Dopa antifungal polypeptide for chelating fungus intracellular free iron sterilization and application of dopa antifungal polypeptide

Abstract

The invention discloses a dopa antifungal polypeptide for chelating free iron in fungal cells for sterilization and application thereof, and the dopa antifungal polypeptide is composed of basic amino acid and dopa amino acid which are respectively responsible for penetrating through fungi to enter the fungal cells and chelating iron ions. The polypeptide can also modify various chemical groups, such as lipophilic groups, assembling groups and the like to obtain corresponding derivatives which have high antifungal activity. By chelating free iron in fungal cells, synthesis of iron-sulfur clusters and various downstream life processes are blocked, so that fungal death due to metabolic failure is caused. The dopa antifungal polypeptide is wide in antibacterial spectrum, has no obvious toxicity to mammalian cells and symbiotic flora, and is not easy to induce drug resistance. The dopa antifungal polypeptide provided by the invention can be used for antifungal pharmaceutical preparations, medical instrument antibacterial coatings and daily disinfection and protection products, and provides an innovative strategy for solving the problem of clinical fungal infection.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine, and particularly relates to a kind of chelated intracellular free iron bactericidal dopa antifungal polypeptide and application thereof, the polypeptide is through specific plunder intracellular iron ion of fungi, interferes with mitochondrial respiratory function, thereby efficiently inhibits or kills a variety of pathogenic fungi, is suitable for the field such as pharmaceutical preparation, antifungal coating and daily disinfection and protection. BACKGROUND

[0002] Fungal infection is an important public health problem worldwide, especially in immunodeficient patients and drug-resistant strains, which makes the limitations of existing antifungal drugs increasingly prominent. Currently, the commonly used drugs (such as polyenes, azoles and echinocandins) mainly act by destroying fungal cell membranes or inhibiting cell wall synthesis, but have the following problems: 1) serious drug resistance: long-term use leads to a significant increase in drug resistance of fungi (such as Candida and Aspergillus) to azole drugs, increasing the risk of treatment failure; 2) large toxicity and poor selectivity: some broad-spectrum antifungal drugs have non-specific killing of host cells and symbiotic flora, disrupting the microecological balance. Traditional drugs (such as amphotericin B) have significant toxicity to host cells, easily causing adverse reactions such as kidney damage and liver toxicity; 3) single mechanism of action: the mechanism of destroying fungal cell membranes or inhibiting specific enzyme activity is easily avoided by fungi through metabolic pathway adjustment or gene mutation, leading to decreased efficacy.

[0003] In recent years, antibacterial peptides have attracted attention due to their broad-spectrum antibacterial activity and low risk of inducing drug resistance, but traditional antibacterial peptides mainly act by disrupting membranes, which limits their clinical application due to the contradiction between high cytotoxicity and antibacterial activity. Therefore, there is an urgent need to develop new antifungal agents with high selectivity and excellent safety. SUMMARY

[0004] The present application aims to provide a polypeptide composed of dopa amino acids and basic amino acids, wherein the basic amino acids in the molecular structure of the polypeptide are used to enter the intracellular space of fungi, and the dopa amino acids are used to plunder intracellular iron ions of fungi, destroy iron-sulfur cluster synthesis, inhibit mitochondrial respiratory chain function and metabolic processes, thereby exerting bactericidal properties.

[0005] The specific technical solution to achieve the purpose of the present application is as follows:

[0006] A kind of dopa antifungal polypeptide for chelating free iron in fungal cell, characterized by: its molecular sequence is Dopa-A-Dopa-A ……-Dopa-A, represented as (Dopa-A) n;A in its sequence is a basic amino acid with positive charge under physiological environment, which is one of lysine, arginine, ornithine or histidine but not limited to this, used to promote the penetration of the dopa antifungal peptide into the fungal cell membrane;Dopa in its sequence is dopa, as the iron chelating group of the dopa antifungal peptide, chelating free iron in the fungal cell, causing fungal iron deficiency, metabolic collapse and death;The repeat number n of dopa and basic amino acid is 1-50, and the preferred repeat number is 5-20 in terms of cost, activity and safety.

