Use of antibacterial peptide KP4 in preparation of medicine for resisting infection of klebsiella pneumoniae with carbapenem-resistant

Abstract

The application relates to application of an antibacterial peptide KP4 in preparation of a medicine for resisting infection of carbapenem-resistant Klebsiella pneumoniae, and belongs to the technical field of medicines. The application provides an antibacterial peptide, and the amino acid sequence of the antibacterial peptide is shown in any one of SEQ ID NO: 1-10. Ten antibacterial peptides with the amino acid sequences shown in SEQ ID NO: 1-10 are constructed, and are respectively named as KP1, KP3, KP4, KP7, KP8, KP9, KP10, KP17, KP18 and KP19. The antibacterial peptides have certain inhibiting effects on the carbapenem-resistant Klebsiella pneumoniae, and the antibacterial peptide KP4 has the strongest inhibiting effect on the carbapenem-resistant Klebsiella pneumoniae.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, and in particular to the application of the antimicrobial peptide KP4 in the preparation of a drug for treating carbapenem-resistant Klebsiella pneumoniae infection. Background Technology

[0002] Klebsiella pneumoniae ( Klebsiella pneumoniae Klebsiella pneumoniae is an important Gram-negative opportunistic pathogen, known for its thick capsule and polysaccharide biofilm, which constitutes a physical barrier against host immunity and antibiotic treatment. In recent years, carbapenem-resistant Klebsiella pneumoniae has emerged as a significant threat. Klebsiella pneumoniae The global prevalence of CRKP has become a significant challenge for clinical infection control. CRKP strains typically carry carbapenemase genes such as blaKPC and blaNDM, making them resistant to most β-lactam antibiotics, including carbapenems, and often only sensitive to a few "last-line" antibiotics such as polymyxins and tigecycline. However, the increasing hepatotoxicity and nephrotoxicity of these drugs, heterogeneous resistance, and high treatment failure rates have led to a high mortality rate (40%-50%) for CRKP infections, especially bacteremia and pneumonia.

[0003] Antimicrobial peptides are considered one of the most promising antibiotic alternatives due to their broad-spectrum antibacterial activity, rapid bactericidal speed, low cytotoxicity to mammalian cells, and low likelihood of developing resistance. However, their in vivo application faces numerous challenges, such as poor stability and short duration of action due to easy degradation by in vivo proteases, and high synthesis and purification costs (especially for antimicrobial peptides with complex structures), hindering their large-scale production and clinical application. There is an urgent need in this field for a novel anti-infective agent that can effectively penetrate the CRKP capsule and biofilm, possesses both highly efficient bactericidal and potent endotoxin neutralization capabilities, and exhibits good in vivo antimicrobial efficacy to address the treatment challenges of CRKP infection. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of the prior art and provide the application of antimicrobial peptide KP4 in the preparation of drugs for treating carbapenem-resistant Klebsiella pneumoniae infections.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows: In a first aspect, the present invention provides an antimicrobial peptide, the amino acid sequence of which is shown in any one of SEQ ID NO: 1-10.

[0006] This invention constructed 10 antimicrobial peptides with amino acid sequences as shown in SEQ ID NO: 1-10, and named them KP1, KP3, KP4, KP7, KP8, KP9, KP10, KP17, KP18, and KP19, respectively. Experiments demonstrated that these 10 antimicrobial peptides all exhibited inhibitory effects against carbapenem-resistant Klebsiella pneumoniae.

[0007] As a preferred embodiment of the first aspect, the amino acid sequence of the antimicrobial peptide is shown in SEQ ID NO: 3. Experiments have shown that the antimicrobial peptide with the amino acid sequence shown in SEQ ID NO: 3 (i.e., antimicrobial peptide KP4) exhibits the strongest inhibitory effect against carbapenem-resistant Klebsiella pneumoniae, with a MIC of 8-16 μM, while other antimicrobial peptides show less antibacterial activity than KP4. Therefore, antimicrobial peptide KP4 is the preferred embodiment of the present invention.

[0008] Secondly, the present invention provides the use of the antimicrobial peptide in the preparation of a medicament against carbapenem-resistant Klebsiella pneumoniae infection.

[0009] Since the antimicrobial peptide of this invention has a strong inhibitory effect on carbapenem-resistant Klebsiella pneumoniae, it can be used as a drug to treat carbapenem-resistant Klebsiella pneumoniae infection and achieve the purpose of treating lung infection.

