Polypeptides with xanthine oxidase inhibitory activity and their applications

Abstract

The present invention discloses a polypeptide with xanthine oxidase inhibitory activity and its application, belonging to the technical field of bioactive peptides. The amino acid sequence of the polypeptide with xanthine oxidase inhibitory activity is WEWTW, as shown in SEQ ID NO.11. The application of the polypeptide with xanthine oxidase inhibitory activity in the preparation of xanthine oxidase inhibitors. The present invention rationally designed the polypeptide WEWTW, which has strong xanthine oxidase inhibitory activity, and the IC 50 value is 1.73 ± 0.13 mmol / L. Xanthine oxidase inhibitors can be prepared using 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 polypeptide with xanthine oxidase inhibitory activity and its application, belonging to the technical field of bioactive peptides. Background Art

[0002] Hyperuricemia is a disease caused by excessive uric acid levels. The main clinical treatment method is to interfere with the production of uric acid by inhibiting the activity of xanthine oxidase (XO). Currently, bioactive peptides with XO inhibitory activity have been found in marine-derived foods such as tuna, oysters, and Pacific white shrimp, but there are no relevant reports on XO inhibitory peptides derived from Penaeus monodon.

[0003] Penaeus monodon is an animal of the genus Penaeus in the family Penaeidae of the order Decapoda. Also known as grass shrimp and black tiger shrimp, it is widely distributed worldwide and is the second most cultured penaeid shrimp species after Litopenaeus vannamei. Summary of the Invention

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

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

[0006] A polypeptide with xanthine oxidase inhibitory activity, with the amino acid sequence WEWTW, as shown in SEQ ID NO.11.

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

[0008] To develop bioactive peptides with XO inhibitory activity from natural sources and explore their structure-activity relationships, the present invention used papain to enzymatically hydrolyze Penaeus monodon, obtained enzymatically hydrolyzed products with XO inhibitory activity, separated and purified the enzymatically hydrolyzed products, and screened out 4 polypeptides with XO inhibitory activity (FPNPW, WPPQPMPQ, WENWTK, WPGDMDRV). Among them, the polypeptide WENWTK has the best XO inhibitory activity, with an IC 50 value of 2.77 ± 0.06 mmol / L. To obtain a polypeptide with stronger activity, the present invention carried out rational design based on the polypeptide WENWTK, and finally obtained a polypeptide WEWTW with better XO inhibitory activity, with an IC 50 value of 1.73 ± 0.13 mmol / L.

[0009] The present invention rationally designed the polypeptide WEWTW, which has strong xanthine oxidase inhibitory activity, and can be used as a raw material to prepare xanthine oxidase inhibitors. 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. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 : Elution diagram of the enzymatic hydrolysis product on a Sephadex G15 gel chromatography column.

[0012] Figure 2 : Determination results of the XO inhibition rate of each component.

[0013] Figure 3 : Mass spectrometry base peak diagram of component F4.

[0014] Figure 4 : Determination results of the XO inhibition rate and IC 50 value of 4 polypeptides, where A; FPNPW: B: WENWTK; C: WPPQPMPQ; D: WPGDMDRV.

[0015] Figure 5 : 3D interaction diagram of the molecular docking of polypeptide WENWTK with 1N5X.

[0016] Figure 6 : 2D interaction diagram of the molecular docking of polypeptide WENWTK with 1N5X.

[0017] Figure 7 : Absolute value of the binding energy of the molecular docking of 20 common amino acids with 1N5X.

[0018] Figure 8 : Comparison of the absolute value of the binding energy and IC 50 value of 5 polypeptides.

[0019] Figure 9 : 3D interaction diagram of the molecular docking analysis of polypeptide WEWTW with 1N5X.

[0020] Figure 10 : 2D interaction diagram of the molecular docking analysis of polypeptide WEWTW with 1N5X. DETAILED DESCRIPTION OF THE INVENTION

[0021] 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.

[0022] For the instruments, reagents, and materials involved in the following examples, unless otherwise specified, they are all conventional instruments, reagents, and materials existing in the prior art and can be obtained through regular commercial channels. For the experimental methods, detection methods, etc. involved in the following examples, unless otherwise specified, they are all conventional experimental methods and detection methods existing in the prior art.

