Small molecule peptides with xanthine oxidase inhibitory activity and their applications

Abstract

The present invention discloses a small molecule peptide with xanthine oxidase inhibitory activity and its application, belonging to the technical field of bioactive peptides. The amino acid sequence of the small molecule peptide with xanthine oxidase inhibitory activity is YDWRPF, as shown in SEQ ID NO.8. The application of the small molecule peptide with xanthine oxidase inhibitory activity in the preparation of a xanthine oxidase inhibitor. The small molecule peptide YDWRPF of the present invention has strong xanthine oxidase inhibitory activity, and the IC 50 is only 2.73 ± 0.20 mM, and a xanthine oxidase inhibitor can be prepared with it as a raw material. The present invention is of great significance for the treatment of hyperuricemia.

Description

Technical Field

[0001] The present invention relates to a small molecule peptide with xanthine oxidase inhibitory activity and its application, belonging to the technical field of bioactive peptides. Background Art

[0002] Xanthine oxidase (XO) is a key enzyme that controls purine metabolism and uric acid synthesis in the human body. Abnormal elevation of XO activity directly leads to metabolic diseases such as hyperuricemia and gout. Developing XO inhibitors is the key to treating metabolic diseases such as hyperuricemia and gout. Currently, a variety of bioactive peptides with XO inhibitory activity have been found in seafood, dairy products, crops, etc., but there is no relevant report on XO inhibitory peptides derived from Macrobrachium rosenbergii.

[0003] Macrobrachium rosenbergii belongs to the genus Macrobrachium of the family Palaemonidae, is a typical tropical freshwater crustacean, also known as Malaysian giant prawn or large long-armed prawn, and is a major cultured variety of freshwater shrimps in China. Summary of the Invention

[0004] In view of the above-mentioned prior art, the present invention provides a small molecule peptide with xanthine oxidase inhibitory activity and its application, belonging to the technical field of bioactive peptides.

[0005] The present invention is achieved by the following technical solutions:

[0006] A small molecule peptide with xanthine oxidase inhibitory activity, the amino acid sequence is YDWRPF, as shown in SEQ ID NO.8.

[0007] The application of the small molecule peptide with xanthine oxidase inhibitory activity in the preparation of a xanthine oxidase inhibitor.

[0008] To develop bioactive peptides of natural origin with XO inhibitory activity and explore their structure-activity relationships, the present invention uses Macrobrachium rosenbergii as raw material, prepares bioactive peptides by enzymatic hydrolysis, and analyzes their action mechanisms by combining separation and purification and molecular docking techniques. The specific process is as follows: First, the enzymatic hydrolysis conditions are screened and optimized. The suitable enzyme is papain, and the optimized enzymatic hydrolysis conditions are: enzymatic hydrolysis time 3 h, enzyme addition amount 2000 U / g, solid-liquid ratio 1:3, and enzymatic hydrolysis temperature 50 °C. Then, through Sephadex G-15 gel chromatography separation and purification, the fraction X5 with the highest in vitro XO inhibitory rate is obtained. Fraction X5 is analyzed and identified by LC-MS / MS, and a total of 27 peptide segments are obtained. Seven peptide segments with the highest absolute value of Vina score are screened out by molecular docking, synthesized and their activities are verified, and 4 bioactive peptides (GPPGPPAGP, PPGPPA, QDYRPR, and GPPGPA) with in vitro XO inhibitory activity are obtained. Among them, the bioactive peptide QDYRPR (IC 50 value is 6.42 ± 0.31 mM) has the strongest in vitro XO inhibitory activity. Finally, using the bioactive peptide QDYRPR as the original peptide segment, Gly is selected as the backbone amino acid, and aromatic amino acids are selected as replacement amino acids for rational design. Five small peptides (YDWRPF, FDGRPR, WDGRPF, FDFRPR, and FFF) with the highest absolute value of Vina score are obtained, synthesized and their activities are verified. It is found that the IC 50 of the small peptide YDWRPF is 2.73 ± 0.20 mM, and its in vitro XO inhibitory activity is the strongest. The small peptide WDGRPF ranks second, with an IC 50 of 2.96 ± 0.16 mM, both significantly superior to the bioactive peptide QDYRPR.

[0009] The present invention rationally designs the small peptide YDWRPF with significant XO inhibitory activity. It has strong xanthine oxidase inhibitory activity, and its IC 50 is only 2.73 ± 0.20 mM. Xanthine oxidase inhibitors and drugs for treating hyperuricemia can be prepared using it as a raw material. The present invention is of great significance for the treatment of hyperuricemia.

[0010] All the terms and phrases used in the present invention have the general meanings well-known to those skilled in the art. Description of the Drawings

[0011] Figure 1 : Standard curve.

[0012] Figure 2 : Effects of different proteases on the in vitro XO inhibitory rate of enzymatic hydrolysis products. Among them, the significance of differences is represented by the letter annotation method, and the same applies hereinafter.

[0013] Figure 3: Effect of enzymatic hydrolysis time on the in vitro XO inhibitory rate of the enzymatic hydrolysis product.

[0014] Figure 4 : Effect of enzyme dosage on the in vitro XO inhibitory rate of the enzymatic hydrolysis product.

[0015] Figure 5 : Effect of enzymatic hydrolysis pH on the in vitro XO inhibitory rate of the enzymatic hydrolysis product.

[0016] Figure 6 : Effect of solid-liquid ratio on the in vitro XO inhibitory rate of the enzymatic hydrolysis product.

[0017] Figure 7 : Effect of enzymatic hydrolysis temperature on the in vitro XO inhibitory rate of the enzymatic hydrolysis product.

[0018] Figure 8 : Results of Sephadex G-15 gel chromatography.

[0019] Figure 9 : Molecular weight distribution diagrams of each component.

[0020] Figure 10 : Mass spectrometry base peak diagram of component X5.

[0021] Figure 11 : Determination results of the in vitro XO inhibitory rate and IC 50 value of bioactive peptides, where A: GPPGPPAGP; B: PPGPPA; C: QDYRPR; D: GPPGPA.

