A Screening Method and Application of DPP-IV Inhibitory Peptides

Abstract

The present invention discloses a screening method and application of DPP-IV inhibitory peptides, belonging to the technical field of bioactive peptides. Specifically, it involves identifying the potential DPP-IV inhibitory peptide TENEWK in wheat protein by combining computer simulation and experimental verification methods, and using virtual screening technology. Research shows that TENEWK exhibits significant DPP-IV inhibitory activity in vitro and does not show obvious cytotoxicity in Caco-2 cells, indicating its good safety. The present invention successfully screens and verifies the bioactive peptide TENEWK with good DPP-IV inhibitory activity, providing new raw materials and theoretical basis for the development of drugs related to diabetes treatment. At the same time, the present invention provides a new direction for the research and development of natural DPP-IV inhibitors based on wheat protein.

Description

Technical Field

[0001] The present invention belongs to the technical field of bioactive peptides, and particularly relates to a screening method and application of DPP-IV inhibitory peptides. Background Art

[0002] DPP-IV (dipeptidyl peptidase-IV), as an important transmembrane serine protease, plays a key role in glucose metabolism. It regulates insulin secretion and glucose metabolism by degrading glucagon-like peptide-1 (GLP-1) and gastric inhibitory polypeptide (GIP). GLP-1 can promote insulin secretion and inhibit glucagon release, thereby effectively reducing blood glucose levels. However, DPP-IV rapidly degrades GLP-1, limiting its physiological effects. Therefore, inhibiting DPP-IV has become an important strategy in the treatment of type 2 diabetes. Currently, various DPP-IV inhibitors (such as sitagliptin and saxagliptin) are widely used clinically, showing significant efficacy. However, existing small molecule DPP-IV inhibitors also have certain side effects in clinical applications, including gastrointestinal discomfort, increased risk of infection, and potential cardiovascular problems associated with long-term use. These side effects have led to a need for safer and less toxic treatment options. Bioactive peptides, as an emerging therapeutic strategy, have been considered ideal candidates for developing DPP-IV inhibitors due to their low toxicity, good biocompatibility, and significant physiological activities. In particular, peptides derived from plant proteins have gradually shown great potential in pharmacological research in recent years.

[0003] Although multiple studies have explored the development of DPP-IV inhibitory peptides, there are still some limitations in the efficiency of screening peptide molecules and the diversity of screening methods. Therefore, combining virtual screening technology and experimental verification to efficiently and accurately screen peptides with DPP-IV inhibitory activity remains an important challenge in current research. In recent years, peptide screening methods based on deep learning have been proven to be able to efficiently process large-scale data and show high accuracy in predicting the interaction between peptides and targets. This method significantly improves the screening efficiency, enabling us to more quickly identify peptides with DPP-IV inhibitory potential. Summary of the Invention

[0004] The present invention innovatively combines computational simulation and experimental verification to accelerate the screening process and reduce costs. By simulating the enzymatic hydrolysis process of wheat protein and performing virtual screening, peptides with potential DPP-IV inhibitory activity are identified, and their biological activities are further verified through a series of in vitro experiments. Therefore, the method combining theoretical calculation and experimental evaluation can be used as an effective method for screening DPP-IV inhibitory peptides.

[0005] The present invention provides a DPP-IV inhibitory peptide TENEWK screened from wheat protein and its application in the preparation of anti-diabetic drugs.

[0006] To achieve the above object, the present invention adopts the following technical solutions:

[0007] The present invention provides a DPP-IV inhibitory peptide, the amino acid sequence of which is: TENEWK, as shown in SEQ ID NO.1.

[0008] In addition, the present invention also provides the application of the DPP-IV inhibitory peptide TENEWK in the preparation of products for treating diabetes.

[0009] Furthermore, the product is a drug for treating diabetes.

[0010] Furthermore, the drug further comprises one or more pharmaceutically acceptable carriers.

[0011] Furthermore, the pharmaceutical dosage form is selected from injection, solution, emulsion, suspension, suppository, ointment, cream, spray, drop, powder, granule, infusion, capsule, pill, tablet, patch or sustained-release preparation.

[0012] Compared with the prior art, the present invention has the following beneficial effects:

[0013] The object of the present invention is to extract the DPP-IV inhibitory peptide TENEWK from wheat protein hydrolysate and systematically evaluate its DPP-IV inhibitory activity. The implementation of this technology can not only improve the added value of wheat, but also discover the DPP-IV inhibitory peptide TENEWK, which has significant application prospects, especially in the treatment field of diabetes and metabolic diseases.

