CN117138024A - Vancomycin-antibacterial peptide conjugate and application thereof - Google Patents

Abstract

The invention relates to a vancomycin-antimicrobial peptide conjugate and its application. After modifying the N-terminus of the antimicrobial peptide M2 by adding a cysteine, vancomycin is converted into a vancomycin-antimicrobial peptide conjugate through a bifunctional protein cross-linking agent sulfo-SMCC. The conjugation with the antimicrobial peptide modification is carried out through a two-step reaction. Vancomycin-antimicrobial peptide conjugates have significantly improved antibacterial activity compared with vancomycin, antimicrobial peptide M2, and mixtures of vancomycin and antimicrobial peptide M2, overcoming the resistance of Gram-positive bacteria to vancomycin, and Some Gram-negative bacteria also produce antibacterial activity, which increases the antibacterial spectrum of vancomycin. It also has stronger anti-biofilm capabilities, which can kill retained bacteria, inhibit bacterial swarming movement, and form a synergistic effect with antibiotics. Common sterilization.

Description

A kind of vancomycin-antimicrobial peptide conjugate and its application

Technical field

The invention relates to the technical field of biochemistry, and in particular to a vancomycin-antimicrobial peptide conjugate and its application.

Background technique

Since the discovery of antibiotics, they have played the most important role in the field of antibacterial and anti-infection. However, in recent years, with the abuse of traditional antibiotics in medical treatment and animal husbandry, more and more drug-resistant bacteria have continued to emerge, and the world has In the post-antibiotic era, microbial resistance has become a major challenge that seriously threatens human life and health.

According to reports, bacterial infections have become the second leading cause of death in humans worldwide, and infections caused by drug-resistant bacteria such as Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, and Klebsiella pneumoniae are even more of a threat. Human life and health are the top priority. Currently, more than 1 million people worldwide die from drug-resistant bacterial infections every year, and the World Health Organization predicts that this number will climb to 10 million by 2050. Therefore, in the post-antibiotic era, it is urgent to seek new and more effective drugs against drug-resistant bacteria.

Since its discovery in 1952, vancomycin has been widely used to treat serious infections with drug-resistant Staphylococcus aureus and other Gram-positive bacteria. Vancomycin mainly binds to 2 D-alanines of the cell wall peptidoglycan precursor lipopeptide. Combination with acid sequences inhibits cell wall synthesis and leads to bacterial death. However, continuous abuse has led to the emergence of many vancomycin-resistant bacteria. The main resistance mechanism is that the binding target of vancomycin, D-alanine, is replaced by D-lactic acid and D-serine substitution increases the steric hindrance of vancomycin binding to the target, reduces the binding affinity of vancomycin to the target, and reduces bacterial cell membrane permeability, which also leads to bacterial resistance to vancomycin.

Antimicrobial peptides are natural active polypeptides mainly derived from animals, plants and microorganisms in nature. They are short peptides composed of 10-50 amino acids connected through amide bonds. They mainly destroy bacterial cell membrane structures and bind to some intracellular targets. Inhibits DNA and protein synthesis and plays a bactericidal effect. To sum up, antimicrobial peptides have several advantages: wide sources, easy to obtain, strong and broad-spectrum antibacterial activity, unique mechanism, not easy to cause drug resistance, simple structure, and easy to modify. In addition, antimicrobial peptides also have certain immunomodulatory activity. Currently, there are more than 30 antimicrobial peptides in the clinical research stage. Among them, M2 is a derivative peptide derived from frog antimicrobial peptides and is used in the treatment of skin infectious diseases such as psoriasis and diabetic foot. In addition, it also shows good killing effect on viruses and tumor cells. In summary, in the post-antibiotic era, antimicrobial peptides have become the most promising antibacterial and anti-infective drugs to replace traditional antibiotics.

At present, some researchers have noticed that antimicrobial peptides may become a breakthrough in solving the problem of bacterial resistance, and the most commonly used method is the combination of antibiotics and antimicrobial peptides. This combination method can only achieve a simple superposition of the effects of antibiotics and antimicrobial peptides, but cannot solve the problem of bacterial resistance to antibiotics. Therefore, there is an urgent need for a method that can maximize the effectiveness of antimicrobial peptides and antibiotics in the sterilization process.

Contents of the invention

In order to solve the problem of unsatisfactory combined effects of antibiotics and antimicrobial peptides, and to maximize the joint effects of antibiotics and antimicrobial peptides, the present invention provides a vancomycin-antimicrobial peptide conjugate.

The first object of the present invention is to provide a vancomycin-antimicrobial peptide conjugate, which is composed of vancomycin and an antimicrobial peptide with an amino acid sequence as shown in SEQ ID NO.1. The cross-linking agent is covalently cross-linked.

Furthermore, the terminal of the antibacterial peptide is modified with cysteine.

