CN114773437A

CN114773437A - Antibacterial peptide with broad-spectrum bactericidal activity and application thereof

Info

Publication number: CN114773437A
Application number: CN202210392287.1A
Authority: CN
Inventors: 国果; 杨隆兵; 张进; 焦正龙; 孙朝琴; 彭建
Original assignee: Guizhou Medical University
Current assignee: Guizhou Medical University
Priority date: 2022-04-14
Filing date: 2022-04-14
Publication date: 2022-07-22
Anticipated expiration: 2042-04-14
Also published as: CN114773437B; CN117886895A; CN117903254A

Abstract

An antibacterial peptide with broad-spectrum antibacterial activity and its application are disclosed, which features broad-spectrum antibacterial activity, especially the strongest antibacterial activity to cryptococcus neoformans, quick sterilization, not easy induction to generate drug resistance, high safety, no obvious cytotoxicity and high environmental stability.

Description

Antibacterial peptide with broad-spectrum bactericidal activity and application thereof

Technical Field

The invention relates to the technical field of biological medicines, in particular to an antibacterial peptide with broad-spectrum bactericidal activity and application thereof.

Background

The antibacterial peptide is a small molecular protein with antibacterial activity. Since Boman et al, Swedish scientists, isolated the first antimicrobial peptide cecropin from silkworm pupae, over 1000 antimicrobial peptides were found in a broad spectrum of organisms including insects, amphibians, aquatic animals, mammals including humans, even plants and bacteria. Endogenous antibacterial peptides are induced and synthesized in organisms, play an important role in resisting pathogen invasion, and are an important part of organism nonspecific immunity.

The antibacterial activity of the antibacterial peptide has the characteristics of high selectivity, quick sterilization, broad spectrum of action and difficulty in forming resistance, so that the antibacterial peptide becomes an ideal candidate for researching and developing novel antibiotics. At present, the antibacterial peptide is expected to be a potential substitute of antibiotics due to the advantages of wide antibacterial spectrum, unique action mechanism, difficult generation of drug resistance and the like; in recent decades, the antibacterial peptide has attracted extensive attention from researchers at home and abroad, and the antibacterial performance, drug resistance, safety, cytotoxicity and environmental stability of the antibacterial peptide are the key points of research at present.

Disclosure of Invention

In order to solve the defects and shortcomings of the prior art, the invention provides an antibacterial peptide with broad-spectrum bactericidal activity and application thereof.

The technical solution adopted by the invention is as follows: antimicrobial peptides having broad-spectrum bactericidal activity comprising the antimicrobial peptides AMP-20, AMP-29, AMP-32, comprising or consisting of:

(a) the amino acid sequence of SEQ ID NO: 1 to 3; or;

(b) the amino acid sequence of SEQ ID NO: 1 to 3, having at least 80% sequence identity.

The spatial structures of the antibacterial peptides AMP-20, AMP-29 and AMP-32 all present alpha-helix and beta-sheet structures, wherein the ratio of the alpha-helix structures is the largest.

The antibacterial peptides AMP-20, AMP-29 and AMP-32 are alpha helical peptides

The application of the antibacterial peptide in preparing antibacterial drugs.

The antibacterial agent is a broad-spectrum antibacterial agent.

The antibacterial drug has antibacterial activity against cryptococcus neoformans, candida albicans, candida krusei, candida tropicalis resistant to drugs, escherichia coli of gram-negative bacteria and staphylococcus aureus of gram-positive bacteria.

An application of the antibacterial peptide in preparing antibacterial and bactericidal medicines for cryptococcus.

The minimum bactericidal concentration of the antibacterial peptide to cryptococcus is 2-16 mu g/mL.

The beneficial effects of the invention are: the invention provides an antibacterial peptide with broad-spectrum bactericidal activity and application thereof, which has the broad-spectrum antibacterial activity, particularly has the strongest antibacterial activity on cryptococcus, has the characteristic of quick sterilization, is difficult to induce to generate drug resistance and higher safety, has no obvious cytotoxicity effect, and has strong environmental stability.

Drawings

FIG. 1 shows a model for predicting the helical and three-dimensional structures of the novel peptides AMP-20, AMP-29 and AMP-32.

FIG. 2 is a time-kill curve of the novel peptides AMP-20, AMP-29, AMP-32 against Cryptococcus H99.

FIG. 3 shows the induction of drug resistance by Cryptococcus H99 at the concentration of the novel peptide Sub-MIC.

FIG. 4 shows the hemolytic activity of the novel peptides AMP-20, AMP-29 and AMP-32 on human erythrocytes.

