Application of BCAT1 inhibitor in preparation of medicine for treating fibrosis diseases

Abstract

The invention relates to application of a BCAT1 inhibitor in preparation of a medicine for treating fibrosis diseases, in particular to a leucine analogue ERG240 serving as a selective inhibitor of BCAT1 and application of the leucine analogue ERG240 in preparation of a medicine for preventing and treating renal fibrosis caused by unilateral ureteral obstruction. The application has the advantages that molecular docking and in-vivo and in-vitro experiments verify that the small molecule ERG240 is combined with the BCAT1 protein and inhibits the expression activity of the BCAT1, so that kidney fibrosis related diseases induced by unilateral ureteral obstruction are effectively relieved; especially, the ERG240 can relieve kidney pathological changes of renal fibrosis model mice, significantly reduce the expression level of fibrosis related indexes and protect renal tubular epithelial cells in a fibrosis model.

Description

Technical Field

[0001] The present invention relates to the field of medicine, and in particular to application of a BCAT1 inhibitor in preparing medicines for treating fibrotic diseases. Background Art

[0002] Unilateral ureteral obstruction (UUO) is a common urinary system disease. Due to partial or complete obstruction of one ureter, the pressure on the affected kidney increases, urine retention, hydronephrosis, and even renal damage. Clinically, the causes of UUO are diverse, and ureteral stones, tumors, trauma, etc. are common. Its symptoms depend on the degree and duration of obstruction. Long-term obstruction without obvious clinical symptoms may lead to continuous decline in renal function, and eventually progress to CKD or even end-stage kidney disease (ESKD), requiring expensive renal replacement therapy, which puts great economic pressure on families and society. CKD caused by unilateral ureteral obstruction has become a global public health problem, but there is still a lack of ideal intervention methods to inhibit the progression of UUO to CKD after renal damage.

[0003] As the intrinsic cells of the kidney, renal tubular epithelial cells (RTEC) constitute the inner wall of the renal tubules and are responsible for performing key functions such as renal waste excretion, substance reabsorption, and acid-base balance regulation. However, after being stimulated by injury, renal tubular epithelial cells undergo epithelial-mesenchymal transition (EMT), that is, epithelial cells are transformed into interstitial cells with stronger migration ability, secreting a large amount of extracellular matrix components, such as collagen and fibronectin, which accumulate in the renal interstitium to form fibrous tissue to cope with injury and repair. However, excessive or persistent EMT may also lead to pathological fibrosis and impair renal function. Finding effective drugs that can inhibit renal tubular epithelial cell transdifferentiation and abnormal collagen deposition is of great significance for preventing renal fibrosis, protecting renal function, and improving patient prognosis.

[0004] The compound sodium 4-methyl-5-oxohexanoate, referred to as ERG240, has the molecular formula C7H11NaO3 and CAS number 1415683-79-2. ERG240 is a leucine analog that acts as a selective inhibitor of branched-chain amino acid transaminase 1 (BCAT1). BCAT1 is a key enzyme responsible for catalyzing the transamination reaction of branched-chain amino acids such as leucine, isoleucine, and valine. ERG240 can inhibit the activity of the enzyme by competitively binding to the active site of BCAT1 by mimicking the structure of leucine without catalytic reaction. Studies in recent years have shown that BCAT1 is highly expressed in a variety of malignant tumor tissues such as gastric cancer, lung adenocarcinoma, and myeloid leukemia. Inhibiting BCAT1 with ERG240 can reduce the proliferation and survival of tumor cells (Mao L, et al. Theranostics 2021). Studies have also shown that ERG240 can reduce the release of pro-inflammatory cytokines by intervening in the activity of BCAT1, showing potential application value in inflammatory diseases (Papathanassiu AE, et al. Nat Commun. 2017). Although ERG240 has shown potential in a variety of malignant tumors and inflammatory diseases, there have been no reports on the use of ERG240 in the prevention and treatment of chronic kidney disease and its fibrotic diseases. Summary of the invention

[0005] The purpose of the present invention is to overcome the deficiencies in the prior art and provide an application of a BCAT1 inhibitor in the preparation of a drug for treating fibrotic diseases, thereby alleviating the pathological changes of chronic kidney disease caused by UUO, inhibiting the transdifferentiation of renal tubular epithelial cells, reducing the accumulation of extracellular matrix, and protecting renal tubules, thereby playing a role in preventing and treating chronic renal fibrosis.

[0006] The present invention is achieved through the following technical solutions: On the one hand, it provides a use of a BCAT1 inhibitor in the preparation of a drug for treating fibrotic diseases.

