A biomimetic peptide derived from human beta defensin-3 and preparation method and application thereof

Abstract

The application discloses a kind of biomimetic peptides derived from human beta defensin-3 and its preparation method and application.The amino acid sequence of the biomimetic peptide includes the sequence shown as SEQ ID NO.1.By the docking calculation of hBD-3 and EGFR / CD36 receptor, the key hydrogen bond binding residue and active domain are determined, and the receptor recognition function is retained by sequence connection and structure optimization.The biomimetic peptide designed in the application has biological activity, structure controllability and material compatibility, and can be widely applied to epithelial tissue repair, anti-fibrosis regulation and bio-medical material functionalization field.

Description

Technical Field

[0001] This invention belongs to the field of molecular docking and biomedical materials technology, and relates to a biomimetic peptide derived from human β-defensin-3, its preparation method and application, specifically a human β-defensin-3 (hBD-3) biomimetic functional peptide designed based on molecular docking and dynamic simulation, and its application in promoting epithelial tissue regeneration, regulating fibroblast phenotype and inhibiting tissue fibrosis. Background Technology

[0002] Tissue repair is a complex biological process involving multiple stages and cells, with epithelial regeneration and matrix remodeling being key steps determining tissue morphology and functional recovery. In a physiological repair environment, fibroblasts participate in matrix synthesis and remodeling through temporary activation; however, in chronic inflammation, infection, or excessive repair responses, fibroblasts often transform into persistently activated fibroblasts or inflammatory phenotypes, accompanied by excessive collagen deposition and extracellular matrix disorder, leading to tissue scarring and repair failure. This pathological repair process not only affects tissue structural integrity but also significantly reduces functional regeneration efficiency, becoming one of the core problems urgently needing to be solved in the fields of tissue engineering and regenerative medicine.

[0003] Human β-Defensin 3 (hBD-3) is a broad-spectrum antibacterial endogenous polypeptide with potential for immunomodulation and tissue repair. The hBD-3 molecule contains three pairs of complex disulfide bonds and a high density of cationic amino acid residues, which not only leads to high chemical synthesis costs and poor conformational stability, but also makes it prone to losing conformation-dependent bioactivity during immobilization in biomedical materials. Therefore, its translational applications in tissue engineering are significantly limited.

[0004] To overcome these limitations, researchers have recently begun to explore biomimetic modification strategies based on natural antimicrobial peptides. By screening active fragments, optimizing sequences, or simplifying structures, they aim to obtain short peptide molecules with similar biological functions but greater stability and modifiability. However, current modifications largely rely on empirical truncation, lacking systematic receptor binding site analysis and structural verification, resulting in unstable bioactivity or unclear mechanisms of action in the obtained peptides.

[0005] Therefore, there is an urgent need to provide a biomimetic peptide that combines bioactivity, structural controllability, and material compatibility. Summary of the Invention

[0006] To address the shortcomings of existing technologies and practical needs, this invention provides a biomimetic peptide derived from human β-defensin-3, its preparation method, and its applications. The biomimetic peptide designed in this invention possesses bioactivity, structural controllability, and material compatibility, and can be widely applied in the fields of epithelial tissue repair, anti-fibrosis regulation, and functionalization of biomedical materials.

[0007] To achieve this objective, the present invention adopts the following technical solution:

[0008] In a first aspect, the present invention provides a biomimetic peptide derived from human β-defensin-3, wherein the amino acid sequence of the biomimetic peptide includes the sequence shown in SEQ ID NO.1.

[0009] In this invention, by performing docking calculations between hBD-3 and the EGFR / CD36 receptor, key hydrogen-bonded residues and active domains are identified. Through sequence linking and structural optimization, the receptor recognition function is preserved. The biomimetic peptide designed in this invention combines bioactivity, structural controllability, and material compatibility, and can be widely applied in the fields of epithelial tissue repair, anti-fibrosis regulation, and functionalization of biomedical materials.

[0010] SEQ ID NO. 1: YYCRVRGGRSGGRKCSRRKK.

[0011] Preferably, the biomimetic peptide further includes a collagen-binding domain.

[0012] Preferably, the amino acid sequence of the collagen-binding domain includes the sequence shown in SEQ ID NO.2.

[0013] SEQ ID NO.2: TKKTLRT.

