CN110283237A - There is antimicrobial function peptidomimetic and preparation method thereof, composition and application from disease fungus - Google Patents
There is antimicrobial function peptidomimetic and preparation method thereof, composition and application from disease fungus Download PDFInfo
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Abstract
The invention discloses one kind to have antimicrobial function peptidomimetic and preparation method thereof, composition and application from disease fungus.The peptidomimetic prepares peptide fragment IIGGRC by conventional Solid-phase synthesis peptides technology first, pass through the sulfydryl of c-terminus cysteine side chain and small molecule compound 3- sulfydryl -5- methyl-1 again, 2, the sulfydryl of 4- triazole forms covalent disulfide bonds, obtains finally by reversed high-efficient liquid phase chromatogram purification is partly prepared.Pharmacological experiment result of study shows that the peptidomimetic has good inhibiting effect to gram-positive bacteria reference culture and the methicillin-resistant staphylococcus aureus Methicillin-resistant S.aureus being clinically separated.In addition to bacterium, also there is certain inhibiting effect to fungi reference culture Candida albicans ATCC10231, C.krusei ATCC 6258 and Cryptococcus neoformans ATCC 34877 and the disease fungus being clinically separated.Meanwhile the peptidomimetic shows Low haemolysis activity, the stability of hot environment and acid or alkali environment, and manually prepares that convenient, pharmacological activity is clear, with further Development volue and has the potentiality of industrialization.
Description
Technical field
The invention belongs to biomedicine fields, and in particular to one kind has antimicrobial function peptidomimetic from disease fungus
And preparation method thereof, composition and application have the peptidomimetic of antimicrobial function and the system of the peptidomimetic from disease fungus
Preparation Method and drug or/and composition containing the peptidomimetic, and the application as antimicrobial.
Background technique
Pathogenic microorganism has become serious problem to the increase of drug resistance.In the world, every year there are about
700000 people die of drug-resistant microorganism infection.Epidemiological survey the result shows that, the bacterium of multidrug resistance, such as escherichia coli
Bacterium, Klebsiella Pneumoniae, Acinetobacter bauamnnii, methicillin-resistant staphylococcus aureus (MRSA), vancomycin resistance
MRSA, the streptococcus pneumonia (PRSP) of penicillin resistant, vancomycin-resistant enterococcus (VRE) and extensive drug resistance (XDR) tuberculosis branch
The drug resistance of the bacteriums such as bacillus is more prevalent.With bacterium (Salmonella, the campylobacter contained in zoonosis and excrement
Belong to, Escherichia coli and enterococcus spp) drug resistance it is also increasingly severe.
The resistance mechanism of generally acknowledged bacterium mainly has at present: 1. generating the enzyme that can inactivate antimicrobial agents.The fermentoid is
A kind of hydrolase superfamily, it is main to hydrolyze the antibiotic containing beta-lactam ring structure and make its inactivation, including in clinical treatment
Common penicillins, cephalosporins, monobactams and carbapenem antibiotic.Encode the gene of resistance to drug metabolizing enzyme
It can be naturally present in strain chromosome that (referred to as intrinsic resistance is such as present in the gram-Negative bacillus in enteron aisle to penicillin
With natural resistance)；It can also be under the induction of antimicrobial agents by plasmid-mediated or be integrated into strain chromosome and (be known as obtaining
Obtain property drug resistance).2. modifying the target of antimicrobial agents effect.The change of antimicrobial agents target position is a kind of relatively conventional
Resistance mechanism.Under normal conditions, the change of target position from bacterial strain obtain related gene or gene spontaneous mutation and
Selective mutation in the presence of antimicrobial agents.Such as the mutation of RNA polymerase and the mutation of DNA gyrase can be led respectively
Bacterial strain is caused to obtain the resistance to rifamycin and quinolones antimicrobial.3. generating efflux pump to pump antimicrobial agents
Out.It is difficult to treat by the microbial infection of Gram-negative, because they have inherent resistance to many antibiotic.And its resistance
Acquisition independent of Horizontal Gene Transfer and mutation.The two film construction of Gram-negative bacteria and the efflux pump of generation can will resist micro-
Bio-pharmaceutical pumps out, to reduce the concentration of Intracellular drug.Gram-negative bacteria can express many efflux pumps in its film, it
Transport various molecules discharge bacterial cells, a part of in these pumps can transport antibiotic.It is pumped out from bacterium
Antibiotic can reduce intracellular drug concentration, and bacterium is made to survive in the antimicrobial agents of higher external concentration, thus
Lead to the drug resistance of its combating microorganisms drug.4. changing bacterial cytoplasm membrane permeability.Cell wall is low outside gramnegative bacterium
Permeability has been demonstrated that can effectively prevent antimicrobial agents under the antibiotic concentration of low dosage plays a role.Antibiotic passes through
The key mechanism that outer membrane enters thallus includes being responsible for the porin (Omps) of hydrophilic antibiotics diffusion and for hydrophobicity antibiosis
The lipid mediated pathways of element, many bacterial strains drug resistance due to the change that the lipid or protein of outer membrane form, show outer membrane barriers
It plays an important role in whether strains are sensitive, it is right that any structure change of outer membrane protein can influence its significantly
The resistance of antibiotic.In addition, when the beta-lactamase in permeability barrier and periplasmic space has synergistic effect, the drug resistance of bacterial strain
It will become more serious, and may cause pathogen and resistance is generated to third generation cephalosporin.
