Antimicrobial cyclic peptides and their applications

Abstract

This invention discloses three antimicrobial cyclic peptides and their applications in antimicrobial activity. The sequences of the three antimicrobial cyclic peptides are c[ D Phe-Pro-Phe- D Phe‑Asn‑Gln‑Tyr‑Val‑Lys‑Leu], c[ D Phe-Pro-Cha- D Phe‑Asn‑Gln‑Tyr‑Val‑Orn‑Leu], c[Phe‑ D Pro- D Phe-Phe- D Asn- D Gln- D Tyr- D Val- D Lys- D [Leu]. The three antibacterial cyclic peptides of this invention have strong bactericidal properties against bacteria such as Staphylococcus aureus and Bacillus subtilis, and their effects are significantly better than those of penicillin and the known cyclic peptide Tyrocidine A.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to three antimicrobial cyclic peptides and their applications. Background Technology

[0002] Antibiotics are the mainstay of global anti-infective drugs, 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, hold promise for overcoming the resistance problem due to their broad-spectrum antibacterial activity and the diversity of their structures and mechanisms of action. Cyclic peptides have attracted much attention due to their structural rigidity, conformational constraints, and excellent cell penetration and anti-enzymatic degradation capabilities. Currently, anti-infective therapy is the most widely used application area for cyclic peptide drugs. Developing novel, highly effective, and drug-resistant cyclic peptide antibiotics is crucial to meeting 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. It can form a β-type secondary structure, is amphiphilic, and has bactericidal activity against a broad spectrum of Gram-positive bacteria. The specific sequence is c[ D Phe-Pro-Phe- D[Phe-Asn-Gln-Tyr-Val-Orn-Leu] contains two D-configurations of phenylalanine. Structural modifications and activity studies based on Tyrocidine A have been widely reported. One approach is to improve water solubility and reduce toxicity through glycosylation. Patent specification CN102268075B discloses a method for preparing glycopeptide antibiotic Tyrocidine A glycosyl derivatives and their salts using a combination of solid-phase and liquid-phase methods, and synthesized some compounds with significant inhibitory activity against drug-resistant bacteria. On the other hand, there is the adjustment of amino acids in the sequence. For example, in 2002, Walsh's group used chemical enzymes to complete the cyclization of linear peptides and adjusted and screened 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, Guo's group used alanine scanning method to find that alanine substitution has the most significant effect at the 6-Gln position. Replacing the 6-Gln of natural products with cationic amino acids alone can greatly improve the therapeutic index (J. Am. Chem. Soc. 2021, 143, 2736-2740). Hilvert's group used mutant enzymes to incorporate functional groups into Tyrocidine A and further achieved site-selective fluorescent labeling through alkyne-azide cycloaddition reaction to obtain a series of Tyrocidine A analogs. Currently, there is limited research on other positions in Tyrocidine A, such as the 9th and 3rd amino acids, and other strategies need to be developed.

[0004] Summary of the Invention

[0005] To address the aforementioned technical problems and shortcomings in this field, this invention provides three antimicrobial cyclic peptides and their applications, which exhibit strong bactericidal properties against bacteria such as Staphylococcus aureus and Bacillus subtilis, with effects significantly superior to penicillin and the known cyclic peptide Tyrocidine A.

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

[0007]

[0008]

[0009] Literature reports that Tyrocidine A molecules can form amphiphilic β-sheets with curvature (J.Med.Chem.,2017,60,9565-9574), with phenylalanine at position 3 located at the curved tail and ornithine at position 9 located at the center of the fold. Therefore, attempting to replace these two amino acids may yield new cyclic peptides by adjusting their conformation and electronic properties.

[0010] Replacing L-ornithine (L-Orn) at position 9 of Tyrocidine A with L-lysine (L-Lys) yields antimicrobial peptide-1, with the sequence c[ D Phe-Pro-Phe- D [Phe-Asn-Gln-Tyr-Val-Lys-Leu] has the structure shown in equation (I).

