Polypeptide for regulating SGLT2 activity and pharmaceutical application thereof

Abstract

The invention relates to the technical field of biological medicines, in particular to a novel polypeptide for regulating the activity of a sodium glucose cotransporter 2 (SGLT2), which consists of the following four amino acid sequences: SEQ. NO: 1: Asp-Pro-Gly-Leu-Thr (DPGLT), SEQ. NO: 2: His-Cys-Val-Arg-Asn (HCVRN), SEQ. NO: 3: Gly-Met-Ser-Lys-Phe (GMSKF), and SEQ. NO: 4: Tyr-Trp-Arg-His-Glu (YWRHE). The invention also relates to a preparation method of the novel polypeptide for regulating the activity of the SGLT2. Experimental results show that the novel polypeptide can effectively inhibit the activity of SGLT2, regulate the blood glucose level, improve blood glucose control and reduce the blood glucose level.

Description

1. Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and mainly relates to a polypeptide for regulating the activity of SGLT2 and its pharmaceutical applications. 2. Background Art

[0002] The development of molecular biology and proteomics brings new opportunities for diabetes treatment: In recent years, with the rapid development of molecular biology and proteomics, researchers have begun to focus on developing new diabetes treatment methods by targeting specific proteins. This method has high specificity and effectiveness, can precisely treat the pathogenesis of diabetes, and reduce the side effects of traditional treatment methods. SGLT2 is mainly distributed in the S1 segment of the proximal renal tubule and is responsible for approximately 90% of glucose reabsorption. Under normal physiological conditions, SGLT2 retransports the filtered glucose in the glomerulus back into the blood to maintain the balance of glucose in the body. SGLT2 is a low-affinity, high-capacity glucose transporter, and its process of transporting glucose depends on the concentration gradient of sodium ions. In diabetic patients, the activity of SGLT2 is enhanced, resulting in excessive glucose being reabsorbed back into the blood, thus increasing blood glucose levels. This is because the blood glucose level in diabetic patients increases, and the filtered glucose in the glomerulus increases, stimulating the expression and activity of SGLT2 to increase. In addition, insulin resistance and hyperglycemia also affect the regulatory mechanism of SGLT2, leading to enhanced activity of SGLT2. SGLT2 has great potential as a therapeutic target. By inhibiting the activity of SGLT2, the reabsorption of glucose by the kidney can be reduced, and the excretion of glucose in the urine can be increased, thereby lowering blood glucose levels. This treatment method does not depend on insulin, so it is effective for diabetic patients with insulin resistance and insulin deficiency. In addition, SGLT2 inhibitors also have some other advantages, such as reducing body weight, lowering blood pressure, and improving cardiovascular function. SGLT2 inhibitors bind to SGLT2 and inhibit its function of transporting glucose, thereby reducing the reabsorption of glucose by the kidney, increasing the excretion of glucose in the urine, and lowering blood glucose levels. The mechanism of action of SGLT2 inhibitors is different from that of traditional hypoglycemic drugs. It does not depend on the action of insulin, so it is effective for diabetic patients with insulin resistance and insulin deficiency. Currently, a variety of SGLT2 inhibitors have been developed and widely used clinically. These inhibitors include canagliflozin, dapagliflozin, empagliflozin, etc. Different SGLT2 inhibitors have different chemical structures and pharmacological properties, but all have good hypoglycemic effects and safety. The clinical application of SGLT2 inhibitors is becoming more and more extensive and has become an important part of diabetes treatment. SGLT2 inhibitors can be used alone or in combination with other hypoglycemic drugs. In clinical applications, SGLT2 inhibitors have good hypoglycemic effects and safety, can effectively lower blood glucose levels, and reduce the risk of occurrence of diabetic complications. In addition, SGLT2 inhibitors can also improve the cardiovascular function of patients and reduce the risk of occurrence of cardiovascular diseases. In recent years, significant progress has been made in the research on SGLT2 inhibitors.A large number of clinical studies have shown that SGLT2 inhibitors have good hypoglycemic effects and safety, can effectively reduce blood glucose levels, and reduce the risk of developing diabetes complications. In addition, SGLT2 inhibitors can also improve the cardiovascular function of patients and reduce the risk of cardiovascular diseases. These research results provide strong support for the clinical application of SGLT2 inhibitors. Although SGLT2 inhibitors have great potential in the treatment of diabetes, they also face some challenges. First, the mechanism of action of SGLT2 inhibitors is not fully understood and further in-depth research is needed. Second, SGLT2 inhibitors may cause some side effects, such as urinary tract infections, hypotension, diabetic ketoacidosis, etc. In addition, the long-term safety of SGLT2 inhibitors also needs further observation and research. III. Summary of the Invention

