Group of polypeptides with rapid bactericidal activity and application thereof

Abstract

The invention relates to a group of polypeptides with rapid bactericidal activity and application thereof, and the polypeptides are amphiphilic polypeptides and have a cation alpha-helical structure. The polypeptide specifically comprises a C22-01 polypeptide, a C22-02 polypeptide, a C22-03 polypeptide, a C22-04 polypeptide and a C22-05 polypeptide, wherein the sequence of the C22-01 polypeptide is shown as SEQ ID NO.1, the sequence of the C22-02 polypeptide is shown as SEQ ID NO.2, the sequence of the C22-04 polypeptide is shown as SEQ ID NO.3, the sequence of the C22-05 polypeptide is shown as SEQ ID NO.4, and the sequence of the C22-05 polypeptide is shown as SEQ ID NO.5. The carboxyl terminals of the polypeptides are subjected to amidation (-NH2) modification.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medicine, and particularly relates to a group of polypeptides with rapid bactericidal activity and uses thereof. BACKGROUND

[0002] Bacterial drug resistance caused by overuse of antibiotics has become a prominent problem in the field of public health [1] . Antimicrobial peptides are a class of small molecule polypeptides with bactericidal activity, and are a potential development direction of traditional antibiotics in the post-antibiotic era [2,3] . Among them, cationic alpha-helical antimicrobial peptides (Cationic alpha-helical Anti-Microbial Peptide, CαAMP) can rapidly kill bacteria by selectively targeting bacterial membranes and destroying the integrity of the membrane structure, and have good development value [4] .

[0003] In recent years, a series of short antibacterial peptides composed of 13-15 amino acid residues have been identified from the venom glands of toxic animals such as frogs, scorpions and wasps, such as Temporins [5] , Mastoparans [6] , BmKn2 [7] , Lausporin-1 [8] and Hp1404 [9] , etc. However, these natural peptides often have poor antibacterial activity and strong cytotoxicity, which limits their pharmaceutical development value. In these toxic animals, many long-chain antibacterial peptides have also been found

[10] . The molecular weight of long-chain antibacterial peptides is too large, and the development cost is too high, which also limits their pharmaceutical development value.

[0004] CαAMP is a class of amphiphilic molecules, and physicochemical properties such as hydrophobicity and amphiphilicity are important factors affecting their antibacterial activity and toxicity. Through bioinformatics analysis, we found that the amino-terminal truncated peptides of scorpion toxin Con22 have good amphiphilicity and hydrophobicity, and these truncated peptides may have antibacterial activity

[11] .

[0005] Accordingly, we designed antibacterial short peptides based on scorpion toxin Con22, and proposed the present application.

[0006]

Reference

[0007] 1. Frieri, M.; Kumar, K.; Boutin, A. Antibiotic resistance. Journal of infection and public health 2017, 10, 369-378, doi:10.1016 / j.jiph.2016.08.007.

[0008] 2. Ghosh, C.; Sarkar, P.; Issa, R.; Haldar, J. Alternatives to conventional antibiotics in the era of antimicrobial resistance. Trends in microbiology 2019, 27, 323-338, doi:10.1016 / j.tim.2018.12.010.

[0009] 3. Koehbach, J.; Craik, D. J. The vast structural diversity of antimicrobial peptides. Trends in pharmacological sciences 2019, 40, 517-528, doi:10.1016 / j.tips.2019.04.012.

[0010] 4. Ciumac, D.; Gong, H.; Hu, X.; Lu, J. R. Membrane targeting cationic antimicrobial peptides. Journal of colloid and interface science 2019, 537, 163-185, doi:10.1016 / j.jcis.2018.10.103.

[0011] 5. Simmaco, M.; Mignogna, G.; Canofeni, S.; Miele, R.; Mangoni, M. L.; Barra, D. Temporins, antimicrobial peptides from the European red frog Rana temporaria. European journal of biochemistry 1996, 242, 788-792.

[0012] 6. Moreno, M.; Giralt, E. Three valuable peptides from bee and wasp venoms for therapeutic and biotechnological use: melittin, apamin and mastoparan. Toxins 2015, 7, 1126-1150, doi:10.3390 / toxins7041126.