[0011] Replacing the L-phenylalanine (L-Phe) at position 3 of the Tyrocidine A molecule with the non-natural amino acid L-3-cyclohexylalanine (L-Cha) yields antimicrobial peptide-2, with the sequence c[ D Phe-Pro-Cha- D [Phe-Asn-Gln-Tyr-Val-Orn-Leu] has the structure shown in equation (II).

[0012] Furthermore, mirror-image peptides and proteins possess unique structures and functions, offering greater potential in terms of stability, biological activity, and immunogenicity. Therefore, this invention also provides a mirror-image peptide of Tyrocidine A, yielding antimicrobial peptide-3 with the sequence c[Phe- D Pro- D Phe-Phe- D Asn- D Gln- D Tyr- D Val- D Lys- D Leu] has the structure shown in equation (III).

[0013] The three antibacterial cyclic peptides of the present invention can be prepared according to conventional cyclic peptide synthesis methods based on the amino acid sequences given in the present invention.

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

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

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

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

[0018] Thirdly, the present invention provides a medicament suitable for treating and / or preventing bacterial infections, said medicament containing at least one of the antimicrobial cyclic peptides described in the first aspect.

[0019] The bacteria in the drug described in the third aspect may be Gram-positive bacteria and / or Gram-negative bacteria.

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

[0021] The medicines described in the third aspect may also include pharmaceutically acceptable carriers and / or excipients.

[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 as an additive to daily necessities for use.

[0023] Compared with the prior art, the beneficial effects of this invention are as follows:

[0024] This invention provides three antimicrobial cyclic peptides and their applications, which have strong bactericidal properties against bacteria such as Staphylococcus aureus and Bacillus subtilis, and their effects are significantly better than those of penicillin and the known cyclic peptide Tyrocidine A. Attached Figure Description

[0025] Figure 1 The images show the liquid chromatography (LC) and mass spectrometry (MS) spectra of antimicrobial peptide-1.

[0026] Figure 2 The images show the LC and MS spectra of antimicrobial peptide-2.

[0027] Figure 3 The images show the LC and MS spectra of antimicrobial peptide-3.

[0028] Figure 4 The images show the LC and MS spectra of Tyrocidine A.

[0029] Figure 5 The figure shows the experimental test results of antimicrobial peptide-1 with Staphylococcus aureus and Bacillus subtilis.

[0030] Figure 6 The figure shows the experimental test results of antimicrobial peptide-2 with Staphylococcus aureus and Bacillus subtilis.

[0031] Figure 7The figure shows the experimental test results of antimicrobial peptide-3 with Staphylococcus aureus and Bacillus subtilis.

[0032] Figure 8 The figure shows the experimental test results of Tyrocidine A with Staphylococcus aureus and Bacillus subtilis. Detailed Implementation

[0033] The present invention will be further described below with reference to the accompanying drawings and specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0034] Unless otherwise specified, the operating methods in the following examples are generally performed under conventional conditions or as recommended by the manufacturer.

[0035] Preparation method of antimicrobial peptides

[0036] (1) Solid-phase synthesis of linear peptides: 2-chlorotriphenylmethyl chloride (2-CTC resin, 0.98 mmol / g loading) was weighed into a reaction tube and swollen in dichloromethane (DCM) at room temperature for 1 hour. After the solvent was removed, a dichloromethane solution of the first C-terminal amino acid monomer (1.5 equiv) and N,N-diisopropylethylamine (DIEA, 5 equiv) was added. After shaking at room temperature for 1 hour, the resin was washed sequentially with N,N-dimethylformamide (DMF) and DCM. Then, deprotection was performed by adding 20% ​​piperidine / DMF solution, deprotection was performed twice, 10 minutes each time, and then the resin was washed sequentially with DCM and DMF. Then, coupling of the second amino acid was performed 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). After shaking at room temperature for 1 hour, the resin was washed sequentially with DMF and DCM. Repeat the above steps of deprotection, washing, coupling, and washing until the coupling of the last amino acid monomer is completed. Finally, perform N-terminal deprotection by adding 20% ​​piperidine / DMF solution and deprotecting twice, 10 minutes each time. Then wash the resin sequentially with DCM, DMF, and DCM, and dry.

