Antibacterial peptide GT-2 and application thereof

Abstract

The application belongs to the technical field of biological medicine, and particularly relates to an antibacterial peptide GT-2 and application thereof. The amino acid sequence of the antibacterial peptide GT-2 is shown as SEQ ID NO:2. The antibacterial peptide GT-2 provided by the application is obtained by modifying a natural antibacterial peptide GP-1 extracted from Gastrodia elata Blume. The antibacterial peptide GT-2 has the effects of broad-spectrum bacteriostasis, high antibacterial efficiency, low toxicity and high safety. The application provides an effective way for providing a bacteriostatic biological peptide with practicability, high efficiency and safety.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to an antimicrobial peptide GT-2 and its applications. Background Technology

[0002] Natural antimicrobial peptides (AMPs) are an important component of the immune system, widely found in plants, animals, and microorganisms, and possess the ability to kill bacteria, fungi, and some viruses. Compared to traditional antibiotics, antimicrobial peptides primarily exert their effects by disrupting the cell membrane structure of pathogenic microorganisms or regulating the host's immune response. They are less likely to induce drug resistance and exhibit rapid onset of action and diverse target sites. Therefore, antimicrobial peptides are considered one of the most promising alternatives to antibiotics.

[0003] However, while natural antimicrobial peptides (AMPs) possess broad-spectrum antibacterial potential, their practical application is still subject to numerous limitations. First, the antimicrobial spectrum of natural antimicrobial peptides is relatively limited, with insufficient inhibitory ability against drug-resistant pathogens, making it difficult to meet the needs of clinical broad-spectrum antimicrobial drugs. Second, some natural antimicrobial peptides exhibit an unbalanced hydrophilicity-hydrophobicity ratio, resulting in insufficient penetration through bacterial cell membranes, thus affecting antimicrobial efficiency. Furthermore, natural antimicrobial peptides are readily degraded by proteases in vivo, resulting in short half-lives that limit their sustained antimicrobial activity. Some natural antimicrobial peptides also exhibit certain host cell toxicity, such as damaging erythrocytes or hepatocytes, thereby reducing their safety. Finally, the extraction and synthesis processes of natural antimicrobial peptides are typically complex and costly, hindering their large-scale application. These factors collectively constitute significant bottlenecks in the drug development and clinical translation of natural antimicrobial peptides.

[0004] Currently, the optimization of antimicrobial peptide drugs mainly focuses on improving stability, reducing toxicity, expanding the antimicrobial spectrum, and enhancing targeting, especially through chemical modification, nanocarrier delivery, and genetic engineering techniques to improve the performance of existing antimicrobial peptides. However, research on discovering new antimicrobial peptides and optimizing them is relatively limited; more work is concentrated on improving and promoting the application of existing antimicrobial peptides. Therefore, how to provide a naturally sourced antimicrobial peptide with broad-spectrum antibacterial activity, high antimicrobial efficiency, low toxicity, and high safety has become an urgent problem to be solved in this field. Summary of the Invention

[0005] To address the lack of an antimicrobial peptide with few application limitations and good antibacterial effect in the existing technology, this invention provides an antimicrobial peptide GT-2 and its applications, specifically including the following technical solutions:

[0006] The present invention provides an antimicrobial peptide GT-2, the amino acid sequence of which is shown in SEQ ID NO:2.

[0007] The present invention also provides the application of the antimicrobial peptide GT-2 as described above in the preparation of antimicrobial products.

[0008] Preferably, the antimicrobial product includes antibacterial infection products and antifungal infection products.

[0009] Preferably, the bacteria include Gram-negative bacteria and / or Gram-positive bacteria.

[0010] Preferably, the bacteria include one or more of Staphylococcus aureus, methicillin-resistant Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa.

[0011] Preferably, the fungus includes Candida albicans.

[0012] Preferably, the product includes a reagent; the reagent includes a pharmaceutical.

[0013] The present invention also provides an antibacterial product, wherein the active ingredient of the antibacterial product includes the antimicrobial peptide GT-2 as described above.

