Small molecule peptide LCAFNK from sunflower disc and its application in inhibiting xanthine oxidase activity

Abstract

The present invention provides a small molecule peptide LCAFNK in a sunflower disk and its application in inhibiting xanthine oxidase activity, belonging to the technical field of bioactive peptides. The present invention obtains a small molecule peptide LCAFNK in a sunflower disk that may have xanthine oxidase inhibitory activity by a virtual screening method. And through experimental verification, this small molecule peptide has the potential to be a xanthine oxidase inhibitor. The xanthine oxidase inhibitory activity of the small molecule peptide LCAFNK in the present invention is 28.06 mM. The present invention is of great significance for the treatment of hyperuricemia.

Description

Technical Field

[0001] The invention belongs to the technical field of bioactive peptides, and particularly relates to a small molecule peptide LCAFNK from sunflower discs, and application of the small molecule peptide LCAFNK in the preparation of a xanthine oxidase inhibitor. Background Art

[0002] Hyperuricemia is a chronic disease caused by increased uric acid levels in the serum due to impaired purine metabolism or reduced uric acid excretion. Excessive uric acid levels can lead to arthritis and gout, and even cardiovascular disease, metabolic syndrome, hypertension and diabetes. At present, the treatment of hyperuricemia is mainly divided into two approaches, namely reducing the production of uric acid in the body and promoting the excretion of uric acid in the body. Xanthine oxidase (XO) is a key enzyme in the decomposition of purines. It catalyzes hypoxanthine to produce xanthine, and then produces uric acid. Therefore, XO can be used as an important therapeutic target for the treatment of hyperuricemia.

[0003] In recent years, various synthetic XO inhibitors have been developed for the clinical treatment of hyperuricemia, such as allopurinol, febuxostat, etc. However, more and more studies have shown that long-term or large-scale use of synthetic XO inhibitors can cause a certain degree of liver and kidney damage. Therefore, the development of XO inhibitors with less toxic side effects is of great significance for the treatment of hyperuricemia.

[0004] Compared with chemical drugs, peptide drugs are more efficient, safer, and more tolerable. They also have the advantages of higher selectivity and less accumulation in the body. In recent decades, peptides have attracted widespread attention. Traditional XO inhibitory peptide purification methods mainly include gel filtration, ion exchange, reversed-phase high-performance liquid chromatography, etc., which are time-consuming and the purification process is cumbersome. Therefore, there is an urgent need for a simpler and more feasible research method for screening XO inhibitory peptides. Virtual screening refers to the pre-screening of compound molecules on a computer according to pre-set conditions before biological activity screening to identify the small molecules that are most likely to bind to the target, thereby greatly reducing the number of actual screening compounds and improving the discovery efficiency of lead compounds. The ConPLex tool is a tool for predicting the possibility of small molecule binding to protein constructed according to Singh et al. (2023). It uses a pre-trained protein language model and comparative learning technology to accurately predict the binding affinity of peptides to target proteins. Therefore, using computer technology to simulate trypsin hydrolysis and predict the affinity of the decomposed small peptides can be used as a more feasible and effective method to replace traditional enzymatic hydrolysis to screen XO inhibitory peptides. Summary of the invention

[0005] In order to solve the above technical problems, the present invention provides a small molecule peptide LCAFNK in sunflower discs, and its application in inhibiting xanthine oxidase activity. The present invention obtains a small molecule peptide LCAFNK in sunflower discs that may have xanthine oxidase inhibitory activity through a virtual screening method. And through experimental verification, this small molecule peptide has the potential to be a xanthine oxidase inhibitor. The xanthine oxidase inhibitory activity of the small molecule peptide LCAFNK in the present invention is 28.06mM. The present invention is of great significance for the treatment of hyperuricemia.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] One of the purposes of the present invention is to provide a small molecule peptide having xanthine oxidase inhibitory activity, wherein the amino acid sequence of the small molecule peptide is shown as SEQ ID NO. 1.

[0008] The second purpose of the present invention is to provide an application of the small molecule peptide in the preparation of xanthine oxidase inhibitors.

[0009] The third object of the present invention is to provide an application of the small molecule peptide in the preparation of a drug for treating hyperuricemia.

[0010] One aspect of the present invention relates to obtaining a small molecule peptide LCAFNK which may have XO inhibitory activity from sunflower discs by using computer virtual screening technology.

[0011] Another aspect of the present invention relates to verifying the above-mentioned small molecule peptide by using an in vitro xanthine oxidase activity detection experiment, and finding that the small molecule peptide has the potential to be a xanthine oxidase inhibitor.

[0012] Compared with the prior art, the present invention has the following technical effects:

[0013] The present invention obtains a small molecule peptide LCAFNK in sunflower disc that may have xanthine oxidase inhibitory activity through a virtual screening method. And through experimental verification, this small molecule peptide has the potential to be a xanthine oxidase inhibitor. The xanthine oxidase inhibitory activity of the small molecule peptide LCAFNK in the present invention is 28.06 mM. The present invention is of great significance for the treatment of hyperuricemia. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 It is a schematic diagram of the molecular docking results of the polypeptide LCAFNK in Example 1 of the present invention;

[0015] Figure 2 Schematic diagram of the XO inhibitory activity of the small molecule peptide LCAFNK in Example 2 of the present invention. DETAILED DESCRIPTION

[0016] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. Without departing from the spirit and essence of the present invention, modifications or replacements made to the method, steps or conditions of the present invention all belong to the scope of the present invention. The reagents and instruments used in the following examples can all be obtained commercially, and the methods used in the examples are consistent with the conventional methods unless otherwise specified.