Furthermore, the terminal lysine of the antibacterial peptide is acylated.

Further, all the amino acids in the modified antimicrobial peptide are L-type amino acids.

Further, the covalent cross-linking is achieved by the bifunctional protein cross-linking agent Sulfo-SMCC.

Further, the preparation method of the antimicrobial peptide modified body includes the following steps: chemical synthesis using solid-phase synthesis technology according to the amino acid sequence of the antimicrobial peptide modified body, purifying using preparative reverse high-performance liquid chromatography, and using high-resolution high-resolution liquid chromatography. Mass spectrometry was used for molecular weight identification.

Furthermore, vancomycin was coupled with the bifunctional protein cross-linking agent sulfo-SMCC through an amide bond to obtain the modified vancomycin Vm-SMCC, with a molecular weight of 1667.55 Da.

Further, the preparation method of the vancomycin modified Vm-SMCC includes the following steps: using preparative reverse-phase high-performance liquid chromatography for purification, and using high-resolution mass spectrometry for molecular weight identification.

Further, the antimicrobial peptide and the vancomycin modified Vm-SMCC are coupled to obtain the vancomycin-antimicrobial peptide conjugate Vm-M2.

Further, the preparation method of the vancomycin-antimicrobial peptide conjugate Vm-M2 includes the following steps: using preparative reverse-phase high-performance liquid chromatography for purification, and using high-resolution mass spectrometry for molecular weight identification.

The second object of the present invention is to provide the use of vancomycin-antimicrobial peptide conjugates in the preparation of antibacterial agents.

Further, the antibacterial agent is used to inhibit Gram-positive bacteria or Gram-negative bacteria.

Further, the Gram-positive bacteria include but are not limited to Staphylococcus aureus (such as Staphylococcus aureus CMCC26003, Staphylococcus aureus ATCC29213, methicillin-resistant Staphylococcus aureus ATCC43300, Staphylococcus aureus 15772, aureus Staphylococcus 15192, vancomycin-resistant Staphylococcus aureus 52 and vancomycin-resistant Staphylococcus aureus 11), Enterococcus faecalis (such as vancomycin-resistant Enterococcus faecalis ATCC 29212 and Enterococcus faecalis 2), Clostridium perfringens bacteria and Bacillus cereus.

Further, the Gram-negative bacteria include but are not limited to Escherichia coli (such as Escherichia coli ATCC25922 and Escherichia coli CMCC44102), Acinetobacter baumannii (such as Acinetobacter baumannii ATCC19606 and pan-drug resistant Acinetobacter baumannii 1 ), Klebsiella pneumoniae (Klebsiella pneumoniae 9883, pan-drug resistant Klebsiella pneumoniae 2, pan-drug resistant Klebsiella pneumoniae 3, pandrug-resistant Klebsiella pneumoniae 4), Pseudomonas aeruginosa bacteria (such as Pseudomonas aeruginosa ATCC27853, Pseudomonas aeruginosa CMCC10104, Pseudomonas aeruginosa 60357, Pseudomonas aeruginosa 52097 and pan-drug resistant Pseudomonas aeruginosa 5), Shigella flexneri (such as Shigella flexneri ATCC12022 and Shigella flexneri CMCC51571), Salmonella typhimurium (such as Salmonella typhimurium ATCC14028 and Salmonella typhimurium CICC21483), and Vibrio parahaemolyticus (such as Vibrio parahaemolyticus ATCC17802).

The third object of the present invention is to provide the application of vancomycin-antimicrobial peptide conjugates in antibacterial biofilms.

The fourth object of the present invention is to provide an antibacterial pharmaceutical composition, including the vancomycin-antibacterial peptide conjugate and antibiotics.

Further, the antibiotic is selected from one of polymyxin B, ciprofloxacin and gentamicin.

Beneficial effects of the present invention:

(1) The present invention couples vancomycin and antimicrobial peptide M2 through the bifunctional protein cross-linking agent sulfo-SMCC, and the obtained vancomycin-antimicrobial peptide conjugate Vm-M2 greatly improves the antibacterial activity of vancomycin. Effectively overcome the problem of vancomycin resistance of some Gram-positive bacteria. The conjugate Vm-M2 significantly expands the inherent antibacterial spectrum of vancomycin and has strong antibacterial activity against major Gram-negative pathogenic bacteria (including drug-resistant and pan-drug-resistant clinical isolates). In addition, at the same molar concentration, the conjugate exhibited more efficient bactericidal activity than vancomycin, antimicrobial peptide M2, and a mixture of vancomycin and antimicrobial peptide M2.

(2) The vancomycin-antimicrobial peptide conjugate Vm-M2 provided by the present invention is obtained through a two-step coupling reaction, and has convenient synthesis, easy acquisition of raw materials, and low synthesis cost. In addition, the conjugate also has the advantages of low hemolytic activity, high stability, and strong anti-biofilm activity. It can be used in medicine, cosmetics, aquaculture and other fields, greatly expanding the value of downstream applications.

