CN104072579B - 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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- CN104072579B CN104072579B CN201410259060.5A CN201410259060A CN104072579B CN 104072579 B CN104072579 B CN 104072579B CN 201410259060 A CN201410259060 A CN 201410259060A CN 104072579 B CN104072579 B CN 104072579B
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Landscapes
- Peptides Or Proteins (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
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 its derivant and in particular to have antimicrobial antiviral activity small-molecular peptides and
Its activity modifying thing.
Background technology
Conventional antibiotic bacterial drug resistance has become global problems, in the urgent need to developing new antibacterials.For
Solve this problem, antibacterial peptide (antimicrobial peptides amps) has become potential and treats multidrug resistant disease
The infectious disease that substance causes.Antibacterial peptide is the polypeptide with antibacterial activity that organism produces when extraneous pathogenic microorganism is invaded
Material, is generally made up of 12-45 aminoacid, molecular weight about 40kd, is the important component part of organism nonspecific immunity.
Antibacterial peptide primary structure has typical feature, that is, n end be rich in lys and arg isopolarity aminoacid, and c end be rich in ala, val and
The nonpolar amino acids such as gly, mid portion is rich in pro, and therefore antibacterial peptide is mainly existed with cationic form.This quasi-molecule has
Stronger conservative, has that molecular weight is low, good stability, Small side effects, water solublity strong, strong basicity, broad-spectrum antiseptic, no resistance to simultaneously
The advantages of property of medicine, it is distributed widely in mammal, invertebratess, plant and microorganism, natural adaptability can be produced and exempt from
The pathogen of epidemic disease defence invasion.The sequence of antibacterial peptide is different with secondary structure, but is always folded into an amphiphilic conformation
With biological membrane interaction, simultaneous with positive charge so as to can be with negatively charged antibacterial membrane interaction.Therefore, amps pair
Antibacterial, funguses, virus, parasite, tumor have lethal effect, the especially for example resistance to methoxy west of the microorganism of Multiple Classes of Antibiotics drug resistance
Woods staphylococcus aureuses (mrsa), tolerant Pseudomonas aeruginosa and acinetobacter calcoaceticus etc..
The people that appear as of antibacterial peptide find preferable antibacterials and provide new field, therefore antibacterial peptide have huge
Application potential.Since boman et al. was found that antibacterial peptide first in 1972 in fruit bat, subsequent 1981 years steiner et al.
After having successfully been isolated two kinds of antibacterial peptide cecropin-a and cecropin-b from U.S. giant silkworm, at the beginning of cut-off 2014, again
The natural amps finding is more than 2000.In recent years, the development of Computeraided drug design technology, greatly facilitates
The research and development of antibacterial peptide, has had more than ten analog entrance according to natural cationic antibacterial peptide symthesis clinical or clinical
Front experimental stage, typical representative is the antibacterial peptide that the magainins that frog skin extracts is the first enters drug development program,
Magainins drugmaker of the U.S. has developed to filter out all has two kinds of obvious lethal effect to antibacterial, virus and tumor cell
Novel antimicrobial peptide: pexiganan (msi-78) and mai1278 has entered the clinical iii phase and has tested, can predict soon will
Carrying out antibacterial peptide will become the key areas finding new drug.Small peptide gpg that tripep company is developing, is carried out in Europe
I/ii clinical trial phase, treatment finds, virus replication slows down, and shows certain anti-hiv application prospect in more than two weeks；Oral anti-
Bacterium peptide ambicin and isegangn has successfully completed i phase safety evaluatio；Canadian micrologix Biochem Technology, INC.
The clinical research of the serious acne infection of two kinds of antibacterial peptide mbi-226 and mbi-594 entrance ii phases or iii phase, wherein mbi-
226iii clinical trial phase result shows only have minimum irritant reaction in induction period, compares with negative group, accumulates irritant reaction
Less.Anti-hiv antibacterial peptide enfuvritide (t20) of the most noticeable trimeris company successfully listed before the several years.
Many antibiotic develop immunity to drugs at present, and peptides antibiotic substance has the antibacterial mechanisms of uniqueness, to eukaryotic cell
Almost do not act on, the eukaryotic cell only acting on prokaryotic cell and pathological changes occurring, and be not likely to produce drug resistance, therefore not
In long future, these peptides antibiotic substance are likely to become the source of novel antibacterial medicine, have fabulous market development and application
Prospect.
With microbiological development, compared with conventional antibiotic, the unique mechanism of action of amps make it have efficiently and
The advantage of low drug resistance.Although amps has certain advantage, but up to the present less amps medicine reaches clinical practice
Requirement.Because amps haves such problems as bioavailability, toxicity, stability and production cost.In order to improve the attribute of amps,
Medicine can be developed into, rational design is the method for solve problem.
Content of the invention
In order to solve above-mentioned problem, the present invention synthesized the ultrashort antibacterial peptide being made up of 3～7 aminoacid and its
Activity modifying thing, it not only can greatly reduce production cost, also significantly reduces the immunity that may cause after carrying out structure optimization
Originality is reacted.
It is an object of the invention to provide the small-molecular peptides of antimicrobial antiviral activity and its activity modifying thing.
