screening.

The application starts from 5370 small molecular compounds, and screens out the first fifty compounds which are highly combined with LARP6-RRM (RNA recognition sequence of LARP6 protein) through virtual docking; then, fibroblasts were treated with the 50 compounds, and 5 small-molecule compounds were found to have a good effect of inhibiting major proteins (including collagen 1 and collagen 3, wherein collagen 1 is abbreviated as Col1, and its coding mRNA is cola1 and cola 1a 2; collagen 3 is abbreviated as Col3, and its coding mRNA is co3a1) of fibrosis; then, the direct interaction of C188-9, Venetochlax and Bumetaside and LARP6-RRM is found through the analysis of microcalorimetric electrophoresis technology.

Figure 2 illustrates that five compounds have the effect of inhibiting collagen 1 and 3 synthesis.

The first fifty compounds which are screened to have strong binding force with LARP6-RRM are used for treating NIH 3T3 fibroblasts, RNA is extracted after 24 hours, and the transcription expression levels of major fibrous proteins, namely collagen 1 and collagen 3 are detected. The concentrations of the compounds were: t1100, 25. mu.M; T3S0175, 10. mu.M; TL0005, 10. mu.M; T2S1158, 10. mu.M; T6S1525, 10. mu.M; T6S1784, 25. mu.M; t1441, 10. mu.M; T4S0590, 10. mu.M; T4S2164, 10. mu.M; T6S1141, 20. mu.M; T4A2458, 5. mu.M; t0937, 5. mu.M; t1494, 10. mu.M; t2415,1 μ M; t3706,1 μ M; t6909, 10. mu.M; t6956, 1. mu.M; t1823, 10. mu.M; t2660, 1. mu.M; t1091, 10. mu.M; t4619, 10. mu.M; t6591, 10. mu.M; t3464, 30. mu.M; t5032, 10. mu.M; t3043, 5. mu.M; t1829, 2. mu.M; t4650, 10. mu.M; t2119, 1. mu.M; t2405, 20. mu.M; T0182L2, 10. mu.M; t3181, 10. mu.M; t0239,50 μ M; t1778, 2. mu.M; t3078, 10. mu.M; t0108, 2. mu.M; t2126, 2. mu.M; t2676, 2. mu.M; t0213, 15. mu.M; t3620, 10. mu.M; t4353, 10. mu.M; t3175, 10. mu.M; t2377, 10. mu.M; t6205, 1. mu.M; t0137, 10. mu.M; t1325, 10. mu.M; t0749, 10. mu.M; t3156, 25. mu.M; t6152, 10. mu.M; t3193, 10. mu.M; t6088, 10. mu.M; t7079, 10. mu.M. Wherein FIG. 2A is the RNA expression level of the alpha 1 chain of collagen 1 (Col 1a1 for short), FIG. 2B is the RNA expression level of the alpha 2 chain of collagen 1 (Col 1a2 for short), and FIG. 2C is the RNA expression level of the alpha 1 chain of collagen 3 (Col 3a1 for short), P <0.05vs control group.

FIG. 3 illustrates the direct interaction of C188-9, Venetoclax and Bumetanide with LARP 6-RRM.

The LARP6-RRM Protein with MBP (Maltose-Binding Protein, which is a common Protein label) label and a control MBP Protein are expressed and purified, and five small molecules screened from a fluorescence labeling graph are respectively interacted with MBP-LARP6 or the MBP Protein through a micro-calorimetric electrophoresis instrument. FIG. 3A illustrates the interaction of Bumetanide with MBP-LARP6 with an equilibrium dissociation constant Kd of 0.848. mu.M, and FIG. 3B illustrates the absence of Bumetanide interaction with MBP; fig. 3C illustrates that C188-9 interacts with MBP-LARP6 with an equilibrium dissociation constant Kd of 12.5 μ M, and fig. 3D illustrates that C188-9 does not interact with MBP; panel E illustrates the interaction of venetocalax with MBP-LARP6 with an equilibrium dissociation constant Kd of 4.53 μ M, and panel F illustrates no interaction of venetocalax with MBP.

Fig. 4A, 4B, and 4C show the interaction patterns of the three compounds, C188-9, venetocalax and Bumetanide, with LARP6-RRM, respectively.

