Polypeptide and application thereof in preparation of medicine for treating obesity and NAFLD

Abstract

The invention belongs to the field of biological medicine. The technical problem to be solved by the invention is to provide a new choice for treatment of obesity and non-alcoholic fatty liver diseases. According to the technical scheme for solving the technical problem, the novel polypeptide is provided, and the polypeptide contains an ERK2 substrate binding pocket structural domain and has the function of blocking combination of Arg1 and ERK2. The polypeptide disclosed by the invention has the characteristics of simplicity in preparation and obvious treatment effect, and has a very good application prospect.

Description

Technical Field

[0001] The present invention belongs to the field of biological medicines, and in particular relates to a polypeptide and use thereof in preparing medicines for treating obesity and NAFLD. Background Art

[0002] Obesity is a complex disease caused by excessive accumulation or abnormal distribution of body fat due to dysregulation of nutritional metabolism, and is associated with a variety of chronic diseases. About 1.5 billion adults worldwide are overweight due to obesity. The main manifestation of obesity in the liver is non-alcoholic fatty liver disease (NAFLD), which can develop into non-alcoholic fatty liver hepatitis or even primary liver cancer in severe cases.

[0003] NAFLD is characterized by increased liver fat accumulation. However, the pathogenesis of obesity and NAFLD has not been clearly elucidated so far, and there is a lack of effective treatment. Therefore, understanding the molecular mechanism of liver lipid metabolism will provide a valuable research basis for the diagnosis and treatment of obesity and NAFLD.

[0004] It has been reported that limiting specific amino acids may be a potential future treatment for obesity. Amino acids participate in related signaling pathways by interacting with targets of the mTOR complex. When mTORC1 is located at the lysosomal level, it mediates the efflux of essential amino acids such as leucine, isoleucine, methionine, and tryptophan through arginine activation of SLC38A9 (a transmembrane lysosomal transporter). Arginine, as a conditionally essential amino acid in animals, is believed to change the balance between energy intake and expenditure. Arginine demonstrated its anti-adipogenic properties by reducing adipocyte formation and triglyceride (TG) content in bone marrow mesenchymal stem cells. In addition, in young Wistar rats, only arginine deficiency led to significant accumulation of hepatic TG.

[0005] There are two main pathways for the catabolism of arginine, the most important of which is the urea cycle pathway. UC in the liver is the main metabolic pathway that converts excess nitrogen into disposable urea. Arginase is the last enzyme in the UC pathway that converts arginine into ornithine and urea. There are two isoforms of arginase: arginase 1 (Arg1) is localized in the cytoplasm and is mainly expressed in the liver, while arginase 2 is localized in the mitochondria and expressed in extrahepatic tissues. Arg1 is expressed in liver cells other than central veins (about 93%). Congenital deletion of this gene leads to Arg1 deficiency (hyperargininemia), and patients show progressive loss of motor function, spastic quadriplegia, deep tendon hyperreflexia, epilepsy and growth retardation, and may be accompanied by severe liver disease. In a previous study involving healthy adults, in addition to arginase activity, Arg1 levels were positively correlated with body mass index and negatively correlated with L-arginine levels. It was reported that when early obese mice were fed a high fat diet (HFD) for 8 weeks, it was found that although the Arg-1 mRNA level in adipose tissue, aorta or muscle did not change, the Arg-1 mRNA level in the liver increased significantly. In addition, it has also been reported that in HFD-induced obese mice, Arg1 in vascular endothelial cells damages endothelial function through the arginine / nitric oxide (NO) pathway. Summary of the invention

[0006] The technical problem to be solved by the present invention is to provide a new option for the treatment and prevention of obesity and NAFLD.

[0007] The first aspect of the present invention provides a polypeptide, which contains an ERK2 (extracellular signal-regulated protein kinase 2) substrate binding pocket domain and has the function of blocking the binding of Arg1 to ERK2.

[0008] Wherein, the amino acid sequence of the ERK2 substrate binding pocket domain is ATRW (ERK2187-190).