[0003] (I) Structure of the novel polypeptide

[0004] The novel polypeptide provided by the present invention is composed of the following four amino acid sequences:

[0005] SEQ.NO 1: Asp-Pro-Gly-Leu-Thr (DPGLT);

[0006] SEQ.NO 2: His-Cys-Val-Arg-Asn (HCVRN);

[0007] SEQ.NO 3: Gly-Met-Ser-Lys-Phe (GMSKF);

[0008] SEQ.NO 4: Tyr-Trp-Arg-His-Glu (YWRHE).

[0009] These sequences were designed by conducting in-depth research on the structure and function of the target protein SGLT2 and combining binding affinity analysis.

[0010] (II) Screening method of the novel polypeptide

[0011] 1. Establish an SGLT2 protein model

[0012] By using bioinformatics methods, collect the amino acid sequence information of SGLT2, and use protein structure prediction software to construct a three-dimensional structure model of SGLT2. Optimize and verify the model to ensure its accuracy and reliability.

[0013] Virtual screening of the polypeptide library

[0014] Construct a polypeptide library containing a large number of different amino acid sequences. Using molecular docking technology, virtually dock the polypeptides in the polypeptide library with the SGLT2 protein model and calculate the binding affinity. Screen out the polypeptide sequences with higher binding affinity as candidate polypeptides. After preliminary screening, a batch of binding candidate polypeptides were obtained.

[0015] In vitro experiment verification

[0016] Conduct in vitro cell experiments on the candidate polypeptides. Add the candidate polypeptides to the cultured kidney cells and detect the activity of SGLT2 and the glucose reabsorption ability of the cells. Screen out the polypeptides that can significantly inhibit the activity of SGLT2 and reduce glucose reabsorption.

[0017] For example, polypeptide SEQ.NO 1 (DPGLT):

[0018] · Design concept: Based on in-depth analysis of the SGLT2 structure, it is found that there is a specific region on the surface of the SGLT2 protein, which is related to the binding of sodium ions and affects the transport activity of SGLT2. Design DPGLT with a specific amino acid sequence to bind to this region of SGLT2 and regulate its interaction with sodium ions.

[0019] · Mechanism of action: After DPGLT binds to SGLT2, it changes the binding mode of SGLT2 to sodium ions. This regulatory effect reduces the transport activity of SGLT2, thereby reducing the reabsorption of glucose by the kidneys.

[0020] Polypeptide SEQ.NO 2 (HCVRN):

[0021] · Design concept: Considering the key structural region of the SGLT2 protein in the regulation of cellular energy metabolism, design this polypeptide to bind to this region and regulate the energy metabolism signaling pathway related to SGLT2 activity.

[0022] · Mechanism of action: After HCVRN binds to a specific structural region of SGLT2, it regulates the energy level of the cell by affecting the activity of key enzymes in cellular energy metabolism. This regulatory effect indirectly affects the activity of SGLT2, reducing its reabsorption of glucose. At the same time, this polypeptide may regulate the intracellular metabolic state, creating a favorable environment for the regulation of SGLT2 activity.

[0023] Polypeptide SEQ.NO 3 (GMSKF):

[0024] · Design concept: Aim at the key region for the conformational stability of SGLT2 and design this polypeptide to affect the conformational stability of SGLT2 and thus regulate its activity.

[0025] · Mechanism of action: After GMSKF binds to the conformational stability region of SGLT2, it stabilizes the conformation of SGLT2 by forming specific chemical bonds or interactions with specific amino acid residues. This stabilizing effect keeps SGLT2 in a low-activity state, reducing glucose reabsorption. At the same time, it may interact with other intracellular proteins to synergistically promote the functional regulation of SGLT2.