The technical solution used in the present invention is:
There is small-molecular peptides and its activity modifying thing of antimicrobial antiviral activity, the aminoacid sequence 3 of described small-molecular peptides
～7 aminoacid compositions, its amino acid sequence formula is x0-3(r/k/o/dab)x0-1w1-2(r/k/o/dab)0-1x0-1；
Wherein x be selected from aminoacid r, in k, w, i any one；
x0-3Represent aminoacid r, any 0～3 in k, w, i；
x0-1Represent aminoacid r, any 0～1 in k, w, i；
w1-2Represent 1 or 2 aminoacid w；
(r/k/o/dab)0-1Represent 0 or 1 aminoacid r or k or o or dab, wherein dabFor 2,4- diaminourea 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 aminoacid is the aminoacid of l- configuration or d- configuration.
Further, there is small-molecular peptides and its activity modifying thing of antimicrobial antiviral activity, described activity modifying thing
Modificationt part is divided into esterified modification, glycosylation modified, amidatioon modification, Carboxylation modification, phosphorylation modification, esterification to modify, n- acyl
Change modify, modified by disulfide bond cyclisation, the modification that changes into acid-addition salts, dimer, polymer, in conjugatedization modification at least
A kind of.
Further, the group of above-mentioned esterified modification is selected from fatty acid, triterpeness, steroid, phospholipid, glycophospholipin acyl flesh
Alcohol, Phosphatidylserine, PHOSPHATIDYL ETHANOLAMINE, sphingomyelins, phosphatidylcholine, cuorin, phosphatidylinositols, phosphatidic acid, haemolysis
At least one in phosphoglyceride and cholesterol group.
Further, above-mentioned fatty acid is c2～c20 fatty acid.
Further, above-mentioned fatty acid is c8～c20 fatty acid.
Small-molecular peptides and its activity modifying thing eliminate the application in antibacterial, funguses or viral agent in preparation.
Small-molecular peptides and its activity modifying thing are used for processing the antibiosis of antibacterial, funguses or virus infection or pollution in preparation
Application in element, disinfectant or cleaning agent, preservative or packaging material.
Encode the nucleic acid of above-mentioned small-molecular peptides.
The invention has the beneficial effects as follows:
1) amps of the present invention has extensive and effective antibacterial activity to Gram-positive and negative bacteria.Some are many
The antibacterial that peptide also shows multiple patterns except having the potential mechanism destroying membrane stability kills mode.
2) amps of the present invention is except its powerful bactericidal activity, some antibacterial peptides are produced with conventional antibiotic drug combination
Give birth to unpredictalbe synergism, this synergism one side being used in combination can effectively reduce the use agent of antibiotic
Amount, on the other hand can realize more preferable antibacterial effect.
3) in all of antibacterial peptide, these amps of present invention synthesis are the shortest, therefore have and are easily-synthesized and low cost
Advantage, the simultaneously potential medicine developing into various infectious disease.As in the present invention, some amps also have
Resisiting influenza virus, the activity of hiv.
4) the invention provides having the new short lipopeptid of anti-gram positive bacteria, gram-negative bacteria, antivirus action.
Brief description
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 fatty acid and c14 fatty acid modifying；
Fig. 6 is the cytotoxicity in mtt test assessment c10-rkwwk and rkwwk cell (tzm-b1)；
Fig. 7 measures the murder by poisoning speed to mammalian cell for the c10-amp3 (c10-rkwwk) for flow cytometer；
Fig. 8 measures the murder by poisoning speed to mammalian cell for the c10-amp14 (c10-rikwk) for flow cytometer；
Fig. 9 is the sterilization speed to s.aureus (c) for the c10-amp3 and c10-amp14；
Figure 10 is the cooperative effect of the drug combination of c10-amp4, c10-amp3, c10-amp7, rkwwk and neomycin.
Specific embodiment
There is small-molecular peptides and its activity modifying thing of antimicrobial antiviral activity, the aminoacid sequence 3 of described small-molecular peptides
～7 aminoacid compositions, its amino acid sequence formula is x0-3(r/k/o/dab)x0-1w1-2(r/k/o/dab)0-1x0-1；
Wherein x be selected from aminoacid r, in k, w, i any one；
x0-3Represent aminoacid r, any 0～3 in k, w, i；
x0-1Represent aminoacid r, any 0～1 in k, w, i；
w1-2Represent 1 or 2 aminoacid w；
(r/k/o/dab)0-1Represent 0 or 1 aminoacid r or k or o or dab, wherein dabFor 2,4- diaminourea Gamma Amino Butyric Acid；
Because arginine (r), ornithine (o), 2,4- diaminourea Gamma Amino Butyric Acid (dab) and lysine (k) show in physiological conditions and have
Polarity and positively charged property, arginine (r), ornithine (o), 2,4- diaminourea Gamma Amino Butyric Acid (dab) lysine can be replaced
(k).
Preferably, above-mentioned small-molecular peptides and its activity modifying thing institute electrically charged at least+2, sequence control is in 7 aminoacid
Within, can be by increasing r, the aminoacid such as k, w, i is changing electric charge, armaticity and the hydrophobicity of small peptide, and adjusts lysine
The distance between (arginine) can also 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 aminoacid is the aminoacid of l- configuration or d- configuration.