[0013] 7. Luo, X.; Ye, X.; Ding, L.; Zhu, W.; Yi, P.; Zhao, Z.; Gao, H.; Shu, Z.; Li, S.; Sang, M.; et al. Fine-tuning of alkaline residues on the hydrophilic face provides a non-toxic cationic alpha-helical antimicrobial peptide against antibiotic-resistant ESKAPE pathogens. Frontiers in microbiology 2021, 12, 684591, doi:10.3389 / fmicb.2021.684591.

[0014] 8. Zhao, Z.; Zhang, K.; Zhu, W.; Ye, X.; Ding, L.; Jiang, H.; Li, F.; Chen, Z.; Luo, X. Two new cationic alpha-helical peptides identified from the venom gland of Liocheles australasiae possess antimicrobial activity against methicillin-resistant staphylococci. Toxicon: official journal of the International Society on Toxinology 2021, 196, 63-73, doi:10.1016 / j.toxicon.2021.04.002.

[0015] 9. Luo, X.; Ye, X.; Ding, L.; Zhu, W.; Zhao, Z.; Luo, D.; Liu, N.; Sun, L.; Chen, Z. Identification of the scorpion venom-derived antimicrobial peptide Hp1404a as a new antimicrobial agent against carbapenem-resistant Acinetobacter baumannii. Microbial pathogenesis 2021, 157, 104960, doi:10.1016 / j.micpath.2021.104960.

[0016] 10. Harrison, P. L.; Abdel-Rahman, M. A.; Miller, K.; Strong, P. N. Antimicrobial peptides from scorpion venoms. Toxicon : official journal of the International Society on Toxinology 2014, 88, 115-137, doi:10.1016 / j.toxicon.2014.06.006.

[0017] 11. Luna-Ramirez, K.; Quintero-Hernandez, V.; Vargas-Jaimes, L.; Batista, C. V. F.; Winkel, K. D.; Possani, L. D. Characterization of the venom from the Australian scorpion: Molecular mass analysis of components, cDNA sequences and peptides with antimicrobial activity. Toxicon : official journal of the International Society on Toxinology 2013, 63, 44-54, doi:10.1016 / j.toxicon.2012.11.017. SUMMARY

[0018] The present application relates to a group of polypeptides with antibacterial activity, which have rapid bactericidal activity targeting bacterial membrane, and the polypeptides are:

[0019] C22-01 polypeptide, sequence as shown in SEQ ID NO. 1:

[0020] C22-02 polypeptide, sequence as shown in SEQ ID NO. 2:

[0021] C22-03 polypeptide, sequence as shown in SEQ ID NO. 3:

[0022] C22-04 polypeptide, sequence as shown in SEQ ID NO. 4:

[0023] C22-05 polypeptide, sequence as shown in SEQ ID NO. 5:

[0024] The carboxyl end of the polypeptide is modified by amide (-NH2).

[0025] Further, the polypeptide is a cationic alpha-helix structure,

[0026] The hydrophobicity of the polypeptide is between 0.500 and 0.626, the hydrophobic moment is between 0.587 and 0.632, and all have 5 net positive charges.

[0027] The polypeptide is an amphiphilic polypeptide.

[0028] The present application also relates to a drug or pharmaceutical composition comprising the polypeptide or polypeptide group, the drug is an antibacterial infection drug, preferably, the bacteria are drug-resistant bacteria, and the bacteria include but are not limited to Staphylococcus aureus, Enterococcus faecium, Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae or Acinetobacter baumannii.

[0029] Preferably, the drug or pharmaceutical composition has rapid bactericidal activity and can complete the bactericidal function within 60 minutes.

[0030] The drug or pharmaceutical composition comprises a therapeutically effective amount of the polypeptide or polypeptide group and necessary auxiliary materials.

[0031] Preferably, the drug or pharmaceutical composition is an external drug, which is an emulsion, cream or ointment.

[0032] The present application also relates to the use of the polypeptide or polypeptide group in the preparation of an antibacterial preparation, and the antibacterial preparation is used for inhibiting bacterial infection, preferably, the bacteria are drug-resistant bacteria, and the bacteria include but are not limited to Staphylococcus aureus, Enterococcus faecium, Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae or Acinetobacter baumannii.