[0014] Preferably, the biomimetic peptide further includes a linker peptide, the amino acid sequence of which includes any one of the following:

[0015] (1) A flexible linker peptide composed of glycine and serine;

[0016] (2) The sequence is The linker peptide, where n is 1, 2, 3 or 4;

[0017] (3) The sequence is The linker peptide, where n is 1, 2, 3, 4 or 5;

[0018] (4) The sequence is The linker peptide, where n is 1, 2, 3, 4 or 5;

[0019] (5) A linker consisting of fragments, variants or combinations of any of the linker peptides in (1)-(4).

[0020] Preferably, the amino acid sequence of the linker peptide includes the sequence shown in SEQ ID NO.3.

[0021] SEQ ID NO.3: GGGGS.

[0022] Preferably, the amino acid sequence of the biomimetic peptide includes the sequence shown in SEQ ID NO.4.

[0023] SEQ ID NO.4: TKKTLRTGGGGSYYCRVRGGRSGGRKCSRRKK.

[0024] It is understood that the biomimetic peptides described in this invention include biomimetic peptides with arbitrary linked peptide sequences. Different linked peptide sequences result in different corresponding biomimetic peptide sequences, all of which are within the protection scope of this invention.

[0025] Secondly, the present invention provides a method for preparing the biomimetic peptide described in the first aspect, the method comprising the following steps:

[0026] (1) The key binding residue sequence of human β-defensin-3 and its receptor binding fragment was determined by screening through molecular docking and molecular dynamics simulation;

[0027] (2) Using short peptide linkers, the key binding residue sequences of human β-defensin-3 and the receptor binding fragment are spliced ​​together to obtain the splice;

[0028] (3) Synthesize the splice sequence and collagen binding domain sequence, purify, and obtain biomimetic peptides.

[0029] Preferably, the receptor-binding fragment in step (1) includes any one or a combination of at least two of EGFR, CD36, CCR6, TLR1 or TLR2.

[0030] Preferably, the short peptide linker in step (2) includes any one of GG, GS, GSGS or GGGG.

[0031] Preferably, the synthesis in step (3) includes solid-phase peptide synthesis; the purification includes high-performance liquid chromatography purification.

[0032] In this invention, the molecular structure of the biomimetic peptide is modeled using AlphaFold2 and scored using the molecular docking software ZDOCK to obtain the binding status of the biomimetic peptide with the receptor binding fragment.

[0033] Preferably, the preparation method further includes: verifying the receptor binding specificity and function of the obtained biomimetic peptide.

[0034] Preferably, the receptor binding specificity verification includes: using molecular docking scoring to detect the binding kinetic parameters of the biomimetic peptide and the receptor binding fragment.

[0035] Preferably, the functional verification includes: detecting the effect of the biomimetic peptide on cell activity and detecting inflammatory genes by qPCR amplification.

[0036] Thirdly, the present invention provides the application of the biomimetic peptide described in the first aspect in the preparation of pharmaceuticals.

[0037] Fourthly, the present invention provides the application of the biomimetic peptide described in the first aspect in the preparation of biomaterials, wherein the collagen matrix material is used for tissue repair or anti-fibrosis.

[0038] Fifthly, the present invention provides a method for preparing collagen matrix materials, the method comprising: immobilizing the biomimetic peptides described in the first aspect onto the surface of a decellularized matrix or collagen scaffold via collagen binding domains.

[0039] Compared with the prior art, the present invention has the following beneficial effects:

[0040] (1) Emphasis on both structural optimization and functional preservation: Key sequences with high binding activity and stable conformation were screened from hBD-3 through molecular docking and molecular dynamics simulation, while preserving its core immune regulatory region (adding biological activity).

[0041] (2) Good structural stability and material compatibility: The biomimetic peptide molecules of the present invention have a low aggregation tendency and strong surface hydrophilicity, and can be stably bound to the surface of natural matrix materials such as collagen, gelatin, and SIS to achieve long-term biological activity fixation, overcoming the defect that natural hBD-3 is difficult to fix in materials.

[0042] (3) Promote epithelial regeneration: The biomimetic peptides of this invention can significantly promote the migration and proliferation of epithelial tissues, enhance the epithelial barrier formation ability, and accelerate the tissue surface repair process;

[0043] (4) Regulate fibroblast phenotype and inhibit fibrosis: In in vitro experiments, the biomimetic peptide of the present invention can inhibit the phenotype transformation of fibroblasts (HFF-1) and reduce the expression of fibrosis-related proteins such as COL1, COL3, and SPARC.

[0044] (5) Low immunogenicity and excellent biocompatibility: Through sequence optimization and scoring prediction as well as animal experiments, the biomimetic peptide of the present invention has good biocompatibility and application safety.

