Small molecule peptide from takifugu obscurus and application of small molecule peptide

Abstract

The invention discloses a small molecule peptide, which is derived from fugu obscurus, and the amino acid sequence of the small molecule peptide is as shown in Trp-Ala-Ala-Phe-Pro-Pro-Asp-Val-Ala-Gly-Asn, Asp-Asp-Glu-Ile-Phe-Pro-Met or Trp-Tyr-Asp-Asn-Glu-Phe-Gly. The micromolecule peptide with xanthine oxidase inhibitory activity is separated from a fugu obscurus enzymolysis product through a method of combining peptidomics and molecular docking, and experimental research verifies that the micromolecule peptide has the effect of inhibiting the xanthine oxidase activity. Therefore, the takifugu obscurus micromolecule peptide has the potential of being used as a medicine for treating the hyperuricemia and has a relatively good application prospect of preventing the hyperuricemia.

Description

Technical Field

[0001] The present invention relates to a class of small molecule peptides derived from Takifugu obscurus and their use in the preparation of drugs for treating hyperuricemia, belonging to the technical field of bioactive peptides. Background Art

[0002] Hyperuricemia is a metabolic disease caused by purine metabolism disorder. Long-term supersaturation of uric acid will lead to the formation and deposition of monosodium urate crystals, and eventually develop into obstructive nephropathy, kidney stones and gout. Xanthine oxidase is a flavoprotein enzyme containing two molecules of FAD, two molybdenum atoms and eight iron atoms, which exists in various organisms and catalyzes the formation of uric acid from purine substrates in the body. Therefore, reducing uric acid production by inhibiting the activity of xanthine oxidase is the main way to treat hyperuricemia. Although the clinically used drugs allopurinol and febuxostat have excellent inhibitory activity, it has been found that they have relatively large toxic side effects such as allergic reactions, rashes and nephropathy. Therefore, choosing food-derived peptides to inhibit the activity of xanthine oxidase is a better strategy for reducing uric acid. Compared with traditional uric acid-lowering drugs, bioactive peptides have the characteristics of wide source, low toxic side effects and easy absorption.

[0003] Takifugu obscurus, commonly known as pufferfish, is an economically important marine fish in the coastal areas of China (including the eastern coast, the Yellow Sea and the Bohai Sea) and the lower reaches of the Yangtze River. It is famous for its delicious taste and high market value. It is also recorded in "Compendium of Materia Medica" that "the pufferfish has a delicious taste" and "tonifies deficiency, removes dampness, regulates the waist and feet, treats hemorrhoids and kills insects". It is rich in collagen, protein and carbohydrates, especially with a high crude protein content (about 20%), and is a promising source of bioactive peptides.

[0004] Generally, the production of food-derived bioactive peptides has to go through protein separation, peptide preparation and identification. Enzymatic hydrolysis, especially digestive enzyme hydrolysis, is the most widely used method due to its high specificity, low toxicity and mild reaction conditions. Different enzymes have different cleavage sites, thus generating peptides with different functional activities. Compared with traditional separation and purification techniques (such as anion exchange, sephadex gel chromatography and reverse phase high performance liquid chromatography RP-HPLC), peptidomics allows rapid identification of peptide sequences in protein hydrolysates. Due to its simple sample pretreatment without complete separation of peptides, it greatly reduces the time. However, it is difficult to obtain bioactive anti-hyperuricemia peptides from the thousands of peptides identified. By combining peptidomics with molecular docking, the efficiency of screening bioactive peptides can be greatly improved. Summary of the Invention

[0005] The present invention provides a small molecule peptide derived from Takifugu obscurus, and its amino acid sequence is as described in Trp-Ala-Ala-Phe-Pro-Pro-Asp-Val-Ala-Gly-Asn (WAAFPPDVAGN), Asp-Asp-Glu-Ile-Phe-Pro-Met (DDEIFPM) or Trp-Tyr-Asp-Asn-Glu-Phe-Gly (WYDNEFG).