[0022] Figure 12 : Vina scores of 20 common amino acids.

[0023] Figure 13 : Relationship between Vina scores and IC 50 value of various small molecule peptides.

[0024] Figure 14 : 3D interaction diagram of the docking results of small molecule peptide YDWRPF with 1N5X.

[0025] Figure 15 ; 2D interaction diagram of the docking results of small molecule peptide YDWRPF with 1N5X.

[0026] Figure 16 : 3D interaction diagram of the docking results of small molecule peptide FDGRPR with 1N5X.

[0027] Figure 17 : 2D interaction diagram of the docking results of small molecule peptide FDGRPR with 1N5X.

[0028] Figure 18 : 3D interaction diagram of the docking results of small molecule peptide WDGRPF with 1N5X.

[0029] Figure 19 : 2D interaction diagram of the docking result between small molecule peptide WDGRPF and 1N5X.

[0030] Figure 20 : 3D interaction diagram of the docking result between small molecule peptide FDFRPR and 1N5X.

[0031] Figure 21 : 2D interaction diagram of the docking result between small molecule peptide FDFRPR and 1N5X.

[0032] Figure 22 : 3D interaction diagram of the docking result between small molecule peptide FFF and 1N5X.

[0033] Figure 23 : 2D interaction diagram of the docking result between small molecule peptide FFF and 1N5X. Detailed implementation manners

[0034] The present invention will be further described below in conjunction with embodiments. However, the scope of the present invention is not limited to the following embodiments. Those skilled in the art can understand that various changes and modifications can be made to the present invention without departing from the spirit and scope of the present invention.

[0035] The instruments, reagents, and materials involved in the following embodiments are all conventional instruments, reagents, and materials existing in the prior art and can be obtained through regular commercial channels without special instructions. The experimental methods, detection methods, etc. involved in the following embodiments are all conventional experimental methods and detection methods existing in the prior art without special instructions.

[0036] Experiment 1 Screening and optimization of enzymatic hydrolysis conditions

[0037] Macrobrachium rosenbergii was enzymatically hydrolyzed to screen for suitable enzymes and enzymatic hydrolysis conditions to obtain bioactive peptides with XO inhibitory activity. Specifically, see the following. Measurements were taken at least three times, and the data were expressed as mean ± standard deviation.

[0038] 1.1 Pretreatment of Macrobrachium rosenbergii

[0039] Macrobrachium rosenbergii (purchased from Nanbin Farmers' Market, Yazhou Bay, Sanya City, Hainan Province) was taken. After removing the head and shell, the shrimp meat was ground into a homogenate and stored at -20 °C for later use.

[0040] 1.2 Index determination method

[0041] 1.2.1 Determination method of polypeptide concentration

[0042] Take 200 μL of the sample, add 200 μL of trichloroacetic acid (TCA) solution (concentration is 10%, unit g / mL), vortex mix, let stand for 10 min, centrifuge at 4000 rpm for 10 min; take 300 μL of the supernatant, add 200 μL of biuret reagent, mix evenly, let stand for 10 min, centrifuge at 4000 rpm for 10 min, take the supernatant, measure the absorbance at 540 nm, and substitute it into the standard curve to calculate the polypeptide concentration of the sample.

[0043] The biuret reagent is prepared by mixing biuret solution A and biuret solution B in a volume ratio of 3:2. Biuret solution A is a 100 g / L NaOH solution, and biuret solution B is a 10 g / L CuSO4 solution.

[0044] The standard curve is measured and plotted using bovine serum albumin as the standard. The abscissa is the concentration of bovine serum albumin, and the ordinate is the absorbance at 540 nm. The standard curve measured in this experiment is as Figure 1 shown.

[0045] 1.2.2 Method for measuring in vitro XO inhibition rate

[0046] Add 50 μL of the sample to be tested and 50 μL of 0.05 U / mL XO solution into a 96-well plate, incubate at 37 °C for 20 min, add 150 μL of 0.40 mmol / L xanthine solution to initiate the enzymatic reaction, and record the kinetic change of the absorbance of the reaction system at 290 nm within 3 min. Use PBS buffer at pH 7.4 as a control, and calculate the in vitro XO inhibition rate according to the following formula.

[0047]

[0048] In the formula: V0 is the rate of the reaction system in the control group, and Vs is the rate of the reaction system in the sample group.

[0049] 1.3 Screening of proteases

[0050] The selection of proteases plays a decisive role in the enzymatic hydrolysis effect and the functional characteristics of the enzymatic hydrolysis products. Different proteases will generate different enzymatic hydrolysis products due to different action sites and action modes. Therefore, it is necessary to screen suitable proteases. In this experiment, 6 kinds of proteases were screened.

[0051] Take 6 portions of shrimp meat homogenate, 4 g for each portion, and add 12 mL of deionized water according to the solid-liquid ratio of 1:3 (g:mL, the same below). Add papain, acid protease, alkaline protease, neutral protease, flavor protease, and bromelain (all purchased from Nanning Pangbo Bioengineering Co., Ltd.) respectively according to the ratio of 2000 U / g (enzyme activity / shrimp meat homogenate, the same below), and enzymatically hydrolyze for 4 h under the optimal temperature and optimal pH conditions of each protease; boil to inactivate the enzyme for 10 min, cool to room temperature, centrifuge at 4°C and 8000 rpm for 20 min, collect the supernatant, measure the polypeptide concentration, and then measure the in vitro XO inhibition rate under the condition of the same polypeptide concentration.

[0052] The effects of different proteases on the in vitro XO inhibition rate of the enzymatic hydrolysate are shown as Figure 2 follows. It can be seen that the in vitro XO inhibition rates of the enzymatic hydrolysates obtained by papain and alkaline protease hydrolysis are the highest, 53.83% and 54.08% respectively, and are significantly higher than those of other proteases (P < 0.05). Considering that the optimal pH of papain is 6.5, which is closer to neutral, the enzymatic hydrolysis conditions are more stable at this time, and it is easier to generate small molecule short peptides. Therefore, papain is selected as the protease for subsequent enzymatic hydrolysis.