[0017] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meanings as those commonly understood by those skilled in the art of the present invention. The terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0018] The technical solution of the present invention is further described in detail below in conjunction with embodiments.

[0019] Example 1 Rational design of peptide sequences

[0020] The present invention firstly obtains all sunflower (Helianthus annuus) protein sequences from NCBI (National Center for Biotechnology Information), and then performs computer-simulated trypsin hydrolysis on all sunflower proteins based on the principle of trypsin hydrolysis. Trypsin mainly acts on lysine and arginine residues in the peptide chain, hydrolyzing the C-terminal peptide bonds of these residues, thereby breaking down the protein into smaller peptide segments.

[0021] The affinity prediction of all tetrapeptides, pentapeptides and hexapeptides obtained by hydrolysis was performed using the ConPLex tool. The affinity prediction results are shown in Figure 1.

[0022] Table 1 Prediction results of affinity of screened peptides

[0023] Ranking Peptide sequence Serial number Binding affinity scoring Ranking Peptide sequence Serial number Binding affinity scoring 1 LCAFNK SEQ ID NO. 1 0.38295 6 FICAK SEQ ID NO. 6 0.349397 2 LIFCEK SEQ ID NO. 2 0.382042 7 EFACK SEQ ID NO. 7 0.344567 3 LCFEK SEQ ID NO. 3 0.378817 8 ECFAK SEQ ID NO. 8 0.344567 4 FVNAC SEQ ID NO. 4 0.368747 9 EFCK SEQ ID NO. 9 0.344486 5 LFNCEK SEQ ID NO. 5 0.36046 10 EEFCK SEQ ID NO. 10 0.342601

[0024] The present invention uses the AlphaFold Server provided by Alpha Fold 3 to perform molecular docking analysis on the screened peptides and predict their binding positions on the target protein. The binding sites of the peptides and XO are as follows: Figure 1 shown.

[0025] Combined with the prediction results of Alpha Fold3, we can understand in detail how the peptide interacts with the protein, and LCAFNK is a competitive site inhibitor.

[0026] Example 2 In vitro xanthine oxidase activity inhibition experiment of small molecule peptide LCAFNK from sunflower disc

[0027] The sunflower disc polypeptide involved in the present invention is synthesized and provided by Liaoning Newkey Biochemical Technology Co., Ltd.

[0028] The reaction system for xanthine oxidase to catalyze the production of uric acid consists of substrate (xanthine), enzyme, inhibitor and buffer. In this experiment, 1 / 15 M PBS will be used as the reaction system buffer. 1 / 15 M PBS is used to dilute xanthine, and 1 / 5 M PBS is used to dilute xanthine oxidase, so that the reaction concentration of xanthine oxidase in the reaction system is 0.02 U / mL and the reaction concentration of xanthine reaches 0.5 mM.

[0029] Different concentrations of inhibitors were prepared according to the following standards. The concentrations of LCAFNK dilutions were: 32.11, 16.06, 8.03, 4.01, and 2.00 Mm.

[0030] It should be carried out at a constant temperature of 25°C. After 25 minutes of reaction, the absorbance (OD value) of each group of solutions, i.e. each reaction system, is measured at 290 nm using an ultraviolet spectrophotometer, since uric acid has a characteristic absorption peak at 290 nm.

[0031] Substitute the measured absorbance value into the formula:

[0032] Inhibition rate =

[0033] A1: absorbance value at OD290 nm, the system contains inhibitors, xanthine, and xanthine oxidase; A2: absorbance value at OD290 nm, the system contains inhibitors and xanthine; A3: absorbance value at OD290 nm, the system contains xanthine and xanthine oxidase; A4: absorbance value at OD290 nm, the system contains only xanthine.

[0034] The results of the in vitro xanthine oxidase inhibition experiment of the small molecule peptide LCAFNK from sunflower disc are as follows Figure 2 The results show that this small molecule peptide has a certain inhibitory effect on XO. And the xanthine oxidase inhibitory activity IC50 of the small molecule peptide LCAKNK of the present invention is 28.06 mM.

[0035] in conclusion:

[0036] The present invention simulates trypsin hydrolysis by computer technology. The affinity of the peptides obtained by hydrolysis was predicted using the Complex tool. The molecular docking analysis of the screened peptides was performed using the AlphaFold Server provided by Alpha Fold 3 to determine the interaction mode and binding site between the peptide and the protein. The in vitro xanthine oxidase activity experiment verified that the small molecule peptide LCAFNK has the potential to be a xanthine oxidase inhibitor.

[0037] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A small molecule peptide having xanthine oxidase inhibitory activity, characterized in that: Its amino acid sequence is shown as SEQ ID NO.

1.

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

Images