[0014] The present invention combines virtual screening with experimental verification, which greatly improves the efficiency of screening DPP-IV inhibitory peptides, can quickly identify the most potential peptide compounds from a large number of candidate molecules, thus saving a large amount of experimental costs and time, and significantly improving the discovery efficiency of lead peptides. This efficient and economical screening method not only reduces resource waste, but also improves the accuracy and reliability of the screening process. At the same time, by screening bioactive DPP-IV inhibitory peptides from wheat protein, the utilization rate of wheat protein is successfully improved, realizing the value-added utilization of agricultural resources, and providing new ideas for the deep processing of crops such as wheat. This technology not only provides potential solutions for the treatment of diabetes and metabolic diseases, but also can be widely applied to multiple fields such as functional foods, health products and pharmaceutical preparations, with broad market prospects and significant social and economic benefits. Description of the Drawings

[0015] Figure 1 For the inhibitory effect of TENEWK on DPP-IV in Example 1;

[0016] Figure 2 For the IC 50 value of TENEWK on DPP-IV in Example 1;

[0017] Figure 3 For the line graph of the inhibition type of TENEWK on DPP-IV in Example 1;

[0018] Figure 4 For the line graph of the inhibition constant of TENEWK on DPP-IV in Example 1;

[0019] Figure 5 For the graph of the effect of different culture times of TENEWK on the viability of Caco-2 cells in Example 1;

[0020] Figure 6 For the evaluation graph of the effect of different treatment times of TENEWK on the expression of DPP-IV protein in Caco-2 cells in Example 1 (where, : p < 0.0001, : p < 0.001, : p < 0.01, : p < 0.1);

[0021] Figure 7 For the graph of the change in blood glucose of mice before and after gavage in Example 1;

[0022] Figure 8 For the effect of TENEWK on the blood glucose of mice in Example 1; Detailed implementation mode

[0023] For better illustration of the present invention, specific examples are listed as follows. Obviously, the described examples are only a part of the present invention, rather than all examples. Based on the examples in the present invention, other examples obtained by those skilled in the art without creative efforts all fall within the scope of protection of the present invention.

[0024] The technical solution of the present invention will be further described below through the drawings and examples.

[0025] Example 1 (1) Virtual screening of DPP-IV inhibitory peptides

[0026] The present invention first uses the Bio.Entrez module in the python script to batch obtain wheat in the protein database of NCBI ( Triticum aestivum L.Protein sequence. Using the PeptideCutter module on the Expasy website (https: / / web.Expasy.org / peptide_cutter / ), virtual digestion was performed with trypsin as the enzyme to generate peptide fragments of 4 to 6 amino acids. A total of 245,751 polypeptides were obtained and subjected to subsequent screening. The polypeptides were converted to SMILES format, and the FASTA sequence of human DPP-IV protein (UniProt ID: P27487) was obtained from the UniProt database. The ConPlex deep learning algorithm was used to score and rank the interactions between the protein and the polypeptides, predicting the inhibitory potential of the polypeptides against DPP-IV. Subsequently, the toxicity of each peptide was evaluated using the machine learning-based toxicity prediction system ToxinPred, providing a strong basis for subsequent in vitro experiments. The top ten ranking results of the binding scores between the inhibitory peptides and DPP-IV are shown in Table 1. According to the results in Table 1, the inhibitory peptide TENEWK (SEQ ID NO.1) ranked first in terms of scoring and had no toxicity.

[0027] Table 1 Prediction of DPP-IV inhibitory activity of peptides

[0028]

[0029] (2) Determination of DPP-IV inhibitory activity

[0030] The inhibitory activity of the screened DPP-IV inhibitory peptides was determined. The DPP-IV inhibitory peptides were serially diluted (1.0, 2.5, 5.0, 10.0 mM). In the sample group, 10 μL of DPP-IV (final concentration 100 ng / mL), 50 μL of the substrate Gly-Pro-PNA (final concentration 500 μM), and 40 μL of the peptide sample were added. The control group did not contain DPP-IV, and the blank group contained neither DPP-IV nor the peptide sample.

[0031] After the reaction solution was incubated at 37 °C for 30 minutes, the absorbance was measured at a wavelength of 405 nm. All reaction solutions were prepared using Tris-HCl buffer (0.1 M, pH 8.0). The concentration of the converted DPP-IV inhibitory peptide was plotted on the x-axis, and the corresponding DPP-IV inhibition rate was plotted on the y-axis to determine the IC 50 value. The formula for calculating the DPP-IV inhibition rate is as follows:

[0032] DPP-IV inhibition rate (%) = 1 - (OD s - OD c ) / OD b × 100%

[0033] Among them, ODs, ODc, and OD b represent the absorbance of the sample group, control group, and blank group at a wavelength of 405 nm, respectively.