[0053] 1.4 Optimization of enzymatic hydrolysis time

[0054] Take 6 portions of shrimp meat homogenate, 4 g for each portion, add 12 mL of deionized water according to the solid-liquid ratio of 1:3, and adjust the initial pH value to 6.5. Add 0.16 g of papain according to the ratio of 2000 U / g, place it in a water bath shaker, and enzymatically hydrolyze for 1 h, 2 h, 3 h, 4 h, 5 h, and 6 h respectively under the conditions of 50°C and 200 rpm; boil to inactivate the enzyme for 10 min, cool to room temperature, centrifuge at 4°C and 8000 rpm for 20 min, collect the supernatant, measure the polypeptide concentration, and then measure the in vitro XO inhibition rate under the condition of the same polypeptide concentration.

[0055] The effect of enzymatic hydrolysis time on the in vitro XO inhibition rate of the enzymatic hydrolysate is shown as Figure 3As shown, it can be seen that as the enzymatic hydrolysis time increases, the in vitro XO inhibition rate of the enzymatic hydrolysis product shows a trend of first increasing and then decreasing. The reason is that at the beginning, as the enzymatic hydrolysis time increases, the protease acts more fully with the substrate. Subsequently, as the enzymatic hydrolysis reaction proceeds, the peptides produced by enzymatic hydrolysis will compete with the shrimp meat protein for the substrate, thereby reducing the efficiency of the protease. When the enzymatic hydrolysis time is 3 h, the in vitro XO inhibition rate is the highest, reaching 57.35% ± 3.37%, and there is no significant difference compared with the 2 h group (the in vitro XO inhibition rate is 51.74% ± 5.56%) and the 4 h group (the in vitro XO inhibition rate is 52.97% ± 0.94%) (P > 0.05), while there is a significant difference compared with the 1 h group (the in vitro XO inhibition rate is 45.54% ± 4.71%) and the 5 h group (the in vitro XO inhibition rate is 37.92% ± 1.31%) (P < 0.05). Therefore, an enzymatic hydrolysis time of 3 h was selected for subsequent research.

[0056] 1.5 Optimization of enzyme dosage

[0057] Take 5 portions of shrimp meat homogenate, each portion being 4 g, add 12 mL of deionized water according to the solid-liquid ratio of 1:3, and adjust the initial pH value to 6.5. Add 0.08 g, 0.16 g, 0.24 g, 0.32 g, and 0.40 g of papain according to the ratios of 1000 U / g, 2000 U / g, 3000 U / g, 4000 U / g, and 5000 U / g respectively, place it in a water bath shaker, and carry out enzymatic hydrolysis at 50 °C and 200 rpm for 3 h; boil to inactivate the enzyme for 10 min, cool to room temperature, centrifuge at 4 °C and 8000 rpm for 20 min, collect the supernatant, measure the polypeptide concentration, and then measure the in vitro XO inhibition rate under the condition of the same polypeptide concentration.

[0058] The effect of enzyme dosage on the in vitro XO inhibition rate of the enzymatic hydrolysis product is as Figure 4 shown. It can be seen that as the enzyme dosage increases, the in vitro XO inhibition rate of the enzymatic hydrolysis product shows a trend of first increasing and then decreasing. The reason is that at the beginning, the shrimp meat protein is in excess. As the enzyme dosage increases, the protease acts more fully with the substrate. Subsequently, as the enzyme dosage increases, the substrate for the protease becomes the peptides produced, resulting in a decrease in the in vitro XO inhibition rate. When the enzyme dosage is 2000 U / g, the in vitro XO inhibition rate is the highest, reaching 61.05% ± 2.23%, and there is no significant difference compared with the 3000 U / g group (the in vitro XO inhibition rate is 58.24% ± 2.23%) (P > 0.05), while there is a significant difference compared with the 1000 U / g group (the in vitro XO inhibition rate is 42.58% ± 1.64%) and the 4000 U / g group (the in vitro XO inhibition rate is 47.82% ± 2.37%) (P < 0.05). Therefore, an enzyme dosage of 2000 U / g was selected for subsequent research.

[0059] 1.6 Optimization of Enzymolysis pH

[0060] Take 5 portions of shrimp meat homogenate, 4 g for each portion, add 12 mL of deionized water according to the solid-liquid ratio of 1:3, and adjust the initial pH values to 4.5, 5.5, 6.5, 7.5, and 8.5 respectively. Add 0.16 g of papain according to the ratio of 2000 U / g, place it in a water bath shaker, and enzymolyze for 3 h at 50 °C and 200 rpm; boil to inactivate the enzyme for 10 min, cool to room temperature, centrifuge at 4 °C and 8000 rpm for 20 min, collect the supernatant, measure the polypeptide concentration, and then measure the in vitro XO inhibition rate under the condition of the same polypeptide concentration.

[0061] The effect of enzymolysis pH on the in vitro XO inhibition rate of enzymolysis products is as Figure 5 shown. It can be seen that with the increase of pH value, the in vitro XO inhibition rate of enzymolysis products shows a trend of first increasing and then decreasing. The reason is that: the pH value of the enzymolysis system has an important influence on the enzyme activity of protease, and an overly acidic or alkaline system will affect the structure of protease, thus reducing its activity. The optimal pH value of papain is 6.0 - 7.0. When the pH value is 6.5, the in vitro XO inhibition rate of enzymolysis products is the highest, reaching 62.55% ± 0.73%, and there is no significant difference compared with the pH 7.5 group (the in vitro XO inhibition rate is 60.84% ± 0.22%) (P > 0.05), while there are significant differences compared with the pH 5.5 group (the in vitro XO inhibition rate is 53.36% ± 0.39%) and the pH 8.5 group (the in vitro XO inhibition rate is 55.12% ± 2.48%) (P < 0.05). The initial pH value of the shrimp meat homogenate is 7.0, which is between 6.5 and 7.5. For the convenience of subsequent experiments, the initial pH value of the shrimp meat homogenate (i.e., without specifically adjusting the pH) is selected for subsequent research.