[0007] Through the above technical scheme, the BCAT1 inhibitor can specifically bind to and interact with the BCAT1 protein through hydrogen bonds; alleviate the pathological changes of chronic kidney disease caused by UUO; inhibit the transdifferentiation of renal tubular epithelial cells, reduce the deposition of extracellular matrix (ECM) molecules, and protect the renal tubules, thereby playing a role in preventing and treating CKD.

[0008] Furthermore, the BCAT1 inhibitor is a leucine analogue.

[0009] Furthermore, the leucine analog is ERG240.

[0010] Through the above technical scheme, the chemical name of the leucine analogue ERG240 is sodium 4-methyl-5-oxohexanoate, and the molecular formula is C7H 11 NaO3, CAS No. 1415683-79-2, has a chemical structure as shown in the following formula (I):

[0011]

[0012] Leucine is a key substrate in the catalytic process of BCAT1, and its molecular formula is C6H 13 NO2, chemical structure is shown in the following formula (II). As a water-soluble analog of leucine, ERG240 retains the key features of leucine in structure, while adding a polar functional group - carbonyl (=CO), which has higher reactivity. This allows ERG240 to not only effectively compete for the binding of leucine to BCAT1, but also has better water solubility and is easier to be absorbed and utilized by the body.

[0013] Specifically, the main chain of ERG240 is sodium hexanoate, in which the long-chain hydrocarbon (-CH2-CH2-CH2-CH2-CH2-) simulates the hydrophobic side chain of leucine, namely the isobutyl side chain (-CH2-CH(CH3)2). ERG240 retains the active site of leucine and can be inserted into the hydrophobic pocket of BCAT1 to interact with the hydrophobic residues in BCAT1. In addition, ERG240 not only retains the carboxyl group (-COOH) of leucine, but also introduces a highly active carbonyl group (=CO), thereby enhancing the binding efficiency of ERG240 with BCAT1 and enabling it to compete more effectively for the binding site of leucine.

[0014]

[0015] Specifically, leucine forms four hydrogen bonds with three amino acid residues of BCAT1 (Thr-290, Arg-119, Thr-333), while ERG240 forms five hydrogen bonds with four amino acid residues of BCAT1 (Thr-290, Arg-119, Val-289, Lys-222). Thr-290 and Arg-119 are the residues that leucine and ERG240 form hydrogen bonds with, indicating that they are key active sites in the structure of BCAT1. ERG240 forms more hydrogen bonds with BCAT1 than leucine, indicating that ERG240 can occupy the active site of BCAT1 more firmly, thereby preventing the binding of leucine.

[0016] Furthermore, the pharmaceutical dosage of ERG240 is 5-10 mg / kg / d.

[0017] Through the above technical scheme, the medicinal dose of ERG240 can significantly reduce the mRNA expression level of fibrosis-related molecules in renal tissue; in particular, two different doses of ERG240 (5 mg / kg / d and 10 mg / kg / d) can dose-dependently reduce the high expression of fibrosis-related molecules, and the difference is statistically significant ( Figure 5 AF), indicating that ERG240 can inhibit the secretion and production of extracellular matrix.

[0018] Furthermore, the application can reduce the high expression of fibrosis-related markers in mice with renal fibrosis.

[0019] Through the above technical scheme, ERG240 at the medicinal dose can significantly reduce the protein expression level of fibrosis-related molecules in renal tissue. In particular, two different doses of ERG240 (5mg / kg / d and 10mg / kg / d) can dose-dependently reduce the high expression of renal fibrosis markers. Except for the FN expression level in the UUO+ERG240 (5mg / kg / d) group, the differences in the grayscale values ​​of the remaining proteins were statistically significant ( Figure 4 AD), indicating that ERG240 has a significant anti-fibrotic effect.

[0020] Furthermore, the pharmaceutical concentration of ERG240 is 100-300 μM.

[0021] Through the above technical scheme, different concentrations of ERG240 did not affect the viability of mouse renal tubular epithelial cells (TKPT). In particular, different concentrations of ERG240 (50, 100, 150, 200, 250, 300 μM) had no significant effect on the cell viability of TKPT, indicating that ERG240 has a large safety range and these concentrations do not cause cytotoxicity ( Figure 6 ).

