CN104072579A - Small molecule peptide with antibacterial antiviral activity and active modifier of small molecule peptide - Google Patents
Small molecule peptide with antibacterial antiviral activity and active modifier of small molecule peptide Download PDFInfo
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Abstract
The invention discloses a small molecule peptide with antibacterial antiviral activity and an active modifier of the small molecule peptide. An amino acid sequence of the small molecule peptide is formed by 3-7 amino acids; a general formula of the amino acid sequence is X[0-3](R/K/O/D[ab])X[0-1]W[1-2](R/K/O/D[ab])[0-1]X[0-1], wherein X is one of the amino acids R, K, W and I; X[0-3] is any 0-3 of R, K, W and I; X[0-1] is any 0-1 of R, K, W and I; W[1-2] represents one or two Ws; (R/K/O/D[ab])[0-1] represents zero or one amino acid R or K or O or Dab; D[ab] is a 2,4-diamido-butyric acid. The experiment proves that the small molecule peptide or the active modifier thereof disclosed by the invention has good antibacterial antiviral effects, and has important application value in preparation of a drug, a sanitizer, a detergent, a corrosion remover or a packaging material for removing bacteria, fungi or viruses.
Description
Technical field
The present invention relates to novel antimicrobial peptide and derivative thereof, be specifically related to have small-molecular peptides and the activity modifying thing thereof of antimicrobial antiviral activity.
Background technology
Tradition microbiotic bacterial drug resistance has become global problems, the antibacterials that make new advances in the urgent need to exploitation.In order to address this problem, antibacterial peptide (AntimicrobiaL peptides AMPs) has become the potential transmissible disease that multidrug resistant disease substance causes for the treatment of.Antibacterial peptide is the peptide material with anti-microbial activity that organism produces when extraneous pathogenic micro-organism invasion, conventionally 12-45 amino acid, consists of, and the about 40kD of molecular weight, is the important component part of organism non-specific immunity.Antibacterial peptide primary structure has typical feature, and N end is rich in Lys and Arg isopolarity amino acid, and C end is rich in the nonpolar amino acids such as Ala, Val and Gly, and middle portion is rich in Pro, so antibacterial peptide mainly exists with cationic form.This quasi-molecule has stronger conservative property, have that molecular weight is low simultaneously, good stability, side effect little, water-soluble strong, strong basicity, broad-spectrum antimicrobial, the advantage such as have no drug resistance, be distributed widely in Mammals, invertebrates, plant and microorganism, can produce the pathogenic agent of natural adaptive immunity defence invasion.The sequence of antibacterial peptide is different with secondary structure, but is always folded into an amphiphilic conformation and microbial film interaction, simultaneously with positive charge, can interact with electronegative bacterial film.Therefore, AMPs has lethal effect to bacterium, fungi, virus, parasite, tumour, and especially the microorganism of Multiple Classes of Antibiotics resistance is as methicillin-resistant staphylococcus aureus (MRSA), tolerant Pseudomonas aeruginosa and acinetobacter calcoaceticus etc.
The people that appear as of antibacterial peptide find desirable antibacterials new field are provided, so antibacterial peptide has huge application potential.Since first the people such as Boman have found antibacterial peptide in fruit bat in 1972, in 1981 subsequently Steiner Deng Renyoucong U.S. giant silkworms, be successfully separated to after two kinds of antibacterial peptide Cecropin-A and Cecropin-B, at the beginning of cut-off 2014, the natural A MPs having found is over 2000.In recent years, the development of area of computer aided SARS drug design technology, promoted greatly the research and development of antibacterial peptide, existing more than ten enters clinical or preclinical experimental stage according to the synthetic analogue of natural cationic antibacterial peptide, typical representative is that the Magainins that frog skin extracts is the antibacterial peptide that the first enters drug development program, U.S. magainins drugmaker has developed and has filtered out bacterium, virus and tumour cell all have two kinds of novel antimicrobial peptide: Pexiganan (MSI-78) of obvious lethal effect and MAI1278 now to enter the clinical III phase to test, can predict that antibacterial peptide will become the key areas of finding new drug in the near future.The little peptide GPG that TriPep company is developing, has carried out I/II clinical trial phase in Europe, treat two weeks above discoveries, and virus replication slows down, and demonstrates certain anti-HIV application prospect; Perorally administrable antimicrobial peptide Ambicin and Isegangn successfully complete I phase safety evaluation; The clinical study that the serious acne of II phase or III phase of entering two kinds of antibacterial peptide MBI-226 of Canada MicroLogix Biochem Technology, INC. and MBI-594 infect, wherein MBI-226III clinical trial phase result shows, in inductive phase, only has minimum irritant reaction, contrast with feminine gender group, accumulate irritant reaction less.The anti-HIV antibacterial peptide Enfuvritide (T20) of the most noticeable Trimeris company successfully listing before the several years.
Current many microbiotic develop immunity to drugs, peptide class antimicrobial substance has unique antibacterial mechanisms, to almost not effect of eukaryotic cell, only act on the eukaryotic cell of prokaryotic cell prokaryocyte and generation pathology, and be difficult for producing resistance, therefore in the near future, these peptide class antimicrobial substances may become the source of novel antibacterial medicine, have the fabulous market development and application prospect.
Along with microbiological development, to compare with traditional microbiotic, the mechanism of action of AMPs uniqueness makes it have the advantage of efficient and low resistance.Although AMPs has certain advantage, yet up to the present less AMPs medicine reaches the requirement of clinical practice.Because there is the problems such as bioavailability, toxicity, stability and production cost in AMPs.In order to improve the attribute of AMPs, can be developed to medicine, reasonably design is the method for dealing with problems.