[0062] 1.7 Optimization of Solid-Liquid Ratio

[0063] Take 5 portions of shrimp meat homogenate, 4 g for each portion, add 8 mL, 12 mL, 16 mL, 20 mL, and 24 mL of deionized water according to the solid-liquid ratios of 1:2, 1:3, 1:4, 1:5, and 1:6 respectively. Add 0.16 g of papain according to the ratio of 2000 U / g, place it in a water bath shaker, and enzymolyze for 3 h at 50 °C and 200 rpm; boil to inactivate the enzyme for 10 min, cool to room temperature, centrifuge at 4 °C and 8000 rpm for 20 min, collect the supernatant, measure the polypeptide concentration, and then measure the in vitro XO inhibition rate under the condition of the same polypeptide concentration.

[0064] The effect of solid-liquid ratio on the in vitro XO inhibition rate of enzymolysis products is as Figure 6As shown, it can be seen that with the increase of the material-liquid ratio, the in vitro XO inhibition rate of the enzymolysis product shows a trend of first increasing and then decreasing. The reason is that when the substrate concentration is too high, the fluidity of the system is low, resulting in insufficient contact between the protease and the substrate; with the increase of the material-liquid ratio, the substrate concentration is too low, which will also lead to insufficient contact between the protease and the substrate, thus reducing the enzymolysis efficiency. When the material-liquid ratio is 1:3, the in vitro XO inhibition rate of the enzymolysis product is the highest, reaching 61.61% ± 4.25%, and there is no significant difference compared with the 1:4 group (the in vitro XO inhibition rate is 57.09% ± 1.74%) (P > 0.05), while there are significant differences compared with the 1:2 group (the in vitro XO inhibition rate is 48.50% ± 4.68%) and the 1:5 group (the in vitro XO inhibition rate is 53.01% ± 2.43%) (P < 0.05). Therefore, the material-liquid ratio of 1:3 is selected for subsequent research.

[0065] 1.8 Optimization of enzymolysis temperature

[0066] Take 5 portions of shrimp meat homogenate, each portion is 4 g, add 12 mL of deionized water according to the material-liquid ratio of 1:3. Add 0.16 g of papain according to the ratio of 2000 U / g, place it in a water bath shaker, and enzymolyze for 3 h at 200 rpm, 30°C, 40°C, 50°C, 60°C, and 70°C respectively; boil to inactivate the enzyme for 10 min, cool to room temperature, centrifuge at 4°C and 8000 rpm for 20 min, collect the supernatant, measure the polypeptide concentration, and then measure the in vitro XO inhibition rate under the condition of the same polypeptide concentration.

[0067] The effect of enzymolysis temperature on the in vitro XO inhibition rate of the enzymolysis product is as Figure 7 shown. It can be seen that with the increase of the enzymolysis temperature, the in vitro XO inhibition rate of the enzymolysis product shows a trend of first increasing and then decreasing. The reason is that the temperature of the enzymolysis system will affect the activity of the enzyme and the reaction rate. Within a certain range, with the increase of temperature, the catalytic rate of the enzyme accelerates, but when the temperature exceeds the limit, it will instead damage the structure of the protease, thus affecting its activity. When the enzymolysis temperature is 50°C, the in vitro XO inhibition rate of the enzymolysis product is the highest, reaching 61.27% ± 3.76%, and there are significant differences compared with the 40°C group (the in vitro XO inhibition rate is 52.70% ± 3.18%) and the 60°C group (the in vitro XO inhibition rate is 37.75% ± 1.59%) (P < 0.05). Therefore, the enzymolysis temperature of 50°C is selected for subsequent research.

[0068] 1.9 Orthogonal experiment to determine the optimal enzymolysis process

[0069] According to the optimization results of the above single factors, time, enzyme dosage, solid-liquid ratio, and temperature were selected as indicators to design an orthogonal experiment with 4 factors and 3 levels. The optimal enzymatic hydrolysis process was determined by measuring the in vitro XO inhibition rate. The orthogonal experimental design level factor table and experimental results are shown in Table 1, where the magnitude of the range R reflects the influence of each factor on the result.

[0070]

[0071] As can be seen from Table 1, the primary and secondary order of the four factors of time, enzyme dosage, solid-liquid ratio, and temperature on the in vitro XO inhibition rate is: D > A > C > B, that is, temperature has the greatest influence, followed by time and solid-liquid ratio, and enzyme dosage has the least influence. The optimal combination is A2B2C2D2, that is: the enzyme is papain, the enzymatic hydrolysis time is 3 h, the enzyme dosage is 2000 U / g, the solid-liquid ratio is 1:3, and the enzymatic hydrolysis temperature is 50 °C. At this time, the in vitro XO inhibition rate of the enzymatic hydrolysis product is 61.60%.

[0072] Experiment 2 Separation and purification of bioactive peptides with XO inhibitory activity

[0073] 2.1 Sephadex G-15 gel chromatography

[0074] Take 4 g of shrimp meat homogenate, add 12 mL of deionized water according to the solid-liquid ratio of 1:3, add 0.16 g of papain according to the ratio of 2000 U / g, place it in a water bath shaker, and enzymatically hydrolyze for 3 h at 50 °C and 200 rpm; boil to inactivate the enzyme for 10 min, cool to room temperature, centrifuge at 4 °C and 8000 rpm for 20 min, and collect the supernatant, which is the enzymatic hydrolysate.