[0007] Further, the molecular sequence, the arrangement order of dopa and basic amino acid is interval arrangement or block arrangement, specifically: Dopa-A, A-Dopa, Dopa-Dopa-A-A, A-A-Dopa-Dopa, Dopa-Dopa-A or Dopa-A-A;The number ratio of dopa and basic amino acid is 4-1:1-4, preferably, the number ratio of the two is 1:1, interval arrangement.

[0008] Further, the molecular sequence, the arrangement order of dopa and basic amino acid is interval arrangement or block arrangement, specifically: Dopa-A, A-Dopa, Dopa-Dopa-A-A, A-A-Dopa-Dopa, Dopa-Dopa-A or Dopa-A-A;The number ratio of dopa and basic amino acid is 4-1:1-4, preferably, the number ratio of the two is 1:1, interval arrangement.

[0009] Further, the molecular sequence (Dopa-A) n, the N-terminal or C-terminal thereof is modified, respectively to obtain dopa antifungal polypeptide derivatives with the structure of X-Y-(Dopa-A) n or (Dopa-A) n-Y-X;The modification group X is a fatty chain, a methoxycarbonyl-phenylalanine-phenylalanine Fmoc-FF assembly group, a naphthalene dicarboximide-phenylalanine-phenylalanine Nap-F-F group or a fluorescein isothiocyanate group but not limited to this;The modification group X can promote the assembly of the dopa antifungal polypeptide and improve the antibacterial activity of the dopa antifungal polypeptide;Y in the structure represents the connecting bond between the modification group and the N-terminal or C-terminal of the polypeptide, including but not limited to amide bond and disulfide bond.

[0010] The use of the above-mentioned dopa antifungal polypeptide has excellent antibacterial effect on dermatophytes, Candida (Candida), Cryptococcus, Sporonema and Ascomycetes (such as Fusarium).

[0011] The use of the above-mentioned dopa antifungal polypeptide in the preparation of external preparations, antifungal coatings and daily disinfection and protection of fungal scenes.

[0012] Compared with traditional drugs, the Dopa antifungal polypeptide of the present application is easy to induce drug resistance, and has killing performance on clinically azole-resistant fungi.

[0013] The Dopa antifungal polypeptide of the present application has no significant toxicity to mammalian cells, no significant hemolytic performance, and good antifungal selectivity.

[0014] The Dopa antifungal polypeptide of the present application has the following beneficial effects:

[0015] (1) The Dopa antifungal polypeptide of the present application is composed of Dopa and basic amino acids, and the molecular sequence is (Dopa-A)n, wherein Dopa is Dopa, A is a basic amino acid, and n is the number of Dopa-A repeats. The polypeptide has good water solubility, and can enter the fungal cells, chelate intracellular free iron, cause fungal iron deficiency, and cause metabolic collapse to exert antibacterial effect.

[0016] (2) The Dopa antifungal polypeptide of the present application can obtain Dopa antifungal polypeptide derivatives through other modification strategies, which can further improve the activity and reduce the cost of the polypeptide.

[0017] (3) The Dopa antifungal polypeptide of the present application has good antibacterial activity on dermatophytes, Candida (Candida), Cryptococcus, Sporonema, Ascomycetes (such as Fusarium), and has no cytotoxicity to mammalian blood cells and human corneal epithelial cells HCEC. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 Schematic diagram of antibacterial activity of Dopa antifungal polypeptide with terminal modification assembly group;

[0019] Figure 2 Schematic diagram of cytotoxicity of Dopa antifungal polypeptide;

[0020] Figure 3 Schematic diagram of hemolysis rate of Dopa antifungal polypeptide;

[0021] Figure 4 Schematic diagram of antibacterial activity and depolarization effect on fungal cell membrane of control polypeptide without iron chelating ability;

[0022] Figure 5 Schematic diagram of permeability of Dopa antifungal polypeptide to mammalian cells;

[0023] Figure 6 Schematic diagram of localization of Dopa antifungal polypeptide in fungal cells;

[0024] Figure 7 Schematic diagram of iron ion content in fungal cells after treatment with Dopa antifungal polypeptide;

[0025] Figure 8Graph showing the relationship between ATP production of fungal mitochondria and the concentration of dopa antifungal polypeptide;

[0026] Figure 9 Graph showing the viable fungal count and the expression of inflammatory factors in a fungal dermatitis model;

[0027] Figure 10 Graph showing the viable fungal count and the expression of inflammatory factors in a fungal keratitis model;

[0028] Figure 11 Graph showing the safety evaluation of dopa antifungal polypeptide on the cornea. DETAILED DESCRIPTION

[0029] The present application will be described in detail below with reference to the accompanying drawings and examples.