[0034] At a concentration of 10 mM, the inhibition rate of TENEWK was 68.89% ( Figure 1 ), and according to the inhibition rate, the IC 50 value of TENEWK was calculated. The results showed that the IC 50 value of TENEWK was 4.96 mM ( Figure 2 ), verifying the inhibitory activity of TENEWK.

[0035] (3) Inhibition type of DPP-IV inhibitory peptide

[0036] By using different concentrations of Gly-Pro-PNA as substrates (250, 500, 1000, 2000 μM), the reaction rate between the peptide and DPP-IV was measured. The inhibition type of the peptide on DPP-IV was determined by constructing a Lineweaver-Burk double-reciprocal plot. From the Lineweaver-Burk plot, the slope corresponding to a given DPP-IV inhibitor concentration can be obtained.

[0037] As Figure 3 shown, the Lineweaver-Burk plot indicated that TENEWK was a competitive inhibitor of DPP-IV. This was confirmed by the increase in the slope when the peptide concentration increased, indicating an increase in the Km value. However, the change in concentration had no effect on the maximum reaction rate (Vmax) because the intersection points of the reaction rates at different concentrations on the y-axis were the same, indicating that Vmax remained stable.

[0038] The slopes (Km values) of TENEWK at different concentrations were obtained from the Lineweaver-Burk plot and subjected to linear analysis to calculate the Ki value, as Figure 4 shown. The results showed that the Ki value of TENEWK was 1.06 mM.

[0039] (4) Effect of DPP-IV inhibitory peptide on the activity of Caco-2 cells

[0040] Caco-2 cells in the logarithmic growth phase were seeded into 96-well plates at a density of 1×10 4 cells / well (where the well plates contained DMEM medium with 20% fetal bovine serum and 1% penicillin-streptomycin), and cultured in an incubator at 37°C and 5% CO 2 for 48 hours.

[0041] After removing the medium, the experimental group was added with IC 50DPP-IV inhibitory peptides at different concentrations were incubated for 2 hours and 4 hours respectively, and the control group was added with the same dose of culture medium. To evaluate the effect of DPP-IV inhibitory peptides on cell viability, the CCK-8 method was used for detection, and the absorbance was measured at a wavelength of 450 nm. The CCK-8 method was used to evaluate the cytotoxicity of these peptides on Caco-2 cells.

[0042] After being stimulated by TENEWK for 2 hours and 4 hours, the cell viabilities were 80.27% and 80.77% respectively ( Figure 5 ). According to the standard of the CCK-8 method, a cell viability greater than 75% indicates that the compound is non-toxic to cells. The above results show that TENEWK has no cytotoxic effect on Caco-2 cells.

[0043] (5)Expression of DPP-IV in Caco-2 cells

[0044] Caco-2 cells in the logarithmic growth phase were seeded into 96-well plates at a density of 1×10 4 cells / well and cultured for 48 hours. The culture conditions were the same as the set parameters in the above cell viability assay.

[0045] After removing the culture medium, the sample groups were treated with DPP-IV inhibitory peptides at IC 50 concentrations and incubated for 2 hours and 4 hours. The control group was not treated. The cells were lysed with RIPA lysis buffer for 3 minutes, and then centrifuged at 12,000 rpm for 10 minutes at 4°C to collect cell proteins. The protein concentrations under different treatment conditions were determined by the BCA method.

[0046] The proteins were separated by 10% SDS-PAGE gels and transferred to polyvinylidene difluoride (PVDF) membranes. The membranes were blocked with 5% BSA at 37°C for 40 minutes. The primary antibodies against DPP-IV (1:1000) and β-actin (1:20,000) were incubated overnight at 4°C.

[0047] After washing the membranes with TBST, the secondary antibody (1:2000) was added and incubated at 37°C for 40 minutes. The membranes were washed with TBST again. After adding the chemiluminescent substrate, exposure was performed and the results were detected.

[0048] This study further explored the expression of DPP-IV protein in Caco-2 cells ( Figure 6 ). Caco-2 cells were treated with IC 50Caco-2 cells were stimulated with TENEWK at a concentration of for 2 hours and 4 hours. Compared with the control group, after treatment with TENEWK, the expression of DPP-IV protein in Caco-2 cells was significantly decreased. In addition, with the extension of the stimulation time, the inhibitory effect on DPP-IV expression became more obvious. These results indicate that TENEWK can effectively inhibit the expression of DPP-IV.