[0022] In addition, different concentrations of ERG240 can reduce the mRNA expression level of the extracellular matrix in a dose-dependent manner. In particular, ERG240 (100, 200, 300 μM) can reduce the mRNA expression level of the extracellular matrix in a dose-dependent manner, that is, under the stimulation of transforming growth factor-β1 (TGF-β1), the mRNA expression level of extracellular matrix-related factors (Fn and Vim) secreted by TKPT cells increased significantly, and ERG240 can reverse the high expression of Fn and Vim in a dose-dependent manner, and 300 μM ERG240 showed a significant effect in reversing the high expression of Fn and Vim induced by TGF-β1, which was statistically significant and showed good anti-fibrosis potential ( Figure 7 AB).

[0023] Furthermore, the application can inhibit excessive deposition of fibronectin in renal tubular epithelial cells.

[0024] Through the above technical scheme, 300μM concentration of ERG240 can significantly downregulate the high expression of FN in TKPT cells. Under the stimulation of TGF-β1, the deposition of fibrin in TKPT cells increased significantly, and 300μM ERG240 can effectively reduce the high expression of FN and inhibit the fibrin deposition induced by TGF-β1, showing its potential application value in anti-fibrosis treatment ( Figure 8 AB).

[0025] Furthermore, the fibrotic disease is renal fibrosis.

[0026] Furthermore, the fibrotic disease is renal fibrosis caused by unilateral ureteral obstruction.

[0027] Preferably, the drug for treating fibrotic diseases is in the form of an injection.

[0028] Beneficial Effects

[0029] The present invention relates to the use of a BCAT1 inhibitor in the preparation of a drug for treating fibrotic diseases, especially in a drug for chronic kidney disease caused by UUO. ERG240 can bind to BCAT1 protein through hydrogen bonds and be displayed and explained through visualization tools. At therapeutic doses, ERG240 has no significant toxic side effects on the kidneys, liver and heart of mice. In addition, ERG240 can alleviate the pathological changes of chronic kidney disease caused by UUO, inhibit the transdifferentiation of renal tubular epithelial cells, reduce the accumulation of extracellular matrix, and protect renal tubules, thereby playing a role in preventing and treating chronic renal fibrosis. In summary, ERG240 has good development and application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 :The present invention shows that the binding of ERG240 to BCAT1 is more stable than that of leucine. Specifically, through molecular docking simulation, the binding conformations of the ligand leucine and ERG240 to the receptor BCAT1 protein were studied respectively. The results showed that the docking energy of ERG240 to BCAT1 was lower than that to leucine. In addition, leucine formed 4 hydrogen bonds with the three amino acid residues of BCAT1, while ERG240 formed 5 hydrogen bonds with the four amino acid residues of BCAT1.

[0031] Figure 2 :The present invention shows that two therapeutic doses of ERG240 (5mg / kg / d and 10mg / kg / d) have no toxic side effects on mice. Specifically, compared with the sham operation (Sham) group and the UUO group model mice, the treatment of 5mg / kg / d and 10mg / kg / d of ERG240 did not affect the concentrations of serum urea nitrogen (BUN) and serum creatinine (sCr) of the mouse kidney function markers; did not affect the concentrations of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) of the mouse liver function markers; did not affect the concentrations of serum creatine kinase (LDH) and creatine kinase isoenzyme (CK-MB) of the mouse cardiac function markers.

[0032] Figure 3 The present invention embodies the effects of two therapeutic doses of ERG240 (5 mg / kg / d and 10 mg / kg / d) on UUO-induced renal pathological damage in mice. The results of Masson and Sirus red staining showed that 5 mg / kg / d and 10 mg / kg / d of ERG240 can dose-dependently reduce abnormal renal pathological changes in UUO mice and reduce the area of ​​renal interstitial fibrosis.

[0033] Figure 4 The present invention shows that two therapeutic doses of ERG240 (5 mg / kg / d and 10 mg / kg / d) can significantly reduce the protein expression levels of fibrosis-related molecules in renal tissue, such as fibronectin (FN), α-smooth muscle actin (α-SMA), and vimentin.

[0034] Figure 5 The present invention shows that two therapeutic doses of ERG240 (5 mg / kg / d and 10 mg / kg / d) can significantly reduce the mRNA expression levels of fibrosis-related molecules (Vim, Fn, Acta2, Postn, Col1a1, Col3a1) in renal tissue.

[0035] Figure 6The present invention embodies the effect of different concentrations of ERG240 (50, 100, 150, 200, 250, 300 μM) on cell viability in mouse renal tubular epithelial cells (TKPT) cultured in vitro.

[0036] Figure 7 The present invention is embodied in mouse renal tubular epithelial cells (TKPT) cultured in vitro, and different concentrations of ERG240 (100, 200, 300 μM) can dose-dependently reduce the mRNA expression level of the extracellular matrix (Fn, Vim).