[0030] Example 1:

[0031] The preparation method of the dopa antifungal polypeptide is as follows: the antifungal polypeptide is synthesized by a solid-phase synthesis method, and the specific operation steps are as follows:

[0032] Take the appropriate amount (1.0 g) of aminomethyl resin and place it in a reaction column. After weighing the resin, soak it in dichloromethane (DCM) for a few minutes, then wash it with N,N-dimethylformamide (DMF), then remove the DCM, then add a deprotection agent (20 mL of a 20% piperidine / DMF solution, 2 times, 5-10 minutes each time) to remove the 9-fluorenylmethoxycarbonyl (FMOC) protecting group, and then wash with DMF until the color is blue, indicating that the deprotection reaction is successful. Weigh the first amino acid at the carbon end, lysine (K, 532 mg), add the condensing agent HBTU (618 mg), and DMF, and place it in the reaction column for 45-60 minutes. Take a small amount of the resin for an indanetrione colorimetric test, and the test result is a bright yellow solution and a transparent resin with no color. Remove the FMOC protecting group on the lysine K using a deprotection solution (20% piperidine / DMF, 2 times, 10 mL per time, 5-10 minutes), and wash with DMF until the color is blue, indicating that the FMOC has been successfully removed. Continue to weigh the second amino acid at the carbon end, dopa amino acid (Dopa, 525 mg), add the condensing agent HBTU (618 mg), dissolve it in an appropriate amount of DMF, and then place it in the reaction column for 45-60 minutes. After the reaction, take the resin for an indanetrione test, and then remove the Fmoc using a 20% piperidine / DMF solution. Repeat the above condensation reaction and Fmoc removal reaction steps 3 times to obtain (Dopa-K)3. After the reaction is complete, wash and shrink the resin with DCM and MeOH, respectively. (Dopa-K)5, (Dopa-K)7, and (Dopa-K)9 can be synthesized according to the above steps, respectively. Replace lysine K with arginine R and repeat the above reaction 5 times to synthesize (Dopa-R)5. Perform mass spectrometry and high-performance liquid chromatography on the polypeptide product to determine its structure and purity, and the mass spectrometry and high-performance liquid chromatography results are shown in Table 1.

[0033] Table 1

[0034] .

[0035] Example 2

[0036] The preparation method of the different arrangement modes of the dopa antifungal polypeptide described in the application is as follows:

[0037] Take the appropriate amount (1.0 g) of aminomethyl resin and place it in a reaction column. After weighing the resin, soak it in dichloromethane (DCM) for a few minutes, then wash it with N,N-dimethylformamide (DMF), then remove the DCM, then add a deprotection agent (20 mL of a 20% piperidine / DMF solution, 2 times, 5-10 minutes each time) to remove the 9-fluorenylmethoxycarbonyl (FMOC) protecting group, and then wash with DMF until the color is blue, indicating that the deprotection reaction was successful. Weigh the first amino acid at the carbon end, lysine (K, 532 mg), add the condensing agent HBTU (618 mg), and DMF, and place it in the reaction column for 45-60 minutes. Take a small amount of the resin for an indanetrione colorimetric test, and the test result is a bright yellow solution and a transparent resin with no color. Remove the FMOC protecting group on the lysine K using a deprotection solution (20% piperidine / DMF, 2 times, 10 mL per time, 5-10 minutes), and wash with DMF until the color is blue, indicating that the FMOC has been successfully removed. Continue to weigh the second amino acid at the carbon end, dopa amino acid (Dopa, 525 mg), add the condensing agent HBTU (618 mg), dissolve it in an appropriate amount of DMF, and then place it in the reaction column for 45-60 minutes. After the reaction, take the resin for an indanetrione test, and then remove the Fmoc using 20% piperidine / DMF. Continue to weigh the third amino acid at the carbon end, dopa amino acid (Dopa, 525 mg), add the condensing agent HBTU (618 mg), dissolve it in an appropriate amount of DMF, and then place it in the reaction column for 45-60 minutes. After the reaction, take the resin for an indanetrione test, and then remove the Fmoc using 20% piperidine / DMF. Repeat the above condensation reaction and Fmoc removal reaction steps 6 times to obtain (Dopa-Dopa-K)6, and determine the correctness of its structure and purity, as shown in Table 1. Similarly, synthesize (Dopa-K-K)6, and the results are shown in Table 1; and according to similar methods, (Dopa-Dopa-K-K) n, (K-K-Dopa-Dopa) n, etc. can be synthesized in turn.