[0026] Polypeptide SEQ.NO 4 (YWRHE):

[0027] · Design concept: Considering various aspects of the regulation of SGLT2 activity, this polypeptide is designed to comprehensively regulate the activity of SGLT2.

[0028] · Mechanism of action: After YWRHE binds to the specific activity regulation region of SGLT2, it affects the activity and metabolic function of SGLT2 by regulating the chemical properties of certain key amino acid residues inside the SGLT2 protein. It may participate in regulating the intracellular redox balance to ensure that SGLT2 can be effectively regulated when needed, reducing glucose reabsorption.

[0029] (III) Mechanism of action of the novel polypeptide

[0030] 1. Inhibiting SGLT2 activity

[0031] The novel polypeptide can specifically bind to SGLT2, thereby inhibiting its activity. By binding to SGLT2, the novel polypeptide can regulate the intracellular sodium ion binding, energy metabolism signaling pathway, conformational stability, or the chemical properties of key amino acid residues, etc., and then inhibit the activity of SGLT2, reduce the renal reabsorption of glucose, and lower blood glucose levels.

[0032] 1. Regulating blood glucose levels

[0033] The novel polypeptide reduces the reabsorption of glucose and increases the excretion of glucose in urine by inhibiting the activity of SGLT2, thereby lowering blood glucose levels.

[0034] Advantages of the invention:

[0035] 1. High efficiency

[0036] The novel polypeptide can significantly inhibit the activity of the target protein SGLT2 and effectively regulate blood glucose levels.

[0037] 1. Safety

[0038] Verified by in vitro and in vivo experiments, the novel polypeptide has no obvious toxicity at high doses and has no obvious adverse effects on important organs such as the liver and kidneys after long-term use.

[0039] 1. Specificity

[0040] The novel polypeptide has a highly specific binding ability to the target protein SGLT2, reducing interference with other irrelevant proteins and lowering the risk of potential side effects. IV. Specific Embodiments

[0041] Example 1

[0042] (I) Synthesis of Polypeptide

[0043] 1. Selection of Amino Acid Monomers

[0044] Amino acid monomers with Boc protecting groups are used to ensure the activity and stability of amino acids during synthesis.

[0045] The amino acid monomers are sequentially linked to the solid-phase carrier. After each coupling reaction, the Boc group is removed through a deprotection step. A step-by-step synthesis method is adopted to ensure the accuracy and purity of the polypeptide.

[0046] After all the amino acids are linked, they are cleaved from the solid-phase carrier to obtain the crude polypeptide. Its purity and molecular weight are confirmed by high-performance liquid chromatography (HPLC) and mass spectrometry (MS) analysis. Multiple purification methods, such as reverse-phase high-performance liquid chromatography and ion-exchange chromatography, are used to improve the purity of the polypeptide.

[0047] (II) Structure Verification Experiments

[0048] Mass Spectrometry Analysis

[0049] 1. Instrument: A high-resolution mass spectrometer (such as MALDI-TOF MS) is used for analysis.

[0050] 2. Sample Preparation: The synthesized peptide segments are separately dissolved in a mixed solvent of methanol and water to prepare a solution with an appropriate concentration.

[0051] 3. Experimental Results: The mass spectrometry analysis results show that the molecular weights of the peptide segments are consistent with the theoretical values, further confirming the correctness of their structures. By comparing the difference between the measured molecular weight and the theoretical molecular weight, the synthesis accuracy of the polypeptide is evaluated.

[0052] Circular Dichroism (CD) Analysis

[0053] 1. Instrument: A circular dichroism spectrometer is used for analysis.

[0054] 2. Sample Preparation: The synthesized peptide segments are separately dissolved in phosphate buffer to prepare a solution with an appropriate concentration.

[0055] 3. Experimental Results: The circular dichroism analysis results show that the peptide segments have specific secondary structure characteristics, which are consistent with the expected structures. Analyze the secondary structure of the polypeptide, such as α-helix, β-sheet, etc., to understand its structural stability.