[0009] The above-mentioned blocking of the binding of Arg1 to ERK2 refers to mainly binding of the ATRW peptide segment of the polypeptide to Arg1, thereby blocking the binding of Arg1 to ERK2.

[0010] Furthermore, the above polypeptide has 0-20 amino acids connected to the nitrogen terminus and / or carbon terminus of the ATRW domain. Preferably, the above polypeptide has 6-20 amino acids connected to the nitrogen terminus and / or carbon terminus of the ATRW domain.

[0011] The above polypeptide may be a truncated form of the ERK2 protein containing ATRW, and the amino acid sequence of the ERK2 of the ERK2 protein is shown in SEQ ID No.4.

[0012] Alternatively, it is a polypeptide having the same or similar function obtained by replacing and / or deleting and / or adding one or more amino acids in the amino acid sequence of the truncation; the same or similar function is the function of binding to Arg1 to block the binding of Arg1 to ERK2.

[0013] Furthermore, the amino acid sequence of the above-mentioned truncation is RVADPDHDHTGFLTEYVATRW or RVADPDHDHTGFLTEYVATRWYRAPEIMLNS.

[0014] Furthermore, the polypeptide can also form a conjugate with a coupling moiety, wherein the coupling moiety is one or more selected from radionuclides, drugs, toxins, cytokines, enzymes, fluorescein, carrier proteins or biotin.

[0015] The present invention further provides derivatives or chemically modified products of the above polypeptide or fusion peptide.

[0016] The present invention further provides the use of the above polypeptide, the above fusion peptide, the above derivative or chemically modified product in preparing a product for treating, preventing or improving obesity or fatty liver disease.

[0017] The present invention also provides products prepared with the above polypeptide, the above fusion peptide, the above derivative or chemically modified product as the main active ingredient. The product can be a medicine or a health product.

[0018] Furthermore, the dosage form of the above-mentioned product is injection, oral preparation, transdermal preparation or mucosal preparation.

[0019] The beneficial effect of the present invention is that the present invention finds that Arg1 can promote lipid accumulation in hepatocytes and adipocytes and Arg1 mainly exerts its effect by binding to ERK2. Further, the use of Arg1 inhibitors in the preparation of products for preventing, improving or treating obesity or fatty liver disease is provided. At the same time, the polypeptide of the present invention has a clear therapeutic effect and better safety, and is also easier to prepare and obtain. Experiments have shown that the polypeptide of the present invention can effectively play a therapeutic role in the mouse obesity and non-alcoholic fatty liver disease model induced by a high-fat diet, and has a good application prospect. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 , BEC (Arg1 inhibitor) can alleviate liver steatosis and reduce fat accumulation in adipose tissue. A: Body shape of mice after BEC intervention; B: Distribution of adipose tissue in mice; C: Appearance of mouse liver; D: HE image of mouse liver tissue; E, F: Weight of mouse adipose tissue and size of fat cells; G: CT image of abdominal fat in mice.

[0021] Figure 2 , Co-IP, Duolink PLA, and Flag pull-down experiments confirmed that Arg1 can bind to ERK2. A: Co-IP results of wild-type mouse liver tissue; B: Duolink PLA experimental results, red spots indicate binding; C: Flag pull-down results after exogenous ERK2 was expressed in aml12 cells.

[0022] Figure 3 , synthesized peptides to determine the specific binding fragment of ERK2 that binds to Arg1. A: His pull-down experiment after synthesizing multiple peptides of ERK2; B: GST pull-down experiment

[0023] Figure 4 , biosynthesized peptides can alleviate HFD-induced obesity and MAFLD. A: Schematic diagram of the treatment process; B, C: the whole body, adipose tissue, liver appearance and HE staining of mice; D, E: the size of mouse adipocytes and CT results.

