CN110283237B

CN110283237B - Peptide mimic with antimicrobial function from pathogenic fungi, and preparation method, composition and application thereof

Info

Publication number: CN110283237B
Application number: CN201910491963.9A
Authority: CN
Inventors: 沈秉正; 高翔; 高越; 喻研; 曹志贱; 吕志华
Original assignee: Wuhan University WHU
Current assignee: Wuhan University WHU
Priority date: 2019-06-06
Filing date: 2019-06-06
Publication date: 2020-11-17
Anticipated expiration: 2039-06-06
Also published as: CN110283237A

Abstract

The invention discloses a peptide mimic with an antimicrobial function derived from pathogenic fungi, and a preparation method, a composition and application thereof. The peptide-like peptide is prepared into a peptide segment IIGGRC by a conventional polypeptide solid-phase synthesis technology, a covalent disulfide bond is formed by a sulfydryl of a carboxyl-terminal cysteine side chain and a sulfydryl of a small molecular compound 3-sulfydryl-5-methyl-1, 2, 4-triazole, and the peptide-like peptide is obtained by purification through semi-preparative reverse high performance liquid chromatography. The research result of pharmacological experiments shows that the peptide mimic has good inhibition effect on gram-positive bacteria standard strains and clinically separated Methicillin-resistant S.aureus. In addition to bacteria, it also has a certain inhibitory effect on the fungal standard strains Candida albicans ATCC10231, C.krusei ATCC 6258 and Cryptococcus neoformans ATCC 34877 and pathogenic fungi isolated clinically. Meanwhile, the peptide mimic shows low hemolytic activity, high temperature environment and acid-base environment stability, is convenient and fast to prepare artificially, has definite pharmacological activity, and has further development value and industrialization potential.

Description

Peptide mimic with antimicrobial function from pathogenic fungi, and preparation method, composition and application thereof

Technical Field

The invention belongs to the field of biomedicine, and particularly relates to a peptide mimic with an antimicrobial function derived from pathogenic fungi, a preparation method, a composition and application thereof, namely the peptide mimic with the antimicrobial function derived from the pathogenic fungi, a preparation method of the peptide mimic, a medicament or/and a composition containing the peptide mimic, and application of the peptide mimic as an antimicrobial agent.

Background

The increase in resistance of pathogenic microorganisms to drugs has become a serious problem. Around 700000 people die annually from drug-resistant microbial infections worldwide. Epidemiological investigations have shown that multidrug-resistant bacteria such as Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii, methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant MRSA, penicillin-resistant Streptococcus pneumoniae (PRSP), vancomycin-resistant enterococcus (VRE) and extensive drug-resistant (XDR) Mycobacterium tuberculosis are increasingly resistant to drugs. The co-morbidity with humans and animals and the resistance to bacteria (salmonella, campylobacter, escherichia coli and enterococcus) contained in feces are also increasing.