[0038] Example 3

[0039] The (Dopa-A-B) n dopa antifungal polypeptide described in the present application, which inserts any other non-functional amino acid B in the molecular sequence, and a preparation method thereof:

[0040] Take the appropriate amount (1.0 g) of aminomethyl resin and place it in a reaction column. After weighing the resin, soak it in dichloromethane (DCM) for a few minutes, then wash it with N,N-dimethylformamide (DMF), then remove the DCM, then add the deprotection agent (20 mL of 20% piperidine / DMF solution, 2 times, 5-10 minutes each time) to remove the 9-fluorenylmethoxycarbonyl (FMOC) protecting group, and then wash with DMF until the color is blue, indicating that the deprotection reaction is successful. Weigh the first amino acid at the carbon end, tryptophan (W, 516 mg), add the condensing agent HBTU (618 mg), and DMF, and place it in the reaction column for 45-60 minutes. Take a small amount of the resin for an indanetrione colorimetric test, and the test result is a bright yellow solution and a transparent resin with no color. Remove the FMOC protecting group on lysine K with the deprotection solution (20% piperidine / DMF, 2 times, 10 mL per time, 5-10 minutes), and wash with DMF until the color is blue, indicating that the FMOC has been successfully removed. Continue to weigh the second amino acid at the carbon end, lysine (K, 532 mg), add the condensing agent HBTU (618 mg), dissolve it in an appropriate amount of DMF, and then place it in the reaction column for 45-60 minutes. After the reaction, take the resin for an indanetrione test, and then remove the Fmoc with 20% piperidine / DMF. Continue to weigh the third amino acid at the carbon end, dopa amino acid (Dopa, 525 mg), add the condensing agent HBTU (618 mg), dissolve it in an appropriate amount of DMF, and then place it in the reaction column for 45-60 minutes. After the reaction, take the resin for an indanetrione test, and then remove the Fmoc with 20% piperidine / DMF. Repeat the above condensation reaction and Fmoc removal reaction steps 5 times to obtain (Dopa-K-W)5.

[0041] Example 4

[0042] The X-Y-(Dopa-A)n dopa antifungal polypeptide derivative having a modification at its N terminus according to the present application, and a preparation method thereof:

[0043] The synthesis of Fmoc-F-F-CONH-(Dopa-R)2 is provided here. Fmoc-F-F corresponds to X, and Y is the CONH amide bond connecting F and Dopa.

[0044] Take the appropriate amount (1.0 g) of aminomethyl resin and place it in the reaction column. After weighing the resin, soak it in dichloromethane (DCM) for a few minutes, then wash it with N,N-dimethylformamide (DMF), then remove the DCM, then add the deprotection agent (20 mL of 20% piperidine / DMF solution, 2 times, 5-10 minutes each time) to remove the 9-fluorenylmethoxycarbonyl (FMOC) protecting group, then wash with DMF until the color is blue, indicating that the deprotection reaction is successful. Weigh the first amino acid at the carbon end, arginine (R, 527 mg), add the condensing agent HBTU (618 mg), and DMF, and place it in the reaction column for 45-60 minutes. Take a small amount of the resin for the ninhydrin color test reaction, and the test result is a bright yellow solution and a transparent resin without any color. Remove the FMOC protecting group on lysine K with the deprotection solution (20% piperidine / DMF, 2 times, 10 mL per time, 5-10 minutes), and wash with DMF until the color is blue, indicating that the FMOC has been successfully removed. Continue to weigh the second amino acid at the carbon end, dopa amino acid (Dopa, 375 mg), add the condensing agent HBTU (618 mg), dissolve it in an appropriate amount of DMF, and then place it in the reaction column for 45-60 minutes. After the reaction, take the resin for the ninhydrin test, then remove the Fmoc with 20% piperidine / DMF. Continue to weigh the third amino acid at the carbon end, arginine (R, 527 mg), add the condensing agent HBTU (618 mg), dissolve it in an appropriate amount of DMF, and then place it in the reaction column for 45-60 minutes. After the reaction, take the resin for the ninhydrin test, then remove the Fmoc with 20% piperidine / DMF. Weigh the dopa amino acid (Dopa, 375 mg), alanine (F, 325 mg), and alanine (F, 325 mg) in turn, repeat the above condensation reaction and Fmoc removal reaction steps, and the last amino acid, alanine, does not undergo the Fmoc removal step, to obtain Fmoc-F-F-(Dopa-R)2.