Among them, LARP6-RRM protein is represented by a linear connected gray sheet structure, and three small molecular compounds and amino acid residues on the protein interacting with them are represented by rods. The small molecule compound and the interacting amino acid residues are grey and dark grey, respectively; in FIG. 4A, K184, L290, M293 and K294 represent lysine 184, leucine 290, methionine 293 and lysine 294, respectively, of LARR6-RRM protein; in FIG. 4B, D242, I246, I291, M293 and P295 represent aspartic acid at position 242, isoleucine at position 246, isoleucine at position 291, methionine at position 293 and proline at position 295, respectively, of the LARR6-RRM protein; in fig. 4C, M185, D242, I246, R249, and K294 represent methionine at position 185, aspartic acid at position 242, isoleucine at position 246, arginine at position 249, and lysine at position 294, respectively, of LARR6-RRM protein.

Figure 5 illustrates that Bumetanide inhibits the binding of LARP6 to collagen 1 and collagen 3 mRNA.

NIH 3T3 fibroblasts were treated with 2. mu.M Bumetanide, harvested 24 hours later, UV-crosslinked for 5min, and RNA immunoprecipitation was performed with LARP6 antibody. Then, reverse transcription is carried out to obtain cDNA, and mRNA levels of collagen 1 and collagen 3 are detected by RT-PCR; it is shown that Bumetanide significantly reduced the binding of LARP6 to collagen mRNA. P <0.05vs control group.

Figure 6 illustrates that Bumetanide can reduce bleomycin-induced pulmonary fibrosis in mice.

Wherein, fig. 6A is a graph of collagen production with or without Bumetanide in a bleomycin-induced pulmonary fibrosis model in mice by masson trichrome staining, collagen fibers being dark grey (arrows indicate dark grey example); FIG. 6B statistics of collagen production; figure 6C hydroxyproline assays on lung tissue of pulmonary fibrosis mice; hydroxyproline is one of imino acids, is a non-essential amino acid, is one of the main components of collagen tissues, is an amino acid peculiar to collagen, and can reflect the fibrosis degree of tissues.

Figure 7 illustrates that Bumetanide can inhibit the binding of LARP6 to collagen 1 and collagen 3 mRNA in human fibroblasts.

Human fibroblasts MRC-5 were treated with 2. mu.M Bumetanide and/or 10ng/ml TGF-. beta.1 (transforming growth factor-. beta.1), harvested 24 hours later, UV-crosslinked for 5min, and RNA immunoprecipitation was performed with LARP6 antibody. After reverse transcription into cDNA, mRNA levels of collagen 1 and 3 were detected by RT-PCR. Fig. 7A, 7B, and 7C show that TGF- β 1 can increase binding of LARP6 to collagen 1 and collagen 3 mrnas, while Bumetanide can significantly reduce binding of LARP6 to collagen 1 and collagen 3 mrnas and eliminate the increase brought by TGF- β 1. P <0.05vs solvent group. # P <0.01vs control group.

Figure 8 demonstrates that Bumetanide inhibits collagen 1 and collagen 3 production in human fibroblasts.

Among them, FIGS. 8A-C are RT-PCR assays for mRNA levels of collagen 1 and 3 in 2. mu.M Bumetanide and/or 10ng/ml TGF-. beta.1 treated human fibroblasts MRC-5. Figure 8D shows protein levels of collagen 1 and 3 in Bumetanide and/or TGF- β 1 treated human fibroblast MRC-5. P <0.05vs solvent group. # P <0.01vs control group.

Detailed Description

The invention will be further illustrated by reference to specific examples.

The following terms used in the specification and claims have the following general meanings, unless otherwise indicated, and are considered to be within the knowledge of one skilled in the art:

"conserved" refers to an amino acid sequence or nucleic acid sequence involved that has a high similarity or identity to the original sequence, is capable of maintaining the basic structure, biological activity or function of the original sequence, and can be generally obtained by similar amino acid residue substitutions or allelic (degenerate codon) substitutions, etc.