The currently accepted drug resistance mechanisms of bacteria are mainly: (ii) producing an enzyme capable of inactivating the antimicrobial drug. The enzyme is a superfamily of hydrolases, which mainly hydrolyze antibiotics containing beta-lactam ring structures to inactivate the antibiotics, and comprises penicillins, cephalosporins, monocyclic beta-lactams and carbapenems antibiotics which are commonly used in clinical treatment. The gene encoding the drug-resistant enzyme may be naturally present in the strain chromosome (referred to as intrinsic drug resistance, e.g., the gram-negative bacilli present in the gut have natural resistance to penicillin); it can also be plasmid-mediated or integrated into the chromosome of the strain under the induction of antimicrobial drugs (called acquired resistance). ② modifying the target of the antimicrobial drug action. Alteration of the antimicrobial drug target is a relatively common mechanism of resistance. Typically, the alteration of the target is derived from the strain acquiring the gene of interest or spontaneous mutation of the gene as well as selective mutation in the presence of an antimicrobial drug. For example, mutations in RNA polymerase and DNA gyrase may result in strains that acquire resistance to rifamycin and quinolone antimicrobials, respectively. And thirdly, generating an efflux pump to pump out the antimicrobial drug. Infections caused by gram-negative bacteria are difficult to treat because they are inherently resistant to many antibiotics. And resistance is obtained independently of horizontal gene transfer and mutation. The double membrane structure of gram-negative bacteria and the resulting efflux pumps can pump out the antimicrobial drug, thereby reducing the intracellular concentration of the drug. Gram-negative bacteria express a number of efflux pumps in their membranes that transport a wide variety of molecules out of the bacterial cell, some of which can transport antibiotics. Pumping out the antibiotic from the bacteria reduces the intracellular drug concentration, allowing the bacteria to survive higher external concentrations of the antimicrobial drug, resulting in their resistance to the antimicrobial drug. And fourthly, changing the permeability of the bacterial cytoplasmic membrane. The low permeability of the outer cell wall of gram-negative bacteria has been shown to be effective at preventing the antimicrobial drug from acting at low antibiotic concentrations. The key mechanisms by which antibiotics enter the thallus via the outer membrane include porins (Omps), which are responsible for the diffusion of hydrophilic antibiotics, and lipid-mediated pathways for hydrophobic antibiotics, many strains are resistant due to changes in the lipid or protein composition of the outer membrane, suggesting that the outer membrane barrier plays an important role in whether a strain is sensitive to antibiotics, and any structural changes in the outer membrane protein can significantly affect its resistance to antibiotics. Furthermore, when the permeability barrier has a synergistic effect with beta-lactamases in the periplasmic space, the resistance of the strain will become more severe, possibly leading to resistance of the pathogenic bacteria to third generation cephalosporins.

In addition to bacteria, resistance to pathogenic fungi, which have been overlooked, is becoming increasingly important and beginning to be of concern (Science 2018,360: 739-. The rapid emergence of multi-drug resistant pathogenic fungi and the widespread spread of antifungal drug resistant strains also pose significant threats to human health. The impact of fungi on human health is currently rising and the global mortality rate of fungal diseases is now surpassing that of malaria or breast cancer and comparable to that of tuberculosis and aids (Sci trans Med 2012,4(165):165rv 13). Although they are ubiquitous, fungal infections have been largely overlooked in the past relative to other types of infectious diseases.

Human beings first used antifungal drugs, nystatin and polyene antifungal drugs, and were discovered in the 50 s of the 20 th century. Today, systemic fungal bacteriostats and fungicides are used as first line therapeutics for the treatment of diseases associated with fungal infections in humans. However, many of the drugs available only transiently inhibit their proliferation due to the rapid development of fungal resistance to the drugs. Fungal genes are highly plastic and can be rapidly propagated. These properties allow rapid mutant generation under drug-selective pressure.

The first line drugs currently available for the treatment of fungal infections in humans are mainly of four classes: 1. a polyene hydrocarbon. Such as amphotericin B) disrupt the structure of cell membranes by inhibiting fungal membrane sterols (ergosterol); 2. a pyrimidine analog. Such as 5-fluorocytosine (5-FC) blocks pyrimidine metabolism and DNA synthesis; 3. azoles, also the most widely used fungicides. Blocking ergosterol biosynthesis by inhibiting lanosterol 14-alpha-demethylase; 4. echinocandin, the latest antifungal drug. By inhibiting (1-3) -beta-D-glucan synthase and disrupting cell wall biosynthesis.

With the long-term and wide use of the above drugs in clinical antifungal therapy, new multidrug-resistant pathogenic fungi have emerged. Multidrug resistant Candida (Candida auris) was first discovered in 2009 in a patient from japanese ear infections, was resistant to all clinical antifungals (Clin infection Dis 2017,64(2): 134-. There are studies showing that the major mycosis pathogen Candida glabrata (Candida glabrata) is resistant to the emergence of this drug, also mainly due to the frequent use of echinocandins and azole antifungals as preventive drugs (Clin infection Dis 2013,56(12): 1724-1732.).