[0014] Preferably, the effective concentration of antimicrobial peptide GT-2 in the antimicrobial product is ≥4 μg / ml.

[0015] Preferably, the effective concentration of the antimicrobial peptide GT-2 in the antimicrobial product is 4~32μg / ml.

[0016] The beneficial effects of this invention are as follows:

[0017] This invention provides an antimicrobial peptide GT-2, the amino acid sequence of which is shown in SEQ ID NO:2. The antimicrobial peptide GT-2 of this invention is extracted from and modified from the traditional medicinal plant Gastrodia elata. This invention extracts the antimicrobial peptide GP-1 from the Gastrodia elata genome and obtains the antimicrobial peptide GT-2, which possesses both low toxicity and high activity, through sequence optimization of GP-1. This invention breaks through the traditional approach to antimicrobial drug development, utilizing the unique plant resource of Gastrodia elata to explore its potential antimicrobial active components, ultimately obtaining a novel antimicrobial peptide GT-2 with stronger antimicrobial activity and stability. This invention provides a new direction for further research on antimicrobial peptides and an effective approach for their application. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0019] Figure 1 A schematic diagram showing the comparative results of sequence analysis of GP-1 and GT-2 antimicrobial peptides;

[0020] The sequence at the bottom of the image is the amino acid sequence of GP-1, and the sequence at the top of the image is the sequence of GT-2.

[0021] Figure 2 The structural formula of the antimicrobial peptide GP-1;

[0022] Figure 3 The structural formula of the antimicrobial peptide GT-2;

[0023] Figure 4 The secondary structure diagram of GP-1 predicted by AlphaFold3;

[0024] Figure 5 The diagram shows the secondary structure of GT-2 as predicted by AlphaFold3.

[0025] Figure 6 The liquid chromatogram of the antimicrobial peptide GP-1 is shown.

[0026] Figure 7 The liquid chromatogram of antimicrobial peptide GT-2;

[0027] Figure 8 This is the mass spectrum of the antimicrobial peptide GP-1;

[0028] Figure 9 This is the mass spectrum of the antimicrobial peptide GT-2;

[0029] Figure 10 Schematic diagram of the hemolytic effect of rabbit erythrocytes under different treatments;

[0030] Figure 11 The survival rate of HaCAT cells under different concentrations of GT-2 treatment. Detailed Implementation

[0031] The present invention provides an antimicrobial peptide GT-2, the amino acid sequence of which is shown in SEQ ID NO:2.

[0032] SEQ ID NO:2: KVWKILRKIIRIKNK.

[0033] In one embodiment, the antimicrobial peptide GT-2 of the present invention is obtained through rational molecular design based on the natural plant-derived Gastrodia elata antimicrobial peptide GP-1. In one embodiment, the amino acid sequence of the Gastrodia elata antimicrobial peptide GP-1 is shown in SEQ ID NO:1. In one embodiment, the rational molecular design includes: replacing negatively charged or excessively flexible residues with cationic amino acids to form a stable cationic distribution surface, while introducing an appropriate amount of hydrophobic amino acids on the other side of the phase misalignment, thereby constructing a sequence with a regular amphiphilic helical structure; and reducing hemolytic activity, aggregation risk, and chemical instability by removing terminal aromatic residues, cysteine, and long-chain glycine repeating motifs. In one embodiment, the cationic amino acid includes lysine or arginine. In one embodiment, the hydrophobic amino acid includes isoleucine, leucine, or tryptophan.

[0034] This invention also provides the application of antimicrobial peptide GT-2 in the preparation of antimicrobial products.

[0035] In one embodiment, the antimicrobial product includes antifungal products and antibacterial products. In one embodiment, the antibacterial product includes products for treating bacterial infections. In one embodiment, the bacteria include Gram-negative bacteria and / or Gram-positive bacteria. In one embodiment, the Gram-negative bacteria include Staphylococcus aureus and / or methicillin-resistant Staphylococcus aureus. In one embodiment, the Gram-negative bacteria include Escherichia coli and / or Pseudomonas aeruginosa. In one embodiment, the fungus includes Candida albicans. In one embodiment, the product includes a reagent. In one embodiment, the reagent includes a pharmaceutical product.

