XO inhibitory peptide and its application

Abstract

The present invention discloses XO inhibitory peptides and applications thereof, and belongs to the technical field of bioactive peptides. There are 7 XO inhibitory peptides, and their amino acid sequences are shown in SEQ ID NO.1 to 7 respectively. The XO inhibitory peptides are used as or in the preparation of xanthine oxidase inhibitors, and are used as or in the preparation of drugs with the efficacy of inhibiting uric acid levels. The present invention uses molecular docking and BLAST+ software to virtually screen out 15 potential XO inhibitory peptides from the full sequence of white shrimp protein, and uses high performance liquid chromatography to determine the XO inhibitory activity. The results show that 7 small molecule peptides show obvious XO inhibitory activity, among which YNITGW has the best XO inhibitory activity, and IC 50 The value is 9.78±0.13mM, which is potentially more valuable.

Description

Technical Field

[0001] The invention relates to an XO inhibitory peptide and application thereof, belonging to the technical field of bioactive peptides. Background Art

[0002] Hyperuricemia is a disease caused by long-term purine metabolism disorder, and its main manifestation is elevated uric acid. Hyperuricemia can cause complications such as gout and hypertension. It is worth noting that many important enzymes are involved in the production of uric acid. Xanthine oxidase (XO) is one of these key enzymes. It is widely present in various organisms and plays an important role in purine metabolism. Therefore, XO is a key target for the treatment of hyperuricemia. Although traditional therapeutic drugs for hyperuricemia, such as allopurinol and febuxostat, are effective, they all have adverse side effects, including allergies and diarrhea.

[0003] At present, the development of new natural XO inhibitors with high efficiency and few side effects is a hot topic for alleviating hyperuricemia. In recent years, some natural XO inhibitory peptides have attracted people's attention due to their high safety and easy absorption. Many studies have shown that XO inhibitory peptides are small molecule peptides that exist in a variety of foods, such as milk, rice, walnuts, oysters, tuna, etc.

[0004] As traditional methods for obtaining bioactive peptides, enzymatic hydrolysis and separation and purification are time-consuming and inefficient. With the continuous development of bioinformatics, molecular docking virtual screening technology has attracted more and more attention from researchers. Molecular docking can not only be used for virtual screening, but also for analyzing the interaction between ligands and receptors, thus helping to further clarify the structure-activity relationship of bioactive peptides. Therefore, compared with traditional preparation methods, molecular docking virtual screening of bioactive peptides may be a more effective method. Summary of the invention

[0005] In view of the above-mentioned prior art, in order to develop XO inhibitory peptides, the present invention uses molecular docking and BLAST+ software to virtually screen out 7 new XO inhibitory peptides from the complete sequence of Litopenaeus vannamei protein, and clarifies the action sites of their XO inhibitory activity through molecular docking, and determines the inhibitory activity.

[0006] The present invention is achieved through the following technical solutions:

[0007] XO inhibitory peptides include the following 7 types, and their amino acid sequences are:

[0008] YNITGW, as shown in SEQ ID NO.1;

[0009] GDEY, as shown in SEQ ID NO. 2;

[0010] AGDY, as shown in SEQ ID NO. 3;

[0011] PDARG, as shown in SEQ ID NO. 4;

[0012] YGDE, as shown in SEQ ID NO. 5;

[0013] VTGW, as shown in SEQ ID NO. 6;

[0014] EDDDA, as shown in SEQ ID NO.7.

[0015] The above 7 XO inhibitory peptides are used as or in the preparation of xanthine oxidase inhibitors; and are used as or in the preparation of drugs with the effect of inhibiting uric acid levels.

[0016] The XO inhibitory peptide of the present invention is virtually screened from the complete sequence of Penaeus vannamei protein using molecular docking and BLAST+ software, and the process is as follows:

[0017] (1) Docking XO with the selected oligopeptides in the bioactive peptide database: An active peptide database was constructed by reading the literature to find the reported active peptide sequences, and XO (PDB ID: 1N5X) was docked with the oligopeptides in the database.

[0018] (2) Alignment of similar sequences: The potential XO inhibitory peptides obtained above were aligned with the complete protein sequence of Penaeus vannamei (NCBI ID: 10710) using BLAST+ software. After alignment, peptides with similar structures were selected for molecular docking with XO, and the top XO inhibitory peptides were screened out by docking interaction values, and XO inhibitory peptides were synthesized.

[0019] (3) The in vitro XO inhibitory activity of the XO inhibitory peptides screened above was verified by high performance liquid chromatography.

[0020] Using the above method, the present invention screened out 15 potential XO inhibitory peptides, and measured the XO inhibitory activity using high performance liquid chromatography. The results showed that 7 small molecule peptides showed obvious XO inhibitory activity. These small molecule peptides have the potential to be xanthine oxidase inhibitors and functional products for inhibiting uric acid levels, and can be used to prepare drugs with the effect of inhibiting uric acid levels. Among them, the peptide Tyr-Asn-Ile-Thr-Gly-Trp (YNITGW, 755.93Da) has the best XO inhibitory activity, IC 50 The value is 9.78±0.13mM, which is potentially more valuable.

[0021] The present invention is an innovation of the traditional complex XO inhibitory peptide screening method, using molecular informatics technology to virtually screen active peptides from the full protein sequence, thereby improving the screening efficiency of XO inhibitory peptides. The XO inhibitory peptides screened by the present invention have the potential value of preventing hyperuricemia.

[0022] Various terms and phrases used herein have the general meanings that are well known to those skilled in the art. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 : Molecular docking results of peptide and XO, where A, YNITGW; B, GDEY.