Summary of the invention
In order to solve the problem of above-mentioned existence, the present invention has synthesized ultrashort antibacterial peptide and the activity modifying thing thereof being comprised of 3～7 amino acid, and it not only can reduce production cost widely, carries out the immunogenic response that after composition optimizes, also remarkable reduction may cause.
The object of the present invention is to provide small-molecular peptides and the activity modifying thing thereof of antimicrobial antiviral activity.
The technical solution used in the present invention is:
Small-molecular peptides and the activity modifying thing thereof with antimicrobial antiviral activity, 3～7 amino acid of aminoacid sequence of described small-molecular peptides form, and its aminoacid sequence general formula is X
0-3(R/K/O/D
ab) X
0-1w
1-2(R/K/O/D
ab)
0-1x
0-1;
Wherein X is selected from amino acid R, K, W, in I any;
X
0-3represent amino acid R, K, W, in I any 0～3;
X
0-1represent amino acid R, K, W, in I any 0～1;
W
1-2represent 1 or 2 amino acid W;
(R/K/O/D
ab)
0-1represent 0 or 1 amino acid R or K or O or D
ab, D wherein
abit is 2,4-diamino Gamma Amino Butyric Acid.
Further, the aminoacid sequence of above-mentioned small-molecular peptides is selected from the aminoacid sequence shown in SEQ ID NO:1～17.
Further, above-mentioned amino acid is the amino acid of L-configuration or D-form.
Further; small-molecular peptides and the activity modifying thing thereof with antimicrobial antiviral activity, the modificationt part of described activity modifying thing is divided into that esterified modification, glycosylation modified, amidation modification, carboxylic acid modification, phosphorylation modification, esterification are modified, N-acidylate is modified, by disulfide linkage cyclisation, modified, changed at least one in the modification, dimer, polymer, puting together modification of acid salt.
Further, the group of above-mentioned esterified modification is selected from lipid acid, triterpenes, steroid, phosphatide, glycophosphatidyl inositol, phosphatidylserine, phosphatidylethanolamine, sphingophospholipid, phosphatidylcholine, Val, phosphatidylinositols, phosphatidic acid, at least one in lysophosphoglyceride and cholesterol group.
Further, above-mentioned lipid acid is C2～C20 lipid acid.
Further, above-mentioned lipid acid is C8～C20 lipid acid.
Small-molecular peptides and activity modifying thing thereof are eliminated the application in bacterium, fungi or viral agent in preparation.
The application in microbiotic, sterilizing agent or sanitising agent, sanitas or wrapping material for the preparation of processing bacterium, fungi or virus infection or pollution of small-molecular peptides and activity modifying thing thereof.
The encode nucleic acid of above-mentioned small-molecular peptides.
The invention has the beneficial effects as follows:
1) AMPs of the present invention has widely Gram-positive and negative bacteria and effective anti-microbial activity.Some polypeptide also show a plurality of patterns bacterium except having the potential mechanism of destruction membrane stability kills mode.
2) AMPs of the present invention is except its powerful fungicidal activity, some antibacterial peptides and traditional Antibiotic combination medication have produced unpredictalbe synergy, this synergy of combining use can effectively reduce antibiotic using dosage on the one hand, can realize better antibacterial effect on the other hand.
3) at all antibacterial peptides, these AMPs that the present invention synthesizes are the shortest, therefore have the easily synthetic and low advantage of cost, the simultaneously potential medicine that develops into various infectious diseases.As some AMPs in the present invention also has the activity of resisiting influenza virus, HIV.
4) the invention provides there is anti-gram positive organism, the novel short lipopeptid of gram-negative bacteria, antivirus action.
Accompanying drawing explanation
Fig. 1 is the HPLC figure of anti-bacteria and anti-virus small-molecular peptides C10-AMP3 (C10-RKWWK);
Fig. 2 is the MS figure of anti-bacteria and anti-virus small-molecular peptides C10-AMP3 (C10-RKWWK);
Fig. 3 is the HPLC figure of anti-bacteria and anti-virus small-molecular peptides C10-AMP14 (C10-RIKWK);
Fig. 4 is the MS figure of anti-bacteria and anti-virus small-molecular peptides C10-AMP14 (C10-RIKWK);
Fig. 5 is the hemolytic activity of the small-molecular peptides of C10 lipid acid and C14 fatty acid modifying;
Fig. 6 is the cytotoxicity in MTT test assessment C10-RKWWK and RKWWK cell (TZM-b1);
Fig. 7 is the murder by poisoning speed of cells were tested by flow cytometry C10-AMP3 (C10-RKWWK) to mammalian cell;
Fig. 8 is the murder by poisoning speed of cells were tested by flow cytometry C10-AMP14 (C10-RIKWK) to mammalian cell;
Fig. 9 is C10-AMP3 and the sterilization speed of C10-AMP14 to S.aureus (C);
Figure 10 is the synergistic effect of the drug combination of C10-AMP4, C10-AMP3, C10-AMP7, RKWWK and Neomycin.
Embodiment
Small-molecular peptides and the activity modifying thing thereof with antimicrobial antiviral activity, 3～7 amino acid of aminoacid sequence of described small-molecular peptides form, and its aminoacid sequence general formula is X
0-3(R/K/O/D
ab) X
0-1w
1-2(R/K/O/D
ab)
0-1x
0-1;
Wherein X is selected from amino acid R, K, W, in I any;
X
0-3represent amino acid R, K, W, in I any 0～3;
X
0-1represent amino acid R, K, W, in I any 0～1;
W
1-2represent 1 or 2 amino acid W;
(R/K/O/D
ab)
0-1represent 0 or 1 amino acid R or K or O or D
ab, D wherein
abit is 2,4-diamino Gamma Amino Butyric Acid; Since arginine (R), ornithine (O), 2,4-diamino Gamma Amino Butyric Acid (D
ab) and Methionin (K) under physiological condition, show and there is polarity and positively charged character, arginine (R), ornithine (O), 2,4-diamino Gamma Amino Butyric Acid (D
ab) can replace Methionin (K).