Description of the drawings

Figure 1 shows the results of mass spectrometry and HPLC analysis of vancomycin modified Vm-SMCC;

Figure 2 is the mass spectrometry and HPLC analysis results of vancomycin-antimicrobial peptide conjugate Vm-M2;

Figure 3 is a synthesis route diagram of vancomycin-antimicrobial peptide conjugate Vm-M2;

Figure 4 shows the residual bactericidal effect of vancomycin-antimicrobial peptide conjugate Vm-M2;

Figure 5 shows the salt ion stability of vancomycin-antimicrobial peptide conjugate Vm-M2;

Figure 6 shows the effect of vancomycin-antimicrobial peptide conjugate Vm-M2 on bacterial swarming movement.

Detailed ways

The present invention will be further described below in conjunction with the accompanying drawings and specific examples, so that those skilled in the art can better understand and implement the present invention, but the examples are not intended to limit the present invention.

Experimental strains: Clinical isolates were collected from the Affiliated Hospital of Suzhou University.

Example 1: Chemical synthesis of vancomycin-antimicrobial peptide conjugate Vm-M2

Antimicrobial peptide M2 is a modified form of frog antimicrobial peptide, consisting of 22 amino acids, with the sequence: Gly ¹ Ile ² Gly ³ Lys ⁴ Phe ⁵ Leu ⁶ Lys ⁷ Lys ⁸ Ala ⁹ Lys ¹⁰ Lys ¹¹ Phe ¹² Gly ¹³ Lys ¹⁴ Ala ¹⁵ Phe ¹⁶ Val ¹⁷ Lys ¹⁸ Ile ¹⁹ Leu ²⁰ Lys ²¹ Lys ²² (SEQ ID NO. 1), in which the C-terminal lysine is amidated.

In order to facilitate coupling, the modified body C-M2 was designed using molecular modification methods based on the amino acid sequence of frog antimicrobial peptide M2. The amino acid sequence is Cys ¹ Gly ² Ile ³ Gly ⁴ Lys ⁵ Phe ⁶ Leu ⁷ Lys ⁸ Lys ⁹ Ala. ¹⁰ Lys ¹¹ Lys ¹² Phe ¹³ Gly ¹⁴ Lys ¹⁵ Ala ¹⁶ Phe ¹⁷ Val ¹⁸ Lys ¹⁹ Ile ²⁰ Leu ²¹ Lys ²² Lys ²³ (SEQ ID NO.2), in which the C-terminal lysine is amidated and modified. The specific preparation method is as follows :

Ⅰ. According to the above amino acid sequence of C-M2, use an automatic peptide synthesizer (433A, Applied Biosystems) to synthesize its entire sequence, and use HPLC reverse-phase column chromatography to desalt;

Ⅱ. Molecular weight determination uses matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF) for identification;

Ⅲ. Use high performance liquid chromatography (HPLC) to identify the purity of purified C-M2.

The secondary amine of vancomycin and the succinimide ester on the bifunctional protein cross-linker sulfo-SMCC undergo an amide condensation reaction in a weakly alkaline environment to form a stable amide bond. Vancomycin and sulfo-SMCC are used in equal amounts. reaction. Dissolve vancomycin in PBS buffer (pH=8.16). After vancomycin is completely dissolved, use a pH meter to measure the pH value of the solution. If the pH value is lower than 8.16, you need to use NaOH (0.1mol/L) to adjust the pH value to 8.16. Dissolve sulfo-SMCC with an appropriate amount of DMSO, then mix the two, stir and react at room temperature for 3 hours, monitor the reaction with reverse-phase high-performance liquid chromatography, and after the reaction is complete, purify the product with preparative HPLC and perform analysis with high-resolution mass spectrometry. The results of molecular weight identification, liquid phase and mass spectrometry analysis are shown in Figure 1. We named the reaction product obtained in this step: Vm-SMCC.

The maleimide group at the other end of the bifunctional protein cross-linker sulfo-SMCC can react with the sulfhydryl group on the N-terminal cysteine of the antimicrobial peptide in a weakly acidic environment (pH=5.5) to form a stable thioether bond. . Vm-SMCC reacts with antibacterial peptide C-M2 in equal amounts. The two are dissolved in PBS buffer (pH=5.5). After complete dissolution, use a pH meter to measure the pH value of the solution. If the pH is lower than 5.5, NaOH (0.1mol/L) solution needs to be used to adjust the pH to 5.5. The reaction was stirred at room temperature for 3 hours, and the reaction was monitored by reverse-phase high-performance liquid chromatography. After the reaction was complete, the product was purified by preparative HPLC, and the molecular weight was identified by high-resolution mass spectrometry. The liquid chromatography and mass spectrometry analysis results are shown in Figure 2. The final product is named: Vm-M2.