[0045] Example 5

[0046] The X-Y-(Dopa-A)n dopa antifungal polypeptide derivative having a modification at its N terminus according to the present application, and a preparation method thereof:

[0047] Terminal modification of the fatty chain, here the synthesis of C8-S-S-(Dopa-K)5 containing an alkane chain of 8 carbon atoms is provided. C8 corresponds to X, and Y is the S-S disulfide bond connecting C8 and Dopa.

[0048] The synthesis of derivative lipopeptide C8-S-S-(Dopa-K)5 was constructed using a thiol-activated disulfide exchange reaction. First, C8-pyridyl disulfide (C8-Py, 4.9 mg) was dissolved in dichloromethane at 10 mg / mL; meanwhile, 5 mg of cysteine-capped Cys-(Dopa-K)5 polypeptide was dissolved in methanol at 10 mg / mL. The two solutions were mixed at a molar ratio of 4:1 (C8-Py:Cys-(Dopa-K)5) and stirred at room temperature under nitrogen protection overnight. After the reaction was completed, anhydrous ether was added to precipitate the reaction product, and centrifugation was used to remove unreacted lipophilic precursors (such as free C8-Py) and byproducts (such as 2-mercaptopyridine). The resulting precipitate was washed with cold ether 3 times to remove residual organic solvents and impurities. The final product was lyophilized for subsequent characterization. By modifying fatty chains of different lengths Cn (n = 8, 10, 12, 14, 16, 18), different derivative lipopeptides can be obtained.

[0049] Example 6

[0050] Antibacterial performance evaluation of a class of dopa antifungal polypeptides of the present application

[0051] Using C. albicans 10231 as a model, the antifungal activity of several antifungal peptides obtained above was tested. The specific steps are as follows: C. albicans 10231 was resuscitated to the logarithmic phase (2 x 10 7 CFU / mL), then diluted 10000 times to a concentration of 2 x 10 3 CFU / mL, and using a two-fold gradient dilution method, all polypeptides were mixed with the bacterial solution at equal volumes, then incubated at 30 oC. The results showed that the antifungal activity of (Dopa-K)npeptides increased with the increase of the length of the peptides. (Dopa-K)3did not show antifungal activity against C. albicans at the tested concentration range. The minimum inhibition concentration (MIC) of (Dopa-K)5, (Dopa-K)7, and (Dopa-K)9were 64 μg / mL, 4 μg / mL, and 2 μg / mL, respectively. In addition, the antifungal activity of (Dopa-Dopa-K)6and (Dopa-K-K)6were 4 μg / mL and 8 μg / mL, respectively. For the peptides with the same length, the performance was the best when the sequence contained equal proportions of dopa groups and cationic groups. Further, if lysine K was replaced by arginine R, the activity was further improved. As shown in Table 2, the MIC value of (Dopa-R)5was 4 μg / mL, which was 16 times higher than that of (Dopa-K)5(64 μg / mL). The specific antibacterial performance is shown in Table 2.

[0052] Table 2

[0053] .