Preferably, above-mentioned small-molecular peptides and activity modifying thing electrically charged being at least+2 thereof, sequence is controlled in 7 amino acid, can be by increasing R, K, W, the amino acid such as I change electric charge, aromaticity and the hydrophobicity of small peptide, and regulate the distance between Methionin (arginine) also can strengthen the activity of small peptide.
Preferably, the aminoacid sequence of above-mentioned small-molecular peptides is selected from the aminoacid sequence shown in SEQ ID NO:1～17.
Preferably, above-mentioned amino acid is the amino acid of L-configuration or D-form.
Preferably; small-molecular peptides and the activity modifying thing thereof with antimicrobial antiviral activity, the modificationt part of described activity modifying thing is divided into that esterified modification, glycosylation modified, amidation modification, carboxylic acid modification, phosphorylation modification, esterification are modified, N-acidylate is modified, by disulfide linkage cyclisation, modified, changed at least one in the modification, dimer, polymer, puting together modification of acid salt.
Preferably, the group of above-mentioned esterified modification is selected from lipid acid, triterpenes, steroid, phosphatide, glycophosphatidyl inositol, phosphatidylserine, phosphatidylethanolamine, sphingophospholipid, phosphatidylcholine, Val, phosphatidylinositols, phosphatidic acid, at least one in lysophosphoglyceride and cholesterol group.
Preferably, above-mentioned lipid acid is C2～C20 lipid acid.
Preferably, above-mentioned lipid acid is C8～C20 lipid acid.
Preferably, above-mentioned C8-C20 lipid acid is selected from lauric acid, palmitinic acid, tetradecanoic acid, stearic acid, oleic acid, linolic acid, linolenic acid, arachidonic acid, timnodonic acid, erucic acid and eicosanoic acid.
Above-mentioned fatty acid molecule can be attached to any suitable part of antibacterial peptide of the present invention, as aminoterminal, carboxyl terminal or amino and carboxyl two ends etc.
Small-molecular peptides and activity modifying thing thereof are eliminated the application in bacterium, fungi or viral agent in preparation.
The application in microbiotic, sterilizing agent or sanitising agent, sanitas or wrapping material for the preparation of processing bacterium, fungi or virus infection or pollution of small-molecular peptides and activity modifying thing thereof.
The mechanism of the antimicrobial antiviral activity of small-molecular peptides of the present invention and activity modifying thing thereof
In the present invention, synthetic effective AMPs combines the advantage of short lipopeptid and the abundant peptide of RW.Active testing to these a series of antibacterial peptides shows, it comprises gram negative bacillus intestinal bacteria to pathogenic micro-organism, and the microorganisms such as gram-positive cocci streptococcus aureus, Candida albicans, virus are inhibited.Hemolytic experiment shows that it has hypotoxicity, and part AM/Ps has antiviral effect.
The polypeptide that contains R and W is present main new A MPs.Yet except containing cation group and hydrophobic grouping, the importance that contains the antibacterial peptide of arginine and tryptophane has obtained sufficient understanding.Explaining, be because formed the positively charged ion π key between electronegative electronic cloud indoles residue and various cationic amino acid residue, and arginine and Methionin will cause breaking of cytolemma.Based on above discovery, the present invention has synthesized a series of R of containing, K, the ultrashort lipopeptid of W and I.Previous work finds that the activity of these antibacterial peptides can pass through hydrophobicity, and the change of electric charge and both sexes regulates and controls.Therefore, increase electric charge, change the amino acid whose position of polar/non-polar, change space structure snappiness, replace the methods such as hydrophobic/hydrophilic residue and can obtain highly active antibacterial peptide.For obtaining more hydrophobic grouping role in antibacterial peptide, two experiments have been carried out: (a) hydrophobicity of different sequence peptides is by reverse high-efficient liquid phase analysis; (b) hydrophobicity of the peptide of same sequence can be assessed and connect peptidyl various lipid acid (being respectively C10 and C18 lipid acid) end to end.On the basis of these experiments, draw a conclusion, a strong antibacterial peptide of activity is the system of a balance, has a high antibacterial range of optimum hydrophobicity, that is to say, high hydrophobicity not always directly and high-efficiency antimicrobial anti-virus ability proportional.Reduce or increase that hydrophobicity is super goes beyond the scope and will greatly affect its anti-microbial activity. therefore, most of peptides do not have the activity of C14, C10 analogue high after replacing with C18 lipid acid.The reason that produces this phenomenon is that the peptide of high hydrophobicity makes itself and cytolemma be difficult to combination.
Antibacterial peptide of the present invention has common biophysical properties, as positive charge, hydrophobicity and both sexes.These physico-chemical properties all can affect destruction and the permeabilization of cell membrane.In the present invention, find that C10-RIKWK has similar activity with C10-RKWWK, but the mechanism of its sterilization is not identical.。This is that C10-RKWWK can be to gram-positive microorganism and Gram-negative bacteria reason selectively.Yet, can't clearly know now that peptide enters the concrete mechanism of bacterium, may be dissolving film or the integrity of destroying film, thereby act on mutually with the target spot in bacterium.And the interaction of DNA-binding description of test peptide and DNA may relate to disturb the copying of DNA, recombinate, repair, translation etc.C10-KIKRWR has good anti-HIV-1 virus activity, and less to the toxicity of cell, has the potentiality of the test proceeded.C18-KKWK, C18-KWK, C20-KKWK, C20-KWK have the activity of anti-H5N1, have the effect that promotes virus infection when lower concentration.
