Targeting BamA antibacterial peptide as well as preparation method and application thereof

Abstract

The invention discloses a BamA-targeting antibacterial peptide as well as a preparation method and application thereof, and is characterized in that amino acids in a sequence are all D-type amino acids, and the amino acid sequence is as follows: w-r-r-w-r-r-p-w-k-r (dTrp-dArg-dArg-dTrp-dArg-dArg-dPro-dTrp-dLys-dArg). The antibacterial peptide provided by the invention has good antibacterial activity, can be synthesized by adopting an Fmoc solid-phase chemical method, is low in preparation difficulty, and has high affinity with target protein BamA.

Description

Technical Field

[0001] The present invention relates to the technical field of polypeptides, and in particular to an antimicrobial peptide targeting BamA and a preparation method and application thereof. Background Art

[0002] Antibiotic resistance has led to an increase in the number of hospitalizations and deaths. The situation of Gram-negative bacteria is particularly severe. Due to the abuse of antibiotics, many different resistant Gram-negative bacteria have emerged, such as carbapenem-resistant Escherichia coli and multidrug-resistant Acinetobacter baumannii. These superbugs have developed resistance to first-line clinical antibiotics such as carbapenems and fourth-generation cephalosporins. However, the development of traditional antibiotics mainly relies on the modification of the antibiotic structure, and the discovery and development of antibiotics with novel skeleton structures has become increasingly difficult, making it difficult to cope with the increasingly serious problem of drug-resistant bacterial infections. Therefore, new antimicrobial drugs are urgently needed to solve the problem of antibiotic resistance.

[0003] Antimicrobial peptides (AMPs) are natural immune molecules with broad-spectrum antimicrobial activity that can resist the invasion of foreign bacteria. As a new type of antimicrobial drug, it has non-receptor-mediated membrane mechanisms and multiple intramolecular mechanisms, rather than many traditional antibiotics acting on a single specific molecular target, so the possibility of bacteria developing resistance is very low. At the same time, AMPs also show good antibacterial activity against antibiotic-sensitive bacteria. AMPs can also be used as potentiators with existing antibiotics. Its mechanism may be to increase the sensitivity of antibiotics to antibiotic-resistant bacteria and reverse resistance through a unique membrane permeability mechanism and by dissipating the transmembrane electrochemical potential to regulate the efflux pump system. Due to these inherent advantages, AMPs have attracted much attention as potential antibacterial candidates to replace traditional antibiotics.

[0004] In Gram-negative bacteria, outer membrane proteins (OMPs) are very critical proteins for the formation of antibiotic resistance, and one important class of proteins is transmembrane β-barrel proteins (BAM). BAM is a complex protein, including β-barrel protein (BamA) and four lipoproteins (BamB-E), of which BamA is the core component of the BAM complex. With the assistance of BamD, BamA completes the folding of outer membrane proteins and inserts into the outer membrane of Gram-negative bacteria. BamB, BamC and BamE are non-essential lipoproteins, but they help BamA and BamD to fold outer membrane proteins. If the process of folding outer membrane proteins by the BAM complex is blocked, the accumulation of unfolded outer membrane proteins in the periplasm will be toxic to the bacteria. BamA is expressed in Gram-negative bacteria and is a widely existing class of outer membrane proteins. Therefore, BamA is a very promising target in the development of targeted antibacterial drugs. Summary of the invention

[0005] In view of the above technical problems, the purpose of the present invention is to provide an antimicrobial peptide targeting BamA, and the present invention also provides a preparation method and application of the antimicrobial peptide targeting BamA, so that it has good antimicrobial activity and high affinity with the target protein.

[0006] In order to achieve the above-mentioned first purpose, the present invention provides an antimicrobial peptide targeting BamA, characterized in that the amino acids in the sequence are all D-type amino acids, and its amino acid sequence is: wrrwrrpwkr (dTrp-dArg-dArg-dTrp-dArg-dArg-dPro-dTrp-dLys-dArg).

[0007] The structural formula is:

[0008]

[0009] Application of the antimicrobial peptide targeting BamA in the preparation of anti-bacterial infection drugs.

[0010] The use of the antimicrobial peptide targeting BamA in the preparation of antibacterial infection drugs is characterized in that the antimicrobial peptide targeting BamA is used against multidrug-resistant Acinetobacter baumannii (MDRAB), carbapenem-resistant Pseudomonas aeruginosa (CRPA) and standard strains of Acinetobacter baumannii (ATCC19606), Pseudomonas aeruginosa (ATCC27853), Klebsiella pneumoniae (ATCC700603), Escherichia coli (ATCC25922), and Enterobacter cloacae (ATCC13047).