[0056] (III) Identification and Related Research of Target Protein SGLT2

[0057] 1. Identification of SGLT2

[0058] SGLT2 was identified in kidney tissues by proteomics and bioinformatics methods. Immunohistochemistry, Western blot and other techniques were used to verify the expression of SGLT2 in kidney tissues of diabetic patients.

[0059] 1. Through cell experiments and animal experiments, study the regulatory effects of novel polypeptides on SGLT2 activity and blood glucose levels. Methods such as glucose transport assay and analysis of glucose content in urine were used to reveal the action mechanism of novel polypeptides.

[0060] (IV) In Vitro Activity Test

[0061] 1. Cell Culture

[0062] (1) Cell line: Select appropriate kidney cell lines, such as human renal proximal tubular cell line (HK-2 cells), etc., for in vitro experiments.

[0063] (2) Culture conditions: Use a culture medium suitable for cell growth, containing appropriate serum, antibiotics, etc., and culture under suitable temperature and gas environment.

[0064] 1. Peptide Treatment

[0065] (1) Dissolve each synthesized polypeptide in DMSO respectively to prepare solutions with different concentrations.

[0066] (2) Add each polypeptide with different concentrations to the cell culture medium and incubate for a certain period of time.

[0067] 1. Animal Model

[0068] (1) Model: Diabetic mouse model (prepared by methods such as injecting streptozotocin).

[0069] (2) Experimental group: Each polypeptide treatment group, that is, novel polypeptides composed of SEQ.NO 1 (DPGLT), SEQ.NO 2 (HCVRN), SEQ.NO 3 (GMSKF), SEQ.NO 4 (YWRHE) were given respectively, and continuously administered for a certain period of time through appropriate administration routes (such as oral administration, injection, etc.).

[0070] (3) Control group:

[0071] · Normal saline treatment group: Give the same volume of normal saline as the experimental group as a blank control to exclude the influence of non-drug factors such as administration route and injection operation on the experimental results.

[0072] · Positive control group: The group treated with common hypoglycemic drugs. Commonly used hypoglycemic drugs in clinical practice, such as metformin, were selected as positive control drugs. The administration method was also carried out according to the conventional usage method of the drug and the same administration route as the experimental group (such as oral administration or injection, etc.), and the drug was administered continuously for a certain period of time. The dose was determined by converting the clinical recommended dose and the equivalent dose in animal experiments.

[0073] 1. Experimental results

[0074] (1) SGLT2 activity: The activity state of SGLT2 was reflected by detecting the glucose transport ability of cells. The results showed that each polypeptide significantly inhibited the activity of SGLT2. The specific data are as follows:

[0075]

[0076] (2) Glucose content in urine: The effect of the novel polypeptide on glucose excretion was reflected by detecting the glucose content in the cell culture medium. The results showed that each polypeptide significantly increased the excretion of glucose in urine. The specific data are as follows:

[0077]

[0078] (5) Verification in animal models

[0079] 1. Animal models

[0080] (1) Model: Diabetic mouse model (prepared by methods such as injecting streptozotocin).

[0081] (2) Experimental group: Each polypeptide treatment group.

[0082] (3) Control group: The group treated with normal saline.

[0083] (4) Positive control group: The group treated with common hypoglycemic drugs.

[0084] 1. Administration method

[0085] Each polypeptide or normal saline was administered through appropriate administration routes, such as oral administration or injection, and continuously administered for a certain period of time.

[0086] 1. Experimental results

[0087] (1) Blood glucose level: The blood glucose level of mice was detected regularly. The results showed that the blood glucose levels in each polypeptide treatment group were significantly lower than those in the control group and the positive control group. The specific data are as follows:

[0088]

[0089] (2) Kidney function indicators: By detecting the kidney function indicators of mice, such as creatinine, urea nitrogen, etc., the effect of the polypeptide on the kidney was evaluated. The results showed that there were no significant differences in the kidney function indicators of each polypeptide treatment group compared with the control group, indicating that the polypeptide had no obvious adverse effects on the kidney. The specific data are as follows:

[0090]

