Carnosine targeting IIB type activin receptor as well as preparation method and application of carnosine

Abstract

The invention discloses a carnosine targeting a IIB type activin receptor and a preparation method and application of the carnosine, and the amino acid sequence of the carnosine targeting the IIB type activin receptor is as shown in SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3. The value of the equilibrium dissociation constant KD of the carnosine and the IIB type activin receptor ranges from 0.514 [mu] M to 1.91 [mu] M. The method disclosed by the invention adopts a multi-technology integration strategy of in vitro digestion simulation, mass spectrum identification, molecular docking, affinity verification and cell function experiment, and has the advantages of high efficiency and strong targeting property. The invention also aims to provide application of the carnosine in preparation of functional food or medicines for preventing and / or treating sarcopenia and promoting muscle growth.

Description

Technical Field

[0001] This invention relates to the field of food biotechnology, and in particular to a muscle-building peptide targeting type IIB activin receptor (ActRIIB) obtained from chicken breast, its preparation method, and its application in functional foods and products for the intervention of sarcopenia. Background Technology

[0002] Sarcopenia, an age-related syndrome characterized by progressive decline in skeletal muscle mass, strength, and function, has become a major global public health challenge. This condition significantly reduces the quality of life for older adults and increases the risk of falls, disability, and death. Current intervention strategies primarily include exercise training, nutritional support (such as protein and amino acid supplementation), and pharmacological treatment. Among these, agents targeting the myostatin signaling pathway have attracted considerable attention due to their proven muscle-promoting effects. Myostatin, by binding to ActRIIB, activates the downstream Smad2 / 3 signaling pathway, thereby inhibiting the proliferation and differentiation of myoblasts. Therefore, blocking the binding of myostatin to ActRIIB is one of the key strategies for promoting muscle growth.

[0003] Currently, the main agents targeting ActRIIB are monoclonal antibodies, such as bimagrumab. While these antibodies exhibit high affinity and well-defined activity, they suffer from limitations such as large molecular weight, high production costs, and poor oral absorption, restricting their widespread use in everyday functional foods. In contrast, bioactive peptides derived from food proteins offer advantages such as good oral absorption, high safety, and relatively low cost, and are considered potential candidates to replace antibody drugs.

[0004] Chicken breast is an excellent source of protein, but current research on its bioactive peptides mainly focuses on antioxidant and blood pressure-lowering (ACE inhibition) effects. There are no reports of screening for peptides from chicken breast that target ActRIIB and have muscle-building activity. Furthermore, existing methods for screening bioactive peptides from food sources are generally limited and lack a systematic strategy that combines in vitro simulated digestion, efficient identification, computer-aided virtual screening, and in vitro functional validation, resulting in low screening efficiency and weak targeting.

[0005] Therefore, developing a method for efficiently screening ActRIIB muscle-building peptides from chicken breast and clarifying their sequence, mechanism of action, and applications is of great significance for developing novel functional foods or intervention products for sarcopenia. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention aims to provide a series of muscle-building peptides with high affinity and bioactivity targeting ActRIIB. Another objective of this invention is to provide a method for preparing the aforementioned muscle-building peptides, employing a multi-technology integrated strategy of "in vitro digestion simulation - mass spectrometry identification - molecular docking - affinity verification - cell function experiments," which has the advantages of high efficiency and strong targeting. A further objective of this invention is to provide the application of the aforementioned muscle-building peptides in the preparation of functional foods or drugs for the prevention and / or treatment of sarcopenia and the promotion of muscle growth.

[0007] To achieve the above objectives, the present invention adopts the following technical solution: I. This invention provides a muscle-building peptide targeting type IIB activin receptor (ActRIIB), the amino acid sequence of which is shown in SEQ ID NO:1 (KEKLHVYKHIEK), SEQ ID NO:2 (EIKKEEKKEER), or SEQ ID NO:3 (DLENDKQQLDEK), and the biological source is Gallus gallus L.

[0008] Preferably, the equilibrium dissociation constant (KD) of the myotrophic peptide and the ActRIIB receptor is between 0.514 μM and 1.91 μM.

[0009] II. This invention provides a method for preparing the above-mentioned muscle-building peptides, comprising the following steps: (1) Chicken breast pretreatment and in vitro simulated digestion: Fresh chicken breast was taken, washed, cooked and minced, and then statically digested in vitro using the INFOGEST model. The supernatant of digestion products was collected after 120 minutes (I-120) of intestinal digestion.

[0010] (2) Peptide identification and peptide library establishment: Peptides in digestion products were identified by liquid chromatography-mass spectrometry and a peptide library was established. Further optimization was carried out. Peptides in I-120 digest were identified by NanoLC-Orbitrap MS / MS. A peptide library containing multiple peptides was established by searching chicken breast protein database.