[0023] Experiment 1: Isolation and Purification of Polypeptides with XO Inhibitory Activity

[0024] In previous studies, the present invention enzymatically hydrolyzed Penaeus monodon and screened out suitable enzymes and enzymatic hydrolysis conditions: The heads and shells of Penaeus monodon were removed and homogenized, deionized water was added according to a solid-liquid ratio of 1:3 (g:mL), papain was added for enzymatic hydrolysis, the enzyme dosage was 3000 U / g, the enzymatic hydrolysis temperature was 50 °C, the enzymatic hydrolysis time was 6 h, and the enzymatic hydrolysis product had significant XO inhibitory activity.

[0025] In this experiment, the enzymatic hydrolysis product was separated and purified in order to obtain polypeptides with XO inhibitory activity, as detailed below. All measurements were performed in at least three parallels, data processing was calculated using Excel 2021, and data analysis was performed using the LSD method and Duncan method of one-way analysis of variance in SPSS 26.0 to determine statistically different values at a significant level of P < 0.05. The data representation form was mean ± standard deviation, and data visualization was processed using Origin 2021 software.

[0026] 1.1 Gel Filtration Chromatography

[0027] 1.1.1 Preparation of Enzymatic Hydrolysis Solution

[0028] Take 4 g of shrimp meat homogenate, add 12 mL of deionized water according to a solid-liquid ratio of 1:3, add papain according to a ratio of 3000 U / g, place it in a water bath shaker, and enzymatically hydrolyze 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, and collect the supernatant, which is the enzymatic hydrolysis solution.

[0029] 1.1.2 Gel Filtration Chromatography

[0030] Dextran gel G15 was pretreated according to the instructions. At room temperature, the dry powder of dextran gel G15 was mixed with deionized water and continuously stirred to ensure that the gel dry powder was fully dissolved. It was left standing for at least 24 hours to ensure that the gel was fully swollen. The swollen gel was stirred into a suspension state and poured into a chromatography column (16 mm×70 cm) at one time. During the column packing process, it was necessary to ensure that the packing was always in a wet state and the surface of the column bed was always covered with water. After natural sedimentation, the column bed was pressurized with deionized water by means of a constant flow pump. When the height of the packing no longer changed and the reading of the detector was always 0, sample loading could begin. Before sample loading, the sample was pre-filtered through a 0.45-micron membrane. The sample loading concentration was 100 mg / mL, the sample loading volume was 3 mL, the eluent was deionized water, the knob of the constant flow pump was adjusted to set the flow rate of the mobile phase to 1.5 mL / min, and the elution components were collected according to the elution time. With time as the abscissa and the absorbance at 230 nm as the ordinate, an elution diagram of the enzymatic hydrolysate on the dextran G15 gel chromatography column was drawn. Each elution component was freeze-dried, redissolved, and the polypeptide concentration was adjusted to 20 mg / mL, and the XO inhibition rate was measured.

[0031] 1.1.3 Method for measuring XO inhibition rate

[0032] The 0.05 U / mL XO solution and 0.4 mmol / L xanthine solution were pre-incubated in a 37°C constant temperature incubator; 50 μL of the sample and 50 μL of the XO solution were respectively added to a 96-well microplate, mixed evenly, incubated in a 37°C constant temperature incubator for 25 min, taken out, 150 μL of the xanthine solution was added, shaken and mixed evenly, and the kinetic change of the absorbance of the reaction system at 290 nm within 3 min (10 s / time) was recorded, with pH 7.4 phosphate buffer as the blank control. The calculation formula for the XO inhibition rate is: ; where V s represents the initial reaction rate of the sample; V0 represents the initial reaction rate of the blank control.

[0033] 1.1.4 Results

[0034] The elution diagram of the enzymatic hydrolysate on the dextran G15 gel chromatography column is as Figure 1 shown. It can be seen that after the enzymatic hydrolysate was separated by dextran G15 gel chromatography, 5 components (named F1 - F5) were obtained, and the separation effect of each peak was good. The measurement results of the XO inhibition rate of each component are as Figure 2As shown (in the figure, F0 represents the enzymatic hydrolysis product), it can be seen that the XO inhibitory activity of component F4 (XO inhibition rate is 94.34% ± 0.30%) is the best, significantly superior to that of component F1 (XO inhibition rate is 41.84% ± 2.77%), component F2 (XO inhibition rate is 42.43% ± 0.77%), component F3 (XO inhibition rate is 62.83% ± 1.75%) and component F5 (XO inhibition rate is 88.08% ± 0.70%). Therefore, component F4 with the strongest XO inhibitory activity was selected for LC-MS / MS analysis.