Other than bacterium, the drug resistance of previous ignored disease fungus also gets worse and starts to be concerned
(Science 2018,360:739-742).The rapid of multidrug resistant disease fungus occurs and to antifungal drug antibody-resistant bacterium
Wide-scale distribution also constitutes sizable threat to human health.Influence of the fungi to human health at present constantly rises,
The global death rate of fungal disease has been more than malaria or breast cancer, and with the death rate of tuberculosis and AIDS quite (Sci
Transl Med 2012,4(165):165rv13).Although they are ubiquitous, fungal infection is relative to other kinds of biography
It catches an illness and is ignored significantly in the past.
The mankind use antifungal, nystatin and polyene antifungal medicine first, are found the 1950s.It is modern
It, systemic mycosis bacteriostatic agent and fungicide are used as the first-line treatment medication of human treatment's fungal infection related disease.So
And the fast development due to fungi to drug resistance, its increment of the inhibition that existing many drugs can only be of short duration.The gene of fungi
It can breed with highly plastic and rapidly.These attributes make it that can quickly generate mutant under drug selectivity pressure.
The first-line drug for being presently available for treatment mankind's fungal infection mainly has four classes: 1, Polyene Hydrocarbons.Such as anphotericin
B) by inhibiting fungi film sterol (ergosterol) to destroy the structure of cell membrane；2, pyrimidine analogue.Such as 5-flurocytosine (5-
FC pyrimidine metabolic and DNA synthesis) are blocked；3, azole and most widely used fungicide.By inhibiting lanosterol 14- α-
Demethylase blocks the biosynthesis of ergosterol；4, echinocandin is the antifungal of newest one kind.Pass through inhibition
(1-3)-callose synzyme and the biosynthesis for destroying cell wall.
With the long-term extensive use in clinical antifungal therapy of above-mentioned drug, it is true to there is new multidrug resistance cause of disease
Bacterium.The candida albicans (Candida auris) of multidrug resistance in 2009 quilt in a patient from Japanese ear infection for the first time
It was found that all clinical antifungals all drug resistance (Clin Infect Dis 2017,64 (2): 134-140.) and counterweight
Disease care unit constitutes a threat to, because can not be inhibited to kill in normal detergent.Some researches show that main true
The drug resistance of bacteremia pathogen Candida glabrata (Candida glabrata) occurs, and is also primarily due to frequently use spine white
Caused by the use of bacteriums and triazole antifungal agent as preventive usage (Clin Infect Dis 2013,56 (12):
1724-1732.)。
The case where in view of above-mentioned bacterium, antifungal agent resistance, it is necessary to avoid the mechanism of action of existing drug to develop new time
Antifungal activity molecule is selected, inhibits or kill disease fungus by new mechanism of action.Antimicrobial peptide (Anti-
Microbial peptides, AMPs) it is a kind of molecule with sequence and structure diversity, and have extensive antimicrobial
Activity, such as antibacterium, antimycotic and antivirus action.It has now been found that more than more than 3000 antimicrobial polypeptides, but due to
The qualitative defect of natural polypeptides (such as haemolysis, high salt concentration environment is unstable), many activity preferably polypeptide do not have simultaneously
Value as drug development.