In view of the above-mentioned resistance to bacterial and fungal agents, it is necessary to avoid the existing mechanisms of action of drugs to develop new candidate antifungal active molecules, which can inhibit or kill pathogenic fungi. Anti-microbial peptides (AMPs) are a class of molecules with sequence and structural diversity and possess a wide range of antimicrobial activities, such as antibacterial, antifungal and antiviral effects. More than 3000 antimicrobial polypeptides have been found, but many of the polypeptides with better activity are not valuable for drug development due to the defects in the nature of the native polypeptide (e.g., hemolysis, unstable environment with high salt concentration, etc.).

Disclosure of Invention

Aiming at the defects of the prior art, the invention provides a peptide mimic which is derived from pathogenic fungi and has antimicrobial function, a preparation method, a composition and application thereof. The polypeptide has the activity of resisting gram-positive bacteria standard strains, clinical drug-resistant bacteria and fungi standard strains at the concentration level of mu g/mL.

In order to achieve the above objects, according to one aspect of the present invention, there is provided a peptidomimetic having an antimicrobial function derived from a pathogenic fungus, wherein the peptidomimetic is derived from a human pathogenic fungus trichophyton interdigital t.interdigitale protein (GenBank No. EZF36158.1), and has the following sequence:

the peptide segment IIGGRC (amino acid sequence in a square frame) in the full-length protein forms covalent disulfide bonds with the sulfhydryl of a small molecular compound 3-sulfhydryl-5-methyl-1, 2, 4-triazole through the sulfhydryl of a carboxyl-terminal cysteine side chain to be connected, namely, the disulfide bonds are used as a linker to be spliced into molecules with a brand new structure, and the molecular structure is as follows:

the peptidomimetic molecule can be obtained by separating and purifying by reversed phase-high performance liquid chromatography (RP-HPLC), has the theoretical molecular weight of 730.34, has antibacterial and antifungal activity, and is also effective on clinically separated drug-resistant bacteria. The peptide mimic has the advantages of simple preparation method and technical route, strong stability, easy control of product quality and the like.

In another aspect, the present invention provides a method for preparing a peptidomimetic having an antimicrobial function from a pathogenic fungus, comprising the steps of: firstly, preparing a peptide segment IIGGRC by a conventional polypeptide solid phase synthesis technology, forming a covalent disulfide bond by a sulfydryl of a carboxyl-terminal cysteine side chain and a sulfydryl of a small molecular compound 3-sulfydryl-5-methyl-1, 2, 4-triazole, and finally purifying by semi-preparative reverse high performance liquid chromatography to finally obtain a complete peptide-like molecule.

The invention also provides application of the peptide mimic with the antimicrobial function from pathogenic fungi in antimicrobial drugs or/and daily necessities, which is characterized in that: the antimicrobial drug is injection, tablet, sterile powder for injection, powder, granule, capsule, oral liquid, ointment or cream; the drug is introduced into muscle, endothelium, subcutaneous, vein or mucosa tissue by injection, oral administration, nasal drop, eye drop, physical or chemical mediated method; the daily necessities are hand sanitizer, facial cleanser, shampoo, bath foam, feminine care lotion, sanitary towel, sanitary pad, baby diaper, adult care urine pad, mouthwash, toothpaste, perfumed soap, laundry detergent, disinfectant, toilet cleaner, disinfectant paper towel, dressing, bandage or feed.

Further, the microorganism is a clinically isolated gram-positive bacterial strain and/or a clinically isolated fungal strain.

Further, the fungi are candida and cryptococcus.

In another aspect, the present invention provides a composition characterized in that: it comprises the above-mentioned peptide mimic with antimicrobial function derived from pathogenic fungi, or also comprises acceptable salt and/or hydrate and/or solvate and/or acceptable carrier of the peptide mimic.

The invention also provides an application of the composition in preparing antibacterial drugs and/or daily necessities, which is characterized in that: the antibacterial drug is injection, tablet, injectable sterile powder, granule, capsule, oral liquid, paste or cream; the antibacterial drug or the composition is introduced into muscle, endothelium, subcutaneous, vein or mucosa tissues by injection, oral administration, nasal drop, eye drop, physical or chemical mediated method; the daily necessities are hand sanitizer, facial cleanser, shampoo, bath foam, feminine care lotion, sanitary towel, sanitary pad, baby diaper, adult care urine pad, mouthwash, toothpaste, perfumed soap, laundry detergent, disinfectant, toilet cleaner, disinfectant paper towel, dressing, bandage or feed.