[0037] Figure 8 The present invention is embodied in mouse renal tubular epithelial cells (TKPT) cultured in vitro, and therapeutic concentration of ERG240 (300 μM) can significantly downregulate the high expression of FN at the protein level. DETAILED DESCRIPTION

[0038] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with specific embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0039] Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention. The experimental methods in the following embodiments without specifying specific conditions are usually carried out under conventional conditions or under conditions recommended by the manufacturer.

[0040] Unless otherwise specified, the reagents and raw materials used in the examples and comparative examples of the present invention can be obtained through commercial channels.

[0041] (1) Molecular docking

[0042] The three-dimensional crystal structure of BCAT1 (PDB ID: 7NWA) protein was downloaded from the Protein Data Bank (PDB). Water molecules, ligands and other non-essential parts in the protein structure were removed using PyMOL software to optimize the protein structure. The three-dimensional structures of leucine and ERG240 were obtained from the compound database (PubChem). Molecular docking was performed using the AutoDock visual interface program MGTools. The blind docking method generated multiple conformations, and the scoring function evaluated the energy and affinity of the ligand-receptor binding conformation. The results were sorted according to the docking score, and the conformation with the highest score was selected. The docking conformation of the ligand and the target protein was analyzed using PyMOL software to identify key hydrogen bonds, hydrophobic interactions and other important molecular interactions.

[0043] (2) Mouse breeding

[0044] The wild-type C57BL / 6J male mice (8 weeks old, weighing 24-26g) involved in the present invention were purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd. and raised in the SPF-level barrier environment of the Experimental Animal Center of Zhengzhou University, a constant temperature and humidity controlled room, with a light-dark cycle of 12 hours of light and 12 hours of darkness, 5 mice per cage, provided with standard mouse food and free drinking water, and the experiment was carried out after one week of adaptive feeding.

[0045] (3) Establishment of unilateral ureteral obstruction (UUO) model in mice

[0046] Surgical instruments were routinely sterilized by high pressure, and sufficient sterile saline, sterile gauze, and 4-0 silk thread were prepared. Mice were anesthetized with isoflurane, routinely disinfected, and skin prepared. An incision of about 1 cm was made along the left side near the kidney, and the skin and muscle layers were carefully separated to expose the ureter. The ureter was bluntly separated, firmly ligated at the junction of the renal pelvis and ureter, and the muscle layer and skin layer were sutured separately. The wound was sutured with iodine disinfection, and 300 μL of saline was injected subcutaneously in the neck of the mouse for fluid replacement. Except for not ligating the ureter, the remaining operations of the sham group mice were consistent with the model group.

[0047] (4) Drug intervention

[0048] The compound ERG240 involved in the present invention was purchased from MedChemExpress (MCE) in the United States. The solvent used for administration was 2% DMSO diluted to normal saline, and the administration method was intraperitoneal injection. 24 mice were randomly divided into 4 groups (n=6 in each group): a solvent group (Vehicle), a model group (UUO) and two different doses of UUO+ERG240 treatment groups (UUO+ERG240 5mg / kg / d and UUO+ERG240 10mg / kg / d). Pre-administration was performed once 24h and 2h before UUO surgery, respectively, and then the drug was administered once a day at regular intervals, and the administration volume was 0.1mL per 10g body weight. The sham group and the UUO group were injected with the same volume of the above-mentioned solvent, and the mice were euthanized 7 days after modeling.

[0049] (5) Culture and administration of mouse renal tubular epithelial cells (TKPT)

[0050] Mouse renal tubular epithelial cells (TKPT) were cultured in DEME / F12 medium containing 7% (v / v) fetal bovine serum, 0.003% (w / v) insulin, penicillin (100U / mL) and streptomycin (100μg / mL) in a 37°C incubator containing 5% CO2. When the cell growth density reached 90%, the cells were digested with trypsin containing EDTA and evenly seeded in 6-well plates and 12-well plates. When the cell density was 50%, after pretreatment with ERG240 for 2h, 10ng / mL TGF-β1 and the corresponding concentration of ERG240 were co-treated for 24h, and the control group was added with the same volume of solvent.

[0051] (6) Kidney, liver and heart function tests

[0052] The blood of mice was collected and centrifuged to obtain serum. The concentrations of urea nitrogen (BUN), serum creatinine (sCr), alanine aminotransferase (ALT), aspartate aminotransferase (AST), lactate dehydrogenase (LDH) and creatine kinase-isoenzyme (CK-MB) in the serum samples were detected using a serum biochemical analyzer.