Summary of the invention
In view of the shortcomings of the prior art with deficiency, the present invention, which provides one kind, has antimicrobial function from disease fungus
Peptidomimetic and preparation method thereof, composition and application.Polypeptide of the present invention has resisting gram-positive in μ g/mL concentration level
The activity of bacterium reference culture, clinical drug-resistant bacterium and fungi reference culture.
To achieve the above object, one kind that one aspect of the present invention provides is quasi- with antimicrobial function from disease fungus
Peptide, peptidomimetic according to the present invention derive from the albumen (GenBank of people's disease fungus Trichophyton interdigitale T.interdigitale
Number: EZF36158.1), sequence is as follows:
Peptide fragment IIGGRC (amino acid sequence in box) in above-mentioned full-length proteins is passed through into c-terminus cysteine side
The sulfydryl and small molecule compound 3- sulfydryl -5- methyl-1 of chain, the sulfydryl of 2,4- triazoles form covalent disulfide bonds and are connected, i.e.,
It is linker with disulfide bond, splicing becomes the molecule of brand new, and molecular structure is as follows:
Inverted high performance liquid chromatography (Reverse phase-high performance liquid
Chromatography, RP-HPLC) it isolates and purifies and can get the peptidomimetic molecule, theoretical molecular weight 730.34 has anti-
The activity of bacterium and fungi is also effective to the drug-fast bacteria being clinically separated.Above-mentioned peptidomimetic has preparation method technology path simple, surely
It is qualitative strong, the advantages that product quality is easy to control.
Another aspect of the present invention provides a kind of preparation method from disease fungus with antimicrobial function peptidomimetic,
It is characterized in that: peptide fragment IIGGRC being prepared by conventional Solid-phase synthesis peptides technology first, then passes through c-terminus cysteine side
The sulfydryl of the sulfydryl and small molecule compound 3- sulfydryl -5- methyl-1 of chain, 2,4- triazoles forms covalent disulfide bonds, finally by
Reversed high-efficient liquid phase chromatogram purification is partly prepared, complete peptidomimetic molecule is finally obtained.
Another aspect of the present invention also provides one kind has antimicrobial function peptidomimetic antimicrobial from disease fungus
Application in drug or/and daily necessities, it is characterised in that: the antimicrobial agents are injection, tablet, sterile powder for injection
End, pulvis, granule, capsule, oral solution, paste or creme；The drug passes through injection, oral, collunarium, eye drip, physics
Or the method for chemistry mediation imports muscle, endothelium, subcutaneous, vein or mucosal tissue；The daily necessities be hand cleanser, facial cleanser,
Shampoo, bath foam, Feminine care lotion, sanitary napkin, panty liner, diaper, adult nursing diaper, mouthwash, toothpaste, perfume (or spice)
Soap, liquid detergent, thimerosal, toilet cleaner, disinfected paper napkin, dressing, bandage or feed.
Further, the microorganism is the Gram positive bacteria strain being clinically separated and/or the fungi being clinically separated
Bacterial strain.
Further, the fungi is Mycotoruloides and Cryptococcus.
Another aspect of the present invention also provides a kind of composition, it is characterised in that: it includes above-mentioned from disease fungus
With antimicrobial function peptidomimetic or also comprising the acceptable salt of the peptidomimetic and/or hydrate and/or solvate and/or
Acceptable carrier.
Another aspect of the present invention also provides a kind of combinations of the above object answering in preparation antibacterials and/or daily necessities
With, it is characterised in that: antibacterials be injection, tablet, injection sterile powder, injection sterile powder, pulvis,
Granula, capsule, oral solution, paste or creme；The antibacterials or composition pass through injection, oral, collunarium, eye drip, object
The method of reason or chemistry mediation imports muscle, endothelium, subcutaneous, vein or mucosal tissue；The daily necessities are hand cleanser, wash one's face
Milk, shampoo, bath foam, Feminine care lotion, sanitary napkin, panty liner, diaper, adult nursing diaper, mouthwash, tooth
Cream, perfumed soap, liquid detergent, thimerosal, toilet cleaner, disinfected paper napkin, dressing, bandage or feed.
