Antibacterial cyclic peptide and application thereof

Abstract

The invention discloses three antibacterial cyclic peptides and an application thereof in an antibacterial aspect. The sequences of the three kinds of antibacterial cyclic peptides are respectively c [DPhe-Pro-Phe-DPhe-Asn-Gln-Tyr-Val-Lys-Leu], c [DPhe-Pro-Cha-DPhe-Asn-Gln-Tyr-Val-Orn-Leu], and c [Phe-DPhe-DPhe-DAsn-DGln-DDyr-DVal-DLys-DLeu]. The invention further discloses a preparation method of the antibacterial cyclic peptides. The three antibacterial cyclic peptides provided by the invention have relatively strong bactericidal performance on bacteria such as staphylococcus aureus, bacillus subtilis and the like, and the effect is obviously superior to that of penicillin and known cyclic peptide Tyrocidine A.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to three antibacterial cyclic peptides and applications thereof. Background Art

[0002] Antibiotics are the mainstay of anti-infective drugs worldwide, with a market size of tens of billions of dollars. However, their widespread use has led to antibiotic resistance, which has become a serious global public health challenge. In contrast, antimicrobial peptides (AMPs), as a new generation of antibiotics, are expected to overcome the problem of drug resistance due to their broad-spectrum antimicrobial activity and the diversity of their structures and mechanisms of action. Cyclic peptides have attracted much attention due to their rigid structure, restricted conformation, excellent cell permeability and resistance to enzyme degradation. At present, anti-infection is the most widely used field of cyclic peptide drugs. The development of new, highly effective and resistant cyclic peptide antibiotics is crucial to meet the growing market demand for antimicrobial drugs.

[0003] Tyrocidine A (amino acid sequence see SEQ ID NO: 1) is a natural cyclic decapeptide isolated from Bacillus brevis, which can form a β-type secondary structure, has amphiphilicity, and has bactericidal activity against a wide spectrum of Gram-positive bacteria. The specific sequence is c[ D Phe-Pro-Phe- DPhe-Asn-Gln-Tyr-Val-Orn-Leu], containing two D-configuration phenylalanines. Research on the structure modification and activity of Tyrocidine A has been widely reported. On the one hand, glycosylation is used to improve water solubility and reduce toxicity. The patent specification with announcement number CN102268075B discloses a method for preparing glycopeptide antibiotic Tyrocidine A glycosyl derivatives and salts thereof by combining solid phase and liquid phase, and synthesized some compounds with significant inhibitory activity against drug-resistant bacteria. On the other hand, the amino acids in the sequence are adjusted. For example, in 2002, the Walsh group used chemical enzymes to complete the cyclization of linear peptides, and adjusted and screened the D-phenylalanine at positions 1 and 4 to construct a cyclic peptide library. Replacing position 4 with a positive D-amino acid (D-Arg, D-Lys or D-Orn) can produce the best therapeutic index (Nature 2002, 418, 658-661); in 2003, the Guo group used the alanine scanning method to find that alanine substitution at the 6-Gln position has the most obvious effect. Replacing the 6-Gln of the natural product with a cationic amino acid alone can greatly improve the therapeutic index (J.Am.Chem.Soc.2021, 143, 2736-2740); the Hilvert group used mutant enzymes to incorporate functional groups into Tyrocidine A, and further achieved site-selective fluorescent labeling through alkyne-azide cycloaddition reactions to obtain a series of Tyrocidine A analogs. Currently, there are few studies on other positions in Tyrocidine A, such as amino acids at positions 9 and 3, and other strategies need to be developed.

[0004] Summary of the invention

[0005] In view of the above technical problems and the shortcomings in the art, the present invention provides three antibacterial cyclic peptides and their applications, which have strong bactericidal properties against bacteria such as Staphylococcus aureus and Bacillus subtilis, and the effects are significantly better than penicillin and the known cyclic peptide Tyrocidine A.