[0024] Figure 5 , HPLC results of the polypeptide product obtained after purification after expression. DETAILED DESCRIPTION

[0025] Since obesity and NAFLD are closely related to the body's fat metabolism and other metabolic systems, their pathogenesis has not yet been clearly elucidated, and there is a lack of effective treatment methods. The inventors have conducted a lot of research to find new methods to treat obesity and NAFLD, and speculated that Arg1 may have a certain correlation with obesity, but it is not clear whether Arg1 is involved in liver fat metabolism.

[0026] In further studies, the use of Arg1 activity inhibitor (BEC) to treat db / db mice slowed down the weight gain of the mice, reduced liver steatosis, inflammation and lipid droplet accumulation in db / db mice, reduced liver weight and LW / BW, SAT and VAT tissue weight and cell size. Therefore, it was proved that Arg1 does play an important role in promoting liver fat synthesis and fat deposition, independent of the accumulation of metabolites.

[0027] Therefore, the inventors believe that a new technical solution has been established, namely, the use of Arg1 inhibitors in the preparation of drugs for treating obesity or fatty liver disease. After the inventors thought whether the use of small molecule Arg1 activity inhibitors could effectively treat and alleviate obesity and NAFLD, they found in subsequent experiments that long-term feeding of BECs would cause the death of mice. This prompted the inventors to explore the corresponding reasons and find a safer and more effective treatment plan.

[0028] After further studying the mechanism of action of the Arg1 activity inhibitor, the present invention found that Arg1 can bind to the kinase ERK2 in the MAPK signaling pathway, thereby activating ERK2 to promote liver fat synthesis and fat deposition. It was further found that the binding site is located in the substrate binding pocket of ERK2, and ATRW (ERK2 187-190) is the key site for recognition and binding with Arg1.

[0029] On this basis, the present invention provides a technical solution of the first aspect, namely, a polypeptide containing the ATRW domain of ERK2 187-190 and having the function of the ERK2 substrate binding pocket, recognizing and binding to Arg1, thereby inhibiting or blocking the binding of Arg1 to ERK2.

[0030] Based on the above description, a person skilled in the art can reasonably anticipate that in order to make the above polypeptide have the function of the ERK2 substrate binding pocket, recognize and bind to Arg1, a peptide segment can be connected to the nitrogen end and / or carbon end of the ATRW domain, so that the entire polypeptide maintains a suitable spatial structure and ensures smooth binding of the ATRW domain and Arg1. At the same time, the extended peptide segment can also increase its stability in vivo.

[0031] Furthermore, 0-30, no more than 20, or 6-20 amino acids may be connected to the nitrogen end and / or carbon end of the ATRW domain, so that the entire polypeptide maintains a suitable spatial structure to ensure smooth binding of the ATRW domain to Arg1.

[0032] Naturally, in order to better ensure that the above polypeptide can better recognize and bind to Arg1 through the ATRW domain. The above polypeptide is preferably a truncated form of the ERK2 protein containing the ATRW domain. Or it is a polypeptide with the same or similar function obtained by replacing and / or deleting and / or adding one or more amino acids in the amino acid sequence of the above truncation; the same or similar function means that it has the function of blocking the binding of Arg1 to ERK2 by binding to Arg1.

[0033] In the example provided by the present invention, two truncated forms of ERK2 protein with amino acid sequences of RVADPDHDHTGFLTEYVATRW (ERK2170-190) and RVADPDHDHTGFLTEYVATRWYRAPEIMLNS (ERK2170-200) were prepared by genetic engineering methods. Experiments have shown that the truncated form of ERK2 containing the ATRW domain can bind to ERK2 and has the effect of improving obesity and NAFLD in mice.

[0034] It can be seen that the polypeptide provided by the present invention can be used to prepare products for treating, preventing or improving obesity or fatty liver disease. In particular, it can be used for treating, preventing or improving non-alcoholic fatty liver disease. It has a good application prospect.

[0035] The present invention is further described below by way of specific examples, but the following description should not be taken as a further limitation of the present invention.