[0033] Preferably, the antibacterial agent is a human antibacterial agent or a veterinary antibacterial agent.

[0034] Preferably, the antibacterial agent is an external antibacterial agent, which is an emulsion, a cream or a paste.

[0035] Preferably, the antibacterial agent has a rapid bactericidal activity, which can complete the bactericidal function within 60 minutes.

[0036] The beneficial effects of the present application are:

[0037] (1) The scorpion toxin polypeptide derivative C22-02 has a rapid bactericidal activity targeting bacterial plasma membrane, and can effectively inhibit various types of drug-resistant strains;

[0038] (2) The scorpion toxin polypeptide derivative C22-02 has good anti-infection efficacy at the animal level;

[0039] (2) The scorpion toxin polypeptide derivative C22-02 also has high safety;

[0040] In summary, the scorpion toxin polypeptide derivative C22-02 has the potential to be further developed as an anti-infection drug. BRIEF DESCRIPTION OF DRAWINGS

[0041] Figure 1 , Molecular design of Con22-derived peptides, 1A, sequence information, 1B, helical wheel diagram, hydrophobic and basic residues are represented by yellow and blue respectively.

[0042] Figure 2 , Evaluation of hemolytic activity of Con22-derived peptides, red blood cells treated with 1% Triton X-100 were considered as 100% hemolysis, 2A, concentration linear graph, 2B, hemolysis data at 128 ug / ml, all experiments were repeated three times, the data shown are mean ± standard error.

[0043] Figure 3 , Evaluation of cytotoxicity of C22-02 and C22-04, 3A, C22-02 cytotoxicity evaluation; 3B, C22-04 cytotoxicity evaluation, all experiments were repeated three times, the data shown are mean ± standard error.

[0044] Figure 4 , Analysis of bactericidal kinetics of Con22-derived peptide C22-02, the data shown are the mean ± standard error of three independent experiments.

[0045] Figure 5 , Study on the anti-infection effect of C22-02 at the animal level. DETAILED DESCRIPTION

[0046] MATERIALS AND METHODS

[0047] 1. Reagents:

[0048] Mueller-Hinton broth was purchased from Oxoid, agarose was purchased from Biofrox, and propidium iodide was purchased from Themofisher.

[0049] Na2HPO4, KH2PO4, NaCl, and KCl were all analytical pure and purchased from National Pharmaceutical Group Chemical Reagent Co., Ltd.

[0050] 2. Strains:

[0051] The multiple drug-resistant clinical strains used in this study were obtained from the Tahe Hospital and Dongfeng Hospital Affiliated to Hubei Medical College, and the drug resistance of the strains was obtained by the disk diffusion drug sensitivity test.

[0052] 3. Peptide synthesis and bioinformatics analysis:

[0053] The derived peptides of Con22 were synthesized by Shanghai Qiangyao Biotechnology Co., Ltd., and the purity of the peptides was greater than 95% after reverse high-performance liquid chromatography and mass spectrometry analysis.

[0054] 4. Hydrophobicity, hydrophobicity matrix, and charge analysis of Con22-derived peptides were completed on the HeliQuest platform: https: / / heliquest.ipmc.cnrs.fr / .

[0055] 5. Minimum inhibitory concentration determination

[0056] (1) Take 10 mL of sterilized Mueller-Hinton culture solution, add 10 μL of frozen bacteria solution, and incubate the bacteria overnight,

[0057] (2) Take 10 μL of bacteria solution and transfer it to new Mueller-Hinton culture solution, and grow to OD 630 = 0.4,

[0058] (3) Con22-derived peptides were dissolved in 0.9% NaCl for use.

[0059] Take one 96-well plate, dilute the bacteria to a final concentration of 5 x 10 5 CFU / mL, and divide them into 96-well plates, add a series of different concentrations of peptides (1, 2, 4, 8, 16, 32, and 64 μg / mL), and incubate the peptides with the bacteria (37°C, 150 rpm) for 16 hours. Measure the absorbance (OD 630 ) after incubation, and the minimum inhibitory concentration is defined as the minimum peptide concentration with no detectable absorbance change.