"variant" refers to an amino acid sequence or a nucleic acid sequence having one or more amino acid or nucleotide changes, which changes may include insertions, deletions, or substitutions of amino acids or nucleotides in the amino acid sequence or nucleic acid sequence. Variants may have conservative changes, where the substituted amino acid has similar structural or chemical properties as the original amino acid, such as a substitution between leucine and isoleucine, or may have non-conservative changes.

"homologous" includes both complete and partial homology, and when describing a polypeptide, protein or amino acid sequence, refers to having the same or similar structure or function, or having a similar amino acid sequence; in describing nucleic acid sequences, reference is made to nucleic acid sequences which are similar or complementary, and also includes nucleic acid sequences which are expressed in terms of codon preferences of the organism in which the construct is constructed; "homologous" is used in the present invention in a relatively broad sense, for example, to include sequences (amino acid sequences or nucleic acid sequences) having a certain percentage of identity, or to include variants of sequences.

"derivative" when describing a polypeptide, protein or amino acid sequence, refers to a related polypeptide, protein or amino acid sequence derived from the original polypeptide, protein or amino acid sequence, which has similar properties, activities or functions as the original polypeptide, protein or amino acid sequence, for example, the polypeptide, protein or amino acid sequence of the present invention includes such derivatives: (i) the mature polypeptide is fused to another compound, or (ii) an additional amino acid sequence (linker, protein purification tag sequence, cleavage site, etc.) is fused or inserted into the amino acid sequence; etc.; in describing a nucleic acid sequence, it is intended that future cognate sequences derived from the original sequence, which have similar properties, activities, or functions as the original nucleic acid sequence, may include: (i) insertions, deletions, substitutions of one or more bases (preferably substitutions of alleles) in a sequence or gene, either consecutively or at intervals, and which insertions, deletions, substitutions of one or more amino acid residues may or may not be present simultaneously in the same sequence or gene; (ii) one or more bases in the sequence or gene are modified; (iii) a gene encoding an additional amino acid sequence fused or inserted into the sequence or gene; and the like.

The correlation between the three compounds C188-9, Venetocclax and Bumetanamide and fibrotic disease is detailed below by specific experiments and analyses and discussion.

EXAMPLE 1 screening of three Compounds C188-9, Venetocclax and Bumetanamide as a procedure for treating fibrotic diseases

The main implementation process related to the embodiment is as follows:

1) fifty small molecular compounds with strong affinity with LARP6-RRM are screened out through computer simulation;

2) treating NIH 3T3 fibroblasts by using the small molecular compound screened in the step 1), and detecting the change of collagen mRNA by Real-Time PCR;

3) detecting the direct combination of the small molecular compound and the collagen by using the small molecular compound which is screened in the step 2) and influences the synthesis of the collagen by using micro-calorimetric electrophoresis;

the main experimental part involved in this example is as follows:

1. screening out small molecular compounds with strong affinity with LARP6-RRM

The experimental procedure was as follows:

the screening method utilizes 5370 compounds which have biological activity and can cause biological reaction of cells, tissues and even individuals and are prepared from a ceramic biological classically known activity library (cargo number: L4000) to carry out computer virtual docking screening.

2. Real-Time PCR detection of collagen expression

The experimental procedure was as follows:

1) after adding Trizol to the treated NIH 3T3 fibroblasts, the cells were left at room temperature for 5min to be sufficiently lysed (note: at this time, the mixture can be kept at-70 ℃ for a long time);

2) chloroform was added to 200. mu.l of chloroform/ml Trizol, shaken and mixed for 15 seconds, and left at room temperature for 10min (note: disabling the vortex oscillator to avoid genomic DNA fragmentation);

3) centrifuging the sample at 12000g at 4 ℃ for 15 min;

4) carefully suck the upper aqueous phase and transfer it to a new 1.5ml centrifuge tube (RNase free);

5) adding 0.5ml of isopropanol/ml of Trizol into the isopropanol, fully and uniformly mixing, and standing for 5-10min at room temperature;

6) centrifuging the sample at 12000g at 4 deg.C for 10min, removing supernatant, and depositing RNA at the bottom of the tube;

7) adding 75% ethanol (prepared by deionized water treated by DEPC) into 1ml of Trizol of 75% ethanol/ml, gently oscillating the centrifugal tube, and suspending, washing and precipitating;

8) centrifuging the sample at 8000g at 4 deg.C for 5min, and discarding the supernatant as much as possible;

9) air drying at room temperature or vacuum drying for 5-10 min. Note: RNA samples were not dried too much, otherwise difficult to dissolve;

10) 20-50 μ l DEPC H can be used₂Dissolving the RNA sample at 55-60 ℃ for 5-10 min;

11) nano Drop measuring RNA concentration;

12) reverse transcription and RT-PCR reaction conditions: this was done with reference to the instructions of the reverse transcription kit of Takara.