[0011] (3) Molecular docking virtual screening: Using the crystal structure of the ActRIIB receptor as the target, candidate peptides with binding energies below a set threshold are virtually screened from the peptide library using molecular docking technology; more preferably, using the crystal structure of ActRIIB (PDBID: 5NGV) as the target, molecular docking software (such as Discovery Studio) is used to dock peptides in the peptide library to the active site of the receptor, and candidate peptides are screened based on the binding energy and key interactions (such as hydrogen bonding with residues such as Glu94 and Pro117). The grid center coordinates of the molecular docking technology are x = -4.88774, y = 10.2925, z = 15.3631, and the grid radius is 20.6 Å; the set threshold for the binding energy is -195 kcal / mol.

[0012] (4) Affinity and activity verification: The candidate peptides screened in step (3) were synthesized and their actual affinity (KD value) with ActRIIB was determined by surface plasmon resonance (SPR) technology. Cell proliferation experiments and Western blot detection were performed using a mouse myoblast (C2C12) model to verify their proliferative capacity and inhibitory effect on the myostatin Smad2 / 3 signaling pathway.

[0013] III. The present invention provides a composition comprising the above-mentioned muscle-building peptides and a pharmaceutically or food-grade acceptable carrier. This composition may be a pharmaceutical composition or a functional food, for example, in the form of a nutrition bar, protein powder, tablet, capsule, or beverage.

[0014] IV. The present invention provides the use of the above-mentioned muscle-building peptide or a composition containing the above in the preparation of products for the prevention and / or treatment of sarcopenia.

[0015] V. This invention provides the use of the above-mentioned myotrophic peptide or compositions comprising it in the preparation of products for promoting muscle growth, inhibiting muscle atrophy, or improving muscle function. The products exert their effects by inhibiting phosphorylation of the Smad2 / 3 signaling pathway and / or upregulating the expression of the myotroph differentiation factor MyoD.

[0016] Compared with the prior art, the advantages of the present invention are as follows: (1) Targeted and highly active: The three myoblast-building peptides provided by this invention can directly and with high affinity bind to ActRIIB (KD=0.514-1.91μM), thereby effectively blocking the myosin signaling pathway and promoting myoblast proliferation.

[0017] (2) Safe source and easy to absorb: The peptides are derived from chicken breast meat, which is consumed daily, and are obtained by simulating the human digestive process, indicating that they have good biocompatibility and oral absorption potential, avoiding the toxic side effects that may be caused by chemical synthesis or antibody drugs. (3) The screening method is highly efficient and systematic: The "multi-technology integration" screening strategy established in this invention organically combines in vitro digestion, high-throughput identification, computer-aided design and high-throughput verification, which greatly improves the efficiency and success rate of discovering highly active target peptides from complex food matrices.

[0018] (4) Broad application prospects: The muscle-building peptides provided by this invention can be used as key functional ingredients and widely used in the development of nutritional supplements and health foods for middle-aged and elderly people with muscle loss, athletes, etc., which have important market value and social benefits. Attached Figure Description

[0019] Figure 1 This is the overall technical roadmap for the screening and validation process of muscle-building peptides in this invention.

[0020] Figure 2 This figure shows the degree of hydrolysis, peptide concentration, and effects on C2C12 cell viability of digestion products at different time points during simulated in vitro digestion of chicken breast.

[0021] Figure 3 This is a distribution map of the major proteins from which the peptides identified in the digestion products of chicken breast I-120 originate.

[0022] Figure 4 This is a schematic diagram of the complex structure formed by the receptor and the top 10 peptides with the highest binding energy to ActRIIB obtained by molecular docking screening.

[0023] Figure 5 These are surface plasmon resonance (SPR) sensing images and affinity fitting curves of three synthetic peptides (SEQ ID NO: 1, 2, 3) with the ActRIIB receptor.

[0024] Figure 6 The graph shows the effects of three muscle-building peptides at different concentrations on the proliferation rate of C2C12 cells, as well as their regulatory effects on Smad2 / 3 phosphorylation levels and MyoD protein expression levels.

[0025] Figure 7 This is a graph showing the changes in RMSD, Rg, SASA, RMSF, and the number of hydrogen bonds in the complexes of three muscle-building peptides with the ActRIIB receptor during a 100 ns molecular dynamics simulation.

[0026] Figure 8 This is a secondary mass spectrometry identification diagram of three muscle-building peptides (SEQ ID NO:1, 2, 3). Detailed Implementation

[0027] The present invention will be further described in detail below with reference to specific embodiments and accompanying drawings. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions in the art or as recommended by the manufacturer.