Due in pH value, ionic strength and lipophilic difference, the synthetic small peptide of the present invention has different effects from different cytolemma, also has different selectivity.The physicochemical property of tryptophane and Methionin uniqueness make them be suitable as the composition module of antibacterial peptide.Polypeptide of the present invention is the antimicrobial antibacterial peptide forming with arginine (R), Methionin (K), tryptophane (W), Isoleucine (I), one of them amino acid with certain physics and/or chemical property is changed to the amino acid that another has identical chemistry or physical property, for example, arginine (R), ornithine (O), 2,4-diamino Gamma Amino Butyric Acid (D
ab) and Methionin (K) under physiological condition, show and there is polarity and positively charged character, arginine (R), ornithine (O), 2,4-diamino Gamma Amino Butyric Acid (D
ab) can replace Methionin (K).
Generally speaking, the lipopeptid that the present invention contains arginine, Methionin, tryptophane has the activity of wide spectrum, efficient resisting gram-positive bacteria and Gram-negative bacteria.And except having the effect of disrupting biofilm, some peptides have multiple sterilization mode.In the present invention, some peptides and traditional microbiotic coupling can reduce the dosage of sterilization.And new synthetic short lipopeptid is the shortest antibacterial peptide of a class.Because it has easily synthetic, advantage cheaply, and can be developed to the anti-infectious medicine for the treatment of.
Below in conjunction with specific embodiment, the present invention is further illustrated, but be not limited to this.
Embodiment 1:
One, micromolecule polypeptide and activity modifying thing thereof
(1) micromolecule polypeptide and activity modifying thing thereof
It is to take KWWK, KWK as basic motif, by 3-7 the polypeptide that amino acid was formed that the present invention has anti-bacteria and anti-virus small-molecular peptides.The aminoacid sequence general formula of a series of antimicrobial small-molecular peptides that obtain according to primary amino acid sequence KWWK, KWK is X
0-3(R/K/O/D
ab) X
0-1w
1-2(R/K/O/D
ab)
0-1x
0-1; Wherein X is selected from amino acid R, K, W, in I any; X
0-3represent amino acid R, K, W, in I any 0～3; X
0-1represent amino acid R, K, W, in I any 0～1; W
1-2represent 1 or 2 amino acid W; (R/K/O/D
ab)
0-1represent 0 or 1 amino acid R or K or O or D
ab, D wherein
abit is 2,4-diamino Gamma Amino Butyric Acid.Wherein preferably 17 sequences are as shown in table 1 for anti-bacteria and anti-virus effect.
The aminoacid sequence table of the antimicrobial small-molecular peptides of table 1
The activity modifying thing of micromolecule polypeptide refers to above-mentioned micromolecule polypeptide is carried out that esterified modification, glycosylation modified, amidation modification, carboxylic acid modification, phosphorylation modification, esterification are modified, N-acidylate is modified, by disulfide linkage cyclisation, modifies, changes into the modification of acid salt, Dimerized, polymer or puting together modification etc.
(2) micromolecule polypeptide and activity modifying thing thereof is synthetic
Small-molecular peptides is all to use standard 9-fluorene methyl carbonyl Fmoc solid phase synthesis process synthetic by acid amides MHBA resin.The synthetic of sequence is to carry out in ABI433A Peptide synthesizer.Peptide lengthening reaction condition is: take standard HBTU/HOBt as coupling reagent; the dimethyl formamide (DMF) of take is solvent; 2 times of excessive diisopropylethylamine (DIEA), 8 times of excessive amino acid or 10 times of excessive free fatty acidies containing Fmoc blocking group.Peptide is all to use reagent M from resin cracking, and reagent M comprises 87.5% trifluoroacetic acid, 2.5% dithioglycol, and 5% thioanisole and 5% deionized water (3h, room temperature), thick product is deposited in methyl tertiary butyl ether, and identical solvent is washed twice.
Peptide fluorescent mark carries out the coupling process that amino acid is identical and adopts rhodamine as sour solvent, reacts 12 hours.The molecular weight of each peptide confirms by electrospray ionization mass spectrometry (ESI-MS, Waters).
Peptide purification is used RP-HPLC, experiment condition: flow velocity 1mL/min; Moving phase solution A water (0.1% trifluoroacetic acid) solution B acetonitrile (0.1% trifluoroacetic acid); Gradient 15～20%B (2min), 20～60%B (6min), 60～80%B (4min), 80～90%B (4min).The purity of all lipopeptids is 85-90%.
Two, the character of micromolecule polypeptide and activity modifying thing thereof
Table 2 for N end and C14 lipid acid conjugation, C hold amidated small-molecular peptides activity modifying thing as physico-chemical properties such as electric charge (Ch), hydrophobicity (%Hp), average hydrophobicity (MH), iso-electric point (PI), retention time (RT) in RP-HPLC chromatographic column, relative molecular weights (MW).
The name of table 2 antimicrobial polypeptide, sequence and physico-chemical property
A: N end and the C14 lipid acid conjugation of peptide, C holds amidation; B: electric charge (Ch), iso-electric point (PI), hydrophobicity (%Hp) are not used the online computational tool of lipid part based on amidated peptide calculating
https: //www.genscript.com/ssLbin/site2/peptide caLcuLation.cgi; The calculating of c:MH (average hydrophobicity) is used website based on Kyte & DooLittLe model
http:// www.bbcm.univ.trieste.it/rossi/HydroCaLc HydroMCaLc.htmL; D:RT (retention time) unit is min.
As can be seen from Table 2, in retention time is 10～13min, hydrophobicity and retention time are closely related, if the retention time of C14-AMP5 (C14-RWWR) is 12.5min, and the retention time of C14-AMP9 (C14-RWR) is 11.93min, the retention time of visible C14-AMP9 (C14-RWR) is longer than the C14-AMP5 (C14-RWWR) of many tryptophan residues (hydrophobicity is high).
