Native Peptide Research Articles

Thermosensitive and mucoadhesive Xanthan gum-based hydrogel for local release of anti-Candida peptide

A thermoresponsive and mucoadhesive hydrogel has been developed for the local delivery of a novel anti-Candida peptide. This antimicrobial peptide was custom-designed and synthesized, utilizing a natural peptide identified in the hemolymph of the freshwater crayfish Procambarus clarkii as a molecular scaffold. The hydrogel, fabricated from a xanthan gum/poly-N-isopropylacrylamide graft copolymer, demonstrates temperature-dependent viscoelastic properties and a pseudoplastic behaviour, making it suitable for potential administration in various tissues. Moreover, the high stability of the hydrogel (about 9 % weight loss after 24 h of incubation) in physiological fluids as well as its mucoadhesive properties indicate that it could withstand in the application site long enough to perform its intended function. Furthermore, the good cytocompatibility of the hydrogel and the peptide's release profile (approximately 90 % release within the first 24 h), coupled with its efficacy in inhibiting fungal growth (logarithmic reduction of 1.08 compared to the control), validates the prospective application of the formulation in managing mucosal and superficial skin C. albicans infections. This not only addresses concerns related to drug resistance but also establishes the hydrogel as a versatile platform for advanced drug delivery systems aimed at circumventing systemic administration of antifungal drugs for the treatment of superficial skin and mucosal candidiasis.

Peptide-specific chemical language model successfully predicts membrane diffusion of cyclic peptides.

Biological language modeling has significantly advanced the prediction of membrane penetration for small molecule drugs and natural peptides. However, accurately predicting membrane diffusion for peptides with pharmacologically relevant modifications remains a substantial challenge. Here, we introduce PeptideCLM, a peptide-focused chemical language model capable of encoding peptides with chemical modifications, unnatural or non-canonical amino acids, and cyclizations. We assess this model by predicting membrane diffusion of cyclic peptides, demonstrating greater predictive power than existing chemical language models. Our model is versatile, able to be extended beyond membrane diffusion predictions to other target values. Its advantages include the ability to model macromolecules using chemical string notation, a largely unexplored domain, and a simple, flexible architecture that allows for adaptation to any peptide or other macromolecule dataset.

The Discovery and Characterization of a Potent DPP-IV Inhibitory Peptide from Oysters for the Treatment of Type 2 Diabetes Based on Computational and Experimental Studies.

The inhibition of dipeptidyl peptidase-IV (DPP-IV) is a promising approach for regulating the blood glucose levels in patients with type 2 diabetes (T2D). Oysters, rich in functional peptides, contain peptides capable of inhibiting DPP-IV activity. This study aims to identify the hypoglycemic peptides from oysters and investigate their potential anti-T2D targets and mechanisms. This research utilized virtual screening for the peptide selection, followed by in vitro DPP-IV activity assays to validate the chosen peptide. Network pharmacology was employed to identify the potential targets, GO terms, and KEGG pathways. Molecular docking and molecular dynamics simulations were used to provide virtual confirmation. The virtual screening identified LRGFGNPPT as the most promising peptide among the screened oyster peptides. The in vitro studies confirmed its inhibitory effect on DPP-IV activity. Network pharmacology revealed that LRGFGNPPT exerts an anti-T2D effect through multiple targets and signaling pathways. The key hub targets are AKT1, ACE, and REN. Additionally, the molecular docking results showed that LRGFGNPPT exhibited a strong binding affinity with targets like AKT1, ACE, and REN, which was further confirmed by the molecular dynamics simulations showcasing a stable peptide-target interaction. This study highlights the potential of LRGFGNPPT as a natural anti-T2D peptide, providing valuable insights for potential future pharmaceutical or dietary interventions in T2D management.

Bilateral Unsymmetrical Disulfurating Reagent Design for Polysulfide Construction.

Polysulfides are significant compounds in life science, pharmaceutical science, and materials science. Therefore, polysulfide construction is in great demand. The controllable sequential installation of groups on both ends of a S-S motif faces an enormous challenge owing to the reversible nature of the covalent S-S bond. A library was established with two divergent mask groups for bilateral unsymmetrical disulfurating reagents (R1O-SS-SO2R2). Sequential coupling with preferential activation of the S-SO2 bond (37.6 kcal/mol) and controllable activation of the S-O bond (54.8 kcal/mol) in the presence of the S-S bond (62.0 kcal/mol) enabled successive reactions at each end of the S-S motif to afford unsymmetrical disulfides and trisulfides, even for the cross-linkage of natural products, pharmaceuticals, peptides, and a protein (bovine serum albumin).