[0045] (6) Broad application prospects: The biomimetic peptides of the present invention are not only applicable to vaginal tissue regeneration, but can also be extended to the field of epithelial damage repair in the skin, oral cavity, cornea and other areas, and have high transformation potential and industrialization value. Attached Figure Description

[0046] Figure 1 This is a 3D interaction diagram of the docking between the biomimetic peptide and the EGFR molecule;

[0047] Figure 2 This is a 3D interaction diagram of the docking between the biomimetic peptide and the CD36 molecule;

[0048] Figure 3 A 3D interaction diagram of the docking between CBD-biomimetic peptide (CBD-BP) and EGFR molecules;

[0049] Figure 4 A 3D interaction diagram of the docking between CBD-biomimetic peptide (CBD-BP) and CD36 molecules;

[0050] Figure 5 This is a graph showing the qPCR results of BP and CBD-BP in Example 5;

[0051] Figure 6 This is a graph showing cell migration activity.

[0052] Figure 7 The graph shows the qPCR results and cell viability statistics for BP and CBD-BP in Example 7.

[0053] Figure 8 The image shows the results of H&E staining. Black marks represent the control area, and red marks represent the damaged area.

[0054] Figure 9 The image shows the results of smearing with arbutin. Black marks indicate the control area, and red marks indicate the damaged area.

[0055] Figure 10 This is a graph showing the results of tissue immunofluorescence analysis. Detailed Implementation

[0056] To further illustrate the technical means and effects of this invention, the following description, in conjunction with embodiments and accompanying drawings, provides a further explanation of the invention. It is understood that the specific embodiments described herein are merely illustrative of the invention and not intended to limit it.

[0057] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0058] Example 1

[0059] Molecular docking of hBD-3 with EGFR and CD36.

[0060] 1. Structural Preparation

[0061] The structural files of defensin (protein database ID: 1KJ6), EGFR (protein database: 1YY9), and CD36 (protein database: 5LGD) were downloaded directly from the RCSB protein database. Software for visualizing and analyzing protein structures was used to preprocess the defensin, EGFR, and CD36 proteins, removing heteroatoms to obtain the structures of the proteins to be docked.

[0062] 2. Molecular docking

[0063] The protein-protein HDOCK Server program, based on hybridization docking, was used to perform molecular docking of defensins with EGFR and CD36, respectively. After docking, the complex conformation with the highest docking score in each docking result was selected for analysis.

[0064] 3. Experimental Results

[0065] The molecular docking results of hBD-3 and EGFR and hBD-3 and CD36 are shown in Table 1. According to the numerical analysis of the matching score and confidence score, both groups of molecular docking are likely to bind spontaneously (confidence score > 0.7).

[0066] Table 1

[0067]

[0068] Example 2

[0069] Biomimetic peptide BP (SEQ ID NO.1) molecular docking

[0070] To verify the receptor affinity of the designed biomimetic peptide, rigid body docking analysis was performed on the constructed biomimetic peptide BP and target receptors (EGFR, CD36, CCR6, TLR1, and TLR2) using the molecular docking ZDOCK software. The ZDOCK software output file contains a list of candidate binding conformations, where each record describes the three-dimensional spatial pose parameters of a candidate conformation and its corresponding comprehensive score (ZDOCK score). Specifically, each record includes three rotation parameters (describing the three-dimensional rotation of the ligand relative to the initial conformation), three translation parameters (describing the spatial translation of the ligand in the receptor coordinate system), and a comprehensive score. A higher comprehensive score indicates better geometric complementarity and interaction energy of the predicted binding conformation, i.e., a higher predicted affinity between the biomimetic peptide BP and the receptor. The ZDOCK score results are shown in Table 2. The purpose was to verify whether the designed biomimetic peptide BP could form a stable binding with the receptor, rather than to discover new sites; therefore, conformations with high comprehensive scores were considered as computational verification of the affinity of the biomimetic peptide BP and were corroborated by subsequent experimental verification results. The results are as follows Figure 1 and Figure 2 As shown, this invention's biomimetic peptide BP can stably bind to EGFR and CD36.

[0071] Table 2

[0072]

[0073] Example 3

[0074] Construction of the biomimetic peptide CBD-BP (SEQ ID NO.4) sequence.

[0075] 1. Sequence splicing

[0076] The amino acid sequence shown in SEQ ID NO.4 was constructed by splicing together SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3.