Preferably, there is small-molecular peptides and its activity modifying thing of antimicrobial antiviral activity, the repairing of described activity modifying thing
Decorations part is esterified modification, glycosylation modified, amidatioon modifications, Carboxylation modification, phosphorylation modification, esterification are modified, n- acylation
Modify, modified by disulfide bond cyclisation, the modification that changes into acid-addition salts, dimer, polymer, at least one in conjugatedization modification
Kind.
Preferably, the group of above-mentioned esterified modification be selected from fatty acid, triterpeness, steroid, phospholipid, glycophosphatidyl inositol,
Phosphatidylserine, PHOSPHATIDYL ETHANOLAMINE, sphingomyelins, phosphatidylcholine, cuorin, phosphatidylinositols, phosphatidic acid, haemolysis phosphoric acid
At least one in glyceride and cholesterol group.
Preferably, above-mentioned fatty acid is c2～c20 fatty acid.
Preferably, above-mentioned fatty acid is c8～c20 fatty acid.
Preferably, above-mentioned c8-c20 fatty acid be selected from lauric acid, Palmic acid, myristic acid, stearic acid, Oleic acid, linoleic acid,
Linolenic acid, arachidonic acid, eicosapentaenoic acid, erucic acid and arachidic acid.
Above-mentioned fatty acid molecule can be attached to any suitable part of antibacterial peptide of the present invention, such as aminoterminal, c-terminuses or
Amino and carboxyl two ends etc..
Small-molecular peptides and its activity modifying thing eliminate the application in antibacterial, funguses or viral agent in preparation.
Small-molecular peptides and its activity modifying thing are used for processing the antibiosis of antibacterial, funguses or virus infection or pollution in preparation
Application in element, disinfectant or cleaning agent, preservative or packaging material.
The mechanism of the antimicrobial antiviral activity of small-molecular peptides of the present invention and its activity modifying thing
In the present invention, the effective amps of synthesis combines the advantage of the peptide that short lipopeptid and rw enrich.A series of to this
The active testing of antibacterial peptide shows, it includes gram negative bacilli escherichia coli, gram-positive cocci to pathogenic microorganism
The microorganisms such as staphylococcus aureuses, Candida albicans, virus are inhibited.Hemolytic experiment shows that it has low toxicity
Property, and part am/ps has the effect of antiviral.
Polypeptide containing r and w is new amps primarily now.However, except containing cation group and hydrophobic group,
Importance containing arginine and the antibacterial peptide of tryptophan has been obtained for sufficiently recognizing.Explain, be because defining
Cation π key between negatively charged electron cloud indole residue and various cationic amino acid residues, and arginine and bad ammonia
Acid may result in the rupture of cell membrane.Found based on above, the present invention synthesized a series of containing r, the ultrashort lipopeptid of k, 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 is regulated and controled.Therefore, increase
Electric charge, changes the position of polar/non-polar aminoacid, changes space structure pliability, and replacing the methods such as hydrophobic/hydrophilic residue can
To obtain highly active antibacterial peptide.For obtaining more hydrophobic group role in antibacterial peptide, carry out two and tested:
A the hydrophobicity of () difference sequence peptide passes through reverse high-efficient liquid phase analysis；B the hydrophobicity of the peptide of () same sequence can assess connection peptides
Base various fatty acids (respectively c10 and c18 fatty acid) end to end.On the basis of these experiments, draw a conclusion, one
The strong antibacterial peptide of activity is the system of a balance, there is an optimum high antibacterial range of hydrophobicity that is to say, that high hydrophobicity
Not always directly proportional with efficient antibacterial antiviral ability.Decrease or increase hydrophobicity surpass go beyond the scope significantly
Affect its antibacterial activity. therefore, most of peptides are not to have the activity of c14, c10 analog high after the replacement of c18 fatty acid.Produce this
The reason plant phenomenon is that the peptide of high hydrophobicity makes itself and cell membrane be difficult to combine.
Antibacterial peptide of the present invention has common biophysical properties, such as positive charge, hydrophobicity and both sexes.These physicochemical properties
The destruction to cell membrane and permeabilization will be affected.In the present invention, find that c10-rikwk and c10-rkwwk has similar
Activity, but the mechanism of its sterilization then differs..This is that c10-rkwwk can have to gram positive bacteria and gram negative bacteria
Selective reason.However, peptide can't be apparent from now enter the concrete mechanism of antibacterial it may be possible to dissolving film or broken
The integrity of bad film, thus with antibacterial in target spot phase separation.And the interaction of dna-binding description of test peptide and dna
May relate to disturb the duplication of dna, restructuring, reparation, translation etc..C10-kikrwr has preferable anti-hiv -1 virus activity, and
And less to the toxicity of cell, there are the potentiality proceeding to test.C18-kkwk, c18-kwk, c20-kkwk, c20-kwk then have
The activity of anti-h5n1, plays the role of in low concentration to promote virus infection.
Due in ph value, ionic strength and lipophilic difference, the small peptide of present invention synthesis and different cell membrane have different
Effect, also have different selectivitys.The physicochemical properties of tryptophan and lysine uniqueness make them be suitable as antibacterial peptide
Comprising modules.The polypeptide of the present invention is with resisting that arginine (r), lysine (k), tryptophan (w), isoleucine (i) form
The antibacterial peptide of microorganism, the aminoacid that one of them has certain physically and/or chemically characteristic be changed to another have identical
The chemically or physically aminoacid of characteristic, for example, arginine (r), ornithine (o), 2,4- diaminourea Gamma Amino Butyric Acid (dab) and lysine
K () shows in physiological conditions has polarity and positively charged property, arginine (r), ornithine (o), 2,4- diaminourea
Gamma Amino Butyric Acid (dab) lysine (k) can be replaced.
