Design of an antimicrobial peptide and its application in bacterial infection

Abstract

The invention relates to the design of an antimicrobial peptide and its application in bacterial infection, which belongs to the field of biopharmaceutical polypeptide technology, and specifically relates to the design of an antimicrobial peptide through an amino acid replacement strategy and its application in the preparation of a drug for preventing or treating bacterial infection-related diseases. The polypeptide designed in the present invention has a short amino acid sequence, can be prepared efficiently and quickly, has strong antimicrobial activity, a broad antimicrobial spectrum, and weak hemolytic activity against red blood cells. It can be used for the prevention and treatment of diseases caused by various microbial infections, and has a good effect on the treatment of diseases such as skin trauma infection, sepsis, and pneumonia caused by bacterial infection.

Description

Technical Field

[0001] The present invention belongs to the technical field of biopharmaceutical polypeptides, and specifically relates to an antimicrobial peptide obtained by an amino acid replacement modification method in the prevention and treatment of diseases caused by various microbial infections, and in the treatment of skin trauma infections, sepsis, pneumonia caused by bacterial infections, and the like. Background Art

[0002] Bacterial infections are a major threat to human health, and these infections can lead to huge morbidity and mortality. Antibiotics, as the most effective method for treating bacterial infections, face the problems of easy drug resistance and high toxicity. The development of safe and efficient antibacterial molecules is imminent. Antimicrobial peptides have attracted widespread attention in recent years due to their potential therapeutic effects. Compared with traditional antibiotics, antimicrobial peptides are not easy to develop drug resistance, low toxicity, biodiversity and direct aggressiveness, and are considered to be the most promising new generation of antibacterial drugs in the post-antibiotic era. However, there are also problems such as high preparation cost, weak antibacterial activity and strong cytotoxicity. Therefore, it is of great practical significance to modify and transform antimicrobial peptides to improve their drugability. In the patent of this invention, the polypeptide Lyco-13 is used as a template. It is designed by our laboratory based on the wolf spider venom Lycosin-I through a series of amino acid truncation and amino acid replacement strategies. It contains 13 amino acid residues and the sequence is WKAMKIIAKFIAK. It has good antibacterial activity, but it also has shortcomings such as high hemolytic activity and high cytotoxicity. Different from the modification of most long-sequence antimicrobial peptides, the shorter amino acid sequence will greatly affect the formation of the α-helical conformation of the peptide, which is not conducive to the peptide lysing the bacterial membrane, so the antibacterial activity is greatly weakened or even lost. Although Lyco-13 has a short sequence, it still has good antibacterial activity. Based on our systematic study of the Lyco-13 sequence, we have identified 7 sites that can be used for the design of safe and efficient antimicrobial peptides. Based on the short sequence of Lyco-13 and the clear key amino acid residues, and compared with natural amino acids, the side chain groups of non-natural amino acids are diverse, and their physical and chemical properties such as volume, size, hydrophobicity, and charge are different from those of natural amino acid side chain groups. In the present invention, we use non-natural amino acids and combine non-natural amino acid mutations on the basis of Lyco-13 to design a new type of antimicrobial peptides, and these antimicrobial peptides are expected to develop a promising drug for the treatment of bacterial infections. Summary of the invention

[0003] The purpose of the present invention is to provide a design method for a class of antimicrobial peptides and the application of polypeptides with antimicrobial effects in antibacterial aspects.

[0004] The above-mentioned purpose of the invention is achieved through the following technical scheme: taking the short sequence polypeptide Lyco-13 as a template, through unique structure-activity relationship research, an excellent skeleton based on Lyco-13 polypeptide design is provided, and on this basis, single-point and combination site design is carried out for 7 key variable sites by replacing them with non-natural amino acids or natural amino acids, and a class of polypeptide modifications are obtained, which have the characteristics of short sequence, strong activity and low toxicity, can be efficiently prepared by chemical synthesis, have low production cost, and can be used for the development of drugs for preventing or treating bacterial infectious diseases.

[0005] The antimicrobial peptide provided by the present invention is modified from the template peptide Lyco-13. The polypeptide or its pharmaceutically acceptable salt is based on the amino acid sequence of Lyco-13, and the amino acid at a specific position is replaced by a non-natural amino acid or a natural amino acid; it comprises an amino acid sequence shown in the following formula (I):

[0006] Y 1 Y 2 AMY 3 IY 4 AY 5 Y 6 IKY 7 (I) Where: Y 1 -Y 7 Each is independently selected from any amino acid, and Y 1 -Y 7 At least one of the amino acids is a non-natural amino acid, including Dip, Bip, Dap and Dab.

[0007] In some embodiments, the Y 1 and Y 6 Each is independently selected from a derivative of Dip, Bip, Trp or Phe.

[0008] In some embodiments, the Y 2 Selected from derivatives such as Dap, Dab, Lys, His or Arg.