[0091] (VI) Safety assessment

[0092] 1. Acute toxicity test

[0093] By administering a single large dose of the drug, the survival rate and behavioral performance of the mice were observed. The results showed that there was no obvious toxicity of each polypeptide at high doses. The specific data are as follows:

[0094]

[0095] 1. Long-term toxicity test

[0096] By continuously administering the drug for a period of time, indicators such as the body weight, blood routine, liver and kidney functions of the mice were observed. The results showed that there were no obvious adverse reactions of each polypeptide under long-term use. The specific data are as follows:

[0097]

[0098] Data table of comparative synergistic test

[0099] Data table of comparative synergistic test

[0100]

[0101]

[0102] Through the above detailed comparison, the advantages of the novel polypeptide of the present invention in blood glucose control compared with the traditional treatment method (taking metformin as an example) are more clearly demonstrated, further verifying its potential application value in the field of diabetes treatment. In order to verify the effect of the novel polypeptide of the present invention in blood glucose control, a series of experiments were carried out. The experimental results showed that the novel polypeptide could effectively inhibit the activity of SGLT2 and regulate blood glucose levels, improve blood glucose control, and reduce blood glucose levels. At the same time, the polypeptide had no obvious toxicity at high doses and had no obvious adverse effects on important organs such as the liver and kidney under long-term use, and had high safety and reliability.

[0103] Example 2: Novel small molecule peptide derived from wolfberry leaves

[0104] . Novel polypeptide structure

[0105] The amino acid sequence of the novel polypeptide is: SEQ.NO 39: Ala-Ser-Gln-His-Lys-Pro-Thr-Glu-Tyr-Ile (ASQHKPTETI). This sequence was obtained from wolfberry leaf protein through specific enzymatic hydrolysis and subsequent screening processes. Wolfberry leaves are rich in various active ingredients, and small peptides that can regulate the activity of sodium-glucose cotransporter 2 (SGLT2) may exist in the protein hydrolysate, providing a new way for blood glucose regulation.

[0106] . Extraction method

[0107] · Raw material pretreatment: Wash and dry fresh wolfberry leaves, then crush them. Take the wolfberry leaf powder and add Tris-HCl buffer solution with pH 8.3 at a ratio of 1:12 (w / v). Stir and extract at 45 °C for 4.5 hours, and perform ultrasonic assistance for 10 minutes with a power of 240 W every 1.5 hours during this period to promote protein dissolution.

[0108] · Enzymatic hydrolysis process: Centrifuge the extract at 9000 rpm for 18 minutes, take the supernatant and adjust the pH to 7.3. Add trypsin (the mass ratio of enzyme to substrate is 1:130), and perform enzymatic hydrolysis at 38 °C for 7 hours. After the enzymatic hydrolysis is completed, place the reaction solution in a 92 °C water bath to inactivate the enzyme for 12 minutes.

[0109] · Separation and purification: After the enzymatic hydrolysate is concentrated by rotary evaporation, ultrafiltrate it through an ultrafiltration membrane with a molecular weight cut-off of 3500 Da, and collect the retentate. Separate the retentate by gel filtration chromatography (Sephadex G-30), use 0.15 mol / L sodium chloride solution as the eluent, with a flow rate of 0.6 mL / min, and collect the active elution peak. Purify the active peak by reverse-phase high-performance liquid chromatography (RP-HPLC), use a C18 chromatographic column, and perform gradient elution with acetonitrile-water (containing 0.1% trifluoroacetic acid) as the mobile phase, and collect the target polypeptide peak.

[0110] . Synergistic effect test

[0111] · Experimental design: Select a diabetic mouse model and randomly divide it into three groups. The control group is given normal saline, the experimental group is given the novel polypeptide (ASQHKPTETI) of the present invention, and the comparison group is given the common hypoglycemic drug metformin.

[0112] · Experimental results: After six weeks of drug administration, the blood glucose level of the mice in the experimental group was significantly lower than that of the control group and the comparison group. The activity of SGLT2 in the kidney tissue was significantly reduced, and the glucose excretion in the urine was significantly increased. At the same time, the insulin sensitivity of the mice was enhanced, and the serum insulin level decreased, indicating that the utilization efficiency of insulin by the body was improved. This shows that the novel polypeptide has a synergistic effect in reducing blood glucose, inhibiting the activity of SGLT2, increasing glucose excretion, and improving insulin sensitivity.