[0006] In a first aspect, the present invention provides three antimicrobial cyclic peptides, each of which has a structure as shown in any one of the following formulas (I) to (III):

[0007]

[0008]

[0009] Literature reports that Tyrocidine A molecules can form amphiphilic β-folds with curvature (J. Med. Chem., 2017, 60, 9565-9574). Phenylalanine at position 3 is located at the curved tail, and ornithine at position 9 is located at the center of the fold. Therefore, attempts to replace the amino acids at these two positions may result in new cyclic peptides that can be adjusted in terms of conformation and electrical properties.

[0010] The 9-position L-ornithine (L-Orn) in the Tyrocidine A molecule was replaced with L-lysine (L-Lys) to obtain antimicrobial peptide-1, the sequence of which is c[ D Phe-Pro-Phe- D Phe-Asn-Gln-Tyr-Val-Lys-Leu] has the structure shown in formula (I).

[0011] The L-phenylalanine (L-Phe) at position 3 in the Tyrocidine A molecule was replaced with the unnatural amino acid L-3-cyclohexylalanine (L-Cha) to obtain antimicrobial peptide-2, the sequence of which is c[ D Phe-Pro-Cha- D Phe-Asn-Gln-Tyr-Val-Orn-Leu], having the structure shown in formula (II).

[0012] In addition, mirror-image polypeptides and mirror-image proteins have unique structures and functions, and have more possibilities in terms of stability, biological activity, immunogenicity, etc. Therefore, the present invention also provides a mirror-image peptide of Tyrocidine A, and obtains antimicrobial peptide-3, the sequence of which is c[Phe- D Pro- D Phe-Phe- D Asn- D Gln- D Tyr- D Val- D Lys- D Leu], having a structure as shown in formula (III).

[0013] The three antibacterial cyclic peptides of the present invention can be prepared according to the existing conventional cyclic peptide synthesis method based on the amino acid sequence given by the present invention.

[0014] In a second aspect, the present invention provides use of the antibacterial cyclic peptide described in the first aspect in the preparation of a drug for treating and / or preventing bacterial infection.

[0015] In the application described in the second aspect, the bacteria may be Gram-positive bacteria and / or Gram-negative bacteria.

[0016] In the application described in the second aspect, the bacteria may include at least one of Staphylococcus aureus and Bacillus subtilis.

[0017] In the application described in the second aspect, the drug may further include a pharmaceutically acceptable carrier and / or excipient.

[0018] In a third aspect, the present invention provides a drug suitable for treating and / or preventing bacterial infection, wherein the drug contains at least one of the antibacterial cyclic peptides described in the first aspect.

[0019] In the drug of the third aspect, the bacteria may be Gram-positive bacteria and / or Gram-negative bacteria.

[0020] In the drug of the third aspect, the bacteria may include at least one of Staphylococcus aureus and Bacillus subtilis.

[0021] The drug described in the third aspect may further include a pharmaceutically acceptable carrier and / or excipient.

[0022] As a general inventive concept, the present invention provides an antibacterial additive, which includes at least one of the antibacterial cyclic peptides described in the first aspect, and the antibacterial additive is added to daily necessities as an additive for use.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] The invention provides three antibacterial cyclic peptides and applications thereof, which have strong bactericidal properties against bacteria such as Staphylococcus aureus and Bacillus subtilis, and are significantly better than penicillin and known cyclic peptide Tyrocidine A. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 It is the liquid chromatography (LC) and mass spectrometry (MS) diagram of antimicrobial peptide-1.

[0026] Figure 2 LC and MS charts of antimicrobial peptide-2.

[0027] Figure 3 LC and MS charts of antimicrobial peptide-3.

[0028] Figure 4 LC and MS charts of Tyrocidine A.

[0029] Figure 5 This is a graph showing the experimental test results of antimicrobial peptide-1 with Staphylococcus aureus and Bacillus subtilis.

[0030] Figure 6 This is a graph showing the experimental test results of antimicrobial peptide-2 with Staphylococcus aureus and Bacillus subtilis.

[0031] Figure 7This is a graph showing the experimental test results of antimicrobial peptide-3 with Staphylococcus aureus and Bacillus subtilis.