[0053] (7) Masson staining of kidney tissue

[0054] Use the Masson staining kit (Seville, item number G1006) to stain according to the instructions. Mouse kidney tissue was fixed with 4% paraformaldehyde at room temperature for at least 24 hours, dehydrated, paraffin-embedded, and sliced. After baking, dewaxing and hydrating, add solution A (2.5% potassium dichromate) and incubate overnight at room temperature for about 15 hours. The next day, the slices were immersed in solution A and incubated in a 65°C oven for 30 minutes, and rinsed with running water until the yellow color faded. At the same time, solution D (Ponceau acid fuchsin) and solution F (2.5% aniline blue) were preheated in the oven for use. Freshly prepare a mixture of B+C (hematoxylin stain) in a 1:1 ratio, immerse for 1 minute, and rinse with running water. Differentiate with 1% hydrochloric acid alcohol (concentrated hydrochloric acid: anhydrous ethanol = 1:100) for 3 minutes, and rinse with running water. Incubate with solution D for 8 minutes, and rinse with running water. Differentiate in solution E (1% phosphomolybdic acid) for 1 min, discard, do not wash, incubate in solution F for 8-30s to terminate the reaction, and differentiate in 1% glacial acetic acid. Routine dehydration, drying, and sealing were performed, and a clear field of view was collected using an Olympus upright fluorescence microscope.

[0055] (8) Sirius red staining

[0056] Use Sirius Red Staining Kit (Golden Clone, catalog number RS1220) to stain according to the instructions. Tissue sections were dewaxed to water. Sirius Red stain was dripped for 1 hour, during which acidified solution was prepared and diluted to acidified water working solution according to the ratio of acidified water concentrate: double distilled water = 1:100. Discard the staining solution, do not wash with water, and directly immerse in acidified water working solution for 3 seconds. Conventional dehydration, transparent, neutral gum sealing, Olympus upright fluorescence microscope to collect clear field of view.

[0057] (9) Western blot

[0058] Prepare protein lysis buffer containing 1% protease inhibitor and phosphatase inhibitor to fully lyse tissues and cells, collect supernatant after high-speed centrifugation at 4°C. Determine protein concentration using BCA kit (Biyuntian, P0012S) according to the instructions, add 5×SDS-PAGE protein loading buffer (Biyuntian, P0015L) to the remaining supernatant in proportion, mix well and place in a metal bath at 100°C for 10 minutes. Take 30μg protein of equal mass from each sample for SDS-PAGE gel electrophoresis, and completely transfer the separated protein to the PVDF membrane in transfer buffer. The PVDF membrane was blocked in 5% skim milk powder at room temperature for 1 hour and then incubated with primary antibodies (shaking overnight at 4°C), primary antibodies BCAT1 (Abclonal, A16351, 1: 2000), fibronectin1 (FN, Abcam, ab2413, 1: 2000), alpha smooth muscle actin (α-SMA, ProteinTech, 14395-1-AP, 1: 4000), vimentin (Abcam, ab92547, 1: 4000), GAPDH (Abclonal, AC033, 1: 50000), and then incubated with rabbit or mouse antibodies (1: 5000) at room temperature for 1 hour. The antibodies were diluted with TBST. After incubation, ECL chemiluminescent solution was added and the membrane was placed in a gel imaging system for development, and the gray value of the bands was quantified using image J.

[0059] (10) Total RNA extraction and reverse transcription

[0060] Total RNA was extracted using the Trizol method. 1 mL of RNAiso Plus (TaKaRa, 9109) was added to each kidney tissue or cell sample. The sample was fully lysed using a grinder or repeated beating. Chloroform was added to separate RNA. An equal volume of isopropanol was added to precipitate RNA. RNA was washed with 75% ethanol and dissolved in RNase-free water. RNA concentration was measured by spectrophotometry. 1 μg of RNA was immediately reverse transcribed into cDNA. The reaction system was 20 μL, as shown in Table 1.

[0061] Table 1: Reverse transcription reaction system

[0062]

[0063] Reverse transcription conditions: 37°C for 15 min, 85°C for 5 s, maintain at 4°C.

[0064] (11) Quantitative Real-time PCR (RT-PCR)

[0065] SYBR Green PCR technology is used to detect and quantify the target DNA sequence. SYBR Green (NoviZan, Q412) fluorescent dye is added to bind to double-stranded DNA, and the DNA amplification process is monitored by measuring the fluorescence intensity. TM 5. Amplification was performed in a real-time fluorescence quantitative PCR instrument, and the reaction system was 10 μL, see Table 2.