[0036] Experimental materials and main experimental methods used in the examples

[0037] Experimental mouse strains

[0038] Eight-week-old wild male mice (C57BL / 6) were purchased from Sichuan Dashuo Co., Ltd. All mice were male and housed under specific pathogen-free conditions. Db / db mice were purchased from Chengdu Yaokang Co., Ltd. A high-fat diet (HFD) was used to establish the animal model of obesity and obesity-induced NAFLD. Six- to eight-week-old male mice were fed an HFD for 12 weeks.

[0039] The dose of BEC was calculated based on previous studies that reduced arginase activity by 50% in cells isolated from treated animals. 0.012 g of BEC was administered daily by gavage in 100 μl of water to match the dose equivalent in studies exposing mice to BEC in drinking water.

[0040] Cell culture

[0041] The aml12 mouse hepatocyte cell line was maintained in a 1:1 mixture of Dulbecco's modified Eagle's medium and Ham's F12 medium supplemented with 10% fetal bovine serum, 5 μg / mL insulin, 5 μg / mL transferrin, 5 ng / mL selenium, and 40 ng / mL dexamethasone. The cells were maintained at 37°C in a 5% (v / v) CO2 atmosphere and passaged every 3 days.

[0042] Proximity ligation assay

[0043] Aml12 cells were fixed with 3.7% paraformaldehyde and blocked with blocking solution provided by Duolink PLA kit (Millipore Sigma, USA) according to the manufacturer's instructions. In brief, fixed cells were incubated overnight with anti-KRT17 antibody and anti-Plectin antibody. After multiple washes, cells were incubated with PLA probe, ligation solution and amplification solution at 37°C in sequence. Coverslips were mounted and images were examined using a Leica TCS SP5 confocal microscope (Leica Microsystems, Mannheim, Germany).

[0044] microCT measurement of fat mass

[0045] After anesthesia with air-propelled isoflurane (1.5-2%), mice were placed supine in the imaging cell, where animals were anesthetized, warmed, and monitored. Three-dimensional X-ray images were acquired on the CT portion of the Quantum GX microCT imaging system. The total acquisition time was 15 minutes. The tube voltage was set to 80 kV and the current was 32 mA.

[0046] Histology and immunohistochemistry

[0047] Liver specimens were fixed in 10% neutral buffered formalin for 36–48 h. Paraffin sections (4 μm) were prepared and subjected to a series of dewaxing, rehydration, antigen retrieval, and quenching of endogenous peroxidase activity. Sections were incubated with corresponding primary antibodies overnight at 4 °C and with anti-mouse / rabbit secondary antibodies (Dako REAL EnVision Detection System, Glostrup, Denmark) for 1 h at room temperature. Detection was performed using 3,3′-diaminobenzidine substrate. Details of primary antibodies are provided in SI Appendix. H&E and PAS staining were performed by standard methods.

[0048] Detect fat weight

[0049] At the time of sampling, mice were anesthetized and sacrificed with a lethal dose of pentobarbital (120 mg / kg, intraperitoneal). Adipose tissue (visceral and subcutaneous) was collected and weighed on a balance.

[0050] Peptide drug synthesis

[0051] The drug peptide is obtained by biological means. The peptide purity is greater than 85%. Preparation steps: (1) strain preparation: gene synthesis, construction into pGEX4T-1 vector; (2) expression optimization test; (3) large-scale expression purification.

[0052] Experimental Results

[0053] Example 1. Results of the validation experiment that Arg1 promotes lipid accumulation in hepatocytes and adipocytes

[0054] The db / db mice (BEC+db / db) were treated with an inhibitor of Arg1 activity (S-(2-bromoethyl)-L-cysteine, BEC) ( Figure 1). After 8 weeks, BEC+db / db mice had a slow weight gain compared to db / db mice. There was no significant difference in liver function between the two groups. BEC alleviated hepatic steatosis, inflammation, and lipid droplet accumulation in db / db mice, and reduced liver weight and LW / BW, SAT and VAT tissue weights, and cell size. Therefore, Arg1 does play an important role in promoting hepatic lipogenesis and fat deposition, independent of the accumulation of metabolites.

