Antibacterial synergistic peptide targeting bacterial g-quadruplex and application thereof

Abstract

The present application relates to the technical field of biological medicine, and particularly relates to an antibacterial synergistic peptide targeting bacterial G-quadruplex and application thereof. The antibacterial synergistic peptide is an amino acid sequence shown in SEQ ID NO:1, a modified sequence or a pharmaceutically acceptable salt of the polypeptide. The amino acid sequence (GR17) is shown in SEQ ID NO:1. The antibacterial synergistic peptide has low antibacterial activity, but can enter the intracellular bacteria by means of polymyxin antibiotic-mediated bacterial membrane permeabilization, combine with bacterial G-quadruplex and interfere with nucleic acid-related processes and protein synthesis, thereby enhancing the antibacterial activity of polymyxin antibiotics on enterobacteriaceae pathogens such as clinical extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli (E. coli) and the like. E. coli The antibacterial synergistic peptide can also be used in combination with polymyxin B to continuously inhibit bacterial growth and recovery, inhibit bacterial biofilm formation, and improve the treatment effect on lung infection and catheter-related urinary tract infection.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and in particular to an antimicrobial synergistic peptide that targets bacterial G-tetramolecular structures and its applications. Background Technology

[0002] Multidrug-resistant Gram-negative bacterial infections pose a serious threat to clinical anti-infective therapy. Polymyxin B exerts its antibacterial effect by binding to lipopolysaccharides in the outer membrane of Gram-negative bacteria and increasing bacterial membrane permeability, making it an important drug for treating some multidrug-resistant Gram-negative bacterial infections. However, polymyxin B suffers from a narrow therapeutic window and dose-limiting toxicities, which restricts its clinical application.

[0003] Existing polymyxin synergists mostly exert their effects by further perturbing the bacterial outer membrane or cytoplasmic membrane. This type of synergistic mechanism may increase the risk of host cell membrane damage and may be affected by outer membrane remodeling in resistance-associated bacteria. Therefore, it is necessary to develop adjuvants that can utilize polymyxin B-mediated membrane permeability to enter the bacterial cell and enhance antibacterial activity through an intracellular mechanism of action complementary to polymyxin B.

[0004] Non-antimicrobial cationic peptides typically have weak intrinsic antimicrobial activity, but they can enter bacterial cells through membrane permeability mediated by membrane-active antimicrobial agents and act on nucleic acid metabolism, protein synthesis, or stress regulation processes. However, existing non-antimicrobial cationic peptides are mainly obtained through phenotypic screening, and their structure-activity relationships and systematic optimization methods are still imperfect, making it difficult to achieve the rational design of highly effective antimicrobial synergistic peptides.

[0005] G-quadruplexes are non-classical four-stranded structures formed by guanine-rich nucleic acid sequences and participate in bacterial replication, transcription, translation, and stress regulation. Designing cationic peptides that recognize bacterial G-quadruplexes holds promise for transforming polymyxin B-mediated intracellular delivery into synergistic antibacterial action targeting key bacterial nucleic acid processes. Although existing studies have reported the use of G-quadruplex ligands (N-methyl-N-porphyrin IX, NMM) to inhibit bacterial growth alone or in combination with antibacterial drugs (aminoglycoside antibiotics), these techniques mostly employ small-molecule ligands or broad-spectrum nucleic acid-binding molecules. A systematic evaluation of the structural selectivity, intracellular targeting efficiency, and functional effects of bacterial G-quadruplexes after binding is lacking. Furthermore, the structure-activity relationship between G-quadruplex binding capacity and antibacterial synergistic activity remains unclear, hindering predictable molecular optimization. Moreover, current research largely focuses on efficacy validation; the broad-spectrum synergistic effect, membrane toxicity control, and in vivo therapeutic efficacy in multidrug-resistant Gram-negative bacteria require further investigation. Therefore, existing technologies lack a technical solution for systematically optimizing the structure and function of non-antimicrobial cationic peptides and obtaining highly efficient polymyxin-enhancing peptides by using bacterial G-quadruplex recognition as a functional indicator. Summary of the Invention