[0066] Table 2: Real-time PCR reaction system

[0067]

[0068] PCR reaction program: pre-denaturation at 95°C for 10 min, cycle reaction at 95°C for 10 s and 60°C for 30 s for 40 times, and use the instrument default melting curve acquisition program.

[0069] (12) CCK-8 reagent to detect cell viability

[0070] CCK-8 (Cell Counting Kit-8) is a commonly used reagent for detecting cell viability. Cells are evenly inoculated in a 96-well plate. After 24 hours, different concentrations of ERG240 are added and incubated for 24 hours. Then, a culture medium containing 10% CCK-8 reagent (ApexBio, K1018) is added to the cell culture and incubated in a 37°C incubator for 2 hours. The absorbance of each well is measured at a wavelength of 450nm using an ELISA reader. The absorbance value is proportional to the cell activity, and a higher absorbance value indicates a higher cell viability.

[0071] (13) Statistical analysis

[0072] GraphPad Prism 9.00 software was used for statistical analysis. The experimental quantitative results were expressed as mean ± standard error (SEM). The differences between the two groups were compared by t test. The differences between more than two groups and only one variable were compared by one-way analysis of variance (One-way ANOVA). The differences between more than two groups and two variables were compared by two-way analysis of variance (Two-way ANOVA). p < 0.05 was considered statistically significant. Relative to the Sham / Vehicle group, p < 0.05 was marked as *; p < 0.01 was marked as **; p < 0.001 was marked as ***; relative to the UUO / TGF-β1 group, p < 0.05 was marked as #; p < 0.01 was marked as ##; p < 0.001 was marked as ###.

[0073] The present invention is described in detail below in conjunction with specific embodiments:

[0074] Example 1 demonstrates that the binding of ERG240 to BCAT1 is more stable than that of leucine.

[0075] The binding conformations of the ligands leucine and ERG240 with the BCAT1 protein receptor were studied using the MGTools visualization interface program of AutoDock. Through the blind docking method, 50 different conformations were generated, and the binding mode of each protein-ligand complex was scored. Subsequently, the optimal conformation was selected for in-depth analysis based on the docking energy score. In order to present these results more intuitively, PyMOL software was used to visualize and display the protein-ligand interaction.

[0076] Experimental results: The docking energy between BCAT1 and leucine is -5.106 kcal / mol, while the docking energy between BCAT1 and ERG240 is -5.305 kcal / mol. The lower the docking energy, the more stable the binding between the ligand and the receptor. Therefore, the binding between ERG240 and BCAT1 is stronger than that between leucine. Leucine forms four hydrogen bonds with three amino acid residues of BCAT1 (Thr-290, Arg-119, Thr-333). Figure 1 A), while ERG240 forms five hydrogen bonds with four amino acid residues (Thr-290, Arg-119, Val-289, Lys-222) of BCAT1 ( Figure 1 B). Among them, Thr-290 and Arg-119 are the residues that form hydrogen bonds with leucine and ERG240, which indicates that they may be the key active sites in the structure of BCAT1. In addition, ERG240 forms hydrogen bonds with more amino acid residues of BCAT1, indicating that ERG240 can occupy the active site of BCAT1 more firmly, thereby preventing the binding of leucine.

[0077] Example 2 demonstrates that two therapeutic doses of ERG240 (5 mg / kg / d and 10 mg / kg / d) have no toxic side effects on mice.

[0078] Mice were randomly divided into Sham group, UUO group, UUO+ERG240 (5 mg / kg / d) group and UUO+ERG240 (10 mg / kg / d) group and intraperitoneally injected. Pre-administration was performed once 24h and 2h before UUO surgery, and then once a day at a fixed time, with a volume of 0.1 mL per 10 g body weight. The sham group and the UUO group were injected with the same volume of the above-mentioned solvent. The mice were euthanized 7 days after modeling, and the whole blood samples of the mice were collected in centrifuge tubes and centrifuged at 4000rpm for 25min at room temperature. Serum samples were taken to measure the levels of renal function markers blood urea nitrogen (BUN) and serum creatinine (sCr), liver function markers alanine aminotransferase (ALT) and aspartate aminotransferase (AST), and cardiac function markers lactate dehydrogenase (LDH) and creatine kinase-muscle / brain (CK-MB).

[0079] Experimental Results

[0080] The renal function marker BUN ( Figure 2 A) and sCr( Figure 2 B) Liver function marker ALT ( Figure 2 C) and AST( Figure 2 D) LDH, a marker of cardiac function Figure 2 E) and CK-MB( Figure 2 F) levels showed no significant difference, indicating that the above doses had no toxic side effects on the kidneys, liver and heart of mice.