[0036] The present invention also provides an antibacterial product, wherein the active ingredient of the antibacterial product includes the antimicrobial peptide GT-2 as described above.

[0037] In one embodiment, the effective concentration of the antimicrobial peptide GT-2 in the antimicrobial product is ≥4 μg / ml. In another embodiment, the effective concentration of the antimicrobial peptide GT-2 in the antimicrobial product is 4~32 μg / ml. In another embodiment, when the antimicrobial product is used to inhibit Candida albicans, the effective concentration is ≥4 μg / ml. In another embodiment, when the antimicrobial product is used to inhibit Pseudomonas aeruginosa, the effective concentration is ≥8 μg / ml. In another embodiment, when the antimicrobial product is used to inhibit Staphylococcus aureus, the effective concentration is ≥8 μg / ml. In another embodiment, when the antimicrobial product is used to inhibit methicillin-resistant Staphylococcus aureus, the effective concentration is ≥32 μg / ml. In another embodiment, when the antimicrobial product is used to inhibit Escherichia coli, the effective concentration is ≥8 μg / ml.

[0038] To further illustrate the present invention, the following detailed description of an antimicrobial peptide GT-2 and its applications, in conjunction with the accompanying drawings and embodiments, is provided but should not be construed as limiting the scope of protection of the present invention.

[0039] Unless otherwise specified, all reagents and instruments used in this invention are commercially available. Unless otherwise specified, all methods used in this invention are standard practices in the field.

[0040] The cell line used in this invention is: human immortalized keratinocytes (HaCAT) purchased from Procell Life Sciences Co., Ltd. (Wuhan, China).

[0041] The experimental materials and reagents used in this invention are as follows: fetal bovine serum (FBS); DMEM high glucose medium; phosphate-buffered saline (PBS, pH 7.2); penicillin and streptomycin were purchased from Biological Industries (BeitHaemek, Israel); MTS solution was purchased from Promega Biotechnology Co., Ltd. (Beijing, China); MHB medium, rabbit erythrocytes and Triton X-100 were purchased from Yuanye Company (Shanghai, China).

[0042] The instruments used in this invention include: centrifuge (Xiangyi Company), ultra-clean workbench (Suzhou Antai Air Technology Co., Ltd.), 96-well bacterial culture plate (Beijing Lanjieke Technology Co., Ltd.), LED inverted microscope (Leica), low temperature freezer (-80℃) (Haier Special Equipment Co., Ltd.), and enzyme-linked immunosorbent assay (ELISA) reader (Thermo Fisher Scientific).

[0043] The software and software information used in this invention include:

[0044] The source website for the Gastrodia elata genome is the National Genome Science Data Center (https: / / ngdc.cncb.ac.cn / ).

[0045] Peptide activity prediction website: PeptideRanker (http: / / distilldeep.ucd.ie / PeptideRanker / );

[0046] Physicochemical property analysis website: ExPASy (https: / / web.expasy.org / protparam / );

[0047] Hydrophobicity analysis website: HeliQuest (https: / / heliquest.ipmc.cnrs.fr / index.html);

[0048] Cleavage site prediction website: http: / / prosperousplus.unimelb-biotools.cloud.edu.au / ;

[0049] The iAMPCN model source code can be obtained from: https: / / github.com / joy50706 / iAMPCN;

[0050] The DeepAMP model source code can be obtained from: https: / / github.com / jimmyrate / deepAMP.

[0051] Example 1: Screening and Optimization of Antimicrobial Peptides

[0052] Based on publicly available data from the National Genome Science Data Center, Gastrodia elata ( Gastrodia elata The whole genome information was obtained, and the coding sequence was systematically screened using bioinformatics analysis tools. The prosperousplus cleavage site prediction website was used to specifically enzymatically cleave the gastrodin protein sequence to obtain several potential antimicrobial peptide candidate sequences. Subsequently, the antimicrobial activity prediction model iAMPCN with a convolutional neural network (CNN) architecture was used to score and rank the candidate sequences, and the peptide GP-1 with significant antimicrobial potential was screened out. The amino acid sequence of this peptide is shown in SEQ ID NO:1.

