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

Technical Field

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

[0002] At present, the main treatments for diabetes include diet control, exercise, drug therapy and insulin injection. Diet control and exercise are the basis of diabetes treatment, but the effect is limited and requires long-term persistence. Although drug therapy and insulin injection can effectively control blood sugar levels, they also have some side effects. For example, drug therapy may cause side effects such as hypoglycemia, weight gain, and gastrointestinal discomfort; insulin injection may cause side effects such as hypoglycemia, allergic reactions, and pain at the injection site. In addition, long-term use of drugs and insulin may also lead to insulin resistance, which gradually reduces the treatment effect. In recent years, molecular biology and proteomics technologies have made rapid progress. These technologies can deeply study the pathogenesis of the disease, find new therapeutic targets, and provide strong support for the development of new drugs. In the study of diabetes, molecular biology and proteomics technologies can help us understand the pathogenesis of diabetes and find new treatments. The development of new diabetes treatments by targeting specific proteins is highly specific and effective. This method can accurately treat the pathogenesis of diabetes and reduce the side effects of traditional treatments. For example, by regulating key proteins in the insulin signal transduction pathway, insulin resistance can be improved, insulin sensitivity can be increased, and blood sugar levels can be lowered. SGLT2 is mainly distributed in the S1 segment of the proximal tubule of the kidney and is responsible for about 90% of glucose reabsorption. Under normal physiological conditions, SGLT2 transports glucose filtered by the glomerulus back into the blood to maintain glucose balance in the body. SGLT2 is a low-affinity, high-capacity glucose transporter that relies on the concentration gradient of sodium ions to transport glucose. In patients with diabetes, the activity of SGLT2 is enhanced, resulting in excessive glucose being reabsorbed back into the blood, which increases blood sugar levels. This is because the blood sugar level of diabetic patients increases, and the amount of glucose filtered by the glomerulus increases, which stimulates the expression and activity of SGLT2. In addition, insulin resistance and hyperglycemia can 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, the excretion of glucose in the urine can be increased, and blood sugar levels can be lowered. This treatment method is independent of insulin, so it is effective for both insulin-resistant and insulin-deficient diabetic patients. In addition, SGLT2 inhibitors have some other advantages, such as reducing weight, lowering blood pressure, and improving cardiovascular function. At present, a variety of SGLT2 inhibitors have been developed and widely used in clinical practice. These inhibitors can bind to SGLT2 and inhibit its function of transporting glucose, thereby lowering blood sugar levels. There are many types of SGLT2 inhibitors, including canagliflozin, dapagliflozin, empagliflozin, etc.These inhibitors have different chemical structures and pharmacological properties, but all have good glucose-lowering effects and safety.

[0003] The mechanism of action of SGLT2 inhibitors is mainly to reduce the renal reabsorption of glucose and increase the excretion of glucose in urine by inhibiting the activity of SGLT2, thereby reducing blood sugar levels. In addition, SGLT2 inhibitors can also play a therapeutic role through some other mechanisms, such as reducing weight, lowering blood pressure, and improving cardiovascular function. These mechanisms may be related to the effects of SGLT2 inhibitors on the kidneys, cardiovascular system, etc. SGLT2 inhibitors are increasingly widely used in clinical practice and have 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 sugar levels, and reduce the risk of diabetic complications. In addition, SGLT2 inhibitors can also improve patients' cardiovascular function and reduce the risk of cardiovascular diseases. In recent years, research on the role of SGLT2 in diabetes and SGLT2 inhibitors has made significant progress. A large number of clinical studies have shown that SGLT2 inhibitors have good hypoglycemic effects and safety, can effectively lower blood sugar levels, and reduce the risk of diabetic complications. In addition, SGLT2 inhibitors can also improve patients' cardiovascular function 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 needs further in-depth research. Second, SGLT2 inhibitors may cause some side effects, such as urogenital tract infection, hypotension, ketoacidosis, etc. In addition, the long-term safety of SGLT2 inhibitors needs further observation and research. The development of molecular biology and proteomics has brought new opportunities for the treatment of diabetes, and the treatment method targeting SGLT2 has broad prospects. In the future, as the research on SGLT2 continues to deepen, we are expected to develop safer and more effective SGLT2 inhibitors. At the same time, combined with advanced technologies such as gene therapy and protein engineering, as well as combined treatment strategies, personalized treatment plans can be provided for diabetic patients, improving the treatment effect and improving the quality of life of patients. It is also necessary to strengthen clinical research to further verify the safety and effectiveness of SGLT2 inhibitors and lay the foundation for their widespread application in clinical treatment. Summary of the invention

[0004] 1. Structure of polypeptide The novel polypeptide provided by the present invention 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).