Sum it up, the present invention contains arginine, lysine, the lipopeptid of tryptophan have wide spectrum, efficiently anti-gram
Positive bacteria and the activity of gram negative bacteria.And in addition to playing the role of disrupting biofilm, some peptides have multiple sterilization mode.
In the present invention, some peptides can reduce the dosage of sterilization with the combination of traditional antibiotic.And the short lipopeptid newly synthesizing be a class
Short antibacterial peptide.Be easily-synthesized because it has, the advantage of low cost, and treatment anti-infective medicine can be developed into.
With reference to specific embodiment, the present invention is further illustrated, but is not limited thereto.
Embodiment 1:
First, micromolecule polypeptide and its activity modifying thing
(1) micromolecule polypeptide and its activity modifying thing
The present invention have anti-bacteria and anti-virus small-molecular peptides be with kwwk, kwk be basic motif, by 3-7 aminoacid institute group
The polypeptide becoming.A series of aminoacid sequence of the antimicrobial small-molecular peptides being obtained according to primary amino acid sequence kwwk, kwk leads to
Formula is x0-3(r/k/o/dab)x0-1w1-2(r/k/o/dab)0-1x0-1；Wherein x be selected from aminoacid r, in k, w, i any one；x0-3Table
Show aminoacid r, any 0～3 in k, w, i；x0-1Represent aminoacid r, any 0～1 in k, w, i；w1-2Represent 1 or 2
Aminoacid w；(r/k/o/dab)0-1Represent 0 or 1 aminoacid r or k or o or dab, wherein dabFor 2,4- diaminourea Gamma Amino Butyric Acid.
Wherein preferably 17 sequences of anti-bacteria and anti-virus effect are as shown in table 1.
The amino acid sequence table of table 1 antimicrobial small-molecular peptides
The activity modifying thing of micromolecule polypeptide refers to above-mentioned micromolecule polypeptide is carried out esterified modification, glycosylation modified, acyl
Amination modification, Carboxylation modification, phosphorylation modification, esterification modification, n- are acylated and modify, modified, changed into by disulfide bond cyclisation
The modification of acid-addition salts, Dimerized, polymer or conjugatedization modification etc..
(2) synthesis of micromolecule polypeptide and its activity modifying thing
Small-molecular peptides are all to use standard 9- fluorenylmethylcarbonyl fmoc solid phase synthesis process to synthesize by amide mhba resin.
The synthesis of sequence is to carry out in abi433a Peptide synthesizer.Peptide lengthening reaction condition is: with standard hbtu/hobt for coupling
Reagent, with dimethylformamide (dmf) as solvent, the diisopropylethylamine (diea) of 2 times of excess, 8 times of excess containing fmoc protect
The shield aminoacid of group or the free fatty of 10 times of excess.Peptide is all to be cracked from resin using reagent m, and reagent m includes
87.5% trifluoroacetic acid, 2.5% dithioglycol, 5% THIOANISOLE and 5% deionized water (3h, room temperature), crude product is deposited in first
Base tertbutyl ether, identical solvent is washed twice.
Peptide fluorescent labeling carries out aminoacid identical coupling process and adopts rhodamine as sour solvent, reacts 12 hours.
The molecular weight of each peptide passes through electrospray ionization mass spectrometry (esi-ms, waters) and confirms.
Peptide purification uses rp-hplc, experiment condition: flow velocity 1ml/min；Mobile phase solution a water (0.1% trifluoroacetic acid) is molten
Liquid 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 of lipopeptid is 85-90%.
2nd, the property of micromolecule polypeptide and its activity modifying thing
Table 2 is n end and c14 fatty acid conjugation, such as the electric charge (ch) of c end amidated small-molecular peptides activity modifying thing, dredges
Aqueouss (%hp), average hydrophilicity (mh), isoelectric point, IP (pi), the retention time (rt) in rp-hplc chromatographic column, average molecular
The physicochemical properties such as amount (mw).
The name of table 2 antimicrobial polypeptide, sequence and physicochemical property
The n end of a: peptide is conjugated with c14 fatty acid, the amidatioon of c end；B: electric charge (ch), isoelectric point, IP (pi), hydrophobicity (%hp)
Calculated based on amidated peptide and do not use the online calculating instrument of lipid parthttps://www.genscript.com/ sslbin/site2/peptide calculation.cgi；The calculating of c:mh (average hydrophilicity) is based on kyte＆doolittle
Model uses websitehttp://www.bbcm.univ.trieste.it/rossi/hydrocalc hydromcalc.html；
D:rt (retention time) unit is min.
From Table 2, it can be seen that being that in 10～13min, hydrophobicity is closely related with retention time, such as in retention time
The retention time of c14-amp5 (c14-rwwr) is 12.5min, and the retention time of c14-amp9 (c14-rwr) is
11.93min it is seen that c14-amp9 (c14-rwr) retention time than many trp residues (hydrophobicity high) c14-
Amp5 (c14-rwwr) is long.