[0028] Example 1: In vitro simulated digestion of chicken breast and determination of optimal active digestion products 1.1 Chicken breast pretreatment: Purchase fresh chicken breast from the local market, rinse it with distilled water, boil it for 20 minutes, and then mince it with a meat grinder until the particle size is no larger than 3mm.

[0029] 1.2 In vitro simulated digestion: The in vitro digestion model was performed according to the INFOGEST standard static in vitro digestion model established by Brodkorb et al. (2019). The basic procedure is as follows: Figure 1 As shown.

[0030] (1) Oral stage: Add 3.76 mL of simulated saliva (SSF), 0.04 mL of 0.3M calcium chloride solution, 1 mL of α-amylase solution (75 U / mL) and 0.2 mL of water to 5g of minced chicken breast. Mix well and incubate in a 37℃ incubator for 2 minutes.

[0031] (2) Stomach stage: Take all samples after oral digestion, add 7.2 mL of simulated gastric juice (SGF), 0.04 mL of 0.3M calcium chloride solution, 1.92 mL of pepsin solution (2000 U / mL), 0.44 mL of water and 0.4 mL of 1 M hydrochloric acid solution, mix well and incubate at 37°C for 120 minutes with shaking.

[0032] (3) Intestinal stage: Take the sample after gastric digestion and adjust the pH to 7.0 with NaOH solution. Then add 12.8 mL of simulated intestinal fluid (SIF), 0.04 mL of 0.3 M calcium chloride solution, 5 mL of trypsin solution (100 U / mL), and add water to make the total system volume 40 mL. Incubate the mixture at 37°C with continuous shaking for 120 minutes. Immediately after incubation, add 1 M formic acid to terminate the reaction. Centrifuge the resulting mixture at 8000×g for 10 minutes (4°C) and collect the supernatant, which is the digestion product of intestinal digestion for 120 minutes (I-120).

[0033] 1.3 Index Measurement and Result Analysis: The degree of hydrolysis, peptide concentration, molecular weight distribution, and free amino acid composition of the products at different digestion stages (e.g., gastric stage 0, 60, 120 minutes and intestinal stage 0, 60, 120 minutes) were measured, and their effects on the viability of C2C12 myoblasts were evaluated.

[0034] The results are as follows Figure 2 As shown, the degree of hydrolysis and peptide concentration of chicken breast digestion products dynamically changed over time during in vitro simulated digestion. Considering all indicators, the product from 120 minutes of intestinal digestion (I-120) exhibited the highest degree of hydrolysis (65.99% ± 4.00%) and peak peptide concentration (328.06 ± 12.70 μg / mL). Furthermore, this product most significantly enhanced the activity of C2C12 myoblasts (128.15% ± 9.90%). Therefore, I-120 digest was identified as the optimal raw material for subsequent screening of muscle-building peptides.

[0035] like Figure 3 As shown, mass spectrometry identification and source analysis of I-120 digests revealed that the identified peptides mainly originated from myofibrillar proteins, such as myosin heavy chain, actin, troponin, etc., as well as enzymes related to energy metabolism.

[0036] Example 2: Virtual screening and affinity verification of ActRIIB-targeted muscle-building peptides 2.1 Peptide Identification and Peptide Library Construction: I-120 digest was desalted via 3 kDa ultrafiltration and peptide identification was performed using NanoLC-Orbitrap MS / MS. Chromatographic separation was performed using an Acclaim PepMap RSLC C18 column (75 μm × 25 cm). Mobile phase A was 0.1% formic acid aqueous solution, and mobile phase B was 0.1% formic acid acetonitrile solution, with gradient elution (0-30 min, 5%-35% B). Mass spectrometry analysis was performed in positive ion mode, with a Full MS resolution of 70,000 and an MS / MS resolution of 17,500, with a scan range of m / z 350-1800. A chicken breast protein sequence database was searched using PEAKS Studio 8.5 software, identifying 12,635 peptides, which were used to construct a peptide library for virtual screening.

[0037] 2.2 Molecular docking virtual screening: The crystal structure of ActRIIB (PDB ID: 5NGV) was obtained from the Protein Database (PDB), and water molecules and original ligands were removed from the structure using Discovery Studio 2019 software. After minimizing the energy of the three-dimensional structures of peptides in the peptide library using the CHARMM force field, the CDOCKER module was used to dock them to the active site of ActRIIB (grid center coordinates defined: x = -4.88774, y = 10.2925, z = 15.3631; grid radius: 20.6 Å). Based on the docking binding energy (<-195 kcal / mol) and interactions such as hydrogen bonding with key amino acid residues (e.g., Glu94, Pro117), the top 10 candidate peptides were screened.