[0009] In some embodiments, the Y 3 Selected from derivatives such as Dap, Dab, Lys, His or Arg.

[0010] In some embodiments, the Y 4 Selected from derivatives such as Dap, Dab, Lys, His or Arg.

[0011] In some embodiments, the Y 5 Selected from derivatives such as Dap, Dab, Lys, His or Arg.

[0012] In some embodiments, the Y7 Selected from derivatives such as Dap, Dab, Lys, His or Arg.

[0013] In some embodiments, the pharmaceutical composition of the present invention further comprises at least one of a pharmaceutically acceptable carrier, diluent, adjuvant and vehicle.

[0014] In some embodiments, the bacteria include but are not limited to Acinetobacter baumannii, Klebsiella pneumoniae, Escherichia coli and Streptococcus mutans. The bacterial infection includes but is not limited to skin wound infection, sepsis, pneumonia caused by bacterial infection and other diseases.

[0015] In some embodiments, the polypeptide design composition or a pharmaceutically acceptable salt thereof comprises Y of the polypeptide or a pharmaceutically acceptable salt thereof. 1 -Y 7 One of the amino acid sequences formed by any combination of mutation sites.

[0016] The significant innovation of the present invention is that, unlike most other antimicrobial peptides, the polypeptide sequence of the present invention is short, which has the advantage of greatly reducing production costs; more importantly, it can kill bacteria with a broad spectrum, has a larger inhibition zone diameter, and is relatively weak in toxicity to mammalian cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 : Reverse phase high performance liquid chromatography (RP-HPLC) of modified body 1.

[0018] Figure 2 : Mass spectrum of transformant 1.

[0019] Figure 3 : Transformation 1 against Acinetobacter baumannii ( Figure 3 A), Klebsiella pneumoniae ( Figure 3 B) Escherichia coli ( Figure 3 C) and Streptococcus mutans ( Figure 3 D) determination of the minimum inhibitory concentration.

[0020] Figure 4 :Analysis of the hemolytic activity of the modified body 1 on mouse erythrocytes and the toxicity analysis on normal cells, among which Figure 4 A represents the analysis of the hemolytic activity of the modified substance 1 on mouse erythrocytes. Figure 4 B and Figure 4 C represents the toxicity analysis of the modified form 1 and the parent peptide Lyco-13 on normal cells, respectively. DETAILED DESCRIPTION

[0021] The polypeptides disclosed in the present invention, including their salts, may also exist in the form of their hydrates or in the form of containing their solvents (e.g., ethanol, DMSO, etc.), and may be used for crystallization. The compounds disclosed in the present invention may inherently or by design form solvates with pharmaceutically acceptable solvents (including water); therefore, the compounds of the present invention include solvated and unsolvated forms.

[0022] For amino acid replacement of the polypeptide of the present invention, non-natural amino acids can be used to replace the natural amino acids in the polypeptide, and non-natural amino acids include but are not limited to 2,3-diaminopropionic acid (Dap), 3,3-diphenylalanine (Dip), 2,4-diaminobutyric acid (Dab), 2,5-diaminopentanoic acid (Orn) and 4-phenylphenylalanine (Bip).

[0023] Any of the 20 L-amino acids commonly found in peptides synthesized in nature, i.e., the L-isomers of alanine (Ala or A), arginine (Arg or R), asparagine (Asn or N), aspartic acid (Asp or D), cysteine ​​(Cys or C), glutamic acid (Glu or E), glutamine (Glu or Q), glycine (Gly or G), histidine (His or H), isoleucine (Ile or I), leucine (Leu or L), lysine (Lys or K), methionine (Met or M), phenylalanine (Phe or F), proline (Pro or P), serine (Ser or S), threonine (Thr or T), tryptophan (Trp or W), tyrosine (Tyr or Y), and valine (Val or V).

[0024] The term "natural amino acid" refers to any of the 20 L-amino acids commonly found in proteins and peptides found in nature, i.e., the L-isomers of alanine (Ala or A), arginine (Arg or R), asparagine (Asn or N), aspartic acid (Asp or D), cysteine ​​(Cys or C), glutamic acid (Glu or E), glutamine (Glu or Q), glycine (Gly or G), histidine (His or H), isoleucine (Ile or I), leucine (Leu or L), lysine (Lys or K), methionine (Met or M), phenylalanine (Phe or F), proline (Pro or P), serine (Ser or S), threonine (Thr or T), tryptophan (Trp or W), tyrosine (Tyr or Y), and valine (Val or V).

[0025] Example

[0026] Example 1: Preparation of transformant 1 (named Lydp-1).

