With R n Centrosymmetric β-hairpin antimicrobial peptides with KW repeat sequences at corners and their applications

Abstract

The application discloses a kind of central symmetry β-hairpin antibacterial peptide with KW repeat sequence with R n As corner, and application thereof, the antibacterial peptide is with R, RR or RRR as corner, WK repeat unit is alternately arranged in two sides, its structure general formula is (WK) x R n (KW) x -NH2, marked as R n W x ;Wherein, n=1,2,3;X=2,3.In vitro antibacterial experiment and hemolytic activity experiment show that this kind of antibacterial peptide has strong antibacterial activity and almost negligible hemolytic toxicity.After D-type amino acid full replacement of R2W3 sequence, the antibacterial activity of D-R2W3 is further enhanced, the hemolytic toxicity is low, and it can resist the hydrolysis of trypsin and chymotrypsin, has very great potential in relieving antibiotic resistance, and has good application prospect in preparing clinical antibacterial drugs.

Description

Technical Field

[0001] This invention belongs to the field of biochemistry technology, and relates to a class of R n This invention relates to a centrosymmetric β-hairpin antimicrobial peptide with a KW repeat sequence at an angle, and also to the application of this antimicrobial peptide in the preparation of clinical antimicrobial drugs. Background Technology

[0002] The emergence of multidrug-resistant bacteria poses a significant challenge to global public health, making the development of modern antimicrobial drugs urgently require new antimicrobial compounds. In recent years, research on antimicrobial peptides has increased. Antimicrobial peptides exist in various animals, plants, and microorganisms, possessing diverse biological activities such as antibacterial, anti-inflammatory, and immunomodulatory effects. Unlike traditional antibiotics that act on bacterial metabolic pathways, antimicrobial peptides have a powerful disruptive effect on bacterial cell membranes, rapidly killing bacteria by increasing biomembrane permeability, thus reducing the likelihood of drug resistance. However, the long sequences, low activity, high toxicity, and poor stability of natural antimicrobial peptides limit their clinical application. The main secondary structures of antimicrobial peptides include α-helices, β-sheets, and random coils. Some studies have shown that β-sheet antimicrobial peptides exhibit higher selectivity between bactericidal and hemolytic toxicity than α-helical antimicrobial peptides (Jia et al., 2020; Jin et al., 2005; Ma et al., 2014). Summary of the Invention

[0003] One of the objectives of this invention is to provide a class of R n It is a centrosymmetric β-hairpin antimicrobial peptide with a KW repeat sequence at the corner.

[0004] A second objective of this invention is to provide the application of the above-mentioned antimicrobial peptides in the preparation of clinical antimicrobial drugs.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] I. Using R n Structural design for a centrosymmetric β-hairpin antimicrobial peptide with KW repeat sequences at the corner.

[0007] This invention uses R n The centrosymmetric β-hairpin antimicrobial peptide with KW repeat sequences at the corners is based on the β-hairpin structure, with varying numbers of arginine residues as corners, alternating lysine and tryptophan residues on both sides, and amidation at the C-terminus. Its general structural formula is: (WK). x R n (KW) x -NH2, labeled R n W x , where n = 1, 2, 3; x = 2, 3.

[0008] Specifically, the antimicrobial peptide is:

[0009] WKWKRKWKW-NH2, labeled R1W2, has the amino acid sequence shown in SEQ ID No. 1;

[0010] Alternatively: WKWKRRKWKW-NH2, labeled R2W2, with its amino acid sequence shown in SEQ ID No. 2;

[0011] Alternatively: WKWKRRRKWKW-NH2, labeled R3W2, with its amino acid sequence shown in SEQ ID No. 3;

[0012] Alternatively: WKWKWKRKWKWKW-NH2, labeled R1W3, with its amino acid sequence shown in SEQ ID No. 4;

[0013] Alternatively: WKWKWKRRKWKWKW-NH2, labeled R2W3, with its amino acid sequence shown in SEQ ID No. 5;

[0014] Alternatively: WKWKWKRRRKWKWKW-NH2, labeled R3W3, with its amino acid sequence shown in SEQ ID No. 6;

[0015] As a further preferred embodiment of the technical solution of the present invention, the antimicrobial peptide is: WKWKWKRRKWKWKW-NH2, labeled as R2W3, and its amino acid sequence is shown in SEQ ID No. 5.