[0038] like Figure 4 As shown, a three-dimensional structural diagram of the complex formed by the ActRIIB receptor and the top 10 peptides with the highest molecular docking binding energy is presented, which intuitively reflects the binding mode of peptides and receptors.

[0039] 2.3 SPR Affinity Verification: The aforementioned 10 candidate peptides were synthesized by Shanghai ChuTai Biotechnology Co., Ltd. Using a Biacore 3000 instrument and CM5 sensor chip, ActRIIB protein was immobilized on the chip surface via standard amine coupling, with an immobilization level of approximately 8000 response units (RU). Candidate peptide solutions of different concentrations (0.2, 0.39, 0.78, 1.56, 3.125, 6.25 μM) were injected at a flow rate of 30 μL / min, with a binding time of 240 seconds and a dissociation time of 120 seconds. The running buffer was a PBS solution containing 0.01% P2O and 1% DMSO. After dual-reference calibration, the sensor maps were fitted to a 1:1 Langmuir binding model using Biacore evaluation software, and the binding rate constant (kon), dissociation rate constant (koff), and equilibrium dissociation constant (KD) were calculated.

[0040] The results are as follows Figure 5 As shown: SPR sensing plots and fitting curves clearly show that the three peptides have high affinity for ActRIIB: KEKLHVYKHIEK (SEQ ID NO:1, KD = 0.514 μM), EIKKEEKKEER (SEQ ID NO:2, KD = 0.813 μM), and DLENDKQQLDEK (SEQ ID NO:3, KD = 1.91 μM).

[0041] like Figure 8As shown, the three target peptides synthesized by chemical synthesis were confirmed by secondary mass spectrometry (MS / MS). The spectra clearly showed their fragment ion information, confirming that the sequence of the synthesized peptide was consistent with the identification results.

[0042] Example 3: Verification of the cellular activity of myotrophic peptides 3.1 Cell proliferation assay: C2C12 mouse myoblasts were cultured in high-glucose DMEM medium containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin, and incubated at 37°C in a 5% CO2 incubator. When the cells reached 70%-80% confluence, the medium was replaced with medium containing different concentrations (31.25, 62.5, 125, 250, 500 μM) of synthetic peptides for 24 hours. A control group without peptides and a positive control group with IGF-1 were also included. After treatment, 10 μL of CCK-8 reagent was added to each well, and the cells were incubated at 37°C for 1 hour. The absorbance at 450 nm was measured using a microplate reader, and the relative cell proliferation rate was calculated.

[0043] 3.2 Western blot analysis: C2C12 cells were cultured in growth medium until near confluence, then switched to differentiation medium (high glucose DMEM containing 2% horse serum) to induce differentiation. The medium was changed daily for 7 days to form mature myotubes. Differentiated myotubes were treated with the optimal concentration of peptides for 48 hours, and cells were collected and total protein was extracted using RIPA lysis buffer. Equal volumes of protein samples were separated by SDS-PAGE electrophoresis and transferred to PVDF membranes. After blocking with 5% skim milk, the membranes were incubated overnight at 4°C with specific primary antibodies (including p-Smad2 / 3, t-Smad2 / 3, MyoD, and β-actin antibodies). After washing, the membranes were incubated with HRP-labeled secondary antibody at room temperature for 1 hour. Finally, ECL chemiluminescence substrate was used for color development, and the protein bands were analyzed by grayscale using ImageJ software.

[0044] The results are as follows Figure 6 As shown: Figure 6 The study showed the effects of three myotrophic peptides on the proliferation rate of C2C12 cells. All three peptides promoted cell proliferation in a concentration-dependent manner, with SEQ ID NO:1 (KK-12) showing the most significant effect at 500 μM, achieving a cell viability of 143.34% ± 6.92%. Figure 6 Western blot results and statistical analysis of df showed that, compared with the control group, treatment with all three peptides significantly reduced the p-Smad2 / 3 / t-Smad2 / 3 ratio and upregulated the protein expression level of the key myoblast factor MyoD. This confirms that these peptides promote myocyte differentiation by inhibiting the myosin-Smad2 / 3 signaling pathway.