Fig. 1～4 are for N end and C10 lipid acid conjugation, C hold the HPLC of amidated small-molecular peptides activity modifying thing to scheme and mass spectrum (MS) is schemed,
Two, the antibacterial experiment of micromolecule polypeptide and activity modifying thing thereof
Minimal inhibitory concentration (MIC) test
Mainly according to NCCLS (National Committee of Clinical Laboratory Standards) guide, test.Respectively gram-positive microorganism streptococcus aureus (S.aureus, ATCC12600), Gram-negative bacteria intestinal bacteria (E.coLi, ATCC25922) and Candida albicans (C.albicans, ATCC 14053) are cultivated to obtain to 1x10
8the bacterium liquid of CFU/mL, redilution is to 1x10
5cFU/mL.Each small-molecular peptides and activity modifying thing thereof are added respectively in E.coli, S.aureu s and C.albicans bacterium liquid, make the concentration of peptide or derivatives thereof in E.coli and S.aureu s bacterium liquid be diluted to gradually 0.97 μ g/mL from 31.2 μ g/mL, join respectively in E.coli and S.aureus bacterium liquid; Make peptide or derivatives thereof concentration in Candida albicans (C.albicans) bacterium liquid be diluted to gradually 1.98 μ g/mL from 62.5 μ g/mL.Gram-positive microorganism and Gram-negative bacteria are cultivated 17 to 20 hours in 37 ℃ of 50%MH substratum, and fungi is cultivated 20 hours in 30 ℃ of 50%YPD substratum.In addition, positive control experiment is set, adopts respectively Liu Suanyan NEOMYCIN SULPHATE and C16-KGGK (existing antibacterial peptide) to process bacterium E.coLi and S.aureus.Parallel four to six of all experiments independently repeat experiment.Detected result is as shown in table 3,4,5.
The MIC detected result that table 3 is held amidated small-molecular peptides activity modifying thing for N end and C14 lipid acid conjugation, C; Table 4 is the MIC result of the small-molecular peptides activity modifying thing containing C10, C18 lipid acid; Table 5 is the MIC result of little minute peptide AMP-3 (RKWWK) and activity modifying thing thereof; From table 3～table 5, can find out, most small-molecular peptides and activity modifying thing thereof can kill intestinal bacteria and streptococcus aureus effectively, each small-molecular peptides and activity modifying thing thereof are to almost there is no significant difference between the anti-microbial activity of streptococcus aureus, this anti-microbial activity that small-molecular peptides of the present invention and activity modifying thing thereof are described is main relevant with its physico-chemical property, particularly electric charge and hydrophobicity.
Change so electric charge or hydrophobic can affect the anti-microbial activity of small-molecular peptides, as C14-AMP2 (C14-KIWWK) that in C14-AMP1 (C14-KWWK), Isoleucine of insertion obtains to the increase of intestinal bacteria minimal inhibitory concentration (MIC) more than 3 times (table 3); In addition, C10-KWWK (C10-AMP1), C10-RWWR (C10-AMP5) and C14-KWWK (C14-AMP1) all have powerful anti-microbial activity (table 3 and table 4), can their anti-microbial activity change with their order of aminoacid sequence? it is the anti-microbial activity of the small-molecular peptides activity modifying thing of RWRW, KWKW, KKWW that the present invention has detected peptide sequence, as shown in table 3～table 5, the activity modifying thing of all these peptides, the peptide activity modifying thing that particularly C10 modifies shows extensively and effectively antibacterial, and its MIC value is low to moderate 3.9 μ g/mL.Illustrate that its anti-microbial activity is mainly subject to its electric charge and hydrophobic impact, good at identical electric charge and hydrophobic situation, the aminoacid sequence of different order is not obvious on the impact of the anti-microbial activity of small-molecular peptides and activity modifying thing thereof.
Table 3 is the MIC result of the small-molecular peptides activity modifying thing containing C14 lipid acid
From table 3 and table 4, can find out, on the anti-microbial activity of small-molecular peptides and activity modifying thing thereof, there is certain impact in the length of fat chain, N end is respectively with the anti-microbial activity of the small-molecular peptides that linear C10, C14 and C18 lipid acid are combined, the anti-microbial activity of the small-molecular peptides of most of C14 fatty acid modifying than C10 fatty acid modifying a little less than, and being eager to excel than C18 fatty acid modifying.
Table 4 is the MIC result of the small-molecular peptides activity modifying thing containing C10, C18 lipid acid
The MIC result of little minute peptide AMP3 (RKWWK) of table 5 and activity modifying thing thereof
arKWWK does not contain aliphatic chain,
bn end rhodamine mark.
Three, antiviral activity test experience
(1) anti-HIV-1
jRFLthe detection of pseudovirus infection activity
After well-grown U87.CD4.CCR5 cell dissociation, centrifugal collection, then with the piping and druming of 1mL substratum evenly, the cell counting that takes a morsel, is remainingly diluted to 1 * 10 with substratum respectively
5individual/mL, then inoculates 96 orifice plates by cell suspension according to 100 μ L/ holes, and 37 ℃, 5%CO
2overnight incubation under condition, every hole adds small-molecular peptides or its activity modifying thing (50 μ L) and pseudovirus HIV-1 of gradient dilution
jRFL(50 μ L), continue to cultivate after 48h, and every hole adds 100 μ L0.5mg/mL MTT solution, under 37 ℃ of conditions, hatches 4h, under inverted microscope, observes whether there is crystallization, or noly has other impurity to generate, and carries out mark.Centrifugal 96 orifice plates, suck supernatant (should siphon away the blue crystallization that substratum can not siphon away again bottom) gently, and 150 μ L/ holes add DMSO dissolving crystallized.The 10min that vibrates gently, microplate reader reads A
570value.Small-molecular peptides or its activity modifying thing are measured concentration: 200 μ g/mL, 100 μ g/mL, 50 μ g/mL, 25 μ g/mL, 12.5 μ g/mL, 6.25 μ g/mL.