Hemolytic experiment the result shows that, the peptidomimetic with antimicrobial function haemolysis when at concentrations up to 64 μ g/mL
Rate < 5% (Fig. 5), safety with higher.
Above-mentioned peptidomimetic or/and composition with antimicrobial function can be prepared into injection according to conventional preparation method
A variety of dosage forms such as agent, tablet, injection sterile powder, pulvis, granule, capsule, oral solution, paste, creme.This has anti-
The peptidomimetic or composition of microbial function can by injecting, taking orally, collunarium, eye drip, the method that physically or chemically mediates import
Muscle, endothelium, subcutaneous, vein or mucosal tissue, or human body is imported after other material mixings or package.
The invention has the advantages that and effect: it is according to the present invention to have the function of antimicrobial (bacterium and fungi)
Peptidomimetic there is good resisting gram-positive bacteria effect (including the drug-fast bacteria being clinically separated), while can also inhibit Mycotoruloides
It is and highly-safe with the fungi of Cryptococcus；The peptidomimetic is manually prepared conveniently, and production cost is low, is suitble to industrialization extensive raw
It produces；Therefore there is good development prospect in treatment bacterium and fungal infection field；It is of the present invention that there is antimicrobial function
The peptidomimetic of energy can provide new selection to develop new antibacterium, antifungus active substance and its compound preparation.
Detailed description of the invention
Fig. 1 is the HPLC figure of natural polypeptides peptide fragment intermediate II GGRC prepared by the present invention.
Fig. 2 is the mass spectrogram of natural polypeptides peptide fragment intermediate II GGRC prepared by the present invention.
Fig. 3 is the HPLC figure prepared by the present invention with antimicrobial function peptidomimetic.
Fig. 4 is the mass spectrogram prepared by the present invention with antimicrobial function peptidomimetic.
Fig. 5 is the hemolytic activity result that the present invention has antimicrobial function peptidomimetic.
Fig. 6 is that the present invention has Activity Results of the antimicrobial function peptidomimetic in various concentration salt environment.
Fig. 7 is that the present invention has Activity Results of the antimicrobial function peptidomimetic in different temperatures environment.
Fig. 8 is that the present invention has Activity Results of the antimicrobial function peptidomimetic in different pH environment.
Specific embodiment
The present invention is described in further detail below in conjunction with the drawings and specific embodiments.
The preparation of one natural polypeptides peptide fragment intermediate II GGRC of embodiment
In the synthesis of the polypeptide peptide fragment intermediate, N-terminal group is protected with 9-fluorenylmethyloxycarbonyl (FMOC), is selected
4- methyldiphenyl base methylamine resin (4-methyl-benzhydrylamine resinHCl, MBHA resin) is solid phase load
Body, HOBt/DCC are condensing agent, extend peptide chain from carboxyl terminal to amino terminal.With 94% trifluoroacetic acid, 3% water and 3% 3
The mixed liquor (being mass percent) of isopropyl monosilane (TIA) cracks it from MBHA resin.The repeated multiple times precipitating of ether
Afterwards, it is purified through preparative RP-HPLC.Use C18Reverse phase preparative column (20mm × 250mm, 5 μm)；Mobile phase: 1.5 ‰ trifluoro second
Acid, 0%~50% (percent by volume) acetonitrile are mobile phase, and the flow velocity of 0.5mL/min carries out gradient elution.It is examined through RP-HPLC
It surveys, retention time (retention time, RT) is 8.14min, and purity > 95% (Fig. 1) is spare after freeze-drying.Through ESI-
QTOF-MS identification, molecular weight theoretical molecular weight corresponding with standby polypeptide intermediate of drawing up is consistent, m/z 618.6 [M+H]+,
m/z 309.8[M+2H]2+(Fig. 2).
