Soybean engineered polypeptides for the prevention and treatment of cardiovascular disease

Abstract

This invention belongs to the field of biomedical technology and discloses a soybean-modified peptide for the prevention and treatment of cardiovascular diseases. Addressing the problem that the original peptide VAWWMY is easily degraded by enzymes in the gastrointestinal tract and has difficulty achieving a significant lipid-lowering effect in vivo, this invention modifies the amino acid sequence of the original peptide to SAIWMY. The modified peptide SAIWMY has higher cholesterol-lowering activity than the original peptide, is less susceptible to degradation by gastrointestinal enzymes and is more stable, has higher safety than cholestyramine, and is more effective than cholestyramine in lowering cholesterol and triglycerides in the liver, exhibiting better lipid-lowering potential. It can be used to prepare drugs for the prevention or treatment of cardiovascular diseases (such as hyperlipidemia, atherosclerosis, and coronary heart disease), fatty liver disease, and diabetes, and can also be used to prevent or treat obesity.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a novel lipid-lowering polypeptide and its application in the preparation of drugs for the treatment or prevention of obesity and cardiovascular diseases. Background Technology

[0002] Cardiovascular diseases, including hyperlipidemia, hypertension, atherosclerosis, coronary heart disease, and stroke, are among the leading causes of death and disability worldwide. According to the World Health Organization (WHO), cardiovascular diseases cause approximately 18 million deaths annually, accounting for 31% of all deaths globally. With an aging population and changes in unhealthy diets and lifestyles, the incidence of cardiovascular diseases continues to rise, placing a significant burden on healthcare systems and the socioeconomic system. Cholesterol homeostasis is crucial for maintaining good health; the body needs it to participate in cell formation, vitamin production, and the generation of other hormones. However, excessive cholesterol can lead to cardiovascular disease. Bile acids are a class of structurally similar molecules, composed of a hydrophobic steroid moiety and a polar functional group alkyl side chain (forming the flexible hydrophilic portion of the bile acid structure). In mammals, bile acid synthesis is the primary pathway (95%) for cholesterol catabolism. The human liver synthesizes 200-600 mg of bile acids daily, and the same level is excreted in feces to maintain adequate bile acid levels in the body. Bile acid synthesis occurs via two pathways: a neutral pathway and an acidic pathway, forming primary bile acids such as cholic acid (CA) and chenodeoxycholic acid (CDCA). In the classical bile acid metabolism pathway, primary bile acids account for approximately 75% of all bile acids, with CA and CDCA at comparable levels. Before bile acids are transported in the tubules, CA and CDCA undergo N-acylation modification with taurine and glycine to form bile salts, such as taurocholic acid (TCA) and glycocholic acid (GCA). Most primary bile acids are reabsorbed in the terminal ileum, while some unabsorbed ones enter the colon, where they are converted into secondary bile acids by the gut microbiota. CA forms deoxycholic acid (DCA), and CDCA forms lithocholic acid (LCA). The main function of bile acid micelles is to promote the dissolution and absorption of lipids in the intestine. Bile acids, as surfactants, can encapsulate hydrophobic molecules, making them stable in an aqueous environment and allowing them to be absorbed into the bloodstream through the intestinal mucosa. In lipid metabolism, the formation of bile acid micelles is a key step in lipid absorption. If bile acid micelles are disrupted, lipid absorption efficiency decreases, thereby reducing blood lipid levels. On the other hand, bile acids can improve insulin sensitivity and alleviate the development of type 2 diabetes by activating Takeda G protein-coupled receptor 5 (TGR5). Therefore, regulating the solubility of bile acid micelles is an important strategy for developing cardiovascular disease drugs or functional foods.

[0003] Bile acid sequestrants are positively charged, non-absorbable macromolecules that bind to negatively charged bile acids in the intestine. A dose of approximately 16-32g of bile acid sequestrant can remove some bile acids from the enterohepatic circulation, thereby consuming about 40% of the body's bile acids. This promotes the conversion of cholesterol into bile acids and increases the excretion of fecal sterols. For example, the first-line drug cholestyramine intervenes in cholesterol metabolism and activates TGR5 through this mechanism, reducing the risk of diseases such as atherosclerosis, coronary heart disease, and type 2 diabetes. However, bile acid sequestrants can cause varying degrees of side effects, such as constipation and flatulence, leading to low patient adherence.

