XO Inhibitory Peptide AGDY and Its Applications

Abstract

This invention discloses the XO-inhibiting peptide AGDY and its applications, belonging to the field of bioactive peptide technology. The amino acid sequence of the XO-inhibiting peptide AGDY is shown in SEQ ID NO.3. The invention also discloses the application of the XO-inhibiting peptide AGDY in the preparation of xanthine oxidase inhibitors and in the preparation of drugs with the effect of inhibiting uric acid levels. This invention used molecular docking and BLAST+ software to virtually screen 15 potential XO-inhibiting peptides from the complete protein sequence of Litopenaeus vannamei. High-performance liquid chromatography (HPLC) was used to determine the XO-inhibiting activity, and the results showed that 7 small molecule peptides exhibited significant XO-inhibiting activity, possessing potential value in preventing hyperuricemia.

Description

[0001] This invention is a divisional application of CN 116655737 A, the original application number of which is 202310412042.5, the application date of which is April 18, 2023, and the invention title is: XO inhibitory peptide and its application. Technical Field

[0002] This invention relates to an XO-inhibiting peptide AGDY and its applications, belonging to the field of bioactive peptide technology. Background Technology

[0003] Hyperuricemia is a disease caused by long-term purine metabolism disorder, mainly characterized by elevated uric acid levels. Hyperuricemia can lead to complications such as gout and hypertension. Notably, many important enzymes are involved in uric acid production, and xanthine oxidase (XO) is one of these key enzymes. Widely present in various organisms, XO plays a crucial role in purine metabolism, making it a key target for treating hyperuricemia. Traditional treatments for hyperuricemia, such as allopurinol and febuxostat, while effective, have adverse side effects, including allergies and diarrhea.

[0004] Currently, developing novel, highly effective, and low-side-effect natural XO inhibitors is a hot topic in alleviating hyperuricemia. In recent years, some natural XO-inhibiting peptides have attracted attention due to their high safety and easy absorption. Many studies have shown that XO-inhibiting peptides are small molecule peptides found in various foods, such as milk, rice, walnuts, oysters, and tuna.

[0005] Enzymatic hydrolysis and purification are traditional methods for obtaining bioactive peptides, but these methods are time-consuming and inefficient. With the continuous development of bioinformatics, molecular docking virtual screening technology has attracted increasing 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 elucidate the structure-activity relationship of bioactive peptides. Therefore, compared with traditional preparation methods, molecular docking virtual screening of bioactive peptides may be a more efficient approach. Summary of the Invention

[0006] In view of the above-mentioned prior art, the present invention provides an XO inhibitory peptide AGDY and its application.

[0007] This invention is achieved through the following technical solution:

[0008] The XO inhibitory peptide AGDY has the amino acid sequence shown in SEQ ID NO.3.

[0009] Application of the XO inhibitory peptide AGDY in the preparation of xanthine oxidase inhibitors.

[0010] The application of the XO inhibitory peptide AGDY in the preparation of drugs that inhibit uric acid levels.

[0011] The XO inhibitory peptide of this invention was virtually screened from the whole protein sequence of Litopenaeus vannamei using molecular docking and BLAST+ software, as follows:

[0012] (1) XO is docked with oligopeptides selected from the bioactive peptide database: an bioactive peptide database is constructed by reading literature to find reported bioactive peptide sequences, and XO (PDB ID: 1N5X) is docked with oligopeptides in the data.

[0013] (2) Alignment of similar sequences: The potential XO inhibitory peptides obtained above were aligned with the full protein sequence of Litopenaeus vannamei (NCBI ID: 10710) using BLAST+ software. After alignment, polypeptides with similar structures were selected for molecular docking with XO, and the top-ranked XO inhibitory peptides were screened by docking interaction values ​​and synthesized.

[0014] (3) The in vitro XO inhibitory activity of the XO inhibitory peptides screened above was verified by high performance liquid chromatography.

[0015] Using the above method, this invention screened 15 potential XO-inhibiting peptides. The XO-inhibiting activity was determined by high performance liquid chromatography. The results showed that 7 small molecule peptides exhibited significant XO-inhibiting activity. These small molecule peptides have the potential to be functional products that inhibit xanthine oxidase and suppress uric acid levels, and can be used to prepare drugs with the effect of inhibiting uric acid levels.

[0016] This invention represents an innovation in traditional, complex methods for screening XO-inhibiting peptides. It utilizes molecular informatics technology to virtually screen for bioactive peptides from the entire protein sequence, thus improving the efficiency of XO-inhibiting peptide screening. The XO-inhibiting peptides screened by this invention have potential value in preventing hyperuricemia.

[0017] The various terms and phrases used in this invention have their general meanings known to those skilled in the art. Attached Figure Description

[0018] Figure 1 The results of molecular docking between peptides and XO, where A is YNITGW and B is GDEY.

[0019] Figure 2 The results of molecular docking between the polypeptide and XO, where A is AGDY and B is PDARG.

[0020] Figure 3 The results of molecular docking between the peptide and XO, where A is YGDE and B is VTGW.

[0021] Figure 4 The results of molecular docking of peptides and allopurinol with XO, where A is EDDDA and B is allopurinol.