[0011] A method for preparing the antimicrobial peptide targeting BamA is characterized in that: the polypeptide is synthesized and prepared by solid phase synthesis, dichlororesin is selected as a resin carrier, the resin is first swollen and then the protecting group is removed during synthesis, the first amino acid is connected to the resin carrier, and then dehydration condensation is performed, and then operations are performed in sequence according to the amino acid sequence of the designed peptide, the remaining amino acids are connected in sequence, and finally the polypeptide on the resin carrier is cut off, and it is refined, separated and purified using high performance liquid chromatography.

[0012] In the above scheme: a DMF solution containing 20% ​​piperidine by volume is used to remove the Fmoc protecting groups on the dichlororesin and the amino acid.

[0013] The method for preparing the antimicrobial peptide is characterized in that: the polypeptide is synthesized and prepared by solid phase synthesis, dichlororesin is selected as a resin carrier, during the synthesis, the resin is first swelled and then the protecting group is removed, the first amino acid is connected to the resin carrier, and then dehydration condensation is performed, and then operations are performed in sequence according to the amino acid sequence of the designed peptide, the remaining amino acids are connected in sequence, and finally the polypeptide on the resin carrier is cut off, and high performance liquid chromatography is used for refinement, separation and purification, and finally a mass spectrometer is used to identify the structure.

[0014] The antimicrobial peptide targeting BamA is used in the preparation of antimicrobial drugs, toiletries, food preservative additives, medical consumable antimicrobial agents, and cosmetics.

[0015] A large number of studies have shown that positive charge and hydrophobicity are necessary for the biological activity and main mechanism of action of antimicrobial peptides. The stronger the hydrophobicity of the antimicrobial peptide, the stronger its ability to penetrate the cell membrane. The advantageous combinations of amino acid sequences (RKW, RRW, RW) were screened for the bacterial outer membrane protein BamA, and the hydrophobic tryptophan (W) and positively charged arginine (R) were selected as the main amino acids of the antimicrobial peptide for structural design. D-10pep was designed in combination with the amphipathic structure of the antimicrobial peptide. The binding site of BamA protein and antimicrobial peptides is shown in Figure 2. Figure 1 shown.

[0016] The α-helical structure of antimicrobial peptides is the key structure for antimicrobial activity. If D-amino acids are introduced at a single site in the sequence, the α-helical structure will be destroyed, resulting in decreased antimicrobial activity. Therefore, the sequence of 10pep was designed into a D-amino acid sequence with the opposite sequence to obtain D-10pep.

[0017] The present invention designs and transforms natural antimicrobial peptides and performs configuration flipping to obtain D-10pep, introduces D-amino acids to reduce the drug resistance of antimicrobial peptides in drug-resistant strains such as MDRAB and CRPA, and standard strains such as ATCC19606 and ATCC27853, and at the same time makes the antimicrobial peptides have high affinity with the target protein BamA protein. The problem of poor binding of natural AMP to target protein and easy drug resistance is solved, and it has great development potential and clinical application value.

[0018] The antimicrobial peptides provided by the present invention have good antimicrobial activity. These antimicrobial peptides can be synthesized by using the Fmoc solid phase chemistry method, are easy to prepare, and have high affinity with the target protein BamA protein. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is a schematic diagram of the binding of the antimicrobial peptide D-10pep to the target protein BamA.

[0020] Figure 2This is the high performance liquid chromatogram of the antimicrobial peptide D-10pep.

[0021] Figure 3 This is the mass spectrum of the antimicrobial peptide D-10pep.

[0022] Figure 4 This is the SPR binding map of the antimicrobial peptide D-10pep. DETAILED DESCRIPTION

[0023] The present invention will be further described below by way of embodiments and in conjunction with the accompanying drawings:

[0024] Example 1

[0025] Synthesis of antimicrobial peptides

[0026] The target product D-10pep has the sequence

[0027] D-10pep:wrrwrrpwkr

[0028] (dTrp-dArg-dArg-dTrp-dArg-dArg-dPro-dTrp-dLys-dArg).

[0029] The antimicrobial peptides were synthesized artificially according to the standard Fmoc solid phase procedure.