[0055] Example 2. Results of the validation experiment of Arg1 binding to a specific site of ERK2

[0056] To understand how Arg1 directly regulates hepatic lipogenesis, potential proteins interacting with Arg1 in the liver were detected by co-immunoprecipitation (Co-IP) and liquid chromatography-tandem mass spectrometry (LC-MS / MS) analysis. Pathway enrichment analysis of these proteins revealed the most affected signaling pathways.

[0057] Among them, ERK2 (extracellular signal-regulated protein kinase 2) in the MAPK signaling pathway was found to be possibly associated with Arg1. It is reported that ERK2 can affect fat metabolism by reducing liver endoplasmic reticulum stress and inhibiting oxidative stress. Activating ERK2 can improve fatty degeneration in the liver of leptin receptor-deficient mice, while liver-specific ERK2 gene knockout mice showed more severe fatty degeneration when fed with a high-fat / high-sugar diet. ERK2 plays an important role in liver fat metabolism.

[0058] In order to determine whether the effect of Arg1 on liver fat metabolism is related to ERK2, the present invention extracted WT mouse liver tissue protein for Co-IP to determine that ERK2 is a protein that interacts with Arg1 ( Figure 2 A). Next, the relationship between ERK2 and Arg1 was verified by Duolink in situ proximity ligation assay (PLA) and Flag pull-down experiments in aml12 cells. As a negative control, almost no background staining was observed, while Arg1 and ERK2 antibodies detected obvious red spots in aml12 cells ( Figure 2 B) Flag-tagged ERK2 was co-immunoprecipitated by the Arg1 antibody, and endogenous Arg 1 could also be pulled down by the Flag antibody ( Figure 2 C) These results all prove that Arg1 can bind to ERK2.

[0059] In addition, in order to further understand the binding force between the specific peptide segment of ERK2 and Arg1, the present invention constructed GST-peptide and His-Arg1. Figure 3The Co-IP results of A and B represent the binding ability of purified Arg1 to multiple peptides of ERK2. The results suggest that Arg1 can specifically bind to the 170-190 sequence (VADPDHDHTGFLTEYVATRW) containing the ERK2 substrate binding pocket ( Figure 3 ). The experimental results showed that Arg1 can bind to ERK2, and the binding site is located in the substrate binding pocket of ERK2.

[0060] Example 3. Results of the synthesis of peptides mimicking the ERK2 substrate binding pocket

[0061] The polypeptide is prepared by biological expression. The amino acid sequence of the polypeptide to be prepared is: ERK2170-200: RVADPDHDHTGFLTEYVATRWYRAPEIMNLS.

[0062] Specific preparation method: (1) strain preparation: synthesize the coding gene of the desired polypeptide (CGTGTTGCAGATCCAGATCATGATCACACAGGGTTCTTGACAGAGTACGT AGCCACACGTTGGTACAGAGCTCCAGAAATTATGTTGAATTCC, SEQ ID No. 5); construct it into the expression vector pGEX4T-1, and transfect it into Escherichia coli; (2) perform expression optimization test and verify the obtained product; (3) express and purify in large quantities to obtain the target polypeptide.

[0063] Results: The purified product was verified by HPLC (see Figure 5 ), indicating that the target polypeptide with a purity of more than 85% was obtained, and the rest was mainly residual polypeptide, which had no interference with the experiment.

[0064] Example 4. Results of efficacy experiments on peptides improving obesity and NAFLDDE1 in mice by targeting Arg1

[0065] Given that ERK2 involves many signaling pathways, intervention of ERK2 has a greater impact on other related signaling pathways, and Arg1 is used as a target to treat obesity and NAFLD. An attempt was made to feed wild-type mice with a small molecule inhibitor of Arg1 (BEC), but three mice died within 12 weeks. Long-term use of BEC still has obvious side effects and is not suitable for use as a drug.

