Small molecule peptides and their use in the preparation of xanthine oxidase inhibitors

Abstract

The application discloses small molecule peptides and application thereof in preparation of xanthine oxidase inhibitors, and belongs to the technical field of bioactive peptides. The amino acid sequence of the small molecule peptide is YHFKIF or YHYKIF, as shown in SEQ ID NO. 3 or SEQ ID NO. 4. The application of the small molecule peptide in preparation of xanthine oxidase inhibitors. The small molecule peptides YHFKIF, YHYKIF and YHWKIW of the application all have high XO inhibiting activity, and are obviously superior to WHDKIF. Meanwhile, the three small molecule peptides all have good antioxidant activity. The application has important significance for treatment of hyperuricemia.

Description

[0001] This invention is a divisional application of CN118126130A. The original application has the application number 202410545204.7 and the application date is May 6, 2024. The invention is entitled: Small molecule peptides and their application in the preparation of xanthine oxidase inhibitors. Technical Field

[0002] This invention relates to small molecule peptides and their application in the preparation of xanthine oxidase inhibitors, belonging to the field of bioactive peptide technology. Background Technology

[0003] Hyperuricemia is a condition where the level of uric acid in the blood is higher than the normal range. Uric acid is a substance produced by the body's metabolism and is usually metabolized by the liver and excreted. When the body produces too much uric acid, or when the liver is unable to completely metabolize and excrete it, uric acid accumulates in the blood, leading to hyperuricemia. Symptoms of hyperuricemia include pain and swelling, usually occurring in joints such as the big toe, ankle, knee, wrist, and fingers. In addition, symptoms of urinary tract stones may occur, such as lower back pain, frequent urination, urgency, and difficulty urinating. Hyperuricemia can also trigger gout, a type of arthritis caused by uric acid crystals, leading to symptoms such as joint pain, redness, swelling, and fever. Furthermore, hyperuricemia can damage the kidneys, leading to kidney disease.

[0004] Xanthine oxidase (XO) is a key enzyme in the purine metabolism pathway to uric acid, and inhibiting xanthine oxidase with drugs is one of the effective ways to treat hyperuricemia. However, drug treatment often brings many complications to patients, such as kidney stones and cardiovascular diseases. Therefore, the development of easily absorbed xanthine oxidase inhibitory peptides with few toxic side effects is of great significance for the treatment of hyperuricemia.

[0005] Furthermore, studies have shown a correlation between antioxidant activity and uric acid-lowering activity. Antioxidant foods such as vegetables, fruits, fungi, algae, and tubers can, to some extent, help lower uric acid levels because these foods typically contain abundant antioxidants that reduce uric acid production. Therefore, obtaining peptides with both antioxidant and xanthine oxidase inhibitory activities would greatly promote the development of drugs with stronger functional activity. Simultaneously, the activity of xanthine oxidase inhibitory peptides is closely related to their structure; by optimizing the peptide structure, their functional activity can be further enhanced, thus providing raw materials for the development of related drugs for the prevention and treatment of hyperuricemia. Summary of the Invention

[0006] In view of the above-mentioned prior art, the present invention provides small molecule peptides and their application in the preparation of xanthine oxidase inhibitors, belonging to the field of bioactive peptide technology.

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

[0008] A small molecule peptide having the amino acid sequence YHFKIF or YHYKIF, as shown in SEQ ID NO.3 or SEQ ID NO.4.

[0009] Application of the small molecule peptide in the preparation of xanthine oxidase inhibitors.

[0010] In preliminary experiments, this invention screened and obtained the small molecule peptide WHDKIF (as shown in SEQ ID NO. 1) with antioxidant capacity, which also showed inhibitory activity against xanthine oxidase. Based on this, this invention further refined the design to obtain three small molecule peptides with high binding energies. All three peptides exhibit significant inhibitory activity against xanthine oxidase, especially the small molecule peptide YHWKIW, which shows an IC50 inhibitory activity against xanthine oxidase of 100%. 50 The concentration was only 2.76 ± 0.25 mmol / L, showing a significant increase in xanthine oxidase inhibitory activity compared to the small molecule peptide WHDKIF. Therefore, xanthine oxidase inhibitors and drugs for treating hyperuricemia can be prepared using the small molecule peptide YHWKIW as a raw material. This invention is of great significance for the treatment of hyperuricemia.

[0011] The various terms and phrases used in this invention have their general meanings known to those skilled in the art. Detailed Implementation

[0012] 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.

[0013] 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.

[0014] Example 1: Rational Design of Peptide Sequences

[0015] In preliminary experiments, this invention screened for peptides with antioxidant capabilities in royal jelly enzymatic hydrolysates, and obtained the small molecule peptide WHDKIF (described in another invention patent application CN 116064708 A by the applicant of this invention). The binding energy of the small molecule peptide WHDKIF is 38.380 kcal / mol, and its DPPH antioxidant activity IC50 is [missing information]. 50=1.00±0.02mg / mL, and it was determined that it also has inhibitory activity against xanthine oxidase, IC50 = 1.00±0.02mg / mL. 50 =5.98±0.02 mmol / L. To obtain bioactive peptides with better activity, this invention uses the small molecule peptide WHDKIF as a basis and rationally designs the peptide sequence. Specifically, the amino acids at both ends and in the middle of the small molecule peptide GGYGIF are replaced with aromatic amino acids. The small molecule peptides obtained after the replacement are divided into the following four categories: those with aromatic amino acids at both ends and in the middle are denoted as category ①; those with aromatic amino acids in the middle, N-terminus, or C-terminus are denoted as category ②; those with aromatic amino acids at both ends are denoted as category ③; and those with aromatic amino acids in the middle and one end are denoted as category ④.

