CN115010785B - Tetrapeptide with dipeptidyl peptidase-4 inhibitory activity and application thereof - Google Patents

Abstract

The invention discloses a dipeptidyl peptidase-4 (DPP-4) inhibitory peptide derived from yak collagen. The amino acid sequence of the peptide is Met-Gly-Pro-Arg, abbreviated as MGPR. With the help of different online databases, the present invention uses a computer simulation method to carry out multiple rounds of screening, and finally obtains a peptide segment with DPP-4 inhibitory activity. The polypeptide of the present invention can be chemically synthesized by a solid-phase synthesis method, has a purity of >98%, has the characteristics of small molecular weight, convenient artificial synthesis, good water solubility, non-toxicity and high safety. The in vitro DPP‑4 inhibitory activity test showed that the 50% inhibitory concentration (IC ₅₀ ) of the tetrapeptide MGPR on DPP‑4 was 0.225 mg/mL; it can be used to develop functional foods with DPP‑4 inhibitory activity and synthesize hypoglycemic therapeutic drugs.

Description

A tetrapeptide having dipeptidyl peptidase-4 inhibitory activity and its application

Technical Field

The present invention relates to the field of bioactive peptides, in particular to a tetrapeptide having dipeptidyl peptidase-4 inhibitory activity and application thereof.

Background Art

Diabetes is a chronic disease with high incidence and high risk. In recent years, with the development of social economy, people's dietary structure and lifestyle have changed, and the incidence of diabetes, especially type 2 diabetes, has been increasing year by year. It is reported that by 2030, the number of diabetes patients in the world will exceed 400 million, of which type 2 diabetes accounts for the vast majority, accounting for about 90%. Various clinical therapeutic drugs are prone to cause various side effects such as hypoglycemia, hypotension, and liver damage. Therefore, the development of new food-derived active substances for the auxiliary treatment of diabetes has a very important application prospect. Some existing studies have shown that bioactive peptides have the effect of improving diabetes. For example, in the study of Wang Junbo et al., marine collagen peptides can alleviate the structural damage of pancreatic β cells in rats with hyperinsulinemia, increase the secretion of granules, reduce the formation of lipid droplets, and significantly improve the biological activity of insulin; it also has a certain improvement effect on fasting blood sugar and oral glucose tolerance.

Dipeptidyl peptidase-4 (DPP-4) is a serine protease that can cleave the N-terminal Xaa-Pro or Xaa-Ala structure of hormones and other peptides. During the development of diabetes, glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic hormone (GIP) can promote the body's insulin secretion and inhibit the secretion of glucagon, playing an important role in regulating the balance of blood sugar in the human body. Studies have found that DPP-4 can remove two amino acids at the amino termini of GLP-1 and GIP, causing them to lose their physiological functions and affecting blood sugar regulation. Therefore, inhibiting the activity of DPP-4 is beneficial to controlling blood sugar levels, and DPP-4 inhibitors have now become a research hotspot for the treatment of diabetes.

At present, the traditional enzymatic hydrolysis-separation-purification method is mainly used to screen and identify hypoglycemic peptides. The process is cumbersome, time-consuming, requires a lot of manpower and materials, and has a low yield. In addition, due to its segmented activity determination method, some single peptides with strong activity are easily missed. Therefore, it is necessary to find a method for efficiently discovering active peptides to achieve mass preparation.

Summary of the invention

In view of the above problems existing in the prior art, the present invention provides a hypoglycemic tetrapeptide with DPP-4 inhibitory activity and its application. The active peptide involved in the present invention is screened from the yak type I collagen sequence, and the screened peptide is obtained by solid phase synthesis. The DPP-4 and HepG2 (human liver cancer cell) cell insulin resistance model is used as the research object to detect the in vitro hypoglycemic effect of the synthetic peptide. The results show that the peptide has good DPP-4 inhibitory activity, IC ₅₀ is 0.225 mg/mL, and can significantly improve insulin resistance, promote glycogen absorption, and has a certain hypoglycemic effect.