The hemolysis experiment result shows that the peptoid with the antimicrobial function has hemolysis rate of less than 5 percent when the concentration is as high as 64 mu g/mL (figure 5) and has higher safety.

The peptide mimic or/and the composition with the antimicrobial function can be prepared into various dosage forms such as injection, tablets, sterile powder for injection, powder, granules, capsules, oral liquid, ointment, cream and the like according to a conventional preparation method. The peptide mimetic or composition having antimicrobial function can be introduced into muscle, endothelium, subcutaneous, vein or mucosal tissue by injection, oral administration, nasal drop, eye drop, physical or chemical mediated method, or can be mixed or coated with other substances and then introduced into the body.

The invention has the following advantages and effects: the peptide mimics with the antimicrobial (bacteria and fungi) function have good gram-positive bacteria resistance (including clinically separated drug-resistant bacteria), can inhibit fungi of candida and cryptococcus, and are high in safety; the artificial preparation of the peptide mimic is convenient, the production cost is low, and the peptide mimic is suitable for industrial large-scale production; therefore, the compound has good development prospect in the field of treating bacterial and fungal infection; the peptide mimic with the antimicrobial function can provide a new choice for developing new antibacterial and antifungal active substances and compound preparations thereof.

Drawings

FIG. 1 is an HPLC chart of IIGGRC, an intermediate of natural polypeptide peptide fragments prepared by the present invention.

FIG. 2 is a mass spectrum of IIGGRC, an intermediate of natural polypeptide peptide fragment prepared by the present invention.

FIG. 3 is an HPLC chart of a peptide mimic with antimicrobial function prepared according to the present invention.

FIG. 4 is a mass spectrum of a peptide mimic with antimicrobial function prepared according to the present invention.

FIG. 5 shows the results of the hemolytic activity of the peptide mimics having antimicrobial function according to the present invention.

FIG. 6 shows the results of the activity of the peptide mimics with antimicrobial function in salt environments with different concentrations.

FIG. 7 shows the results of the activity of the peptide mimics with antimicrobial function of the invention in different temperature environments.

FIG. 8 shows the results of the activities of the antimicrobial peptide mimics of the present invention in different pH environments.

Detailed Description

The invention is described in further detail below with reference to the figures and specific examples.

Example preparation of intermediate IIGGRC of peptide fragment of native polypeptide

In the synthesis of the polypeptide peptide segment intermediate, 9-Fluorenylmethoxycarbonyl (FMOC) is used for protecting an amino end group, 4-methyldiphenylmethylamine resin (4-methyl-benzylmethylamine resin HCl, MBHA resin) is used as a solid phase carrier, HOBt/DCC is used as a condensing agent, and a peptide chain is extended from a carboxyl terminal to an amino terminal. It was cleaved from the MBHA resin with a mixture of 94% trifluoroacetic acid, 3% water and 3% Triisopropylsilane (TIA), all in mass%. The ether was precipitated repeatedly and purified by preparative RP-HPLC. Use of C₁₈Reverse phase preparation of the column (20 mm. times.250 mm, 5 μm); mobile phase: 1.5 per mill trifluoroacetic acid and 0 to 50 percent (volume percentage) acetonitrile are taken as mobile phases, and gradient elution is carried out at the flow rate of 0.5 mL/min. RP-HPLC detection shows that Retention Time (RT) is 8.14min, and the purity is high>95% (fig. 1), and is ready for use after lyophilization. Identified by ESI-QTOF-MS, the molecular weight of the polypeptide intermediate is consistent with the corresponding theoretical molecular weight of the polypeptide intermediate to be prepared, and M/z is 618.6[ M + H ]]⁺，m/z 309.8[M+2H]²⁺(FIG. 2).