Bilateral Unsymmetrical Disulfurating Reagent Design for Polysulfide Construction

AbstractPolysulfides are significant compounds in life science, pharmaceutical science, and materials science. Therefore, polysulfide construction is in great demand. The controllable sequential installation of groups on both ends of a S−S motif faces an enormous challenge owing to the reversible nature of the covalent S−S bond. A library was established with two divergent mask groups for bilateral unsymmetrical disulfurating reagents (R1O−SS−SO2R2). Sequential coupling with preferential activation of the S−SO2 bond (37.6 kcal/mol) and controllable activation of the S−O bond (54.8 kcal/mol) in the presence of the S−S bond (62.0 kcal/mol) enabled successive reactions at each end of the S−S motif to afford unsymmetrical disulfides and trisulfides, even for the cross‐linkage of natural products, pharmaceuticals, peptides, and a protein (bovine serum albumin).

Lactoferrin Binds through Its N-Terminus to the Receptor-Binding Domain of the SARS-CoV-2 Spike Protein.

Since Coronavirus disease 2019 (COVID-19) still presents a considerable threat, it is beneficial to provide therapeutic supplements against it. In this respect, glycoprotein lactoferrin (LF) and lactoferricin (LFC), a natural bioactive peptide yielded upon digestion from the N-terminus of LF, are of utmost interest, since both have been shown to reduce infections of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), the virus responsible for COVID-19, in particular via blockade of the virus priming and binding. Here, we, by means of biochemical and biophysical methods, reveal that LF directly binds to the S-protein of SARS-CoV-2. We determined thermodynamic and kinetic characteristics of the complex formation and mapped the mutual binding sites involved in this interaction, namely the N-terminal region of LF and the receptor-binding domain of the S-protein (RBD). These results may not only explain many of the observed protective effects of LF and LFC in SARS-CoV-2 infection but may also be instrumental in proposing potent and cost-effective supplemental tools in the management of COVID-19.

Abstract Mo042: Poison Peptides May Contribute to Dysregulation of SERCA in Heart Failure

Proteolysis increases in heart disease, resulting in an accumulation of degraded membrane protein fragments in heart cells. Previous studies have shown that diverse hydrophobic alpha helices can bind and non-specifically inhibit the cardiac calcium pump SERCA. Thus, we hypothesized that partially degraded fragments of transmembrane proteins may directly inhibit SERCA or compete with native peptide regulators of the pump, like such as phospholamban. The resulting dysregulation of SERCA may contribute to calcium mishandling in heart failure. To test these hypotheses, purified cell membranes from non-failing or dilated cardiomyopathy human hearts were subjected to SDS-PAGE and mass spectrometry to identify protein fragments smaller than 26 kD. 1800 proteins were detected, of which 92 were upregulated >3 fold in the failing heart samples, consistent with increased production of small fragments from these membrane proteins. 9 of the upregulated fragments containing transmembrane segments were selected for further analysis based on sequence hydrophobicity and structural similarity to endogenous micropeptide regulators of SERCA. Of these 9 fragments, 6 showed ER localization, suggesting potential for a toxic interaction with SERCA. The relative binding of these candidates to SERCA was quantified with FRET microscopy. Fragments of three membrane proteins (SEC61B, VAMP3, VAMP8) showed an avid interaction with SERCA. To measure SERCA inhibition, we utilized a calcium activity assay was performed usingin which HEK293T cells were transfected with SERCA, ryanodine receptor, candidate peptides and rCepia as an ER calcium indicator. ER-calcium level was normalized to minimum fluorescence after caffeine addition and an ionomycin induced maximum fluorescence after addition of ionomycin and calcium and caffeine induced minimum. Unregulated Candidate poison peptides were compared to SERCA alone and SERCA-PLB phospholamban controls were also performed. . SEC61B demonstrated the a strongest inhibition, most (similar to phospholamban, ) while VAMP3 and VAMP8’s effect was more modest. This type of non-nativeThus, we propose accumulation of toxic transmembrane peptide fragments may inhibition could be contributinginhibit SERCA and contribute to the calcium mishandling seen in heart failure. Future experiments will be performed to quantify the relative abundance of the fragments compared to endogenous regulatory micropeptides.