[0035] 1.2 LC-MS / MS Analysis

[0036] 1.2.1 Peptide Desalting

[0037] Take the sample, add it to an appropriate amount of trifluoroacetic acid (TFA) solution (concentration is 0.1%, unit g / mL), mix well, centrifuge at 20000g 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, collect the filtrate. Use C18 StageTip for desalting treatment and vacuum drying. The dried peptide is redissolved with formic acid (FA) solution (concentration is 0.1%, unit g / mL), measure its concentration, and prepare for LC-MS / MS analysis.

[0038] 1.2.2 LC-MS / MS Analysis

[0039] Take an appropriate amount of peptide and perform chromatographic analysis using a nano-flow Easy nLC 1200 chromatographic system. The buffers used are solution A (0.1% formic acid solution, concentration unit g / mL) and solution B (80% acetonitrile solution, volume ratio). Equilibrate the chromatographic column with 100% solution A, inject the sample into the trapping column (100 μm × 20 mm, 5 μm, C18), and then perform gradient separation through the analytical column (75 μm × 150 mm, 3 μm, C18) at a flow rate of 300 nL / min.

[0040] The liquid phase separation gradient is as follows: 0 - 2 min, the linear gradient of solution B is from 2% to 5%; 2 - 44 min, the linear gradient of solution B is from 5% to 28%; 44 - 51 min, the linear gradient of solution B is from 28% to 40%; 51 - 53 min, the linear gradient of solution B is from 40% to 100%; 53 - 60 min, solution B is maintained at 100%.

[0041] After peptide separation, data-dependent acquisition (DDA) mass spectrometry analysis was performed using a Q-Exactive Plus mass spectrometer. The analysis duration was 60 min, the detection mode was positive ion, the parent ion scanning range was 350 - 1800 m / z, the resolution of the first-level mass spectrometry was 70000 @m / z 200, the AGC target was 3e6, and the first-level Maximum IT was 50 ms. The second-level mass spectrometry analysis of peptides was acquired according to the following method: after each full scan, the second-level mass spectrometry spectra of 20 parent ions with the highest intensities were triggered for acquisition. The resolution of the second-level mass spectrometry was 17000 @m / z 200, the AGC target was 1e5, the second-level Maximum IT was 50 ms, the MS2 Activation Type was HCD, the Isolation window was 1.6 m / z, and the Normalized collision energy was 28.

[0042] The mass spectrometry base peak chromatogram of component F4 is as Figure 3 shown.

[0043] 1.2.3 Database search

[0044] For the Figure 3 peaks that appeared at different times, the mass spectrometry database search software used was MaxQuant 2.0.1.0.

[0045] After database search, 151 peptides were obtained. The shortest peptide was a pentapeptide, the longest peptide consisted of 23 amino acids, and the number distributions of octapeptides, nonapeptides, decapeptides, and undecapeptides were relatively large.

[0046] 1.3 Prediction of the basic properties of peptides

[0047] Based on the peptide sequences obtained from the mass spectrometry identification results, these peptide sequences were preliminarily screened using a toxicity prediction website (https: / / webs.iiitd.edu.in / raghava / toxinpred / index.html) and a bioactivity scoring tool (http: / / distilldeep.ucd.ie / PeptideRanker / ). Peptides with predicted non-toxicity and an activity score greater than 0.5 were selected. There were a total of 30 peptide segments with a potential activity score above 0.5.