[0081] Example 3 demonstrates that two therapeutic doses of ERG240 (5 mg / kg / d and 10 mg / kg / d) can alleviate renal pathological changes in UUO mice.

[0082] The modeling and administration methods of the mice in the sham group, UUO group, UUO+ERG240 (5 mg / kg / d) group and UUO+ERG240 (10 mg / kg / d) group were the same as those in Example 2. The mice were killed after the last administration, and the renal tissues were fixed, embedded, sectioned, and stained with Masson and Sirius red.

[0083] Experimental Results

[0084] Masson staining is a classic histological staining method used to show the structure of cells and extracellular matrix (such as collagen fibers) in tissues. Under 200x and 400x optical microscopes, compared with the Sham group, extensive collagen deposition was observed in the renal tubular interstitium of UUO mice, accompanied by atrophy, dilation, necrosis and glomerular sclerosis of the renal tubules. However, two different doses of ERG240 (5mg / kg / d and 10mg / kg / d) can dose-dependently reduce the area of ​​renal tissue fibrosis in UUO mice, and the renal interstitial collagen deposition was reduced by 31.3% (p<0.05) and 51.5% (p<0.001), respectively, and the damaged renal structure was significantly restored ( Figure 3 AB).

[0085] Sirius red staining is mainly used to identify and quantify collagen fibers in connective tissue. Sirius red is a red azo dye that can bind to specific structures in collagen fibers, making the collagen fibers appear bright red under a microscope. Observation under an optical microscope at 200 times and 400 times showed abnormal collagen deposition in the tubular interstitium of UUO mice compared with the Sham group, with obvious dilation of the renal tubules and the formation of tubular urine. However, two different doses of ERG240 (5 mg / kg / d and 10 mg / kg / d) also dose-dependently reduced the area of ​​renal tissue fibrosis in UUO mice, reducing it by 24.07% and 39.2%, respectively ( Figure 3 CD).

[0086] Example 4 demonstrates that two therapeutic doses of ERG240 (5 mg / kg / d and 10 mg / kg / d) can significantly reduce the protein expression levels of fibrosis-related molecules in UUO kidney tissue.

[0087] The modeling and administration methods of the mice in the sham group, UUO group, UUO+ERG240 (5 mg / kg / d) group and UUO+ERG240 (10 mg / kg / d) group were the same as those in Example 2. The mice were killed after the last administration, and about 20 mg of mouse kidney tissue was taken. RIPA lysis buffer containing protease inhibitors and phosphatase inhibitors was added in proportion to extract total tissue protein. Western blot was used to detect the protein expression levels of fibrosis molecules FN, Vimentin, and α-SMA.

[0088] Experimental Results

[0089] Compared with the Sham group, the protein expressions of fibrosis-related molecules FN, Vimentin, and α-SMA in the renal tissues of mice injured by UUO were significantly increased, and the differences were statistically significant; and two different doses of ERG240 (5 mg / kg / d and 10 mg / kg / d) could dose-dependently reduce the high expression of fibrosis indicators. Except for the FN expression level in the UUO+ERG240 (5 mg / kg / d) group, the differences in the grayscale values ​​of the remaining proteins were statistically significant ( Figure 4 AD), indicating that ERG240 has a significant anti-fibrotic effect.

[0090] Example 5 demonstrates that two therapeutic doses of ERG240 (5 mg / kg / d and 10 mg / kg / d) can significantly reduce the mRNA expression levels of fibrosis-related molecules in UUO kidney tissue.

[0091] The modeling and administration methods of mice in the sham group, UUO group, UUO+ERG240 (5 mg / kg / d) group and UUO+ERG240 (10 mg / kg / d) group were the same as those in Example 2. The mice were killed after the last administration, and the kidney tissues of the mice were taken. 1 mL of RNAiso Plus was added and fully lysed with a grinder. The mRNA expression levels of fibrosis-related molecules (Vim, Fn, Acta2, Postn, Col1a1, Col3a1) were detected by qT-PCR.

[0092] Experimental Results

[0093] Compared with the Sham group, the mRNA expressions of Vim, Fn, Acta2, Postn, Col1a1, and Col3a1 in the renal tissues of mice after UUO injury were significantly increased, and the difference was statistically significant; and two different doses of ERG240 (5 mg / kg / d and 10 mg / kg / d) could dose-dependently reduce the high expression of the above fibrosis indicators, and the difference was statistically significant ( Figure 5 AF), indicating that ERG240 can inhibit the secretion and production of fibrotic extracellular matrix.