The technical solution of the present invention is as follows:

A tetrapeptide having dipeptidyl peptidase-4 inhibitory activity, wherein the tetrapeptide is abbreviated as MGPR and the amino acid sequence of the tetrapeptide is Met-Gly-Pro-Arg.

The molecular weight of the tetrapeptide is 459.56 Da.

The tetrapeptide is a single-chain linear structure, in the form of white powder, and is easily soluble in water.

The tetrapeptide is derived from yak collagen.

The inhibitory activity IC ₅₀ of the tetrapeptide against dipeptidyl peptidase-4 is 0.225 mg/mL.

The tetrapeptide was chemically synthesized by solid phase synthesis with a purity of >98%.

Further, the solid phase synthesis method is:

According to the characteristics of the amino acid sequence Met-Gly-Pro-Arg, the carboxyl group of Met is first connected to a resin in the form of a covalent bond, and then the amino group of Met and the carboxyl group of Gly are hydrated. After treatment, Pro, the amino group of Gly and the carboxyl group of Pro are added to react, and then the last Arg amino acid is added. After the reaction, the resin is removed and purified by high performance liquid chromatography to obtain the target polypeptide MGPR.

A pharmaceutical composition comprising the tetrapeptide having dipeptidyl peptidase-4 inhibitory activity.

An application of the tetrapeptide having dipeptidyl peptidase-4 inhibitory activity is used for preparing medicine for preventing and treating diabetes.

An application of the tetrapeptide having dipeptidyl peptidase-4 inhibitory activity is used for preparing health food for preventing and treating diabetes.

The present invention uses bioinformatics methods and various online active peptide databases to screen new hypoglycemic peptides from yak collagen. First, yak collagen is virtually enzymatically hydrolyzed to obtain enzymatic peptide segments, from which 2 to 5 peptides that have not been reported are screened, and then the bioactivity score, molecular weight, water solubility, ADMET (absorption, distribution, metabolism, excretion, toxicity) and other properties of the screened sequences are predicted, and molecular docking is performed using Discovery studio 2019 software.

The results showed that the tetrapeptide MGPR has good water solubility, is non-toxic, has good intestinal absorption and blood-brain barrier permeability, and can tightly bind to the key active site of DPP-4. The selected active peptides were synthesized by solid phase synthesis, and the in vitro DPP-4 inhibitory activity of the synthetic peptides was verified.

Advantages and technical effects of the present invention

1. The present invention uses computer-aided technology to perform virtual screening of directional binding peptides. Compared with the traditional enzymatic method for preparing active peptides, the method used in the present invention not only reduces the screening intensity and shortens the research and development cycle, but also increases the probability of successful screening.

2. Through searching of online databases, it was found that the peptide sequence described in the present invention has not been reported in any paper. The tetrapeptide provided by the present invention is a novel food-derived hypoglycemic peptide.

3. The present invention synthesized the tetrapeptide MGPR by solid phase synthesis and tested the in vitro DPP-4 inhibitory activity of the synthetic peptide. The results showed that the synthetic peptide has potential blood sugar lowering ability.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 is a molecular docking diagram of the tetrapeptide MGPR and DPP-4;

FIG2 is a high performance liquid chromatogram of the synthetic polypeptide MGPR;

FIG3 is a mass spectrum of the synthetic polypeptide MGPR;

FIG4 shows the inhibitory activity of the synthetic peptide MGPR against DPP-4;

FIG5 shows the effect of the synthetic peptide MGPR on glucose uptake in HepG2 cells.

DETAILED DESCRIPTION

The present invention is described in detail below in conjunction with the accompanying drawings and embodiments.

Embodiment 1:

Virtual screening of active peptides:

1. Acquisition of yak collagen sequence

The collagen sequences from yaks were searched in the protein database of the National Center for Biotechnology Information (NCBI), and finally two chains of yak type I collagen sequences were selected: the alpha-1(I) chain (Accession: ELR60286.1) and the alpha-2(I) chain (Accession: ELR46121.1).

