Screening method and application of DPP-IV inhibitory peptide

Abstract

The invention discloses a screening method and application of a DPP-IV inhibitory peptide, and belongs to the technical field of bioactive peptides. Specifically, the invention relates to a method combining computer simulation and experimental verification, and a virtual screening technology is adopted to identify the potential DPP-IV inhibitory peptide TENEWK in wheat protein. Researches show that the TENEWK shows remarkable DPP-IV (dipeptidyl peptidase-IV) inhibitory activity in vitro and does not show obvious cytotoxicity in Caco-2 cells, so that the TENEWK has good safety. According to the invention, the bioactive peptide TENEWK with good DPP-IV inhibitory activity is successfully screened and verified, and a new raw material and a theoretical basis are provided for development of related drugs for treating diabetes mellitus. Meanwhile, the invention provides a new direction for research and development of a natural DPP-IV inhibitor based on wheat protein.

Description

Technical Field

[0001] The present invention belongs to the technical field of bioactive peptides, and in particular relates to a screening method and application of a DPP-IV inhibitory peptide. 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 peptide (GIP). GLP-1 can promote insulin secretion and inhibit the release of glucagon, thereby effectively lowering blood glucose levels. However, DPP-IV rapidly degrades GLP-1, resulting in limited physiological effects. Therefore, inhibiting DPP-IV has become an important strategy in the treatment of type 2 diabetes. Currently, there are a variety of DPP-IV inhibitors on the market (such as sitagliptin and saxagliptin) that are widely used in clinical practice and show 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 cardiovascular problems that may occur with long-term use. These side effects have triggered the need for safer and less toxic treatment options. As an emerging therapeutic strategy, bioactive peptides have been considered ideal candidates for the development of DPP-IV inhibitors due to their low toxicity, good biocompatibility, and significant physiological activity. In particular, peptides derived from plant proteins have gradually shown great potential in pharmacological research in recent years.

[0003] Although many 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, deep learning-based peptide screening methods have been shown 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, allowing us to more quickly identify peptides with DPP-IV inhibitory potential. Summary of the invention

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

[0005] The invention provides a DPP-IV inhibitory peptide TENEWK screened from wheat protein and application of the peptide in preparing an anti-diabetic drug.

[0006] To achieve the above object, the present invention adopts the following technical solutions: The present invention provides a DPP-IV inhibitory peptide, whose amino acid sequence is: TENEWK, as shown in SEQ ID NO.1.

[0007] In addition, the present invention also provides the use of the DPP-IV inhibitory peptide TENEWK in the preparation of a product for treating diabetes.

[0008] Furthermore, the product is a drug for treating diabetes.

[0009] Furthermore, the drug also contains one or more pharmaceutically acceptable carriers.

[0010] Furthermore, the drug 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.

[0011] Compared with the prior art, the present invention has the following beneficial effects: The purpose of the present invention is to extract 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 increase the added value of wheat, but also discover DPP-IV inhibitory peptide TENEWK, which has significant application prospects, especially in the treatment of diabetes and metabolic diseases.

[0012] The present invention greatly improves the efficiency of DPP-IV inhibitory peptide screening by combining virtual screening with experimental verification, and can quickly identify the most promising peptide compounds from a large number of candidate molecules, thereby saving a lot of experimental costs and time, and significantly improving the efficiency of lead peptide discovery. 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 out biologically active DPP-IV inhibitory peptides from wheat protein, the utilization rate of wheat protein is successfully improved, the value-added utilization of agricultural resources is achieved, and new ideas are provided for the deep processing of crops such as wheat. This technology not only provides a potential solution for the treatment of diabetes and metabolic diseases, but can also be widely used in multiple fields such as functional foods, health products and pharmaceutical preparations, with broad market prospects and significant social and economic benefits. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1The inhibitory effect of TENEWK on DPP-IV in Example 1; Figure 2 is the IC of TENEWK to DPP-IV in Example 1 50 value; Figure 3 It is a line graph of the inhibition type of TENEWK on DPP-IV in Example 1; Figure 4 It is a line graph of the inhibition constant of TENEWK on DPP-IV in Example 1; Figure 5 This is a graph showing the effect of different TENEWK culture times on Caco-2 cell viability in Example 1; Figure 6 This is a graph evaluating the effect of different TENEWK treatment times on DPP-IV protein expression in Caco-2 cells in Example 1 (wherein, : p<0.0001, : p<0.001, : p<0.01, :p<0.1); Figure 7 This is a graph showing changes in blood sugar in mice before and after intragastric administration in Example 1; Figure 8 The effect of TENEWK on blood glucose in mice in Example 1; DETAILED DESCRIPTION

[0014] In order to better illustrate the present invention, the following embodiments are listed. Obviously, the described embodiments are only a part of the present invention, not all embodiments. Based on the embodiments in the present invention, other embodiments obtained by those skilled in the art without creative work are within the scope of protection of the present invention.