FIG. 5 is a graph showing the cytotoxicity of the novel peptides AMP-20, AMP-29, AMP-32 against HK-2, HepG2, LO2, RAW 264.7.

FIG. 6 shows the in vivo toxicity of H99 to larvae of Chilo suppressalis.

FIG. 7 is a graph of the in vivo treatment of the new peptides AMP-20, AMP-29, AMP-32 in a greater wax moth infection model; wherein 1X 10 is used⁸CFU/mL Cryptococcus neoformans H99 infected Chilo suppressalis larvae and treated with 3 neopeptides and fluconazole and amphotericin B, respectively. (A) Time schematic of drug treatment of galleria mellonella infection model. (B) Toxicity of the drug to larvae. (C) Survival of larvae in each group after drug treatment. (D) The population of worms from 2 replicates (n-3) was sacrificed 24h for each replicate and 3 larvae per group were homogenized and plated and colonies were counted to determine the CFU for each larva. (E) Representative photographs of larvae in the 5 th sky white control group (PBS Only), model group (H99Only), and drug treatment groups (AMP-20, AMP-29, AMP-32, FLC, and AMB). This experiment was conductedA set of 2 replicates of survival plots (n-32) was performed and for each replicate a panel of 16 larvae per treatment was monitored for survival over 7 days.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Through the arrangement of network databases, documents, monographs and patents, 5775 polypeptide sequences with antifungal activity are collected, 5775 negative sequences with the same length distribution as the polypeptides and without the antifungal activity are randomly extracted from UniProt as reference, and a discrimination model is established for the antifungal polypeptides through a support vector machine. Our built anti-fluent webserver (http:// www.chemoinfolab.com/an tifungal) has achieved free open sharing of the model, which has received 1000 visits and 200 data submissions three months open. We searched unknown polypeptide sequences with the length of 11-75 amino acids from UniProt, 3445312 sequences in total, and used the model to discriminate and screen the unknown functional sequences for antifungal activity, and combined with our self-defined antifungal index (AFI) to determine a batch of polypeptide sequences with possible antifungal activity. Approximately 50 of the polypeptides were synthesized by solid phase synthesis, and the antifungal activity was verified in the laboratory, and finally 3 novel peptides AMP-20(YFPPAKRLLNWAKKKVGIKKKKK), AMP-29(EKKPWARLRFKFKLLKGLAKKMK), and AMP-32(LKKLAGKIYRVLAKL) having broad-spectrum antibacterial activity and particularly excellent cryptococcus resisting activity were selected from these polypeptides.

Example 1

Structural analysis of 3 novel peptides

The distribution of hydrophilic and hydrophobic amino acids, as well as the average hydrophobicity and average hydrophobic moment, in the amino acid sequences of the peptides Mm-AMP1, Dv-AMP1, Nc-AMP1 were analyzed using NetWheels (http:// lbqp. un. br/NetWheels /) online software. Meanwhile, the spatial structures (tertiary structures) of the peptides AMP-20, AMP-29 and AMP-32 are simulated by using protein spatial structure simulation software I-TASSER (https:// zhanggroup. org/I-TASSER /). As shown in FIG. 1, the polar amino acids and the nonpolar amino acids of AMP-20 were distributed uniformly, and the average hydrophobicity was 0.129 and the average hydrophobic moment was 0.203. The polar and nonpolar amino acids of AMP-29 were uniformly distributed, and had an average hydrophobicity of 0.201 and an average hydrophobic moment of 0.360. AMP-32 had a relatively small number of polar amino acids, a majority of non-polar amino acids, an average hydrophobicity of 0.429, and an average hydrophobic moment of 0.738. The spatial structure of the peptides AMP-20, AMP-29 and AMP-32 showed that the spatial structure of the 3 new peptides exhibited α -helical and β -sheet structures, in which the ratio of α -helical structures was largest and the 3 new peptides were α -helical peptides.

Example 2

The novel peptides have a broad spectrum of antibacterial activity and outstanding activity against cryptococcus neoformans

To determine the drug sensitivity of 3 new peptides to bacteria and fungi, we used the micro-liquid dilution method to measure the Minimum Inhibitory Concentration (MIC) and the minimum bactericidal concentration (MFC) values, respectively. The results showed that MIC values for AMP-20, AMP-29, AMP-32 to Cryptococcus neoformans H99 and KN99 were 4. mu.g/mL, and 2. mu.g/mL, respectively; MFC values were 4. mu.g/mL, 8. mu.g/mL, and 2. mu.g/mL. The MIC of the peptide for Candida albicans, Candida krusei and even drug-resistant Candida tropicalis is 16-32 mu g/mL, and the peptides have strong effects on fungi and show certain antibacterial activity on gram-negative bacteria Escherichia coli (MIC 8-32 mu g/mL) and gram-positive bacteria Staphylococcus aureus (MIC 64-128 mu g/mL). Laboratory results confirmed that these peptides are broad-spectrum antimicrobial peptides with the strongest antimicrobial activity against cryptococcus (table 1).