[0006] The present invention uses enzymatic hydrolysis and polypeptide group technology to screen for peptides with xanthine oxidase (XOD) inhibitory activity in the enzymatic hydrolysate of Takifugu obscurus, and determines the action sites of the screened small molecule peptides on XOD inhibitory activity through molecular docking and molecular dynamics, and obtains its inhibition mode and half inhibitory concentration.

[0007] Another object of the present invention is to apply the above small molecule peptide in the preparation of drugs for treating hyperuricemia.

[0008] The composition (or active ingredient) of the drug for treating hyperuricemia in the present invention is a small molecule peptide, and one or more pharmaceutically acceptable excipients can also be added, or it can be compounded with other active ingredients to exert a therapeutic effect; in addition to being made into tablets, the drug can also be made into various pharmaceutically acceptable forms such as pills, powders, capsules, granules, oral liquids and injections.

[0009] Advantages and technical effects of the present invention:

[0010] The present invention separates and obtains small molecule peptides with xanthine oxidase inhibitory activity from the enzymatic hydrolysate of Takifugu obscurus by combining peptide group technology and molecular docking, and verifies through experimental research that the small molecule peptides have the effect of inhibiting xanthine oxidase activity. Therefore, the small molecule peptides of Takifugu obscurus have the potential to be used as drugs for treating hyperuricemia. Description of the drawings

[0011] Figure 1 It is a schematic diagram of the detection results of the inhibitory activity of different enzymatic hydrolysates on xanthine oxidase;

[0012] Figure 2 It is a schematic diagram of the interaction site between peptide WAAFPPDVAGN and xanthine oxidase;

[0013] Figure 3 It is a schematic diagram of the interaction site between peptide DDEIFPM and xanthine oxidase;

[0014] Figure 4 It is a schematic diagram of the interaction site between peptide WYDNEFG and xanthine oxidase;

[0015] Figure 5Inhibitory curves of three peptides against xanthine oxidase;

[0016] Figure 6 Graph of the Lineweaver - Burk analysis results for peptide WAAFPPDVAGN;

[0017] Figure 7 Graph of the Lineweaver - Burk analysis results for peptide DDEIFPM;

[0018] Figure 8 Graph of the Lineweaver - Burk analysis results for peptide WYDNEFG;

[0019] Figure 9 RMSD results of three peptides;

[0020] Figure 10 Gibbs free energy results for peptide WAAFPPDVAGN;

[0021] Figure 11 RMSF results of three peptides;

[0022] Figure 12 Gibbs free energy results for peptide DDEIFPM;

[0023] Figure 13 Rg value results of three peptides;

[0024] Figure 14 Gibbs free energy results for peptide WYDNEF. Specific implementation mode

[0025] 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 examples. 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. The instruments, reagents, and materials involved in the following examples 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 examples are all conventional experimental methods and detection methods existing in the prior art without special instructions.

[0026] Example 1: Preparation of enzymatic hydrolysate

[0027] 1. Thaw, skin, and eviscerate the Takifugu obscurus purchased from Jingjiang Tunzhijie Food Co., Ltd., and then freeze and package it in portions. During the experiment, take out the frozen sample, cut it into small pieces, and grind it with a meat grinder;

[0028] 2. Add the minced Takifugu obscurus and ultrapure water (1:2.5, g:mL) to a reactor, add alkaline protease (200,000 U / g), and perform water bath oscillation at 55 °C and pH 7.95 - 8.05 for 5 h; after enzymatic hydrolysis is completed, inactivate the enzyme in a 90 °C water bath for 10 min to obtain an enzymatic hydrolysate. Centrifuge the enzymatic hydrolysate at 4500 g for 10 min, take the supernatant, filter and sterilize it through a 0.22 μm filter membrane, and then filter the sterilized product through a 1 kDa microporous membrane ultrafiltration tube to obtain an alkaline protease enzymatic hydrolysis product containing components >1 kDa, an alkaline protease enzymatic hydrolysis product containing components <1 kDa, and freeze-dry to obtain peptide powder;