[0113] Example 3: A Novel Small Molecule Peptide Extracted from Mulberry Root Bark

[0114] . Novel Polypeptide Structure

[0115] The amino acid sequence of the novel polypeptide is: SEQ.NO 40: Gly-Glu-Asn-Cys-Leu-Ile-Arg-Pro-Thr-Val (GENCLIRPV). This sequence was determined by enzymatic hydrolysis, separation, and activity screening of mulberry root bark protein. The unique amino acid composition of mulberry root bark protein may produce small molecule peptides with regulatory functions on SGLT2 activity after specific enzymatic hydrolysis.

[0116] . Extraction Method

[0117] · Pretreatment: Wash, dry, and crush mulberry root bark, soak it in sodium hydroxide solution with pH 9.0, so that the ratio of mulberry root bark powder to the solution is 1:10 (w / v), stir and extract at 50 °C for 4 hours, and perform intermittent ultrasound (power 220W, ultrasound time 10 minutes) during this period to promote protein dissolution.

[0118] · Enzymatic Hydrolysis and Separation: Centrifuge the extract at 8500 rpm for 16 minutes to obtain the supernatant, and adjust the pH to 7.5. Add alkaline protease (the mass ratio of enzyme to substrate is 1:110), and perform enzymatic hydrolysis at 45 °C for 6 hours. After the enzymatic hydrolysis is completed, heat to 95 °C to inactivate the enzyme for 10 minutes. Ultrafilter the enzymatic hydrolysate through an ultrafiltration membrane with a molecular weight cut-off of 3200 Da, and collect the retentate. The retentate is separated by ion exchange chromatography (CM-Sepharose FF), eluted with a gradient of 0-0.6 mol / L sodium chloride solution, and the active peak is collected. The active peak is further purified by reverse-phase high performance liquid chromatography (RP-HPLC), using a C18 column, eluted with a gradient of methanol-water (containing 0.1% formic acid) as the mobile phase, and the target polypeptide peak is collected.

[0119] . Synergistic Effect Test

[0120] · Experimental Design: Use a diabetic rat model, and set up a control group, an experimental group of the novel polypeptide (GENCLIRPV), and a comparison group of positive control (the commonly used hypoglycemic drug gliclazide).

[0121] · Experimental Results: After seven weeks of drug administration, the blood glucose level of the rats in the experimental group was significantly lower than that of the control group and the comparison group. The expression level of SGLT2 gene in kidney tissue was significantly reduced, and the content of SGLT2 protein detected by Western blotting decreased. At the same time, the level of glycated hemoglobin (HbA1c) in the blood decreased, indicating good long-term blood glucose control. In addition, blood lipid levels (such as triglycerides and total cholesterol) were also improved, suggesting that this polypeptide may also have a regulatory effect on dyslipidemia complicated with diabetes. This indicates that this novel polypeptide has a synergistic effect in reducing blood glucose, inhibiting SGLT2 expression, improving long-term blood glucose control, and regulating blood lipids.

[0122] Example 4: A Novel Small Molecule Peptide Based on Blackcurrant Seed Extract

[0123] . Novel Polypeptide Structure

[0124] The amino acid sequence of the novel polypeptide is: SEQ.NO 41: Thr-Pro-Gln-Lys-His-Gly-Ala-Ser-Phe-Met (TPQKHGASFM). This sequence was screened from the enzymatic hydrolysate of proteins in blackcurrant seeds. Blackcurrant seeds are rich in high-quality protein, and small peptides that may regulate the activity of SGLT2 may be produced after enzymatic hydrolysis of the protein.

[0125] . Extraction Method

[0126] · Raw material preparation: Wash, dry and crush blackcurrant seeds, defat them by refluxing with petroleum ether twice, 2 hours each time, to remove the grease. Add the defatted blackcurrant seed powder to a phosphate buffer solution with a pH of 7.8 at a ratio of 1:12.5 (w / v), stir and extract at 42°C for 3 hours, while performing ultrasonic assistance (power 200W, ultrasonic time 8 minutes) to fully dissolve the protein.