[0048] Then, the physicochemical properties of the polypeptide amino acid sequence, including isoelectric point, instability coefficient, and total average hydrophilicity (Gravy), were simply analyzed using the ProtParam tool (https: / / www.expasy.org / ) on the ExPASy website. The water solubility of the polypeptide was analyzed using the peptide property calculator (Proteomics tools (innovagen.com)) on the Innovagen website. The allopurinol and these 30 peptide segments were respectively docked with XO using the Autodock Vina algorithm. The results showed that the binding energy of allopurinol was negative (-7.2 Kcal / mol), indicating that the docking site of XO was set correctly. A negative binding energy indicates that the ligand can successfully dock with the macromolecular receptor XO. The smaller the negative value, the lower the energy required for the ligand to dock with the receptor, and the more likely it is for the peptide to obtain a stable conformation when binding to XO.

[0049] The sequences, property predictions, and molecular docking binding energies of the 30 peptide segments are shown in Table 1.

[0050]

[0051] Note: The total average hydrophilicity is the ratio of the sum of the hydrophilic values of all amino acid sequences to the number of amino acids. The larger the negative value, the stronger the hydrophilicity; the larger the positive value, the stronger the hydrophobicity. For the instability coefficient index, generally, if it is less than 40, it indicates stability; if it is greater than 40, it is unstable.

[0052] Experiment 2 Rational Design Study of Bioactive Peptides with XO Inhibitory Activity

[0053] 2.1 Synthesis and Activity Verification of Peptide Segments

[0054] The 6 peptide segments with the smallest binding energies screened in Experiment 1 were: FPNPW, WPPQPMPQ, WENWTK, WPGDMDRV, GPAGPIGPTG, VGPIGPRGPAG, and their amino acid sequences are shown in SEQ ID NO.1 - 6 in sequence. These 6 polypeptides were commissioned to be synthesized by Shanghai Sangon Biotech Co., Ltd. with a synthesis purity of 95%, and desalting treatment was carried out. The synthesized polypeptide powder was redissolved with deionized water to prepare a certain concentration, and the XO inhibition rate was measured. The IC 50 value was calculated by the non - linear regression method by integrating the inhibitor concentration and its average inhibition rate.

[0055] The results showed that for 2 polypeptides (GPAGPIGPTG, VGPIGPRGPAG), the inhibition rate of XO did not exceed 50% at a concentration of 10 mg / mL; 4 polypeptides (FPNPW, WPPQPMPQ, WENWTK, WPGDMDRV) showed relatively obvious XO inhibitory activity. The measurement results of the XO inhibition rate and IC 50 values are as Figure 4 shown. The IC 50 value of polypeptide FPNPW was 4.06 ± 0.15 mmol / L, the IC 50 value of polypeptide WPPQPMPQ was 6.23 ± 0.06 mmol / L, the IC 50 value of polypeptide WENWTK was 2.77 ± 0.06 mmol / L, and the IC 50 value of polypeptide WPGDMDRV was 3.96 ± 0.09 mmol / L, showing significant differences. The IC 50 values of these 4 polypeptides were significantly lower than those of the xanthine oxidase inhibitory peptide ACECD (13.40 mmol / L) obtained from skipjack hydrolysate by Zhong et al., but far less than the inhibitory activity of allopurinol (0.016 ± 0.00032 mmol / L) against XO. They may have insufficient potential for drug development in the treatment of hyperuricemia, but can contribute to a certain extent as functional food-derived peptides in improving hyperuricemia and gout, and have relatively less toxic and side effects on human health compared to drugs. All 4 of these polypeptides contain tryptophan, indicating that the presence of tryptophan can affect the exertion of the inhibitory activity of the polypeptide against XO, which may be due to the structural similarity between the indole group in tryptophan and the drug allopurinol.

[0056] To obtain XO inhibitory peptides with better activity, the present invention attempts to perform rational design based on polypeptide WENWTK, as described below.

[0057] 2.2 Molecular docking of peptide segments

[0058] 2.2.1 Treatment of receptor and ligand

[0059] Download the crystal structure of xanthine oxidase (PDB ID: 1N5X) from the PDB database (https: / / www.rcsb.org) as the protein receptor, import it into pymol, delete one of the parent chains (chain B) and the bound ligand (TEI), and save this macromolecule in pdbqt format; then use Autodock Tools to open the macromolecule in pdbqt format, and perform operations such as dehydration, hydrogenation, atom AD4 formatting, and charge calculation in sequence, and save it in pdb format, that is, obtain the ideal receptor for molecular docking.