ExamplesIIPreparation of peptide mimics with antimicrobial function from pathogenic fungi

The intermediate peptide segment IIGGRC prepared in the example 1 and a small molecular compound 3-mercapto-5-methyl-1, 2, 4-triazole are used as raw materials. Taking 1mg of intermediate peptide IIGGRC and 0.2mg of 3-mercapto-5-methyl-1, 2, 4-triazole, dissolving the intermediate peptide IIGGRC and the 3-mercapto-5-methyl-1, 2, 4-triazole by using 1mL of Tris-HCl buffer solution with the concentration of 0.15M (the pH value is 8.4), and accelerating the dissolution of the intermediate peptide and the small molecular compound by using a liquid transfer gun to gently suck the solution. The EP tube containing the polypeptide solution was incubated in a constant temperature shaker at 30. + -. 2 ℃ and 80rpm for about 36 h. Centrifuging at room temperature 10000rmp for 4min, and collecting supernatant. Purification by preparative RP-HPLC. Use of C₁₈Reverse phase preparation of the column (20 mm. times.250 mm, 5 μm); mobile phase: 0.15 percent of trifluoroacetic acid and 0 to 55 percent (volume percentage) of acetonitrile are taken as mobile phases, and gradient elution is carried out at the flow rate of 0.5 mL/min. RP-HPLC detection shows that RT is 10.24min, and its purity is>95％(FIG. 3), lyophilized for use. Identified by ESI-QTOF-MS, the molecular weight of the peptide is consistent with the corresponding theoretical molecular weight of the quasi-peptide, M/z 731.2[ M + H ]]⁺，m/z 366.4[M+2H]²⁺(FIG. 4).

EXAMPLE III determination of antimicrobial Activity derived from fungal pathogenic peptidomimetics

Has good inhibitory effect on gram-positive bacteria standard strain (such as Staphylococcus aureus ATCC25922, Neisseria gonorrhoeae ATCC49226) and clinically-isolated Methicillin-resistant Staphylococcus aureus (Methicillin-resistant S. aureus). In addition to bacteria, it also has strong inhibitory activity against the fungal standard strains Candida albicans ATCC10231, C.kruseiATCC 6258 and Cryptococcus neoformans ATCC 34877.

The standard strains used were: staphyloccus aureus ATCC25922, Neissria gonorreea ATCC49226, Candida albicans ATCC10231, C.krusei ATCC 6258 and Cryptococcus neoformans ATCC 34877 were purchased from the Chinese Type Culture Collection (China Center of Type Culture Collection, CCTCC). Clinically isolated strains: aureus, Methicillin-resistant s.aureus, Candida albicans were provided by the national hospital inspection medicine center of wuhan university. The antimicrobial activity derived from the pathogenic fungal peptidomimetics is characterized by the MIC value by determining the Minimum Inhibitory Concentration (MIC) using a two-fold dilution method.

The specific method comprises the following steps:

respectively inoculating bacteria to be detected into sterilized Luria-Bertani (LB) solid culture medium plates by using a three-region streaking method; the fungus to be tested was inoculated into sterilized Yeast Extract Peptone Dextrose (YPD) solid medium plates by three-zone streaking method. Inversely culturing the bacteria in a constant temperature incubator at 37 ℃ for 14 hours; the fungus is inversely cultured for 48-96 hours in a constant temperature incubator at 30 ℃. Single colonies were picked with an inoculating loop and transferred to a sterilized liquid medium (LB liquid medium for bacteria; YPD liquid medium for fungi), respectively, and cultured to logarithmic phase under conditions of 37 deg.C (bacteria) or 30 deg.C (fungi) and 150rpm with shaking. Measuring absorbance (OD) of bacteria solution at 600nm wavelength with ultraviolet spectrophotometer₆₀₀) According to 1OD 1 × 10⁹The conversion relation of CFU/mL is that the culture mediums of the standard strain and the clinical drug-resistant strain are respectively diluted to (1-2) multiplied by 10⁵CFU/mL。

Firstly, 100 mu L of sterilized liquid LB culture medium (bacteria) or liquid YPD culture medium (fungi) is added into a sterile 96-well plate; then adding 100 mu L of peptoid solution with the concentration of 256 mu g/mL dissolved by the sterilized LB culture medium into the 1 st hole, uniformly mixing, adding 100 mu L into the 2 nd hole, finally sucking 100 mu L from the 9 th hole, discarding, and sequentially diluting twice; finally, adding the solution with the concentration of (1-2) multiplied by 10 into each hole⁵The diluted bacterial solution of CFU/mL is 100 mu L, and the mixture is mixed evenly, and the final concentration of the peptide mimic in each hole is 128 mu g/mL, 64 mu g/mL, 32 mu g/mL, 16 mu g/mL, 8 mu g/mL, 4 mu g/mL, 2 mu g/mL, 1 mu g/mL, 0.5 mu g/mL. Wells without peptoids added served as negative controls.