2. Simulated gastrointestinal enzymatic hydrolysis

ExPASy PeptideCutter (https://web.expasy.org/peptide_cutter/) was used to simulate the enzymatic hydrolysis of yak collagen sequence. Two digestive enzymes, pepsin (pH1.3) (EC 3.4.23.1) and trypsin (EC 3.4.21.4), were selected to simulate the enzymatic hydrolysis of human gastrointestinal digestion. 2 to 5 peptides were screened from the peptides produced by enzymatic hydrolysis and compared with the online database to screen out unreported peptides for the next step of virtual screening.

3. Prediction of biological activity, water solubility, and ADMET properties.

The PeptideRanker (http://distilldeep.ucd.ie/PeptideRanker/) program was used to evaluate the bioactivity potential of the screened active peptide fragments, and the peptides with high scores were screened out. The molecular weight and isoelectric point of the peptides were predicted using the pI/Mw tool of the online software Expasy (http://web.expasy.org/compute_pi/), the net charge and hydrophobicity of the peptides were predicted by Pepdraw (http://www.tulane.edu/~biochem/WW/PepDraw), and the water solubility of the peptides was predicted using the "Peptide property calculator" function in the Innovagen program (http://www.innovagen.com/proteomics-tools). ADMETlab2.0 (https://admetmesh.scbdd.com/) was used to predict its ADMET properties, mainly focusing on human intestinal aabsorption (HIA), blood-brain barrier penetration (BBB) and acute oral toxicity as analysis indicators. Finally, five peptides GHR, GIR, PGPK, PGMK, MGPR and PGPR were selected with good water solubility and ADMET properties. The results are shown in Table 1.

Table 1 Bioactivity scores, physicochemical properties and ADMET property predictions

4. Molecular docking screening of DPP-4 inhibitory peptides

Discovery studio2019 software was used to perform molecular docking between the screened peptides and DPP-4 (PDB ID: 5J3J), and finally a tetrapeptide MGPR with the best docking effect was selected. Existing studies have shown that DPP-4 has three key active pockets. The S1 active pocket is composed of Tyr547, Ser630, Tyr631, Val656, Trp659, Tyr662, Asn710, Val711 and His740, and the S2 is composed of Glu205, Glu206 and Tyr662. Ser209, Arg358 and Phe357 constitute the S3 pocket. The amino acid sites of these three active pockets are important targets for screening DPP-4 inhibitors. The docking results showed that the tetrapeptide MGPR can bind tightly to Ser630, His740, Tyr547 in the S1 active pocket of DPP-4 and Glu205, Glu206 in the S2 pocket, forming hydrogen bonds and electrostatic interactions. In addition, it can also bind to the amino acid residues His126 and Arg125 of DPP-4 (Figure 1). Finally, the tetrapeptide screened by solid phase synthesis was used to verify the activity of the synthesized peptide in vitro.

Embodiment 2:

Solid-phase synthesis method for screening active peptides

The Fmoc solid phase synthesis method was used. According to the characteristics of the amino acid sequence Met-Gly-Pro-Arg, the carboxyl group of Met was first connected to a resin in the form of a covalent bond, and then the amino group of Met and the carboxyl group of Gly were hydrated. After treatment, Pro, the amino group of Gly and the carboxyl group of Pro were added to react, and then the last Arg amino acid was added. After the reaction, the resin was removed to obtain the target polypeptide MGPR. The HPLC was used for purification. The column model was Kromasil C18, the size was 4.6*250 mm, 5 μm, the mobile phase A was acetonitrile containing 0.1% trifluoroacetic acid (TFA); the mobile phase B was water containing 0.1% TFA; the flow rate was 1.0 mL/min, and the detection wavelength was 220 nm. The purity was required to reach more than 98% (as shown in Figure 2 and Table 2), and the structure was identified by MS (as shown in Figure 3).

Table 2

As shown in Figure 3, the purity of the tetrapeptide MGPR is 98.11%, which meets the purity requirements of synthetic peptides.

Embodiment 3:

In vitro inhibitory activity assay of synthetic peptide MGPR on DPP-4

The Sigma DPP-4 inhibitor screening kit (MAK203) was used for detection.