[0027] The chemical structure of the modified body is as follows:

[0028]

[0029] The modified body was efficiently prepared by peptide solid phase chemical synthesis, and a polypeptide with 13 amino acid residues with good antibacterial performance and low hemolytic activity was obtained by testing the minimum inhibitory concentration (MIC) against Acinetobacter baumannii, Escherichia coli and Streptococcus mutans and the hemolytic activity against mouse erythrocytes, and its theoretical molecular weight was 1621.8398Da. Further antibacterial performance characterization showed that the antibacterial activity of the polypeptide in Example 1 was increased by 4 times, the hemolytic activity was reduced by 2 times, and the cytotoxic activity was weakened by nearly 3 times compared with the parent peptide Lyco-13, indicating that it is a lead molecule of antimicrobial peptide drugs for the treatment of bacterial infections.

[0030] Research Methods

[0031] 1. Solid-phase peptide chemical synthesis of transformant 1

[0032] The present invention adopts Fmoc solid phase peptide synthesis method to synthesize linear peptides, that is, Rink resin is used as a carrier during synthesis, and the peptides are synthesized in order from C-terminus to N-terminus, and Fmoc (9-fluorenylmethoxycarbonyl) is used as the amino protecting group of the amino acid. After the last amino acid coupling is completed, the newly synthesized peptide is cleaved from the Rink resin with a cleavage solution, and then precipitated with ice ether and identified by mass spectrometry. According to the peptide synthesis scale of 0.1mmol, the specific steps are as follows:

[0033] (1) Resin expansion

[0034] Weigh 0.1 mmol of Rink resin and put it into a synthesis tube, add 3 mL of DMF, swell for 1 h and filter out the DMF in the tube.

[0035] (2) Deprotection of resin:

[0036] 3 mL of 20% piperidine was added to the expanded resin and deprotected on a rotator for 7 minutes. The piperidine was then removed and 3 mL of 20% piperidine was added to the rotator for a second deprotection for 8 minutes. After deprotection, the piperidine was removed and washed with DMF 8 times.

[0037] (3) Activation of amino acids

[0038] While the first deprotection is being carried out, the amino acid is activated. Take one portion of HATU and one portion of HOBT, add 0.75 mL of N-methylmorpholine to each, mix the two and add them to the pre-weighed amino acid, mix on a rotary mixer, and the activation time is 15-20 minutes.

[0039] (4) Amino Acid Coupling

[0040] Add the activated amino acid to the resin in step (2), shake to mix the resin and amino acid solution thoroughly, and carry out coupling reaction on a rotator for 1 hour.

[0041] (5) Peptide chain extension

[0042] Repeat steps (2)-(4) until the last amino acid coupling reaction is completed.

[0043] (6) Peptide chain cleavage

[0044] After the last amino acid coupling reaction is completed, use 20% piperidine to deprotect, then wash with DMF 8 times, and wash with anhydrous methanol 4-5 times. After washing, drain and add 6mL of lysis solution. React in a rotary mixer for 2h. Collect the lysis solution in a 50mL centrifuge tube, add ice ether to the lysis solution for precipitation twice, and the lower precipitate obtained is the crude polypeptide. The crude polypeptide after precipitation is placed in a fume hood for 2-3 minutes to volatilize the ether contained in the tube, and then seal and store at 4℃.

[0045] 2. Isolation and purification of transformant 1 ( Figure 1 )

[0046] The cleaved polypeptide crude product was dissolved in an appropriate amount of ultrapure water, centrifuged at 10000rpm for 10min, and then filtered with a 0.22μm filter head. RP-HPLC chromatography purification was performed on a semi-preparative reversed-phase high performance liquid chromatograph (Hanbang) using a C18 reversed-phase column (10mm×250mm, 5μm, Yuexu), with a detection wavelength of 215nm or 280nm, a column temperature of 25±5°C, phase A (H2O+0.1% TFA), phase B (ACN+0.1% TFA). The concentration range of phase B was 10%-54%, the elution gradient was 2% / min, the elution rate was 3ml / min, and the collected target peak was freeze-dried.

[0047] 3. Determination of molecular weight of modified 1 ( Figure 2 )

[0048] The molecular weight of the peptide was determined using an AB SCIEX-TOF / TOFTM 5800 mass spectrometer produced by ABI, USA, using MALDI-TOF for detection. 1 μL of the peptide sample was spotted on the sample plate, dried at room temperature, and the operation was repeated 3-4 times, and then 1 μL of CCA saturated solution was spotted to cover the sample.

[0049] 4. Activity Assay of Transformant 1

[0050] (1) Minimum inhibitory concentration (MIC) determination Figure 3 )

[0051] The MIC of the peptide was determined by broth microdilution method. When the bacteria grew to the logarithmic phase in liquid culture medium, the bacterial solution was diluted to 1×10 7 CFU / mL, add 1 μL of diluted bacterial solution to a 96-well plate, dilute the polypeptide in the culture medium until the final concentration is 1-128 μg / mL. Add 99 μL of different concentrations of polypeptide dilution solution to a 96-well plate, and place the 96-well plate in an anaerobic incubator at 37°C for 18-24 hours. Use a microplate reader (BioTek Inc, USA) to detect the absorbance of the above 96-well plate at 600 nm. According to the absorbance OD 600 Calculate the inhibition rate of peptides on bacteria.