[0016] Furthermore, by replacing all amino acids in the R2W3 sequence with D-type amino acids, a new antimicrobial peptide was obtained, the structural formula of which is as follows: D W D K D W D K D W D K D R D R D K D W D K D W D K D W-NH2, labeled D-R2W3, has the amino acid sequence D-Trp-D-Lys-D-Trp-D-Lys-D-Trp-D-Lys-D-Arg-D-Arg-D-Lys-D-Trp-D-Lys-D-Trp-D-Lys-D-Trp-D-Lys-D-Trp-NH2.

[0017] The above uses R nThe centrosymmetric β-hairpin antimicrobial peptides with KW repeating sequences at the corners were all prepared using the classical solid-phase synthesis method.

[0018] II. Using R n Application of centrosymmetric β-hairpin antimicrobial peptides with KW repeat sequences at corners in the preparation of clinical antimicrobial drugs

[0019] 1. In vitro antibacterial test

[0020] To evaluate the antimicrobial activity of the antimicrobial peptides of this invention, the minimum inhibitory concentration (MIC) of the antimicrobial peptides was determined using the microbroth dilution method. A series of gradient antimicrobial peptides were prepared using the two-fold dilution method, and then diluted with bacterial diluents (~10). 5 Equal volumes of peptides (CFU / mL) were incubated at 37℃ for 18 hours, and the lowest peptide concentration at which no bacterial growth was observed was determined as the minimum inhibitory concentration (MIC). Streptomycin, polymyxin B, and kanamycin were used as positive controls. The experiment was performed in three independent replicates, with each replicate in triplicate. The results are shown in Table 1.

[0021] Table 1. Minimum inhibitory concentrations (μM) of the antimicrobial peptides of this invention against common standard strains.

[0022]

[0023]

[0024] Table 1 shows that the antimicrobial peptides of the present invention have a strong inhibitory effect on Gram-positive bacteria, represented by Staphylococcus aureus, Bacillus subtilis and Staphylococcus epidermidis, as well as on Gram-negative bacteria, represented by Klebsiella pneumoniae, Escherichia coli and Pseudomonas aeruginosa, demonstrating broad-spectrum antimicrobial activity.

[0025] 2. Hemolytic activity test

[0026] To evaluate the toxicity of the antimicrobial peptide of this invention to normal mammalian cells, the hemolysis of the antimicrobial peptide after co-incubation with mouse erythrocytes at 37°C for 1 hour was measured. The structure is shown below. Figure 1 .

[0027] Figure 1 The results showed that the hemolysis rate of 256 μM R1W3 after incubation with mouse erythrocytes for 1 h was less than 10%, and the hemolysis rate of the other antimicrobial peptides at 512 μM was still less than 10%, which was much higher than the concentration at which the antimicrobial peptides exerted their antimicrobial activity, indicating that these antimicrobial peptides had high biocompatibility.

[0028] 3. Treatment Index

[0029] To evaluate the efficacy and safety of the antimicrobial peptides of this invention, their therapeutic index, i.e., MHC, was calculated. 10 With GM MIC The ratio of MHC10 : Peptide concentration that causes 10% hemolysis; GM MIC The present invention represents the geometric mean of the MICs of the antimicrobial peptides against six bacteria. If the hemolytic activity of the antimicrobial peptide at the highest tested concentration of 512 μM is less than 10%, its MHC value is considered when calculating the therapeutic index. 10 Calculated based on 1024.

[0030] Table 2. Therapeutic index of the antimicrobial peptides of this invention

[0031]

[0032]

[0033] Table 2 shows that the antimicrobial peptides of the present invention all have high therapeutic indices. Among the first six antimicrobial peptides, R2W3 has the highest therapeutic index, reaching 203.2. However, antimicrobial peptides composed of L-amino acids have poor stability, so D-amino acids were replaced to obtain D-R2W3. Unexpectedly, D-R2W3 maintained low toxicity while also enhancing antimicrobial activity. Therefore, D-R2W3 has the highest therapeutic index, namely 322.5.

[0034] 4. Determination of enzymatic hydrolysis stability

[0035] To evaluate the enzyme resistance of the antimicrobial peptide of this invention, the antimicrobial peptide was incubated with different concentrations of trypsin or chymotrypsin for 1 hour, then inactivated at 60°C for 15 minutes. The inhibitory effect of the antimicrobial peptide on bacterial growth was then measured. The final concentration of the antimicrobial peptide was 2×MIC. The results are shown in [Figure number missing]. Figure 2 .