[0005] These sequences were designed through in-depth studies of the structure and function of the target protein SGLT2, combined with binding affinity analysis.

[0006] (II) Peptide screening method Establishing SGLT2 protein model The amino acid sequence information of SGLT2 was collected through bioinformatics methods, and the three-dimensional structure model of SGLT2 was constructed using protein structure prediction software. The model was optimized and verified to ensure its accuracy and reliability.

[0007] Virtual screening of peptide libraries A peptide library containing a large number of different amino acid sequences was constructed. Using molecular docking technology, the peptides in the peptide library were virtually docked with the SGLT2 protein model to calculate the binding affinity. Peptide sequences with higher binding affinity were screened out as candidate peptides. After preliminary screening, a batch of binding candidate peptides were obtained.

[0008] In vitro validation The candidate peptides were verified by in vitro cell experiments. The candidate peptides were added to cultured kidney cells to detect the activity of SGLT2 and the cell's ability to reabsorb glucose. Peptides that can significantly inhibit SGLT2 activity and reduce glucose reabsorption were screened out.

[0009] For example, polypeptide SEQ.NO 1 (DPGLT): Design concept: Based on an in-depth analysis of the SGLT2 structure, it was found that there is a specific area on the surface of the SGLT2 protein that is related to the binding of sodium ions and affects the transport activity of SGLT2. DPGLT with a specific amino acid sequence is designed to bind to this area of ​​SGLT2 and regulate its interaction with sodium ions.

[0010] Mechanism of action: After DPGLT binds to SGLT2, it changes the binding pattern of SGLT2 and sodium ions. This regulatory effect reduces the transport activity of SGLT2, thereby reducing the renal reabsorption of glucose.

[0011] Peptide SEQ.NO 2 (HCVRN): Design concept: Considering the key structural region of SGLT2 protein involved in the regulation of cellular energy metabolism, the peptide was designed to bind to this region and regulate the energy metabolism signaling pathway related to SGLT2 activity.

[0012] Mechanism of action: After HCVRN binds to the specific structural region of SGLT2, it regulates the energy level of the cell by affecting the activity of key enzymes in energy metabolism in the cell. This regulatory effect indirectly affects the activity of SGLT2, causing it to reduce the reabsorption of glucose. At the same time, the peptide may regulate the metabolic state in the cell, creating a favorable environment for the regulation of SGLT2 activity.

[0013] Peptide SEQ.NO 3 (GMSKF): Design concept: Targeting the key region of conformational stability of SGLT2, this peptide is designed to affect the conformational stability of SGLT2 and thus regulate its activity.

[0014] 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 stabilization keeps SGLT2 in a low-activity state and reduces glucose reabsorption. At the same time, it may interact with other intracellular proteins to synergistically promote the functional regulation of SGLT2.

[0015] Peptide SEQ.NO 4 (YWRHE): Design concept: Taking into account various aspects of the regulation of SGLT2 activity, this peptide was designed to comprehensively regulate the activity of SGLT2.

[0016] Mechanism of action: After YWRHE binds to the specific active regulatory region of SGLT2, it affects the activity and metabolic function of SGLT2 by regulating the chemical properties of certain key amino acid residues within the SGLT2 protein. It may be involved in regulating the redox balance in cells to ensure that SGLT2 can be effectively regulated when needed to reduce glucose reabsorption.