[0049] (6)Effect of TENEWK on blood glucose in mice

[0050] Kunming mice at 8 weeks of age and weighing about 20 g were selected. After one week of adaptive feeding, they were intraperitoneally injected with STZ solution at a concentration of 5 mg / ml every morning, at a dose of 35 mg / kg per day according to body weight, and supplemented with a high-fat and high-sugar diet to induce the establishment of a type 2 diabetes mouse model. After continuous injection for three days, the blood glucose of the mice rose to 23.43 mmol / L (≥11.1 mmol / L), indicating successful modeling. The mice were randomly divided into 6 groups according to body weight and blood glucose value: normal group (Normal), model group (Model), low-dose TENEWK administration group (20 mg / kg, TENEWK), medium-dose administration group (40 mg / kg, TENEWK), high-dose administration group (80 mg / kg, TENEWK), and positive drug sitagliptin phosphate group (20 mg / kg, Sitagliptin). The mice were given drugs or normal saline by gavage according to their groups, and the gavage was carried out at the same time every afternoon in the same order for 8 consecutive weeks. The normal group and the model group were given normal saline by gavage at the same dose.

[0051] The average blood glucose concentration after film formation was 24.27 mmol / L. After continuous gavage for 8 weeks, the blood glucose concentration of the mice in the model group was 29.47 mmol / L. The blood glucose concentration of the mice in the TENEWK gavage group decreased significantly compared with that before gavage, and the decrease in the high-dose group (80 mg / kg) was more obvious, dropping to 19.24 mmol / L. The blood glucose concentration of the sitagliptin phosphate administration group decreased significantly compared with that before gavage, dropping to 18.43 mmol / L ( Figure 7 )

[0052] After successfully establishing a type 2 diabetic mouse model, the mice were given intragastric administration for 8 consecutive weeks. From the first day of intragastric administration, the fasting blood glucose concentration was measured once a week. After fasting for 8 h without water deprivation, the blood glucose concentration of the mice was detected by tail vein blood sampling. The results showed that the blood glucose level of the normal group mice remained basically unchanged after 8 weeks of intragastric administration of normal saline, while the blood glucose concentration of the type 2 diabetic mice increased slightly in the first 2 weeks before intragastric administration and decreased significantly from the 3rd week. Compared with the model group, the blood glucose concentration of the mice in the low and medium dose (20 mg / kg, 40 mg / kg) intragastric administration groups of TENEWK decreased slightly, and the blood glucose concentration of the mice in the high dose intragastric administration group (80 mg / kg) decreased significantly, from 24.53 mmol / L to 19.2 mmol / L ( Figure 8 ). This result indicates that the DPP-IV inhibitory peptide TENEWK can regulate blood glucose in diabetic mice.

[0053] The present invention designs a preparation method for screening the DPP-IV inhibitory peptide TENEWK from wheat and its application in the treatment of diabetes, aiming to solve the side effect problems of existing DPP-IV inhibitors in clinical applications and provide new ideas for the development of DPP-IV inhibitors. By combining computer simulation and experimental verification methods, virtual screening technology is used to identify the potential DPP-IV inhibitory peptide TENEWK in wheat protein. The implementation of this method not only significantly improves the screening efficiency of DPP-IV inhibitory peptides but also reduces the experimental cost. Research shows that TENEWK exhibits significant DPP-IV inhibitory activity in vitro and does not show obvious cytotoxicity in Caco-2 cells, indicating its good safety. In addition, TENEWK can effectively inhibit the expression of DPP-IV in Caco-2 cells, further verifying its potential as a DPP-IV inhibitor. Animal experiments also show that TENEWK can regulate blood glucose in diabetic mice. The present invention successfully screens and verifies the bioactive peptide TENEWK with good DPP-IV inhibitory activity, providing new raw materials and theoretical basis for the development of drugs related to diabetes treatment. At the same time, the present invention provides a new direction for the research and development of natural DPP-IV inhibitors based on wheat protein.

[0054] In summary, the present invention not only has high application prospects and social benefits but also provides new ideas for the further research and development of DPP-IV inhibitors, having important scientific significance and economic value.

[0055] The above-described embodiments are only used to describe the preferred embodiments of the present invention and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the claims of the present invention.

Claims

1. A DPP-IV inhibitory peptide, characterized in that Its amino acid sequence is: TENEWK, as shown in SEQ ID NO.

1.

2. Use of the DPP-IV inhibitory peptide TENEWK according to claim 1 in the preparation of a drug for treating diabetes.

3. The use according to claim 2, characterized in that: The medicine further comprises one or more pharmaceutically acceptable carriers.

4. The use according to claim 3, characterized in that: The pharmaceutical dosage form is selected from injection, solution, emulsion, suspension, suppository, ointment, cream, spray, drop, powder, granule, granule, capsule, pill, tablet, patch or sustained-release preparation.

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