[0060] Use KingDraw to convert the polypeptide sequence into a polypeptide structural formula, then convert the molecular structural formula into a three-dimensional structure in KingDraw3D, perform MM2 energy minimization processing on it, save it in mol2 format, and finally obtain an ideal ligand for molecular docking.

[0061] 2.2.2 Molecular Docking of Receptor and Ligand

[0062] Import the processed receptor and ligand into PyRx for docking. Based on the position of the original inhibitor ligand (TEI), set the center coordinates of the docking box ( x = 96.6635, y = 54.963, z = 39.4334). The size of the docking box is 40*40*40, and the number of docking times is 8 times. Docking the polypeptide ligand with 1N5X one by one, and screening based on the binding energy (Kcal / mol) obtained by the Autodock Vina algorithm in PyRx. The smaller the binding energy (negative value), the better the docking effect of the polypeptide ligand with 1N5X.

[0063] 2.2.3 Analysis of Molecular Docking Results

[0064] Use Discovery Studio 4.5 (DS) to explore the interaction mechanism between the polypeptide and xanthine oxidase. Open the polypeptide file and 1N5X file output by the Autodock Vina algorithm in PyRx using Pymol software, and export them as ligand-receptor binding molecules, saving the file in pdb format; open this binding molecule file using Discovery Studio 4.5, where Receptor and Ligand are the receptor and ligand respectively, and visualization can be selectively performed. Observe the spatial position relationship between the polypeptide small molecule and 1N5X with the help of the "Display receptor surfaces" function, and use "Show2D Diagram" to show the interaction types and interaction distances between the polypeptide small molecule and 1N5X. Based on the docking conformation results, analyze the influence on the conformational stability of the complex from factors such as hydrogen bonds, alkyl interactions, van der Waals forces, and other interactions.

[0065] The 3D interaction diagram of the molecular docking of polypeptide WENWTK with 1N5X is as Figure 5 shown, and the 2D interaction diagram of the molecular docking of polypeptide WENWTK with 1N5X is as Figure 6 shown. As Figure 5 can be seen, the tryptophan (W) at the N-terminus of the peptide segment can completely enter the active pocket of 1N5X, fully occupy a part of the active center of 1N5X, and thus hinder the entry of xanthine into the space of the active center of 1N5X. AsFigure 6 It can be seen that the positive charge carried by the amide bond formed by the side-chain amino group of lysine (K) at the C-terminus of the peptide segment and the carboxyl group of threonine (T) forms an attractive charge interaction with Glu-879 and Glu-1143 of 1N5X. The polypeptide WENWTK contacts His-875 and Thr-1010 of XO through carbon-hydrogen bond interactions. There is a conventional hydrogen bond interaction between the polypeptide and the amino acids around the 1N5X pocket, formed by -NH in the N-terminal tryptophan and Glu-802. The -CH3 in the polypeptide forms 4 alkyl interactions with Ala-1078, Ala-1079, Pro-1012, and Val-1011 of 1N5X. Phe-1009, Phe-914, and Phe-1013 form 3 π-alkyl interactions with the tryptophan on the polypeptide. In addition, the polypeptide forms van der Waals interactions with the amino acids around the 1N5X pocket such as Arg-880, Ser-780, Glu-711, Leu-712, Phe-1142, Ser-1141, Tyr-1140, His-875, Leu-648, Leu-1014, Pro-1076, Thr-1010, and Arg-880.

[0066] 2.3 Rational design

[0067] 2.3.1 Molecular docking of 20 common amino acids with XO

[0068] The 20 common amino acids were docked with XO one by one, and the absolute values of the binding energies obtained by the Autodock Vina algorithm were sorted. The higher the absolute value of the binding energy, the better the molecular docking result.