[0037] (2) Linear peptide cleavage: Prepare cleavage reagent (20% hexafluoroisopropanol / DCM), add it to dry resin, shake at room temperature for 0.5 h, repeat once, combine the filtrates and collect them in centrifuge tubes, remove the solvent by rotary evaporation to obtain crude product.

[0038] (3) Cyclation of linear peptides: The crude linear peptide product was dissolved in DMF (0.005M). After complete dissolution, HATU (2 equiv) and DIEA (3-6 equiv) were added sequentially, and the reaction was carried out 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 the crude cyclic peptide product. Acetonitrile was added to dissolve the product, and the insoluble solids were removed by centrifugation. The filtrate was collected and evaporated to dryness for 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, and shake at room temperature for 0.5-2 hours. Monitor the deprotection process using LCMS. After complete deprotection, dry the solvent with an air pump, purify the product using reversed-phase preparative HPLC, and lyophilize to obtain the pure product. Antimicrobial peptides 1-3 and Tyrocidine A are all white solids with a purity >95%.

[0040] (5) Detection of cyclic peptide samples: The prepared cyclic peptide products were analyzed by LC-MS. The detection results are shown in the figure. Figure 1-4 .

[0041] In this invention, the minimum inhibitory concentration (MIC) of cyclic peptide samples was determined using the 96-well plate microdilution method to evaluate the antibacterial activity of cyclic peptides. Staphylococcus aureus (ATCC29213) and Bacillus subtilis (ATCC6633) were selected as standard strains, and penicillin was used as a control. Figure 5-8 The experimental test results are presented.

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

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

[0044] 3) MIC Test: Dilute the bacterial culture to 10⁶ CFU / mL using LB liquid medium. Dilute the sample stock solution to a 1.024 mg / mL sample-medium mixture. Dilute DMSO 10-fold. Prepare a 1.024 mg / mL penicillin solution. Add 100 μL of LB liquid medium to wells 2 through 12 of a 96-well plate. Add 200 μL of the 1.024 mg / mL sample-medium mixture to well 1. Then, add 100 μL of the sample-medium mixture from well 1 to well 2, mix well, add 100 μL of the mixed sample-medium mixture to well 3, and so on. Dilute the sample solution twofold. Finally, discard 100 μL of the mixed sample-medium mixture from well 12. Repeat the above steps using the 1.024 mg / mL penicillin solution instead of the sample-medium mixture. The above steps were repeated using diluted DMSO instead of the sample-culture medium mixture as a control group, with DMSO comprising 5% in the first well.

[0045] Add 100 μL of diluted bacterial culture to each well of a 96-well plate, resulting in an initial concentration of 512 μg / mL for both the sample and penicillin. Incubate the 96-well plate at 37°C for 24 hours and record the MIC values.

[0046] Table 1 shows the minimum inhibitory concentrations of the antimicrobial peptides 1-3 of the present invention, as well as 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 2μg / mL 128 μg / mL 2μg / mL >512μg / mL 256 μg / mL Bacillus subtilis MIC 1μg / mL 32μg / mL 2μg / mL 128 μg / mL 128 μg / mL

[0049] In Table 1, a smaller MIC value indicates a stronger antibacterial ability. The results show that the three antimicrobial cyclic peptides designed in this invention exhibit significant antibacterial activity against Staphylococcus aureus and Bacillus subtilis, with effects superior to penicillin, especially antimicrobial peptide-1 and antimicrobial peptide-3.

[0050] Furthermore, it should be understood that after reading the above description of the present invention, those skilled in the art can make various alterations or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

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

2. The use of the antimicrobial cyclic peptide according to claim 1 in the preparation of medicaments for treating and / or preventing bacterial infections, characterized in that, The bacteria are at least one of Staphylococcus aureus and Bacillus subtilis.

3. The application according to claim 2, characterized in that, The drug also includes pharmaceutically acceptable carriers and / or excipients.

4. A medicine suitable for treating and / or preventing bacterial infections, characterized in that, The drug contains at least one of the antimicrobial cyclic peptides of claim 1, and the bacteria is at least one of Staphylococcus aureus and Bacillus subtilis.

5. The drug according to claim 4, characterized in that, The drug also includes pharmaceutically acceptable carriers and / or excipients.

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

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