[0052] (2) Hemolytic activity assay Figure 4 )

[0053] Extract fresh mouse red blood cells, dilute 25 times with PBS buffer (pH = 7.4) to prepare about 4% mouse red blood cell suspension. Prepare different concentrations of polypeptide solutions with PBS solution and place them in a 1.5mL sterile centrifuge tube with a volume of 200μL. Add 200μL of the above mouse red blood cell suspension to this tube. Incubate at 37℃ for 1 hour, centrifuge at 3500rpm for 5 minutes, collect 100μL of supernatant on a 96-well plate, and measure the absorbance at 490nm with an enzyme reader (BioTek, USA). Use an untreated red blood cell suspension and a red blood cell suspension treated with 0.1% Triton X-100 in an equal volume of PBS solution as negative control and positive control, respectively.

[0054] (3) Cytotoxicity assay Figure 4 )

[0055] L929 cells were cultured in 96-well plates using DMEM containing 10% FBS and 1% antibiotics. Each well contained approximately 1×10 5 After culturing the cells in a carbon dioxide incubator for 24 hours, different concentrations of peptides were added to the wells and incubated for another 24 hours under the same conditions. CCK-8 solution was then added to continue incubation for 1-4 hours to determine cell viability. The A 450 The absorbance at nm was measured and the IC of peptides on cells was calculated using GraphPad Prism 10 software. 50 value.

[0056] Experimental Results

[0057] After solid phase chemical synthesis of modified compound 1, it was separated by reverse phase high performance liquid chromatography (RP-HPLC) and identified by mass spectrometry. Figure 1 and Figure 2As shown, its purity is >95%, and the experimentally determined molecular weight is 1621.8005Da, which is consistent with the theoretical molecular weight, indicating that it can be efficiently prepared by chemical synthesis. Figure 3 As shown, the antibacterial experiment of the present invention fully shows that the antimicrobial peptide modified body 1 of the present invention can kill a variety of bacteria, including Acinetobacter baumannii, Klebsiella pneumoniae, Escherichia coli and Streptococcus mutans, and the MIC of the action is 4μg / mL. 100μM modified body 1 has no obvious activity on mouse red blood cells, indicating that its hemolytic activity is low ( Figure 4 A). Compared with the template peptide Lyco-13, the MIC of the modified peptide 1 was reduced by 4 times, and the hemolytic activity was reduced by about 2 times. The toxicity experiment on mouse epithelial fibroblast L929 cells showed that the half effective inhibitory concentration (IC 50 ) is 19.21μM( Figure 4 B), the results show that compared with the parent peptide, the activity of the polypeptide in the example on L929 cells is reduced by nearly 3 times, indicating that its toxicity is reduced. And the hemolytic activity is about 10 times different from its MIC data, showing a certain selective antibacterial activity. In summary, the modified body 1 has the potential to develop a new generation of antibacterial drugs.

[0058] The preferred embodiments of the present invention have been specifically described above, but the present invention is not limited to the described embodiments. Those skilled in the art may make various equivalent modifications or substitutions without violating the spirit of the present invention. These equivalent modifications or substitutions are all included in the scope defined by the claims of this application.

Claims

1. A polypeptide or a pharmaceutically acceptable salt thereof, characterized in that: The polypeptide or its pharmaceutically acceptable salt is based on the amino acid sequence of Lyco-13, and the amino acid at a specific position is replaced by a natural amino acid or a non-natural amino acid; the sequence of the obtained antimicrobial peptide modification is: WDapAMDapIDipADapDipIKDap.

2. The polypeptide or pharmaceutically acceptable salt thereof according to claim 1, characterized in that: Further comprising a pharmaceutically acceptable carrier.

3. The polypeptide or pharmaceutically acceptable salt thereof according to claim 1, characterized in that: Further comprising a pharmaceutically acceptable diluent.

4. The polypeptide or pharmaceutically acceptable salt thereof according to claim 1, characterized in that: It further comprises a pharmaceutically acceptable adjuvant.

5. The polypeptide or pharmaceutically acceptable salt thereof according to claim 1, characterized in that: Further comprising a pharmaceutically acceptable vehicle.

6. The polypeptide or pharmaceutically acceptable salt thereof according to claims 1-5, characterized in that: The antimicrobial peptide drug can be prepared to prevent or treat bacterial infectious diseases, and the bacteria are Acinetobacter baumannii, Klebsiella pneumoniae, Escherichia coli and Streptococcus mutans.

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