Fig. 1～4 are n end and c10 fatty acid conjugation, the hplc figure of c end amidated small-molecular peptides activity modifying thing and matter
Spectrum (ms) figure,
2nd, the antibacterial experiment of micromolecule polypeptide and its activity modifying thing
Minimal inhibitory concentration (mic) is tested
Tested essentially according to nccls (National Committee of Clinical Laboratory Standards) guide.Respectively by gram sun
Property bacterium staphylococcus aureuses (s.aureus, atcc12600), gram negative bacteria escherichia coli (e.coli, atcc25922)
Cultivate to obtain 1x10 with Candida albicans (c.albicans, atcc 14053)8The bacterium solution of cfu/ml, then it is diluted to 1x105cfu/
ml.Each small-molecular peptides and its activity modifying thing are separately added in e.coli, s.aureu s and c.albicans bacterium solution, make
In e.coli and s.aureu s bacterium solution, the concentration of peptide or derivatives thereof is gradually diluted to 0.97 μ g/ml from 31.2 μ g/ml, respectively
It is added in e.coli and s.aureus bacterium solution；Make in Candida albicans (c.albicans) bacterium solution peptide or derivatives thereof concentration from
62.5 μ g/ml are gradually diluted to 1.98 μ g/ml.Gram positive bacteria and gram negative bacteria are trained in 37 DEG C of 50%mh culture medium
Support 17 to 20 hours, funguses cultivate 20 hours in 30 DEG C of 50%ypd culture medium.In addition, setting positive control experiment, point
Cai Yong not neomycin and c16-kggk (existing antibacterial peptide) process antibacterial e.coli and s.aureus.All experiments parallel four are arrived
Six are independently repeated to test.Testing result is as shown in table 3,4,5.
Table 3 is n end and c14 fatty acid conjugation, the mic testing result of c end amidated small-molecular peptides activity modifying thing；Table
4 is the mic result of the small-molecular peptides activity modifying thing containing c10, c18 fatty acid；Table 5 is little point of peptide amp-3 (rkwwk) and its lives
The mic result of sex modification thing；As can be seen that most small-molecular peptides and its activity modifying thing can be effective from table 3～table 5
Kill escherichia coli and staphylococcus aureuses, each small-molecular peptides and its activity modifying thing are that staphylococcus aureuses are resisted
Almost it is not significantly different between bacterium activity, the small-molecular peptides of this explanation present invention and its antibacterial activity of activity modifying thing are main
Relevant with its physicochemical property, particularly electric charge and hydrophobicity.
So change electric charge or the hydrophobic antibacterial activity that can affect small-molecular peptides, in such as c14-amp1 (c14-kwwk)
The c14-amp2 (c14-kiwwk) that one isoleucine of insertion obtains increased 3 to escherichia coli minimal inhibitory concentration (mic)
Again many (tables 3)；In addition, c10-kwwk (c10-amp1), c10-rwwr (c10-amp5) and c14-kwwk (c14-amp1) all have
There is powerful antibacterial activity (table 3 and table 4), whether their antibacterial activity can change with the order of their aminoacid sequence
?？The present invention have detected the antibacterial activity of the small-molecular peptides activity modifying thing that peptide sequence is rwrw, kwkw, kkww, such as table 3～table
Shown in 5, the activity modifying thing of this peptides all, the peptide activity trim that particularly c10 modifies shows extensively effective antibacterial,
Its mic value as little as 3.9 μ g/ml.Illustrate its antibacterial activity mainly be subject to its electric charge and hydrophobic affect, identical electric charge and dredge
The situation of aqueouss is good, and the impact to small-molecular peptides and its antibacterial activity of activity modifying thing of the aminoacid sequence of different order is failed to understand
Aobvious.
Table 3 is the mic result of the small-molecular peptides activity modifying thing containing c14 fatty acid
As can be seen that the length of fat chain exists to the antibacterial activity of small-molecular peptides and its activity modifying thing from table 3 and table 4
Certain impact, in the antibacterial activity of the small-molecular peptides that n end is combined with linear c10, c14 and c18 fatty acid respectively, most of
The antibacterial activity of the small-molecular peptides of c14 fatty acid modifying is weaker than c10 fatty acid modifying, and wanting than c18 fatty acid modifying
By force.
Table 4 is the mic result of the small-molecular peptides activity modifying thing containing c10, c18 fatty acid
5 little points of peptide amp3 (rkwwk) of table and its mic result of activity modifying thing
aRkwwk does not contain aliphatic chain,bN uses at end rhodamine labelling.
3rd, antiviral activity test experience
(1) anti-hiv -1jrflThe detection of pseudoviruss infection activity
After well-grown u87.cd4.ccr5 cell dissociation, it is collected by centrifugation, then use the piping and druming of 1ml culture medium uniformly, take
A small amount of cell counting, remaining is diluted to 1 × 10 with culture medium respectively5Then cell suspension connect by individual/ml according to 100 μ l/ holes
Kind 96 orifice plates, 37 DEG C, 5%co2Under the conditions of overnight incubation, every hole adds small-molecular peptides or its activity modifying thing of gradient dilution
(50 μ l) and pseudoviruss hiv-1jrfl(50 μ l), after continuing culture 48h, every hole addition 100 μ l0.5mg/ml mtt solution, 37 DEG C
Under the conditions of be incubated 4h, observe under inverted microscope and separate out either with or without crystallization, or whether have other impurity to generate, carry out labelling.