[0006] To address the problems of unclear structure-activity relationships, limited antibacterial synergistic activity, and difficulty in systematic optimization of existing non-antimicrobial cationic peptides, this invention provides an antimicrobial synergistic peptide targeting bacterial G-quadruplexes and its application.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows: An antimicrobial synergistic peptide targeting bacterial G-quadruplexes, wherein the antimicrobial synergistic peptide is the amino acid sequence shown in SEQ ID NO:1, a modified sequence, or a pharmaceutically acceptable salt of the polypeptide; The amino acid sequence (GR17) is shown in SEQ ID NO:1, and its amino acid sequence is: Arg 1 –Arg 2 –Ala 3 –Gly 4 –Arg 5 –Ser 6 –Arg 7 –Gly 8 –Arg 9 –Ser 10 –Arg 11 –Gly 12 –Arg 13 –Pro 14 –Pro 15 –Gly 16 –Arg 17 The theoretical average molecular weight of this amino acid is approximately 1935.19 Da. The modified sequence is the amino acid sequence shown in SEQ ID NO:2-4; The amino acid sequence (GF17) is shown in SEQ ID NO:2, and its amino acid sequence is: Arg 1 –Arg 2 –Ala 3 –Gly 4 –Arg 5 –Ser 6 –Arg 7 –Gly 8 –Arg 9 –Ser 10 –Arg 11 –Gly 12 –Arg 13 –Pro 14 –Phe 15 –Gly 16 –Arg 17 The theoretical average molecular weight of this amino acid is approximately 1985.23 Da. The amino acid sequence (CGF1) is a disulfide bond formed between the cysteine ​​residue at position 1 and position 19, as shown in SEQ ID NO:3; that is, its linear amino acid sequence is as shown in SEQ ID NO:3: Cys 1 –Arg 2 –Arg 3 –Ala 4 –Gly 5 –Arg 6 –Ser 7 –Arg 8 –Gly 9 –Arg 10 –Ser 11 –Arg 12 –Gly 13 –Arg 14 –Pro 15 –Phe 16 –Gly 17 –Arg 18 –Cys 19 The theoretical average molecular weight of this amino acid is approximately 2189.50 Da. The amino acid sequence (CGF2) is shown in SEQ ID NO:4, where a disulfide bond is formed between the 5th and 15th cysteine ​​residues; that is, its linear amino acid sequence is shown in SEQ ID NO:4: Cys 1 –Arg 2 –Arg 3 –Ala 4 –Gly 5 –Arg 6 –Ser 7 –Arg 8 –Gly 9 –Arg 10 –Ser 11 –Arg 12 –Gly 13 –Arg 14 –Cys 15 –Pro 16 –Phe 17 –Gly 18 –Arg 19 The theoretical average molecular weight of this amino acid is approximately 2189.50 Da.

[0008] The antimicrobial synergistic peptide is CGF1 and / or CGF2.

[0009] The application of the aforementioned antimicrobial synergistic peptide targeting bacterial G-tetramolecular structures, wherein the antimicrobial synergistic peptide is used as a synergist for antimicrobial substances.

[0010] The antibacterial substance is polymyxin B.

[0011] An antimicrobial pharmaceutical composition comprising an active ingredient and an adjuvant, wherein the active ingredient contains the aforementioned antimicrobial synergistic peptide. The adjuvant may be a pharmaceutically acceptable carrier, excipient, or diluent.

[0012] The active ingredients are the antimicrobial synergistic peptide and polymyxin antibiotics (concentration of 25 μM + 1 / 4 × MIC).

[0013] One application is the use of the antimicrobial synergistic peptide or the composition in the preparation of a medicament for the prevention and / or treatment of Gram-negative bacterial infections or drug-resistant Gram-negative bacterial lung infections.

[0014] One application is the use of the antimicrobial synergistic peptide or the composition in the preparation of a medicament for inhibiting the formation of Gram-negative bacterial biofilms, reducing biofilm-associated bacterial load, or preventing the formation of biofilms on the surface of medical devices.

[0015] The Gram-negative bacteria are selected from at least one of Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii, and Pseudomonas aeruginosa; The Gram-negative bacteria are drug-resistant Escherichia coli that produce extended-spectrum β-lactamase.

[0016] Advantages of this invention: This invention provides an antimicrobial synergistic peptide with low intrinsic antimicrobial activity, but which can enter the bacterial cell via bacterial membrane permeability mediated by polymyxin antibiotics. It binds to bacterial G-quadruplexes and interferes with nucleic acid-related processes and protein synthesis, thereby enhancing the effectiveness of polymyxin antibiotics against clinically produced extended-spectrum β-lactamase (ESBL)-producing *Escherichia coli*. E. coli This invention exhibits antibacterial activity against Enterobacteriaceae pathogens such as [list of pathogens]. Furthermore, the antimicrobial synergistic peptide described in this invention, when used in combination with polymyxin B, can continuously inhibit bacterial growth and resuscitation, suppress bacterial biofilm formation, and improve the therapeutic effect on pulmonary infections and catheter-related urinary tract infections. The antimicrobial synergistic peptide of this invention provides a novel peptide adjuvant for enhancing the efficacy of polymyxin antibiotics and treating infections caused by drug-resistant Gram-negative bacteria. Attached Figure Description

[0017] Figure 1 Synergistic antibacterial curves of different antimicrobial synergistic peptides and polymyxin B provided in embodiments of the present invention.

[0018] Figure 2 The image shows the survival rate of mice with lung infection as provided in an embodiment of the present invention. Detailed Implementation

[0019] The following examples further illustrate specific embodiments of the present invention. It should be noted that the specific embodiments described herein are merely for illustration and explanation and are not intended to limit the scope of the present invention.