In order to further confirm the activity of the new peptide on cryptococcus neoformans, test strains of the cryptococcus neoformans are expanded, MIC and MFC of the cryptococcus neoformans on other 16 clinical strains are continuously determined, the result is still revived, and the MIC values of the three peptides are respectively 4-8 mu g/mL, 4-16 mu g/mL and 2-4 mu g/mL; MFC values were 4-8. mu.g/mL, 8-16. mu.g/mL and 2-4. mu.g/mL, respectively (Table 2). The antibacterial activity of AMP-20 and AMP-29 is equivalent to that of fluconazole and caspofungin which are common clinical antifungal drugs, and is stronger than that of 5-fluorouracil; AMP-32 was most prominent and the anti-cryptococcus effect was stronger than the above 3 antibiotics, thus confirming the prominent anti-cryptococcus activity of the novel peptide.

TABLE 1 antimicrobial Spectrometry of novel peptides AMP-20, AMP-29 and AMP-32

Note: "-" indicates not determined

TABLE 2 MIC and MFC values for the novel peptides AMP-20, AMP-29, AMP-32 against Cryptococcus neoformans

Example 3

The new peptide can exert bactericidal action within 2h

The antibacterial peptide has the functions of bacteriostasis and sterilization, and in order to determine whether the new peptide has the effect of killing fungi, the time-sterilization curve of the new peptide to cryptococcus is determined. The results show that 3 new peptides have bactericidal activity against cryptococcus neoformans H99 within 2H (FIG. 2), while fluconazole can only inhibit the growth of cryptococcus, and amphotericin can achieve bactericidal effect within 4H. The above results suggest that the novel peptides AMP-20, AMP-29 and AMP-32 have a more rapid fungicidal activity against Cryptococcus neoformans.

Example 4

The new peptide is not easy to induce drug resistance

The characteristic advantage of the antibacterial peptide is that the antibacterial peptide is not easy to generate drug resistance. To determine whether the neopeptides are resistant, we induced resistance by repeated treatment of cryptococcus with sub-MIC concentrations of the neopeptides. As shown in FIG. 3, in the 10-generation MIC test, the sensitivity of 3 new peptides to cryptococcus neoformans H99 remained relatively stable, and no drug resistance phenomenon was generated, indicating that the new peptides AMP-20, AMP-29 and AMP-32 are not easy to induce drug resistance.

Example 5

The novel peptide has high safety

To preliminarily define the safety of the 3 novel peptides, we tested their hemolytic, cytotoxic and serum stability. We incubated the new peptides at different concentrations for 1h with human erythrocytes at a final concentration of 2% respectively, with 10mM PBS and 1% Triton-X100 as negative and positive controls. The results show that 3 peptides at a concentration of 64. mu.g/mL did not show significant hemolysis, HC, in human erythrocytes₅₀The values were 202.96, 136.51, and 141.41. mu.g/mL, respectively. Only when the peptide concentration is>At 64. mu.g/mL, a slight hemolytic effect appeared (FIG. 4).

To clarify what were the cytotoxicity of the novel peptides AMP-20, AMP-29 and AMP-32? The cytotoxicity determination of the novel peptide is determined by a CCK8 method by taking human renal proximal tubular epithelial cells HK-2, human liver cancer cells HepG2, human normal liver cells LO2 and mouse macrophage RAW264.7 as test cells. The results are shown in FIG. 5, where 3 new peptides showed no significant cytotoxic effect on HK-2, HepG2, LO2 and RAW264.7 at 32. mu.g/mL, IC₅₀The values are shown in Table 3.

TABLE 3 IC of the novel peptides AMP-20, AMP-29, AMP-32 on HK-2, HepG2, LO2 and RAW264.7 cells₅₀Value of

To initially evaluate the stability of the novel peptides in human serum and in different environments, the MIC changes of the novel peptides in different environments were determined using a microdilution method. As a result, it was found that none of 10% human serum, 150mM NaCl and 4.5mM KCl had any effect on the activity of the novel peptide. Surprisingly, the antibacterial activity of the new peptide is obviously enhanced in the 10% human serum environment, the MIC is changed from 2-4 to MIC <2, and the antibacterial peptide is supposed to play a role in the immune protein in the serum, so that the antibacterial activity of the antibacterial peptide is further enhanced. However, as with the other polypeptides, the novel peptides were trypsin sensitive and were readily inactivated at 1mg/mL trypsin (Table 4).