It is centrifuged 96 orifice plates, gently suck supernatant (the blue crystallization that culture medium can not siphon away bottom again should be siphoned away), 150 μ l/ holes add
Dmso is dissolving crystallized.Gently vibrate 10min, microplate reader reads a570Value.Small-molecular peptides or its activity modifying thing mensure concentration are:
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.
As shown in table 6, wherein, ic50 refers to the ability of antibacterial peptide suppressing virus replication to testing result, is to suppress
Peptide concentration during 50% virus replication.Cc50 just refers to the toxicity to cell for the antibacterial peptide, is that peptide when making 50% cell death is dense
Degree.It can be seen that c10-kikrwr, kiwwk, kikrwr, c10-kiwwk are respectively provided with the effect of anti-hiv virus, especially
C10-kikrwr, its ic50 concentration is 37.241 ± 1.421 μ g/ml, has good inhibiting effect to hiv virus, and to thin
The toxic action of born of the same parents is weak, and cc50 concentration is in more than 200 μ g/ml.
The anti-hiv result of table 6 c10-kikrwr, kiwwk, kikrwr, c10-kiwwk
(2) detection of anti-h5n1 pseudoviruss infection activity
It is 1x10 that 100 μ l concentration are accessed in every hole of 96 porocyte culture plates5The mdck cell of individual/ml, 5%co2, 37 DEG C
Overnight incubation；With culture medium, pseudoviruss doubling dilution is become 6 dilution factors, take 50 μ l to add in 96 porocyte culture plates, then plus
Enter small-molecular peptides or its activity modifying thing (50 μ l) of the present invention, each dilution factor (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.Setting cell controls, are not added with pseudoviruss.
In 5%co2, after culture 48h under the conditions of 37 DEG C, discard culture medium, 200ml/ hole, pbs washs 1 time.According to luciferase detection
Kit specification, the infection ability of detection pseudoviruss, concrete detection operation is as follows:
A. every hole adds 50 μ l lysates, jiggles, and stands 20min at room temperature, takes 409l cell pyrolysis liquid to add
To in the flat luciferase detection plate of 96 holes；
B. every hole adds 60 μ l luciferase substrate, with genios pro type tecan microplate reader detection luciferase chemistry
Luminous value.Using 2.5 times of cell control well values of chemiluminescence as cutoff value, values of chemiluminescence is accordingly to be regarded as sun more than cutoff
Property.
Testing result is as shown in table 7, it can be seen that kkwk-c18, kkwk-c18, kkwk-c20, kkwk-c20 have
The activity of preferable anti-avian influenza virus, especially kkwk-c18, its ic50 concentration is only 8.874 μ g/ml.
The anti-h5n1 activity of table 7 c18-kkwk, c18-kwk, c20-kkwk, c20-kwk
4th, Study of cytotoxicity
(1) hemolytic activity
Facing critically important problem in exploitation antibacterial peptide as clinically useful medicine is the toxicity to host cell, resists
Bacterium peptide allows for distinguishing mammal and bacterial cell.In current research, the toxicity evaluation of peptide generally utilizes the mankind red
Its hemolytic activity of cytoscopy.
The maximally effective peptide of this hemolytic activity experimental selection, with fresh human erythrocyte (erythrocyte), is reported according to existing information
Method be measured.Fresh human erythrocyte's (erythrocyte) is washed 3 times with pbs, and 10% respin is in pbs.Average external volume (100 μ
L) solution of red blood cells is added to the small-molecular peptides of the present invention comprising equal volume in 96 orifice plates in plate or its activity modifying thing
Pbs solution, concentration is respectively 62.5,125,250 μ g/ml, and 37 DEG C of hatching 1h, 3000rpm are centrifuged 15min.Then at 570nm
The absorbance (50 μ l96 orifice plate) of the measurement supernatant.With pbs and 10%triton x-100 as hemolytic experiment feminine gender and
Positive control.Percent hemolysis (hemolysis rate) are calculated as follows:
Percent hemolysis %=[(abpeptide-abpbs)/(abtriton-abpbs)] x100%
Fig. 5 is the percent hemolysis of the small-molecular peptides of c10 fatty acid and c14 fatty acid modifying, it can be seen that big portion
The hemolytic of the small-molecular peptides of point c10 fatty acid modifying and the few small-molecular peptides of big portion (w) containing tryptophan quantity is low.Haemolysis effect
May be related to the length of the quantity of tryptophan and position and aliphatic chain.Trp residue is more, higher hemolytic, e.g.,
C14-amp12 (c14-kwk) and c14-amp1 (c14-kwwk), wherein, under conditions of 125 μ g/ml concentration, c14-amp12
(c14-kwk) holding blood effect is 11%, and the haemolysis effect containing the c14-amp1 (c14-kwwk) of 2 trp residues reaches
56%；However, c14-amp-16 (c14-kwkw) hemolytic toxicity containing 2 trp residues under identical concentration is but
17%, this explanation tryptophan position also can affect the hemolytic activity of antibacterial peptide and its derivant；Additionally, being 250 μ g/ml in concentration
When, all c10-amp12 containing a trp residue (c10-kwk), c14-amp12 (c14-kwk), c10-amp14 (c10-
Rikwk) and c14-amp14 (c14-rikwk) hemolytic activity be respectively ＜ 10%, 19%, ＜ 10%, 20%, this explanation is anti-
The blood dissolubility of bacterium peptide and its derivant is also relevant with the length of aliphatic chain.In addition, from c10-amp3 (c10-rkwwk), c10-
As can be seen that increasing the electrically charged energy of polypeptide in the hemolytic data of amp1 (c10-kwwk) and c10-amp4 (c10-rikwwk)
Be conducive to distinguishing neutral mammalian cell membrane and negatively charged bacterial cell membrane.Generally speaking, peptide is to human erythrocyte
Hemolytic toxicity is adjustable by changing position, charging property and the hydrophobicity of their the physical-chemical parameters particularly amino acid residue.