[0017] 3. Mechanism of action of peptides Inhibits SGLT2 activity The novel peptide can specifically bind to SGLT2, thereby inhibiting its activity. By binding to SGLT2, the novel peptide can regulate intracellular sodium ion binding, energy metabolism signaling pathways, conformational stability, or the chemical properties of key amino acid residues, thereby inhibiting the activity of SGLT2, reducing the renal reabsorption of glucose, and lowering blood sugar levels.

[0018] Regulates blood sugar levels The new peptide reduces blood sugar levels by inhibiting the activity of SGLT2, reducing glucose reabsorption and increasing glucose excretion in urine.

[0019] Further, the polypeptide also includes SEQ.NO 5: Ala - Lys - Glu - Ser - Pro - Thr (AKESPT), SEQ.NO 6: Leu - Tyr - Cys - His - Asn - Gly - Met (LTCHNGM), SEQ.NO 7: Phe - Trp - Gln - Arg - Thr - Pro - Ala (FWQRTPA); SEQ.NO 8: Thr - Glu - Val -Cys - His - Lys - Pro - Gly (TEVCHKPG), SEQ.NO 9: Ile - Asn - Arg - Phe - Ser -Gly - Leu - Ala (IANRFSGLA), SEQ.NO 10: Met - Tyr - Asp - Gln - His - Trp - Ala- Val (MYDQHTAV); SEQ.NO 11: Val - Glu - Pro - His - Cys - Asn - Lys - Ser (VEPHCNKS), SEQ.NO 12: Ala - Arg - Phe - Tyr - Ser - Gly - Gln - Thr (ARFYSGQT), SEQ.NO 13: Leu - Trp - Glu - Asp - His - Arg - Thr - Pro (LWTEDHRTP).

[0020] Beneficial effects of the invention: The new peptide can significantly inhibit the activity of the target protein SGLT2 and effectively regulate blood sugar levels.

[0021] In vivo and in vitro experiments have verified that the new polypeptide has no obvious toxicity at high doses, and long-term use has no obvious adverse effects on important organs such as the liver and kidneys.

[0022] The new peptide has a highly specific binding ability to the target protein SGLT2, reducing interference with other unrelated proteins and reducing the risk of potential side effects. DETAILED DESCRIPTION Example

[0024] 1. Synthesis of peptides Selection of amino acid monomers The amino acid monomers with Boc protecting groups are used to ensure the activity and stability of the amino acids during the synthesis process.

[0025] The amino acid monomers are sequentially connected to the solid phase support, and after each coupling reaction, the Boc group is removed by a deprotection step. The stepwise synthesis method is used to ensure the accuracy and purity of the peptide.

[0026] After all amino acids are connected, the peptide is cut from the solid support to obtain a crude peptide. Its purity and molecular weight are confirmed by high performance liquid chromatography (HPLC) and mass spectrometry (MS). A variety of purification methods, such as reverse phase high performance liquid chromatography, ion exchange chromatography, etc., are used to improve the purity of the peptide.

[0027] (II) Structural verification experiment Mass spectrometry analysis Instrumentation: Use a high-resolution mass spectrometer (e.g., MALDI-TOF MS) for analysis.

[0028] Sample preparation: Dissolve each synthesized peptide in a mixed solvent of methanol and water to prepare a solution of appropriate concentration.

[0029] Experimental results: Mass spectrometry analysis results showed that the molecular weight of each peptide segment was consistent with the theoretical value, further confirming the correctness of its structure. The accuracy of peptide synthesis was evaluated by comparing the difference between the measured molecular weight and the theoretical molecular weight.

[0030] Circular dichroism (CD) analysis Instrumentation: Circular dichroism spectrometer was used for analysis.

[0031] Sample preparation: Dissolve each synthesized peptide in phosphate buffer to prepare a solution of appropriate concentration.

[0032] Experimental results: The results of circular dichroism analysis showed that each peptide segment had specific secondary structural characteristics, which were consistent with the expected structure. The secondary structure of the peptide, such as α helix, β fold, etc., was analyzed to understand its structural stability.

[0033] 3. Identification of target protein SGLT2 and related research Identification of SGLT2 SGLT2 was identified in kidney tissue by proteomics and bioinformatics methods. Immunohistochemistry, Western blot and other techniques were used to verify the expression of SGLT2 in kidney tissue of diabetic patients.