Detected result is as shown in table 6, and wherein, IC50 refers to that antibacterial peptide suppresses the ability of virus replication, is the peptide concentration in the time of can suppressing 50% virus replication.CC50 just refers to the toxicity of antibacterial peptide to cell, is the peptide concentration while making 50% necrocytosis.Therefrom can find out that C10-KIKRWR, KIWWK, KIKRWR, C10-KIWWK all have the effect of anti HIV-1 virus, especially C10-KIKRWR, its IC50 concentration is 37.241 ± 1.421 μ g/mL, HIV virus is had to good restraining effect, and a little less than the toxic action of cell, CC50 concentration is more than 200 μ g/mL.
The anti-HIV result of table 6 C10-KIKRWR, KIWWK, KIKRWR, C10-KIWWK
(2) detection of anti-H5N1 pseudovirus infection activity
It is 1x10 that 100 μ L concentration are accessed in every hole of 96 porocyte culture plates
5the mdck cell of individual/mL, 5%CO
2, 37 ℃ of overnight incubation; With substratum, pseudovirus doubling dilution is become to 6 extent of dilution, getting 50 μ L adds in 96 porocyte culture plates, add again small-molecular peptides of the present invention or its activity modifying thing (50 μ L), each extent of dilution (200 μ g/mL, 100 μ g/mL, 50 μ g/mL, 25 μ g/mL, 12.5 μ g/mL, 6.25 μ g/mL) repeat 3 holes, final volume is 200 μ L.Cell contrast is set, does not add pseudovirus.At 5%CO
2, under 37 ℃ of conditions, cultivate after 48h, discard substratum, 200mL/ hole, PBS washing 1 time.According to luciferase detection kit specification sheets, detect the infection ability of pseudovirus, concrete detection operation is as follows:
A. every hole adds 50 μ L lysates, jiggles, and at room temperature standing 20min, gets 409L cell pyrolysis liquid and join in the 96 flat luciferase check-out consoles in hole;
B. every hole adds 60 μ L luciferase substrates, by Genios Pro type Tecan microplate reader, detects luciferase values of chemiluminescence.Using 2.5 times of cell control well values of chemiluminescence as cutoff value, and values of chemiluminescence is greater than cutoff and is all considered as the positive.
Detected result is as shown in table 7, therefrom can find out that KKWK-C18, KKWK-C18, KKWK-C20, KKWK-C20 have the activity of good anti-avian influenza virus, KKWK-C18 especially, and its IC50 concentration is only 8.874 μ g/mL.
The anti-H5N1 of table 7 C18-KKWK, C18-KWK, C20-KKWK, C20-KWK is active
Four, Study of cytotoxicity
(1) hemolytic activity
At exploitation antibacterial peptide, as useful clinically medicine, facing very important problem is the toxicity to host cell, and antibacterial peptide must can be distinguished Mammals and bacterial cell.In current research, the toxicity evaluation of peptide utilizes human erythrocyte to check its hemolytic activity conventionally.
The most effective peptide of this hemolytic activity experimental selection, with fresh human erythrocyte (red blood corpuscle), is measured according to the method for existing information report.Fresh human erythrocyte (red blood corpuscle) washes 3 times with PBS, and 10% is heavily spun in PBS.Average-volume (100 μ L) solution of red blood cells is added in 96 orifice plates, comprises the small-molecular peptides of the present invention of equal volume or the PBS solution of its activity modifying thing in plate, and concentration is respectively 62.5,125,250 μ g/mL, 37 ℃ of hatching 1h, the centrifugal 15min of 3000rpm.Then at 570nm place, measure the absorbancy (50 μ L96 orifice plate) of supernatant liquid.Feminine gender and positive control with PBS and 10%Triton X-100 as hemolytic experiment.Haemolysis per-cent (Hemolysis rate) is calculated as follows:
Haemolysis per-cent %=[(Ab
peptide-Ab
pBS)/(Ab
triton-Ab
pBS)] x100%
Fig. 5 is the haemolysis per-cent of the small-molecular peptides of C10 lipid acid and C14 fatty acid modifying, therefrom can find out that the small-molecular peptides of most of C10 fatty acid modifying and large portion are low containing the hemolytic of the few small-molecular peptides of tryptophane (W) quantity.Haemolysis effect may be relevant with the length of position and aliphatic chain to the quantity of tryptophane.Tryptophan residue is more, higher hemolytic, as, C14-AMP12 (C14-KWK) and C14-AMP1 (C14-KWWK), wherein, under the condition of 125 μ g/mL concentration, it is 11% that C14-AMP12 (C14-KWK) holds blood effect, and reaches 56% containing the haemolysis effect of the C14-AMP1 (C14-KWWK) of 2 tryptophan residues; Yet C14-AMP-16 (C14-KWKW) hemolytic toxicity containing 2 tryptophan residues under identical concentration is but 17%, this explanation tryptophane position also can affect the hemolytic activity of antibacterial peptide and derivative thereof; In addition, when concentration is 250 μ g/mL, the hemolytic activity that all contains C10-AMP12 (C10-KWK), C14-AMP12 (C14-KWK), C10-AMP14 (C10-RIKWK) and the C14-AMP14 (C14-RIKWK) of a tryptophan residue is respectively < 10%, 19%, < 10%, 20%, and this explanation antibacterial peptide and the blood dissolubility of derivative thereof are also relevant with the length of aliphatic chain.In addition, from the hemolytic data of C10-AMP3 (C10-RKWWK), C10-AMP1 (C10-KWWK) and C10-AMP4 (C10-RIKWWK), can find out, what increase polypeptide electrically chargedly can be conducive to distinguish neutral mammalian cell membrane and electronegative bacterial cell membrane.Generally speaking, peptide is to adjustable their the physical-chemical parameters of change particularly position, charging property and the hydrophobicity of amino-acid residue passed through of the hemolytic toxicity of human erythrocyte.