(2) mtt measures cytotoxicity
It is 1x10 that 100 μ l concentration are accessed in every hole of 96 porocyte culture plates5The tzm-b1 cell of individual/ml, 5%co2, 37
DEG C overnight incubation, adds 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.Centrifugation abandoning supernatant, by specification adds mtt, then is incubated 4h in 37 DEG C.
Finally, discard mtt solution, add dmso dissolving first a ceremonial jade-ladle, used in libation crystal, survey its trap in 570nm.The percentages of cell survival rate
Calculating formula is: at/ac × 100 (absorbance of absorbance ac: the matched group of at: reaction group).Each data is expressed as three solely
Vertical repetition.
The potential cytotoxicity of antibacterial peptide in order to assess the present invention further and assessment they to protokaryon and mammal
The selectivity of cell, this experiment test cell toxicant to mankind's tzm-b1 cell for the c10-amp3 (c10-rkwwk) and rkwwk
Property.Obviously, under 125 μ g/ml concentration, c10-amp3 can cause cell death, because its cc50 is 115 μ g/ml (table 4), and
Even if there is not cytotoxicity (Fig. 6) at concentrations up to 500 μ g/ml in rkwwk yet.
(3) flow cytometry cytotoxicity:
Above-mentioned mtt cell toxicity test have detected the hatching of antibacterial peptide and mammalian cell more than 48 hours after, antibacterial peptide is to the food in one's mouth
The toxic action of newborn zooblast, this experiment study further within the short time (1h and 2h) antibacterial peptide to mammalian cell and
The toxic action of antibacterial.
Tzm-b1 cell incubated overnight, abandoning supernatant in the high sugar dmem culture medium containing 10% Ox blood serum, then use pbs
Wash 2 times, add trypsin-edta (respectively 0.05% and 0.02%w/v) to be digested.It is subsequently added into containing 10% Ox blood serum
High sugar dmem culture medium terminate digestion, 800rpm is centrifuged 5min.It is eventually adding the high sugar dmem culture medium containing 10% Ox blood serum
Diluting cells are to 2 × 105cells/ml.C10-amp3 (c10-rkwwk) and c10-amp14 (c10-rikwk) is separately added into
Process 1h and 2h in cell, make the concentration of c10-amp3 and c10-amp14 be 40 μ g/ml.Test addition iodine in last cell
Change the third pyridine (10 μ g/ml), 4 DEG C of avoid light places 10min, measure the survival ability of tzm-b1 cell with facscalibur streaming instrument.
As shown in Figure 7 and Figure 8, as can be seen from the figure the tzm-b1 cell more than 99% for the result is not little two for measurement result
When interior death, the small-molecular peptides of the present invention and its activity modifying thing toxic action to mammalian cell at short notice is described
Very weak.
The mensure of sterilization speed: concentration be 1 × 105Amps (c10-amp3 is added in the s.aureus bacterium solution of cfu/ml
And c10-amp14), make amps concentration be 40 μ g/ml (10 times of mic are excessive), be incubated 0,60,120min at 25 DEG C respectively altogether,
Take the bacterial suspension of 10 μ l respectively in ice bath, press 1: 50 dilution with culture medium, after 37 DEG C of aerobic overnight incubation, measure titre.
Measurement result is as shown in Figure 9.
From Fig. 7～9 as can be seen that c10-amp3 and c10-amp14 of same concentrations to be respectively acting on mammal thin
After born of the same parents and antibacterial (s.aureus) 1h, 2h, the survival ability Flow cytometry of tzm-b1 cell, result is more than 99%
The not death (Fig. 7 and Fig. 8) in two hours of tzm-b1 cell, and the staphylococcus aureuses more than 95% are killed entirely after 1h
(Fig. 9).Illustrate that the small-molecular peptides of the present invention and its activity modifying thing are non-to the toxic action of mammalian cell at short notice
Often weak, and there is good toxic action to antibacterial.
5th, cooperative effect analysis
Three effective peptide c10-amp4 (c10-rikwwk), c10-amp7 (c10-kikrwwr), c10-amp3 (c10-
Rkwwk) and rkwwk (without fat chain) respectively with neomycin (neomycin) synergy.The antibacterial of 5 μ l is added in 96 orifice plates
Peptide solution, is from left to right diluted to different concentration (3.12 to 0.097 μ g/ml), adds the neomycin solution of 10 μ l, finally
Add the bacterial suspension of 100 μ l, after being incubated 17h altogether at 37 DEG C, the antibacterial effect of detection each group.Each Data duplication two
Secondary.