[0054] Example 7

[0055] Antibacterial activity evaluation of X-Y-(Dopa-A)n dopa antifungal peptides with N-terminal modification assembly groups

[0056] The antibacterial activity of dopa antifungal peptide derivatives was also tested according to the two-fold gradient dilution method. The modification of Fmoc-F-F or Nap-F-F groups can facilitate the assembly of dopa antifungal peptides. It can be seen that the Fmoc-F-F capped polypeptides all form fiber structures (Figure Figure 1 A) ), which can further improve the antibacterial performance of (Dopa-A)n. Only one Dopa-R repeat unit can obtain an antibacterial activity with a MIC value of 8 μg / mL (as shown in Figure 1 B) ).

[0057] Example 8

[0058] Antibacterial activity evaluation of X-Y-(Dopa-A)n or (Dopa-A)n-Y-X dopa antifungal lipopeptides with N-terminal or C-terminal modification of fatty chains

[0059] The polypeptides with fatty alkyl chain modification were evaluated for their activity in the same way as above. The modification of lipophilic alkyl chain can further improve the antibacterial activity of iron-chelating polypeptides. As shown in Table 3, the unmodified (Dopa-K)1, (Dopa-K)2, (Dopa-K)3 and (Dopa-K)4 did not show significant activity (MIC > 128 μg / mL) at a concentration higher than 128 μg / mL, but after modification of C12 fatty chain at their N-terminus, the MIC values were all 4 μg / mL, and the antibacterial activity was significantly enhanced. Similarly, (Dopa-K)5 had a MIC of 64 μg / mL in unmodified state, but after modification of C8, C10 or C14 fatty chain at its N-terminus, the antibacterial activity was increased by 8 times (MIC value decreased to 8 μg / mL). In addition, modification of C12 fatty chain at the C-terminus of (Dopa-K)5 also greatly improved the activity, with a MIC value of 4 μg / mL. These results show that the introduction of fatty chain modification at the N-terminus or C-terminus can significantly enhance the antibacterial activity of the series of polypeptides.

[0060] Table 3

[0061] .

[0062] Example 9

[0063] Antibacterial activity of Dopa antifungal polypeptides against other species

[0064] The Dopa antifungal polypeptides not only have antibacterial activity against Candida albicans, but also have good antibacterial activity against other species of Candida, such as Candida krusei and Candida parapsilosis. In addition, they also have antibacterial activity against Cryptococcus neoformans and various Fusarium species, and the corresponding MIC values are shown in Table 4.

[0065] Table 4

[0066] .

[0067] Example 10

[0068] Drug resistance test

[0069] Candida albicans 10231 in the logarithmic growth phase was diluted to 2 x 10 3CFU / mL, followed by mixing with (XK)9 at a final concentration of 0.5xMIC in SD medium and incubated at 37°C for 24 hours. The treated bacterial solution was inoculated on SD agar plates, and the surviving strains were collected for labeling and entering the next round of circulation. The collected colonies stimulated by sub-concentration drug at different circulation numbers were tested for their antibacterial performance. The same procedure was repeated with fluconazole as a control. As shown in Table 5, after up to 16 rounds of treatment, no significant increase in drug resistance was detected in the (Dopa-K)9 treatment group, while the antibacterial activity of the fluconazole control group decreased by 64-fold, and drug-resistant strains appeared.

[0070] Table 5

[0071] .

[0072] Example 11

[0073] Cytotoxicity and hemolytic test

[0074] Next, the biosafety, antibacterial mechanism, and therapeutic potential of (Dopa-K)9 were studied.

[0075] The safety of the obtained antifungal polypeptide was comprehensively evaluated by cytotoxicity test and hemolytic test. For the cytotoxicity test: mouse embryonic fibroblasts NIH 3T3 were seeded in 96-well plates at a density of 10 4 cells / well until the cell density reached 80%, then treated with (Dopa-K)9 at a concentration gradient (2-512 μg / mL) for 24 hours, and the cell viability was determined by CCK8 method, with 6 repeats in each group. The cytotoxicity data are shown in Figure 2 (Dopa-K)9 at 256 μg / mL, the cell survival rate was still higher than 50%.