The synthesis roadmap is shown in Figure 3.

Example 2: Pharmacological experiments on vancomycin-antimicrobial peptide conjugate Vm-M2

1. Determination of Vm-M2 antibacterial activity:

(1) Determination of Vm-M2 minimum inhibitory concentration (Minimum Inhibitory Concentration) (2-fold dilution method).

11 strains of Gram-positive bacteria and 18 strains of Gram-negative bacteria were selected for the MIC determination test. The test strains were inoculated into MH liquid culture medium (Meilun Biotechnology), cultured with shaking at 37°C to the logarithmic growth phase, and then inoculated with fresh MH. Liquid medium: Dilute the culture medium cultured to logarithmic growth phase to 2×10 ⁵ cfu/mL for later use.

Pre-add 90 μL of MH liquid culture medium to each well of a sterile 96-well plate, and then add 10 μL of vancomycin, M2 and Vm that were diluted to a certain concentration with MH liquid culture medium and filtered through a 0.22 μm pore filter membrane into the first well. -M2 sample solution. In addition, a physical mixing group of vancomycin and antimicrobial peptide M2 is also set up. The system is 80μL MH+10μL vancomycin+10μL M2. After mixing, 50μL is added to the second well and diluted sequentially ( Table 1), aspirate 50 μL from well 8 and discard.

Table 1 Dilution method

Mix each of the above tubes and place them at 37°C, incubate with slow shaking at 110 rpm for 16 hours, and measure the light absorption at a wavelength of 600 nm. The minimum inhibitory concentration is the lowest sample concentration at which no bacterial growth can be seen.

Table 2 Antibacterial activity of vancomycin-antimicrobial peptide conjugate Vm-M2

MIC: Minimum inhibitory concentration. The above results are the average of three independent repeated experiments.

It can be seen from Table 2 that the conjugate Vm-M2 has stronger and broader-spectrum antibacterial activity than vancomycin alone and antimicrobial peptide alone. For Gram-positive bacteria, Vm-M2 overcomes the vancomycin resistance of Staphylococcus aureus and Enterococcus faecalis, and many Gram-negative bacteria that are not sensitive to vancomycin, especially some pan-drug resistant bacteria. Clinically isolated strains, the conjugate Vm-M2 also showed strong antibacterial activity, and its antibacterial activity was higher than that of the antibacterial peptide M2 alone. At the same time, a physical mixing group of vancomycin and antimicrobial peptides was used as a control. The results showed that physical mixing alone did not produce stronger antibacterial activity, which further demonstrated that chemical coupling produced better antibacterial effects. The GM value is the arithmetic mean of the MIC values of antibacterial drugs against test bacteria. The lower the GM value, the stronger the antibacterial activity. As shown in Table 2, the GM value of vancomycin is 56.48 μM and the GM value of antimicrobial peptide M2 is 8.66. μM, the GM value of the physical mixing group is 4.40 μM, and the GM value of the conjugate Vm-M2 is 2.60 μM. Therefore, it can be considered that the overall antibacterial activity of the conjugate is increased by 21.72 times compared to vancomycin, and compared to the antibacterial peptide M2 alone It is also improved by 3.33 times, which is 1.69 times higher than that of the physical hybrid group. This shows that the conjugate Vm-M2 has excellent antibacterial activity and clinical application value.

(2) Bactericidal kinetics of vancomycin-antimicrobial peptide conjugate Vm-M2

Escherichia coli ATCC25922 and Staphylococcus aureus CMCC26003 were cultured in MH liquid medium at 37°C for 6 hours, and then diluted with fresh MH liquid medium into a bacterial suspension of 10 ⁶ cfu/mL. Vancomycin, M2 and Vm-M2 samples dissolved in sterile deionized water were added to the bacterial suspension to a final concentration of 4 μM. Place the administered bacterial liquid in an incubator at 37°C and incubate with shaking in an incubator. Take 10 μL of bacterial liquid and dilute it 1000 times at 0, 15, 30, 60, 120 and 180 minutes, and then take 50 μL of the diluted bacterial liquid and apply it on Spread onto MH solid medium, incubate overnight in a 37°C incubator and count colonies. Because vancomycin is ineffective against Gram-negative bacteria, polymyxin B was used as a positive control and sterilized PBS as a negative control for E. coli ATCC25922.