[0077] 2. Biomimetic peptide CBD-BP molecular docking

[0078] To verify the receptor affinity of the designed biomimetic peptide, rigid body docking analysis was performed on the constructed biomimetic peptide CBD-BP with target receptors (EGFR, CD36, CCR6, TLR1, and TLR2) using ZDOCK software. The ZDOCK output file contains a list of candidate binding conformations, where each record describes the three-dimensional spatial pose parameters of a candidate conformation and its corresponding comprehensive score (ZDOCK score). Specifically, each record includes three rotation parameters (describing the three-dimensional rotation of the ligand relative to the initial conformation), three translation parameters (describing the spatial translation of the ligand in the receptor coordinate system), and a comprehensive score. A higher comprehensive score indicates better geometric complementarity and interaction energy of the predicted binding conformation, i.e., a higher predicted affinity between the biomimetic peptide CBD-BP and the receptor. The ZDOCK score results are shown in Table 3. The objective was to verify whether the designed biomimetic peptide CBD-BP could form a stable binding with the receptor, rather than to discover new sites; therefore, the high-scoring conformation of ZDOCK was used as the computational validation basis for the affinity of the biomimetic peptide CBD-BP, and was corroborated by subsequent experimental results. The results are as follows: Figure 3 and Figure 4 As shown, this demonstrates that the biomimetic peptide CBD-BP of the present invention can stably bind to EGFR and CD36.

[0079] Table 3

[0080]

[0081] Example 4

[0082] Plasmon resonance detection of biomimetic peptides (CBD-BP and BP) binding to multiple receptors.

[0083] Ligand conjugation was performed according to the instructions provided with the amino-coupling kit. The chip channels were activated with 11.5 mg / mL N-hydroxysuccinimide (NHS) and 75 mg / mL 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) (1:1). Then, the diluted ligand (10 mM NaAc, pH 4.0) was injected into the activated channels at a flow rate of 10 μL / min for conjugation. Finally, 1 M ethanolamine was injected to seal the channels. The analytes were serially diluted with running buffer. At a flow rate of 30 μL / min, the serially diluted analytes were injected into the channels to bind to the conjugated ligands on the chip for 180 s. The dissociation time was also set to 180 s. Repeated injections and conjugation cycles were performed for different concentrations of analyte. Finally, the data were analyzed using a Biacore T200 molecular interaction analysis system.

[0084] The results, as shown in Table 4, indicate that the biomimetic peptides (CBD-BP and BP) do not function through a single high-affinity receptor, but rather exhibit moderate affinity binding to multiple immune and metabolic-related receptors, suggesting that they may participate in inflammatory responses and tissue microenvironment remodeling through multi-receptor synergistic regulation.

[0085] Table 4

[0086]

[0087] Example 5

[0088] Immunosuppressive activity.

[0089] To verify the immunomodulatory effects of the designed biomimetic peptides (BP and CBD-BP) on macrophages, this study used M0 type RAW 264.7 macrophages as an in vitro model. Cells were cultured to 50 weeks / well (six-well plate) using standard methods, stimulated with LPS and IFN-γ, and then incubated with the biomimetic peptides (10 μg / mL) for 24 hours. The control group consisted of untreated M0 type RAW264.7 cells. After treatment, the expression of classical inflammatory factors and phenotypic markers was detected by qRT-PCR. The results are as follows: Figure 5 As shown, compared with the model group, the expression of M1-related markers Tnf, Il6, and Cd86 was significantly reduced in the biomimetic peptide (BP and CBD-BP) treatment groups, while the expression of M2-related markers Arg, Il10, and Cd206 was increased. This indicates that the designed biomimetic peptides (BP and CBD-BP) can regulate the immunophenotype of macrophages, inhibit inflammatory responses, and affect phenotypic polarization. These results demonstrate that the biomimetic peptides (BP and CBD-BP) designed in this invention have immunomodulatory effects on M0-type macrophages in an in vitro model, providing experimental evidence for their application in tissue repair and anti-inflammation.

[0090] Example 6

[0091] Cell migration activity.

[0092] To verify the effect of the designed biomimetic peptides (BP and CBD-BP) on fibroblast migration, HFF-1 fibroblast cells were used as in vitro experimental cells in this embodiment. Cells were cultured to 50 w / well (6-well plate) using conventional methods, and then the biomimetic peptides (BP and CBD-BP) (concentration 10 μg / mL) were added and incubated for 24 hours. The experimental control group consisted of cells without the addition of biomimetic peptides. Cell migration was observed at 0, 6, 12, and 24 hours, and the healing rate was calculated. The results are as follows: Figure 6 As shown, (BP and CBD-BP) can significantly promote cell migration, and the healing rate is significantly different from that of the control group.