[0036] Figure 2 The results showed that peptides R1W2, R2W2, R3W2, R1W3, R2W3, and R3W3, composed of L-amino acids, exhibited poor stability and lost their antibacterial activity after incubation with lower concentrations of enzymes. R2W3, which had the highest therapeutic index, lost its ability to inhibit Staphylococcus aureus after incubation with trypsin at 2 μg / mL or higher for 1 hour; and lost its ability to inhibit Staphylococcus aureus growth after incubation with chymotrypsin at 20 μg / mL or higher. In contrast, D-R2W3 completely inhibited bacterial growth even after incubation with the highest concentrations of trypsin or chymotrypsin, indicating that D-R2W3 has very high enzymatic stability.

[0037] This invention uses R, RR, or RRR as corners to alternately arrange WK repeating units on both sides, resulting in a class of centrosymmetric β-hairpin antimicrobial peptides R. n W xIn vitro antibacterial and hemolytic activity assays showed that this class of antimicrobial peptides possesses potent antibacterial activity and negligible hemolytic toxicity. Further enhancement of antibacterial activity and low hemolytic toxicity were achieved by completely replacing the D-type amino acids in the R2W3 sequence. It also exhibits resistance to hydrolysis by trypsin and chymotrypsin, demonstrating significant potential in alleviating antibiotic resistance and promising applications in the preparation of clinical antimicrobial drugs. Attached Figure Description

[0038] Figure 1 The results show the hemolytic activity of the antimicrobial peptide of this invention on mouse erythrocytes after incubation for 1 hour.

[0039] Figure 2 The antimicrobial activity of the antimicrobial peptide of this invention against Staphylococcus aureus after incubation with chymotrypsin or trypsin for 1 hour;

[0040] Figure 3 This is the mass spectrum of the antimicrobial peptide R1W2 of the present invention;

[0041] Figure 4 This is the mass spectrum of the precursor peptide R2W2 of this invention;

[0042] Figure 5 This is the mass spectrum of the antimicrobial peptide R3W2 of the present invention;

[0043] Figure 6 This is the mass spectrum of the antimicrobial peptide R1W3 of the present invention;

[0044] Figure 7 This is the mass spectrum of the antimicrobial peptide R2W3 of the present invention;

[0045] Figure 8 This is the mass spectrum of the antimicrobial peptide R3W3 of this invention;

[0046] Figure 9 This is the mass spectrum of the antimicrobial peptide D-R2W3 of the present invention. Detailed Implementation

[0047] The synthesis of the antimicrobial peptides of the present invention, which have broad-spectrum antibacterial activity, low toxicity, and resistance to enzymatic hydrolysis, will be further illustrated below through specific embodiments.

[0048] Example 1: Synthesis of antimicrobial peptide D-R2W3

[0049] (1) Resin activation and pretreatment

[0050] Weigh MBHA resin and add it to a polypeptide solid-phase synthesizer. After swelling in DCM for 30 minutes and washing with DMF, identify the resin using the ninhydrin colorimetric method. If it is colorless, the resin is qualified.

[0051] (2) Synthesis of Fmoc-D-R2W3-MBHA

[0052] The Fmoc protecting group was removed using a DMF solution containing 20% ​​piperidine. The indole test resin turned blue-purple, indicating successful deprotection and exposure of the amino group. After washing four times with DMF, 3 times the amount of D-Trp, 3 times the amount of HOBt, HBTU, and 6 times the amount of DIEA were dissolved in DMF and added to the synthesizer. The mixture was stirred and condensed for 1 hour. After the reaction time was reached, the mixture was washed three times with DMF. The indole test resin turned colorless and transparent, indicating successful condensation, yielding Fmoc-D-Trp-MBHA.

[0053] Following the method described above, D-Lys, D-Trp, D-Lys, D-Trp, D-Lys, D-Arg, D-Arg, D-Lys, D-Trp, D-Lys, D-Trp, D-Lys, D-Trp are condensed sequentially to obtain Fmoc-D-Trp-D-Lys-D-Trp-D-Lys-D-Trp-D-Lys-D-Arg-D-Arg-D-Lys-D-Trp-D-Lys-D-Trp-D-Lys-D-Trp-MBHA.