[0010] Thirdly, the present invention provides a drug for combating carbapenem-resistant Klebsiella pneumoniae infection, the drug comprising the antimicrobial peptide described in the first aspect.

[0011] As a preferred embodiment of the third aspect, the drug further includes pharmaceutically acceptable excipients.

[0012] As a preferred embodiment of the third aspect, the excipients include fillers, binders, disintegrants, lubricants, antioxidants, and coating materials.

[0013] As a preferred embodiment of the third aspect, the filler is at least one of lactose, microcrystalline cellulose, and starch.

[0014] As a preferred embodiment of the third aspect, the disintegrant is at least one of sodium carboxymethyl starch, low-substituted hydroxypropyl cellulose, and cross-linked polyvinylpyrrolidone.

[0015] As a preferred embodiment of the third aspect, the lubricant is at least one of magnesium stearate, micronized silica gel, and talc.

[0016] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention constructs a series of novel antimicrobial peptides with different amino acid sequences, exhibiting antimicrobial activity against carbapenem-resistant Klebsiella pneumoniae, thus demonstrating efficacy against carbapenem-resistant Klebsiella pneumoniae infection. Among them, antimicrobial peptide KP4 exhibits the strongest antimicrobial activity. Compared with polymyxin B, antimicrobial peptide KP4 shows significantly better therapeutic effects. This invention enriches the antimicrobial peptide library against carbapenem-resistant Klebsiella pneumoniae. Attached Figure Description

[0017] Figure 1 This is a schematic diagram of the molecular structure of the antimicrobial peptide KP4; Figure 2 A schematic diagram illustrating the concentration-dependent binding of antimicrobial peptide KP4 to lipopolysaccharide; Figure 3 A schematic diagram showing the results of antimicrobial peptide KP4 treatment for carbapenem-resistant Klebsiella pneumoniae acute lung infection (A: comparison of lung bacterial content; B: comparison of blood bacterial content; C: comparison of inflammatory factor TNF-α levels). Detailed Implementation

[0018] To better illustrate the purpose, technical solution, and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments.

[0019] Example 1: Investigation of the antimicrobial activity of different antimicrobial peptides against carbapenem-resistant Klebsiella pneumoniae This embodiment uses in vitro antibacterial experiments to investigate the antibacterial effects of different antimicrobial peptides (KP1, KP3, KP4, KP8, KP9, KP10, KP17, KP178, and KP19) on carbapenem-resistant Klebsiella pneumoniae standard strains (ATCC BAA 2146, 1705, and 3063). The amino acid sequences of the antimicrobial peptides are shown in Table 1. Table 1. Amino acid sequences of a series of antimicrobial peptides Note: The symbol “()” in the table represents a branch.

[0020] 1. Experimental Method: CRKP strains stored at -20℃ were streaked onto MHA (Mueller-Hinton Agar) solid medium and cultured at 37℃. Single colonies were then inoculated into MHB (Mueller-Hinton Broth) liquid medium. The cultures transferred to MHB were cultured at 37℃ and 220 rpm until the logarithmic growth phase. The concentration was adjusted to OD using MHB liquid medium. 600 nm=0.4; finally, further dilution was performed with MHB solution to obtain a bacterial suspension with a concentration of OD=0.4.

[0021] In 96-well plates, the 2.56 mM antimicrobial peptide was serially diluted in 0.2% BSA solution to 1-64 μM (specific antimicrobial peptide concentrations: 1 μM, 2 μM, 4 μM, 8 μM, 16 μM, 32 μM, 64 μM), with three replicates of the antimicrobial peptide per plate.

[0022] Add 50 µL of the prepared bacterial culture to the wells of a 96-well plate containing the antimicrobial peptide. In the last row, inoculate 50 µL of the bacterial culture into 6 wells without antimicrobial peptide as a positive control. Add 50 µL of fresh MHB medium to the remaining 6 wells without any antimicrobial peptide as a negative control. After thorough mixing, incubate at 37°C for 24 h.

[0023] Under normal growth conditions in both the negative and positive control wells, the clarified wells treated with the minimum inhibitory concentration of the antimicrobial peptide were visually observed after 24 hours to determine the corresponding minimum inhibitory concentration (MIC) of the antimicrobial peptide series. The experiment was performed in three independent replicates.