[0015] The technical solution of the present invention is further described below through the accompanying drawings and embodiments.

[0016] Example 1 (1) Virtual screening of DPP-IV inhibitory peptides The present invention first uses the Bio.Entrez module in the python script to batch obtain wheat ( Triticum aestivum L.) protein sequence. The PeptideCutter module on the Expasy website (https: / / web.Expasy.org / peptide_cutter / ) was used to perform virtual digestion with trypsin to generate peptide fragments of 4 to 6 amino acids. A total of 245,751 peptides were obtained and subsequently screened. The peptides were converted into 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 proteins and peptides to predict the inhibitory potential of peptides on DPP-IV. Subsequently, the machine learning prediction toxicity scoring system ToxinPred was used to evaluate the toxicity of each peptide, and it provided a strong basis for subsequent in vitro experiments. The top ten ranking results of the inhibitory peptide and DPP-IV binding scores are shown in Table 1. According to the results in Table 1, the inhibitory peptide TENEWK (SEQ ID NO.1) ranked first and had no toxicity.

[0017] Table 1 Prediction of peptide DPP-IV inhibitory activity

[0018] (2) Determination of DPP-IV inhibitory activity The inhibitory activity of the screened DPP-IV inhibitory peptides was determined. The DPP-IV inhibitory peptides were diluted in a gradient (1.0, 2.5, 5.0, 10.0 mM). In the sample group, 10 μL of DPP-IV (final concentration of 100 ng / mL), 50 μL of substrate Gly-Pro-PNA (final concentration of 500 μM), and 40 μL of peptide samples were added. The control group did not contain DPP-IV, and the blank group contained neither DPP-IV nor peptide samples.

[0019] 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 converted DPP-IV inhibitory peptide concentration was plotted on the x-axis and the corresponding DPP-IV inhibition rate was plotted on the y-axis to determine the IC 50 The DPP-IV inhibition rate is calculated as follows: DPP-IV inhibition rate (%) = 1 - (OD s -OD c ) / OD b × 100% 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.

[0020] At a concentration of 10 mM, the inhibition rate of TENEWK was 68.89% ( Figure 1 ), based on the inhibition rate, the IC of TENEWK was calculated 50 The results show that TENEWK's IC 50 The value is 4.96 mM ( Figure 2 ), verifying the inhibitory activity of TENEWK.

[0021] (3) Inhibition type of DPP-IV inhibitory peptide The reaction rate between the peptide and DPP-IV was measured by using different concentrations of Gly-Pro-PNA as substrate (250, 500, 1000, 2000 μM). The type of inhibition 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.

[0022] like Figure 3 As shown, the Lineweaver-Burk plot indicates that TENEWK is a competitive inhibitor of DPP-IV. This is confirmed by the increase in slope as the peptide concentration increases, indicating an increase in the Km value. However, the concentration change has no effect on the maximum reaction rate (Vmax), because the reaction rates at different concentrations intersect the same point on the y-axis, indicating that Vmax remains stable.

[0023] The slopes (Km values) of TENEWK at different concentrations were obtained from the Lineweaver-Burk plot, and linear analysis was performed to calculate the Ki values, as shown in Figure 4 The results showed that the TENEWK Ki value was 1.06 mM.

[0024] (4) Effects of DPP-IV inhibitory peptide on Caco-2 cell activity Caco-2 cells in the logarithmic growth phase were cultured at 1×10 4 The cells were seeded at a density of 10 cells / well into a 96-well plate (the plate contained DMEM medium containing 20% ​​fetal bovine serum and 1% penicillin-streptomycin) and cultured in an incubator at 37°C and 5% CO2 for 48 hours.

[0025] After removing the culture medium, IC 50The DPP-IV inhibitory peptides were incubated for 2 hours and 4 hours, respectively, and the control group was added with the same dose of culture medium. In order to evaluate the effect of DPP-IV inhibitory peptides on cell activity, the CCK-8 method was used for detection, and the absorbance was measured at a wavelength of 450 nm. The cytotoxicity of these peptides on Caco-2 cells was evaluated using the CCK-8 method.

[0026] After TENEWK stimulation for 2 hours and 4 hours, the cell viability was 80.27% and 80.77%, respectively ( Figure 5 ). According to the standard of CCK-8 method, cell activity 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.