Figure 10 is two dimension screening antibacterial peptide cooperative effect, is from left to right the Concentraton gradient (μ g/ml) of peptide, and from top to bottom
It is the Concentraton gradient (μ g/ml) of neomycin.In Figure 10, top is divided into c10-amp4 (c10-rikwwk) and neomycin, c10-amp3
(c10-rkwwk) and neomycin collective effect, bottom is c10-amp7 (c10-kikrwwr) and neomycin, rkwwk and newly mould
The collective effect of element.In figure italic lattice represent that the two achieves cooperative effect.
As can be seen from Figure 10 the small-molecular peptides in the present invention or its activity modifying thing can also be with related antibiotic
It is used in combination and produces unpredictalbe synergism, i.e. obtained antibacterial effect ratio when being used in combination of both antibacterials
With more preferably, this synergism one side being used in combination can reduce the use agent of antibiotic to the effect that this two medicine is used alone
Amount, on the other hand can realize more preferable antibacterial effect.
<110>Nanfang Medical Univ
<120>there is small-molecular peptides and its activity modifying thing of antimicrobial antiviral activity
<130>
<160> 17
<170> patentin version 3.5
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Claims (2)
1. small-molecular peptides and its activity modifying thing eliminate the application in bacterial agents in preparation；
Described small-molecular peptides and its activity modifying thing are c10-rikwwk, c10-kikrwwr or c10-rkwwk, described c10
For c10 fatty acid, wherein c10 sign carbon chain lengths；
Neomycin is also contained in described medicament.
2. small-molecular peptides and its activity modifying thing are used for processing antibiotic, disinfectant or the cleaning of bacterium infection or pollution in preparation
Application in agent, preservative or packaging material；
Its activity modifying thing of described small-molecular peptides is c10-rikwwk, c10-kikrwwr or c10-rkwwk；Described c10 is
C10 fatty acid, wherein c10 characterize carbon chain lengths；
Neomycin is also contained in described antibiotic, disinfectant or cleaning agent, preservative or packaging material.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410259060.5A CN104072579B (en) | 2014-06-11 | 2014-06-11 | Small molecule peptide with antibacterial antiviral activity and active modifier of small molecule peptide |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
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| CN201410259060.5A CN104072579B (en) | 2014-06-11 | 2014-06-11 | Small molecule peptide with antibacterial antiviral activity and active modifier of small molecule peptide |
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| CN104628869B (en) * | 2015-01-28 | 2018-04-03 | 南方医科大学 | One kind has the fusion peptide derivant of antibacterial and anti-influenza virus activity concurrently |
| 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 |
| EP3512867B1 (en) * | 2016-09-15 | 2020-12-16 | DSM IP Assets B.V. | Novel compounds |
| EP3434287B1 (en) * | 2017-07-25 | 2021-01-27 | Arta Peptidion S.r.l.s. | Short and ultra-short antimicrobial lipopeptides and use thereof |
| CN109320585B (en) * | 2018-09-06 | 2021-10-26 | 南方医科大学 | Short lipopeptide with specific anti-acne propionibacterium effect and anti-inflammatory effect |
| CN110664988B (en) * | 2019-11-06 | 2022-09-30 | 山东省农业科学院奶牛研究中心 | Application of H-Lys-Trp-Lys-OH in preparation of medicine for resisting bovine intestinal viruses |
| CN110938112A (en) * | 2019-12-10 | 2020-03-31 | 倪京满 | Fatty acid modified ultrashort-sequence antibacterial peptide analogue and application thereof |
| CN111574619B (en) * | 2020-05-06 | 2022-03-25 | 中国农业科学院饲料研究所 | Lipopeptide Lin-Lf4NH2And Lin-Lf5NH2And uses thereof |
| CN116284243A (en) * | 2021-04-09 | 2023-06-23 | 倪京满 | Alpha helix antibacterial short peptide 3RI4F with high antibacterial activity and low toxicity and application thereof |
| CN115043925B (en) * | 2022-04-29 | 2023-08-11 | 苏州大学 | Modified antibacterial peptide oNCM and application thereof |
| CN116332777A (en) * | 2023-02-20 | 2023-06-27 | 华中科技大学 | Diaryl benzyl methylamine compound, preparation and application as carrier in synthesizing polypeptide |
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| CN1116427A (en) * | 1993-10-14 | 1996-02-07 | 生化学工业株式会社 | Polypeptide and anti-hiv agent prepared therefrom |
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| CN1116427A (en) * | 1993-10-14 | 1996-02-07 | 生化学工业株式会社 | Polypeptide and anti-hiv agent prepared therefrom |
Non-Patent Citations (2)
| Title |
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| Tuning the antimicrobial pharmacophore to enable discovery of short lipopeptides with multiple modes of action;Yuxin Fang et al.;《European Journal of Medicinal Chemistry》;20140604;第83卷;第36页左栏第1段，第37页左栏第2段至右栏第2段，第38页右栏第7段至第39页左栏第4段，第42页右栏第3段至第44页左栏第3段，表1-4，图3 * |
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