The reaction system for removing genomic DNA is shown in the following table:

TABLE 1 removal reaction System for genomic DNA

Reacting at 42 deg.C for 2min (or at room temperature for 5 min)^*b)。

A: in a 20 mul reverse transcription reaction system,

the method can use 1 μ g Total RNA at most;

b: the reaction time at room temperature can be prolonged to 30 minutes;

the reverse transcription reaction system is shown in the following table:

TABLE 2 reverse transcription reaction System

The reaction was carried out at 37 ℃ for 15min, followed by 85 ℃ for 5 s.

The quantitative PCR reaction system is shown in the following table:

TABLE 3 quantitative PCR reaction System

Reaction procedure: carrying out reaction at 95 ℃ for 5min to complete the pre-denaturation reaction; PCR was performed for 40 cycles, each cycle including denaturation of the template by incubation at 95 ℃ for 25s, incubation at 60 ℃ for 25s to facilitate primer binding to template, completion of the extension reaction at 72 ℃ for 30s and recording of fluorescence readings. The results are shown in FIGS. 2A-2C.

3. Micro-calorimetric electrophoresis detection of interaction of protein and small molecule

The experimental procedure was as follows:

1) the recombinant protein MBP-LARP6 protein or MBP control protein was purified using an AKTA Rapid protein liquid chromatography system, and the protein was dissolved in 20mM HEPES (pH 7.5) and 100mM NaCl buffer.

2) The MBP-LARP6 protein and the MBP control protein were fluorescently labeled using the NT 647 labeling kit (NanoTemper Technologies, Munchen, Germany).

3) The assay was performed in a buffer containing 20mM HEPES (pH 7.5), 100mM NaCl and 0.05% Tween-20.

4) After a short incubation period, the samples were loaded into MST nt.115 standard glass capillaries.

5) Before each experiment, it was confirmed that the sample did not adhere to the capillary wall and that the sample did not aggregate.

6) The labeled MBP-LARP6 protein was incubated at a constant concentration (1 XM) and the ligand (different small molecule compound) concentration was serially diluted two-fold to 16 different concentrations (from 1 XM to 0.03 nM).

7) Sixteen titration series of ligands were mixed with labeled MBP-LARP6 to generate the final reaction mixture.

Fluorescence analysis was carried out on a Monolith NT.115 apparatus (NanoTemper technology, Munchen, Germany). The MST power is 20% and the excitation power is 75%. The measurement protocol was 5s pre-fluorescence, 20s MST,5s post-fluorescence, 25s delayed. Kd values were calculated by NT analysis software (NanoTemper Technologies).

The results are shown in FIGS. 3A-F.

And (3) knotting: in the experimental process of this example, we screened a library of small molecules with known activities of ceramic organisms, and screened molecules that could bind strongly to the RRM domain of LARP6 by high-throughput virtual docking screening with the RRM domain of LARP6 protein as docking target. Next, the first fifty small molecule compounds with high docking scores were selected, and the NIH 3T3 fibroblast cell line was treated, and five small molecule compounds of C188-9 (cat # T4650), Venetocalax (cat # T2119), AT7519 (cat # T1778) Bumetanide (cat # T0108) and Antidulafungin (cat # T6088) were found to reduce the mRNA levels of collagen 1 and collagen 3 AT the cellular level by RT-PCR detection. Then, further by MST technology, the purified LARP6-RRM protein and five small molecules are subjected to direct interaction analysis, and three compounds of C188-9, Venetocalax and Bumetanide are found to be capable of directly interacting with LARP 6-RRM. Therefore, we speculate that three compounds, namely C188-9, Venetochlax and Bumetanide, have the anti-fibrosis effect, and later experiments prove that Bumetanide is taken as a representative molecule and is verified in a mouse pulmonary fibrosis model and human fibroblasts.