[0075] Perform gel filtration chromatography on the enzymatic hydrolysate: First, rinse Sephadex G-15 with distilled water multiple times to remove gel fragment particles, and then soak it in distilled water for at least 24 h and stir continuously to achieve full swelling until the gel volume no longer changes; when the packing is in a suspended state in water, pour the mixture into a 16 mm × 70 cm chromatography column. After packing the column, first let the pure water in the column flow out by gravity sedimentation to avoid generating bubbles or faults, and then continue to rinse with pure water and pressurize with a pump for 1 h until the packing height no longer changes. The loading concentration of the enzymatic hydrolysate is 100 mg / mL, the loading volume is 3 mL, the flow rate of the mobile phase is 1.2 - 1.4 mL / min. Plot the schematic diagram of the Sephadex G-15 gel chromatography results with time as the abscissa and the absorbance value at 230 nm as the ordinate; collect each eluted fraction, freeze-dry, redissolve, adjust the polypeptide concentration to 10 mg / mL, and measure the in vitro XO inhibition rate.

[0076] The schematic diagram of the Sephadex G-15 gel chromatography results is as Figure 8As shown, it can be seen that Sephadex G-15 gel chromatography separated the bioactive peptides in the enzymatic hydrolysate into 5 components (named X1-X5), and the separation effect of each peak was good. The in vitro XO inhibition rate determination results of each component are shown in Table 2 (MES in the table represents the enzymatic hydrolysate). It can be seen that the in vitro XO inhibition rate of component X5 was the highest, at 47.07% ± 6.76%, and was significantly higher than that of other components (P < 0.05). This indicates that the bioactive peptides with XO inhibitory activity were mainly in the molecular weight range of X5.

[0077]

[0078] 2.2 Molecular weight distribution determination

[0079] The molecular weights of the above components (X1-X5) were detected by HPLC, and the parameters were as follows: liquid chromatography analysis column: TSKgel G2000SWXL; mobile phase: water:acetonitrile:trifluoroacetic acid = 55:45:0.1, volume ratio; detection wavelength: 220 nm; sample loading concentration: 1 mg / mL; flow rate: 0.5 mL / min; sample loading volume: 20 μL. A standard curve was made based on the relationship between the retention time of the standards (L-reduced glutathione, oxidized glutathione, aprotinin, and cytochrome C) and the logarithm of the molecular weight: y = -0.2024*x + 6.5526, R 2 = 0.9964, and then the molecular weight distribution of each component was calculated according to the standard curve.

[0080] The molecular weight distribution diagrams of each component are as Figure 9 shown. It can be seen that MES was mostly composed of parts with a molecular weight less than 1 Kda, accounting for 69.47% of the total peak area. The higher the proportion of small molecules in the component with a later elution time, and the part with a molecular weight less than 1 KDa in component X5 accounted for 96.68% of the total peak area.

[0081] 2.3 LC-MS / MS analysis

[0082] The above component X5 was subjected to LC-MS / MS analysis, and the steps were as follows:

[0083] (1) Peptide segment treatment: Take the sample, add it to an appropriate amount of trifluoroacetic acid (TFA) solution (concentration 0.1%, unit g / mL), mix well, centrifuge at 20000 g for 5 min, transfer the supernatant to a 10 KD ultrafiltration tube, and centrifuge at 12000 g for 15 min; add 200 μL of TFA solution, and centrifuge twice under the same conditions, and collect the filtrate. Use C18 StageTip for desalting treatment and vacuum drying. The dried peptide segments were dissolved in formic acid (FA) solution (concentration 0.1%, unit g / mL), and its concentration was measured to prepare for LC-MS / MS analysis.

[0084] (2)LC-MS / MS analysis

[0085] Take an appropriate amount of peptide segments and separate them through an Easy nLC 1200 chromatography system (Thermo Scientific) with a nano-flow rate. The buffers used are buffer A (0.1% formic acid solution, concentration unit g / mL) and buffer B (80% acetonitrile solution, volume ratio). Equilibrate the chromatographic column with 100% buffer A. The sample first enters the Trap Column (100 μm * 20 mm, 5 μm, C18, Dr. Maisch GmbH), and then undergoes gradient separation through the analytical column (75 μm * 150 mm, 3 μm, C18, Dr. Maisch GmbH) at a flow rate of 300 nl / min. The liquid phase gradient is as follows: 0 - 2 min, buffer B linearly increases from 2% to 5%; 2 - 44 min, buffer B increases from 5% to 28%; 44 - 51 min, buffer B increases from 28% to 40%; 51 - 53 min, buffer B increases from 40% to 100%; 53 - 60 min, buffer B remains at 100%.

[0086] The separated peptide segments are subjected to data-dependent acquisition (DDA) mass spectrometry analysis using a Q-Exactive HF-X mass spectrometer (Thermo Scientific). The analysis time is 60 min, the detection mode is positive ion, the precursor ion scan range is 350 - 1800 m / z, the first-stage mass spectrometry resolution is 60000 @ m / z 200, the AGC target is 3e6, and the first-stage Maximum IT is 50 ms. After each full scan, 20 precursor ions with the highest intensities are selected for the second-stage mass spectrometry spectrum (MS2 scan). The second-stage mass spectrometry resolution is 15000 @ m / z 200, the AGC target is 1e5, the second-stage Maximum IT is 50 ms, MS2 Activation Type: HCD, Isolation window: 1.6 m / z, Normalized collision energy: 28.

[0087] The mass spectrometry base peak chromatogram of component X5 is as Figure 10 shown.

[0088] (3)Database search

[0089] Search the peaks that appear at different times in Figure 10 using the mass spectrometry database search software MaxQuant 2.4.24.0.

[0090] After searching the library, 27 peptide segments were obtained (as shown in Table 3). Most of the peptide segments carried only 1 or 2 charges, without modifications such as methylation and acetylation, and most of the peptide segments were short in length, resulting in a molecular weight of less than 1 KDa. All 27 peptide segments contained hydrophobic amino acids: valine (V), leucine (L), isoleucine (I), alanine (A), and methionine (M).

[0091] Experiment 3 Rational Design Study of Bioactive Peptides with XO Inhibitory Activity

[0092] 3.1 Molecular Docking of Peptide Segments

[0093] 3.1.1 Receptor Modification

[0094] Download the 3D structure of 1N5X containing the ligand febuxostat (TEI) from the PDB database (https: / / www.rcsb.org / ). Delete the B chain in 1N5X through the PyMOL software, and at the same time delete the febuxostat ligand molecule in the sequence. Save the processed molecule in PDB format. Then use the Autodock software to dehydrate and hydrogenate the receptor 1N5X, calculate the gasteiger charge (4.41), and finally attribute all atoms to the AD4 type and save it in PDBQT format.