[0053] Building upon GP-1, the DeepAMP antimicrobial peptide optimization platform, driven by deep learning, was used. This model takes peptides with low antimicrobial activity as input and outputs analogs with high antimicrobial activity and broad-spectrum resistance. Combined with extensive pre-trained feature sensitivity analysis results, targeted optimization was performed at key amino acid sites affecting activity and toxicity.

[0054] The targeted optimization method operates as follows: A multi-objective scoring and sequence search method is used to rationally replace and rearrange the charged and hydrophobic sites of proteins or peptides. Negatively charged or overly flexible residues are replaced with cationic amino acids (such as lysine and arginine) to form a stable cationic distribution surface. Simultaneously, an appropriate amount of hydrophobic amino acids (such as isoleucine, leucine, and tryptophan) are introduced on the phase-shifted side to construct a sequence with a regular amphiphilic helical structure. Furthermore, by removing terminal aromatic residues, cysteine, and long-chain glycine repeating motifs, hemolytic activity, aggregation risk, and chemical instability are reduced. Finally, the improved antimicrobial peptide GT-2 is obtained, and its amino acid sequence is shown in SEQ ID NO:2.

[0055] The amino acid sequences of the structure-optimized antimicrobial peptides obtained through the above methods are shown in Table 1, and their physicochemical properties are shown in Table 2.

[0056] Table 1. Amino acid sequences of antimicrobial peptides optimized by deep learning

[0057]

[0058] Table 2 Physicochemical properties of antimicrobial peptides

[0059]

[0060] As shown in Table 2, compared with GP-1, the net charge of GT-2 increased from 0 to +7, indicating a significantly enhanced electrostatic binding ability with negatively charged bacterial membranes. The hydrophobic moment increased from 0.234 to 0.612, indicating enhanced amphiphilicity of its α-helical structure, which facilitates peptide penetration through bacterial membranes and results in a broad-spectrum antibacterial effect. The gravy value changed from positive (0.133) to negative (-0.460), reflecting an overall increase in the hydrophilicity of the peptide, which helps improve water solubility and affinity for bacterial targets, thereby enhancing antibacterial efficiency. These changes in physicochemical properties are closely related to the significant improvement in antibacterial activity and are consistent with the theoretical basis of the mechanism of action of antimicrobial peptides.

[0061] The peptide sequences of GP-1 (SEQ ID NO:1) and GT-2 (SEQ ID NO:2) were compared and plotted. The results are shown below. Figure 1 As shown. The chemical structural formulas of GP-1 and GT-2 are as follows. Figures 2-3 As shown. The secondary structures of GP-1 and GT-2 predicted by AlphaFold3 are as follows: Figure 4 and 5 As shown.

[0062] Depend on Figures 2-5 As can be seen, the polypeptides described in this invention are all linear chain structures formed by the sequential linkage of natural amino acids through peptide bonds (–CO–NH–). Their main chain consists of repeating –NH–CH(R)–CO– groups, where R is the characteristic side chain of each amino acid.

[0063] The chemical structure of the original sequence GP-1, VVWEGGGKGGGNCCF, contains valine (Val) with a hydrophobic isopropyl group, tryptophan (Trp) with an indole ring, glutamic acid (Glu) with a carboxyl group, numerous glycine (Gly) without side chains, lysine (Lys) with a long-chain primary amine, cysteine ​​(Cys) with a thiol group, and phenylalanine (Phe) with a benzene ring. Overall, it exhibits high flexibility and contains sites that can potentially form disulfide bonds.

[0064] The optimized sequence GT-2, KVWKILRKIIRIKNK, is composed of multiple positively charged lysine (Lys) and arginine (Arg) residues, along with alkyl hydrophobic side chains of isoleucine (Ile), leucine (Leu), valine (Val), and aromatic ring tryptophan (Trp), supplemented by an amide side chain of asparagine (Asn). This sequence exhibits a clear alternating arrangement of cationic and hydrophobic residues, possessing the potential to form an amphiphilic helical conformation while avoiding the instability caused by cysteine ​​and long chains of glycine residues.