Table 3 Sterilization speed of vancomycin, M2 and Vm-M2 (E. coli ATCC25922)

Table 4 Bactericidal speed of vancomycin, M2 and Vm-M2 (Staphylococcus aureus CMCC26003)

As shown in Tables 3 and 4, for Staphylococcus aureus CMCC26003, the conjugate Vm-M2 showed extremely fast sterilization speed. It could kill all living bacteria within 30 minutes. The antimicrobial peptide M2 was still effective at 120 minutes. There are still a small amount of viable bacteria. Vancomycin's sterilization speed is very slow. It cannot effectively kill bacteria within 180 minutes and can only inhibit bacterial growth. For E. coli ATCC25922, the conjugate Vm-M2 exhibited a bactericidal rate comparable to the positive control polymyxin B, killing all bacteria within 30 minutes. Vancomycin, on the other hand, does not have any bactericidal or bacteriostatic activity.

(3)Residual bacteria experiment

After being hit by antibacterial drugs, the vast majority of bacteria will die quickly, but there are still a small number of subpopulations that cannot be completely killed. Unlike traditional drug-resistant bacteria, they have not undergone changes in their genetic sequences. , this subgroup of bacteria is called retention bacteria, which is a dormant phenotypic variation of bacteria. Although drugs can bind to their targets, they cannot exert a killing effect on bacteria. Therefore, retention bacteria are resistant to almost all antibacterial drugs. Tolerance, but its resistance characteristics cannot be passed on to offspring. After collection and reculture, its resistance characteristics disappear and the MIC value does not change significantly. In recent years, more and more clinical cases have shown that retained bacteria are an important reason for the persistence of chronic infectious diseases and the recurrence of symptoms. Research on the production mechanism, prevention, and removal of retained bacteria has also received increasing attention. In order to test the killing effect of the conjugate Vm-M2 on retained bacteria, Staphylococcus aureus CMCC26003 was selected and cultured in a 37°C shaker at 200rpm for 6 hours until the logarithmic phase of growth, and diluted to 10 ⁸ CFU with MH liquid medium /mL, add 900 μL into a centrifuge tube, add ciprofloxacin (CIP) with a final concentration of 20 times the MIC to the bacterial solution, and incubate on a shaking table at 37°C for 8 hours, at 0h, 2h, 4h, 6h, 8h, respectively. Take 10 μL of bacterial solution and dilute it 1000 times, and take 50 μL of the diluted bacterial solution and apply it to the plate. At the 8h time point, take out 300 μL of bacterial liquid, centrifuge and discard the culture medium, wash 3 times with sterile PBS, remove ciprofloxacin, resuspend with 300 μL MH liquid culture medium, divide into 3 equal parts, 100 μL per tube, add final solution respectively. Vancomycin, M2 and Vm-M2 samples with a concentration of 4 μM were then sampled and diluted 1000 times at the 24h and 48h time points, plated, and placed in a 37°C incubator for 16h, and the plates were taken out for colony counting. This experiment also set up a physical mixing group in which vancomycin and antimicrobial peptide M2 were co-incubated at 4 μM each.

It can be seen from the experimental results in Figure 4 that after 8 hours of incubation with ciprofloxacin, stable and resistant bacteria were formed. If ciprofloxacin was continued to be incubated for 48 hours, the number of viable bacteria remained almost unchanged. The bactericidal effect of antibiotic peptide M2 on retained bacteria was not significant. After incubation for 48 hours, the number of retained bacterial colonies only decreased by 1 order of magnitude. Antimicrobial peptide M2 had a stronger bactericidal effect on retained bacteria than vancomycin. After incubation for 48 hours, the number of retained bacteria The number of bacterial colonies decreased by 3 orders of magnitude. The bactericidal effect of the physical mixture group on retained bacteria was similar to that of the antimicrobial peptide M2 alone. The conjugate Vm-M2 showed a strong bactericidal effect on retained bacteria. The number of bacterial colonies after incubation for 48 hours decreased. More than 4 orders of magnitude, statistical analysis showed that when the drug was administered and incubated for 48 hours, there was a significant difference in the number of retained bacterial colonies in the conjugate group and the vancomycin alone group, the antimicrobial peptide alone group and the physical mixture group. Therefore, the bactericidal effect of the conjugate on retained bacteria was significantly stronger than that of vancomycin and antimicrobial peptide M2 alone, and the bactericidal effect of the physical mixture of vancomycin and antimicrobial peptides on retained bacteria was not as good as that of the conjugate group.

2. Determination of hemolytic activity of vancomycin-antimicrobial peptide conjugate Vm-M2

The collected human blood was mixed with Aldrin's solution for anticoagulation, washed twice with physiological saline and resuspended into a suspension of 10 ⁷ -10 ⁸ cells/mL. The above-mentioned diluted red blood cell suspension was mixed with M2 and Vm-M2 samples dissolved in physiological saline, incubated at 37°C for 30 min, and then centrifuged at 1000 rpm for 5 min. The absorbance value of the supernatant was measured at 540 nm. Use normal saline for the negative control, and use Triton X-100 for the positive control. The hemolysis percentage is calculated according to the following formula: Hemolysis percentage H% = A _sample - A _{negative control} /A _{positive control} × 100%. The results are shown in Table 5.