[0020] In the following examples, the antimicrobial synergistic peptides were obtained by modifying the non-antimicrobial cationic peptide AW2 (patent application number: CN202310367170.2) as the initial modified backbone. The G-quadruplex-binding peptide RGG25 was used as a comparative peptide.

[0021] Example 1 Discovery, modification routes and preparation of antimicrobial synergistic peptides (1) The amino acid composition and physicochemical properties of AW2 and RGG25 were analyzed using the DBAASP database. The binding ability of bacterial G-quadruplexes and their antibacterial synergistic activity when used in combination with polymyxin B were compared by ITC isothermal titration and antibacterial synergistic index experiments. The results showed that RGG25 contained locally distributed aromatic residues in its flexible cationic skeleton and had moderately enhanced hydrophobicity, and its polymyxin B synergistic activity was better than that of AW2. Based on the above results, AW2 was modified by sequence truncation, alanine scanning, aromatic amino acid substitution, and conformational cyclization (Table 1).

[0022] (2) Based on the interaction force analysis of the ITC isothermal titration results of AW2 and bacterial G-quadruplex HPG2, the binding of AW2 and HPG2 mainly depends on the first nineteen amino acids starting from the N-terminus. The C-terminal region, which has a low contribution to G-quadruplex binding, was removed, and the peptides were truncated bidirectionally. By comparing the G-quadruplex binding ability and polymyxin B synergistic activity of each truncated peptide, the peptide GR17 with the amino acid sequence RRAGRSRGRSRGRPPGR, i.e., SEQ ID NO:1, was obtained. The non-arginine residues in GR17 were scanned for alanine, and the proline at position 15 was identified as a highly tolerant modification site. This site was replaced with phenylalanine to obtain GF17, with the amino acid sequence RRAGRSRGRSRGRPFGR, i.e., SEQ ID NO:2.

[0023] (3) According to the docking results of AW2 and HPG2 molecules using PyMOL software, it was shown that the binding force of GF17 to G4 was significantly improved after modification. Subsequently, the above steps were used to further restrict the conformational freedom of GF17, and two disulfide bond cyclized peptides were designed based on it. The linear amino acid sequence of CGF1 is CRRAGRSRGRSRGRPFGRC, i.e., SEQ ID NO:3, in which a disulfide bond is formed between cysteine ​​at position 1 and position 19; the linear amino acid sequence of CGF2 is RRAGCRSRGRSRGRCPFGR, i.e., SEQ ID NO:4, in which a disulfide bond is formed between cysteine ​​at position 5 and position 15. CGF1 provides conformational constraint on the overall polypeptide backbone, while CGF2 provides local conformational constraint on the central region rich in arginine and glycine.

[0024] (4) Chemical synthesis method of antibacterial synergistic peptides GR17, GF17, CGF1, and CGF2: Based on the modified amino acid sequence, their full sequences are synthesized using an automated peptide synthesizer (433A, Applied Biosystems). The peptides are then purified by HPLC reverse-phase column chromatography after desalting. Alternatively, synthesis can be outsourced to a company. Cyclization reaction: The linear crude peptides of CGF1 and CGF2, each containing two free cysteine ​​residues, are dissolved in an oxidative cyclization buffer, and the peptide concentration is controlled within a range favorable for intramolecular disulfide bond formation. The reaction solution is adjusted to neutral or weakly alkaline, and the reaction is stirred at room temperature to allow the Cys group of CGF1 to cyclize. 1 With Cys 19 Intramolecular disulfide bonds are formed between them, making the Cys of CGF2... 5 With Cys 15 Intramolecular disulfide bonds are formed between them.

[0025] (5) The purity of the purified peptides was identified by high performance liquid chromatography (HPLC), the molecular weight was determined by matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF), the isoelectric point was determined by isoelectric focusing electrophoresis, and the amino acid sequence structure was determined by an automated amino acid sequencer. The peptides were dissolved in PBS (NaCl 137 mmol / L, KCl 2.7 mmol / L, Na2HPO4 4.3 mmol / L, KH2PO4 1.4 mmol / L, pH approximately 7.4) for subsequent biochemical analysis.

[0026] The GR17 consists of 17 amino acid residues, with a theoretical average molecular weight of approximately 1935.19 Da. Its amino acid sequence is shown in SEQ ID NO:1: Arg 1 –Arg 2 –Ala 3 –Gly 4 –Arg 5 –Ser6 –Arg 7 –Gly 8 –Arg 9 –Ser 10 –Arg 11 –Gly 12 –Arg 13 –Pro 14 –Pro 15 –Gly 16 –Arg 17 The GF17 consists of 17 amino acid residues, with a theoretical average molecular weight of approximately 1985.23 Da. Its amino acid sequence is: Arg 1 –Arg 2 –Ala 3 –Gly 4 –Arg 5 –Ser 6 –Arg 7 –Gly 8 –Arg 9 –Ser 10 –Arg 11 –Gly 12 –Arg 13 –Pro 14 –Phe 15 –Gly 16 –Arg 17 (SEQ ID NO:2).