[0045] Example 4: Molecular dynamics simulation analysis of peptide-ActRIIB complex To investigate the dynamic stability of the binding of peptides to ActRIIB, molecular dynamics simulations of the complexes formed by three peptides (SEQ ID NO: 1, 2, 3) and ActRIIB were performed for 100 ns using GROMACS 2020.6 software. The initial structure was derived from the optimal conformation obtained through molecular docking. The system used an Amber99sb-ildn force field, placed within a cubic periodic box of the TIP3P water model, and sodium ions were added to neutralize the system charge. Energy minimization was performed first to eliminate unfavorable spatial conflicts, followed by equilibriuming of the NVT ensemble (300 K, 100 ps) and the NPT ensemble (1 bar, 200 ps). Finally, a 100 ns production simulation was performed with a time step of 2 fs, and the trajectory was saved every 10 ps. The root mean square deviation (RMSD), radius of gyration (Rg), solvent accessible surface area (SASA), root mean square fluctuation (RMSF), and number of intermolecular hydrogen bonds were analyzed using GROMACS built-in tools.

[0046] The results are as follows Figure 7 As shown: (1) Figure 7 a (RMSD): The RMSD values ​​of the three peptide-receptor complexes tend to stabilize in the later stages of the simulation, indicating that the complexes have reached an equilibrium state with minimal conformational fluctuations.

[0047] (2) Figure 7 b (Rg): The radius of gyration (Rg) of the complex remained stable throughout the simulation, indicating that the overall structure of the complex was compact and did not undergo significant loosening or denaturation.

[0048] (3) Figure 7 c (SASA): After peptide binding, the solvent-accessible surface area (SASA) of the receptor-ligand complex is reduced compared to the unbound receptor, indicating that the binding interface is well buried. The SASA reduction is most significant for the complex corresponding to SEQ ID NO:1.

[0049] (4) Figure 7 d (RMSF): The RMSF of the ActRIIB receptor is low in the region of residues 0-120, which contains key binding residues predicted by molecular docking, indicating that these regions become more stable due to peptide binding.

[0050] (5) Figure 7 e (hydrogen bonds): This shows the change in the number of hydrogen bonds formed between the peptide and ActRIIB over time during the simulation, indicating that hydrogen bond interactions are an important force in stabilizing the complex.

[0051] In summary, molecular dynamics simulations have confirmed from a dynamic perspective that these three myotrophic peptides can form stable complexes with the ActRIIB receptor. This, along with the experimental results of molecular docking and SPR, further supports their feasibility as targeted inhibitors.

Claims

1. A myotrophic peptide targeting type IIB activin receptor, characterized in that, The amino acid sequence of the muscle-building peptide is shown in SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:

3.

2. The myotrophic peptide targeting type IIB activin receptor according to claim 1, characterized in that, The equilibrium dissociation constant KD value of the myotrophic peptide and the type IIB activin receptor ranges from 0.514 μM to 1.91 μM.

3. A method for preparing the muscle-building peptide as described in claim 1 or 2, characterized in that, Includes the following steps: (1) Obtain the digestion products of chicken breast raw material after in vitro simulated gastrointestinal digestion; (2) Peptides in the digestion products were identified by liquid chromatography-mass spectrometry and a peptide library was established; (3) Using the crystal structure of type IIB activin receptor as the target, candidate peptides with binding energies lower than a set threshold are virtually screened from the peptide library by molecular docking technology. (4) Based on the candidate peptides, the target peptides are synthesized, and their affinity for type IIB activin receptors is verified by surface plasmon resonance technology. Peptides with muscle-building activity are screened out, which are muscle-building peptides targeting type IIB activin receptors.

4. The preparation method according to claim 3, characterized in that, In step (1), the in vitro simulated gastrointestinal digestion was performed using the INFOGEST static digestion model.

5. The preparation method according to claim 3, characterized in that, In step (3), the crystal structure of the type IIB activin receptor is PDB ID: 5NGV; The grid center coordinates for the molecular docking technique are x = -4.88774, y = 10.2925, z = 15.3631, and the grid radius is 20.6 Å. The set threshold for the binding energy is -195 kcal / mol.

6. A composition, characterized in that, It contains the muscle-building peptide as described in claim 1 or 2.

7. The composition according to claim 6, characterized in that, The composition is a pharmaceutical composition or a functional food. The functional food is available in the form of nutrition bars, protein powder, tablets, or capsules.

8. The use of the muscle-building peptide according to claim 1 or 2, or the composition according to claim 6 or 7, in the preparation of products for the prevention and / or treatment of sarcopenia.

9. The use of the myotrophic peptide according to claim 1 or 2, or the composition according to claim 6 or 7, in the preparation of products for promoting myoblast proliferation, inhibiting muscle atrophy, or improving muscle function.

10. The application according to claim 8 or 9, characterized in that, The product works by inhibiting phosphorylation of the Smad2 / 3 signaling pathway and / or upregulating the expression of myoblast differentiation factor MyoD.

Images