Table 5 Hemolytic activity of M2 and Vm-M2

The results show that when the sample concentration is close to 8 times the GM (arithmetic mean of MIC) value, the hemolysis percentage of Vm-M2 is 8.16%. At the same concentration, the hemolysis percentage of M2 is 13.45%, indicating that the conjugate Vm-M2 is more effective than the individual antimicrobial peptide. M2 has lower hemolytic activity and is less likely to cause rupture and lysis of mammalian red blood cells. Especially within the range of antibacterial activity, it is highly safe.

3. Experimental study on the salt ion stability of vancomycin-antimicrobial peptide conjugate Vm-M2

There are many common salt ions in the human body. These salt ions are indispensable for maintaining normal life activities of the human body. Most of these salt ions are positively charged. The conjugate Vm-M2 is positively charged because it is coupled with antimicrobial peptides, and bacteria The lipid component of the cell membrane is negatively charged. Studies have shown that positively charged salt ions will compete with antimicrobial peptides to bind to the bacterial cell membrane, causing the antibacterial activity of the antimicrobial peptides to weaken or disappear. Therefore, the salt ions of the detection conjugate Vm-M2 are stable. The resistance is necessary. Escherichia coli ATCC25922 and vancomycin-resistant Staphylococcus aureus VRSA52 were selected, respectively, and cultured in MH liquid medium (Suzhou Alpha Biotechnology Co., Ltd.) at 37°C for 12 hours. Then they were diluted to 10 ⁵ CFU/ml with MH liquid medium containing physiological salt ion concentration (NaCl 150mM, KCl 4.5mM, CaCl ₂ 2mM, MgCl ₂ 1mM, ZnCl ₂ 8μM, FeCl ₃ 4μM, NH ₄ Cl 6μM). Vm-M2 samples with different concentration gradients were prepared using MH liquid culture medium containing corresponding salt ion concentrations. The MIC values of Vm-M2 against Escherichia coli ATCC25922 and vancomycin-resistant Staphylococcus aureus VRSA52 were determined using the 2-fold dilution method to determine the effects of different salt ions on the antibacterial activity of vancomycin-antimicrobial peptide conjugates.

As shown in Figure 5, the conjugate Vm-M2 has strong salt ion tolerance. Under the reference human physiological salt ion concentration (NaCl 150mM, KCl 4.5mM, CaCl ₂ 2mM, MgCl ₂ 1mM, ZnCl ₂ 8μM, FeCl ₃ 4μM, NH ₄ Cl 6μM), the antibacterial activity of the conjugate Vm-M2 remains basically unchanged. (MIC value does not increase more than double).

Example 3: Effect of vancomycin-antimicrobial peptide conjugate Vm-M2 on bacterial swarming effect

Bacterial swarming movement is a flagella-dependent migration movement of bacteria from the inoculation point to the surroundings in a group manner on the surface of the culture medium. It is a self-adaptive response behavior of bacteria to their environment, which is of great significance to bacterial resistance and biofilm formation processes. . In order to study the effect of the conjugate Vm-M2 on the bacterial swarming effect, we conducted the following experiments with culture media: tryptone (10g/L), NaCl (10g/L), yeast extract (5g/L), agar ( 0.3%), dissolve each component in deionized water, and after high-temperature sterilization, add vancomycin, M2 and Vm-M2 with final concentrations of 0.5 μM, 1 μM, and 2 μM while hot, as well as vancomycin and antibacterial agents with the same final concentration. For the peptide physical mixing group, add an equal volume of PBS buffer as a blank control, place it in a six-well plate and cool it for later use. Take Escherichia coli ATCC25922 and Staphylococcus aureus CMCC26003, and use MH liquid culture medium (Suzhou Alpha Biotechnology Co., Ltd.) respectively. Cultivate at 37°C for 6 hours to the logarithmic phase of growth, dilute it with MH liquid medium to 10 ⁶ CFU/mL, pipet 5 μL, drop it on the cooled solid medium, and then place it in a 37°C incubator for 24 hours and take it out. Take pictures and measure the colony area using imageJ software.

As shown in Figure 6, for E. coli ATCC25922, the colony area of the vancomycin group was not significantly different from that of the PBS blank control group, and it had no inhibitory effect on the swarming movement of the tested bacteria. For Staphylococcus aureus CMCC26003, vancomycin had no inhibitory effect on the colony movement of the tested bacteria. The inhibitory effect on swarming motility is dose-dependent, but its inhibitory effect is weaker than that of the antimicrobial peptide M2 alone and the conjugate Vm-M2. In addition, for the above two strains of test bacteria, at the same dosage concentration (2 μM), the conjugate Vm The colony area of the -M2 group was significantly smaller than the antimicrobial peptide M2 administration group alone and the physical mixing group. It is worth noting that when the Vm-M2 administration concentration was 2 μM, no colonies of E. coli ATCC25922 were observed on the culture medium. , so the colony area is 0, which may be because all E. coli have been killed at this concentration. To sum up, the conjugate Vm-M2 can effectively block the signal transduction between bacterial cells and effectively inhibit the clustering of bacteria. Motile behavior, which will help inhibit the development of biofilm and drug resistance, further illustrates the huge application value of the conjugate Vm-M2.