[0069] The absolute values of the binding energies of the 20 common amino acids docked with 1N5X are as Figure 7 shown. Among all the amino acids, the aromatic amino acids have the best docking effect with the XO molecule. Among the aromatic amino acids, tryptophan has the highest absolute value of the binding energy for docking with the XO molecule, followed by tyrosine and phenylalanine. This may be because the molecular structures of these aromatic amino acids contain benzene ring structures, and the presence of the benzene ring structure helps to form π-π interactions with the key amino acid Phe-914 in the active pocket of XO, stabilizing the binding of the polypeptide to XO and hindering the oxidation of xanthine by XO. Therefore, when optimizing the sequence, aromatic amino acids (tryptophan, phenylalanine, and tyrosine) with a relatively high contribution to the inhibitory activity of XO were selected for amino acid substitution to design different types of polypeptides for molecular docking with XO, and glycine was selected to assist in sequence optimization because glycine has a simple structure and a relatively short chain length, and has a weak influence on the configuration of aromatic amino acids.

[0070] 2.3.2 Sequence design of different types of peptides

[0071] (1)Aromatic amino acids replace the N-terminus, middle, and C-terminus of the polypeptide

[0072] Based on the polypeptide WENWTK, aromatic amino acids are used to replace the amino acids at key positions such as the N-terminus, middle, and C-terminus of this sequence one by one. The replacement methods can be divided into two categories: single replacement (such as FENFTF) and mixed replacement (such as WENFTY), which are called Class ① and Class ② polypeptides. The sequences of Class ① and Class ② polypeptides and their binding energies for docking with the 1N5X molecule are shown in Table 2. The binding energy of the polypeptide FENFTF is -8.9 kcal / mol, and the binding energy of the polypeptide FENFTY is -9.1 kcal / mol.

[0073]

[0074] (2)Aromatic amino acids replace only the N-terminus or the middle or the C-terminus of the polypeptide

[0075] Based on the polypeptide WENWTK, aromatic amino acids are used to replace only one amino acid at a key position such as the N-terminus, middle, and C-terminus of this sequence. If the amino acid at the un-replaced position is an aromatic amino acid, it is replaced by glycine. The replacement methods can be divided into three categories: only N-terminus replacement (such as WENGTK), only middle position replacement (such as GENWTK), and only C-terminus replacement (such as GENGTW), which are called Class ③, Class ④, and Class ⑤ polypeptides. The sequences of Class ③, Class ④, and Class ⑤ polypeptides and their binding energies for docking with the 1N5X molecule are shown in Table 3. The binding energy of the polypeptide FENGTK is -7.8 kcal / mol.

[0076]

[0077] (3)Aromatic amino acids replace only the N-terminus and the C-terminus of the polypeptide

[0078] Based on the polypeptide WENWTK, the amino acids at the N-terminus and C-terminus positions of this sequence are replaced with aromatic amino acids. If the amino acid at the un-replaced position is an aromatic amino acid, it is replaced by glycine. The replacement methods can be divided into two categories: single replacement and mixed replacement, which are called Class ⑥ and Class ⑦ polypeptides. The sequences of Class ⑥ and Class ⑦ polypeptides and their binding energies for docking with the 1N5X molecule are shown in Table 4. The binding energy of the polypeptide WENGTF is -8.5 kcal / mol.

[0079]

[0080] (4)Aromatic amino acids replace the amino acids at the middle and any one end of the polypeptide

[0081] Based on the polypeptide WENWTK, the amino acids at the middle position and any position at either end (N-terminus or C-terminus) of this peptide sequence are replaced with aromatic amino acids. If the amino acid at the non-replaced position is an aromatic amino acid, it is replaced with glycine (G). The replacement methods can be divided into two categories: N-terminus and middle replacement (such as FENFTK), and C-terminus and middle replacement (such as WENWTF), which are called Class ⑧ and Class ⑨ polypeptides. The sequences of Class ⑧ and Class ⑨ polypeptides and their binding energies in docking with the 1N5X molecule are shown in Table 5. The binding energy of polypeptide WENWTK is -8.6 kcal / mol, and the binding energy of WENFTK is -8.1 kcal / mol.

[0082]

[0083] (5)Optimize the sequence length of the polypeptide

[0084] From the results of the molecular docking binding energies of the polypeptides obtained by replacing aromatic amino acids at different positions above, it can be found that the number and position of tryptophan can affect the binding effect of the peptide segment with 1N5X. Therefore, based on the polypeptide WENWTK, a single tryptophan replacement is first carried out (the C-terminal amino acid is replaced with tryptophan), and then the sequence length is optimized. The sequences of the polypeptides with optimized length and their binding energies in docking with the 1N5X molecule are shown in Table 6. It can be found that from WENWTW to WEWTW, the binding energy of the peptide segment in docking with the molecule changes from -7.3 kcal / mol to -9.7 kcal / mol. After removing the aspartic acid at the third position at the N-terminus of WENWTW, the binding effect is significantly improved. However, for the inhibitory activity against XO, it still needs to be further verified after synthesis.