[0016] ① There are 27 types of small molecule peptides as follows: FHFKIF, WHWKIW, YHYKIY, WHYKIF, WHYKIW, WHYKIY, WHFKIF, WHFKIY, WHFKIW, WHWKIF, WHWKIY, YHFKIF, YHFK IY, YHFKIW, YHYKIF, YHYKIW, YHWKIF, YHWKIY, YHWKIW, FHFKIY, FHFKIW, FHYKIF, FHYKIY, FHYKIW, FHWKIF, FHWKIY, FHWKIW.

[0017] ② There are 9 types of small molecule peptides, among which GHFKIG, GHYKIG, and GHWKIG have aromatic amino acids only in the middle; YHDKIG, WHDKIG, and FHDKIG have aromatic amino acids only in the N-terminus; and GHDKIF, GHDKIY, and GHDKIW have aromatic amino acids only in the C-terminus.

[0018] ③ There are 8 types of small molecule peptides: FHDKIF, FHDKIY, FHDKIW, YHDKIF, YHDKIY, YHDKIW, WHDKIY, and WHDKIW.

[0019] ④ There are 18 types of small molecule peptides, among which the following have aromatic amino acids only in the middle and N-terminus: FHFKIG, FHYKIG, FHWKIG, YHFKIG, YHYKIG, YHWKIG, WHFKIG, WHYKIG, WHWKIG; and the following have aromatic amino acids only in the middle and C-terminus: GHFKIF, GHFKIY, GHFKIW, GHYKIF, GHYKIY, GHYKIW, GHWKIF, GHWKIY, GHWKIW.

[0020] Example 2 Molecular docking

[0021] A xanthine oxidase inhibitory peptide library was established based on the series of peptides designed in Example 1 above. Autodock 1.5.7 molecular docking software was used as the peptide screening tool. The key sites 914Phe and 1009Phe of the xanthine oxidase PDB structure 1N5X, which are reported above, and the active center molybdenum cofactor (MOS3004) were defined as docking sites. The docking box size was set to the default value, and the number of docking attempts was set to 10. The average absolute value of the binding energy from the 10 docking attempts was used as the final key indicator for peptide screening.

[0022] Results: Each type of small molecule peptide in Example 1 was docked with XO, and the docking results are shown in Tables 1, 2, 3, and 4. Based on the Vina score, the three active peptides with the highest binding energies were selected: YHFKIF, YHYKIF, and YHWKIW. The amino acid sequence of YHWKIW is shown in SEQ ID NO.2, the amino acid sequence of YHFKIF is shown in SEQ ID NO.3, and the amino acid sequence of YHYKIF is shown in SEQ ID NO.4.

[0023] Table 1 ① Screening of small molecule peptides

[0024]

[0025] Table 2 ② Screening of small molecule peptides

[0026]

[0027] Table 3 ③ Screening of small molecule peptides

[0028]

[0029] Table 4 Screening of Class ④ Small Molecule Peptides

[0030]

[0031] Example 3 Synthesis and Activity Assay of Small Molecule Peptides

[0032] (1) Synthesis of small peptides

[0033] The small molecule peptides WHDKIF, YHFKIF, YHYKIF, and YHWKIW were all synthesized by Sangon Biotech (Shanghai) Co., Ltd. using the Fmoc solid-phase synthesis method.

[0034] (2) Activity assay

[0035] The synthesized small peptides were prepared into solutions with a concentration of 3 mg / mL using PBS buffer (pH 7.4). The in vitro XO inhibition rates of these four small peptides were then measured, and the IC50 was calculated based on the regression line between the in vitro XO inhibition rate and the concentration of the substances. 50 value.

[0036] The in vitro XO inhibition rate was determined as follows: Xanthine solution (1.5 mmol / L) and xanthine oxidase solution (0.1 U / mL) were prepared using PBS buffer solution at pH 7.4. 125 μL of sample, 150 μL of PBS buffer, and 100 μL of xanthine oxidase solution were added to a 1.5 mL centrifuge tube. The mixture was incubated at 37 °C for 15 min, then 125 μL of xanthine solution was added, and incubation continued for 20 min. Finally, 100 μL of hydrochloric acid (1 mol / L) was added to terminate the reaction. Controls were set up with no sample, no xanthine oxidase, no xanthine oxidase, and a sample. The absorbance at 290 nm was measured.

[0037] The formula for calculating the in vitro XO inhibition rate is as follows:

[0038] In vitro XO inhibition rate (%) = [(1-(Abs1-Abs3) / (Abs2-Abs4)]×100%.

[0039] Wherein, Abs1: absorbance of the assay system containing xanthine, xanthine oxidase and sample; Abs2: absorbance of the assay system containing xanthine and xanthine oxidase but without sample; Abs3: absorbance of the assay system containing xanthine substrate and sample but without xanthine oxidase; Abs4: absorbance of the assay system containing xanthine but without xanthine oxidase and sample.

[0040] The relevant properties and activity verification results of small molecule peptides are shown in Table 5.

[0041] Table 5. Relevant properties and activity verification results of small molecule peptides

[0042]

[0043] Note: Due to the randomness of molecular docking results, the Vina score shown in the table is the average of the highest values ​​of 10 docking results (negative values).

[0044] As shown in Table 5, the small molecule peptides YHFKIF, YHYKIF, and YHWKIW all exhibited high XO inhibitory activity, significantly superior to WHDKIF, especially YHWKIW, whose xanthine oxidase inhibitory activity IC50 was significantly higher. 50 The concentration was only 2.76 ± 0.25 mmol / L. Furthermore, all three small peptides exhibited good antioxidant activity.

[0045] 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. A small molecule peptide, characterized in that: The amino acid sequence is YHFKIF or YHYKIF, as shown in SEQ ID NO. 3 or SEQ ID NO.

4.

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

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