Example 4: Study on the anti-biofilm activity of vancomycin-antimicrobial peptide conjugate Vm-M2

1. Biofilm scavenging activity assay

Take out the stored strains from the -80°C refrigerator, quickly thaw them in a 37°C water bath, dip a little liquid into the inoculating loop, and draw lines in four areas in a zigzag shape on the LB solid medium, starting from the end of the previous line. , cultivate at a constant temperature of 37°C until a single colony grows; pick a single colony in a sterile liquid MHB medium, and culture it with shaking at 37°C and 200 rpm until the logarithmic growth phase; detect the concentration of the bacterial solution and dilute it to 1×10 ⁷ CFU/mL . Add 100 μL of the above bacterial solution to a sterile 96-well plate and incubate at 37°C for 48 hours to form a biofilm. Aspirate the bacterial fluid from each well and wash three times with PBS. Add 100 μL of diluted vancomycin, M2 and conjugate Vm-M2 samples to each well. The physical mixing group is 50 μL vancomycin + 50 μL M2. The final concentration of the sample is 1 μM, 2 μM, 4 μM and 8 μM. After adding the sample, place it at 37 Incubate at constant temperature for 24 hours. Add crystal violet dyeing solution (0.1%) to each well. After dyeing for 30 minutes, aspirate the dyeing solution, wash three times with sterile PBS, and air-dry in an ultra-clean bench. Add 100 μL of absolute ethanol to each well and let it stand for 20 minutes to dissolve the crystal violet. Detect the OD value at a UV wavelength of 560nm, and set up three parallel experiments. Use the following formula to calculate the percentage of biofilm formation (BiofilmRetention%, BR%): BR (%) = 100% - [100% × (F ₀ -F _drug )/F ₀ ]. PBS was selected as a negative control in this experiment. The measured value is regarded as the maximum biofilm residual amount. Biofilm Retention%, BR% is the remaining percentage of biofilm, _F0 is the absorbance value of the PBS-treated group, and _Fdrug is the absorbance value of the drug-treated group.

Table 6 Biofilm-clearing activities of vancomycin, M2 and Vm-M2 (E. coli ATCC25922)

Table 7 Biofilm-clearing activities of vancomycin, M2 and Vm-M2 (Staphylococcus aureus VRSA52)

2. Biofilm inhibitory activity assay

Take out the stored strains from the -80°C refrigerator, quickly melt them in a 37°C water bath, dip a little liquid into a zigzag pattern on the LB solid medium, and cultivate at a constant temperature of 37°C until a single colony grows; pick a single colony without The bacteria were cultured in liquid MHB culture medium at 37°C with shaking at 200 rpm to the logarithmic growth phase; the concentration of the bacterial solution was detected and diluted to 2×10 ⁷ CFU/mL. Add 50 μL of the above bacterial solution to the sterile 96-well plate, and add 50 μL of diluted vancomycin, M2 and conjugate Vm-M2 samples to each well. The physical mixing group is 25 μL vancomycin + 25 μL M2, and the final concentration of the sample is 1 μM. , 2μM, 4μM and 8μM, incubate at 37℃ for 48h. Aspirate the bacterial liquid from each well, wash it three times with PBS, and place the plate on a clean bench to ventilate and dry it. Add crystal violet dyeing solution (0.1%) to each well. After dyeing for 30 minutes, aspirate the dyeing solution, wash three times with sterile PBS, and air-dry in an ultra-clean bench. Add 100 μL of absolute ethanol to each well and let it stand for 20 minutes to dissolve the crystal violet. Detect the OD value under UV wavelength 560nm. Calculate the percentage of biofilm formation (Biofilm Formation%, BF%) using the following formula: BF (%) = 100% - [100% × (F ₀ -F _drug )/F ₀ ], where PBS (F ₀ ) is the negative control , the measured value is regarded as the maximum amount of biofilm formation. Biofilm Formation%, BF% is the percentage of biofilm formation, and F _drug is the absorbance value of the drug treatment group.