[0066] Next, the potential therapeutic value of inhibiting the specific binding of Arg1 to ERK2 in obesity and NAFLD was studied. Based on the binding of the ERK2 substrate binding pocket to Arg1, a peptide was obtained by biosynthesis using the method of Example 3. Compared with chemical drugs, the peptide is more effective, safer, and more tolerable. It also has the advantages of high selectivity and less accumulation in the body. The amino acid sequence of the synthesized peptide is: ERK2170-200:RVADPDHDHTGFLTEYVATRWYRAPEIMLNS. .

[0067] After 8 weeks of HFD induction, WT mice were treated with peptide drugs. 50 μg / kg of drug peptide was injected into the tail vein of mice every day for 4 weeks ( Figure 4 A). After peptide treatment, obese mice recovered liver function and lost weight. At the same time, liver macroscopic and liver section HE showed that HFD-induced mouse livers showed vacuolar degeneration and fat deposition. The vacuolar degeneration and fat deposition in the liver of mice treated with drug peptides were significantly reduced. After drug peptide treatment, the lipid accumulation in the liver of mice was reduced. Compared with control mice, the peptide reduced the fat weight in SAT (average from 0.6 to 0.2) and VAT (average from 1.8g to 0.63). The area of ​​SAT and VAT adipocytes decreased, nearly 2 times and 1 times, respectively, and the proportion of adipose tissue in SAT (14%-9%) and VAT (24%-14%) was also significantly reduced ( Figure 4 ), which indicates that the peptide reduces HFD-induced liver fat synthesis in mice. Therefore, it can be determined that the peptide can inhibit liver lipogenesis by targeting Arg1 and alleviate obesity and NAFLD.

Claims

1. A polypeptide, characterized in that: A polypeptide containing an ERK2 substrate binding pocket domain; and having the function of blocking the binding of Arg1 to ERK2.

2. The polypeptide according to claim 1, characterized in that The amino acid sequence of the ERK2 substrate binding pocket domain is ATRW (SEQ ID No. 1).

3. The polypeptide according to claim 1, characterized in that The blocking of the binding of Arg1 to ERK2 refers to mainly binding of the ATRW peptide segment of the polypeptide to Arg1, thereby blocking the binding of Arg1 to ERK2.

4. The polypeptide according to claim 4, characterized in that There are 0-20 amino acids connected to the nitrogen-terminus and / or carbon-terminus of the ATRW domain.

5. The polypeptide according to claim 8, characterized in that The polypeptide is a truncated form of the ERK2 protein containing ATRW, and the base sequence and corresponding amino acid sequence of the ERK2 protein are (SEQ ID No. 1); Or it is a polypeptide with the same or similar function obtained by replacing and / or deleting and / or adding one or several amino acids in the amino acid sequence of the above truncation; the same or similar function is the function of binding to Arg1 to block the binding of Arg1 to ERK2.

6. The polypeptide according to claim 7, characterized in that The amino acid sequence of the truncation is: RVADPDHDHTGFLTEYVATRW(SEQ ID No.2); Or: RVADPDHDHTGFLTEYVATRWYRAPEIMLNS (SEQ ID No. 3).

7. A derivative or chemically modified product of the polypeptide according to any one of claims 1 to 6.

8. Use of the polypeptide according to any one of claims 1 to 6, the derivative or chemically modified product according to claim 7 in the preparation of a product for treating, preventing or improving obesity or fatty liver disease; further, the fatty liver disease is non-alcoholic fatty liver disease.

9. Products for treating, preventing or improving obesity or fatty liver disease, characterized in that: Contains the polypeptide according to any one of claims 1 to 6, or the derivative or chemically modified product according to claim 7 as a main active ingredient.

10. The product according to claim 22, characterized in that: The product is a medicine or a health product; further, the fatty liver disease is non-alcoholic fatty liver disease; further, the dosage form of the medicine or health product is an injection, an oral preparation, a transdermal preparation or a mucosal preparation.

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