[0027] The CGF1 consists of 19 amino acid residues, with a theoretical average molecular weight of approximately 2189.50 Da, and its amino acid sequence is: Cys 1 –Arg 2 –Arg 3 –Ala 4 –Gly 5 –Arg 6 –Ser 7 –Arg 8 –Gly 9 –Arg 10 –Ser 11 –Arg 12 –Gly 13 –Arg 14 –Pro 15 –Phe 16 –Gly 17 –Arg 18 –Cys 19 (SEQ ID NO:3).

[0028] The CGF2 consists of 19 amino acid residues, with a theoretical average molecular weight of approximately 2189.50 Da, and its amino acid sequence is: Cys 1 –Arg 2 –Arg 3 –Ala 4 –Gly 5 –Arg 6 –Ser 7 –Arg 8 –Gly 9 –Arg 10 –Ser 11 –Arg 12 –Gly 13 –Arg 14 –Cys 15 –Pro 16 –Phe 17 –Gly 18 –Arg 19 (SEQ ID NO:4).

[0029] Table 1. Modification routes and key parameters of antimicrobial synergistic peptides

[0030] Example 2 The antibacterial activity of the aforementioned antimicrobial synergistic peptides GR17, GF17, CGF1, and CGF2 was determined. Each microbial strain listed in Table 2 was inoculated into MH liquid medium (Oxoid, UK) and cultured at 37°C with shaking until the logarithmic growth phase. Each bacterial culture was then diluted to 2 × 10⁻⁶ with fresh MH liquid medium. 5 Prepare CFU / mL for later use. Add 100 µL of MH liquid medium to each well of a sterile 96-well plate (Thermo, USA). Then, add 100 µL of GR17, GF17, CGF1, and CGF2 sample solutions diluted with MH liquid medium (final concentration 100 μg / mL) to the first well. Mix well, then add 100 µL to the second well, and so on, diluting by half each time. Discard 100 µL from the ninth well. The tenth well serves as a control.

[0031] The 96-well plates were incubated at 37°C with slow shaking for 18 hours. The light absorption was measured at a wavelength of 600 nm, and the minimum inhibitory concentration (MIC) of the samples against different microorganisms was calculated. The results are shown in Table 2.

[0032] As shown in Table 2, the GR17, GF17, CGF1, and CGF2 peptides themselves have no antibacterial activity against Gram-positive bacteria, Gram-negative bacteria, and fungi. Their MIC values ​​are all greater than 200 μg / mL.

[0033] Table 2. Autoantibacterial activity of antimicrobial synergistic peptides

[0034] Note that the labels ATCC and CMCC after the strain name represent standard strains, sourced from ATCC (American Center for Type Culture Collection) and CMCC (China Center for Medical Bacteriological Collection), respectively. All other bacteria were clinically isolated from the Department of Laboratory Medicine, Zunyi First People's Hospital, Zunyi City, Guizhou Province, donated by Dr. Yao Shifei and described in the following literature.

[0035] 1. Zhang P, Ye Z, Dou H, Li S, Jiao X, Wang Y. Identification and characterization of broad-spectrum cathelicidin family antimicrobial peptides from the pygmy sperm whale Kogia breviceps. Biochem Pharmacol. 2026 Mar;245:117675. 2. Ye P, Yang Y, Qu Y, Yang W, Tan J, Zhang C, Sun D, ​​Zhang J, ZhaoW, Guo S, Song L, Hou T, Zhang Z, Tang Y, Limjunyawong N, Xu J, Dong S, DouC, Luo F. LL-37 and bisphosphonate co-delivery 3D-scaffold with antimicrobialand antiresorptive activities for bone regeneration. Int J Biol Macromol.2024 Oct;277(Pt 1):134091. Example 3 The synergistic antibacterial activity of each antimicrobial synergistic peptide listed in Table 1 above with polymyxin B was determined: This experiment used clinically isolated β-lactam antibiotic-resistant Gram-negative Escherichia coli R2. This E. coli R2 was cultured in MH liquid medium (Oxoid, UK) at 37°C for 12 hours, and then diluted with fresh MH liquid medium to a concentration of 1 × 10⁻⁶. 6 CFU / mL bacterial suspension. In a 96-well plate (Thermo, USA), 80 μL of bacterial suspension was added to an 8×8 grid. In 8 columns, 10 μL of polymyxin B sample dissolved in sterile deionized water was added to the bacterial suspension to a final concentration of 8 × MIC (18.72 µg / mL), then diluted 2 / 3 to 0.25 × MIC (18.72 µg / mL). In the other 8 rows, in addition to polymyxin B, an antimicrobial synergistic peptide was added to a final peptide concentration of 200 μg / mL, then diluted 2 / 3 to 3.1 μg / mL. Specific addition ratios are shown in Table 4. The bacterial suspensions with added samples were incubated at 37°C with shaking. The synergistic index was then determined using a microplate reader (Teikoku Spark, Switzerland). In this experiment, PBS alone was used as the positive control (with bacteria but no drug, and an equal volume of PBS buffer was added), and sterile deionized water was used as the negative control (without bacteria or drug, and only sterile MH medium was used as the negative control) (see Table 3).