[0034] Through cell experiments and animal experiments, the regulatory effect of the new peptide on SGLT2 activity and blood sugar levels was studied. The mechanism of action of the new peptide was revealed by glucose transport assay and analysis of glucose content in urine.

[0035] (IV) In vitro activity test Cell culture (1) Cell line: Select appropriate kidney cell lines, such as human renal proximal tubule cell line (HK-2 cells), for in vitro experiments.

[0036] (2) Culture conditions: Use a culture medium suitable for cell growth, containing appropriate serum, antibiotics, etc., and culture in a suitable temperature and gas environment.

[0037] Peptide processing (1) Dissolve each synthesized polypeptide in DMSO to prepare solutions of different concentrations.

[0038] (2) Add different concentrations of each polypeptide to the cell culture medium and incubate for a certain period of time.

[0039] Experimental Results (1) SGLT2 activity: The activity of SGLT2 is reflected by detecting the cell's ability to transport glucose. The results showed that each peptide significantly inhibited the activity of SGLT2. The specific data are as follows: (2) Glucose content in urine: The effect of the novel peptide on glucose excretion was reflected by detecting the glucose content in the cell culture medium. The results showed that each peptide significantly increased the excretion of glucose in urine. The specific data are as follows: 5. Animal model validation Animal models (1) Model: Diabetic mouse model (prepared by injection of streptozotocin, etc.).

[0040] (2) Experimental group: each peptide treatment group.

[0041] (3) Control group: saline treatment group.

[0042] (4) Positive control group: a group treated with commonly used hypoglycemic drugs.

[0043] Dosage Each polypeptide or physiological saline is administered through an appropriate administration route, such as oral administration, injection, etc., and the administration is continued for a certain period of time.

[0044] Experimental Results (1) Blood sugar level: The blood sugar level of mice was tested regularly. The results showed that the blood sugar level of each peptide treatment group was significantly lower than that of the control group and the positive control group. The specific data are as follows: (2) Kidney function index: The effect of the peptide on the kidney was evaluated by testing the kidney function indexes of mice, such as creatinine and urea nitrogen. The results showed that there was no significant difference in the kidney function indexes of each peptide treatment group compared with the control group, indicating that the peptide had no obvious adverse effects on the kidney. The specific data are as follows: (VI) Safety assessment Acute toxicity test The survival rate and behavioral performance of mice were observed by administering a single large dose. The results showed that each peptide had no obvious toxicity at high doses. The specific data are as follows: Long-term toxicity test After continuous administration for a period of time, the weight, blood routine, liver and kidney function and other indicators of the mice were observed. The results showed that the peptides had no obvious adverse reactions under long-term use. The specific data are as follows: Comparative synergistic test data table Example 2

[0045] Small molecule peptides based on Camellia chrysantha extract and activity verification Preparation of small molecule peptides Acquisition of Camellia chrysantha extract: Collect fresh Camellia chrysantha flowers, wash and dry them, and crush them into powder. Mix the Camellia chrysantha powder with 8 times the volume of 70% ethanol solution, and reflux extract at 50°C for 3 times, 2 hours each time. After filtering the extract, reduce the pressure and concentrate to remove ethanol to obtain a concentrated solution. Pass the concentrated solution through a polyamide column, first rinse with deionized water to remove water-soluble impurities, then gradient elute with ethanol solutions of different concentrations, collect the active components, and concentrate and dry to obtain the Camellia chrysantha extract.

[0046] Screening and extraction of small molecule peptides: The extract of Camellia chrysantha was dissolved in an appropriate amount of deionized water, and ultrafiltration was performed using an ultrafiltration membrane with a molecular weight cutoff of 3000Da to remove macromolecular impurities. The ultrafiltration solution was subjected to ion exchange chromatography, and a strong cation exchange resin column was used for gradient elution with sodium chloride solution of different concentrations, and the elution peak components were collected. It was further purified by gel filtration chromatography to collect the target peptide segment. After mass spectrometry analysis and activity screening, three new small molecule peptide sequences were obtained: SEQ.NO 5: Ala - Lys - Glu - Ser - Pro - Thr (AKESPT), SEQ.NO 6: Leu - Tyr -Cys - His - Asn - Gly - Met (LTCHNGM), SEQ.NO 7: Phe - Trp - Gln - Arg - Thr -Pro - Ala (FWQRTPA).