[0095] 3.1.2 Ligand Processing

[0096] Draw the structural formula of the polypeptide (i.e., the 27 peptide segments obtained in Experiment 2) through the software KingDraw, then convert the structural formula of the polypeptide into the three-dimensional spatial result of the polypeptide, and finally save it in mol2 format after optimization by the MM2 force field.

[0097] 3.1.3 Molecular Docking

[0098] Perform molecular docking of the modified receptor 1N5X and the ligand polypeptide through the software PyRx to simulate the interaction between the receptor and the ligand. According to the position of febuxostat in the bound state in the receptor 1N5X, set the center coordinates of the docking box to x = 96.663, y = 54.963, z = 39.433, the box size to 40×40×40 grid points, the number of docking times to 8 times, and other parameters to default values. Through molecular docking with the receptor 1N5X, obtain the Vina scores of 27 peptide segments, and predict the related properties (isoelectric point and toxicity) of the peptide segments through the websites Innovagen Tool and ToxinPred.

[0099] The sequences of 27 peptide segments, their Vina scores, and the predicted results of related properties are shown in Table 3. It can be seen that the absolute values of the Vina scores of the peptide segments with more amino acid residues (the higher the absolute value, the stronger the binding ability) are relatively low. This may be because their spatial configurations are relatively large, making it difficult for them to bind to the active pocket of the receptor.

[0100]

[0101] 3.2 Synthesis and verification of peptide segments

[0102] According to the above Vina scores, 7 bioactive peptides with the highest binding energy were screened out, namely: GPPGPAGPPV, GPPGPPAGP, PPGPPA, QDYRPR, GPPGPA, SDALVLQ, and GPPGPPG. Their amino acid sequences are shown in SEQ ID NO.1 - 7.

[0103] Shanghai Sangon Biotech Co., Ltd. was commissioned to perform Fmoc solid-phase synthesis on these 7 peptide segments. The synthesis amount of each peptide segment was 15 mg, and the purity of the synthesized polypeptide was 95%. The polypeptide was configured into solutions with concentrations of 0, 2, 4, 6, 8, and 10 mg / mL respectively, and their in vitro XO inhibition rates and IC 50 values were measured. The results showed that the bioactive peptides GPPGPPAGP, PPGPPA, QDYRPR, and GPPGPA all had in vitro XO inhibitory activities. The measurement results of the in vitro XO inhibition rates and IC 50 values of the bioactive peptides are as Figure 11 shown. The IC 50 value of the bioactive peptide GPPGPPAGP was 7.91 ± 0.16 mM, the IC 50 value of the bioactive peptide PPGPPA was 9.47 ± 0.12 mM, the IC 50 value of the bioactive peptide QDYRPR was 6.42 ± 0.31 mM, and the IC 50 value of the bioactive peptide GPPGPA was 8.76 ± 0.56 mM. It can be seen that the IC50 value of the bioactive peptide QDYRPR was the lowest and its activity was the strongest, but it was still not ideal enough. To obtain bioactive peptides with better activity, the present invention attempted to perform rational design based on the bioactive peptide QDYRPR, as described below.

[0104] 3.3 Selection of backbone amino acids and substituted amino acids

[0105] Twenty common amino acids (i.e., the 20 amino acids that make up human proteins) were processed using the same method as in 3.1.2, and then molecular docking was performed with the receptor 1N5X. The selection of backbone amino acids and substituted amino acids for rational design was determined according to the Vina scores.

[0106] By performing molecular docking with the receptor 1N5X, the Vina scores of 20 common amino acids were obtained. The Vina scores of the 20 common amino acids are as follows: Figure 12 As shown, it can be seen that the absolute values of the Vina scores of aromatic amino acids (Trp, Tyr, and Phe) are the highest, among which Trp is the highest, Tyr is the second highest, and Phe is the lowest. The absolute value of the Vina score of Gly is the lowest, probably because its structure is the simplest.

[0107] 3.4 Rational design

[0108] 3.4.1 Based on the results of 3.3 above, Gly was selected as the backbone amino acid of the peptide segment, and aromatic amino acids (Trp, Tyr, and Phe) were selected as the replacement amino acids for the rational design of the bioactive peptide QDYRPR. The steps are as follows:

[0109] (1) Design of single or mixed replacement of N-terminal, C-terminal, and middle positions with aromatic amino acids: Gln at the N-terminal, Arg at the C-terminal, and Tyr in the middle of the bioactive peptide QDYRPR were replaced. After replacement, two types of small peptides, namely single amino acid replacement (such as FDFRPF) and mixed amino acid replacement (such as YDWRPF), were obtained, which are called type ① and type ② small peptides. The type ① and type ② small peptides were respectively subjected to molecular docking with the receptor 1N5X to obtain their Vina scores. The sequences and Vina scores of the type ① and type ② small peptides are shown in Table 4.

[0110]

[0111] As can be seen from Table 4, the absolute value of the Vina score of the peptide sequence with single replacement of Phe in this category of design is the highest, followed by that with single replacement of Trp, and the lowest with single replacement of Tyr. Among the mixed replacements, the peptide sequence with the highest absolute value of the Vina score is YDWRPF, and its absolute value of the Vina score is 9.9. And when there are 2 Phes in the peptide chain, the absolute values of the Vina scores of the peptide segments with Phe at the N-terminal and C-terminal are higher than those at the middle position and one end.