Use of protease substrate specificity screening in the rational design of selective protease inhibitors with unnatural amino acids: Application to HGFA, matriptase, and hepsin.

Inhibition of the proteolytic processing of hepatocyte growth factor (HGF) and macrophage stimulating protein (MSP) is an attractive approach for the drug discovery of novel anticancer therapeutics which prevent tumor progression and metastasis. Here, we utilized an improved and expanded version of positional scanning of substrate combinatorial libraries (PS-SCL) technique called HyCoSuL to optimize peptidomimetic inhibitors of the HGF/MSP activating serine proteases, HGFA, matriptase, and hepsin. These inhibitors have an electrophilic ketone serine trapping warhead and thus form a reversible covalent bond to the protease. We demonstrate that by varying the P2, P3, and P4 positions of the inhibitor with unnatural amino acids based on the protease substrate preferences learned from HyCoSuL, we can predictably modify the potency and selectivity of the inhibitor. We identified the tetrapeptide JH-1144 (8) as a single digit nM inhibitor of HGFA, matriptase and hepsin with excellent selectivity over Factor Xa and thrombin. These unnatural peptides have increased metabolic stability relative to natural peptides of similar structure. The tripeptide inhibitor PK-1-89 (2) has excellent pharmacokinetics in mice with good compound exposure out to 24 h. In addition, we obtained an X-ray structure of the inhibitor MM1132 (15) bound to matriptase revealing an interesting binding conformation useful for future inhibitor design.

Ferredoxin: A novel antimicrobial peptide derived from the black scraper (Thamnaconus modestus)

Ferredoxin (FDX) is a highly conserved iron-sulfur protein that participates in redox reactions and plays an important role as an electron transport protein in biological processes. However, its function in marine fish remains unclear. We identified two ferrodoxin proteins, FDX1 and FDX2, from black scraper (Thamnaconus modestus) to confirm their genetic structures and expression profiles and to investigate their antimicrobial activity properties by fabricating them with antimicrobial peptides based on sequences. The two TmFDXs mRNAs were most abundant in peripheral blood leukocytes of healthy T. modestus. After artificial infection with Vibrio anguillarum, a major pathogen of T. modestus, TmFDX1 mRNA was significantly upregulated in the gills, heart, intestines, kidneys, liver, and spleen, but was consistently downregulated in the brain. The expression levels of TmFDX2 mRNA were significantly upregulated in the heart, intestines, kidneys, liver, and spleen; however, no significant changes in expression were observed in the brain or gills. Based on the 2Fe-2S ferredoxin-type iron-sulfur-binding domain sequence, two peptides (pFDX1 and pFDX2) were synthesized. The bactericidal effect, biofilm formation inhibition, and gDNA-binding activity of these peptides were investigated. These findings highlight the potential as a natural peptide candidate for TmFDXs.

Exploring the Potential of Cyclic Peptidyl Antitumor Agents Derived from Natural Macrocyclic Peptide Phakellistatin 13.

The exploration of novel anticancer compounds based on natural cyclopeptides has emerged as a pivotal paradigm in the contemporary advancement of macrocyclic pharmaceuticals. Phakellistatin 13 is a cycloheptapeptide derived from the brown snubby sponge and exhibits remarkable antitumor activity. In this study, we have designed and synthesized a series of chiral cyclopeptides incorporating the rigid isoindolinone moiety at various sites within the natural cycloheptapeptide Phakellistatin 13, with the aim of investigating conformationally constrained cyclopeptides as potential antitumor agents. Cyclopeptide 3, comprising alternating l-/d-amino acid residues, exhibited promising antihepatocellular carcinoma effects. Detailed biological experiments have revealed that Phakellistatin 13 analogs effectively inhibit the proliferation of tumor cells and induce apoptosis and autophagy, while also causing cell cycle arrest through the modulation of the p53 and mitogen-activated protein kinase (MAPK) signaling pathway. This study not only provides valuable insights into chemical structural modifications but also contributes to a deeper understanding of the biological mechanisms underlying the development of natural cyclopeptide-based drugs.

Design and Discovery of New Collagen V-Derived FGF2-Blocking Natural Peptides Inhibiting Lung Squamous Cell Carcinoma In Vitro and In Vivo.