[0029] 3. The method is the same as in step 2, except that papain (800,000 U / g) is added, and enzymatic hydrolysis is carried out at 55 °C and pH 6.95 - 7.05 to obtain papain enzymatic hydrolysis peptide powder containing components >1 kDa and papain enzymatic hydrolysis peptide powder containing components <1 kDa;

[0030] 4. The method is the same as in step 2, except that trypsin (250,000 U / g) is added, and enzymatic hydrolysis is carried out at 37 °C and pH 7.95 - 8.05 to obtain trypsin enzymatic hydrolysis peptide powder containing components >1 kDa and trypsin enzymatic hydrolysis peptide powder containing components <1 kDa.

[0031] Example 2: Detection of xanthine oxidase (XOD) inhibitory activity of the enzymatic hydrolysate in Example 1

[0032] 1. Add the peptide powder prepared in Example 1 to 1× phosphate buffer to obtain a peptide-containing liquid at 10 mg / mL. Take 100 μL of the peptide-containing liquid (or 1× phosphate buffer) and add it to 100 μL of 1× phosphate buffer (or 1× phosphate buffer) containing 0.01 U / mL XOD, shake for 30 s, incubate at 25 °C for 5 min, add 150 μL of 0.48 mM xanthine solution, gently shake the mixture again for 30 s, and after incubating at 25 °C for 40 min, measure the absorbance at 290 nm, and calculate the XOD inhibitory activity using the following formula:

[0033] Δ sample = (A1 40min - A2 40min ) - (A1 0min - A2 0min )

[0034] Δ control = (A3 40min - A4 40min ) - (A3 0min - A4 0min )

[0035]

[0036] A1: Peptide powder + XOD + Xanthine; A2: Peptide powder + Buffer solution + Xanthine; A3: Buffer solution + XOD + Xanthine; A4: Buffer solution + Buffer solution + Xanthine;

[0037] The results are as Figure 1 shown. The results show that the alkaline protease hydrolysis products containing >1 kDa components and the papain hydrolysis products containing >1 kDa components have good inhibitory effects on XOD, and the inhibition rates reach 99.91 ± 0.33% and 96.75 ± 1.70%, respectively.

[0038] Example 3: Peptide identification, screening, and synthesis

[0039] The alkaline protease hydrolysis products containing >1 kDa components and the papain hydrolysis products containing >1 kDa components in Example 2 were selected for identification. The nano liquid chromatography-tandem mass spectrometry (nanoLC-MS / MS) method was used to determine the peptide sequences in the hydrolysis products. The peptide was analyzed by coupling a nano ultra-high performance liquid chromatograph (Evosep one, Denmark) with a timsTOF Pro2 mass spectrometer (Bruker, Germany) equipped with a nanoelectrospray ion source. The separation process was carried out on a reversed-phase chromatographic column (PePSep C18, 1.9 μm, 150 μm × 15 cm, Bruker, Germany). The mass spectrometer used the aSEF mode of data-dependent acquisition (DDA), and the DDA data was collected using SpectroMine software (Biognosys, Switzerland). The scanning range was from 100 - 1700 m / z. During the PASEF MS / MS scan, the collision energy increased linearly with the ion mobility, from 20 eV (1 / K0 = 0.6 Vs / cm2) to 59 eV (1 / K0 = 1.6 Vs / cm2). The raw data files were searched against a database using the Pulsar search engine in SpectroMine (4.2.230428.52329; Biognosys AG) software. After the search, qualitative analysis was performed, and 5781 peptides were identified from the alkaline protease hydrolysis products and 532 peptides were identified from the papain hydrolysis products, and they were subjected to docking screening.

[0040] The XOD crystal protein (PDB: 1N5X) used for docking was downloaded from the Protein Data Bank (PDB). Molecular docking was performed using the Libdock module in Discovery Studio (DS) 2019 software. At the same time, the docking coordinates were set as x = 96.6635, y = 54.963, z = 39.4334, and the docking radius was Finally, the optimal conformation was obtained through DS2019, and the Libdock score was used as the criterion for predicting the optimal binding position of the peptide to xanthine oxidase (XOD).