[0076] For the hemolytic test: 4% blood cell suspension was prepared, then mixed with (Dopa-K)9 at a concentration of 31.25-4000 μg / mL in equal volume, incubated at 37 o C for 1 hour, then centrifuged at a speed of 3000 rpm, and the obtained supernatant was detected for absorbance at 405 nm, while PBS and 0.5% Triton were set as negative and positive controls, respectively. The hemolysis rate was calculated according to the formula: hemolysis% = (OD 405 (Dopa-K)9-OD 405 negative) / (OD 405 positive-OD 405 negative) x 100. The results are shown in Figure 3

[0077] Example 12 ​

[0078] Studies on the interaction with the membrane verify the non-simple membrane-rupturing bactericidal mechanism

[0079] In order to study the antibacterial mechanism of (Dopa-K)9, the polyphenol hydroxyl groups in the molecular structure thereof were gradually reduced, and (YK)9 and (FK)9 were designed. The antibacterial activity showed that the antibacterial performance of the control polypeptides (FK)9 and (YK)9 decreased with the decrease in the number of hydroxyl groups in the Dopa structure, and the corresponding MIC values were 16 μg / mL and >128 μg / mL, respectively.

[0080] The disturbance of the antifungal peptide to the fungal cell membrane was evaluated by detecting the potential change and permeability of the fungal cell membrane, respectively. The specific membrane depolarization evaluation operation steps were as follows: Candida albicans 10231 (1×10 7 CFU / mL) and the membrane polarization fluorescent probe 3,3'-dipropylthiadicarbocyanine DiSC3(5) (1 μM) were incubated in HEPES buffer for 5 minutes, 4 μg / mL (Dopa-K)9 was added, and the fluorescence intensity change was detected by a multifunctional enzyme label instrument (λex = 622 nm, λem = 670 nm). The results evaluated are shown in Figure 4 (Dopa-K)9 treatment group fluorescence intensity decreased significantly, indicating membrane potential depolarization. At the same time, (YK)9 and (FK)9 with higher hydrophobicity also showed higher membrane depolarization ability, but the antibacterial performance was lower, indicating that (XK)9 did not simply play an antibacterial role by disturbing the fungal membrane potential.

[0081] The membrane permeability evaluation operation steps were as follows: the mouse fibroblasts were cultured to a cell density of about 80%, and (FK)9, (YK)9 and (Dopa-K)9 were added at a concentration of 512 μg / mL, respectively. After 1 hour of incubation, the cells were stained with propidium iodide (PI) and photographed under fluorescence, and the results are shown in Figure 5 (Dopa-K)9, indicating that (FK)9 caused higher cell permeability, but did not bring higher bactericidal activity, which was contrary to the traditional membrane-rupturing antibacterial mechanism, and also proved that (Dopa-K)9 did not kill bacteria by the mechanism of destroying the fungal cell membrane, which was different from the traditional antibacterial peptide membrane-rupturing mechanism. The activity needs to be compared here, and the membrane-rupturing data is also added.

[0082] Example 13

[0083] Localization of antifungal peptides in fungi

[0084] Through the above analysis, the antibacterial peptide does not kill bacteria by the membrane breaking mechanism, and the interaction between the peptide and the fungus is continuously observed. The N-terminal of the peptide is modified with FITC to label fluorescence, and then the localization of the material in the fungus is observed by laser confocal microscopy. As shown in Figure 6 , it can be seen that the material can penetrate the cell wall and cell membrane of the fungus, and then enter the intracellular material of the fungus.

[0085] Example 14

[0086] Effect of Dopa antifungal polypeptide on iron content in fungus

[0087] C. albicans 10231 was then incubated with 4 μg / mL (Dopa-K)9 for 3 hours, and then lysed and ground, and the intracellular material was extracted, and the iron content was detected using an iron colorimetric kit. The results are shown in Figure 7 , which show that the intracellular iron content of the (Dopa-K)9-treated group is significantly lower than that of the untreated group. Through the above comprehensive analysis, (Dopa-K)9 has high iron chelating ability, and significantly reduces the intracellular iron content of the fungus.