[0024] Figure 2 : Molecular docking results of peptide and XO, where A, AGDY; B, PDARG.

[0025] Figure 3 : Molecular docking results of peptide and XO, including A, YGDE; B, VTGW.

[0026] Figure 4 : Molecular docking results of peptide and allopurinol with XO, where A, EDDDA; B, allopurinol.

[0027] 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

[0028] The present invention will be further described below in conjunction with the embodiments. However, the scope of the present invention is not limited to the following embodiments. It will be appreciated by those skilled in the art that various changes and modifications may be made to the present invention without departing from the spirit and scope of the present invention.

[0029] The instruments, reagents, and materials involved in the following examples, unless otherwise specified, are all conventional instruments, reagents, and materials available in the prior art and can be obtained through regular commercial channels. The experimental methods, detection methods, etc. involved in the following examples, unless otherwise specified, are all conventional experimental methods and detection methods available in the prior art.

[0030] Example 1 Molecular docking of selected oligopeptides from the bioactive peptide database with XO

[0031] By consulting the literature, the present invention constructed a bioactive peptide database containing 5795 active peptides. Based on the principle that active peptides are generally small molecule sequences, 1330 oligopeptides in the database were screened out, the oligopeptides were molecularly docked with XO, and the docking energy value of the initial ligand TEI was used as the threshold to screen potential XO inhibitory peptides.

[0032] Through docking, 653 potential XO inhibitory peptides were screened.

[0033] ChemDraw 19.0 and CDOCKER were used as peptide drawing and docking software, respectively. The docking coordinates were x = 96.6635, y = 54.963, z = 39.4334, and the docking radius was Other parameters were kept at default values. For each ligand, the docking software was used to generate 10 optimal poses, and the binding degree was determined by the value of -CDOCKER ENERGY, and peptides with energy values ​​higher than the docking energy value of the initial ligand TEI were screened.

[0034] Example 2 BLAST comparison of similar sequences

[0035] The 653 potential XO inhibitory peptides obtained in Example 1 were aligned with the complete protein sequence of Penaeus vannamei using BLAST+ software. The sequence alignment conditions were: value was set to 50, num_threads was set to 2, and window_size was set to 40. After sequence alignment, 1671 peptides with similar structures to the 653 potential XO inhibitory peptides were screened and docked with XO. For each ligand, 10 optimal poses were generated using docking software, and the degree of binding was determined by the value of -CDOCKER INTERACTIONENERGY. The 15 peptides with the highest energy values ​​were screened (the sequences of the peptides are shown in Table 1), and these peptides were synthesized by Sangon Biotech (Shanghai) Co., Ltd. using the Fmoc solid phase synthesis method.

[0036] Example 3 Verification of in vitro XO inhibitory activity of active peptides by high performance liquid chromatography

[0037] Take the dissolved polypeptide sample, add it to 100mmol / L PBS buffer (pH 7.4), add xanthine with a final concentration of 0.7mmol / L and 0.15U / mL XO, incubate at 37℃ for 15min, and add 1mol / L hydrochloric acid to terminate the reaction.

[0038] The chromatographic column was Agilent XDB C18 (250×4.6 mm, 5 μm), the mobile phase was 85% 10 mmol / L NH 4 H 2 PO aqueous solution and 15% methanol, and the flow rate was 1.0 mL / min.

[0039] The uric acid content in the final mixture was determined by 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] × 100%.

[0040] The results are shown in Table 1.

[0041] Table 1 Information of the screened peptides

[0042]

[0043] Note: (1) Due to the randomness of molecular docking results, the scores shown in the table are the average of the 10 best poses;

[0044] (2) The toxicity of the peptide was calculated by http: / / crdd.osdd.net / raghava / / toxinpred / ;

[0045] (3) “-” indicates no activity was detected.

[0046] As shown in Table 1, peptides YNITGW (755.93Da), GDEY (482.49Da), AGDY (424.45Da), PDARG (514.53Da), YGDE (482.49Da), VTGW (452.51Da) and EDDDA (563.48Da) showed XO inhibitory activity, among which the IC 50 The lowest value indicates the highest activity.

[0047] In order to further explore the reasons why XO inhibitory peptides play a role, the active sites of these seven peptides were further analyzed, and allopurinol was used as a positive control. The docking results with XO are as follows Figures 1 to 4 The molecular docking results showed that traditional hydrogen bonding, attractive charge interaction, π-π stacking and hydrophobic interaction played an important role in the interaction between XO inhibitory peptide and the key residues Glu 802, Glu 1261 and Arg 880 of XO. In addition, the IC 50 The value is significantly lower than that of the other six peptides, indicating that YNITGW has higher inhibitory activity. This is because YNITGW, as a hexapeptide with Trp at the C-terminus, is the peptide that interacts most with the important amino acids of XO among the seven XO inhibitory peptides. In addition, the molecular docking results also show that YNITGW docks at the surface entrance of the XO hydrophobic channel, preventing the substrate from entering the active center, thereby inhibiting XO activity.

[0048] The above examples are provided to those skilled in the art to fully disclose and describe how to make and use the claimed embodiments, and are not intended to limit the scope of the disclosure herein. Modifications that are obvious to those skilled in the art will fall within the scope of the appended claims.

Claims

1. A XO inhibitory peptide, characterized in that Its amino acid sequence is: YNITGW, as shown in SEQ ID NO.

1.

2. Use of the XO inhibitory peptide according to claim 1 in the preparation of a medicament for treating hyperuricemia.

Images