Table 4 stability assay of the novel peptides in different environments

Example 6

The new peptide treatment can remarkably reduce the death rate and the fungal load of the larvae infected by the galleria mellonella

In vitro we have confirmed that all 3 new peptides have strong activity against cryptococcus, but it is unknown whether they exert antibacterial effect in vivo. In order to preliminarily evaluate the in vivo antibacterial effect of the new peptide, cryptococcus H99 is used as an infection strain, the toxicity of the new peptide to the larvae of the greater wax moth and the time-survival curve of the greater wax moth infected with the cryptococcus H99 are firstly measured, on the basis, a cryptococcus infection model of the larvae of the greater wax moth is constructed, the survival rate of each group of larvae is recorded after the intervention treatment by using the new peptide with different concentrations, and the fungal load of the larvae is measured by a colony counting method.

The infection concentration of cryptococcus H99 galleria mellonella larva is groped, and the result shows that the larva is injected with 5 multiplied by 10⁸All deaths within 48H after CFU/mL H99, 1.0X 10⁸CFU/mL group died completely within 96h, while 0.5X 10⁸The CFU/mL H99 group still had 10% of larvae alive after 168H (FIG. 6). The result of the research is combined with the related literature, and 1.0 multiplied by 10 is selected⁸CFU/mL group was used as the infectious dose for the subsequent experiment.

After the new peptides with different concentrations are injected into the galleria mellonella larvae, the AMP-20 and the AMP-29 do not show obvious toxicity to the larvae in the range of 8-64 mg/kg, the survival rate is 100%, and the survival rate of the 64mg/kg AMP-32 group larvae is 90% (figure 7-B). In infection experiments, 100% of the larvae in the model group died on day 4, whereas 3 peptides were effective in prolonging the survival time of infected larvae, with more than 80% of larvae in the 32mg/kg group surviving to day 5 and more than 35% surviving to day 7 (FIGS. 7-C, 7-E). The polypide fungal burden results showed a significant reduction in the bacterial load (P <0.01) for all peptide-treated groups compared to the model group (fig. 7-D). The results suggest that 3 new peptides can exert antibacterial activity in infected larvae, can obviously improve the survival rate of the infected larvae and reduce the bacterial load of the larvae.

The skilled person should know that: although the invention has been described in terms of the above specific embodiments, the inventive concept is not limited thereto and any modification applying the inventive concept is intended to be included within the scope of the patent claims.

The above description is only a preferred embodiment of the present invention, and the protection scope of the present invention is not limited to the above embodiments, and all technical solutions belonging to the idea of the present invention belong to the protection scope of the present invention. It should be noted that modifications and adaptations to those skilled in the art without departing from the principles of the present invention should also be considered as within the scope of the present invention.

Sequence listing

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Claims

1. Antimicrobial peptides having broad-spectrum bactericidal activity comprising the antimicrobial peptides AMP-20, AMP-29, AMP-32, comprising or consisting of:

(a) SEQ ID NO: 1 to 3; or;

(b) SEQ ID NO: 1 to 3, having at least 80% sequence identity.

2. The peptides of claim 1, wherein the spatial structure of the peptides AMP-20, AMP-29, AMP-32 exhibits both α -helical and β -sheet structures, and the ratio of α -helical structures is the largest.

3. The antimicrobial peptide having broad-spectrum bactericidal activity according to claim 2, wherein the antimicrobial peptides AMP-20, AMP-29, AMP-32 are alpha helical peptides.

4. Use of the antimicrobial peptide of claim 1 for the preparation of an antimicrobial medicament.

5. The use of claim 4, wherein said antibacterial agent is a broad spectrum antibacterial agent.

6. The use of claim 5, wherein said antibacterial agent has antibacterial activity against Cryptococcus neoformans, Candida albicans, Candida krusei, Candida tropicalis, Escherichia coli, Staphylococcus aureus, which are gram-negative bacteria.

7. Use of the antimicrobial peptide of claim 1 for the preparation of a medicament for the antimicrobial and bactericidal treatment of cryptococcus.

8. The use according to claim 7, wherein the antimicrobial peptide has a minimum bactericidal concentration against cryptococcus from 2 to 16 μ g/mL.