[0094] Example 6 demonstrates that different concentrations of ERG240 do not affect the viability of mouse renal tubular epithelial cells (TKPT).

[0095] TKPT cells with good growth status were evenly inoculated in a 96-well plate. After 24 hours, ERG240 with a final concentration of 50, 100, 150, 200, 250, and 300 μM was added to the serum-free medium, and the same volume of solvent was added to the control group. After 24 hours, the culture medium containing 10% CCK-8 reagent was added to the cell culture, incubated in a 37°C incubator for 2 hours, and the absorbance of each well was measured at a wavelength of 450 nm using an enzyme reader.

[0096] Experimental Results

[0097] Compared with the Vehicle group, different concentrations of ERG240 (50, 100, 150, 200, 250, 300 μM) had no significant effect on the cell viability of TKPT, indicating that ERG240 has a large safety range and these concentrations do not cause cytotoxicity ( Figure 6 ).

[0098] Example 7 demonstrates that different concentrations of ERG240 (100, 200, 300 μM) can dose-dependently reduce the mRNA expression level of the extracellular matrix.

[0099] In order to observe the effect of different concentrations of ERG240 on the secretion and production of extracellular matrix of renal tubular epithelial cells, TGF-β1 was used to stimulate TKPT in vitro in this study to simulate the response of renal tubular epithelial cells in the process of fibrosis. TKPT cells with good growth status were evenly seeded in 12-well plates. After 24 hours, ERG240 with a final concentration of 100, 200 and 300 μM was added to serum-free medium for pretreatment for 2 hours, and then TGF-β1 with a final concentration of 10 ng / mL was added. After 24 hours, the cells were collected to extract total RNA, and the mRNA expression of Fn and Vim was detected by qT-PCR.

[0100] Experimental Results

[0101] Under the stimulation of TGF-β1, the mRNA expression levels of extracellular matrix-related factors (Fn and Vim) secreted by TKPT cells were significantly increased, and ERG240 could reverse the high expression of Fn and Vim in a dose-dependent manner. Among them, 300μM ERG240 showed a significant effect in reversing the high expression of Fn and Vim induced by TGF-β1, which was statistically significant and showed good anti-fibrosis potential ( Figure 7 AB).

[0102] Example 8 demonstrates that therapeutic concentration of ERG240 (300 μM) can significantly downregulate the high expression of fibronectin level in TKPT cells.

[0103] In order to observe the effect of ERG240 (300 μM) with anti-fibrotic potential on the production and deposition of extracellular matrix of renal tubular epithelial cells, TGF-β1 was used to stimulate TKPT in this study. TKPT cells with good growth status were evenly seeded in 6-well plates. After 24 hours, ERG240 with a final concentration of 300 μM was added to serum-free medium for pretreatment for 2 hours, and then TGF-β1 with a final concentration of 10 ng / mL was added for co-stimulation for 24 hours. The cells were collected to extract total protein, and the protein expression of FN was detected by Western blot.

[0104] Experimental Results

[0105] Under the stimulation of TGF-β1, the deposition of FN in TKPT cells increased significantly, while 300 μM ERG240 could effectively reduce the high expression of FN and inhibit TGF-β1-induced fibrin deposition, showing its potential application value in anti-fibrosis therapy ( Figure 8 AB).

[0106] Finally, it should be noted that the above is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, it is still possible for those skilled in the art to modify the technical solutions described in the aforementioned embodiments or to make equivalent substitutions for some of the technical features therein. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. Use of a BCAT1 inhibitor in the preparation of a drug for treating fibrotic diseases.

2. The use according to claim 1, characterized in that: The BCAT1 inhibitor is a leucine analog.

3. The use according to claim 2, characterized in that: The leucine analog is ERG240.

4. The use according to claim 3, characterized in that: The pharmaceutical dosage of ERG240 is 5-10 mg / kg / d.

5. The use according to claim 4, characterized in that: The application can reduce the high expression of fibrosis-related markers in mice with renal fibrosis.

6. The use according to claim 3, characterized in that: The pharmaceutical concentration of ERG240 is 100-300 μM.

7. The use according to claim 6, characterized in that: The application can inhibit the excessive deposition of fibronectin (FN) in renal tubular epithelial cells.

8. The use according to any one of claims 1 to 7, characterized in that: The fibrotic disease is renal fibrosis.

9. The use according to claim 8, characterized in that: The fibrotic disease is renal fibrosis caused by unilateral ureteral obstruction.

10. The use according to claim 9, characterized in that: The dosage form of the medicine for treating fibrotic diseases is injection.

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