[0060] 6. Hemolytic activity experiment

[0061] (1) Fresh human red blood cells (from healthy blood donors of Taihe Hospital and Dongfeng Hospital of Hubei Medical College) were diluted in 0.9% NaCl solution, and centrifuged and rinsed three times (1000 x g, 5 min) until the supernatant became colorless.

[0062] (2) The red blood cells were diluted in 0.9% NaCl solution to a final concentration of 2% (v / v), and different concentrations of polypeptides (0, 16, 32, 64, 128 and 256 μg / mL) were added, and incubated at 37°C for 1 hour. The cells were centrifuged (2000 x g, 5 min), and the supernatant was removed and the absorbance value (OD 540 ) was measured to evaluate the hemolysis rate of the polypeptides.

[0063] (3) Human red blood cells treated with 1% Triton X-100 solution (100% hemolysis) or 0.9% NaCl solution (0% hemolysis) were used as controls.

[0064] 7. Bactericidal kinetics curve determination

[0065] The bacteria were prepared as described above (minimum inhibitory concentration determination test).

[0066] (1) The bacterial solution was diluted to a final concentration of 5 x 10 630 CFU / mL, 5

[0067] (2) The bacteria were incubated (37°C, 150 rpm) with 0 x, 1 x, 2 x and 4 x MIC of polypeptides, respectively,

[0068] (3) At 5, 15, 30, 60 and 120 min, a certain amount of bacterial solution was taken for gradient dilution, and 100 μL of the diluted bacterial solution was evenly spread on solid culture medium, and incubated overnight to count the number of viable bacteria.

[0069] 8. Bacterial membrane permeability experiment

[0070] The bacteria were prepared as described above (minimum inhibitory concentration determination test).

[0071] (1) The bacterial solution was centrifuged (2000 x g, 4°C) and rinsed three times with PBS buffer, and the bacterial solution was diluted to OD 630 = 0.1 (1 x 10 630 CFU / mL), 7

[0072] ​​(2) Add 2 μM final concentration of propidium iodide to the bacteria solution. Immediately after adding 0x, lx, 2x and 4x MIC of the polypeptide, place the plate in a microplate reader (Molecular Devices SpectraMax i3x) and set the excitation wavelength at 535 nm and the emission wavelength at 617 nm.

[0073] 9. Cytotoxicity test

[0074] (1) Take human normal liver cells L02 as the test object. After cell recovery, inoculate 8 x 105 cells / well in a 96-well plate and incubate for 24 hours. 3

[0075] (2) Add a series of polypeptides with different concentrations (2.5, 5, 10, 20 and 40 μg / mL) and incubate for 24 hours.

[0076] (3) Add CCK-8 reagent and measure the absorbance (OD 450 ) to evaluate the cytotoxicity of the polypeptide.

[0077] (4) The cell survival rate % is defined as: cell survival rate % = (As - Ab) / (Ac - Ab) x 100%, wherein As is the absorbance of the experimental well, Ac is the absorbance of the control well, and Ab is the absorbance of the blank well.

[0078] 10. Animal level anti-infection efficacy test

[0079] (1) Take healthy ICR mice (male, 7-8 weeks old, 28-30 g) as the research object.

[0080] (2) Take 25 mice and randomly divide them into 5 groups. Inject 0.5 mL of S. aureus 4188 with a bacterial number of 5 x 105 CFU / mL into each mouse by intraperitoneal injection. 7

[0081] (3) After half an hour, inject the polypeptide into the mice in the experimental group at a dose of 2 mg / kg, 5 mg / kg and 10 mg / kg, respectively. Inject the same volume of PBS solution into the mice in the positive control group, and do not treat the mice in the blank control group.

[0082] (4) After 4 hours, sacrifice the mice, take the peritoneal fluid, plate it, incubate overnight, and count the number of colonies.

[0083] Example 1, Molecular design and structure analysis of Con22-derived peptides

[0084] Five derived peptides were designed based on the template of scorpion toxin Con22 (Figure-1A), and their physicochemical properties were analyzed (Table-1).

[0085] ​​Table-1. Amino acid sequence of Con22 derived peptides and their physico-chemical property analysis.

