Small molecule peptide efgmggw and its use in the preparation of xanthine oxidase inhibitors

Abstract

The application discloses a small molecule peptide EFGMGGW, and an amino acid sequence of the small molecule peptide is shown as SEQ ID NO.2.The application discloses an application of the small molecule peptide in preparation of a xanthine oxidase inhibitor and an application of the small molecule peptide in preparation of a drug for treating or preventing hyperuricemia.Through experimental research, six small molecule peptides with xanthine oxidase inhibiting activity are screened from enzymatic hydrolysate of Penaeus vannamei, and the small molecule peptides have the potential of being used as xanthine oxidase inhibitors, wherein EFGMGGW shows higher xanthine oxidase inhibiting activity, and has greater potential value in preventing hyperuricemia.

Description

[0001] The application is a divisional application of CN 115385986 A, the original application number is 202211239499.2, the application date is October 11, 2022, and the invention name is: Small molecule peptide with xanthine oxidase inhibitory activity and its application. TECHNICAL FIELD

[0002] The application relates to a small molecule peptide EFGMGGW and application thereof in preparation of a xanthine oxidase inhibitor, and belongs to the technical field of bioactive peptides. BACKGROUND

[0003] Hyperuricemia is a common metabolic disease caused by purine metabolism disorder, characterized by elevated serum uric acid levels. Excess uric acid levels can lead to arthritis and gout, and even cardiovascular disease, hypertension and type II diabetes, etc. The production and metabolism of uric acid is a complex process, and enzymes involved in purine metabolism can regulate the production of uric acid in the body, and uric acid transporters regulate the excretion and reabsorption of uric acid. Xanthine oxidase (XO) is a key enzyme in purine catabolism, which catalyzes the generation of xanthine from hypoxanthine, and then generates uric acid, and high levels of XO can also cause uric acid to deposit in the body, and XO inhibitors can block the biosynthesis of uric acid. Therefore, XO is an important therapeutic target for treating hyperuricemia.

[0004] In recent years, various synthetic XO inhibitors such as allopurinol, febuxostat, etc. have been developed for clinical treatment of hyperuricemia. Although synthetic XO inhibitors are very effective as anti-hyperuricemia drugs, they inevitably cause various adverse side effects, such as allergic reactions and elevated blood pressure, and even cardiovascular disease and chronic kidney disease, etc. Therefore, there is an urgent need to explore or develop XO inhibitors with fewer adverse side effects.

[0005] In recent decades, polypeptides have attracted extensive attention due to their easy absorption, non-toxicity, high specificity and various biological activities. More and more bioactive peptides, such as ACE inhibitory peptides and antibacterial peptides, have been obtained from aquatic products. However, there are few studies on obtaining XO inhibitory peptides from aquatic products. In addition, traditional XO inhibitory peptide purification methods mainly include gel filtration, ion exchange, reverse phase high performance liquid chromatography, etc., which are time-consuming and complicated, so there is an urgent need for a more simple and feasible method for screening XO inhibitory peptides. With the continuous progress of science and technology, the research on active peptide screening assisted by high-performance computers has gradually developed, such as the use of molecular docking technology, which can not only reduce the screening intensity and screening cycle, but also improve the success probability of screening. However, the use of computer simulation of protein enzymolysis is limited by the length of the protein sequence, and is not suitable for the enzymolysis of proteins with longer sequences, which greatly reduces the efficiency of screening XO inhibitory peptides. Therefore, the combination of enzymolysis and molecular docking may be a more feasible and effective method to replace the traditional enzymolysis for screening XO inhibitory peptides. SUMMARY

[0006] In view of the above prior art, the present application provides a small molecule peptide with xanthine oxidase inhibitory activity and its application. The present application mainly uses molecular docking technology to screen XO inhibitory peptides from shrimp enzymolysis products, and elucidates the action site of its high XO inhibitory activity through molecular docking to determine its half-inhibitory concentration.

[0007] The present application is realized by the following technical solutions:

[0008] The small molecule peptide EFGMGGW has an amino acid sequence as shown in SEQ ID NO. 2.

[0009] The application of the above-mentioned small molecule peptide in the preparation of xanthine oxidase inhibitors; in the preparation of drugs for treating or preventing hyperuricemia.

[0010] Through experimental research, the present application has screened 6 small molecule peptides with xanthine oxidase inhibitory activity from the enzymolysis liquid (pepsin, trypsin, alpha-chymotrypsin) of Penaeus vannamei, which have the potential to be used as functional products of xanthine oxidase inhibitors and uric acid level inhibitors, and can be used for preparing drugs with uric acid level inhibiting efficacy. Among them, AEAQMWR, EFGMGGW and AGGINLAR show higher XO inhibitory activity, and their potential value in preventing hyperuricemia may be greater. The present application is an innovation of the traditional complex XO inhibitory peptide screening method, which adopts a screening method combining traditional enzymolysis identification and molecular docking, thereby improving the work efficiency of XO inhibitory peptide screening.