(2) MTT measures cytotoxicity
It is 1x10 that 100 μ L concentration are accessed in every hole of 96 porocyte culture plates
5the TZM-b1 cell of individual/mL, 5%CO
2, 37 ℃ of overnight incubation, add AMPs and mammalian cell (TZM-b1) jointly to hatch 48h.AMPs concentration as shown in Figure 5,500,250,125,62.5,31.2,15.6 μ g/mL.Centrifugal abandoning supernatant, by specification adds MTT, then hatches 4h in 37 ℃.Finally, discard MTT solution, add DMSO to dissolve first a ceremonial jade-ladle, used in libation crystal, at 570nm, survey its optical density.The percentage calculation formula of cell survival rate is: AT/AC * 100 (AT: the absorbance A C of reaction group: the absorbancy of control group).Each data is expressed as three independent repetitions.
For their selectivity to protokaryon and mammalian cell of the potential cytotoxicity of further assessment antibacterial peptide of the present invention and assessment, this experiment test C10-AMP3 (C10-RKWWK) and the cytotoxicity of RKWWK to mankind TZM-b1 cell.Obviously, under 125 μ g/mL concentration, C10-AMP3 can cause necrocytosis, because its CC50 is 115 μ g/mL (table 4), even and if RKWWK cytotoxicity (Fig. 6) does not occur in concentration yet up to 500 μ g/mL.
(3) flow cytometry cytotoxicity:
MT reconnaissance T cell toxicity test has detected antibacterial peptide and mammalian cell hatching surpasses after 48 hours, the toxic action of antibacterial peptide to mammalian cell, further research antibacterial peptide toxic action to mammalian cell and bacterium within the short period of time (1h and 2h) of this experiment.
TZM-b1 cell is containing incubated overnight in the DMEM in high glucose substratum of 10% bovine serum, and abandoning supernatant, then wash 2 times with PBS adds trypsin-EDTA (be respectively 0.05% and 0.02%w/v) to digest.Then add the DMEM in high glucose substratum containing 10% bovine serum to stop digestion, the centrifugal 5min of 800rpm.Finally add DMEM in high glucose substratum diluting cells to 2 * 10 containing 10% bovine serum
5ceLLs/mL.C10-AMP3 (C10-RKWWK) and C10-AMP14 (C10-RIKWK) are added respectively in cell and process 1h and 2h, make the concentration of C10-AMP3 and C10-AMP14 be 40 μ g/mL.Test in last cell and add propidium iodide (10 μ g/mL), 4 ℃ of lucifuges are placed 10min, measure the viability of TZM-b1 cell with FACSCaLibur streaming instrument.
As shown in Figure 7 and Figure 8, it is dead in two hours that as can be seen from the figure result surpasses 99% TZM-b1 cell to measurement result, a little less than illustrating that at short notice small-molecular peptides of the present invention and activity modifying thing thereof are to the toxic action very of mammalian cell.
The mensuration of sterilization speed: be 1 * 10 in concentration
5in the S.aureus bacterium liquid of CFU/mL, add AMPs (C10-AMP3 and C10-AMP14), making AMPs concentration is 40 μ g/mL (10 times of MIC are excessive), at 25 ℃, hatch altogether 0 respectively, 60,120min, in ice bath, get respectively the bacterial suspension of 10 μ L, with substratum, by dilution in 1: 50, after 37 ℃ of aerobic overnight incubation, measure titre.Measurement result as shown in Figure 9.
From Fig. 7～9, can find out, the C10-AMP3 of same concentrations and C10-AMP14 act on respectively after mammalian cell and bacterium (S.aureus) 1h, 2h, the viability Flow cytometry of TZM-b1 cell, result surpasses 99% TZM-b1 cell not in two hours dead (Fig. 7 and Fig. 8), and after 1h, surpasses 95% streptococcus aureus be entirely killed (Fig. 9).A little less than illustrating that at short notice small-molecular peptides of the present invention and activity modifying thing thereof are to the toxic action very of mammalian cell, and bacterium is had to good toxic action.
Five, synergistic effect analysis
Three effective PEPC 10-AMP4 (C10-RIKWWK), C10-AMP7 (C10-KIKRWWR), C10-AMP3 (C10-RKWWK) and RKWWK (containing fat chain) respectively with Liu Suanyan NEOMYCIN SULPHATE (Neomycin) combined action.The antibacterial peptide solution that adds 5 μ L in 96 orifice plates, from left to right be diluted to different concentration (3.12 to 0.097 μ g/mL), then add the Liu Suanyan NEOMYCIN SULPHATE solution of 10 μ L, finally add the bacterial suspension of 100 μ L, at 37 ℃, hatch altogether after 17h, detect the antibacterial effect of each group.Each Data duplication twice.
Figure 10 is two dimension screening antibacterial peptide synergistic effect, is from left to right the concentration gradient (μ g/mL) of peptide, and is the concentration gradient (μ g/mL) of Liu Suanyan NEOMYCIN SULPHATE from top to bottom.In Figure 10, top is divided into C10-AMP4 (C10-RIKWWK) and Liu Suanyan NEOMYCIN SULPHATE, the acting in conjunction of C10-AMP3 (C10-RKWWK) and Liu Suanyan NEOMYCIN SULPHATE, and bottom is C10-AMP7 (C10-KIKRWWR) and Liu Suanyan NEOMYCIN SULPHATE, the acting in conjunction of RKWWK and Liu Suanyan NEOMYCIN SULPHATE.In figure, italic lattice represent that the two has realized synergistic effect.