[0027] (5) Expression of DPP-IV in Caco-2 cells Caco-2 cells in the logarithmic growth phase were counted at 1×10 4 The cells were seeded into a 96-well plate at a density of 10 cells / well and cultured for 48 hours. The culture conditions were consistent with the parameters set in the above cell activity assay.

[0028] After removing the culture medium, the sample groups were treated with IC 50 The cells were treated with DPP-IV inhibitory peptides at different concentrations and incubated for 2 and 4 hours. The control group was not treated. The cells were lysed with RIPA lysis buffer for 3 minutes, followed by centrifugation at 12,000 rpm for 10 minutes at 4°C to collect cell proteins. The protein concentration under different treatment conditions was determined by BCA method.

[0029] Proteins were separated by 10% SDS-PAGE gel and transferred to polyvinylidene fluoride (PVDF) membranes. The membranes were blocked with 5% BSA for 40 minutes at 37°C. Primary antibodies against DPP-IV (1:1000) and β-actin (1:20,000) were incubated overnight at 4°C.

[0030] After washing the membrane with TBST, add the secondary antibody (1:2000) and incubate at 37°C for 40 minutes. Wash the membrane with TBST again. After adding the chemiluminescent substrate, expose and detect the results.

[0031] This study further explored the expression of DPP-IV protein in Caco-2 cells ( Figure 6 ). Caco-2 cells were treated with IC 50The concentration of TENEWK was stimulated for 2 hours and 4 hours. Compared with the control group, the expression of DPP-IV protein in Caco-2 cells was significantly reduced after TENEWK treatment. In addition, the inhibitory effect on DPP-IV expression became more obvious with the extension of stimulation time. These results indicate that TENEWK can effectively inhibit the expression of DPP-IV.

[0032] (6) Effect of TENEWK on blood glucose in mice Eight-week-old Kunming mice weighing about 20g were selected. After one week of adaptive feeding, STZ solution with a concentration of 5mg / ml was intraperitoneally injected every morning, and 35mg / kg was injected every day according to body weight. The type 2 diabetes mouse model was induced by feeding with high-fat and high-sugar feed. After three consecutive injections, the blood glucose of the mice rose to 23.43mmol / L (≥11.1mmol / L), indicating successful modeling. The mice were randomly divided into 6 groups according to body weight and blood glucose values: normal group (Normal), model group (Model), TENEWK low-dose administration group (20mg / kg, TENEWK), medium-dose administration group (40mg / kg, TENEWK), high-dose administration group (80mg / kg, TENEWK) and positive drug Sitagliptin phosphate group (20mg / kg, Sitagliptin). According to the grouping of mice, drugs or saline were gavaged, and gavage was performed at the same time in the afternoon every day in the same order for 8 consecutive weeks. The normal group and the model group were gavaged with the same dose of saline.

[0033] The average blood glucose concentration after film formation was 24.27mmol / L. After 8 weeks of continuous intragastric administration, the blood glucose concentration of mice in the model group was 29.47mmol / L. The blood glucose concentration of mice in the TENEWK intragastric administration group was significantly lower than that before intragastric administration, and the blood glucose concentration of mice in the high-dose group (80mg / kg) decreased significantly, down to 19.24mmol / L. The blood glucose concentration of the sitagliptin phosphate administration group decreased significantly compared with that before intragastric administration, down to 18.43 mmol / L ( Figure 7 ).

[0034] After the type 2 diabetes mouse model was successfully established, the mice were gavaged for 8 consecutive weeks. Starting from the first day of gavage, the fasting blood glucose concentration was measured once a week. After fasting but not water deprivation for 8 hours, the blood glucose concentration of the mice was detected by blood sampling from the tail vein. The results showed that the blood glucose level of the normal group mice remained basically unchanged after gavage of normal saline for 8 weeks, while the blood glucose concentration of the type 2 diabetes mice increased slightly in the first 2 weeks of gavage, and began to decrease significantly in the third week. Compared with the model group, the blood glucose concentration of the mice in the low and medium doses (20 mg / kg, 40 mg / kg) of TENEWK gavage groups decreased slightly, and the blood glucose concentration of the mice in the high dose gavage 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.

[0035] The present invention designs a preparation method for screening DPP-IV inhibitory peptide TENEWK from wheat and its application in the treatment of diabetes, aiming to solve the side effect problem 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 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. Studies have shown that TENEWK exhibits significant DPP-IV inhibitory activity in vitro and does not show obvious cytotoxicity in Caco-2 cells, showing 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 sugar 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.

[0036] 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, and has important scientific significance and economic value.

[0037] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all 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 products for treating diabetes.

3. The use according to claim 2, characterized in that: The product is a medicine for treating diabetes.

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

5. The use according to claim 4, characterized in that: The drug 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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