[0054] (3) Peptide cleavage

[0055] The obtained Fmoc-D-Trp-D-Lys-D-Trp-D-Lys-D-Trp-D-Lys-D-Arg-D-Arg-D-Lys-D-Trp-D-Lys-D-Trp-D-Lys-D-Trp-MBHA was deprotected with a DMF solution containing 20% ​​piperidine. The resin was then washed successively with DCM and methanol, and thoroughly dried under vacuum. 10 mL of a cleavage reagent (TFA:Tris:water = 95:2.5:2.5 (v:v:v)) was added, and the mixture was reacted for 3 h. After extraction with diethyl ether, the mixture was freeze-dried.

[0056] (4) Peptide purification

[0057] The RP-HPLC purification conditions were: mobile phase A: 0.1% TFA / acetonitrile, mobile phase B: 0.1% TFA / water, using linear gradient elution. The target peak eluent was collected, lyophilized, and the antimicrobial peptide D-R2W3 was obtained. Its mass spectrum is shown below. Figure 9 As shown.

[0058] Example 2: Synthesis of antimicrobial peptide R2W3

[0059] (1) Resin activation and pretreatment

[0060] Same as Example 1.

[0061] (2) Synthesis of Fmoc-R2W3-MBHA

[0062] The Fmoc protecting group was removed using a DMF solution containing 20% ​​piperidine. The indole test resin turned blue-purple, indicating successful deprotection and exposure of the amino group. After washing four times with DMF, 3 times the amount of Trp, 3 times the amount of HOBt, HBTU, and 6 times the amount of DIEA were dissolved in DMF and added to the synthesizer. The mixture was stirred and condensed for 1 hour. After the reaction time was reached, the mixture was washed three times with DMF. The indole test resin turned colorless and transparent, indicating successful condensation, yielding Fmoc-Trp-MBHA.

[0063] Following the above method, Lys, Trp, Lys, Trp, Lys, Arg, Arg, Lys, Trp, Lys, Trp, Lys, Trp are condensed sequentially to obtain Fmoc-Trp-Lys-Trp-Lys-Trp-Lys-Arg-Arg-Lys-Trp-Lys-Trp-Lys-Trp-MBHA.

[0064] (3) Peptide cleavage

[0065] Same as Example 1.

[0066] (4) Peptide purification

[0067] As in Example 1, the antimicrobial peptide R2W3 was obtained, and its mass spectrum is shown below. Figure 7 As shown.

[0068] Example 3: Synthesis of antimicrobial peptide R1W2

[0069] (1) Resin activation and pretreatment

[0070] Same as Example 1.

[0071] (2) Synthesis of Fmoc-R1W2-MBHA

[0072] The synthesis of Fmoc-Trp-MBHA is the same as in Example 2.

[0073] Following the above method, Lys, Trp, Lys, Arg, Lys, Trp, Lys, Trp are condensed sequentially to obtain Fmoc-Trp-Lys-Trp-Lys-Arg-Lys-Trp-Lys-Trp-MBHA.

[0074] (3) Peptide cleavage

[0075] Same as Example 1.

[0076] (4) Peptide purification

[0077] As in Example 1, the antimicrobial peptide R1W2 was obtained, and its mass spectrum is shown below. Figure 3 As shown.

[0078] Example 4: Synthesis of antimicrobial peptide R2W2

[0079] (1) Resin activation and pretreatment

[0080] Same as Example 1.

[0081] (2) Synthesis of Fmoc-R2W2-MBHA

[0082] The synthesis of Fmoc-Trp-MBHA is the same as in Example 2.

[0083] Following the above method, Lys, Trp, Lys, Arg, Arg, Lys, Trp, Lys, Trp are condensed sequentially to obtain Fmoc-Trp-Lys-Trp-Lys-Arg-Arg-Lys-Trp-Lys-Trp-MBHA.

[0084] (3) Peptide cleavage

[0085] Same as Example 1.

[0086] (4) Peptide purification

[0087] As in Example 1, the antimicrobial peptide R2W2 was obtained, and its mass spectrum is shown below. Figure 4 As shown.

[0088] Example 5: Synthesis of antimicrobial peptide R3W2

[0089] (1) Resin activation and pretreatment

[0090] Same as Example 1.

[0091] (2) Synthesis of Fmoc-R3W2-MBHA

[0092] The synthesis of Fmoc-Trp-MBHA is the same as in Example 2.

[0093] Following the above method, Lys, Trp, Lys, Arg, Arg, Arg, Lys, Trp, Lys, Trp are condensed sequentially to obtain Fmoc-Trp-Lys-Trp-Lys-Arg-Arg-Arg-Lys-Trp-Lys-Trp-MBHA.

[0094] (3) Peptide cleavage

[0095] Same as Example 1.