[0036] The antibacterial rate of the drug % = A ( 药物组-阴性对照药物组 ) / A （阳性对照PBS组-阴性空白对照组） ×100% The antimicrobial synergy index is calculated as follows: Drug concentration in cells with a synergistic drug inhibition rate higher than 90% / Polymyxin B drug concentration in cells with an inhibition rate higher than 90% + Drug concentration in cells with a synergistic drug inhibition rate higher than 90% / Antimicrobial synergistic peptide drug concentration in cells with an inhibition rate higher than 90%.

[0037] A synergy index of ≤0.5 indicates synergy.

[0038] The results are shown in Table 3. The FIC index of the parent peptide AW2 combined with polymyxin B was 0.48. After removing the C-terminal region of AW2, which contributed less to G-quadruplex binding, the FIC index of RR19 decreased to 0.41, indicating that it still maintained good synergistic antibacterial activity. However, after further N-terminal truncation, the FIC indices of RR17, RR15, RR13, RR11, and RR9 increased to 0.53, 0.72, 1.03, 1.07, and 2.00, respectively, suggesting that excessive removal of the N-terminal arginine-rich region would significantly weaken the synergistic effect. Among the C-terminal truncated series, the FIC index of GR17 was 0.47, still maintaining synergistic activity, while the FIC indices of PP15, GR13, SR11, and GR9, obtained by further truncation, increased to 0.67, 0.78, 1.28, and 1.38, respectively. Therefore, GR17 was selected as the subsequent modification backbone. After scanning the alanine residues in GR17, the FIC indices of all mutant peptides were generally higher than those of GR17. Among them, the FIC index of GR17-15A was 0.51, which was closest to that of GR17, indicating that the proline at position 15 has high tolerance to substitution. Replacing this site with phenylalanine yielded GF17, whose FIC index decreased to 0.35. After disulfide cyclization of GF17, the FIC indices of CGF1 and CGF2 decreased to 0.32 and 0.18, respectively. Among them, the locally cyclized peptide CGF2 showed the strongest polymyxin B synergistic activity.

[0039] Table 3 Antimicrobial Synergistic Index

[0040] Table 4. Schematic diagram of the antibacterial synergy index experiment.

[0041] The results are shown in Table 3. The FIC index of the parent peptide AW2 combined with polymyxin B was 0.48. After removing the C-terminal region of AW2, which contributed less to G-quadruplex binding, the FIC index of RR19 decreased to 0.41, indicating that it still maintained good synergistic antibacterial activity. However, after further N-terminal truncation, the FIC indices of RR17, RR15, RR13, RR11, and RR9 increased to 0.53, 0.72, 1.03, 1.07, and 2.00, respectively, suggesting that excessive removal of the N-terminal arginine-rich region would significantly weaken the synergistic effect. Among the C-terminal truncated series, the FIC index of GR17 was 0.47, still maintaining synergistic activity, while the FIC indices of PP15, GR13, SR11, and GR9, obtained by further truncation, increased to 0.67, 0.78, 1.28, and 1.38, respectively. Therefore, GR17 was selected as the subsequent modification backbone. After scanning the alanine residues in GR17, the FIC indices of all mutant peptides were generally higher than those of GR17. Among them, the FIC index of GR17-15A was 0.51, which was closest to that of GR17, indicating that the proline at position 15 has high tolerance to substitution. Replacing this site with phenylalanine yielded GF17, whose FIC index decreased to 0.35. After disulfide cyclization of GF17, the FIC indices of CGF1 and CGF2 decreased to 0.32 and 0.18, respectively. Among them, the locally cyclized peptide CGF2 showed the strongest polymyxin B synergistic activity.

[0042] Example 4 The above-mentioned antimicrobial synergistic peptides GR17, GF17, CGF1, and CGF2, together with polymyxin B, synergistically inhibit bacterial growth. In this embodiment, *Escherichia coli* R2, a clinically isolated Gram-negative bacterium resistant to β-lactam antibiotics, was selected. The bactericidal effect of the antimicrobial synergistic peptides GR17, GF17, CGF1, and CGF2 combined with polymyxin B was determined. The bacterial concentration was adjusted to 1 × 10⁻⁶. 7 CFU / mL (80 μL) of antimicrobial synergistic peptide and polymyxin B (antibiotic) sample dissolved in sterile deionized water (volume ratio 1:1) was added to the bacterial suspension to a final concentration of 1 / 4 × MIC (0.59 µg / mL). In addition to the antibiotic, the antimicrobial synergistic peptide was added to a final peptide concentration of 100 µg / mL. The bacterial suspension with the added sample was incubated at 37°C with shaking. The absorbance at 600 nm was measured using a multi-functional microplate reader (Teikoku Spark, Switzerland) from 0 to 18 hours. After 18 hours, the bacterial colonies were revived by diluting the sample 1:100 with fresh culture medium. This experiment used separate PBS as a positive control (bacteria added but no drug, with an equal volume of PBS buffer) and a blank control (no bacteria or drug added, only sterile MH medium as a negative control).