EmbodimentTwoThere is the preparation of antimicrobial function peptidomimetic from disease fungus
With the intermediate peptide fragment IIGGRC and small molecule compound 3- sulfydryl -5- methyl-1 prepared in above-described embodiment 1,
2,4- triazole is raw material.1mg intermediate peptide fragment IIGGRC and 0.2mg 3- sulfydryl -5- methyl-1 is taken, 2,4- triazoles are used
1mL concentration is the Tris-HCl buffer solution of 0.15M (pH value 8.4), accelerates intermediate with the soft pressure-vaccum solution of liquid-transfering gun
Peptide fragment and small molecule compound dissolution.The EP pipe for filling polypeptide solution is put into constant-temperature table in 30 ± 2 DEG C, 80rpm is incubated for
About 36h.In room temperature 10000rmp high speed centrifugation 4min, supernatant is taken.It is purified through preparative RP-HPLC.Use C18Reverse phase preparation
Column (20mm × 250mm, 5 μm)；Mobile phase: 0.15% trifluoroacetic acid, 0%~55% (percent by volume) acetonitrile are mobile phase,
The flow velocity of 0.5mL/min carries out gradient elution.It detects, RT 10.24min, purity > 95% (Fig. 3), is lyophilized through RP-HPLC
It is spare afterwards.It is identified through ESI-QTOF-MS, molecular weight theoretical molecular weight corresponding with standby peptidomimetic of drawing up is consistent, 731.2 [M of m/z
+H]+, m/z 366.4 [M+2H]2+(Fig. 4).
Embodiment three is measured from the antimicrobial acivity of disease fungus peptidomimetic
To gram-positive bacteria reference culture (such as staphylococcus aureus Staphylococcus aureus ATCC
25922, NEISSERIA GONORRHOEAE Neissria gonorrhoeae ATCC49226) and the methicillin-resistant staphylococcus Portugal that is clinically separated
Grape coccus Methicillin-resistant S.aureus has good inhibiting effect.In addition to bacterium, to fungi standard
Bacterial strain Candida albicans ATCC10231,6258 C.kruseiATCC and Cryptococcus neoformans
ATCC 34877 also has stronger inhibitory activity.
The reference culture used: Staphylococcus aureus ATCC 25922, Neissria gonorrhoeae
ATCC49226, Candida albicans ATCC10231, C.krusei ATCC 6258 and Cryptococcus
Neoformans ATCC 34877 is purchased from China typical culture collection center (China Center of Type Culture
Collection,CCTCC).The bacterial strain being clinically separated: S.aureus, Methicillin-resistant S. aureus,
Candida albicans is provided by the People's Hospital, Wuhan University laboratory medicine center.It is minimum antibacterial using coubling dilution measurement
Concentration (Minimum Inhibitory Concentration, MIC) should derive from disease fungus peptidomimetic by MIC value characterization
Antimicrobial acivity.
The specific method is as follows:
Tested bacteria is inoculated in Luria-Bertani (LB) solid medium tablets of sterilizing respectively with three zoning collimation methods
In；Fungi to be measured is inoculated in the Yeast Extract Peptone Dextrose (YPD) of sterilizing respectively with three zoning collimation methods
In solid medium tablets.Bacterium is inverted culture 14 hours in 37 DEG C of constant incubators；Fungi is in 30 DEG C of constant incubators
It is inverted culture 48~96 hours.(bacterium LB liquid is transferred in the fluid nutrient medium of sterilizing respectively with oese picking single colonie
Body culture medium；Fungi YPD fluid nutrient medium), earthquake is trained under the conditions of 37 DEG C (bacteriums) or 30 DEG C of (fungi), 150rpm
It supports to logarithmic growth phase.With the absorbance value (OD of bacterium solution at ultraviolet specrophotometer measurement 600nm wavelength600), according to 1OD=1
×109Reference culture and clinical drug-resistant strain cultures are diluted to (1~2) × 10 respectively by the conversion relation of CFU/mL5
CFU/mL。
The LB liquid medium (bacterium) or liquid YPD medium of 100 μ L sterilizing is first added in sterile 96 orifice plate first
(fungi) culture medium；Next the 100 μ L of peptidomimetic solution that the concentration dissolved with the LB culture medium of sterilizing is 256 μ g/mL is added
Into the 1st hole, 100 μ L is taken to be added in the 2nd hole after mixing, 100 μ L finally are sucked out from the 9th hole and discard, successively doubling dilution；
It is (1~2) × 10 that concentration is finally added into each hole5The 100 μ L of bacterium solution that CFU/mL has diluted is uniformly mixed, peptidomimetic in each hole
Final concentration of 128 μ g/mL, 64 μ g/mL, 32 μ g/mL, 16 μ g/mL, 8 μ g/mL, 4 μ g/mL, 2 μ g/mL, 1 μ g/mL, 0.5 μ g/
mL.Each hole of peptidomimetic is not added as negative control.