[0004] The VAWWMY (V-valine, A-alanine, W-tryptophan, W-tryptophan, M-methionine, Y-tyrosine) peptide in soybean globulin possesses cholesterol-lowering bioactivity. VAWWMY peptides can significantly reduce bile acid micelle solubility in vitro, thereby inhibiting cholesterol absorption. Furthermore, the in vitro binding capacity of VAWWMY peptides to bile acids is comparable to that of the first-line lipid-lowering drug cholestyramine. However, when VAWWMY peptides are taken orally, their structure is easily affected by trypsin degradation in the gastrointestinal tract. The enzymatically hydrolyzed VAW and WMY peptides significantly reduce their ability to disrupt bile acid micelles, making it difficult for them to exert a significant lipid-lowering effect in vivo. Therefore, this invention modifies the amino acid sequence of VAWWMY peptides to improve their stability and cholesterol-lowering activity in the gastrointestinal tract, making them better suited for the development of drugs for the prevention and treatment of cardiovascular diseases. Summary of the Invention

[0005] This invention designs and synthesizes a polypeptide SAIWMY (S-serine, A-alanine, I-isoleucine, W-tryptophan, M-methionine, Y-tyrosine). This polypeptide has good bile acid micelle disruption ability and is not easily degraded by intestinal trypsin. In addition, it has a significant in vivo lipid-lowering effect, providing a new approach for the treatment of cardiovascular diseases.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] A soybean-modified polypeptide with the amino acid sequence SAIWMY.

[0008] The modified peptide SAIWMY has the following applications:

[0009] (1) Preparation of cholesterol-lowering products;

[0010] (2) Preparation of triglyceride-lowering products;

[0011] (3) Prepare drugs for the prevention or treatment of hyperlipidemia, atherosclerosis or coronary heart disease;

[0012] (4) To prepare drugs for the prevention or treatment of fatty liver disease;

[0013] (5) Prepare products for the prevention or treatment of obesity;

[0014] Preferably, the soybean-modified polypeptide is an oral preparation.

[0015] Compared with the prior art, the present invention has the following beneficial effects:

[0016] At the molecular level, the modified peptide SAIWMY exhibits stronger bile acid micelle disruption ability than the original peptide VAWWMY at different concentrations and is not degraded by intestinal enzymes. Figure 3 At the cellular level, the modified peptide SAIWMY exhibits lower toxicity than the currently used positive control drug cholestyramine. Figure 4 In short-term animal studies, the modified peptide SAIWMY significantly inhibited the absorption of fluorescent cholesterol in the animal intestine, thereby reducing the level of fluorescent cholesterol in the animal serum and achieving a lipid-lowering effect. Its effect was comparable to that of the original peptide VAWWMY and the positive control drug cholestyramine. Figure 5 In long-term animal studies, the modified peptide SAIWMY significantly reduced serum cholesterol levels, with efficacy comparable to cholestyramine. SAIWMY also reduced liver cholesterol and triglyceride levels in animals, with a more significant effect than the positive control drug cholestyramine. Under long-term intervention, compared to the model group and the cholestyramine group, the modified peptide SAIWMY significantly reduced animal body weight. Figure 6 ). Attached Figure Description

[0017] Figure 1 High-performance liquid chromatography (HPLC) chromatogram of the modified peptide SAIWMY.

[0018] Figure 2 Mass spectrum of the modified peptide SAIWMY.

[0019] Figure 3 To modify the bile acid micelle solubility of the peptide SAIWMY.

[0020] Figure 4 To investigate the effect of the modified peptide SAIWMY on the viability of Caco2 cells.

[0021] Figure 5 To investigate the effect of the modified peptide SAIWMY on the absorption of fluorescent cholesterol in hamsters.

[0022] Figure 6 To investigate the regulatory effect of the peptide SAIWMY on lipids in hamsters on a long-term high-cholesterol diet. Detailed Implementation

[0023] Example 1: Synthesis of the modified peptide SAIWMY

[0024] (1) Swelling resin: Weigh 600mg of dichloromethane resin and add it to the cleaned and dried fully automatic peptide synthesizer. First, add an appropriate amount of dichloromethane (DCM) to soak for 5 minutes to allow the resin to swell fully. Then, add 7mL of N,N-dimethylformamide (DMF) to wash the resin 3 times.