[0011] The various terms and phrases used in the present application have the general meanings known to those skilled in the art. BRIEF DESCRIPTION OF DRAWINGS

[0012] Figure 1 Figure 2: XO inhibitory activity of enzymatic hydrolysate and different molecular weight fractions obtained by ultrafiltration of the enzymatic hydrolysate, wherein A: enzymatic hydrolysate; B: fraction B; C: fraction C; D: fraction D.

[0013] Figure 2 Figure 3: Purification of fraction B using Sephadex G-15 gel chromatography column (5 elution peaks were obtained).

[0014] Figure 3 Figure 4: XO inhibitory activity of fractions (B1, B2, B3, B4, B5) obtained after purification by Sephadex gel column.

[0015] Figure 4 Figure 5: LC-MS / MS spectrum of B-3 fraction.

[0016] Figure 5 Figure 6: Molecular docking results of three polypeptides, wherein A-C: molecular docking results of three polypeptides AEAQMWR, EFGMGGW and AGGINLAR with XO, green represents Hydrogen Bond; light blue represents Carbon Hydrogen Bond; orange represents Attractive Charge, Pi-Cation and Salt-Bridge; red represents Unfavorable Donor-Donor; pink represents Alkyl and Pi-Alkyl; rose red represents Pi-Pi Stacked. DETAILED DESCRIPTION

[0017] The application will be further described below with reference to the following examples. However, the scope of the application is not limited to the following examples. Those skilled in the art can understand that various changes and modifications can be made to the application without departing from the spirit and scope of the application.

[0018] The instruments, reagents, materials involved in the following examples, if not specifically stated, are conventional instruments, reagents, materials already existing in the prior art, which can be obtained through regular commercial channels. The experimental methods, detection methods, etc. involved in the following examples, if not specifically stated, are conventional experimental methods, detection methods already existing in the prior art.

[0019] Example 1 Simulated gastrointestinal digestion of Penaeus vannamei

[0020] The shrimp meat of Penaeus vannamei was mixed with water at a ratio of 1:9 (w / v, g / ml) and its pH was adjusted to 3.0. 1.5% (w / w) of pepsin was added and the reaction was carried out at 37°C for 4h; then the pH of the enzymatic solution was adjusted to 6.5, and 1% (w / w) of trypsin and α-chymotrypsin were added, and the reaction was continued at 37°C for 1.5h; the enzyme was inactivated by boiling for 10 min, and the enzymatic solution was obtained and stored by freeze-drying.

[0021] Example 2: In vitro XO inhibitory activity of the active peptide was verified by high performance liquid chromatography

[0022] The sample or 100 mmol / L PBS buffer (pH 7.4) was added with xanthine at a final concentration of 0.7 mmol / L and XO at a final concentration of 0.15 U / mL, and incubated at 37°C for 15 min, and then 1 mol / L hydrochloric acid was added to terminate the reaction.

[0023] The chromatographic column was Agilent XDB C 18 (250 mm x 4.6 mm, 5 μm), the mobile phase was 85% 10 mmol / L aqueous solution and 15% methanol, and the flow rate was 1.0 mL / min.

[0024] The content of uric acid in the final mixture was determined by the absorbance at 290 nm. The XO inhibitory activity in the enzymatic reaction was calculated as: [(blank uric acid content - sample uric acid content) / blank uric acid content] x 100%.

[0025] Example 3: Ultrafiltration separation of Penaeus vannamei enzymatic solution and activity determination

[0026] The enzymatic solution was separated by two ultrafiltration membranes with molecular weights of 10 kDa and 5 kDa, and the XO inhibitory activity of the components with different molecular weights was determined, and the results are shown in Table 1. Figure 1 The enzymatic solution (component A) was ultrafiltrated into three components (components B, C and D), wherein component B (<5 kDa) had higher XO inhibitory activity (50.46±0.68%, IC 50 =19.82±0.27 mg / mL), which was higher than that of component C (5-10 kDa) and component D (10 kDa).

[0027] Example 4: Sephadex gel filtration chromatography and activity analysis of components

[0028] Sephadex G-15 was boiled in a proper amount of ultrapure water for 2h to remove floating scum and impurities; then ultrapure water was added and stirred, and then the upper layer was removed. The above operation was repeated until there was no impurity in the upper layer. The excess water in the upper layer of the gel was absorbed, and the filler was introduced into a 16 mm x 80 cm chromatographic column with a glass rod to avoid stratification and air bubbles. After the column was filled, it was washed with pure water until the height of the chromatographic column no longer changed.

[0029] The component B determined in Example 3 was used as sample for the loading. The loading concentration and volume were 50 mg / mL, 5 mL; the component of the collected peak (the mobile phase was ultrapure water, the elution rate was 2 mL / min) was collected, and the activity of different components was determined after lyophilization.

[0030] The separation results of the enzymatic hydrolysate by gel chromatography column are shown in Figure 2 The elution peaks B1-B5 were collected in sequence, and the polypeptide solution was prepared after lyophilization. The XO inhibition rate of each component was determined, and the results are shown in Figure 3 The elution peak B-3 had the highest inhibition rate, and therefore the elution peak B-3 was selected for the next experiment.