The above each group is in 37 DEG C shake culture 12~14 hours (bacteriums)；In 30 DEG C shake culture 24~48 hours, measurement
Wavelength is the absorbance value at 600nm.Minimal inhibitory concentration (MIC), which takes, can't detect bacterium or fungi growth (OD600≤0.05)
Hole in polypeptide final concentration minimum.Bacterium solution is muddy in negative control hole, and the absorbance OD at 600nm600>
2.5.This has antimicrobial and immunological regulation bifunctional polypeptides antimicrobial acivity result such as tables 1 described in this patent.
Table 1 has antimicrobial and immunological regulation bifunctional polypeptides antimicrobial acivity
It can be seen that the peptidomimetic according to the present invention from disease fungus with comparatively ideal from the result in table 1
Antibacterial and antifungal activities.For all reference cultures including bacterium and fungi, the μ of MIC≤16 g/mL.For
It is 32 μ g/mL that clinical drug-resistant bacterium MRSA, which also has very strong inhibiting effect MIC value, to the Candida being clinically separated
The relatively weak MIC value of the inhibiting effect of C.albicans is 64 μ g/mL.
Example IV from disease fungus there is the hemolytic activity of antimicrobial function peptidomimetic to measure
New blood is obtained from healthy donors, suspension is made after separating human red blood cells, is negative right with sterile saline
According to, using 1% Triton X-100 as positive control, measure the hemolytic activity of the bifunctional polypeptides.
Specific step is as follows: collecting whole blood with EDTA anticoagulant tube, gently overturning keeps blood sufficiently anticoagulant, in room temperature 600rpm
It is centrifuged 4min, upper plasma is discarded and retains lower layer's red blood cell；The sterile saline of 4 times of volumes is added, gently overturning makes to be centrifuged
Pipe makes the red blood cell suspension of bottom, and 600rpm is centrifuged 4min at room temperature, discards supernatant the red blood cell for retaining and precipitating, and repeats
Operation 3 times, until supernatant is colourless；Red blood cell is made to the cell suspension of 2% (v/v) concentration with sterile saline；With
Sterile saline the peptidomimetic is dissolved and is made into 128 μ g/mL, 64 μ g/mL, 32 μ g/mL, 16 μ g/mL, 8 μ g/mL, 4 μ g/mL,
The solution of 2 μ g/mL and 1 μ g/mL concentration；The 100 μ L bifunctional polypeptides solution and 100 μ L red blood cell suspensions are mixed and added
Enter into 96 orifice plates, the final concentration of polypeptide be respectively 64 μ g/mL, 32 μ g/mL, 16 μ g/mL, 8 μ g/mL, 4 μ g/mL, 2 μ g/mL,
1 μ g/mL and 0.5 μ g/mL；Negative control group is the red blood cell to be suspended with sterile physiological salt, and positive control is with containing 1%
The red blood cell that the sterile physiological salt of TritonX-100 suspends；Sample is put into constant-temperature table, 100rpm is incubated at 37 DEG C
60min；Sample is centrifuged 5min with 3000rpm in room temperature, takes 100 μ L supernatants to be transferred in another 96 orifice plate in every hole；With
All-wave length microplate reader measures the absorbance at 490nm wavelength, and is calculated by the following formula percentage of hemolysis, and H is in formula
Absorbance at 490nm wavelength: hemolysis rate (%)=(Hsample-Hnegative)/(Hpositive-Hnegative) × 100%, such as Fig. 5
It is shown.
The result shows that of the present invention there are antimicrobial and immunological regulation bifunctional polypeptides to be up to 64 μ in final concentration
Do not occur apparent haemocylolysis (hemolysis rate < 5%) when g/mL yet.
Embodiment five has activity of the antimicrobial function peptidomimetic in various concentration salt environment
By it is of the present invention from disease fungus have antimicrobial function peptidomimetic use respectively 0mM, 75 mM,
The NaCl solution dissolution of 150mM, 300mM, 450mM and 600mM sterilizing, and it is made into the peptidomimetic solution that concentration is 128 μ g/mL.It presses
According to the method in embodiment 1, N.gonorrhoeae ATCC49226 and C.albicans ATCC10231 the measurement present invention are chosen
It is described that there is antibacterium and antimycotic activity of the antimicrobial function peptidomimetic in various concentration from disease fungus, as a result such as
Shown in Fig. 6.