[0025] (2) Adding the first amino acid: Dissolve Fmoc-protected tyrosine Y in DMF at a resin-to-amino acid equivalent ratio of 1:0.6 and add it to the resin. Add N,N-diisopropylethylamine (DIEA) at 3-5 times the amino acid equivalent, mix gently, and then add to the reactor. React at room temperature for 1 hour. Add methanol to end-cap unreacted dichloro sites and react for 30 minutes. After the reaction, wash the resin three times with DMF and dry the resin.

[0026] (3) Adding the second amino acid: Peptide synthesis proceeds from the C-terminus to the N-terminus. Weigh 600 mg of methionine M, add 2 mL each of condensing agent DIEA and benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate (HBTU), and microwave at 45°C for 300 s to carry out the condensation reaction. Drain the liquid, add 7 mL of DMF, and wash three times with the system, each wash lasting 25 s. Drain the washing liquid. Add 7 mL of deprotecting agent piperidine, and microwave at 45°C for 300 s to carry out the condensation reaction. Drain the liquid, add 7 mL of DMF, and wash three times with the system, each wash lasting 25 s. Drain the washing liquid.

[0027] (4) Subsequent amino acid addition: The method of addition is the same as the process of adding the second amino acid, until all amino acids are added.

[0028] (5) Resin lysis and drying: First, wash the resin with 6 mL of methanol and vacuum dry for 2 min. Add 8 mL of lysis buffer (containing 90% TFA, 2.5% phenol, 2.5% EDT, 2.5% benzyl mercaptan, and 2.5% diethyl ether), and lyse at 30 °C for 3 h. Add 40 mL of ice-cold diethyl ether to precipitate, shake well, and centrifuge for 2 min. Remove the supernatant, resuspend in ice-cold diethyl ether, and centrifuge once more to obtain the crude peptide.

[0029] (6) Crude product purification: The peptides were purified using a Shimadzu LC-20AP preparative high-performance liquid chromatograph. The peptides were weighed, dissolved in a measured amount of 70% acetonitrile aqueous solution by sonication, filtered through a 0.45 μm filter membrane, and then loaded onto the sample. A quantitative loop was inserted into the sample injector, and the crude peptide solution was loaded at a flow rate of 12 ml / min. Gradient elution was performed according to the following procedure:

[0030] Column: 20mm × 250mm 10μM C18 reverse-flow silica column

[0031] Mobile phase A: 0.1% TFA aqueous solution

[0032] Mobile phase B: 0.1% TFA acetonitrile solution

[0033] Gradient elution program: 0-25 min 5-65% mobile phase B; 25-30 min 95% mobile phase B; 30-35 min 5% mobile phase B; detection wavelength: 220 nm.

[0034] (7) Purity Identification: After freeze-drying, the purified peptides were subjected to purity identification using a Shimadzu LC-20AB analytical high-performance liquid chromatography (HPLC) system. The conditions were as follows:

[0035] Column: Inertsil ODS-3 4.6×250mm

[0036] Mobile phase A: 0.065% TFA aqueous solution

[0037] Mobile phase B: 0.05% TFA acetonitrile solution

[0038] Gradient elution program: 0-25 min 5-65% mobile phase B; 25-27 min 95% mobile phase B; 27-35 min 5% mobile phase B; detection wavelength: 220 nm.

[0039] The chromatogram of the peptide detected by HPLC is as follows: Figure 1 As shown, a major absorption peak is observed at a retention time of 17.900 min at a wavelength of 220 nm, indicating a purity of 98.95%. The secondary spectrum of the peptide, identified by a Shimadzu LCMS 2020 mass spectrometer, is shown below. Figure 2 As shown, the strongest ion signal is found at a mass-to-charge ratio of 770.3, which can be identified as the main detectable component.