Shake culturing the above groups at 37 ℃ for 12-14 hours (bacteria); shake-culturing at 30 ℃ for 24-48 hours, and measuring the absorbance value at the wavelength of 600 nm. The Minimum Inhibitory Concentration (MIC) is determined such that no bacterial or fungal growth (OD) is detected₆₀₀≦ 0.05) for the lowest final concentration of polypeptide in the wells. The bacteria solution in the negative control well is turbid, and the absorbance OD at 600nm is₆₀₀Is greater than 2.5. The results of the antimicrobial activity of the bifunctional polypeptides having antimicrobial and immunomodulatory properties described in this patent are shown in Table 1.

TABLE 1 antimicrobial Activity of polypeptides with antimicrobial and immunomodulatory Functions

As can be seen from the results in Table 1, the peptidomimetics derived from pathogenic fungi according to the present invention have more desirable antibacterial and antifungal activities. For all standard strains, including bacteria and fungi, the MIC is 16. mu.g/mL or less. The clinical drug-resistant bacteria MRSA also has a strong inhibitory effect MIC value of 32 mug/mL, and the clinical separated Candida fungus C.albicans has a relatively weak inhibitory effect MIC value of 64 mug/mL.

EXAMPLE four determination of hemolytic Activity of peptidomimetics having antimicrobial function derived from pathogenic fungi

Obtaining fresh blood from a healthy donor, separating human red blood cells to prepare a suspension, and determining the hemolytic activity of the bifunctional polypeptide by using sterilized physiological saline as a negative control and using 1% Triton X-100 as a positive control.

The method comprises the following specific steps: collecting whole blood with EDTA anticoagulation tube, slightly inverting to fully anticoagulate blood, centrifuging at room temperature of 600rpm for 4min, discarding upper layer plasma and retaining lower layer red blood cells; adding 4 times volume of sterilized normal saline, slightly inverting to make the centrifuge tube suspend the bottom red blood cells, centrifuging at room temperature at 600rpm for 4min, discarding the supernatant, retaining the precipitated red blood cells, repeating the operation for 3 times until the supernatant is colorless; preparing erythrocyte into 2% (v/v) cell suspension by using sterilized normal saline; dissolving the peptide mimic with sterilized normal saline to prepare solutions with the concentrations of 128 mug/mL, 64 mug/mL, 32 mug/mL, 16 mug/mL, 8 mug/mL, 4 mug/mL, 2 mug/mL and 1 mug/mL; mixing 100 μ L of the bifunctional polypeptide solution and 100 μ L of the erythrocyte suspension and adding to a 96-well plate, the final concentration of the polypeptide being 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, respectively; the negative control group is red blood cells suspended by sterilized physiological salt, and the positive control group is red blood cells suspended by sterilized physiological salt containing 1% TritonX-100; putting the sample into a constant temperature shaking table, and incubating for 60min at 37 ℃ and 100 rpm; the samples were centrifuged at 3000rpm for 5min at room temperature, and 100. mu.L of supernatant per well was transferred to another 96 well plate; the absorbance at 490nm was measured with a full wavelength microplate reader and the percentage of hemolysis was calculated by the following formula, where H is the absorbance at 490 nm: hemolysis rate (%) - (H)_sample-H_negative)/(H_positive-H_negative) X 100%, as shown in FIG. 5.

The results show that the polypeptide with the functions of resisting the microorganisms and regulating the immunity still has no obvious hemolysis (hemolysis rate is less than 5%) when the final concentration is as high as 64 mu g/mL.