It is in Fig. 6 statistics indicate that, it is of the present invention to resist micro- life from disease fungus peptidomimetic with the raising of salinity
Object activity reduces, but entire change is little.In salinity environment in physiological conditions (about 150 mM), to what is measured
The MIC value of two kinds of reference cultures and the result in embodiment 1 are unchanged, and the peptidomimetic is antimicrobial under the conditions of illustrating physiological salt concentration
It is activity stabilized.
Embodiment six has activity of the antimicrobial function peptidomimetic in different hot environments
There is the sterilizing PBS solution dissolution of the peptidomimetic of antimicrobial function from disease fungus by of the present invention,
And it is made into the peptidomimetic solution that concentration is 128 μ g/mL, respectively in 25 DEG C, 35 DEG C, 45 DEG C, 55 DEG C, 65 DEG C and 75 DEG C isoperibols
It is middle to be incubated for 2 hours.According to the method in embodiment 1, N. gonorrhoeae ATCC49226 and C.albicans is chosen
ATCC10231 measurement is of the present invention from disease fungus to there is antimicrobial function peptidomimetic to be incubated under different temperatures environment
Antibacterium and antimycotic activity afterwards, as a result as shown in Figure 7.
It is in Fig. 7 statistics indicate that, MIC value of the peptidomimetic to the two kinds of reference cultures measured within the scope of 25 DEG C~55 DEG C
It is unchanged with the result in embodiment 1.It is of the present invention micro- from resisting for disease fungus peptidomimetic but as temperature continues to increase
Bioactivity decreases.The peptidomimetic is to the MIC value of the above-mentioned two plants of bacterium of inhibition respectively than reality after being incubated for 2 hours in 65 DEG C of environment
Apply 2 times and 2 times that the MIC value in example 1 increases respectively；The peptidomimetic is to inhibition above-mentioned two after being incubated for 1 hour in 75 DEG C of environment
Increased respectively than the MIC value in embodiment 1 respectively 4 times and 2 times of MIC value of strain bacterium.The above results show institute of the present invention
The peptidomimetic being related to still has stronger antimicrobial acivity in the environment of higher temperature.
Embodiment seven has activity of the antimicrobial function peptidomimetic in different pH environment
From disease fungus there is the peptidomimetic of antimicrobial function to use HCl or NaOH tune respectively for of the present invention
PH value is saved, the sterilizing PBS solution that pH value is respectively 3,5,7,9,11 is made into, is dissolved with the sterilizing PBS solution of different pH values,
And it is made into the peptidomimetic solution that concentration is 128 μ g/mL.According to the method in embodiment 1, N.gonorrhoeae is chosen
ATCC49226 and C.albicans ATCC10231 measurement is of the present invention quasi- with antimicrobial function from disease fungus
Antibacterium and antimycotic activity of the peptide under different pH values pH environment, as a result as shown in Figure 8.
It is in Fig. 8 statistics indicate that, in ± 2 range of physiological pH (physiological pH is about 7.4) its to the two kinds of standards measured
The MIC value of bacterial strain and the result in embodiment 1 are unchanged.But with the reduction of pH value (pH=3) or (pH=11) is increased, this
The invention antimicrobial acivity from disease fungus peptidomimetic decreases.Wherein the peptidomimetic is more sensitive to acidic environment,
Inhibit the MIC values of above-mentioned two plants of bacterium respectively than 8 times of pH=7 duration and 4 times when pH=3, when pH=11 inhibits above-mentioned two plants of bacterium
MIC value respectively than 4 times of pH=7 duration and 2 times.The above results show peptidomimetic according to the present invention in acid and alkalinity
There is certain stability in environment.
Eight tablet of embodiment
Take peptidomimetic 0.15g, starch 3g, the dextrin 3g from disease fungus with antimicrobial function of the present invention
Mixing, the pharmaceutical grade polyvinylpyrrolidone (PVP) that mass concentration is 35% is adhesive, granulation, and tabletting both obtains tablet.