[0127] · Enzymatic hydrolysis and purification: Centrifuge the extract at 8000 rpm for 13 minutes to obtain the supernatant, and adjust the pH to 7.0. Add papain (the mass ratio of enzyme to substrate is 1:120), and perform enzymatic hydrolysis at 42°C for 5 hours. After the enzymatic hydrolysis is completed, heat to 85°C to inactivate the enzyme for 8 minutes. After the enzymatic hydrolysate is concentrated by rotary evaporation, ultrafiltration is performed through an ultrafiltration membrane with a molecular weight cut-off of 3000 Da, and the retentate is collected. The retentate is separated by hydrophobic interaction chromatography (Phenyl-Sepharose 6FF), eluted with an ammonium sulfate concentration gradient (2 - 0 mol / L), and the active components are collected. The active components are further purified by reverse-phase high-performance liquid chromatography (RP-HPLC), using a C18 column, and gradient eluted with acetonitrile-water (containing 0.1% trifluoroacetic acid) as the mobile phase, and the target polypeptide peak is collected.

[0128] . Synergistic Experiment

[0129] · Experimental design: Select a diabetic rabbit model, and divide it into a control group, an experimental group of the novel polypeptide (TPQKHGASFM), and a comparison group of traditional therapeutic drugs (such as acarbose).

[0130] · Experimental results: After eight weeks of drug administration, the blood glucose level of the experimental group rabbits was significantly lower than that of the control group and the comparison group. The results of the oral glucose tolerance test (OGTT) showed that after glucose administration, the increase in blood glucose of the experimental group rabbits was significantly smaller than that of the control group and the comparison group, and the time for blood glucose to return to normal levels was shorter. At the same time, the activity of SGLT2 in the kidney tissue was significantly inhibited, and the transport function of SGLT2 on the cell membrane was weakened. In addition, the insulin secretion function was improved to a certain extent, and the function of pancreatic islet β cells was restored. This indicates that the novel polypeptide has a synergistic effect in improving glucose tolerance, inhibiting SGLT2 activity, and promoting insulin secretion.

[0131] Example 5: A novel small molecule peptide extracted from dandelion roots

[0132] . Novel polypeptide structure

[0133] The amino acid sequence of the novel polypeptide is: SEQ.NO 42: Val-Asn-Glu-Pro-His-Lys-Ala-Tyr-Trp-Thr (VNEPHKAYWT). This sequence was obtained by extracting, enzymatically digesting, and screening the activity of dandelion root proteins. Dandelion roots are rich in various nutrients, and the proteins in them may produce small molecule peptides with the ability to regulate SGLT2 activity after enzymatic digestion.

[0134] . Extraction method

[0135] · Treatment of dandelion roots: Wash and air-dry fresh dandelion roots, then homogenize them with a homogenizer. Add the homogenate to Tris-HCl buffer at pH 8.8 at a ratio of 1:10.5 (w / v), and stir and extract at 40°C for 4.6 hours, with ultrasonic assistance (power 210W, ultrasonic time 11 minutes) during this period to promote the dissolution of dandelion root proteins.

[0136] · Enzymatic digestion and separation: Centrifuge the extract at 8200 rpm for 14 minutes to obtain the supernatant, and adjust the pH to 7.2. Add neutral protease (the mass ratio of enzyme to substrate is 1:120), and enzymatically digest at 40°C for 6.5 hours. After the enzymatic digestion is completed, heat to 90°C to inactivate the enzyme for 9 minutes. Ultrafilter the enzymatic hydrolysate through an ultrafiltration membrane with a molecular weight cut-off of 4000 Da, and collect the retentate. The retentate is separated by ion exchange chromatography (DEAE-Sepharose FF), eluted with a gradient of 0 - 0.5 mol / L sodium chloride solution, and the active peak is collected. The active peak is further purified by reverse-phase high-performance liquid chromatography (RP-HPLC), using a C18 column, and eluted with a gradient of methanol-water (containing 0.1% formic acid) as the mobile phase, and the target polypeptide peak is collected.