[0112] (2) Design of replacement of N-terminal, C-terminal, or middle position with aromatic amino acids: Gln at the N-terminal, Arg at the C-terminal, or Tyr in the middle of the bioactive peptide QDYRPR were replaced. After replacement, three types of small peptides, namely replacement of N-terminal amino acid (such as FDGRPR), replacement of C-terminal amino acid (such as QDGRPF), and replacement of middle amino acid (such as QDFRPR), were obtained, which are called type ③, type ④, and type ⑤ small peptides. The type ③, type ④, and type ⑤ small peptides were respectively subjected to molecular docking with the receptor 1N5X to obtain their Vina scores. The sequences and Vina scores of the type ③, type ④, and type ⑤ small peptides are shown in Table 5.

[0113]

[0114] As can be seen from Table 5, the peptide sequence with the highest absolute value of the Vina score is FDGRPR, and its absolute value of the Vina score is 7.9. When replacing at the same position, the peptide sequence replaced with Phe has the highest absolute value of the Vina score, followed by the sequence replaced with Trp alone, and the lowest is the sequence replaced with Tyr alone. And when replacing with the same aromatic amino acid, the absolute value of the Vina score of the peptide with the aromatic amino acid replacing the N-terminus is the highest, followed by that of the peptide with the aromatic amino acid replacing the C-terminus, and the lowest is the peptide with the aromatic amino acid replacing the middle position.

[0115] (3)Design of replacing the N-terminal and C-terminal positions with aromatic amino acids: Gln at the N-terminus and Arg at the C-terminus of the bioactive peptide QDYRPR were replaced. After replacement, peptide segments with aromatic amino acids only at the N-terminus and C-terminus (such as WDGRPF) can be obtained, which are called type ⑥ small molecule peptides. The type ⑥ small molecule peptides were respectively docked with the receptor 1N5X to obtain their Vina scores. The sequences and Vina scores of the type ⑥ small molecule peptides are shown in Table 6.

[0116]

[0117] As can be seen from Table 6, the peptide sequence with the highest absolute value of the Vina score is WDGRPF, and its absolute value of the Vina score is 9.5. Compared with the sequences that replace only the N-terminus or C-terminus, the absolute value of the Vina score of the sequence that replaces both ends simultaneously is higher.

[0118] (4)Design of replacing the middle and one end positions with aromatic amino acids: Tyr in the middle of the bioactive peptide QDYRPR, and Gln at the N-terminus or Arg at the C-terminus were replaced. After replacement, peptide segments with aromatic amino acids only at the middle and one end positions can be obtained, which are divided into two types of small molecule peptides, single amino acid replacement and mixed amino acid replacement, called type ⑦ and type ⑧ small molecule peptides. The type ⑦ and type ⑧ small molecule peptides were respectively docked with the receptor 1N5X to obtain their Vina scores. The sequences and Vina scores of the type ⑦ and type ⑧ small molecule peptides are shown in Table 7.

[0119]

[0120] As can be seen from Table 7, the peptide sequence with the highest absolute value of the Vina score is FDFRPR, and its absolute value of the Vina score is 8.5. When the number of aromatic amino acids replaced is the same, the absolute value of the Vina score of the peptide segment with the aromatic amino acid at the N-terminal position is higher than that of the peptide segment with the aromatic amino acid at the C-terminal position.

[0121] (5)Effect of the number of amino acids in the peptide segment: Through the above research, it was found that Phe plays an important role in the Vina score of peptide molecular docking. Therefore, using the peptide FDFRPF with Phe replaced singly as the original peptide, small molecule peptides with different peptide chain lengths were designed, called class ⑨ small molecule peptides. The class ⑨ small molecule peptides were respectively subjected to molecular docking with the receptor 1N5X to obtain their Vina scores. The sequences and Vina scores of the class ⑨ small molecule peptides are shown in Table 8.

[0122]

[0123] As can be seen from Table 8, when the number of amino acids changes from 5 to 3, that is, from FDFRF to FFF, the absolute value of the Vina score increases from 8.5 to 9.1.

[0124] 3.4.2 Analysis of the Vina score of small molecule peptides

[0125] Synthesize the small molecule peptides with the highest absolute value of the Vina score in each category of the above rational design (that is, the small molecule peptide YDWRPF in Table 4, whose amino acid sequence is shown in SEQ ID NO.8; the small molecule peptide FDGRPR in Table 5, whose amino acid sequence is shown in SEQ ID NO.9; the small molecule peptide WDGRPF in Table 6, whose amino acid sequence is shown in SEQ ID NO.10; the small molecule peptide FDFRPR in Table 7, whose amino acid sequence is shown in SEQ ID NO.11; the small molecule peptide FFF in Table 8, whose amino acid sequence is shown in SEQ ID NO.12;), and verify their in vitro XO inhibitory activity (determine the IC 50 value).

[0126] Results: The relationship between the Vina scores of various small molecule peptides and the IC 50 value is as Figure 13 shown. The IC 50 of the small molecule peptide YDWRPF is 2.73 ± 0.20 mM, the IC 50 of the small molecule peptide WDGRPF is 2.96 ± 0.16 mM, the IC 50 of the small molecule peptide FFF is 7.63 ± 0.24 mM, the IC 50 of the small molecule peptide FDFRPR is 9.67 ± 0.02 mM, and the IC 50 of the small molecule peptide FDGRPR is 11.12 ± 0.07 mM. The analysis results show that there is a significant structure-activity relationship between the Vina score and biological activity. From Figure 13It can be seen that the higher the absolute value of the Vina score, the stronger the in vitro XO inhibitory activity of the corresponding polypeptide. There is an obvious positive correlation between the two. Moreover, the in vitro XO inhibitory activities of the small peptides YDWRPF, WDGRPF, and FFF with aromatic amino acids at both ends are significantly higher than those of the small peptides FDFRPR and FDGRPR with aromatic amino acids at only one end.