[0024] 2. Experimental Results: The specific experimental results are shown in Table 2: Table 2 shows the minimum inhibitory concentrations (MICs) of a series of antimicrobial peptides. Table 2 shows the minimum inhibitory concentrations (MICs) of 10 antimicrobial peptides against carbapenem-resistant Klebsiella pneumoniae (ATCC BAA 2146, 1705, and 3063). The results show that antimicrobial peptide KP4 has stable and strong inhibitory activity against three carbapenem-resistant Klebsiella pneumoniae strains (MIC of 8-16 μM), making it a superior candidate antimicrobial peptide for anti-carbapenem-resistant Klebsiella pneumoniae.

[0025] Example 2: Preparation of antimicrobial peptide KP4 This embodiment uses peptide solid-phase synthesis technology to prepare antimicrobial peptide KP4. The specific steps are as follows: 1. Weigh 2-CTC resin, soak in DCM for 1 hour to swell, and wash 3 times with DMF; 2. Take 1 eq of the first protected amino acid and 1.5 eq of DIEA, and then react with the resin in DMF as solvent for 2 hours; 3. Drain the resin, clean with DMF 3 times, add methanol + DIEA to the end cap for 1 hour; 4. Clean the resin, remove Fmoc with 20% piperidine DMF solution, and react for 10 min × 2 times; 5. Wash the resin, add 3 eq of the second amino acid, 3 eq of HOBT, and 3 eq of DIC, using DMF as solvent, and react for 1.5 hours. 6. Repeat steps 4-5 until the last amino acid residue at the N-terminus is attached, and remove the Fmoc from the N-terminus; 7. Wash the resin and dry it. 8. 95% TFA + 2% Tis + 2% EDT + 1% H2O (mass percentage) cleaves the resin and peptide side chain protecting groups, reacts for 2 hours; 9. Filter the resin, wash the filtrate with ice-cold ether, centrifuge and keep the precipitate, which is the crude product; 10. Purify by liquid chromatography and freeze-dry to obtain antimicrobial peptide KP4.

[0026] Table 3 Comparison of Abbreviations and Chinese Names Example 3: Experimental Investigation on the Binding of Antimicrobial Peptide KP4 with Lipopolysaccharide of Carbapenem-Resistant Klebsiella pneumoniae This embodiment employs a lipopolysaccharide (LPS) binding experiment to investigate the interaction between the antimicrobial peptide KP4 and the cell membrane of carbapenem-resistant Klebsiella pneumoniae. The experimental principle is as follows: the lipid A portion of the LPS molecule can be modified with a fluorescent dye (such as BODIPYFL) to form a fluorescent LPS probe. When LPS binds to KP4, KP4 may alter the structure of LPS through electrostatic or hydrophobic interactions, displacing the fluorescent dye originally bound to LPS and re-exposing it to the aqueous phase. Upon returning to the polar aqueous environment, the fluorescence intensity of the fluorescent dye significantly decreases. Measuring the change in fluorescence intensity before and after binding determines whether LPS and KP4 have bound.

[0027] 1. Experimental Method: LPS was extracted from Klebsiella pneumoniae ATCC BAA 2146 using an LPS kit. The concentration of LPS was adjusted to 5 mg / mL with Tris-HCl solution, and then further diluted to 50 µg / mL with Tris-HCl solution. BODIPY-BC dye with a stock solution concentration of 5 mg / mL was added at a ratio of 1:1000, and the mixture was incubated at room temperature in the dark for 4 hours before use.

[0028] The 2.56 mM antimicrobial peptide was diluted to different concentrations (1 µM, 2 µM, 4 µM, 8 µM, 16 µM, 32 µM, 64 µM, 128 µM) with Tris-HCl solution in 96-well plates, with three replicates of the antimicrobial peptide per plate.

[0029] Add 50 µL of the prepared LPS solution to the wells containing the antimicrobial peptide in a 96-well plate. In the last row, add three wells containing 5 µg / mL polymyxin B solution as positive controls. Add 50 µL of LPS solution to the remaining three wells (without any antimicrobial peptide) as negative controls. Mix thoroughly and incubate at room temperature for 1 h. Measure the fluorescence value using a microplate reader at an excitation wavelength of 580 nm and an emission wavelength of 620 nm (580 / 620 nm is the brightest working window for the dye; using the peak wavelength maximizes signal intensity and improves detection sensitivity). The experiment was performed three independent replicates.

[0030] %ΔF=(F-F0) / (F1-F0)×100% Wherein, ΔF is the relative fluorescence value; F is the fluorescence value obtained by detection; F1 is the fluorescence value of the positive control; and F0 is the fluorescence value of the negative control.

