Xod inhibiting peptides derived from sheep milk and methods of making and using the same

Abstract

To address the problem that "existing XOD-inhibiting peptides are mostly derived from cow's milk or non-dairy proteins, and their peptide activity varies greatly, binding stability is limited, and mechanisms are unclear, thus restricting their application in the prevention and treatment of hyperuricemia and related metabolic diseases," this invention provides a group of XOD-inhibiting peptides derived from sheep milk, their preparation methods, and applications. The inhibiting peptides include the peptide sequences GPGGAW and FGER. In vitro enzyme activity assays show that the above peptides can effectively inhibit XOD activity and reduce uric acid production. The sheep milk-derived XOD-inhibiting peptides provided by this invention have the characteristics of clear origin, simple structure, and high safety, and can be used in functional foods, nutritional supplements, and dietary interventions, providing a novel active substance basis for the prevention and treatment of hyperuricemia and related metabolic diseases.

Description

Technical Field

[0001] This invention relates to the field of bioactive peptides and functional food technology, specifically to a group of XOD-inhibiting peptides derived from sheep milk and their preparation methods and applications. Background Technology

[0002] Xanthine oxidase (XOD) is a key rate-limiting enzyme in purine metabolism, catalyzing the conversion of hypoxanthine and xanthine into uric acid, accompanied by the generation of reactive oxygen species (ROS). Abnormally elevated XOD activity is not only the direct biochemical basis of hyperuricemia and gout, but also closely related to oxidative stress, inflammatory responses, and kidney damage. Therefore, inhibiting XOD activity is considered an important strategy for intervening in hyperuricemia and related metabolic diseases. Currently, commonly used XOD inhibitors in clinical practice mainly include allopurinol and its derivatives, febuxostat, and other synthetic small-molecule drugs. Although these drugs have good efficacy in lowering serum uric acid levels, long-term or high-dose use can easily cause safety issues such as skin rashes, liver and kidney toxicity, and cardiovascular adverse reactions. Some patients also have poor tolerance to them, limiting their widespread use in people with chronic hyperuricemia. Therefore, developing safer, naturally derived XOD inhibitors is of great significance.

[0003] In recent years, bioactive peptides obtained from the enzymatic hydrolysis of food proteins have attracted attention in the field of metabolic disease intervention due to their small molecular weight, easy absorption, and good biocompatibility. Existing studies have shown that short peptides derived from milk, aquatic, and plant proteins can inhibit the catalytic activity of XOD by binding to the active site of XOD. However, current technologies still have shortcomings in terms of source, structural characteristics, and mechanism of action: First, XOD-inhibiting peptides are mostly concentrated in cow's milk or non-dairy proteins, and there is a lack of systematic research on XOD-inhibiting peptides from sheep milk protein as a new dairy resource; second, some peptides have insufficient binding stability, resulting in limited inhibition efficiency; third, most studies remain at the activity screening stage, lacking in-depth elucidation of the peptide-enzyme binding mechanism, which limits their further application. Sheep milk protein, as a new dairy resource, has unique advantages in terms of amino acid composition, digestibility, and the abundance of potentially bioactive short peptides, providing a new avenue for developing highly active, natural, and safe functional peptides. However, no functional peptides derived from sheep milk protein with a defined amino acid sequence and capable of effectively inhibiting XOD activity have been publicly reported to date, and related technologies still need breakthroughs.

[0004] Therefore, there is an urgent need to provide a functional peptide derived from sheep milk protein that has a clear source, high safety, clear structure, and good XOD inhibitory activity, so as to provide technical support for the natural intervention of hyperuricemia and related metabolic diseases, and at the same time expand the innovative application potential of sheep milk as a new dairy resource in functional foods and health products. Summary of the Invention

[0005] To address the problem that "existing XOD inhibitory peptides are mostly derived from cow's milk or non-dairy proteins, and their peptide activities vary greatly, binding stability is limited, and mechanisms are unclear, thus restricting their application in the prevention and treatment of hyperuricemia and related metabolic diseases," this invention aims to provide a group of XOD inhibitory peptides derived from sheep milk, their preparation methods, and applications. The inhibitory peptides include the peptide sequences GPGGAW and FGER. In vitro enzyme activity assays show that these peptides can effectively inhibit XOD activity and reduce uric acid production. Network pharmacology analysis further reveals their potential targets and related signaling pathways. The sheep milk-derived XOD inhibitory peptides provided by this invention have the characteristics of clear origin, simple structure, and high safety, and can be used in functional foods, nutritional supplements, and dietary interventions, providing a novel active substance basis for hyperuricemia and related metabolic diseases, and have broad application prospects.