Nine injection freeze-dried powder of embodiment
Peptidomimetic 1.5g, the mannitol 22.5g from disease fungus with antimicrobial function of the present invention is taken,
It is placed in container, adds appropriate PBS buffer solution (0.1M, pH7.4) to dissolve, inject water to 400 mL, shake up, add 8~10g needle
It with active carbon, is stirred at room temperature 30~60 minutes, coarse filtration, with 0.22 μm of membrane filtration degerming, packing, every bottle of 1mL, using quick-frozen
Method cools down 10~15 DEG C per minute, is cooled to -45 DEG C, maintains 2.5h, vacuumizes, under vacuum conditions slowly heating, heating
2~10 DEG C per hour of speed, temperature stops heating when rising to 30 DEG C, takes out, seals after temperature near room temperature, must both freeze
Dry powder injection.
Claims (7)
1. one kind has antimicrobial function peptidomimetic from disease fungus, it is characterised in that: the peptidomimetic has antimicrobial work
Property, molecular structure are as follows:
2. a kind of prepare the method from disease fungus as described in claim 1 with antimicrobial function peptidomimetic, feature
It is: peptide fragment IIGGRC is prepared by conventional Solid-phase synthesis peptides technology first, then passes through c-terminus cysteine side chain
The sulfydryl of sulfydryl and small molecule compound 3- sulfydryl -5- methyl-1,2,4- triazoles forms covalent disulfide bonds, makes finally by half
Standby reversed high-efficient liquid phase chromatogram purification, finally obtains complete peptidomimetic molecule.
3. it is a kind of as described in claim 1 from disease fungus have antimicrobial function peptidomimetic antimicrobial agents or/
With the application in daily necessities, it is characterised in that: the antimicrobial agents are injection, tablet, injection sterile powder, injection
With aseptic powdery, pulvis, granule, capsule, oral solution, paste or creme；The drug passes through injection, oral, collunarium, drop
Eye, the method physically or chemically mediated import muscle, endothelium, subcutaneous, vein or mucosal tissue；The daily necessities be hand cleanser,
Facial cleanser, shampoo, bath foam, Feminine care lotion, sanitary napkin, panty liner, diaper, adult nursing diaper, mouthwash,
Toothpaste, perfumed soap, liquid detergent, thimerosal, toilet cleaner, disinfected paper napkin, dressing, bandage or feed.
4. have antimicrobial function peptidomimetic in antimicrobial agents from disease fungus according to claim 3 or/and
Application in daily necessities, it is characterised in that: the microorganism is the Gram positive bacteria strain being clinically separated and/or clinical point
From fungal bacterial strain.
5. have antimicrobial function peptidomimetic in antimicrobial agents from disease fungus according to claim 4 or/and
Application in daily necessities, it is characterised in that: the fungi is Mycotoruloides and Cryptococcus.
6. a kind of composition, it is characterised in that: it includes that the disease fungus described in claim 1 that derives from has antimicrobial function
Energy peptidomimetic also includes the acceptable salt of the peptidomimetic and/or hydrate and/or solvate and/or acceptable carrier.
7. a kind of application of composition as claimed in claim 6 in preparation antibacterials and/or daily necessities, feature exist
In: the antibacterials are injection, tablet, injection sterile powder, injection sterile powder, pulvis, granule, capsule
Agent, oral solution, paste or creme；The antibacterials or composition are by injecting, taking orally, collunarium, eye drip, be physically or chemically situated between
The method led imports muscle, endothelium, subcutaneous, vein or mucosal tissue；The daily necessities are hand cleanser, facial cleanser, shampoo, wash one's hair
Bath foam, Feminine care lotion, sanitary napkin, panty liner, diaper, adult nursing diaper, mouthwash, toothpaste, perfumed soap, laundry
Liquid, thimerosal, toilet cleaner, disinfected paper napkin, dressing, bandage or feed.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113583098A (en) * | 2021-07-09 | 2021-11-02 | 武汉大学 | Cyclic peptide mimetic from fungus and preparation method and application thereof |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113583098A (en) * | 2021-07-09 | 2021-11-02 | 武汉大学 | Cyclic peptide mimetic from fungus and preparation method and application thereof |
| CN113583098B (en) * | 2021-07-09 | 2023-03-14 | 武汉大学 | Cyclic peptide mimetic from fungus and preparation method and application thereof |
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| CN110283237B (en) | 2020-11-17 |
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