[0031] 2. Experimental Results: Experimental results are as follows Figure 2 As shown, the antimicrobial peptide KP4 exhibits a concentration-dependent binding to LPS; the higher the concentration of antimicrobial peptide KP4, the greater the degree of binding to LPS. This indicates that the antimicrobial peptide KP4 of the present invention can bind to the lipopolysaccharide of carbapenem-resistant Klebsiella pneumoniae.

[0032] Example 4: Validation of antimicrobial peptide KP4 in treating carbapenem-resistant Klebsiella pneumoniae acute pulmonary infection. 1. Experimental Method: Thirty-two 8-week-old female C57BL / 6 mice were divided into four groups of eight each: PBS group: PBS buffer (phosphate buffer solution); No treatment group: ATCC BAA 2146 infection + normal saline; Polymyxin treatment group: ATCC BAA 2146 infection + polymyxin 2.5 mg / kg; Antimicrobial peptide KP4 treatment group: ATCC BAA 2146 infection + antimicrobial peptide KP4 10mg / kg; After centrifuging the logarithmic growth phase ATCC BAA 2146 bacterial culture at 4000 rpm for 5 min, the bacterial cells were collected, washed twice with PBS, and resuspended in PBS to a concentration of 1×10⁻⁶. 8 CFU / mL.

[0033] Mice other than those in the PBS group were given 50 μL of CRKP suspension via intranasal instillation, while the PBS group received an equal volume of PBS buffer. Mice were then intraperitoneally injected with antimicrobial peptide KP4 10 mg / kg, polymyxin 2.5 mg / kg, and an equal volume of physiological saline at 1 h, 13 h, 25 h, and 37 h post-CRKP infection (12 h intervals, for a total of 48 h).

[0034] Mice were euthanized 48 hours after infection, and peripheral blood was collected. 1 mL was used for bacterial counting and routine blood tests. The remaining blood was centrifuged at 1500g for 15 min at 4°C to collect serum and measure the levels of inflammatory factors. The lungs and spleen were removed, washed in excess physiological saline, and a small portion of the sample was fixed in 10% neutral formalin for pathological section observation. The remaining sample was homogenized and diluted a series of times, then cultured on LB agar plates and counted.

[0035] 2. Experimental Results: Depend on Figure 3 The results show that the lungs of mice in the untreated group ( Figure 3 A) and blood ( Figure 3 B) bacterial load at 10 5 Antimicrobial peptide KP4 significantly reduced bacterial load (p<0.05). Both antimicrobial peptide KP4 and polymyxin B reduced serum inflammatory factor TNF-α levels. Figure 3 C), achieving an anti-inflammatory effect. And from Figure 3 As shown in Figure A, the bacterial load in the lungs of the antimicrobial peptide KP4 group was significantly lower than that of the polymyxin B group, indicating that the therapeutic effect of antimicrobial peptide KP4 is superior to that of polymyxin B. These experimental results suggest that antimicrobial peptide KP4 possesses both antibacterial and immunomodulatory effects.

[0036] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. An antimicrobial peptide, characterized in that, The amino acid sequence of the antimicrobial peptide is shown in any one of SEQ ID NO: 1-10.

2. The antimicrobial peptide according to claim 1, characterized in that, The amino acid sequence of the antimicrobial peptide is shown in SEQ ID NO:

3.

3. The use of the antimicrobial peptide as described in claim 1 or 2 in the preparation of a medicament for treating carbapenem-resistant Klebsiella pneumoniae infection.

4. A drug for treating carbapenem-resistant Klebsiella pneumoniae infection, characterized in that, The drug includes the antimicrobial peptide as described in claim 1 or 2.

5. The drug as described in claim 4, characterized in that, The drug also includes pharmaceutically acceptable excipients.

6. The drug as described in claim 5, characterized in that, The excipients include fillers, binders, disintegrants, lubricants, antioxidants, and coating materials.

7. The drug as described in claim 6, characterized in that, The filler is at least one of lactose, microcrystalline cellulose, and starch.

8. The drug as described in claim 6, characterized in that, The disintegrant is at least one of sodium carboxymethyl starch, low-substituted hydroxypropyl cellulose, and crosporovinylpyrrolidone.

9. The drug as described in claim 6, characterized in that, The lubricant is at least one of magnesium stearate, micronized silica gel, and talc.

10. The medicament as claimed in claim 6, characterized in that, The antioxidant is at least one of vitamin C, glutathione, and acetylcysteine.

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