[0065] The GT-2 sequence significantly enhances its net charge and isoelectric point by introducing multiple basic amino acids (such as lysine and arginine), thereby strengthening its electrostatic interaction with the bacterial membrane. Simultaneously, the well-placed hydrophobic residues (such as tryptophan and isoleucine) within the sequence contribute to stabilizing its α-helix structure and facilitating peptide insertion into the cell membrane. Figures 4-5 As shown, the GT-2 obtained by optimizing GP-1 in this invention helps stabilize the α-helix of the peptide. Combined with the HeliQuest analysis results, GT-2 exhibits a higher hydrophobic moment, indicating that it has a stronger amphiphilic arrangement, which is conducive to the formation of transmembrane channels in bacterial membranes, thereby enhancing its antibacterial efficacy.

[0066] In summary, GT-2 has achieved a significant improvement in antibacterial properties by optimizing its peptide sequence while retaining the original structural advantages.

[0067] Example 2 Antibacterial Activity Test

[0068] The antimicrobial peptides were synthesized by Nanjing Genscript Biotech Co., Ltd. The synthesized GP-1 and GT-2 antimicrobial peptides were purified by high-performance liquid chromatography (HPLC), and their molecular weight and purity were verified by mass spectrometry (MS). The results are as follows: Figures 6-9 As shown.

[0069] according to Figures 6-9 As can be seen, the HPLC showed a single main peak and the MS molecular weight was consistent with the theoretical value, which preliminarily confirms that the GP-1 and GT-2 antimicrobial peptides were successfully synthesized and the purity reached more than 95%.

[0070] According to CLSI standards, the minimum inhibitory concentrations (MICs) of GP-1 and GT-2 against Staphylococcus aureus, MRSA, Escherichia coli, Pseudomonas aeruginosa, and Candida albicans were determined using the microbroth dilution method. Bacterial culture in the logarithmic growth phase was added to 96-well bacterial culture plates. Antimicrobial peptides were added to each well in a concentration gradient (μg / ml) to achieve final concentrations of 128, 64, 32, 16, 8, 4, 2, 1, and 0.5 μg / ml, respectively. Negative control wells without antimicrobial peptides were also included. The 96-well plates were incubated at 37°C for 16–20 h. The minimum inhibitory concentration (MIC) was determined in wells where no visible bacterial growth or precipitation was observed. Each antimicrobial peptide was tested three times to ensure data reliability and reproducibility.

[0071] Table 3 Antimicrobial activity of antimicrobial peptides

[0072]

[0073] As shown in Table 3, GT-2 exhibited significantly enhanced antibacterial activity against a variety of tested strains, particularly demonstrating highly effective inhibition against both Gram-positive and Gram-negative bacteria. Its minimum inhibitory concentration (MIC) was significantly lower than that of the natural antimicrobial peptide GP-1. This significant activity enhancement validates the positive effect of structural optimization in enhancing antimicrobial efficacy, indicating that GT-2 has the potential to become a next-generation antimicrobial peptide candidate. Furthermore, GT-2 also exhibited good antifungal activity, with particularly outstanding inhibitory effects against Candida albicans. This broad-spectrum antimicrobial capability is closely related to its structural optimization, further supporting the crucial role of sequence optimization design in antimicrobial peptide development.

[0074] Example 3 Hemolysis Experiment

[0075] Rabbit erythrocyte suspension at a concentration of 5% (adjusted with PBS) was mixed with an equal volume of antimicrobial peptide GT-2 (final concentration 500 μg / ml, 3 wells per concentration) and incubated at 37°C for 1 h. An equal volume of PBS or 0.5% Triton X-100 was used as the negative and positive controls, respectively. After centrifugation at 3000 rpm for 10 min at 25°C, the supernatant was collected. The appearance of rabbit erythrocyte suspensions from different groups after centrifugation was as follows: Figure 10 As shown, absorbance was monitored at a wavelength of 415 nm. The formula for calculating the hemolysis rate is as follows:

[0076] Hemolysis rate (%) = [(A sample [-A0) / (A1-A0)]×100%;

[0077] Where A sample OD containing antimicrobial peptide GT-2 solution 415 nm; A0 is the OD of PBS.415 nm; A1 is the OD of 0.5% Triton X-100 415 nm.