[0032] Figure 8 This is a graph showing the test results of Tyrocidine A against Staphylococcus aureus and Bacillus subtilis. DETAILED DESCRIPTION

[0033] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and are not used to limit the scope of the present invention.

[0034] The operating methods in the following examples without specifying specific conditions are usually carried out under conventional conditions or conditions recommended by the manufacturers.

[0035] Method for preparing antimicrobial peptides

[0036] (1) Linear peptide solid phase synthesis: 2-Chlorotriphenylmethyl chloride (2-CTC resin, 0.98 mmol / g loading) was weighed in a reaction tube and dichloromethane (DCM) was added to swell at room temperature for 1 hour. After the solvent was drained, the first amino acid monomer at the C-terminus (1.5 equiv) and N,N-diisopropylethylamine (DIEA, 5 equiv) in dichloromethane solution were added. After shaking at room temperature for 1 hour, the resin was washed with N,N-dimethylformamide (DMF) and DCM in turn. Subsequently, Fmoc protection was removed by adding 20% ​​piperidine / DMF solution, deprotection was performed twice, each time for 10 minutes, and then the resin was washed with DCM and DMF in turn. Subsequently, the second amino acid was coupled by adding the activated second amino acid monomer DMF solution (including 5 equiv of amino acid monomer, 4.75 equiv of condensing agent HBTU and 5 equiv of DIEA), shaking at room temperature for 1 hour, and then the resin was washed with DMF and DCM in turn. Repeat the above steps of Fmoc deprotection, washing, coupling, and washing until the last amino acid monomer is coupled. Finally, N-terminal deprotection is performed by adding 20% ​​piperidine / DMF solution and deprotection is performed twice, each time for 10 minutes, and then the resin is washed with DCM, DMF, and DCM in sequence and dried.

[0037] (2) Linear peptide cleavage: Prepare a cleavage reagent (20% hexafluoroisopropanol / DCM), add it to the dry resin, and react at room temperature with shaking for 0.5 h. Repeat once, combine the filtrate and collect it in a centrifuge tube, and remove the solvent by rotary evaporation to obtain a crude product.

[0038] (3) Cyclization of linear peptide: The crude linear peptide was dissolved in DMF (0.005 M). After complete dissolution, HATU (2 equiv) and DIEA (3-6 equiv) were added in sequence and reacted at room temperature for 2 hours. The cyclization process was monitored by LCMS. After the reaction was complete, the solvent was removed by vacuum distillation to obtain a crude cyclic peptide. Acetonitrile was added to dissolve it, and the insoluble solid was removed by centrifugation. The filtrate was collected and dried to carry out the next step of deprotection.

[0039] (4) Deprotection of cyclic peptide side chains: Prepare a deprotection solution (trifluoroacetic acid / triisopropylsilane / DCM=50 / 5 / 45), add it to the crude cyclic peptide product, shake at room temperature for 0.5-2 hours, and monitor the deprotection process by LCMS. After the deprotection is complete, the solvent is blown dry by an air pump, and the product is purified by reverse phase preparative HPLC and freeze-dried to obtain a pure product. Antimicrobial peptides 1-3 and Tyrocidine A are both white solids with a purity of >95%.

[0040] (5) Cyclic peptide sample detection: The prepared cyclic peptide product was subjected to LC-MS analysis. The detection results are shown in Figure 1-4 .

[0041] In the present invention, the 96-well plate microdilution method is used to determine the minimum inhibitory concentration (MIC) of Yangping for the cyclic peptide sample to evaluate the antibacterial activity of the cyclic peptide. Staphylococcus aureus (number: ATCC29213) and Bacillus subtilis (number: ATCC6633) are selected as standard strains, and penicillin is used as a control. Figure 5-8 The experimental test results are presented.

[0042] 1) Preparation of bacterial suspension: Take three 12 mL bacterial culture tubes, add 3 mL LB liquid medium to each, pick single colonies from the solid culture medium of Staphylococcus aureus and Bacillus subtilis and add them to the liquid culture medium, and use the other tube as a blank control. Place in a constant temperature shaker (37°C, 200 rpm) and shake culture overnight (15 hours).