[0022] 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; and rose red represents Pi-Pi Stacked. Detailed Implementation

[0023] The present invention will be further described below with reference to embodiments. However, the scope of the present invention is not limited to the following embodiments. Those skilled in the art will understand that various changes and modifications can be made to the present invention without departing from the spirit and scope thereof.

[0024] Unless otherwise specified, the instruments, reagents, and materials used in the following embodiments are all conventional instruments, reagents, and materials already available in the prior art and can be obtained through legitimate commercial channels. Unless otherwise specified, the experimental methods and detection methods used in the following embodiments are all conventional experimental methods and detection methods already available in the prior art.

[0025] Example 1: Molecular docking of oligopeptides selected from the bioactive peptide database with XO.

[0026] By reviewing the literature, this invention constructed a bioactive peptide database containing 5795 bioactive peptides. Based on the principle that bioactive peptides are generally small molecule sequences, 1330 oligopeptides were screened from the database. The oligopeptides were molecularly docked with XO, and the docking energy value of the initial ligand TEI was used as a threshold to screen potential XO inhibitory peptides.

[0027] Through docking, 653 potential XO inhibitory peptides were obtained through screening.

[0028] ChemDraw 19.0 and CDOCKER were used as the peptide mapping and docking software, respectively. The docking coordinates were x=96.6635, y=54.963, z=39.4334, and the docking radius was 15 Å. Other parameters were kept at their default values. For each ligand, 10 optimal poses were generated using the docking software, and the binding strength was determined by the -CDOCKER ENERGY value. Peptides with docking energies higher than the initial ligand TEI were selected.

[0029] Example 2: BLAST alignment of similar sequences

[0030] The 653 potential XO inhibitory peptides obtained in Example 1 were sequence aligned with the full-length protein sequences of Litopenaeus vannamei using BLAST+ software. The alignment conditions were: value set to 50, num_threads set to 2, and window_size set to 40. After alignment, 1671 peptides with structures similar to the 653 potential XO inhibitory peptides were selected for XO docking. For each ligand, 10 optimal poses were generated using docking software, and the binding strength was determined by the -CDOCKER INTERACTIONENERGY value. The 15 peptides with the highest energy values ​​were selected (the sequences of the peptides are shown in Table 1). These peptides were synthesized by Sangon Biotech (Shanghai) Co., Ltd. using the Fmoc solid-phase synthesis method.

[0031] Example 3: Verification of the in vitro XO inhibitory activity of the active peptide using high performance liquid chromatography.

[0032] Take the dissolved peptide sample and add it to 100 mmol / L PBS buffer (pH 7.4). Add xanthine to a final concentration of 0.7 mmol / L and XO to 0.15 U / mL. Incubate at 37°C for 15 min. Add 1 mol / L hydrochloric acid to terminate the reaction.

[0033] The chromatographic column was an Agilent XDB C18 (250 × 4.6 mm, 5 μm), the mobile phase was 85% 10 mmol / L NH4H2PO aqueous solution and 15% methanol, and the flow rate was 1.0 mL / min.

[0034] 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%.

[0035] The results are shown in Table 1.

[0036]

[0037] Note: (1) Due to the randomness of molecular docking results, the scores shown in the table are the average of 10 best poses;

[0038] (2) The toxicity of the peptide was calculated using http: / / crdd.osdd.net / raghava / / toxinpred / ;

[0039] (3) "-" indicates that no activity was detected.

[0040] As shown in Table 1, peptides YNITGW (755.93 Da), GDEY (482.49 Da), AGDY (424.45 Da), PDARG (514.53 Da), YGDE (482.49 Da), VTGW (452.51 Da), and EDDDA (563.48 Da) exhibited XO inhibitory activity. Among them, the IC50 of peptide YNITGW was [missing information]. 50 The lowest value indicates the highest activity. The amino acid sequences of these seven polypeptides are shown in SEQ ID NO. 1–7, respectively.

[0041] To further investigate the mechanism of action of XO-inhibiting peptides, active site analysis was performed on these seven peptides, using allopurinol as a positive control. The docking results with XO are as follows: Figures 1-4 As shown. Molecular docking results indicate that conventional hydrogen bonding, mutual attraction of charges, π-π superposition, and hydrophobic interactions play important roles in the interaction between the XO repressor peptide and the key residues of XO, Glu 802, Glu 1261, and Arg 880. Furthermore, the IC50 of the peptide YNITGW... 50 The value was significantly lower than that of the other six peptides, indicating that YNITGW has higher inhibitory activity. This is because YNITGW, as a hexapeptide with a C-terminus of Trp, is the peptide that interacts most frequently with the important amino acids of XO among the seven XO inhibitory peptides. In addition, molecular docking results also showed that YNITGW docks at the surface inlet of the XO hydrophobic channel, preventing the substrate from entering the active site, thereby inhibiting XO activity.

[0042] The above embodiments are provided to those skilled in the art to fully disclose and describe how the claimed implementations can be carried out and used, and are not intended to limit the scope of the disclosure herein. Modifications that will be obvious to those skilled in the art will be within the scope of the appended claims.

Claims

1. An XO-inhibiting peptide AGDY, characterized in that: Its amino acid sequence is shown in SEQ ID NO.

3.

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

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