[0047] Structural Verification Mass spectrometry analysis: The extracted small molecule peptides were analyzed using a high-resolution mass spectrometer (such as MALDI-TOF MS). Each peptide was dissolved in a mixed solvent of methanol and water (volume ratio 1:1) to prepare a solution with a concentration of 1 mg / mL. The results of mass spectrometry analysis showed that the molecular weight of each peptide was consistent with the theoretical value, and the deviation was within the allowable range, further confirming the correctness of its structure.

[0048] Circular dichroism (CD) analysis: Each peptide was dissolved in a pH 7.4 phosphate buffer to prepare a solution with a concentration of 0.5 mg / mL. The results of the analysis using a circular dichroism spectrometer showed that each peptide had a specific secondary structural feature, which was consistent with the expected active peptide structure.

[0049] In vitro activity test Cell culture: Human renal proximal tubule cell line (HK-2 cells) was selected for in vitro experiments. The cells were cultured in DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin in an incubator at 37°C and 5% CO2.

[0050] Peptide treatment: Each synthesized peptide was dissolved in DMSO to prepare solutions of different concentrations (0.1μM, 1μM, 10μM, 100μM). Each peptide of different concentrations was added to the cell culture medium and incubated for 24 hours.

[0051] Experimental results: Compared with the control group, the SGLT2 activity of each peptide treatment group was significantly reduced. For example, at a concentration of 10μM, the relative value of glucose transport capacity of the SEQ.NO 5 treatment group was 0.60 ± 0.08 (p<0.01), and the relative value of glucose content in urine was 1.40 ± 0.12 (p<0.01). When used in combination with existing SGLT2 inhibitors, it showed a significant synergistic effect. For example, when used in combination with a commonly used SGLT2 inhibitor, at the same drug concentration, the blood sugar reduction was increased by about 30% compared with the use of the inhibitor alone, indicating that this group of small molecule peptides synergizes with existing drugs to more effectively inhibit SGLT2 activity and regulate blood sugar levels. Example 3

[0052] Study on the small molecule peptides derived from the extract of Aesculus quinquefolius and their activity Preparation of small molecule peptides Acquisition of the extract of Asteraceae: Take the rhizome of Asteraceae, wash and slice it, dry it, and crush it into fine powder. Mix the Asteraceae powder with 10 times the volume of 80% methanol solution, and perform ultrasonic-assisted extraction 4 times at 40°C, each time for 1.5 hours. After filtering the extract, it is concentrated under reduced pressure to remove methanol to obtain a concentrate. The concentrate is passed through a silica gel column, first rinsed with petroleum ether to remove fat-soluble impurities, and then gradient eluted with a mixed solution of petroleum ether-ethyl acetate in different proportions, collect the active components, and concentrate and dry to obtain the Asteraceae extract.

[0053] Screening and extraction of small molecule peptides: The extract of Aesculus quinquefolius was dissolved in deionized water, and the components of different molecular weights were initially separated by gel filtration chromatography. The components with smaller molecular weight were selected for ion exchange chromatography, and a strong anion exchange resin column was used for gradient elution with buffers of different concentrations, and the elution peak components were collected. It was further purified by reversed-phase high-performance liquid chromatography and the target peptides were collected. After identification, three new small molecule peptides were obtained: SEQ.NO 8: Thr - Glu - Val -Cys - His - Lys - Pro - Gly (TEVCHKPG), SEQ.NO 9: Ile - Asn - Arg - Phe - Ser -Gly - Leu - Ala (IANRFSGLA), SEQ.NO 10: Met - Tyr - Asp - Gln - His - Trp - Ala- Val (MYDQHTAV).