[0086] Peptides Sequences a ]] AA b ]] MW c (Da)]]> Z d ]]> <h> e ]]> < / h> ​ <μH> f ]]> C22-01 IWS WIKKTAKKVW-NH2 13 1673.06 5 0.626 0.632 C22-02 GIWSWIKKTAKKVW-NH2 14 1730.12 5 0.581 0.587 C22-03 [ IWS WIKKTAKKVWN-NH2 ] 14 1787.17 5 0.539 0.629 C22-04 GIWSWIKKTAKKVWN-NH2 15 1844.22 5 0.503 0.587 C22-05 IWS WIKKT AKKVWNS-NH2 15 1874.25 5 0.500 0.587

[0087] a. C-terminus of all peptides is amidated;

[0088] b. AA, number of amino acids;

[0089] c. MW, molecular weight determined by mass spectrometry;

[0090] d. z, net charge determined at pH 7.4;

[0091] e. <h>, average hydrophobicity;

[0092] f. <μH>, hydrophobic moment.

[0093] Analysis by HeliQuest platform showed that,

[0094] (1) The hydrophobicity of Con22-derived peptides was between 0.500 and 0.626, the hydrophobic moment was between 0.587 and 0.632, and all of them had 5 net positive charges (Table-1).

[0095] (2) The helical wheel projection showed that Con22-derived peptides were all amphipathic peptides Figure 1 B).

[0096] Example 2, Functional evaluation of Con22-derived peptides

[0097] To study the antibacterial activity of Con22-derived peptides, we synthesized Con22-derived peptides and selected the following representative strains: Staphylococcus aureus S. aureus ATCC29213, Enterococcus faecalis E. faecalis ATCC29212, Escherichia coli E. coli ATCC25922, Pseudomonas aeruginosa P. aeruginosa ATCC27853, Klebsiella pneumoniae K. pneumoniae ATCC700603, and Acinetobacter baumannii A. baumannii ATCC19606, and determined the minimum inhibitory concentration (MIC) of Con22-derived peptides against the above pathogenic bacteria (Table-2) and hemolytic activity, and the results showed that:

[0098] (1) C22-02 and C22-04 have the lowest minimum inhibitory concentration values and the strongest antibacterial activity .

[0099] (2) The hemolytic activity of the five derived peptides was low, and the safety was good.

[0100] Table-2, Antimicrobial activity and hemolytic toxicity of Con22-derived peptides against standard strains.

[0101]

[0102] To further study the antibacterial activity of Con22-derived peptides against clinically drug-resistant strains, we collected clinically drug-resistant strains from the affiliated hospital of our unit, and found that C22-02 and C22-04 had strong antibacterial activity against clinically drug-resistant strains (Table-3).

[0103] Table-3, Evaluation of the antibacterial activity of Con22-derived peptides against clinically drug-resistant strains.

[0104]

[0105] MDR: Multi-Drug resistant, multi-drug resistant,

[0106] PRSA: Penicillin-resistant Staphylococcus aureus, penicillin-resistant Staphylococcus aureus,

[0107] MRSA: Methicillin resistant Staphylococcus aureus, methicillin-resistant Staphylococcus aureus,

[0108] ESBL: Extended-Spectrum β-Lactamases, extended-spectrum β-lactamase,

[0109] CRE: Carbapenem resistant, carbapenem-resistant.

[0110] In order to study the hemolytic toxicity of Con22 derived peptides, the present application uses anticoagulant human red blood cells to find that the hemolytic toxicity of Con22 derived peptides is low, and the HC 10 (the concentration of polypeptide causing 10% hemolysis) is greater than 128 μg / mL Figure 2 Table-2).

[0111] In view of the stronger antibacterial activity of C22-02 and C22-04, the cytotoxicity (CCK-8 method) of C22-02 and C22-04 is studied by taking human normal liver cells L02 as the detection object. The results show that C22-02 and C22-04 have no obvious cytotoxicity at 64 μg / mL Figure 3 ).

[0112] Accordingly, the present application invents two antibacterial peptides C22-02 and C22-04 which have strong antibacterial activity and low hemolysis and cytotoxicity.

[0113] The analysis of the molecular structure and physicochemical properties of the Con22 derived peptides of the present application shows that the Con22 derived peptides belong to cationic alpha-helix antibacterial peptides. A large number of studies show that cationic alpha-helix antibacterial peptides target and destroy bacterial plasma membranes to kill bacteria, and have rapid bactericidal kinetic characteristics.