EXAMPLE five Activity of antimicrobial functional peptidomimetics in salt environments of varying concentrations

The peptide mimics which are derived from pathogenic fungi and have the antimicrobial function are dissolved by 0mM, 75mM, 150mM, 300mM, 450mM and 600mM sterilized NaCl solutions respectively to prepare peptide mimic solutions with the concentration of 128 mu g/mL. The antibacterial and antifungal activities of the antimicrobial functional peptidomimetics derived from pathogenic fungi of the present invention at various concentrations were determined according to the method of example 1 using N.gonorrhoeae ATCC49226 and C.albicans ATCC10231, and the results are shown in FIG. 6.

The data in FIG. 6 show that the antimicrobial activity derived from a pathogenic fungal peptidomimetic according to the invention decreases with increasing salt concentration, but does not change much overall. The MIC values of the two standard strains measured in the saline concentration environment (about 150mM) under physiological conditions were unchanged from the results in example 1, indicating that the peptidomimetic antimicrobial activity is stable under physiological saline concentration conditions.

Example six Activity of peptidomimetics with antimicrobial function in various high temperature environments

The peptide mimic which is derived from pathogenic fungi and has the antimicrobial function is dissolved by a sterilized PBS solution to prepare a peptide mimic solution with the concentration of 128 mug/mL, and the peptide mimic solution is respectively incubated for 2 hours in constant temperature environments of 25 ℃, 35 ℃, 45 ℃, 55 ℃, 65 ℃ and 75 ℃. The antibacterial and antifungal activities of the pathogenic fungus-derived antimicrobial functional peptidomimetics of the present invention after incubation under different temperature environments were determined according to the method of example 1 using N.gonorrhoeae ATCC49226 and C.albicans ATCC10231, and the results are shown in FIG. 7.

The data in FIG. 7 show that the MIC values of the peptidomimetic for the two standard strains determined in the 25 ℃ to 55 ℃ range were unchanged from the results in example 1. However, as the temperature continues to increase, the antimicrobial activity of the peptides derived from pathogenic fungi of the present invention decreases. After 2 hours of incubation in the environment of 65 ℃, the MIC values of the peptide mimics for inhibiting the two bacteria are respectively increased by 2 times and 2 times compared with the MIC values in example 1; after incubation for 1 hour at 75 ℃, the MIC values of the peptide mimics for inhibiting the two bacteria are respectively increased by 4 times and 2 times compared with the MIC values in example 1. The above results indicate that the peptide mimics of the present invention have strong antimicrobial activity in higher temperature environments.

EXAMPLE seven Activity of antimicrobial functional peptidomimetics in different pH environments

The peptoid with the antimicrobial function from pathogenic fungi is respectively adjusted in pH value by HCl or NaOH to prepare sterilized PBS solutions with pH values of 3,5, 7, 9 and 11, and the sterilization PBS solutions with different pH values are used for dissolving to prepare the peptoid solution with the concentration of 128 mug/mL. The antibacterial and antifungal activities of the antimicrobial functional peptidomimetics derived from pathogenic fungi of the present invention under various pH values were determined according to the method of example 1 using N.gonorrhoeae ATCC49226 and C.albicans ATCC10231, and the results are shown in FIG. 8.

The data in FIG. 8 show that there is no change in the measured MIC values for the two standard strains from the results in example 1 over a physiological pH range. + -. 2 (physiological pH of about 7.4). However, the antimicrobial activity of the peptides derived from pathogenic fungi according to the invention decreases with decreasing pH (pH 3) or increasing pH (pH 11). Wherein the peptide mimic is more sensitive to acidic environment, and inhibits MIC values of the two bacteria respectively 8 times and 4 times of pH7 at pH 3, and inhibits MIC values of the two bacteria respectively 4 times and 2 times of pH7 at pH 11. The above results show that the peptide mimics have certain stability in acidic and alkaline environments.

EXAMPLE eight tablets

0.15g of the peptoid with the antimicrobial function from the pathogenic fungi, 3g of starch and 3g of dextrin are mixed, medicinal grade polyvinylpyrrolidone (PVP) with the mass concentration of 35% is taken as a binding agent, and the mixture is granulated and tabletted to obtain the tablet.