[0043] Antibacterial rate of drug % = A （药物组-阴性对照药物组） / A （阳性对照PBS组-阴性空白对照组） ×100% The results are as follows Figure 1 As shown, compared with the control group, AW2 / polymyxin B and GR17 / polymyxin B only exhibited limited synergistic antibacterial effects, and the bacteria could gradually recover growth during the treatment period. GF17 / polymyxin B could further delay bacterial growth, but after 18 hours of treatment, the bacteria could still recover after being diluted 1:100 to fresh culture medium. In contrast, CGF1 / polymyxin B and CGF2 / polymyxin B both continuously inhibited bacterial growth during the initial 18 hours of treatment, and no significant growth recovery was observed after dilution and recovery.

[0044] Example 5 The above-mentioned antimicrobial synergistic peptides GR17, GF17, CGF1, and CGF2 were obtained for cytotoxicity. The in vitro cytotoxicity of antimicrobial synergistic peptides GR17, GF17, CGF1, and CGF2 was determined using Raw 264.7 (mouse macrophage cell line) and HacaT (human immortalized keratinocytes) via the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method.

[0045] The above cell cultures were conducted in DMEM complete medium containing 10% fetal bovine serum (Corning Life Sciences (Wujiang) Co., Ltd.) and 100 U / mL streptomycin-penicillin (Shanghai Beyotime Biotechnology Co., Ltd.). Before the formal experiment, the cells were seeded in 96-well plates (2×10⁶ cells / well). 4 Cells were cultured overnight in 96-well plates, then different concentrations of antimicrobial synergistic peptides were added, and the plates were incubated at 37°C in a 5% CO2 cell culture incubator for 24 hours. After incubation, 20 µL of 5 mg / mL MTT was added to each well of the 96-well plate, and the plates were incubated for another 4 hours. The supernatant was discarded, and DMSO was added. The absorbance was measured at 490 nm (see Tables 5 and 6). The zeroing group received only DMEM medium, and the negative control group received only cells without drug administration. The cytotoxicity percentage C% = A. 样品 -A 调零组 / A 阴性对照 -A 调零组 ×100%.

[0046] The experimental results are shown in Tables 5-6. When the concentration of GR17, GF17, CGF1 and CGF2 was 200 µg / mL, the cell viability of Raw264.7 cells was the lowest at 64.25%, while the viability of the other three cell lines was higher than 70%. This indicates that the antimicrobial synergistic peptides have very weak cytotoxicity to mammalian cells even at high concentrations, showing great application potential.

[0047] Table 5. Cytotoxic activity of the antimicrobial synergistic peptide Raw264.7

[0048] Table 6. Cytotoxic Activity of the Antimicrobial Synergistic Peptide HacaT

[0049] Example 6 The above-obtained antimicrobial synergistic peptides GR17, GF17, CGF1, and CGF2 were used for biofilm assays with polymyxin B. Biofilm clearance capabilities of antimicrobial synergistic peptides GR17, GF17, CGF1, and CGF2 and polymyxin B were determined: Crystal violet staining was used to evaluate the clearance capabilities of the antimicrobial synergistic peptides and polymyxin B against *E. coli* R2 biofilm. The strains were cultured in BHI liquid medium (Oxoid, UK) at 37°C for 12 hours, and then diluted with fresh medium to a concentration of 10⁻⁶. 7 CFU / mL bacterial suspension was added to each well of a 96-well plate, 100 µL of the suspension was added, and the plate was incubated at 37°C for 48 hours. After removing the supernatant, the plate was washed three times with PBS, and then 100 µL of (50 μg / mL potentiating peptide + 1 × MIC polymyxin B) was added sequentially for incubation for another 24 hours. After incubation, the supernatant was removed, the plate was washed with PBS, fixed with 50 µL of methanol, and after drying, 0.1% crystal violet was added for staining. Finally, the staining solution was discarded, and the crystal violet was dissolved in anhydrous ethanol. The absorbance was measured at 600 nm. The zeroing group only received culture medium, and the negative control group only received bacterial suspension without drug administration. Biofilm survival rate (%) = A 样品 -A 调零组 / A 阴性对照 -A 调零组 ×100% The biofilm inhibitory activity of antimicrobial synergistic peptides and polymyxin B was determined: Crystal violet staining was used to evaluate their inhibitory activity against *E. coli* R2 biofilm. The strains were cultured in BHI liquid medium (Oxoid, UK) at 37°C for 12 hours, and then diluted with fresh medium to a concentration of 1 × 10⁻⁶. 8 CFU / mL bacterial suspension was added to each well of a 96-well plate at 50 µL, along with 50 µL of a synergistic peptide (50 μg / mL + 1 × MIC polymyxin B). The plates were incubated together at 37°C for 48 hours. The remaining steps were the same as for biofilm removal. The zero-conditioning group received only culture medium, while the negative control group received only bacterial suspension without any drug administration. Biofilm formation rate (%) = A 样品 -A 调零组 / A 阴性对照 -A 调零组 ×100%, Table 7. Effects of antimicrobial synergistic peptides and polymyxin B on biofilm clearance and inhibition.