[0041] Through the docking scores, three peptide segments, namely WAAFPPDVAGN, DDEIFPM, and WYDNEFG, were selected, with scores of 194.432, 161.988, and 157.389 respectively. The schematic diagram of the interaction sites with xanthine oxidase is as Figure 2 , Figure 3 and Figure 4 shown below.

[0042] WAAFPPDVAGN can form conventional hydrogen bonds, carbon-hydrogen bonds, Pi-Pi stacking, Pi-Pi T-shaped interactions, Pi-alkyl interactions, and Pi-sulfur interactions with the key sites Mos3004, Glu802, and Phe469, Leu712, Leu710, Lys713, His875, Ser876, Glu879, Phe914, Phe1009, Ala1078, Ala1079, Phe1142, and Phe1143; DDEIFPM can form Pi-alkyl, Pi-σ interactions, carbon-hydrogen bonds, conventional hydrogen bonds, salt bridges, alkyl, and Pi-anion interactions with the key sites Glu802 and Phe649, His875, Phe1143, Gly709, Ser710, Arg871, Leu873, Ser876, Val1011, Leu1014, Pro1076, Phe1142; WYDNEFG can form Pi-alkyl, carbon-hydrogen bonds, salt bridges, conventional hydrogen bonds, and Pi-Pi T-shaped interactions with the sites Leu648, Phe649, Arg871, Leu873, His875, Glu879, Leu900, Pro1012, Leu1014, Pro1076, Met1118. In summary, WAAFPPDVAGN, DDEIFPM, and WYDNEFG mainly inhibit the activity of xanthine oxidase by interacting with the key sites.

[0043] WAAFPPDVAGN, DDEIFPM, and WYDNEFG were selected for synthesis. The Fmoc-Pro-Wang resin was swollen in dimethylformamide (DMF) (20 mL) for 1 hour. The suspension was filtered. A 20% piperidine solution in DMF (20 mL) was added to the resin to remove the Fmoc protecting group. The suspension was kept at room temperature for 0.5 hour while nitrogen gas was bubbled through it. The mixture was filtered, and the resin was washed with DMF (6 times × 20 mL). Fmoc-Thr(tBu)-OH (0.3 mmol) was pre-activated with N,N'-diisopropylcarbodiimide (DIC) (0.3 mmol) and hydroxybenzotriazole (HOBt) (0.3 mmol) in 10 mL of DMF, and then the activated mixture was added to the resin. The reaction was carried out under a nitrogen atmosphere. The Kaiser ninhydrin test was used to indicate the completion of the reaction. After the reaction was completed, the suspension was filtered, and the resin was washed with DMF (3 times × 20 mL). Subsequently, all amino acid couplings were completed using the same method.

[0044] A cleavage solution (90% trifluoroacetic acid: 5% triisopropylsilane: 5% water) was added to the peptidyl resin. The suspension was shaken for 3.5 hours and then filtered. Diethyl ether (2000 mL) was added to the filtrate to precipitate the peptide. The mixture was centrifuged, and the ether layer was decanted. The peptide was washed three times with ether and dried under vacuum overnight to obtain a small molecule peptide with the amino acid sequence shown in SEQ ID NO:1 - SEQ ID NO:3.

[0045] Example 4: Determination of the half-inhibitory concentration and inhibition type of the small molecule peptide

[0046] 1. The small molecule peptide from Example 3 was prepared into peptide solutions with concentrations of 0.5 mg / mL, 1 mg / mL, 2 mg / mL, 4 mg / mL, and 8 mg / mL using distilled water. Referring to the inhibition activity determination method in Example 2, an inhibition curve was plotted, and the IC50 was calculated using the Analyze Data module of GraphPad Prism 8.0. The results are shown in Figure 5 , Table 1;

[0047] 2. Determination of the inhibition type

[0048] For the peptide WAAFPPDVAGN, solutions with concentrations of 0 mg / mL, 1 mg / mL, and 2 mg / mL were prepared, and xanthine solutions with concentrations of 0.24 mM, 0.48 mM, and 0.96 mM were prepared. Referring to the inhibition activity determination method in Example 2, the enzyme reaction rate v was calculated, and a Lineweaver - Burk plot was drawn to analyze its inhibition type. Figure 6 The results showed that the inhibition type was a partial non - competitive inhibition mode.

