Modification of a soybean polypeptide and its use in the prevention and treatment of cardiovascular diseases

Abstract

The application belongs to the technical field of biological pharmacy, and discloses a soybean polypeptide modification and application thereof in preventing and treating cardiovascular diseases. In view of the problem that the original polypeptide VAWWMY is prone to enzymatic hydrolysis in the gastrointestinal tract and is difficult to play a significant cholesterol-lowering effect in the body, the amino acid sequence of the original polypeptide is modified, and the amino acid sequence of the modified peptide is VCIWMY. The modified peptide is not prone to be degraded by gastrointestinal enzymes and is more stable, the bile acid binding capacity and the bile acid micelle destruction capacity of the modified peptide are significantly better than those of the original peptide, the modified peptide is safe and non-toxic, has a small molecular weight, can be artificially synthesized, and has a significant in-vivo cholesterol-lowering effect, and the effect is equivalent to that of cholestyramine, a first-line drug. Therefore, the modified peptide VCIWMY can be used for preparing a drug for preventing or treating cardiovascular diseases (such as hyperlipidemia, atherosclerosis, coronary heart disease, etc.).

Description

Technical Field

[0001] This invention belongs to the field of biopharmaceutical technology, specifically relating to a modified polypeptide and its application in the preparation of drugs for the prevention and treatment of cardiovascular diseases. Background Technology

[0002] Hyperlipidemia is a common metabolic disease characterized by lipid metabolism disorders, with complications including atherosclerosis, renal insufficiency, and hypertension. Regulating bile acid and cholesterol homeostasis is crucial for preventing and treating cardiovascular diseases associated with hyperlipidemia. Cholestyramine, a high-molecular-weight bile acid chelator, lowers cholesterol levels by binding to bile acids in the intestines, inhibiting their enterohepatic circulation, and promoting cholesterol metabolism into bile acids. It can also reduce blood glucose levels by decreasing hepatic glucose production, thereby reducing the risk of atherosclerosis, coronary heart disease, and type 2 diabetes. However, cholestyramine is typically taken as a powder mixed with liquid, resulting in poor taste and low patient compliance. It can also cause gastrointestinal side effects such as constipation, and long-term use may interfere with the absorption of fat-soluble vitamins, limiting its clinical application. Therefore, there is an urgent need to develop a novel, safe, orally administered cholesterol-lowering drug.

[0003] Naturally derived bioactive peptides have always been a focus of drug research due to their high bioactivity, strong targeting, and lack of toxic side effects. Soybean peptide VAWWMY, extracted from plants, is a bioactive peptide that can bind to bile acids and has cholesterol-lowering effects in vivo. However, VAWWMY peptides are easily degraded by gastrointestinal proteases and have a short half-life, making it difficult to achieve the corresponding therapeutic concentrations in vivo, thus reducing efficacy. With the increasing prevalence of hyperlipidemia, the development of a safe and stable soybean peptide analogue is urgently needed.

[0004] Therefore, this invention designs and modifies the amino acid sequence of VAWWMY polypeptide to improve its stability in the gastrointestinal tract without affecting its cholesterol-lowering activity, which will help to make soybean polypeptides more widely used. Summary of the Invention

[0005] The main objective of this invention is to obtain a new polypeptide, VCIWMY, by modifying the existing VAWWMY polypeptide sequence. Compared to the original VAWWMY polypeptide, the modified peptide is less susceptible to degradation by gastrointestinal proteases, exhibits more efficient bile acid binding and bile acid micelle disruption capabilities, and demonstrates significant in vivo cholesterol-lowering effects, providing new possibilities for the development of cholesterol-lowering products.

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

[0007] An active polypeptide identified from soybean contains six amino acids with a molecular weight of 855.01. Its specific amino acid sequence is Val-Ala-Trp-Trp-Met-Tyr, i.e., VAWWMY. VAWWMY is degraded by trypsin in the intestine into VAW and WMY. The short peptides after enzymatic hydrolysis show a significantly reduced binding affinity to the six bile acids compared to VAWWMY. Studies have shown that bile acid-binding peptides are typically rich in W, Y, F, L, I, and V amino acid residues. Based on the characteristics of trypsin hydrolysis, polypeptides containing the IWMY sequence are not degraded by intestinal proteases between the I and W amino acids. Therefore, VAWWMY was modified while retaining the IWMY sequence. The main difference in sequence between the modified peptide VCIWMY and the original polypeptide is the difference in the second and third amino acid sequences. It contains six amino acids with a molecular weight of 814.03, and its specific amino acid sequence is Val-Cys-Ile-Trp-Met-Tyr, i.e., VCIWMY.