Example 2 Bumetanide inhibits bleomycin-induced pulmonary fibrosis in mice.

The main experimental part involved in this example is as follows:

1. RIP-PCR detection of collagen gene expression

1) Cells were treated with Bumetanide or solvent control.

2) Cells in 10cm dishes were cross-linked in ice and irradiated with UV at 400mJ/cm2 for 5 minutes in 1ml of cold PBS.

3) PBS was discarded, 500ul of cold wash buffer (1 XPBS, 0.1% SDS, 0.5% NP-40, 0.5% sodium deoxycholate) containing 200U/ml RNase inhibitor (Takara) and protease inhibitor cocktail (Roche) was added, and ice-coated for 10 minutes.

4) The cells were scraped off and placed in a 1.5ml tube.

5) Centrifuge at 16000g for 20 min at 4 ℃.

6) RQ I (Promega) was added to a final concentration of 1U/mol/l, incubated in a water bath at 37 ℃ for 5 minutes and then cooled on ice for 5 minutes.

6) For co-immunoprecipitation, 5ul of antibody or control IgG was incubated overnight at 4 ℃ on a Ferris wheel.

7) Immunoprecipitation was incubated with protein A or protein G beads for 3 hours at 4 ℃.

8) In RIP buffer (20mM Tris-Cl, pH 7.4,2000mM NaCl,1mM EDTA,0.3Triton X-100, 5% glycerol)

In the middle, 4 times of washing at 4 ℃.

9) RNA was recovered using TRIzol (Invitrogen).

The RT-PCR method was the same as the PCR assay in example 1, and the results are shown in FIG. 5.

2. Establishment of mouse pulmonary fibrosis model

1) Adult male mice of 6-8 weeks were randomly divided into two groups.

2) Mice to be used were anesthetized by intraperitoneal injection of alfuzin.

3) The mouse neck was depilated with depilatory cream, followed by sterilization of the depilated site with 75% medical alcohol.

4) And (3) cutting a small opening of 2-3 cm along the midline of the neck by using a sterilized scalpel, and exposing the submaxillary gland.

5) The submaxillary gland of the mouse is stripped to both sides with a cotton swab, exposing the air outlet pipe.

6) Bleomycin was injected into the trachea of mice at a dose of 3.5mg/kg (dissolved in normal saline) using an insulin syringe.

7) The wound is sutured with 5-0 silk medical suture.

8) Bumetanide was injected subcutaneously at a dose of 2mg/kg, and the control group was injected with an equal amount of DMSO. Injections were continued for 14 days.

9) After 14 days, mice were sacrificed and lung tissue was removed for paraffin embedding or hydroxyproline assay.

3. Tissue section masson pine trichrome staining

1) Fixing lung tissues by using a neutral formaldehyde solution, and slicing by using paraffin wax to obtain 4-6 mu m slices.

2) The lung tissue paraffin sections are sequentially placed in xylene I15 min → xylene II 15min → xylene: anhydrous ethanol is 1: 12 min; then placing in 100% ethanol I for 5min → 100% ethanol II for 5min → 80% ethanol for 5min → distilled water for 5 min.

3) And fixing the Bouin liquid for 10-15 min.

4) Harris hematoxylin is stained for 4-5 min, and washed by running water for 2 min.

5) And (3) differentiating by 0.5% hydrochloric acid alcohol for 10-30 s, and washing for 5min by running water.

6) Masson composite staining solution for 4-5 min.

7) Slightly rinsed with 0.2% aqueous acetic acid.

8) And 5% phosphomolybdic acid is differentiated for 5-10 min.

9) Slightly rinsed with 0.2% aqueous acetic acid.

10) And 2% aniline blue solution for 10-30 s.

11) Washing with absolute ethanol, naturally drying, and sealing with neutral gum.

12) The microscopic field of view was taken to analyze tissue remodeling and the results are shown in figure 6A.

13) Collagen area was counted using imageJ software and the results are shown in figure 6B.