[0093] Example 7

[0094] Applications in tissue regeneration.

[0095] To evaluate the bioactivity of the designed biomimetic peptides (BP and CBD-BP) in normal fibroblasts, this example used cultured human normal fibroblasts in vitro. Cells were cultured to 50 w / well (six-well plate) using standard methods, and then 10 μg / mL of the biomimetic peptides (BP and CBD-BP) were added and incubated for 24 hours. The control group consisted of untreated cells. After treatment, the expression of extracellular matrix-related and vascular-related genes was detected by qPCR. Results are as follows: Figure 7 As shown, compared with the control group, the expression levels of COL1, COL3, VEGFA, and SPARC were all upregulated in the biomimetic peptide (BP and CBD-BP) treatment groups, suggesting their potential activity in regulating extracellular matrix synthesis and angiogenesis-related functions. Simultaneously, cell proliferation assays (CCK-8) showed that the biomimetic peptides (BP and CBD-BP) promoted the proliferation of normal fibroblasts, exhibiting activity similar to that of hBD-3.

[0096] In summary, this embodiment demonstrates that the designed biomimetic peptides (BP and CBD-BP) can not only upregulate ECM and vascular-related genes in normal fibroblasts, but also promote cell proliferation, which may have a promoting effect on angiogenesis and extracellular matrix remodeling, providing experimental evidence for their application in tissue repair and regeneration.

[0097] Example 8

[0098] Application of hBD-3 in animal models.

[0099] Epithelial cell regeneration was assessed using H&E staining. By day 7, untreated tissue showed localized irregular epithelial coverage, with loose columnar epithelium in the basal layer, presumably immature epithelium, and irregular cell arrangement, failing to restore the typical squamous epithelial layering characteristics, indicating delayed or impaired wound healing. In contrast, the group with added biomimetic peptide CBD-BP showed dense columnar epithelial coverage in the basal layer and exhibited papillary regeneration, indicating that scaffold implantation significantly accelerated early epithelial repair. Figure 8 ).

[0100] The results of the masson staining are as follows: Figure 9 As shown, the collagen fibers in the group treated with the biomimetic peptide CBD-BP showed no abnormal deposition and were structurally very similar to normal vaginal tissue. In contrast, the untreated group exhibited excessive collagen accumulation and disordered arrangement, with loosely distributed and irregularly oriented fibers.

[0101] Example 9

[0102] Tissue immunofluorescence analysis.

[0103] To further analyze the regeneration of muscle and blood vessels after tissue regeneration, immunofluorescence staining was used to stain smooth muscle cells (α-SMA labeled), endothelial cells (CD31 labeled), and cell nuclei (DAPI) in the tissue, followed by confocal imaging. The results are as follows: Figure 10 As shown, the biomimetic peptide CBD-BP group exhibited significant muscle and blood vessel regeneration, further demonstrating that the biomimetic peptide CBD-BP possesses bioactivity consistent with hBD-3.

[0104] In summary, the biomimetic peptides (BP and CBD-BP) designed in this invention possess bioactivity, structural controllability, and material compatibility, and can be widely applied in the fields of epithelial tissue repair, anti-fibrosis regulation, and functionalization of biomedical materials.

[0105] The applicant declares that the detailed method of the present invention is illustrated by the above embodiments, but the present invention is not limited to the above detailed method, that is, it does not mean that the present invention must rely on the above detailed method to be implemented. Those skilled in the art should understand that any improvements to the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

Claims

1. A biomimetic peptide derived from human β-defensin-3, characterized in that, The amino acid sequence of the biomimetic peptide is the sequence shown in SEQ ID NO.

1.

2. A biomimetic peptide derived from human β-defensin-3, characterized in that, The amino acid sequence of the biomimetic peptide is the sequence shown in SEQ ID NO.

4.

3. The use of the biomimetic peptide according to claim 1 or 2 in the preparation of a medicament for skin healing.

4. The use of the biomimetic peptide according to claim 1 or 2 in the preparation of biomaterials for skin healing.

5. A method for preparing collagen matrix materials, characterized in that, The method includes: immobilizing the biomimetic peptide of claim 1 or 2 onto the surface of a decellularized matrix or collagen scaffold via a collagen binding domain.

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