[0008] Based on the amino acid sequence of the modified peptide VCIWMY, it was chemically synthesized using the Fomc solid-phase peptide synthesis method. The complete sequence was then desalted using HPLC reversed-phase chromatography, achieving a purity of 96.2%. The specific production process of the modified peptide is as follows:

[0009] (1) Swelling resin: Dichloro resin was swollen in a 24-channel fully automated microwave synthesizer;

[0010] (2) Add the first amino acid: Weigh out tyrosine Y, dissolve it in DMF and add it to the resin. At the same time, add DIEA and mix it with the resin to react. Add methanol to seal the end and wash the resin.

[0011] (3) Adding the second amino acid: The synthesis proceeds from the C-terminus to the N-terminus of the peptide chain. After adding the first amino acid in step (2), the resin is protected, washed, and the second amino acid, methionine M, DIEA and HBTU, are added in proportion. The reaction is carried out and washed. After adding the deprotecting agent and reacting, the mixture is washed again.

[0012] (4) Add subsequent amino acids: The specific method is the same as the process of adding the second amino acid, until all amino acids are added;

[0013] (5) Resin drying and transfer: Wash the resin with methanol and transfer it to the vacuum drying table for vacuum drying.

[0014] (6) Resin lysis and drying: Add lysis buffer, lyse at 30°C for 3 hours, then add ice-cold ether for precipitation; shake well and centrifuge, remove supernatant, the obtained crude polypeptide solid is resuspended in ice-cold ether and centrifuged again to obtain crude polypeptide.

[0015] (7) Purification and quality control of crude polypeptide: The crude polypeptide was purified by RP-HPLC, then lyophilized, and small samples were taken for QC testing.

[0016] Application of the modified peptide VCIWMY in cholesterol lowering: Experiments have confirmed that the binding capacity of the modified peptide VCIWMY to bile acids is comparable to that of cholestyramine, a clinically used lipid-lowering drug, and is superior to that of VAWWMY. Animal experiments have also demonstrated that the modified peptide VCIWMY can significantly reduce the absorption of fluorescent cholesterol (NBD-cholesterol) in hamsters, achieving a cholesterol-lowering effect, and its efficacy is comparable to that of cholestyramine.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] Compared to the original peptide VAWWMY, the modified peptide VCIWMY designed in this invention exhibits better stability, is less susceptible to degradation by trypsin in the intestine, and demonstrates significantly superior bile acid binding capacity and bile acid micelle disruption ability. Compared to cholestyramine, a first-line clinical drug for treating hypercholesterolemia, the modified peptide designed in this invention is safe and non-toxic according to cytotoxicity tests, has a small molecular weight, can be synthesized artificially, and possesses significant in vivo cholesterol-lowering effects, with efficacy comparable to that of the first-line drug cholestyramine. Attached Figure Description

[0019] Figure 1 HPLC chromatogram of the modified peptide VCIWMY.

[0020] Figure 2 LC-MS identification pattern of the modified peptide VCIWMY.

[0021] Figure 3 Results of bile acid binding capacity assay for short peptides after enzymatic hydrolysis of the original peptide VAWWMY. (a) Bile acid (CA) binding capacity, (b) Chenodeoxycholic acid (CDCA) binding capacity, (c) Taurocholic acid (TCA) binding capacity, (d) Glycinecholic acid (GCA) binding capacity, (e) Deoxycholic acid (DCA) binding capacity, and (f) Lithocholic acid (LCA) binding capacity.

[0022] Figure 4 Results of bile acid binding capacity and cholesterol micelle solubility assays for the modified peptide VCIWMY. (a) Bile acid (CA) binding capacity, (b) Deoxycholic acid (DCA) binding capacity, (c) Chenodeoxycholic acid (CDCA) binding capacity, and (d) Bile acid micelle solubility.

[0023] Figure 5 The results show the cytotoxicity assays for different concentrations of peptides.

[0024] Figure 6 The results show that the modified peptide VCIWMY inhibits the absorption of NBD-cholesterol in serum in vivo. Detailed Implementation

[0025] To better understand the cholesterol-lowering effect of the modified peptides described in this invention, the invention is further described through the following specific examples.