[0040] Example 2: Bile acid micelle solubility of modified peptide SAIWMY

[0041] Solutions of 2 mM cholesterol, 4 mM oleic acid, 2.4 mM lecithin, and 2 mM monoacylglycerol were prepared using methanol. 250 μL of each solution was thoroughly mixed, concentrated by rotary freezing, and then added to a 4 mM taurocholic acid (TCA) solution prepared with 1 mL of PBS. The mixture was sonicated for 20 min and incubated at 37°C for 24 h. Different concentrations (0, 312.5, 625, 1250, 2500, 5000 μM) of peptides (SAIWMY, VAWWMY, VAW, or WMY) and cholestyramine were prepared using PBS. Bile acid micelles and peptide solutions or cholestyramine were mixed at a 1:1 volume ratio and reacted at 37°C and 250 rpm for 1 h. The mixture was then centrifuged at 15000 × g for 30 min, and the cholesterol concentration in the supernatant was measured. The solubility of bile acid micelles after peptide or cholestyramine treatment was calculated. Figure 3As shown, the bile acid micelle solubility of the short peptides VAW and WMY after enzymatic hydrolysis of the original peptide VAWWMY was significantly higher than that before hydrolysis, indicating that the bile acid micelle disruption ability of the original peptide was significantly reduced after enzymatic hydrolysis. The bile acid micelle solubility of the modified peptide SAIWMY at different concentrations was lower than that of the original peptide, indicating that the modified peptide had a greater ability to disrupt bile acid micelles than the original peptide. This suggests that the activity of the modified peptide SAIWMY was improved compared to before modification, and at high concentrations, its bile acid micelle solubility was comparable to that of cholestyramine.

[0042] Example 3: Effect of modified peptide SAIWMY on Caco2 cell viability

[0043] (1) Take Caco-2 cells in the logarithmic growth phase, digest, centrifuge, resuspend, and plate them in 96-well plates (1×10⁶ cells / wells). 4 (100 μL per well) and cultured overnight in a cell culture incubator for 12 h.

[0044] (2) Discard the old culture medium in the dish, and add cell culture medium containing different concentrations (0, 9.765625, 19.53125, 39.0625, 78.125, 156.25, 312.5, 625, 1250, 2500, 5000 μM) of polypeptide (VAWWMY or SAIWMY) or positive control drug cholestyramine to each group, and incubate overnight in a cell culture incubator for 24 h.

[0045] (3) Discard the old culture medium in the plate, add 90 μL of cell culture medium to each well, add 10 μL of CCK8 reagent to each well under light-protected conditions, mix well and incubate in a cell culture incubator at 37℃ for 1 h, measure the absorbance value at 450 nm wavelength using an ELISA reader and calculate cell viability.

[0046] The results are as follows Figure 4 As shown, the positive control drug cholestyramine at 156.25 μM resulted in cell viability of less than 80%, while the original peptide VAWWMY and the modified peptide SAIWMY showed no significant toxicity at 5 mM and below, and had little effect on Caco-2 cell viability. This indicates that the cytotoxicity of the original peptide VAWWMY and the modified peptide SAIWMY was significantly lower than that of the positive control drug cholestyramine.

[0047] Example 4: Effect of modified peptide SAIWMY on fluorescent cholesterol uptake in hamsters

[0048] One hundred male golden Syrian hamsters (4 weeks old, weighing 100-120 grams) were used as an animal model. After one week of acclimatization feeding, they were randomly divided into groups. Food was removed from their cheek pouches, and they were acclimatized to gavage using a 12-gauge gavage syringe with an empty gavage tube. After restraint, one ear was exposed, iodine was applied, and an ear tag was placed at the base of the ear using ear-tag pliers. They were kept hungry but allowed free access to water for 18 hours. Gavage treatment:

[0049] Control group: 200 μL corn oil;

[0050] Model group: 200 μL corn oil + 0.5 mg NBD-cholesterol (fluorescent cholesterol);

[0051] Cholestyramine group: 200μL corn oil + 0.5mg NBD-cholesterol + 50mg cholestyramine;

[0052] Peptide group: 200μL corn oil + 0.5mg NBD-cholesterol + 50mg peptide (VAWWMY or SAIWMY).