[0043] 2) Cyclic peptide sample processing and grouping: Weigh the cyclic peptide samples separately and sterilize them under ultraviolet light for 30 minutes. Add dimethyl sulfoxide (DMSO) to the samples to prepare a 10.24 mg / mL sample stock solution for later use. Set up 3 parallel groups for each sample, and set up a penicillin group and a control group.

[0044] 3) MIC test: Dilute the bacterial solution to 106 CFU / mL with LB liquid culture medium, dilute the sample stock solution to a 1.024 mg / mL sample-culture medium mixture, dilute DMSO 10 times, and prepare penicillin to a 1.024 mg / mL penicillin solution for use. Add 100 μL LB liquid culture medium to the second to twelfth wells of the 96-well plate, take 200 μL of the 1.024 mg / mL sample-culture medium mixture and add it to the first well, then take 100 μL of the sample-culture medium mixture from the first well and add it to the second well, mix well, take 100 μL of the mixed sample-culture medium mixture and add it to the third well, mix well... and so on, dilute the sample solution by two times, and finally take 100 μL of the mixed sample-culture medium mixture from the twelfth well and discard it. Replace the sample-culture medium mixture with the 1.024 mg / mL penicillin solution and repeat the above steps. The sample-medium mixture was replaced with diluted DMSO, and the above steps were repeated as a control group, with the DMSO ratio in the first well being 5%.

[0045] 100 μL of diluted bacterial solution was added to each well of the 96-well plate, that is, the initial concentration of the sample and penicillin was 512 μg / mL. The 96-well plate was placed in a constant temperature incubator at 37°C for 24 hours, and the MIC value was recorded.

[0046] Table 1 shows the minimum inhibitory concentrations of the antimicrobial peptides 1 to 3 of the present invention, Tyrocidine A and penicillin (control).

[0047] Table 1

[0048] Antimicrobial peptide-1 Antimicrobial peptide-2 Antimicrobial peptide 3 Tyrocidine A Penicillin (control) Staphylococcus aureus MIC 2 μg / mL 128 μg / mL 2 μg / mL >512 mcg / mL 256 μg / mL Bacillus subtilis MIC 1 μg / mL 32 μg / mL 2 μg / mL 128 μg / mL 128 μg / mL

[0049] In Table 1, the smaller the MIC value, the stronger the antibacterial ability. The results show that the three antibacterial cyclic peptides designed in the present invention have obvious antibacterial ability against Staphylococcus aureus and Bacillus subtilis, and the effect is better than penicillin, especially antimicrobial peptide-1 and antimicrobial peptide-3 have excellent performance.

[0050] In addition, it should be understood that after reading the above description of the present invention, those skilled in the art may make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

1. An antimicrobial cyclic peptide, characterized in that: The antibacterial cyclic peptide has a structure as shown in any one of the following formulas (I) to (III):

2. Use of the antibacterial cyclic peptide according to claim 1 in the preparation of a drug for treating and / or preventing bacterial infection.

3. The use according to claim 2, characterized in that: The bacteria are Gram-positive bacteria and / or Gram-negative bacteria.

4. The use according to claim 2, characterized in that: The bacteria include at least one of Staphylococcus aureus and Bacillus subtilis.

5. The use according to claim 2, characterized in that: The medicine further comprises a pharmaceutically acceptable carrier and / or excipient.

6. A drug suitable for treating and / or preventing bacterial infection, characterized in that: The drug contains at least one of the antimicrobial cyclic peptides according to claim 1.

7. The drug according to claim 6, characterized in that The bacteria are Gram-positive bacteria and / or Gram-negative bacteria.

8. The drug according to claim 6, characterized in that The bacteria include at least one of Staphylococcus aureus and Bacillus subtilis.

9. The drug according to claim 6, characterized in that The medicine further comprises a pharmaceutically acceptable carrier and / or excipient.

10. An antibacterial additive, characterized in that: The antibacterial additive comprises at least one of the antibacterial cyclic peptides according to claim 1, and the antibacterial additive is added to daily necessities as an additive for use.

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