[0026] Example 1: Modification and preparation of soybean polypeptide VAWWMY

[0027] The modified peptide described in this invention contains 6 amino acids with a molecular weight of 814.03. The specific amino acid sequence is Val-Cys-Ile-Trp-Met-Tyr, i.e., VCIWMY. The main difference between the modified peptide and the original peptide VAWWMY is the difference in the amino acid sequences at positions 2 and 3.

[0028] 1. Preparation method

[0029] The modified peptide VCIWMY described in this invention was prepared using the Fomc solid-phase peptide synthesis method via a fully automated microwave synthesizer. The specific preparation process is as follows:

[0030] (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.

[0031] (2) Add the first amino acid: Dissolve Fmoc-protected tyrosine Y in DMF according to the resin: amino acid equivalent ratio of 1:0.6 and add it to the resin; add N,N-diisopropylethylamine (DIEA) at 5 times the amino acid equivalent, mix gently and add it to the reactor, and react at room temperature for 1 h; add methanol to end unreacted dichloro sites, react for 30 min, wash the resin 3 times with DMF after the reaction, and dry the resin.

[0032] (3) Adding the second amino acid: The synthesis of the peptide chain 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 cycle and wash 3 times, each time for 25 s, and drain the washing liquid; add 7 mL of deprotecting agent piperidine (PIP), and microwave at 45°C for 300 s to carry out the condensation reaction; drain the liquid, add 7 mL of DMF and cycle and wash 3 times, each time for 25 s, and drain the washing liquid;

[0033] (4) Subsequent amino acid addition: The method of addition is the same as the process of adding the second amino acid above, until all amino acids are added;

[0034] (5) Resin lysis and drying: First, add 6 mL of methanol to wash the resin and vacuum dry for 2 min; add 8 mL of lysis buffer (containing 92% trifluoroacetic acid, 3% phenol, 2% 1,2-ethylenedithiol, 2% benzyl mercaptan, and 1% diethyl ether), lyse at 30℃ 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 to obtain crude polypeptide;

[0035] (6) Crude product purification: The peptide was purified using a Shimadzu LC-20AP preparative high-performance liquid chromatograph. The peptide was weighed, dissolved in 70% acetonitrile aqueous solution by sonication, filtered through a 0.45 μm filter membrane, and then loaded onto the sample. The crude peptide solution was injected into a quantitative loop using an injector at a flow rate of 12 mL / min. The sample was equilibrated with 98% mobile phase A (0.1% TFA aqueous solution) for 7 min, and gradient elution was performed according to the following procedure:

[0036] Column: 10μm C18 reversed-phase silica column

[0037] Mobile phase A: 0.1% TFA aqueous solution

[0038] Mobile phase B: 0.1% TFA acetonitrile solution

[0039] 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; flow rate 1 mL / min; detection wavelength 220 nm;

[0040] (7) Quality control testing: After lyophilization, the purified peptides were subjected to purity identification using a Shimadzu LC-2030 analytical high-performance liquid chromatography system under the following conditions:

[0041] Column: Inertsil ODS-SP 4.6×250mm

[0042] Mobile phase A: 0.065% TFA aqueous solution

[0043] Mobile phase B: 0.05% TFA acetonitrile solution

[0044] Gradient elution program: 0-25 min 5-65% mobile phase B; 25-27 min 95% mobile phase B; 27-35 min 95-5% mobile phase B; flow rate 1 mL / min; detection wavelength 220 nm.

[0045] 2. Preparation Results

[0046] The chromatogram of the peptide detected by HPLC is as follows: Figure 1As shown, a major absorption peak is observed at a retention time of 13.817 min at a wavelength of 220 nm, indicating a purity of 96.2%. The primary mass spectrum of the peptide, identified by a Shimadzu LCMS2020 mass spectrometer, is shown below. Figure 2 As shown, the strongest ion signal is found at a mass-to-charge ratio of 814.5, which can be identified as the main detectable component.