[0030] The synthesized product was purified by high-purity silica gel column (SHIMADZU Inertsil ODS-SP) using the following mobile phase:

[0031] Phase A: 0.1% trifluoroacetic acid aqueous solution

[0032] Phase B: 0.1% trifluoroacetic acid in acetonitrile

[0033] 1. Activation and pretreatment of resin

[0034] 0.47 g of dichlororesin (0.43 mmol / g) was accurately weighed and placed in a peptide solid phase synthesizer. After swelling with dichloromethane solution for 30 min, the resin was tested by ninhydrin colorimetry and it was colorless and transparent, indicating that the resin was normal.

[0035] 2. Synthesis of peptide chains

[0036] The above-mentioned normal dichlororesin was subjected to DMF solution containing 20% ​​piperidine by volume to remove the Fmoc protecting group. The resin was blue-purple when tested by ninhydrin colorimetry, indicating that the protecting group had been removed. Fmoc-Trp-OH (212 mg), HOBT (1-hydroxybenzotriazole 81 mg), HBTU (O-benzotriazole-tetramethyluronium hexafluorophosphate 228 mg), DIEA (diisopropylethylamine 0.2 mL) were added to 5-10 mL of 4% ethanol and 1% ethanol. The mixture was dissolved in DMF (N, N-dimethylformamide) and mixed evenly, added to the synthesizer and mixed with the dichlororesin from which the Fmoc protecting group was removed, and the condensation reaction was carried out for 1 hour; the resin was colorless and transparent when tested by ninhydrin colorimetric method, indicating that the condensation reaction was successful to obtain Fmoc-Trp-resin; the method was the same as above, and the subsequent amino acids were condensed in sequence: Fmoc-Arg(pbf)-OH(212mg), Fmoc-Arg(pbf)-OH(212mg), Fmoc-Trp( The amount of HOBT, HBTU and DIEA is the same as above. The peptide chain obtained above was deprotected by using a DCM (dichloromethane) solution containing 1% TFA (trifluoroacetic acid) by volume, and the resin was blue-purple when tested by ninhydrin colorimetry.

[0037] 3. Peptide cleavage

[0038] The peptide chain obtained in the above step was cleaved with a mixed solution of TFA (trifluoroacetic acid), triisopropylsilane and water in a volume ratio of 9.5:0.25:0.25 as a cleavage reagent, extracted with ice ether and water, and freeze-dried to obtain a crude peptide lyophilized powder.

[0039] 4. Purification

[0040] The crude peptide freeze-dried powder obtained by freeze drying was separated and purified by high-purity silica gel chromatography column (SHIMADZU Inertsil ODS-SP), and the effluent was collected and freeze-dried. The HPLC chromatogram is shown in Figure 2 The purification conditions were as follows: mobile phase A: 0.1% trifluoroacetic acid in water, mobile phase B: 0.1% trifluoroacetic acid in acetonitrile, elution.

[0041] 5. Structural identification

[0042] 0.1 mg of crude peptide lyophilized powder was dissolved in 50% acetonitrile solution, and the structure was identified by triple quadrupole liquid chromatography-mass spectrometry. The molecular weight was measured to be 1745.18. The mass spectrum is shown in Figure 3 .

[0043] Experimental Example 1

[0044] Antimicrobial activity test of minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of antimicrobial peptides

[0045] Standard bacteria: Acinetobacter baumannii (ATCC19606), Pseudomonas aeruginosa (ATCC27853), Klebsiella pneumoniae (ATCC700603), Escherichia coli (ATCC25922), Enterobacter cloacae (ATCC13047).

[0046] Drug-resistant strains: multidrug-resistant Acinetobacter baumannii (MDRAB), carbapenem-resistant Pseudomonas aeruginosa (CRPA). The activity test method is briefly described as follows:

[0047] The revived bacterial solution was inoculated on LB solid medium by streaking method and incubated in a 37°C incubator overnight. Then the colonies on the plate were diluted with physiological saline to 4×10 8 CFU / mL, and then diluted to 4×10 5 CFU / mL. Add 100μl of antimicrobial peptide solution containing different concentrations (256μg / mL, 128μg / mL, 64μg / mL, 32μg / mL, 16μg / mL, 8μg / mL, 4μg / mL, 2μg / mL, 1μg / mL, 0.5μg / mL) to a 96-well plate, and then add 100μl of bacterial solution diluted with MHB medium to each well. To avoid edge effect, the outermost wells are used as blank wells. Imipenem (IPM) was used as the positive control, and sterile water was used as the negative control. After incubation at 37°C for 18-24h, the absorbance of each well at 600nm was measured with an enzyme reader. After determining the MIC, the liquid in the wells without bacterial growth was aspirated and applied to LB solid culture medium, and incubated at 37°C overnight. The lowest concentration without bacterial growth is the minimum bactericidal concentration (MBC). The experiment was repeated three times.