[0050] The results are shown in Table 7. The combined use of GR17, GF17, CGF1, and CGF2 with polymyxin B showed limited effectiveness in clearing existing biofilms, with biofilm survival rates ranging from 93.32% to 105.64%. In contrast, each combination exhibited varying degrees of inhibition on biofilm formation. The biofilm survival rates after treatment with GR17 / polymyxin B and GF17 / polymyxin B were 90.24% and 85.68%, respectively, while CGF1 / polymyxin B further reduced the survival rate to 68.84%, and CGF2 / polymyxin B further reduced it to 42.11%.

[0051] Example 7 CGF1 and CGF2 and polymyxin B in mice: hepatotoxicity and nephrotoxicity To evaluate the in vivo safety of the finally modified antimicrobial synergistic peptides, BALB / c mice were randomly divided into groups and injected with CGF1 or CGF2 via the tail vein, respectively. The dosage of each peptide was 40 mg / kg. Additional groups were established, including a peptide-low-dose polymyxin B combination group, a polymyxin B monotherapy group, and a solvent control group. The peptide-polymyxin B combination group received 40 mg / kg peptide and 2.5 mg / kg polymyxin B, while the polymyxin B monotherapy doses were 5 mg / kg and 10 mg / kg, respectively. Blood was collected from mice 24 hours after administration, and serum was separated. Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels were measured to evaluate liver function; serum creatinine (CREA) and blood urea nitrogen (BUN) levels were measured to evaluate kidney function.

[0052] Table 8. Hepatotoxicity and nephrotoxicity of antimicrobial synergistic peptides and polymyxin B

[0053] The results showed that, after administration of high-dose CGF1 and CGF2 alone, no significant abnormal increases were observed in serum ALT, AST, CREA, and BUN in mice. When CGF1 or CGF2 was combined with a low-dose polymyxin B treatment, the overall liver and kidney function indicators were close to those of the solvent control group, and the changes were less pronounced than in the high-dose polymyxin B treatment group. These results indicate that CGF1 and CGF2 did not cause significant acute liver and kidney damage at the measured doses, and their combined use with a low-dose polymyxin B showed good acute in vivo tolerability.

[0054] Example 8 CGF1 and CGF2 and their effects on polymyxin B in mice against ductal biofilm formation A mouse model of catheter-related urinary tract infection was established using drug-resistant Escherichia coli R2. A sterile silicone catheter fragment was implanted into the mouse bladder via the urethra, and the following day, 50 μL of a 1 × 10⁻⁶ concentration was inoculated via the urethra. 8 CFU / mL bacterial suspension. After infection, mice were treated with 200 μL of CGF1 / polymyxin B, CGF2 / polymyxin B (10 mg / kg (peptide) + 5 mg / kg (polymyxin B)), and polymyxin B (5 mg / kg), respectively. Each drug was administered intraperitoneally once daily for 3 consecutive days.

[0055] On day 14 post-infection, the bladder catheter was removed and gently washed with phosphate-buffered saline to remove unattached bacteria. The catheter was then stained with crystal violet solution. After washing away unbound dye, ethanol or acetic acid solution was added to dissolve the crystal violet bound to the catheter biofilm, and the absorbance at 595 nm was measured to evaluate the amount of biofilm on the catheter surface.

[0056] Table 9. Inhibitory effects of antimicrobial synergistic peptides and polymyxin B on duct biofilm formation.

[0057] The results showed that a large amount of biofilm formed on the surface of the catheter in the solvent control group. After treatment with CGF1 and polymyxin B, the amount of biofilm on the catheter surface was significantly reduced to below 20%; after treatment with CGF2 and polymyxin B, the amount of residual biofilm was further reduced, and the inhibitory effect was stronger than that of the polymyxin B treatment group alone.

[0058] Example 9 Protective effect of CGF1 and CGF2 combined with polymyxin B against lung infection in mice An immunosuppressed mouse model was established by cyclophosphamide pretreatment, followed by intratracheal inoculation with 1 × 10⁻⁶ cyclophosphamide. 7 An acute pulmonary infection model was established using CFU-producing extended-spectrum β-lactamase *Escherichia coli* R2. Two hours after infection, mice were administered the following solvents via tail vein: CGF1+polymyxin B, CGF2+polymyxin B, and polymyxin B. The combined peptide and polymyxin B group received 5 mg / kg peptide and 1.25 mg / kg polymyxin B, while the positive control group received a high concentration of polymyxin B, with each individual dose being 5 mg / kg. Mice were observed for 36 hours post-administration, and survival was recorded (see [link to relevant documentation]). Figure 2 ).