[0137] . Synergistic test

[0138] · Experimental design: A diabetic mouse model was used, and a control group, an experimental group of a novel polypeptide (VNEPHKAYWT), and a comparative group of the commonly used hypoglycemic drug repaglinide were set up.

[0139] ● Experimental results: After five weeks of drug administration, the blood glucose levels of the mice in the experimental group were significantly lower than those in the control group and the comparative group. Immunohistochemical analysis showed that the expression of SGLT2 in the kidney tissue was significantly reduced and the distribution pattern was changed. At the same time, glycogen synthesis in the liver tissue increased, and the activities of key enzymes in gluconeogenesis (such as phosphoenolpyruvate carboxykinase) decreased, indicating that the ability of the liver to regulate blood glucose was improved. In addition, the weight gain of the mice tended to be normal, avoiding abnormal weight changes caused by diabetes. This indicates that the novel polypeptide has a synergistic effect in reducing blood glucose, inhibiting SGLT2 expression, regulating liver glucose metabolism, and improving body weight.

Claims

1. A novel polypeptide for regulating the activity of sodium-glucose cotransporter 2 (SGLT2), characterized in that, It consists of the following four amino acid sequences: SEQ.NO 1: Asp - Pro - Gly - Leu - Thr (DPGLT), SEQ.NO 2: His - Cys - Val - Arg - Asn (HCVRN), SEQ.NO 3: Gly - Met - Ser - Lys - Phe (GMSKF), SEQ.NO 4: Tyr - Trp - Arg - His - Glu (YWRHE).

2. The novel polypeptide according to claim 1, characterized in that, The polypeptide further includes SEQ.NO 39: Ala - Ser - Gln - His - Lys - Pro - Thr - Glu - Tyr - Ile (ASQHKPTETI), SEQ.NO 40: Gly - Glu - Asn - Cys - Leu - Ile - Arg - Pro - Thr - Val (GENCLIRPV), SEQ.NO 41: Thr - Pro - Gln - Lys - His - Gly - Ala - Ser - Phe - Met (TPQKHGASFM), SEQ.NO 42: Val - Asn - Glu - Pro - His - Lys - Ala - Tyr - Trp - Thr (VNEPHKAYWT).

3. A screening method for a novel polypeptide, characterized in that It includes the following steps: establishing an SGLT2 protein model, collecting the amino acid sequence information of SGLT2 by bioinformatics methods, constructing a three - dimensional structure model of SGLT2 using protein structure prediction software and optimizing and validating it; constructing a polypeptide library containing a large number of different amino acid sequences, using molecular docking technology to perform virtual docking of the polypeptides in the polypeptide library with the SGLT2 protein model, calculating the binding affinity, and screening out the polypeptide sequences with higher binding affinity as candidate polypeptides; performing in vitro cell experiments on the candidate polypeptides to detect the activity of SGLT2 and the glucose reabsorption ability of the cells, and screening out the polypeptides that can significantly inhibit the activity of SGLT2 and reduce glucose reabsorption.

4. Use of the novel polypeptide according to claim 1 in inhibiting SGLT2 activity, characterized in that, The novel polypeptide can specifically bind to SGLT2, and by regulating the binding of sodium ions, energy metabolism signaling pathways, conformational stability or the chemical properties of key amino acid residues in the cell, etc., it can further inhibit the activity of SGLT2, reduce the reabsorption of glucose by the kidney, and lower the blood glucose level.

5. Use of the novel polypeptide according to claim 1 in regulating blood glucose levels, characterized in that, The novel polypeptide reduces the reabsorption of glucose by inhibiting the activity of SGLT2, increases the excretion of glucose in the urine, thereby lowering the blood glucose level.

6. A method for synthesizing a novel polypeptide, characterized in that, Amino acid monomers using Boc protecting groups are sequentially linked to a solid-phase support. After each coupling reaction, the Boc group is removed through a deprotection step. The stepwise synthesis method is used to ensure the accuracy and purity of the polypeptide. After all the amino acids are linked, the crude polypeptide is cleaved from the solid-phase support. Its purity and molecular weight are confirmed by high-performance liquid chromatography (HPLC) and mass spectrometry (MS), and various purification methods are used to improve the purity of the polypeptide.

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