[0078] Table 4 Hemolysis rate of antimicrobial peptides

[0079]

[0080] The hemolysis rates across different groups showed that even at a high concentration of 500 μg / ml, the hemolysis rate of the antimicrobial peptide GT-2 was only 0.49%, significantly better than that of traditional antimicrobial peptides, confirming the advantages of GT-2 in terms of biocompatibility. Combined with its highly effective antimicrobial activity and extremely low cytotoxicity, GT-2 not only demonstrates great potential as a novel antimicrobial agent but also provides a reliable candidate molecule for developing safer and more effective anti-infective treatments.

[0081] Example 4 Safety Experiment

[0082] The toxicity of antimicrobial peptides to mammalian cells was detected using the MTS assay; HaCAT cells were cultured at 1 × 10⁻⁶ cells per well. 4 Cells were seeded at a density of [missing information - likely a specific density] in 96-well plates. Cells were pretreated with different concentrations of GT-2 (100, 50, 25, 12.5, 6.25, 3.125, 1.563, 0.781 μg / ml, with each concentration treated in triplicate) at 37°C in a 5% CO2 incubator for 24 h. Then, 100 µL of MTS working solution was added to each well to create different sample groups. A control group was used, consisting of cells without GT-2 but with an equal volume of PBS. After incubating both sample and control groups at 37°C for 2 h, the absorbance of each well was monitored at 490 nm. Simultaneously, a blank control group was prepared using MTS+ medium and containing no HaCAT cells, and its OD value was measured as the OD value. 空白 Use the FlexStaton™ 3 multimode microplate reader to measure absorbance at 490 nm and calculate cell viability using the following formula. The results are as follows: Figure 11 As shown.

[0083] Cell viability (%) = (OD) 样品 -OD 空白 ) / (OD 对照组平均值 -OD 空白 The absorbance of each well was monitored at a wavelength of 490 nm.

[0084] from Figure 11As can be seen, when the GT-2 concentration reached 100 μg / ml, the survival rate of HaCAT cells still exceeded 95%, and it had no negative effect on cell proliferation and activity. This indicates that within the tested concentration range, this antimicrobial peptide has almost no toxic effect on mammalian cells. This result further demonstrates the good biocompatibility of GT-2, providing reliable support for its safety in clinical applications, and thus enabling further research and development as a biological antimicrobial peptide.

[0085] In summary, this invention provides an antimicrobial peptide GT-2 that combines the advantages of broad-spectrum antibacterial effect, high antibacterial efficiency, good antibacterial activity, extremely low probability of hemolysis, and high biocompatibility. This invention provides an effective approach for in-depth research, development, and utilization of biological antimicrobial peptides.

[0086] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, not all embodiments. People can obtain other embodiments without creative effort, as shown in these embodiments, and these embodiments all fall within the protection scope of the present invention.

Claims

1. An antimicrobial peptide GT-2, characterized in that, The amino acid sequence of the antimicrobial peptide GT-2 is shown in SEQ ID NO:

2.

2. The application of the antimicrobial peptide GT-2 as described in claim 1 in the preparation of antimicrobial products, characterized in that, The antibacterial products are antibacterial and antifungal products; The bacteria are one or more of Staphylococcus aureus, methicillin-resistant Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa. The fungus in question is Candida albicans.

3. The application as described in claim 2, characterized in that, The product includes reagents; the reagents include pharmaceuticals.

4. An antibacterial product, characterized in that, The active ingredient of the antibacterial product includes the antimicrobial peptide GT-2 as described in claim 1.

5. The antibacterial product as described in claim 4, characterized in that, The effective concentration of antimicrobial peptide GT-2 in the antimicrobial product is ≥4 μg / ml.

6. The antibacterial product as described in claim 5, characterized in that, The effective concentration of the antimicrobial peptide GT-2 in the antimicrobial product is 4~32μg / ml.

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