As can be seen from Figure 10 the small-molecular peptides in the present invention or its activity modifying thing can also be used and produce unpredictalbe synergy with relevant Antibiotic combination, be these two kinds of antibacterials combine while using resulting antibacterial effect than the effect of the only use of this two prescription with better, this synergy of combining use can reduce antibiotic using dosage on the one hand, can realize better antibacterial effect on the other hand.
<110> Nanfang Medical Univ
<120> has small-molecular peptides and the activity modifying thing thereof of antimicrobial antiviral activity
<130>
<160> 17
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Claims (9)
1. small-molecular peptides and the activity modifying thing thereof with antimicrobial antiviral activity, is characterized in that: 3～7 amino acid of aminoacid sequence of described small-molecular peptides form, and its aminoacid sequence general formula is X
0-3(R/K/O/D
ab) X
0-1w
1-2(R/K/O/D
ab)
0-1x
0-1;
Wherein X is selected from amino acid R, K, W, in I any;
X
0-3represent amino acid R, K, W, in I any 0～3;
X
0-1represent amino acid R, K, W, in I any 0～1;
W
1-2represent 1 or 2 amino acid W;
(R/K/O/D
ab)
0-1represent 0 or 1 amino acid R or K or O or D
ab, D wherein
abit is 2,4-diamino Gamma Amino Butyric Acid.
2. small-molecular peptides and the activity modifying thing thereof with antimicrobial antiviral activity according to claim 1, is characterized in that: the aminoacid sequence of described small-molecular peptides is selected from the aminoacid sequence shown in SEQ ID NO:1～17.
3. small-molecular peptides and the activity modifying thing thereof with antimicrobial antiviral activity according to claim 1 and 2, is characterized in that: described amino acid is the amino acid of L-configuration or D-form.
4. small-molecular peptides and the activity modifying thing thereof with antimicrobial antiviral activity according to claim 1 and 2, is characterized in that: the modificationt part of described activity modifying thing is divided into that esterified modification, glycosylation modified, amidation modification, carboxylic acid modification, phosphorylation modification, esterification are modified, N-acidylate is modified, by disulfide linkage cyclisation, modified, changed at least one in the modification, dimer, polymer, puting together modification of acid salt.
5. small-molecular peptides and the activity modifying thing thereof with antimicrobial antiviral activity according to claim 4, is characterized in that: the group of described esterified modification is selected from lipid acid, triterpenes, steroid, phosphatide, glycophosphatidyl inositol, phosphatidylserine, phosphatidylethanolamine, sphingophospholipid, phosphatidylcholine, Val, phosphatidylinositols, phosphatidic acid, at least one in lysophosphoglyceride and cholesterol group.
6. the small-molecular peptides derivative with antimicrobial antiviral activity according to claim 5, is characterized in that: described lipid acid is C2～C20 lipid acid.
7. in claim 1-6, arbitrary described small-molecular peptides and activity modifying thing thereof are eliminated the application in bacterium, fungi or viral agent in preparation.
8. the small-molecular peptides described in any one and activity modifying thing thereof the application in microbiotic, sterilizing agent or sanitising agent, sanitas or wrapping material for the preparation of processing bacterium, fungi or virus infection or pollution in claim 1-6.
9. the nucleic acid of small-molecular peptides described in coding claim 1.
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| CN104628869A (en) * | 2015-01-28 | 2015-05-20 | 南方医科大学 | Fusion peptide derivatives with antibacterial anti-influenza virus activities |
| WO2017190619A1 (en) * | 2016-05-03 | 2017-11-09 | 重程投资管理（上海）有限公司 | Chemosynthetic cyclo-heptamodified peptide capable of inhibiting toxin of staphylococcus aureus and use thereof |
| EP3434287A1 (en) * | 2017-07-25 | 2019-01-30 | Arta Peptidion S.r.l.s. | Short and ultra-short antimicrobial lipopeptides and use thereof |
| CN109320585A (en) * | 2018-09-06 | 2019-02-12 | 南方医科大学 | A kind of short lipopeptid with specificity anti-acne Propionibacterium effect and anti-inflammatory effect |
| JP2019529376A (en) * | 2016-09-15 | 2019-10-17 | ディーエスエム アイピー アセッツ ビー．ブイ．Ｄｓｍ Ｉｐ Ａｓｓｅｔｓ Ｂ．Ｖ． | New compounds |
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| CN104628869A (en) * | 2015-01-28 | 2015-05-20 | 南方医科大学 | Fusion peptide derivatives with antibacterial anti-influenza virus activities |
| WO2017190619A1 (en) * | 2016-05-03 | 2017-11-09 | 重程投资管理（上海）有限公司 | Chemosynthetic cyclo-heptamodified peptide capable of inhibiting toxin of staphylococcus aureus and use thereof |
| US10905735B2 (en) | 2016-05-03 | 2021-02-02 | Zhongcheng Investment Management (Shanghai) Co., Ltd | Chemosynthetic cyclo-hepta modified peptide capable of inhibiting toxin of Staphylococcus aureus and use thereof |
| JP2019529376A (en) * | 2016-09-15 | 2019-10-17 | ディーエスエム アイピー アセッツ ビー．ブイ．Ｄｓｍ Ｉｐ Ａｓｓｅｔｓ Ｂ．Ｖ． | New compounds |
| JP6996042B2 (en) | 2016-09-15 | 2022-01-17 | ディーエスエム アイピー アセッツ ビー．ブイ． | New compound |
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| CN110938112A (en) * | 2019-12-10 | 2020-03-31 | 倪京满 | Fatty acid modified ultrashort-sequence antibacterial peptide analogue and application thereof |
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