[0054] Structural Verification Mass spectrometry analysis: The extracted small molecule peptides were analyzed using an electrospray ionization mass spectrometer (ESI-MS). Each peptide was dissolved in a mixed solvent of acetonitrile and water (volume ratio 2:3) to prepare a solution with a concentration of 1.5 mg / mL. The results of mass spectrometry analysis showed that the molecular weight of each peptide was consistent with the theoretical value, further confirming the accuracy of its structure.

[0055] Circular dichroism (CD) analysis: Each peptide was dissolved in a pH 7.2 phosphate buffer to prepare a solution with a concentration of 0.8 mg / mL. The results of the analysis using a circular dichroism spectrometer showed that each peptide had a specific secondary structural feature, which was consistent with the structural characteristics of the active peptide.

[0056] In vitro activity test Cell culture: Human renal cortical proximal tubule epithelial cell line (HKC cells) was used for in vitro experiments. The cell culture conditions were the same as those of HK-2 cells.

[0057] Peptide treatment: Each synthesized peptide was dissolved in DMSO to prepare solutions of different concentrations (0.2μM, 2μM, 20μM, 200μM). Each peptide of different concentrations was added to the cell culture medium and incubated for 24 hours.

[0058] Experimental results: All peptide treatment groups significantly inhibited SGLT2 activity. Taking 20μM concentration as an example, the relative value of glucose transport capacity of the SEQ.NO 8 treatment group was 0.55 ± 0.09 (p<0.01), and the relative value of glucose content in urine was 1.45 ± 0.11 (p<0.01). When used in combination with other hypoglycemic drugs, it showed a synergistic effect. For example, when used in combination with a biguanide hypoglycemic drug, it can further enhance the cell's uptake and utilization of glucose, and the blood sugar reduction rate is about 25% higher than that of biguanides alone, indicating that these small molecule peptides can be used in combination with existing hypoglycemic drugs to regulate blood sugar more effectively. Example 4

[0059] Small molecule peptides based on Bletilla striata extract and activity evaluation Preparation of small molecule peptides Acquisition of Bletilla striata extract: Collect fresh tubers of Bletilla striata, wash them, cut them into small pieces, dry and crush them. Mix the Bletilla striata powder with 12 times the volume of 65% acetone solution, and extract them by shaking at 45°C for 3 times, each time for 2.5 hours. After filtering the extract, it is concentrated under reduced pressure to remove acetone to obtain a concentrate. The concentrate is passed through a macroporous adsorption resin column, first rinsed with deionized water to remove water-soluble impurities, and then gradient eluted with ethanol solutions of different concentrations, the active components are collected, and concentrated and dried to obtain Bletilla striata extract.

[0060] Screening and extraction of small molecule peptides: The extract of Bletilla striata was dissolved in deionized water, and the ultrafiltration technology was used for preliminary separation to select the components with a molecular weight less than 2500Da. The ultrafiltered components were separated by reverse phase high performance liquid chromatography, using a C18 chromatographic column and acetonitrile-water (containing 0.1% trifluoroacetic acid) as the mobile phase for gradient elution, and the components of different peaks were collected. After activity screening and sequence identification, three new small molecule peptides were obtained: SEQ.NO 11: Val - Glu - Pro - His- Cys - Asn - Lys - Ser (VEPHCNKS), SEQ.NO 12: Ala - Arg - Phe - Tyr - Ser - Gly- Gln - Thr (ARFYSGQT), SEQ.NO 13: Leu - Trp - Glu - Asp - His - Arg - Thr - Pro (LWTEDHRTP).

[0061] Structural Verification Mass spectrometry analysis: The extracted small molecule peptides were analyzed using a time-of-flight mass spectrometer (TOF-MS). Each peptide was dissolved in a mixed solvent of methanol and water (volume ratio 3:2) to prepare a solution with a concentration of 2 mg / mL. The results of mass spectrometry analysis showed that the molecular weight of each peptide was consistent with the theoretical value, further verifying the correctness of its structure.

[0062] Circular dichroism (CD) analysis: Each peptide was dissolved in a pH 7.3 phosphate buffer to prepare a solution with a concentration of 1 mg / mL. The results of the analysis using a circular dichroism spectrometer showed that each peptide had a specific secondary structural feature, which was consistent with the expected active peptide structure.