[0047] Example 2: Detection of the bile acid binding capacity of peptides

[0048] 1. Detection Method

[0049] The total bile acid concentration in samples was determined by enzymatic colorimetric assay using a total bile acid assay kit. A 10 mM polypeptide solution (VAWWMY, VAW, WMY, or VCIWMY) or cholestyramine solution was prepared using PBS buffer at pH 7.4. Six bile acid standards (cholic acid CA, chenodeoxycholic acid CDCA, taurocholic acid TCA, glycocholic acid GCA, deoxycholic acid DCA, and lithocholic acid LCA) were added to each well. The mixture was incubated at 37°C and 250 rpm for 2 h, followed by centrifugation at 15000 × g for 20 min, and the supernatant was collected. 2.5 μL of PBS (blank control), bile acid standards, and the supernatant to be tested were added to each well sequentially, followed by 180 μL of reagent R1. The mixture was incubated at 37°C and 250 rpm for 5 min. Finally, 60 μL of reagent R2 was added, and the mixture was incubated at 37°C and 250 rpm for 1 min. The absorbance A0 was measured at 405 nm using a multi-functional microplate reader. After incubation at 37°C for 3 min, the absorbance A1 was read again. The bile acid content of the system was calculated as follows:

[0050]

[0051] The bile acid binding capacity is calculated as follows:

[0052]

[0053] 2. Test Results

[0054] The bile acid binding capacity of the original peptide VAWWMY and its enzymatically hydrolyzed short peptides, such as Figure 3 As shown, the binding affinity of the short peptides VAW and WMY to six bile acids (CA, CDCA, TCA, GCA, DCA, and LCA) was significantly lower than that of the original peptide VAWWMY. This indicates that the binding affinity of the short peptides after enzymatic hydrolysis of the original peptide to bile acids was significantly reduced. The bile acid binding affinity of the modified peptide VCIWMY is shown in the figure. Figure 4 As shown, the binding affinity of the modified peptide to bile acid (CA) is comparable to that of the positive control drugs cholestyramine and VAWWMY. Figure 4 a) The modified peptide's binding affinity for deoxycholic acid (DCA) is comparable to that of the positive control drug cholestyramine, but significantly higher than that of VAWWMY. Figure 4b) The modified peptide VCIWMY's binding affinity for chenodeoxycholic acid (CDCA) was comparable to that of the positive control drug cholestyramine, and significantly higher than that of VAWWMY. Figure 4 c). This indicates that the modified peptide VCIWMY has a good modification effect, not only having a similar effect to cholestyramine, but also having a significantly better bile acid binding capacity than the original peptide VAWWMY.

[0055] Example 3: Determination of bile acid micelle solubility of polypeptides

[0056] 1. Detection Method

[0057] Solutions of 2 mM cholesterol, 4 mM oleic acid, 2.4 mM lecithin, and 2 mM monoacylglycerol were prepared separately using methanol. 250 μL of each solution was thoroughly mixed. After complete evaporation of methanol by vacuum freeze-drying, 1 mL of 13.2 mM taurocholic acid (TCA) solution prepared in PBS was added, and the mixture was sonicated for 20 min and incubated at 37°C for 24 h to prepare bile acid micelle solutions. Different concentrations (0, 312.5, 625, 1250, 2500, 5000 μM) of peptide solutions (VAWWMY or VCIWMY) and cholestyramine solution were prepared using PBS buffer. Equal volumes of bile acid micelles were added, and the mixture was reacted at 37°C and 250 rpm for 1 h, followed by centrifugation at 15000 × g for 30 min. The cholesterol content in the supernatant was determined using a total cholesterol kit to characterize the solubility of the bile acid micelles.

[0058] 2. Test Results

[0059] Bile acid micelles play a role in dissolving and transporting lipids (such as cholesterol) in the small intestine. Higher bile acid micelle solubility promotes cholesterol absorption, while lower solubility indicates reduced cholesterol absorption, thus lowering blood cholesterol levels. Peptides or cholestyramine can disrupt the formation of bile acid micelles, reducing their solubility and consequently decreasing cholesterol absorption. The results of bile acid micelle solubility testing for modified peptides are shown below. Figure 4 As shown in Figure d, the experiment indicates that the concentration of the peptide is inversely proportional to the solubility of bile acid micelles. At different dosages, the solubility of the modified peptide in bile acid micelles was lower than that of the original peptide, meaning that the modified peptide's ability to disrupt bile acid micelles is superior to that of the original peptide. At concentrations below 1.25 mM, the solubility of the modified peptide VCIWMY in bile acid micelles was slightly higher than that of the cholestyramine group, indicating that the modified peptide's ability to disrupt bile acid micelles was lower than that of cholestyramine. However, at concentrations of 1.25-5 mM, the modified peptide achieved a bile acid micelle solubility comparable to that of cholestyramine. This demonstrates that the modified peptide possesses a bile acid micelle-disrupting ability comparable to that of cholestyramine, and its effect is better than that of the original peptide VAWWMY.