[0049] For the peptide DDEIFPM, prepare solutions with concentrations of 0 mg / mL, 4 mg / mL, and 8 mg / mL, and prepare xanthine solutions with concentrations of 0.24 mM, 0.48 mM, 0.72 mM, and 0.96 mM. Refer to the inhibitory activity assay method in Example 2, calculate the enzyme reaction rate v, plot the Lineweaver - Burk plot, and analyze its inhibition type. Figure 7 The results showed that the inhibition type was a hyperbolic non - competitive inhibition mode.

[0050] For the peptide WYDNEFG, prepare solutions with concentrations of 0 mg / mL, 2 mg / mL, and 4 mg / mL, and prepare xanthine solutions with concentrations of 0.24 mM, 0.48 mM, 0.72 mM, and 0.96 mM. Refer to the inhibitory activity assay method in Example 2, calculate the enzyme reaction rate v, plot the Lineweaver - Burk plot, and analyze its inhibition type. Figure 8 The results showed that no obvious inhibition type was observed.

[0051] 3. Toxicity prediction was achieved through the online website https: / / webs.iiitd.edu.in / raghava / toxinpred / , and the results are shown in Table 1;

[0052] Table 1

[0053]

[0054] Example 5: Molecular Dynamics Simulation of the Small - Molecule Peptide in Example 3

[0055] The molecular dynamics simulation of the xanthine - peptide complex was carried out for 100 nanoseconds (ns) using Gromacs v2022.03 software, and the CHARMM36 force field was adopted. For the peptide part, we used the GAFF (Generalized Amber Force Field) force field in AmberTools22 software. These complexes were dissolved in the three - point transferable intermolecular potential (TIP3P) solvent, ensuring that the protein atoms were at least 1.2 nanometers away from the edge of the water box. Meanwhile, by adding Na + and Cl -(At a concentration of 0.154 M) to simulate the charge of the system. To achieve the stability of the system, we used the steepest descent algorithm for energy minimization (EM). Subsequently, the solute was placed in an isothermal and isochoric (NVT) system, and the system was gradually heated from 0 K to 300 K, and then equilibrated in an isothermal and isobaric (NPT) system at a temperature of 300 K and a pressure of 1 Bar. Finally, 100 ns of molecular dynamics simulations were performed on these complexes. Based on the results of the molecular dynamics simulations, we estimated the root mean square deviation (RMSD), root mean square fluctuation (RMSF), and radius of gyration (Rg). Using the RMSD and Rg values, the Gibbs free energy was calculated by Gromacs v2022.03 software. The RMSD results of the three peptides and the Gibbs free energy of WAAFPPDVAGN are as Figure 9 and 10 shown. The RMSF results of the three peptides and the Gibbs free energy of DDEIFPM are as Figure 11 and 12 shown. The Rg value results of the three peptides and the Gibbs free energy of WYDNEF are as Figure 13 and 14 shown; The three peptides WAAFPPDVAGN, DDEIFPM, and WYDNEF with the XOD complex showed good stability. By comparing the Gibbs free energy diagrams, WAAFPPDVAGN has a sharp peak, indicating a more stable binding to XOD.

Claims

1. A small molecule peptide, derived from Takifugu obscurus, has an amino acid sequence as shown by Trp-Ala-Ala-Phe-Pro-Pro-Asp-Val-Ala-Gly-Asn, Asp-Asp-Glu-Ile-Phe-Pro-Met or Trp-Tyr-Asp-Asn-Glu-Phe-Gly.

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

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