[0031] LC-MS / MS mass spectrum identification of the B-3 component peptide segment

[0032] (1) Polypeptide extraction

[0033] After desalination, the B-3 component peptide segment was vacuum dried, reconstituted with 0.1% trifluoroacetic acid solution, and the peptide segment concentration was determined for LC-MS analysis.

[0034] (2) LC-MS / MS analysis

[0035] Peptide detection was performed using an EASY-nLC 1200 system (Thermo Fisher Scientific) and a Q-exactive HF-X mass spectrometer (Thermo Scientific). Peptides were loaded into an in-house packed C18 capillary trap column (100 μm x 20 mm, 5 μm) and separated with a C18 separation column (75 μm x 150 mm, 3 μm). The A mobile phase was 0.1% formic acid (v / v) in water, and the B mobile phase was 0.1% formic acid (v / v) and 80% acetonitrile (v / v) in water. The gradient was: 2% to 5% B mobile phase 0 to 2 min, 5% to 28% B mobile phase 2 to 44 min, 28% to 40% B mobile phase 44 to 51 min, 40% to 100% B mobile phase 51 to 53 min, B mobile phase held at 100% 53 to 60 min. The scan range was set to 300 to 1800 m / z, the primary MS resolution was 60000 @ m / z 200, the AGC target was 3e6, and the primary maximum IT was 50 MS. The secondary MS resolution was 15000 @ m / z 200, the AGC target was 1e5, the secondary 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. The raw files from mass spectrometry were searched against the Uniprot Protein Database using MaxQuant 1.6.1.0, and finally 150 proteomes and 585 peptides were obtained. The sample mass spectrometry Basepeak graph is shown in FIG. 1. Figure 4

[0036] Example 6 Peptide screening, synthesis and verification

[0037] (1) Treatment of ligand

[0038] The molecular structure of the polypeptide was drawn using Chem3D Pro 14.0 software and saved as a mol2 file.

[0039] (2) Treatment of receptor

[0040] The crystal structure 1N5X of XO was downloaded from the PDB database, and the B chain in 1N5X was deleted using the CDOCKER of Discovery Studio (DS) 2019 software, and the ligand Tenofovir (TEI) was isolated and saved for later use.

[0041] (3) Molecular docking

[0042] Molecular docking was performed using the CDOCKER module of Discovery Studio (DS) 2019 software. The docking coordinates were x = 96.6635, y = 54.963, z = 39.4334, and the docking radius was ​Other parameters remain default. For each ligand, 10 best poses were generated using DS2019 software, and the binding degree was determined by the values of -CDOCKER ENERGY and -CDOCKER INTERACTION ENERGY, and 15 polypeptides with the highest energy values were screened, and their XO inhibitory activities were verified by synthesis, and the results are shown in Table 1.

[0043] Table 1 Polypeptide information screened

[0044]

[0045] Note: ① Since the molecular docking results have randomness, the scores shown in the table are the average values of 10 best poses;

[0046] ② The toxicity of the polypeptide was calculated by http: / / crdd.osdd.net / raghava / / toxinpred / ;

[0047] ③ "-" represents no activity detected.

[0048] (4) Polypeptide synthesis and activity verification

[0049] The 6 peptide segments with XO inhibitory activity in Table 1 were synthesized by GenScript Biotech (Shanghai) Co., Ltd. using the Fmoc solid-phase synthesis method. The polypeptide activity verification was performed according to the method in Example 2. Among them, the amino acid sequence of AEAQMWR is shown in SEQ ID NO. 1; the amino acid sequence of EFGMGGW is shown in SEQ ID NO. 2; the amino acid sequence of AGGINLAR is shown in SEQ ID NO. 3; the amino acid sequence of MAFGDKF is shown in SEQ ID NO. 4; the amino acid sequence of RWPGDMDR is shown in SEQ ID NO. 5; and the amino acid sequence of FNHHMF is shown in SEQ ID NO. 6.

[0050] As shown in Table 1, the 6 peptide segments all have XO inhibitory activity, among which AEAQMWR, EFGMGGW and AGGINLAR show higher XO inhibitory activity, so it is decided to further analyze the active sites of these 3 polypeptides.

[0051] The molecular docking results show that Figure 5 ), traditional hydrogen bonding, mutual attraction charge interaction and salt bridge play an important role in the interaction between the XO inhibitory peptide and the key residues Glu802, Glu1261 and Arg880 of XO. In addition, the activity of the polypeptide AEAQMWR is higher than that of other peptides, which may be related to the connection between AEAQMWR and the key metal atom Mos3004 in XO.

[0052] The foregoing examples are provided to give the skilled person in the art complete disclosure and description of how to make and use the claimed embodiments, and are not intended to limit the scope of what is disclosed herein. Modifications apparent to those skilled in the art are to be within the scope of the claims.

Claims

1. The small molecule peptide EFGMGGW characterized in that, The amino acid sequence thereof is shown as SEQ ID NO.

2.

2. Use of the small molecule peptide EFGMGGW in the preparation of a drug for treating hyperuricemia according to claim 1.

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