[0060] Example 4: Cytotoxicity detection of peptides

[0061] 1. Detection Method

[0062] Take Caco-2 cells in the logarithmic growth phase and use 1×10 4 Cells were evenly seeded per well in a 96-well plate and cultured at 37°C for 24 h in 5% CO2. Cells were then treated with freshly prepared polypeptide solutions (VAWWMY or VCIWMY) or cholestyramine solution at different concentrations for 24 h. WST-1 reagent was added at a ratio of 10 μL / 100 μL of cell culture, gently mixed, and the cells were incubated in the dark for another 2 h. Absorbance was measured at 450 nm using a microplate reader, and cell viability was calculated.

[0063] 2. Test Results

[0064] Cytotoxic effects of modified peptides, such as Figure 5 As shown, compared with the control group, neither the original peptide VAWWMY nor the modified peptide VCIWMY exhibited significant cytotoxicity to Caco-2 cells at concentrations below 5 mM, while cholestyramine showed significant cytotoxicity with cell viability below 80% at a concentration of 156.25 μM. This indicates that both the modified peptide and the original peptide are safe and non-toxic compared to cholestyramine at higher doses.

[0065] Example 5: Evaluation of the in vivo cholesterol-lowering efficacy of the peptide

[0066] 1. Detection Method

[0067] An acute animal experiment was conducted using 4-week-old male Syrian golden hamsters (n=40) of clean-grade breed as an animal model to inhibit cholesterol absorption in vivo. The hamsters were fasted for 18 hours but had unrestricted water intake. They were randomly divided into 4 groups of 10 hamsters each based on their body weight. The treatment and control groups were set up as follows:

[0068] Control group: 200 μL corn oil;

[0069] Model group: 200μL corn oil + NBD-cholesterol;

[0070] Cholestyramine group: 200μL corn oil + NBD-cholesterol + cholestyramine;

[0071] Peptide group: 200μL corn oil + NBD-cholesterol + modified peptide VCIWMY.

[0072] The drug was administered via gavage in the form of bile acid micelles. One and a half hours after administration, the patient was anesthetized and euthanized. Blood was collected by enucleation, allowed to stand at room temperature for 30 minutes, and then centrifuged at 3000 rpm for 15 minutes to separate serum. 20 μL of serum was taken and 500 μL of a cholesterol extraction buffer prepared in a ratio of isopropanol:n-heptane:sulfuric acid (volume ratio) of 80:19:16 was added. The buffer was allowed to stand for 2 minutes, then centrifuged at 2000×g for 5 minutes. 200 μL of the extract was transferred to a black ELISA plate, and the fluorescence value of NBD-cholesterol was detected using a multi-mode microplate reader with an excitation wavelength of 465 nm and a detection wavelength of 535 nm.

[0073] 2. Experimental Results

[0074] Results of acute animal experiments on the in vivo cholesterol-lowering effects of modified peptides are as follows: Figure 6 As shown, compared with the control group, the serum NBD-cholesterol level in the model group was significantly increased, indicating successful model establishment. Compared with the model group, serum NBD-cholesterol level decreased significantly after cholestyramine treatment, and the cholesterol level in the peptide group was also significantly lower than that in the model group, with effects comparable to cholestyramine treatment. This indicates that the modified peptide VCIWMY has the same cholesterol-lowering effect as cholestyramine.

[0075] In summary, the modified peptide VCIWMY exhibits significantly superior cholesterol-lowering efficacy compared to the original peptide VAWWMY and comparable to the first-line drug cholestyramine at both the molecular and animal levels. Specifically, the modified peptide VCIWMY demonstrates higher cholesterol-lowering activity than the original peptide, is less susceptible to intestinal enzyme degradation and is more stable, and has a higher safety profile than cholestyramine. This suggests that the modified peptide VCIWMY has a similar mechanism of action to cholestyramine and can be used to prevent or treat indications related to cholestyramine, including hyperlipidemia, atherosclerosis, and coronary heart disease.

Claims

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

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 drugs for the prevention or treatment of hyperlipidemia, atherosclerosis or coronary heart disease.

4. The application according to claim 2 or 3, characterized in that, The polypeptide is an oral preparation.

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