[0006] To achieve the above objectives, the present invention provides the following technical solution: This invention first provides a method for screening XOD inhibitory peptides derived from sheep milk. The method is characterized by using sheep milk protein as raw material to obtain potential peptides through in vitro enzymatic hydrolysis simulation; screening the XOD inhibitory potential of candidate peptides using an artificial intelligence prediction model; and performing molecular docking and molecular dynamics simulation on the target peptides and XOD active sites to analyze the binding conformation and stability.

[0007] A group of XOD-inhibiting peptides derived from sheep milk, screened using the above method, includes the following peptide sequences: Peptide A: GPGGAW; Peptide B: FGER.

[0008] The method for preparing the above-mentioned XOD inhibitory peptide is characterized by being prepared by solid-phase peptide synthesis, having a purity ≥95%, and being used alone or in combination with peptide A and peptide B.

[0009] This invention also provides the application of the above-mentioned XOD inhibitory peptide in the preparation of XOD inhibitors and in reducing the production of uric acid in vivo.

[0010] Furthermore, the present invention also provides the application of the above-mentioned XOD inhibitory peptide in the preparation of products for the prevention, treatment or assistance in improving hyperuricemia or gout-related symptoms.

[0011] Furthermore, the present invention also provides the application of the above-mentioned XOD inhibitory peptide in the preparation of functional foods, nutritional supplements, health foods or dietary intervention products that have the effect of reducing uric acid production.

[0012] The aforementioned functional foods or nutritional supplements may take the form of dairy products, dairy beverages, protein beverages, powders, tablets, or capsules.

[0013] The present invention also provides a composition comprising the XOD-inhibiting peptide and a food or pharmaceutically acceptable carrier, wherein the composition may contain peptide A and peptide B simultaneously or individually.

[0014] The technical effects of this invention are: 1. The peptide is clearly derived from sheep milk protein, highlighting the innovation of the new dairy resource; moreover, the peptide has a simple structure, high safety, and is easy to synthesize and apply artificially; 2. By combining artificial intelligence prediction, molecular docking, and molecular dynamics simulation, we ensure that the peptide has high affinity and stable binding to the XOD active site; 3. It can be used alone or in combination, expanding the development space for functional foods and natural XOD inhibitors; it is expected to provide a natural, safe and effective new intervention method for hyperuricemia and related metabolic diseases. Attached Figure Description

[0015] Figure 1 Flowchart of XOD inhibitory peptide screening based on machine learning and molecular docking; Figure 2 : Ranking of high-affinity peptides obtained through screening; Figure 3 : In vitro XOD inhibitory activity of FGER; Figure 4 : In vitro XOD inhibitory activity of GPGGAW; Figure 5 Effects of FGER on the secondary structure of xanthine oxidase; Figure 6 Effects of GPGGAW on the secondary structure of xanthine oxidase; Figure 7 Molecular dynamics simulation stability analysis of the FGER-XOD complex; Figure 8 Molecular dynamics simulation stability analysis of the GPGGAW-XOD complex; Figure 9 The amino acid binding pattern of FGER with xanthine oxidase; Figure 10 The amino acid binding pattern of GPGGAW with xanthine oxidase; Figure 11 Schematic diagram of network pharmacology analysis of potential targets and related signaling pathways of sheep milk XOD inhibitory peptides; the left figure is a potential target diagram; the right figure is a bubble diagram of the KEGG pathway. Detailed Implementation

[0016] To make the objectives, technical solutions, and advantages of this invention clearer, the specific embodiments of this invention will be described in detail below with reference to the accompanying drawings. The following embodiments are used to further illustrate this invention, but should not be construed as limiting it. Modifications or substitutions made to this invention without departing from its spirit and substance are all within the scope of protection of this invention.

[0017] I. Design Principles of the Invention Based on the following core design principles, this invention has successfully screened peptides with novel structures and highly efficient inhibitory activity from sheep milk: 1. Target structure-driven peptide sequence design principle: Based on the structural features of XOD active center being a hydrophobic cavity containing key polar residues, short peptides that simultaneously possess an aromatic hydrophobic core and the ability to form directional hydrogen bonds / salt bridges are most likely to achieve efficient and stable binding.

[0018] 2. Functional Prediction-Structure Validation Screening Strategy: To efficiently identify targets from a massive amount of enzymatically digested fragments, a step-by-step screening approach is adopted. First, a machine learning model is used for rapid initial functional screening based on global physicochemical characteristics; then, molecular docking is used to rigorously evaluate the three-dimensional structural complementarity with the target.

[0019] 3. Validation of Dynamic Action and Conformation Effect: Efficient inhibition requires not only static affinity but also dynamic binding stability and perturbation of the enzyme's functional conformation. In the validation phase, molecular dynamics simulations confirmed the dynamic stability of binding, and circular dichroism spectroscopy was used to detect its induction of changes in the secondary structure of XOD, thus establishing a complete inhibition mechanism.