[0096] (4) Peptide purification

[0097] Same as in Example 1, antimicrobial peptide R3W2 was obtained, and its mass spectrum is shown below. Figure 5 As shown.

[0098] Example 6: Synthesis of antimicrobial peptide R1W3

[0099] (1) Resin activation and pretreatment

[0100] Same as Example 1.

[0101] (2) Synthesis of Fmoc-R1W3-MBHA

[0102] The synthesis of Fmoc-Trp-MBHA is the same as in Example 2.

[0103] Following the above method, Lys, Trp, Lys, Trp, Lys, Arg, Lys, Trp, Lys, Trp, Lys, Trp are condensed sequentially to obtain Fmoc-Trp-Lys-Trp-Lys-Trp-Lys-Arg-Lys-Trp-Lys-Trp-Lys-Trp-MBHA.

[0104] (3) Peptide cleavage

[0105] Same as Example 1.

[0106] (4) Peptide purification

[0107] As in Example 1, the antimicrobial peptide R1W3 was obtained, and its mass spectrum is shown below. Figure 6 As shown.

[0108] Example 7: Synthesis of antimicrobial peptide R3W3

[0109] (1) Resin activation and pretreatment

[0110] Same as Example 1.

[0111] (2) Synthesis of Fmoc-R3W3-MBHA

[0112] The synthesis of Fmoc-Trp-MBHA is the same as in Example 2.

[0113] Following the above method, Lys, Trp, Lys, Trp, Lys, Arg, Arg, Arg, Lys, Trp, Lys, Trp, Lys, Trp are condensed sequentially to obtain Fmoc-Trp-Lys-Trp-Lys-Trp-Lys-Arg-Arg-Arg-Lys-Trp-Lys-Trp-Lys-Trp-MBHA.

[0114] (3) Peptide cleavage

[0115] Same as Example 1.

[0116] (4) Peptide purification

[0117] As in Example 1, the antimicrobial peptide R3W3 was obtained, and its mass spectrum is shown below. Figure 8 As shown.

Claims

1. A type of R n It is a centrosymmetric β-hairpin type antimicrobial peptide with a KW repeat sequence at an angle, characterized by: The antimicrobial peptide is based on a β-hairpin structure, with varying numbers of arginine residues as turns, alternating lysine and tryptophan residues on both sides, and is obtained by C-terminal amidation; the antimicrobial peptide is: WKWKRKWKW-NH2, labeled R1W2, has the amino acid sequence shown in SEQ ID No. 1; Alternatively: WKWKRRKWKW-NH2, labeled R2W2, with its amino acid sequence shown in SEQ ID No. 2; Alternatively: WKWKRRRKWKW-NH2, labeled R3W2, with its amino acid sequence shown in SEQ ID No. 3; Alternatively: WKWKWKRKWKWKW-NH2, labeled R1W3, with its amino acid sequence shown in SEQ ID No. 4; Alternatively: WKWKWKRRKWKWKW-NH2, labeled R2W3, with its amino acid sequence shown in SEQ ID No. 5; Alternatively: WKWKWKRRRKWKWKW-NH2, labeled R3W3, with its amino acid sequence shown in SEQ ID No.

6.

2. A type of R as described in claim 1 n It is a centrosymmetric β-hairpin type antimicrobial peptide with a KW repeating sequence at an angle, characterized in that, The antimicrobial peptide is: WKWKWKRRKWKWKW-NH2, labeled R2W3, and its amino acid sequence is shown in SEQ ID No.

5.

3. A type of R as described in claim 1 n A centrosymmetric β-hairpin antimicrobial peptide with a KW repeating sequence at an angle, characterized by: The antimicrobial peptide is obtained by replacing all amino acids in the R2W3 sequence with D-type amino acids, and its structural formula is as follows: D W D K D W D K D W D K D R D R D K D W D K D W D K D W-NH2, labeled as D-R2W3.

4. A type of R as described in any one of claims 1-3 n The application of a centrosymmetric β-hairpin antimicrobial peptide with a KW repeating sequence at a turning angle in the preparation of clinical antimicrobial drugs is characterized by, The antibacterial drug is an anti-Gram-positive bacteria drug and / or an anti-Gram-negative bacteria drug; the Gram-positive bacteria are Staphylococcus aureus, Bacillus subtilis or Staphylococcus epidermidis, and the Gram-negative bacteria are Klebsiella pneumoniae, Escherichia coli and Pseudomonas aeruginosa.

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