[0114] In order to verify whether the Con22 derived peptides target bacterial plasma membranes to kill bacteria rapidly, the present application carries out bactericidal kinetic experiments Figure 4 with C22-02 as a representative. The results show that: C22-02 has fast bactericidal activity, consistent with the properties of cationic α-helix antibacterial peptides targeting and destroying bacterial plasma membranes to kill bacteria Figure 5 .

[0115] To further study the anti-infection activity of C22-02, an ICR mouse was used to establish a peritonitis model of S. aureus infection, and an animal experiment of C22-02 against drug-resistant S. aureus infection was carried out. It was found that C22-02 had good anti-infection activity. ​

[0116] In summary, the scorpion toxin polypeptide derivative C22-02 has rapid bactericidal activity targeting bacterial plasma membranes, and has good anti-infection efficacy at the animal level. This is an important basis for the polypeptide C22-02 to resist multi-drug resistant bacteria.

[0117] Finally, it should be noted that the above examples are only used to help those skilled in the art to understand the essence of the present application, and are not used to limit the protection scope of the present application.​< / h>

Claims

1. A group of polypeptides having antibacterial activity, characterized in that, The polypeptide has rapid bactericidal activity of targeting bacterial plasma membrane, and the polypeptide is: C22-01 polypeptide, the sequence is shown as SEQ ID NO. 1: C22-02 polypeptide, the sequence is shown as SEQ ID NO. 2: C22-03 polypeptide, the sequence is shown as SEQ ID NO. 3: C22-04 polypeptide, the sequence is shown as SEQ ID NO. 4: C22-05 polypeptide, the sequence is shown as SEQ ID NO. 5: The carboxyl end of the polypeptide is modified by amide (-NH2).

2. The polypeptide of claim 1, wherein, The polypeptide is an amphiphilic polypeptide with cationic α-helix structure.

3. The polypeptide of claim 1 or 2, characterized in that, The hydrophobicity of the polypeptide is between 0.500 and 0.626, the hydrophobicity moment is between 0.587 and 0.632, and all have 5 net positive charges.

4. A medicine or a pharmaceutical composition comprising the polypeptide or the polypeptide group according to any one of claims 1-3.

5. The medicament or pharmaceutical composition according to claim 4, characterized in that, The medicine is an antibacterial infection medicine. Preferably, the medicine or the pharmaceutical composition has rapid bactericidal activity and can complete the bactericidal function within 60 minutes. Preferably, the bacteria are drug-resistant bacteria, and the bacteria include but are not limited to Staphylococcus aureus, Enterococcus faecium, Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae or Acinetobacter baumannii.

6. The medicament or pharmaceutical composition according to claim 4 or 5, characterized in that, The medicine or the pharmaceutical composition comprises a therapeutically effective amount of the polypeptide or the polypeptide group and necessary excipients.

7. The medicament or pharmaceutical composition according to any one of claims 4-6, wherein, The medicine or the pharmaceutical composition is an external medicine; preferably, the external medicine is an emulsion, a cream or a paste.

8. Use of the polypeptide or the polypeptide group according to any one of claims 1-3 in the preparation of an antibacterial preparation. Preferably, the antibacterial preparation has rapid bactericidal activity and can complete the bactericidal function within 60 minutes. Preferably, the bacteria are drug-resistant bacteria, and the bacteria include but are not limited to Staphylococcus aureus, Enterococcus faecium, Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae or Acinetobacter baumannii.

9. Use according to claim 8, characterized in that, The antibacterial agent is a human antibacterial agent or a veterinary antibacterial agent; preferably, the antibacterial agent is an external antibacterial agent, and more preferably, the external antibacterial agent is an emulsion, a cream or a paste.

10. An antibacterial agent comprising the polypeptide or the polypeptide group according to any one of claims 1-3; the antibacterial agent is a human antibacterial agent or a veterinary antibacterial agent. Preferably, the antibacterial agent has rapid bactericidal activity and can complete the bactericidal function within 60 minutes. Preferably, the antibacterial agent is an external antibacterial agent, and more preferably, the external antibacterial agent is an emulsion, a cream or a paste.

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