[0020] Based on the above principles, peptides FGER and GPGGAW met the design expectations, and the final experiment verified their excellent inhibitory activity and clear conformational perturbation effect.

[0021] II. The present invention will be further described in detail below with reference to specific embodiments.

[0022] Unless otherwise specified, the experimental methods and detection methods involved in the following embodiments are all conventional experimental methods and detection methods that already exist in the prior art.

[0023] Example 1: Screening and identification of XOD-inhibiting peptides derived from goat milk protein 1.1 Construction of Virtual Peptide Library The flowchart for screening and identification of XOD inhibitory peptides FGER and GPGGAW is as follows: Figure 1As shown, the amino acid sequences of eight major proteins in goat milk were obtained from the UniProt database, including κ-casein (P02669), αs1-casein (P04653), αs2-casein (P04654), β-casein (P11839), α-lactalbumin (P09462), β-lactoglobulin (P67976), serum albumin (P14639), and lactoferrin (R9QXS6). Using the BIOPEP-UWM database, single-enzyme and combined enzymatic digestions (129 patterns in total) of nine proteases (trypsin, pepsin, chymotrypsin, elastase, proteinase K, subtilisin, papain, bromelain, and figase) were simulated to obtain a virtual peptide library containing a large number of potentially active fragments.

[0024] 1.2 Preliminary Screening of Bioactive Peptides Based on Machine Learning A balanced dataset was constructed by collecting 202 experimentally validated XOD-inhibiting peptides as positive samples and an equal number of non-inhibiting peptides as negative samples. Seven amino acid descriptors (including VHSE, Z-Scale, etc.) were used to feature-encode the peptide sequences. Ensemble learning algorithms such as CatBoost were employed for model training and optimization. Through 5-fold cross-validation, the prediction model built using the VHSE descriptor combined with the CatBoost algorithm was determined to be the best performing. This model was applied to predict a virtual peptide library, identifying 538 high-confidence candidate peptides with an XOD inhibition probability greater than 0.8.

[0025] 1.3 Fine Screening Based on Molecular Docking Using the crystal structure of human xanthine oxidase (PDB ID: 3NVW) as the target, molecular docking was performed on the aforementioned 538 candidate peptides using AutoDock Vina. The center of the docking grid was set as the coordinate of the active pocket center. Peptides with binding energies ≤ -8.0 kcal / mol were selected as high-affinity candidate molecules. Figure 2 As shown, peptides FGER and GPGGAW exhibit excellent binding potential, with predicted binding energies of -9.6 kcal / mol and -9.5 kcal / mol, respectively.

[0026] Example 2: Chemical Synthesis and Purity Identification of Peptides The target peptides FGER and GPGGAW were synthesized using the standard Fmoc solid-phase synthesis method. After synthesis, they were purified by high-performance liquid chromatography (HPLC) and identified by electrospray ionization mass spectrometry (ESI-MS). Mass spectrometry analysis showed that their purity was ≥95%, meeting the requirements for subsequent bioactivity testing.

[0027] Example 3: In vitro XOD inhibitory activity assay of peptides The inhibitory activity of the peptides was determined using the WST-8 XOD activity assay kit. Different concentrations of FGER and GPGGAW peptide solutions (2, 4, 6, 8, and 10 mg / mL) were pre-incubated with XOD enzyme solution at a final concentration of 10 mU / mL at 37°C for 10 minutes. Then, xanthine was added to initiate the reaction, and absorbance changes were detected at 450 nm. A peptide-free sample was used as a blank control (100% enzyme activity), and the inhibition rate at each concentration was calculated. The results showed that the inhibition of XOD by FGER and GPGGAW was significantly dose-dependent. Figure 3 and Figure 4 The half-inhibitory concentration (IC50) of FGER was calculated using dose-response curves. 50 The value is 16.90 ± 0.14 mM, and the IC of GPGGAW is... 50 The value was 15.51 ± 0.35 mM, proving that both have clear XOD inhibitory activity.

[0028] Example 4: Effect of peptides on the secondary structure of XOD protein The effect of FGER and GPGGAW binding on the conformation of XOD was investigated using circular dichroism spectroscopy. XOD and peptides were mixed at a molar ratio of 1:10 and incubated, then scanned in the far-ultraviolet region of 190-260 nm (see [reference]). Figure 5-6 The results showed that compared with the spectrum of free XOD, the XOD-peptide complex exhibited decreased negative ellipticity values ​​at 222 nm and 208 nm, along with changes in the 200-205 nm region. This indicates that the α-helix content of XOD decreased relatively after peptide binding, while the random coil content increased. This suggests that the binding of FGER and GPGGAW may induce local conformational relaxation or unfolding in the active pocket region of XOD, thereby interfering with its catalytic function, explaining its inhibitory mechanism at the structural level.