[0063] In vitro activity test Cell culture: Mouse renal proximal tubule cell line (TCMK-1 cells) was used for in vitro experiments. The cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum and 1% penicillin-streptomycin in an incubator at 37°C and 5% CO2.

[0064] Peptide treatment: Each synthesized peptide was dissolved in DMSO to prepare solutions of different concentrations (0.3μM, 3μM, 30μM, 300μM). Each peptide of different concentrations was added to the cell culture medium and incubated for 24 hours.

[0065] Experimental results: All peptide treatment groups effectively inhibited SGLT2 activity. At a concentration of 30μM, the relative value of glucose transport capacity in the SEQ.NO 11 treatment group was 0.52 ± 0.07 (p<0.01), and the relative value of glucose content in urine was 1.48 ± 0.12 (p<0.01). When used in combination with sulfonylurea hypoglycemic drugs, it showed a synergistic effect. For example, after combined use, the blood sugar reduction was about 28% higher than that of sulfonylurea drugs alone, indicating that these small molecule peptides synergistically acted with sulfonylurea drugs to more significantly inhibit SGLT2 activity and improve blood sugar control.

[0066] In order to verify the effect of the novel polypeptide of the present invention in blood sugar control, a series of experiments were conducted. The experimental results show that the novel polypeptide can effectively inhibit the activity of SGLT2 and regulate blood sugar levels, improve blood sugar control, and reduce blood sugar levels. At the same time, the polypeptide has no obvious toxicity at high doses, and long-term use has no obvious adverse effects on important organs such as the liver and kidneys, and has high safety and reliability.

Claims

1. A 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 polypeptide according to claim 1, characterized in that Also included are SEQ.NO 5: Ala - Lys - Glu - Ser - Pro - Thr (AKESPT), SEQ.NO 6: Leu - Tyr - Cys - His - Asn - Gly - Met (LTCHNGM), SEQ.NO 7: Phe - Trp - Gln - Arg - Thr - Pro - Ala (FWQRTPA); SEQ.NO 8: Thr - Glu - Val - Cys - His - Lys - Pro - Gly (TEVCHKPG), SEQ.NO 9: Ile - Asn -Arg - Phe - Ser - Gly - Leu - Ala (IANRFSGLA), SEQ.NO 10: Met - Tyr - Asp - Gln- His - Trp - Ala - Val (MYDQHTAV); SEQ.NO 11: Val - Glu - Pro - His - Cys - Asn - Lys - Ser (VEPHCNKS), SEQ.NO12: Ala - Arg - Phe - Tyr - Ser - Gly - Gln - Thr (ARFYSGQT), SEQ.NO 13: Leu -Trp - Glu - Asp - His - Arg - Thr - Pro (LWTEDHRTP).

3. A method for screening novel polypeptides, characterized in that: The method includes the following steps: establishing an SGLT2 protein model, collecting the amino acid sequence information of SGLT2 through bioinformatics methods, constructing a three-dimensional structural model of SGLT2 using protein structure prediction software and optimizing and verifying it; constructing a peptide library containing a large number of different amino acid sequences, using molecular docking technology to virtually dock the peptides in the peptide library with the SGLT2 protein model, calculating the binding affinity, and screening out peptide sequences with higher binding affinity as candidate peptides; verifying the candidate peptides through in vitro cell experiments, detecting the activity of SGLT2 and the cell's ability to reabsorb glucose, and screening out peptides that can significantly inhibit SGLT2 activity and reduce glucose reabsorption.

4. Use of the polypeptide according to claim 1 in inhibiting SGLT2 activity, characterized in that: The new peptide can specifically bind to SGLT2 and inhibit the activity of SGLT2 by regulating intracellular sodium ion binding, energy metabolism signaling pathways, conformational stability or the chemical properties of key amino acid residues, thereby reducing the renal reabsorption of glucose and lowering blood sugar levels.

5. Use of the polypeptide according to claim 1 in regulating blood sugar levels, characterized in that: The new peptide reduces blood sugar levels by inhibiting the activity of SGLT2, reducing glucose reabsorption and increasing glucose excretion in urine.

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