Aberrant FGF2/FGFR signaling is implicated in lung squamous cell carcinoma (LSCC), posing treatment challenges due to the lack of targeted therapeutic options. Designing drugs that block FGF2 signaling presents a promising strategy different from traditional kinase inhibitors. We previously reported a ColVα1-derived fragment, HEPV (127AA), that inhibits FGF2-induced angiogenesis. However, its large size may limit therapeutic application. This study combines rational peptide design, molecular dynamics simulations, knowledge-based prediction, and GUV and FRET assays to identify smaller peptides with FGF2-blocking properties. We synthesized two novel peptides, HBS-P1 (45AA) and HBS-P2 (66AA), that retained the heparin-binding site. Both peptides demonstrated anti-LSCC and antiangiogenesis properties in cell viability and microvessel network induction assays. In two LSCC subcutaneous models, HBS-P1, with its affinity for FGF2 and enhanced penetration ability, demonstrated substantial therapeutic potential without apparent toxicities. Our study provides the first evidence supporting the development of collagen V-derived natural peptides as FGF2-blocking agents for LSCC treatment.

Natural zinc-rich oyster peptides ameliorate DSS-induced colitis via antioxidation, anti-inflammation, intestinal barrier repair, microbiota modulation, and SCFA accumulation

Natural oyster zinc-rich peptide (OZP) was extracted and purified. The higher zinc concentration in natural OZP may be closely related to the high zinc affinity of amino acids containing Lys, His, Cys, Val, Arg and Ser residues. FTIR analysis indicated that amino, amide, carboxyl and carbonyl groups may contribute to the binding sites for zinc and peptides. Administration of OZP ameliorated the symptoms of DSS-induced ulcerative colitis (UC) in mice, including weight loss, colonic damage, mucosal edema, goblet cell exhaustion and crypt disappearance. Natural OZP suppressed oxidative stress by significantly reducing the level of NO, MDA and MPO as well as increasing SOD activity. The anti-inflammatory effects of OZP were associated with the inhibition of TLR4/NF-κB pathways by diminishing NF-κB, TLR4, COX-2, iNOS, TNF-α, IL-6 and IFN-γ and elevating IL-10. By promoting mucus secretion, repairing goblet cells and enhancing the expression of tight junction proteins including occludin, claudin-1 and ZO-1, natural OZP maintained the intestinal barrier function. Furthermore, natural OZP regulated the microbial composition by remarkedly enhancing the beneficial microbial abundance like Faecalibaculum, Odoribacter and Anaerostipes, which in turn led to a significant increase in SCFA production. Collectively, natural OZP exerted the synergistic capacity to protect against DSS-induced UC by improving antioxidation and anti-inflammation, repairing intestinal barrier and modulating intestinal microbiota and SCFA.

Effects of alloferon and its analogues on reproduction and development of the Tenebrio molitor beetle

As the most numerous group of animals on Earth, insects are found in almost every ecosystem. Their useful role in the environment is priceless; however, for humans, their presence may be considered negative or even harmful. For years, people have been trying to control the number of pests by using synthetic insecticides, which eventually causes an increased level of resistance to applied compounds. The effects of synthetic insecticides have encouraged researchers to search for alternatives and thus develop safe compounds with high specificity. Using knowledge about the physiology of insects and the functionality of compounds of insect origin, a new class of bioinsecticides called peptidomimetics, which are appropriately modified insect analogues, was created. One promising compound that might be successfully modified is the thirteen amino acid peptide alloferon (HGVSGHGQHGVHG), which is obtained from the hemolymph of the blue blowfly Calliphora vicinia. Our research aimed to understand the physiological properties of alloferon and the activity of its peptidomimetics, which will provide the possibility of using alloferon or its analogues in the pharmaceutical industry, as a drug or adjuvant, or in agriculture as a bioinsecticide. We used alloferon and its three peptidomimetics, which are conjugates of the native peptide with three unsaturated fatty acids with various chain lengths: caprylic, myristic, and palmitic. We tested their effects on the morphology and activity of the reproductive system and the embryogenesis of the Tenebrio molitor beetle. We found that the tested compounds influenced the growth and maturation of ovaries and the expression level of the vitellogenin gene. The tested compounds also influenced the process of egg laying, embryogenesis, and offspring hatching, showing that alloferon might be a good peptide for the synthesis of effective bioinsecticides or biopharmaceuticals.