[0127] 3.4.3 Molecular docking analysis of small peptides

[0128] The docking result of the small peptide YDWRPF with 1N5X is as Figure 14 shown. As Figure 14 can be seen, the benzene ring structure of Tyr at the N-terminus of the small peptide YDWRPF is stably bound within the active pocket of 1N5X. This spatial arrangement provides a channel for xanthine to enter the enzyme catalytic center, enabling it to be smoothly converted into uric acid through the metabolic pathway. The small peptide YDWRPF forms hydrogen bond interactions with the amino acid residues Tyr1140, Asn768, Thr1010, Ser876, Asp872, and Glu1261 of XO. Moreover, the benzene ring structure of Tyr in the small peptide YDWRPF forms π-alkyl interactions with the amino acid residue Ala1078, one π-π stacking interaction with each of the residues Phe914 and Phe1009, and a π-σ interaction with the residue Ala1079. The indole structure of Trp forms π-alkyl interactions with the amino acid residue Leu648, and the benzene ring structure of Phe forms one π-π stacking interaction with the residue Phe1142. Compared with the original peptide segment QDYRPR, the benzene ring stacking interactions of the designed small peptide YDWRPF with the aromatic amino acids on 1N5X increase. This enhanced intermolecular interaction may optimize the substrate binding conformation and ultimately improve its catalytic activity. This may be the reason why the in vitro XO inhibitory activity of the small peptide YDWRPF is significantly higher than (P < 0.05) that of the peptide segment QDYRPR.

[0129] The docking result of the small peptide FDGRPR with 1N5X is as Figure 15 shown. As Figure 15 can be seen, the benzene ring structure of Phe at the N-terminus of the small peptide FDGRPR can be firmly embedded within the active pocket of 1N5X. The small peptide FDGRPR forms hydrogen bond interactions with the amino acid residues Ser876, Glu879, and Tyr1140 of XO. Moreover, the benzene ring of Phe in the small peptide FDGRPR forms π-alkyl interactions with the amino acid residues His875 and Ala1078, one π-π stacking interaction with the residue Phe914, and a π-σ interaction with the residue Ala1079.

[0130] The docking results of the small molecule peptide WDGRPF with 1N5X are as follows Figure 16 shown. It can be seen from Figure 16 that there is an aromatic amino acid Trp at the N-terminus of the small molecule peptide WDGRPF, which contains a unique indole structure, and there is an aromatic amino acid Phe at the C-terminus of the peptide segment, and its side chain contains a benzene ring structure, resulting in the ability of the small molecule peptide WDGRPF to produce benzene ring interactions with the benzene rings of the aromatic amino acids around the 1N5X protein. The small molecule peptide WDGRPF forms hydrogen bond interactions with the amino acid residues Glu802, Glu879 and Tyr1010 of XO. Moreover, the indole structure of Trp in the small molecule peptide WDGRPF forms a π-alkyl interaction with the amino acid residue Ala1078, forms 2 π-π stacking interactions with the residues Phe914 and Phe1009, forms a π-σ interaction with the residue Ala1079, and the benzene ring structure of Phe forms 1 π-π stacking interaction with the amino acid residue Phe1142 and forms a π-σ interaction with the residue Leu712. Compared with the original peptide segment QDYRPR, the π-π stacking interaction of the small molecule peptide WDGRPF with the aromatic amino acids on the surface of the 1N5X protein is significantly enhanced (P<0.05), which may be the reason why the in vitro XO inhibitory activity of the small molecule peptide WDGRPF is significantly better than that of the peptide segment QDYRPR.

[0131] The docking results of the small molecule peptide FDFRPR with 1N5X are as follows Figure 17 shown. It can be seen from Figure 17 that the benzene ring of the aromatic amino acid Phe existing in the middle of the small molecule peptide FDFRPR produces interactions with the benzene rings of the aromatic amino acids around the 1N5X protein. The small molecule peptide FDFRPR forms hydrogen bond interactions with the amino acid residues Glu879, Tyr1140, Ser1141, Phe1142 and Glu1143 of XO. Moreover, the benzene rings of Phe at the N-terminus and in the middle of the small molecule peptide FDFRPR form π-alkyl interactions with the amino acid residues Leu1014, Pro1076 and Arg871, form 1 π-π stacking interaction with the residue Phe649, and form a π-σ interaction with the residue Leu648.

[0132] The docking results of the small molecule peptide FFF with 1N5X are as follows Figure 18 shown. It can be seen from Figure 18It can be seen that the benzene ring structure of Phe at the N-terminus of the small molecule peptide FFF can be embedded in the active pocket of 1N5X and interact with the benzene rings of the surrounding aromatic amino acids. The small molecule peptide FFF forms a hydrogen bond interaction with the amino acid residue Thr1010 of XO, and the benzene ring of Phe in the small molecule peptide FFF forms a π-alkyl interaction with the amino acid residues Leu648, Leu1014, Pro1076, and Ala1078, and forms a π-π stacking interaction with the residue Phe914, and forms a π-σ interaction with the residues Leu648 and Ala1079.

[0133] The results of the above molecular docking show that during the rational design of active peptides, when the interaction between the peptide segment and the amino acid residues Phe914, Phe1009, and Phe1142 in the 1N5X protein gradually increases, its in vitro XO inhibitory activity is stronger. And when the peptide segment forms more π-π stacking interactions with the amino acid residues of XO, the Vina score of the peptide segment is higher and the XO inhibitory activity is stronger. A polypeptide composed of alternating simple amino acids (Gly) and aromatic amino acids (Trp, Tyr, and Phe), when it has an appropriate chain length, not only shows a higher Vina score but also exhibits good in vitro XO inhibitory activity. Research shows that the XO inhibitory activity of polypeptides mainly depends on the type, position, and quantity of specific amino acids, and the arrangement order and synergistic effect also play a key role.

[0134] The above embodiments are provided to fully disclose and describe how to implement and use the claimed embodiments to those skilled in the art, rather than to limit the scope disclosed herein. Modifications that are obvious to those skilled in the art will be within the scope of the appended claims.

Claims

1. A small molecule peptide with xanthine oxidase inhibitory activity, characterized in that: The amino acid sequence is YDWRPF, as shown in SEQ ID NO.

8.

2. Use of the small molecule peptide with xanthine oxidase inhibitory activity according to claim 1 in the preparation of a xanthine oxidase inhibitor.

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