Table 8 Biofilm inhibitory activity of vancomycin, M2, Vm-M2 (E. coli ATCC25922)

Table 9 Biofilm inhibitory activity of vancomycin, M2 and Vm-M2 (Staphylococcus aureus VRSA52)

As shown in Tables 6, 7, 8, and 9, for E. coli ATCC25922, vancomycin-resistant Staphylococcus aureus VRSA52, vancomycin has no biofilm clearance and inhibitory activity, and both antimicrobial peptide M2 and conjugate Vm-M2 show Concentration-dependent anti-biofilm activity. Interestingly, at the same dosage concentration, the conjugate Vm-M2 showed stronger biofilm clearance and inhibitory activity than the antibacterial peptide M2 alone. The anti-biofilm activity of the physical mixture group was also Less than the conjugate group. Therefore, it can be considered that the conjugate Vm-M2 has good application potential in chronic refractory diseases caused by bacterial infections.

Example 5: Determination of the synergistic antibacterial effect of vancomycin-antimicrobial peptide conjugate Vm-M2 and traditional antibiotics

Weigh the antibiotics (meropenem, polymyxin B, ciprofloxacin and gentamicin), dissolve them into a 2 mg/mL solution with sterile water, dilute them in sequence to obtain a concentration of 8-1/64 MIC, add A total of 11 concentrations of the solvent are used for future use. The conjugate Vm-M2 is prepared with sterile water to a concentration of 4×MIC, and then diluted again until it reaches 4-1/16 MIC. A total of 8 concentrations of the solvent are added for use. Dilute the bacterial solution to 5×10 ⁵ CFU/mL for later use. Add 90 μL of diluted bacterial solution to the 96-well plate; add antibiotics to the bacteria, 5 μL per well, one concentration in each column, 11 columns in total: add conjugate Vm-M2 to the bacterial solution, 5 μL per well, each One concentration per line, 8 lines in total; find MICA, MICB, A,,B to calculate FIC, FIC=FMICA+FMICB=A/MICA+B/MICB (A, B represent the concentration at the best collection point of the combination of two drugs , MICA, MICB represent the MIC when the two drugs are used alone), there is synergy when FIC＜0.5, there is antagonism when FIC＞4 (including 4), and the two drugs have additive effects when 0.5＜FIC＜2 (including 0.5). When 2＜FIC＜4 (including 2), the two drugs have unrelated effects, and the dosing method is as shown in the table below.

Table 10 Synergistic antibacterial effect of Vm-M2 and traditional antibiotics

Escherichia coli ATCC25922 and vancomycin-resistant Staphylococcus aureus VRSA52 were selected as test bacteria to detect the synergistic antibacterial effects of the above four clinical first-line antibiotics and the conjugate Vm-M2. According to the experimental results, the conjugate has synergistic effects with other antibiotics except for meropenem, which may be related to the inherent mechanism of action of different antibiotics. Overall, the conjugate has a synergistic antibacterial effect with most of the antibiotics used in clinical first-line applications, demonstrating its great potential in clinical combination therapy.

The above-described embodiments are only preferred embodiments to fully illustrate the present invention, and the protection scope of the present invention is not limited thereto. Equivalent substitutions or transformations made by those skilled in the art on the basis of the present invention are within the protection scope of the present invention. The protection scope of the present invention shall be determined by the claims.

Claims (10)

1. A vancomycin-antibacterial peptide conjugate, characterized in that: the vancomycin-antibacterial peptide conjugate is obtained by covalent crosslinking of vancomycin and antibacterial peptide with an amino acid sequence shown as SEQ ID NO. 1.

2. The vancomycin-antibacterial peptide conjugate of claim 1, wherein: the end of the antibacterial peptide is modified with cysteine.

3. The vancomycin-antibacterial peptide conjugate of claim 2, wherein: the covalent crosslinking is achieved by the bifunctional protein crosslinking agent Sulfo-SMCC.

4. Use of a vancomycin-antibacterial peptide conjugate according to any one of claims 1-3 for the preparation of an antibacterial agent.

5. The use according to claim 4, characterized in that: the antibacterial agent is used for inhibiting gram-positive bacteria or gram-negative bacteria.

6. The use according to claim 5, characterized in that: the gram positive bacteria comprise at least one of staphylococcus aureus, enterococcus faecalis, clostridium perfringens and bacillus cereus.

7. The use according to claim 5, characterized in that: the gram negative bacteria include at least one of Escherichia coli, acinetobacter baumannii, klebsiella pneumoniae, pseudomonas aeruginosa, shigella flexneri, salmonella typhimurium and Vibrio parahaemolyticus.

8. Use of a vancomycin-antibacterial peptide conjugate according to claims 1-3 for antibacterial biofilms.

9. An antibacterial pharmaceutical composition characterized in that: the antibacterial pharmaceutical composition comprises the vancomycin-antibacterial peptide conjugate of claims 1-3 and an antibiotic.

10. The antimicrobial pharmaceutical composition according to claim 9, wherein: the antibiotic is selected from one of polymyxin B, ciprofloxacin and gentamicin.