[0029] Example 5: Molecular dynamics simulation study of peptide-XOD interaction Based on the FGER-XOD and GPGGAW-XOD complex structures obtained through molecular docking, the AMBER ff14SB force field was used to describe protein and peptide parameters, while the cofactor (Mo-pt molybdenum pterin center) parameters were generated by the GAFF force field. The complexes were placed in a 10 Å cubic box, solvated using TIP3P water molecules, and 0.15 mol / L NaCl was added to neutralize the charge and simulate physiological ionic strength. First, a 2500-step steepest descent energy minimization method was performed (step size 0.01 ps). Then, the system was gradually heated from 0 K to 300 K using the NVT and NPT ensembles (Berendsen hot bath and pressure bath, coupling time constants of 0.1 ps and 1.0 ps, ​​respectively). Formal simulations employed periodic boundary conditions, a time step of 2 fs, SHAKE constraint for all hydrogen bonds, PME to handle long-range electrostatic interactions, a van der Waals cutoff of 10 Å, and a total duration of 200 ns, with one frame of trajectory saved every 10 ps. All simulations were performed using the GROMACS 2024 software package. The root mean square deviation of the complex reached equilibrium and remained stable after 50 ns, indicating that the binding conformation is reliable under dynamic conditions. Figure 7-8 The key hydrogen bonds identified during docking (such as Arg4-Asp1191, Phe1-Gln767, etc.) remained stable for most of the simulation time. Figure 9-10 ).

[0030] Example 6: Analysis of the potential pathways of XOD inhibitory peptides based on network pharmacology Using the chemical structures of FGER and GPGGAW as search terms, potential therapeutic targets were predicted using SwissTargetPrediction. After merging and deduplicating, target profiles for each peptide were obtained. Human targets related to hyperuricemia and gout were retrieved from the GeneCards database, and their intersection with the predicted targets was used to obtain candidate therapeutic targets. These intersection targets were then imported into the STRING database to construct proteins. Protein-protein interaction networks were visualized and topologically analyzed using Cytoscape. The target interaction networks reveal (e.g.) Figure 11 (See the left side of the figure). The high correlation between the targets indicates that the XOD inhibitory peptide may participate in the regulation of related biological processes through multi-target synergistic effects.

[0031] Pathway enrichment analysis of candidate targets was performed using the DAVID 6.8 database (https: / / david.ncifcrf.gov). The list of intersecting target genes was uploaded to the DAVID database, with the species set to "Homo sapiens". The "KEGG_PATHWAY" database was selected for analysis in the functional annotation tool. The hypergeometric test was used for statistical analysis, with a p-value < 0.05 as the screening criterion for statistically significant pathway enrichment. After the enrichment analysis, to further improve the reliability of the results, the Benjamini-Hochberg (BH) method was used to perform multiple tests to correct the p-values, and the false discovery rate (FDR) was calculated. An FDR < 0.05 was used as the key indicator for screening enriched pathways. The results showed (e.g.) Figure 11 (See right side of the figure). The target sites are mainly enriched in pathways related to purine metabolism, inflammatory response, and signal transduction. The above analysis results suggest that sheep milk inhibitory peptides, in addition to inhibiting xanthine oxidase activity, may participate in the regulation of hyperuricemia-related physiological processes through a multi-pathway synergistic mechanism, supporting their rationale as xanthine oxidase inhibitory peptides at the systemic level.

[0032] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A screening method for a group of XOD-inhibiting peptides derived from sheep milk, characterized in that, Using sheep milk protein as raw material, potential peptides were obtained through in vitro enzymatic hydrolysis simulation; the XOD inhibition potential of candidate peptides was screened using an artificial intelligence prediction model; molecular docking and molecular dynamics simulations were performed on the target peptides and XOD active sites to analyze the binding conformation and stability.

2. A group of XOD-inhibiting peptides derived from sheep milk, characterized in that the peptides... A: GPGGAW; Peptide B: FGER, used alone or in combination, obtained by the screening method described in claim 1.

3. The method for preparing the XOD inhibitory peptide according to claim 2, characterized in that, Prepared by solid-phase peptide synthesis.

4. The application of the XOD inhibitory peptide according to claim 2 in the preparation of XOD inhibitors and the reduction of uric acid production in vivo.

5. The use of the XOD inhibitory peptide according to claim 2 in the preparation of products for the prevention, treatment or assistance in improving symptoms of hyperuricemia or gout.

6. The use of the XOD inhibitory peptide according to claim 2 in the preparation of functional foods, nutritional supplements, health foods or dietary intervention products that reduce uric acid production.

7. The application as described in claim 6, characterized in that, The functional foods or nutritional supplements are in the form of dairy products, dairy beverages, protein beverages, powders, tablets, or capsules.

8. A composition comprising the XOD-inhibiting peptide of claim 2 and a food- or pharmaceutically acceptable carrier, wherein the composition may contain peptide A and peptide B simultaneously or individually.

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