And (3) knotting: the above results demonstrate that Bumetanide can reduce the binding of LARP6 to collagen mRNA (fig. 5), and is effective in alleviating bleomycin-induced pulmonary fibrosis model in mice (fig. 6).

Example 3 Bumetanide inhibits collagen synthesis in human fibroblasts.

The main experimental part involved in this example is as follows:

1. RIP-PCR detection of LARP 6-binding collagen mRNA levels

The method was the same as the RIP-PCR assay in example 2, and the results are shown in FIGS. 7A-C.

2. RT-PCR (reverse transcription-polymerase chain reaction) for detecting influence of Bumetanide on collagen mRNA production

The procedure was the same as for RT-PCR in example 1, and the results are shown in FIGS. 8A-C.

And (3) knotting: the above results indicate that Bumetanide also has a function of inhibiting collagen production in human-derived fibroblasts.

By combining the above embodiments, the invention starts from a natural active compound library, is screened by computer simulation, is repeatedly researched at a cell animal level, and finds that in C188-9, Venetocalax and Bumetaside can be referred to as a new potential medicament for clinically treating fibrotic diseases.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and should not be taken as limiting the invention, so that any modifications, equivalents, improvements and the like, which are within the spirit and principle of the present invention, should be included in the scope of the present invention.

Claims

The application of at least one compound of C188-9, Venetocalax and Bumetanide in preparing medicine for preventing and treating fibrosis diseases;

wherein, the structural formulas of C188-9 (formula I), Venetoclax (formula II) and Bumetanide (formula III) are respectively as follows:
2. the use according to claim 1, wherein the functional product is a product or potential substance capable of producing a beneficial effect of treating, alleviating, inhibiting, regulating the occurrence, development and/or progression of a fibrotic disease in a body organ; the functional product is a single preparation or a composition containing effective dose of preparation components.
3. The use according to claim 1, wherein the functional product targets the RRM region of the LARP6 protein.
4. Use according to claim 1, wherein the functional product is adapted to function as one or more of:

i) reducing the expression of collagen in fibroblasts;

ii) inhibits the binding of LARP6 to collagen mRNA in fibroblasts.
5. The use of claim 1, wherein the fibrotic disease comprises pulmonary fibrosis, liver fibrosis, kidney fibrosis, cardiac fibrosis, endometrial fibrosis, ocular fibrosis, pancreatic fibrosis, a fibroproliferative disease of the spleen, a fibrotic disease of the bone marrow or a disease induced by fibrosis.
6. The use of claim 5, wherein the pulmonary fibrotic disease is drug induced pulmonary fibrosis, wherein the drug induced pulmonary fibrosis is sarcoidosis, pneumoconiosis, hypersensitivity pneumonitis or radiation induced pulmonary fibrosis, pulmonary fibrosis of unknown etiology, and diseases induced by pulmonary fibrosis; the hepatic fibrosis is viral hepatitis, alcoholic hepatitis, autoimmune diseases, fatty liver, malnutrition, chronic congestive heart failure or liver fibrosis caused by drugs, liver fibrosis of unknown etiology, and diseases induced by hepatic fibrosis; the renal fibrosis is renal fibrosis caused by hypertension, glomerulonephritis, systemic lupus erythematosus, scleroderma, renal transplant rejection, pyelonephritis, renal calculus, hyperlipidemia, diabetes, hyperuricemia and hypercalciuria, renal fibrosis with unknown etiology, and diseases induced by renal fibrosis; the cardiac fibrosis is cardiac fibrosis, cardiac remodeling and cardiac hypertrophy caused by ischemic heart disease, hypertension, viral myocarditis, metabolic cardiomyopathy, keshan disease, dilated cardiomyopathy, hypertrophic cardiomyopathy, restrictive cardiomyopathy or arrhythmia, cardiac fibrosis with unknown etiology, and diseases induced by cardiac fibrosis; the endometrial fibrosis is endometrial fibrosis lesion caused by different reasons, and diseases induced by the endometrial fibrosis; the ocular fibrosis is a retinal fibroproliferative disease of the eye caused by ocular trauma, ocular surgery or diabetes, and a disease induced by ocular fibrosis.