[0053] Four zirconium beads were added to the gavage before administration. The mixture was broken up at 6 Hz for 10 seconds, with a 10-second interval, and repeated 10 times to ensure complete mixing. 1.5 hours later, the patient was anesthetized with isoflurane, and blood was collected by enucleation. The blood was left at room temperature for at least 30 minutes, then centrifuged at 3000 rpm for 15 minutes, and the supernatant was collected. This process was repeated twice. 20 μL of serum was taken and mixed with 500 μL of cholesterol extraction buffer (isopropanol: n-heptane: 125 mM sulfuric acid, volume ratio = 80:19:16). After centrifugation, 200 μL was added to a black ELISA plate for detection (excitation wavelength 465 nm, detection wavelength 535 nm). Figure 5 As shown, compared with the model group, the modified peptide SAIWMY, the original peptide VAWWMY, and cholestyramine can significantly reduce the NBD-cholesterol levels in hamster serum, jejunal tissue, and duodenal tissue. Moreover, the effect of the modified peptide SAIWMY is comparable to that of the original peptide VAWWMY and the positive control drug cholestyramine.

[0054] Example 5: The regulatory effect of modified peptide SAIWMY on lipids in hamsters on a long-term high-cholesterol diet.

[0055] This experiment used 50 male golden Syrian hamsters (7-8 weeks old, weighing 100-120 grams) as animal models, randomly divided into 5 groups. The control group was fed a normal diet; the model group was fed a normal diet containing 0.15% high cholesterol; the peptide group was fed a normal diet containing 0.15% high cholesterol plus 1% modified peptide SAIWMY; and the cholestyramine group was fed a normal diet containing 0.15% high cholesterol plus 1% cholestyramine. The diet was changed weekly, and the weight of added and remaining food was recorded each time. Food intake was calculated, and body weight was measured. At week 5, blood was collected from the orbital rim, and the hamsters were euthanized. Serum was obtained by centrifugation, and total cholesterol was measured. Liver tissue was harvested, washed with physiological saline, and dried. 10 mg of central liver tissue was used to detect cholesterol and triglyceride levels. Figure 6As shown, compared to the control group, the serum cholesterol level in the model group was significantly increased, indicating successful model establishment. Compared to the model group, the modified peptide SAIWMY significantly reduced serum cholesterol levels and was comparable in efficacy to cholestyramine. In tissues, SAIWMY reduced liver cholesterol levels, while cholestyramine did not show a significant reduction, indicating that the modified peptide SAIWMY was superior to cholestyramine in reducing liver cholesterol levels. Regarding triglycerides, the modified peptide SAIWMY significantly reduced liver triglyceride levels, and its effect was more significant than that of the positive control drug cholestyramine. Furthermore, under long-term intervention, compared to the model group and the cholestyramine group, the modified peptide SAIWMY significantly reduced animal body weight, suggesting that the modified peptide SAIWMY can be used to treat obesity.

[0056] In summary, the modified peptide SAIWMY exhibits significantly superior efficacy compared to the original peptide VAWWMY and the first-line drug cholestyramine at both the molecular and animal levels. Specifically, the modified peptide SAIWMY demonstrates higher cholesterol-lowering activity, is less susceptible to intestinal enzyme degradation and is more stable, exhibits higher safety than cholestyramine, and is more effective than cholestyramine in lowering cholesterol and triglycerides in the liver. This indicates that the modified peptide SAIWMY possesses a similar mechanism of action to cholestyramine, with better lipid-lowering potential, and can be used to treat indications related to cholestyramine, including hyperlipidemia and atherosclerosis. Furthermore, it can be used for the prevention or treatment of fatty liver disease and obesity.

Claims

1. A soybean-modified polypeptide, characterized in that, The amino acid sequence of the polypeptide is SAIWMY.

2. The use of the polypeptide of claim 1 in the preparation of cholesterol-lowering products.

3. The use of the polypeptide of claim 1 in the preparation of triglyceride-lowering products.

4. The use of the polypeptide of claim 1 in the preparation of drugs for the prevention or treatment of hyperlipidemia, atherosclerosis or coronary heart disease.

5. The use of the polypeptide of claim 1 in the preparation of a drug for the prevention or treatment of fatty liver disease.

6. The use of the polypeptide of claim 1 in the preparation of products for the prevention or treatment of obesity.

7. The application according to any one of claims 2 to 6, characterized in that, The soybean-modified polypeptide is an oral preparation.

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