[0085]

[0086] 2.3.3 Synthesis and activity verification of the designed peptides

[0087] The above 5 polypeptides with relatively high absolute values of binding energy are: polypeptide FENFTY, polypeptide FENGTK, polypeptide WENGTF, polypeptide WENFTK, and polypeptide WEWTW, and their amino acid sequences are shown in SEQ ID NO.7 - 11 in sequence.

[0088] Synthesize the above 5 polypeptides, and measure the XO inhibition rate and IC 50 value. The comparison of the absolute values of the binding energies and IC 50 values of the 5 polypeptides is as Figure 8 shown. The IC 50 value of polypeptide WENGTF is 5.62 ± 0.33 mmol / L, the IC 50 value of polypeptide WENFTK is 4.87 ± 0.08 mmol / L, and the IC 50The value was 1.73 ± 0.13 mmol / L. The results showed that the XO inhibitory activity of polypeptide WEWTW was the strongest, which was consistent with the predicted binding energy results obtained from molecular docking. However, the XO inhibitory activity of polypeptide FENETY could not be used as a good XO inhibitory peptide as predicted by the binding energy of molecular docking. This indicated that using the binding energy of molecular docking for screening XO inhibitory peptides was not absolute. This might be because the entropy effect was difficult to accurately estimate in actual reactions, so there would be a certain deviation between the molecular docking binding energy data and the in vitro activity verification experiment.

[0089] 2.3.4 Molecular Docking of Designed Peptides

[0090] The 3D interaction diagram of the molecular docking analysis of polypeptide WEWTW and 1N5X is as Figure 9 shown, and the 2D interaction diagram of the molecular docking analysis of polypeptide WEWTW and 1N5X is as Figure 10 shown. As can be seen from the figure, the tryptophan at the N-terminus of polypeptide WEWTW can be completely embedded in the active pocket, not only forming alkyl interactions with Ala-1079 and Ala-1078 around the active pocket, but also forming π-alkyl interactions with Phe-914 and Phe-1009; the tryptophan in the middle position of the polypeptide forms π-alkyl interactions with Phe-649 and His-875; the polypeptide forms 2 conventional hydrogen bond interactions with the amino acid residues Glu-802 and Glu-879 around the active pocket, and forms 2 carbon-hydrogen bond interactions with His-875 and Thr-1010; in addition, the polypeptide also forms van der Waals interactions with dozens of amino acids.

[0091] From the interaction diagram of the above polypeptide and 1N5X, it can be found that amino acids with a benzene ring structure will preferentially occupy the active pocket region of 1N5X, thereby affecting the catalytic reaction of xanthine. Xanthine may form interactions with the key residues Phe914, Arg880, Thr1010, and Glu802 of XO, while the polypeptide preferentially forms interactions with these amino acid residues. When the types and numbers of formed interactions are different, it will affect the conformational change of XO, thereby affecting the catalysis of xanthine. This explains the possible reasons for the different binding energies and the different XO inhibitory activities when the polypeptide is docked with 1N5X. In the in vitro XO inhibitory activity experiment, the polypeptide WEWTW obtained by sequence optimization showed a significant improvement in XO inhibitory activity compared with the initial polypeptide WENWTK, indicating that the number of tryptophans in the polypeptide and the overall length of the peptide segment can make the polypeptide show different XO inhibitory activities. Reasonable substitution of amino acids at key positions and adjustment of the peptide segment sequence length can be effective means to improve the XO inhibitory activity of the polypeptide.

[0092] The above embodiments are provided to those skilled in the art to fully disclose and describe how to implement and use the claimed embodiments, and not 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 polypeptide with xanthine oxidase inhibitory activity, characterized in that: The amino acid sequence is WEWTW, as shown in SEQ ID NO.

11.

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

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