[0048] Table 1 Antimicrobial activity of antimicrobial peptide D-10pep

[0049]

[0050] The results are shown in Table 1. Except for slightly weaker antibacterial activity against ATCC13047 and ATCC19606, the antimicrobial peptide D-10pep has strong antibacterial activity against other common Gram-negative bacteria and drug-resistant strains.

[0051] Experimental Example 2

[0052] 1. SPR affinity detection

[0053] The affinity of D-10pep and BamA was analyzed by Biacore X100. The molecular weight of DR-10pep was 1745.18 MW. BamA protein was purchased from Shanghai Taoshu Company. First, sodium acetate buffer with an optimal pH of 4.5 was used to couple BamA protein to the CM5 chip by amine coupling method. Then, D-10pep was diluted with PBS to a certain concentration (10μg / mL, 5μg / mL, 2.5μg / mL, 1.25μg / mL, 0.625μg / mL, 0.3125μg / mL, 0.106μg / mL) and flowed through the surface of the CM5 chip fixed with BamA at a flow rate of 30μL / min. The binding time was 120s and the dissociation time was 600s. Finally, the RU value was recorded, and the affinity constants of the two were evaluated by BIA evaluation analysis software.

[0054] 2. SPR affinity analysis

[0055] SPR biosensor is an optical biosensor technology that can be used to study the binding affinity and kinetics of different biomolecules, such as antigen-antibody, DNA-protein, small molecule-protein, enzyme-substrate and enzyme-inhibitor interactions. In this study, we used Biacore X100 to explore the binding affinity of the antimicrobial peptide D-10pep to the BamA protein. We found that the response values ​​at an antimicrobial peptide concentration of 10 μg / mL were approximately 141 RU ( Figure 4 ), where the KD value of D-10pep and BamA was 6.7035×10 -7 M, Experimental results show that D-10pep has a high affinity for BamA.

[0056] The present invention is not limited to the above-mentioned embodiments. Those skilled in the art will appreciate that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and purpose of the present invention. The scope of the present invention is defined by the claims and their equivalents.

Claims

1. An antimicrobial peptide targeting BamA, characterized in that: The amino acids in the sequence are all D-type amino acids, and its amino acid sequence is: wrrwrrpwkr (dTrp-dArg-dArg-dTrp-dArg-dArg-dPro-dTrp-dLys-dArg).

2. The antimicrobial peptide targeting BamA according to claim 1, characterized in that: The structural formula is:

3. Use of the antimicrobial peptide targeting BamA according to claim 1 in the preparation of antibacterial infection drugs.

4. The use of the antimicrobial peptide targeting BamA according to claim 3 in the preparation of an antibacterial infection drug, characterized in that: The antimicrobial peptide targeting BamA is used in multidrug-resistant Acinetobacter baumannii (MDRAB), carbapenem-resistant Pseudomonas aeruginosa (CRPA) and standard strains of Acinetobacter baumannii (ATCC19606), Pseudomonas aeruginosa (ATCC27853), Klebsiella pneumoniae (ATCC700603), Escherichia coli (ATCC25922), and Enterobacter cloacae (ATCC13047).

5. A method for preparing the antimicrobial peptide targeting BamA according to claim 1, characterized in that: The polypeptide is prepared by solid phase synthesis, and dichlororesin is selected as the resin carrier. During the synthesis, the resin is first swelled and then the protecting group is removed, and the first amino acid is connected to the resin carrier, and then dehydration condensation is performed. Then, operations are performed in sequence according to the amino acid sequence of the designed peptide, and the remaining amino acids are connected in sequence. Finally, the polypeptide on the resin carrier is cut off and refined, separated and purified using high performance liquid chromatography.

6. The method for preparing the antimicrobial peptide targeting BamA according to claim 5, characterized in that: The Fmoc protecting groups on the dichlororesin and the amino acid were removed using a DMF solution containing 20% ​​piperidine by volume.

7. Use of the antimicrobial peptide targeting BamA according to claim 1 in the preparation of antimicrobial drugs, toiletries, food preservative additives, medical consumables antimicrobial agents, and cosmetics.

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