[0059] The results showed that all mice in the solvent control group died within 12 hours after infection; the final survival rates of the polymyxin B alone treatment group and the AW2 / polymyxin B treatment group were approximately 50% and 62.5%, respectively. In contrast, no mice in the CGF1+polymyxin B and CGF2+polymyxin B treatment groups died during the 36-hour observation period, with a survival rate of 100%.

[0060] The above-described embodiments are merely preferred embodiments provided to fully illustrate the present invention, and the scope of protection of the present invention is not limited thereto. Equivalent substitutions or modifications made by those skilled in the art based on the present invention are all within the scope of protection of the present invention. The scope of protection of the present invention is defined by the claims.

Claims

1. An antimicrobial synergistic peptide targeting bacterial G-quadruplexes, characterized in that, The antimicrobial synergistic peptide is the amino acid sequence shown in SEQ ID NO:1, a modified sequence, or a pharmaceutically acceptable salt of the polypeptide. The amino acid sequence (GR17) is shown in SEQ ID NO:1, and its amino acid sequence is: Arg 1 –Arg 2 –Ala 3 –Gly 4 –Arg 5 –Ser 6 –Arg 7 –Gly 8 –Arg 9 –Ser 10 –Arg 11 –Gly 12 –Arg 13 –Pro 14 –Pro 15 –Gly 16 –Arg 17 .

2. The antimicrobial synergistic peptide targeting bacterial G-tetramole according to claim 1, characterized in that, The modified sequence is any amino acid sequence shown in SEQ ID NO:2-4; The amino acid sequence (GF17) is shown in SEQ ID NO:2, and its amino acid sequence is: Arg 1 –Arg 2 –Ala 3 –Gly 4 –Arg 5 –Ser 6 –Arg 7 –Gly 8 –Arg 9 –Ser 10 –Arg 11 –Gly 12 –Arg 13 –Pro 14 –Phe 15 –Gly 16 –Arg 17 ; The amino acid sequence (CGF1) is shown in SEQ ID NO:1, where a disulfide bond is formed between the cysteine ​​residue at position 1 and the cysteine ​​residue at position 19; that is, its linear amino acid sequence is shown in SEQ ID NO:3: Cys 1 –Arg 2 –Arg 3 –Ala 4 –Gly 5 –Arg 6 –Ser 7 –Arg 8 –Gly 9 –Arg 10 –Ser 11 –Arg 12 –Gly 13 –Arg 14 –Pro 15 –Phe 16 –Gly 17 –Arg 18 –Cys 19 ; The amino acid sequence (CGF2) is shown in SEQ ID NO:1, where a disulfide bond is formed between the 5th and 15th cysteine ​​residues; that is, its linear amino acid sequence is shown in SEQ ID NO:4: Cys 1 –Arg 2 –Arg 3 –Ala 4 –Gly 5 –Arg 6 –Ser 7 –Arg 8 –Gly 9 –Arg 10 –Ser 11 –Arg 12 –Gly 13 –Arg 14 –Cys 15 –Pro 16 –Phe 17 –Gly 18 –Arg 19 .

3. The antimicrobial synergistic peptide targeting bacterial G-quadruplexes according to claim 1 or 2, characterized in that, The antimicrobial synergistic peptide is CGF1 and / or CGF2.

4. The application of the antimicrobial synergistic peptide targeting bacterial G-quadruplexes as described in claim 1, characterized in that, The application of the antimicrobial synergistic peptide as a synergist for antimicrobial substances.

5. The application of the antimicrobial synergistic peptide targeting bacterial G-quadruplexes according to claim 4, characterized in that, The antibacterial substance is polymyxin B.

6. An antibacterial drug composition, characterized in that, The composition comprises an active ingredient and an adjuvant, wherein the active ingredient contains the antibacterial synergistic peptide as described in claim 1.

7. The antibacterial pharmaceutical composition according to claim 5, characterized in that, The active ingredients are the antimicrobial synergistic peptide and polymyxin antibiotics as described in claim 1 (concentration of 25 μM + 1 / 4 × MIC).

8. An application according to claim 4 or 6, characterized in that: The use of the antimicrobial synergistic peptide of claim 1 or the composition of claim 6 in the preparation of a medicament for the prevention and / or treatment of Gram-negative bacterial infections or drug-resistant Gram-negative bacterial lung infections.

9. An application according to claim 4 or 6, characterized in that: The use of the antimicrobial synergistic peptide of claim 1 or the composition of claim 6 in the preparation of a medicament for inhibiting biofilm formation by Gram-negative bacteria, reducing biofilm-associated bacterial load, or preventing biofilm formation on the surface of medical devices.

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