Preparation of reagents

(1) Substrate solution: dilute the original 200 μL with buffer to 2.5 mL and aliquot for use.

(2) Enzyme solution: dilute the original 100 μL with buffer to 5 mL and aliquot for use.

(3) Positive inhibitor (sitagliptin): dilute the original 50 μL with buffer to 0.5 mL and dispense for use.

(4) Sample solution: Prepare sample solutions with gradient concentrations using buffer.

The specific implementation steps are as follows: the reaction container is a black 96-well plate, 50 μL of enzyme solution and 25 μL of sample solution are added to the well plate, the sample solution is replaced by buffer in the enzyme control group, and the sample solution is replaced by positive inhibitor in the positive control group, and the reaction is carried out at 37°C for 10 minutes. Finally, 25 μL of substrate solution is added, and the fluorescence FLU (FLU, λex＝360/λem＝460) is measured every 1 minute during the 15-30 minutes after the reaction. A fluorescence value/time (min) curve is drawn, two time points (T ₁ and T ₂ ) are selected within the linear range of the curve graph, and the slope of each well between T ₁ and T ₂ is obtained. The FLU (FLU ₁ and FLU ₂ ) is determined each time, and they are used to determine the slope of the graph (ΔFLU/minute).

Slope = (FLU ₂ – FLU ₁ ) / (T ₂ – T ₁ ) = FLU/minute

Relative inhibition rate (%) = (slope of control group – slope of experimental group) / slope of control group × 100%

According to the calculation results, an inhibition rate-concentration curve was drawn and the IC ₅₀ value was calculated.

As can be seen from Figure 4, the tetrapeptide MGPR has good DPP-4 inhibitory activity, with an IC ₅₀ value of 0.225 mg/mL.

Embodiment 4:

Effects of synthetic peptide MGPR on HepG2 cell insulin resistance model

Establishment of insulin resistance model

HepG2 cells in the logarithmic growth phase were taken, the culture medium was discarded and washed once with PBS, the cells were digested with trypsin to make a cell suspension, and the cell suspension with a density of about 1×10 ⁵ /mL was inoculated into a 96-well plate, 100 μL was added to each well, and the plate was placed in a cell culture incubator at 37°C and 5% CO ₂ for 12 h. The culture medium was discarded, and 18 mmol/L glucosamine was added to induce the culture for 18 h to establish an insulin resistance model.

After the model was established, the mice were divided into groups. The blank group (not treated with glucosamine) and the model group (treated with glucosamine) were added with 100 μL of complete culture medium respectively; the drug-treated groups were added with 100 μL of peptide solutions of different concentrations (MGPR: 25, 50, 100, 200, 400 μmol/L). Rosiglitazone at 10 μmol/L was used as a positive control. After 24 h of drug treatment, the supernatant was taken and the glucose content in the supernatant was detected by glucose oxidase-peroxidase coupling method (GOD-POD). The specific operation method was referred to the instructions of the glucose kit. Glucose consumption was calculated as follows:

Glucose consumption = glucose content of blank group - glucose content of experimental group

The experimental results showed that the glucose uptake of the model group cells after 18 h of treatment with glucosamine was 38.4% lower than that of the blank control group, indicating that the insulin resistance model was successfully established. After 24 h of treatment with the tetrapeptide MGPR, the glucose uptake ability of the cells was detected. As shown in Figure 5, the active peptide MGPR improved glucose uptake in a dose-dependent manner, among which 400 μmol/L concentration of MGPR could significantly promote the glucose uptake of the model cells, which was increased by 35.6% compared with the model group.

Finally, it should be noted that the above examples are only some specific embodiments of the present invention. Obviously, the present invention is not limited to the above examples, and there are many variations. All variations directly derived or associated with the contents disclosed in the present invention should be considered as the protection scope of the present invention.

Claims (1)

1. The application of tetrapeptides with dipeptidyl peptidase-4 inhibitory activity is characterized in that the tetrapeptides are used for preparing medicines for preventing and treating diabetes;

the tetrapeptide is abbreviated as MGPR, and the amino acid sequence of the tetrapeptide is Met-Gly-Pro-Arg.

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