Trifluoroacetic Acid Mediated Additive-Free Late-Stage Native Peptide Cyclization to Form Disulfide Mimetics via Thioketalization with Ketones.

Peptide cyclization is often used to introduce conformational rigidity and to enhance the physiological stability of the peptide. This study presents a novel late-stage cyclization method for creating thioketal cyclic peptides from bis-cysteine peptides and drugs. Symmetrical cyclic ketones and acetone were found to react with bis-cysteine unprotected peptides efficiently to form thioketal linkages in trifluoroacetic acid (TFA) without any other additive. The attractive features of this method include high chemoselectivity, operational simplicity, and robustness. In addition, TFA as the reaction solvent can dissolve any unprotected peptide. As a showcase, the dimethyl thioketal versions of lanreotide and octreotide were prepared and evaluated, both of which showed much improved reductive stability and comparable activity.

Excreted/Secreted 15-kDa Proteins and Serine Protease Peptides from Haemonchus contortus Act as Immune-Response Enhancers in Lambs.

This study assessed the immunoprotective effect in lambs of a native excreted/secreted 15-kDa protein and two synthesised S28 peptides derived from the infective transitory larvae (xL3) and adult stages (AS) of Haemonchus contortus. Twenty-two Pelibuey lambs were divided into negative and positive control groups, as well as immunised lamb groups, with 100 µg of the 15-kDa native protein (15kDaNP) and S28 peptides (S28P). The eggs per gram (EPG) and haematocrit were measured, and AS were counted and morphologically measured. To assess the immunoprotection in lambs, indirect enzyme-linked immunosorbent assays and relative expression analyses of immune cytokines were performed using serum and abomasal samples. Our results showed a 72.28% reduction in adult worms (AW) in the 15kDaNP-immunised group, achieving a high clinical response with 41% haematocrit and low EPG values (436 ± 661). Conversely, the S28P group achieved the highest IgG levels (2.125 ± 0.880 OD), with AW exhibiting the greatest body length (p > 0.05) and upregulation of the IL5 and FCεR1A genes associated with nematode control. The 15kDaNP group showed increased expression of genes related to nematode control and anti-inflammatory responses, including IL4, IL5, IL6, and IL13 (p < 0.05). The S28P and 15kDaNP should be explored as potential vaccines against sheep haemonchosis.

Assessment of the impact of whey protein hydrolysate on myofibrillar proteins in surimi during repeated freeze–thaw cycles: Quality enhancement and antifreeze potential

The quality of surimi, widely used in processed seafood, is compromised by freeze–thaw cycles, leading to protein denaturation and oxidative degradation. The objective of this study is to explore the effects of adding natural whey peptide hydrolysate (WPH) on the myofibrillar proteins of repeatedly freeze–thawed surimi. Results indicated surimi treated with 15% WPH exhibited only a 128% increase in surface hydrophobicity and a maximum peroxide value of 7.84 μg/kg, significantly lower than the control group. Additionally, salt-soluble protein content, emulsification activity, and stability decreased with the increase in freeze–thaw cycles. With a 15% WPH offering the most significant protective effect, evidenced by reductions of only 25.02%, 42.52% and 37.02% in salt-soluble protein content, emulsification activity, and stability, respectively. These outcomes demonstrate that WPH effectively reduces protein denaturation during repeated freeze–thaw processes. Future research should explore the molecular mechanisms underlying WPH's protective effects and evaluate their applicability in other food systems.

A Natural Bioactive Peptide from Pinctada fucata Pearls Can Be Used as a Potential Inhibitor of the Interaction between SARS-CoV-2 and ACE2 against COVID-19.

The frequent occurrence of viral infections poses a serious threat to human life. Identifying effective antiviral components is urgent. In China, pearls have been important traditional medicinal ingredients since ancient times, exhibiting various therapeutic properties, including detoxification properties. In this study, a peptide, KKCH, which acts against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), was derived from Pinctada fucata pearls. Molecular docking showed that it bound to the same pocket of the SARS-CoV-2 S protein and cell surface target angiotensin-converting enzyme II (ACE2). The function of KKCH was analyzed through surface plasmon resonance (SPR), Enzyme-Linked Immunosorbent Assays, immunofluorescence, and simulation methods using the SARS-CoV-2 pseudovirus and live virus. The results showed that KKCH had a good affinity for ACE2 (KD = 6.24 × 10-7 M) and could inhibit the binding of the S1 protein to ACE2 via competitive binding. As a natural peptide, KKCH inhibited the binding of the SARS-CoV-2 S1 protein to the surface of human BEAS-2B and HEK293T cells. Moreover, viral experiments confirmed the antiviral activity of KKCH against both the SARS-CoV-2 spike pseudovirus and SARS-CoV-2 live virus, with half-maximal inhibitory concentration (IC50) values of 398.1 μM and 462.4 μM, respectively. This study provides new insights and potential avenues for the prevention and treatment of SARS-CoV-2 infections.