EXAMPLE nine lyophilized powder for injection

Taking 1.5g of the peptide mimic which is derived from pathogenic fungi and has an antimicrobial function and 22.5g of mannitol, putting the peptide mimic in a container, adding a proper amount of PBS buffer solution (0.1M, pH7.4) for dissolving, adding water for injection to 400mL, shaking up, adding 8-10 g of active carbon for injection, stirring for 30-60 minutes at room temperature, roughly filtering, filtering and sterilizing by using a 0.22 mu M filter membrane, subpackaging, taking 1mL of each bottle, adopting a quick-freezing method, cooling to 10-15 ℃ per minute, cooling to-45 ℃, maintaining for 2.5 hours, vacuumizing, slowly heating at a vacuum state, stopping heating at a speed of 2-10 ℃ per hour, heating to 30 ℃, taking out after the temperature approaches the room temperature, capping and sealing to obtain the freeze-dried powder injection.

Claims

1. A peptide mimic with antimicrobial activity derived from a pathogenic fungus, wherein: the peptide mimic has antimicrobial activity, and the molecular structure of the peptide mimic is as follows:

2. a method for preparing a peptidomimetic having antimicrobial function from a pathogenic fungus according to claim 1, wherein the peptidomimetic comprises: firstly, preparing a peptide segment IIGGRC by a conventional polypeptide solid phase synthesis technology, forming a covalent disulfide bond by a sulfydryl of a carboxyl-terminal cysteine side chain and a sulfydryl of a small molecular compound 3-sulfydryl-5-methyl-1, 2, 4-triazole, and finally purifying by semi-preparative reverse high performance liquid chromatography to finally obtain a complete peptide-like molecule.

3. The use of the peptide mimics with antimicrobial activity derived from pathogenic fungi of claim 1 in the preparation of antimicrobial drugs and/or daily necessities, wherein the peptide mimics with antimicrobial activity are as follows: the antimicrobial drug is injection, tablet, sterile powder for injection, powder, granule, capsule, oral liquid, ointment or cream; the drug is introduced into muscle, endothelium, subcutaneous, vein or mucosa tissue by injection, oral administration, nasal drop, eye drop, physical or chemical mediated method; the daily necessities are hand sanitizer, facial cleanser, shampoo, bath foam, feminine care lotion, sanitary towel, sanitary pad, baby diaper, adult care urine pad, mouthwash, toothpaste, perfumed soap, laundry detergent, disinfectant, toilet cleaner, disinfectant paper towel, dressing, bandage or feed.

4. The use of the peptide mimics with antimicrobial function from pathogenic fungi of claim 3 in the preparation of antimicrobial drugs and/or daily necessities, wherein: the microorganism is a clinically isolated gram-positive bacterial strain and/or a clinically isolated fungal strain.

5. The use of the peptide mimics with antimicrobial function from pathogenic fungi of claim 4 in the preparation of antimicrobial drugs and/or daily necessities, wherein: the fungi are Candida and Cryptococcus.

6. A composition characterized by: it comprises the peptide mimic with antimicrobial function derived from pathogenic fungi according to claim 1 or further comprises acceptable salt and/or hydrate and/or solvate and/or acceptable carrier of the peptide mimic.

7. Use of a composition according to claim 6 for the preparation of an antimicrobial medicament and/or a daily product, characterized in that: the antimicrobial drug is injection, tablet, sterile powder for injection, powder, granule, capsule, oral liquid, ointment or cream; the antimicrobial drug or composition is introduced into muscle, endothelial, subcutaneous, intravenous or mucosal tissue by injection, oral administration, nasal drop, eye drop, physical or chemical mediated method; the daily necessities are hand sanitizer, facial cleanser, shampoo, bath foam, feminine care lotion, sanitary towel, sanitary pad, baby diaper, adult care urine pad, mouthwash, toothpaste, perfumed soap, laundry detergent, disinfectant, toilet cleaner, disinfectant paper towel, dressing, bandage or feed; the microorganism is clinically separated gram-positive bacterial strain and/or clinically separated candida and cryptococcus strain.