Structural analysis of peptide identified from the 2KRR domain of the nucleolin protein with a c-Myc G4 structure using biophysical and biochemical methods.

For the first time, the c-Myc G4 structure is reported to be stabilized by binding of the peptide (derived from the 2KRR domain of the nucleolin protein called the Nu peptide) in the loop region of the G-quadruplex structure by stacking interactions. CD results showed the formation of parallel G4 structure in the presence of 100 mM Na+ or 100 mM K+ with the appearance of two isodichroic points at 229 nm, 254 nm and 252 nm in the presence of 100 mM Na+ or 100 mM K+, respectively. In addition, in UV thermal and CD melting studies, we observed drastic changes with an increase in the hyperchromicity at a DNA : peptide ratio of 1 : 50. On titrating the Nu peptide with c-Myc G4, we calculated the value of binding constant (K a) by plotting fluorescence intensity and DNA concentration as 0.1369 ± 0.008 μM and 0.1277 ± 0.073 μM in Na+ and K+, respectively, which confirms the strong association of Nu peptide with c-Myc G4. The Nu peptide showed preferential cytotoxicity against MDA-MB-231 cells with IC50 values of 5.020 μM and 5.501 μM after 72 and 96 hours. This approach suggests a novel strategy to target G4 structure using natural key peptide segments derived from G4 stabilizing protein.

Unlocking the Potential: PEGylation and Molecular Weight Reduction of Ionenes for Enhanced Antifungal Activity and Biocompatibility.

Numerous synthetic polymers, imitating natural antimicrobial peptides, have demonstrated potent antimicrobial activity, positioning them as potential candidates for new antimicrobial drugs. However, the high activity of these molecules often comes at the cost of elevated toxicity against eukaryotic organisms. In this study, a series of cationic ionenes with varying molecular weights to assess the influence of polymer chain length on ionene activity is investigated. To enhance polymer antimicrobial activity and limit toxicity a PEG side chain is introduced into the repeating unit. The resulting molecules consistently exhibited high activity against three model organisms: E. coli, S. aureus and C. albicans. The incorporation of side PEG chain improves antifungal properties and biocompatibility, regardless of molecular weight. The most important finding of this work is that the reduction of polymer molecular mass led to increased antifungal activity and reduced cytotoxicity against HMF and MRC-5 cell lines simultaneously. As a result, the best-performing molecules reported herein displayed minimal inhibitory concentrations (MIC) as low as 2 and 0.0625µgmL1 for C. albicans and C. tropicalis respectively, demonstrating exceptional selectivity. It is plausible that some of described herein molecules can serve as potential lead candidates for new antifungal drugs.

Novel antioxidant peptides from soybean protein by employing computational and experimental methods and their mechanisms of oxidative stress resistance

Antioxidant peptides have the potential to play a crucial role in mitigating and managing a variety range of diseases associated with oxidative stress. Soybean protein is an important source of antioxidant peptides. Here we employ computational and experimental methods to screen and identify five antioxidant peptides (Gly-Ala, Gly-Val-Pro-Tyr, Pro-His, Pro-Lys, and Ser-Gly-Pro) derived from soybean protein, and explore their potential targets and mechanisms of action. All five peptides demonstrated potent antioxidant activity, effectively scavenging superoxide radicals and protecting cells from H₂O₂-induced oxidative damage. These peptides exhibited multi-target effects on pathways implicated in oxidative stress, cardiovascular disease, and cancer. Notably, our results reveal that these peptides can stably interact with the key target nitric oxide synthase (NOS2) through Glu377, a critical residue of NOS2, thereby inhibiting its activity and potentially mitigating oxidative stress and inflammation. This study confirms the function of soybean peptide sequence that has not been previously disclosed, and provides a more specific theoretical basis for the development of natural antioxidant peptides.