[0088] Example 15

[0089] Mitochondrial dysfunction

[0090] Iron is the most abundant trace element in all living organisms, and is a core participant in redox reactions. It can form iron-sulfur clusters and heme cofactors, and is widely involved in mitochondrial energy metabolism, thereby providing energy for the organism. In order to verify the effect of (Dopa-K)9 on mitochondria by depriving the fungus of iron elements, the respiratory metabolism of mitochondria was detected, and the production of ATP was significantly reduced, indicating that the respiratory metabolism of mitochondria was significantly inhibited. As shown in Figure 8 , the amount of ATP produced gradually decreased as the amount of (Dopa-K)9 increased, and when (Dopa-K)9 reached 8 μg / mL, the amount of ATP produced decreased by 40%. This result shows that (Dopa-K)9 hinders the respiratory function of the mitochondrial of the fungus, and inhibits the energy metabolism in the fungus.

[0091] Example 16

[0092] Treatment model of fungal dermatitis

[0093] Further verification of the treatment of the obtained antifungal peptide (Dopa-K)9 on the infection of drug-resistant bacteria in vivo, a skin infection model of clinically isolated drug-resistant bacteria was constructed. The specific steps are as follows: BALB / c mice (female, 20 g) were infected with drug-resistant C. albicans A52 (2 x 10 7CFU), topical application of (Dopa-K) 9 (1 mg / mL, once daily) for 4 days. Four days later, the mice were sacrificed by cervical dislocation and the infected skin was collected for evaluation of the effect by colony counting and histopathological analysis.

[0094] Results are shown in Figure 9 a-b, the skin fungal load was reduced by 95% and the expression of proinflammatory factors was significantly reduced in the (Dopa-K) 9 group. It was proved that (Dopa-K) 9 had a significant therapeutic effect on skin infection caused by drug-resistant fungi.

[0095] Example 17

[0096] Fungal keratitis treatment model

[0097] To further evaluate the therapeutic activity of the anti-fungal peptides obtained in the present application in vivo, a Candida albicans-induced rabbit fungal keratitis model was constructed. The specific steps are as follows: logarithmic phase of the infection Candida albicans 10231 (2 x 10 5 CFU) was injected into the corneal stroma of the right eye of New Zealand white rabbits (female, 2 kg in weight, n = 5) using a 33G fine needle. After 24 h, local white hyphae were observed, indicating that the modeling was successful. Subsequently, (Dopa-K) 9 (1 mg / mL, 6 times daily) was applied topically, and continuous treatment was performed for 7 days. The mice were sacrificed by ear vein perfusion, and then the reduction of fungal load was evaluated by colony counting, and the production of inflammatory factors was detected by qPCR to comprehensively evaluate the therapeutic effect.

[0098] After treatment with (Dopa-K) 9, the degree of infection of the corneal tissue was significantly improved, and the appearance showed that the degree of corneal swelling was reduced, the corneal stroma was relatively transparent, there was no obvious turbidity, and there was no obvious secretion. In addition, the number of viable bacteria was reduced to 32.4% of the untreated group (as shown in Figure 10 a), and the content of inflammatory factors produced was significantly reduced, proving that (Dopa-K) 9 has good therapeutic effect in the in vivo disease model (as shown in Figure 10 b).

[0099] Example 18

[0100] Safety evaluation

[0101] (Dopa-K) 9 (1 mg / mL, 6 times daily) was added to the rabbit eye, and continuous administration was performed for 7 days, and the drug dosage and frequency were consistent with those in the treatment of the infection model. Before administration and 7 days after administration, the appearance was photographed, the sodium fluorescein staining was photographed (5 mg / mL, 20 μL), and the apoptosis staining of the corneal tissue was performed, and the results are shown in Figure 11No significant apoptotic signals were found in any of the groups, demonstrating that (Dopa-K)9 is well tolerated by corneal tissue at the dose range used.

Claims

1. A type of dopa antifungal polypeptide that chelates and kills fungal intracellular free iron, characterized in that, Its molecular sequence is Dopa-A-Dopa-A……-Dopa-A, denoted by (Dopa-A)n; the A in its sequence is a positively charged basic amino acid under physiological conditions, which is one of lysine, arginine, ornithine, or histidine, used to promote penetration of the fungal cell membrane and entry into the cell; the Dopa in its sequence is dopa, which acts as an iron ion chelating group to chelate free iron in the fungal cell, causing iron deficiency, metabolic collapse, and death of the fungus; The repeat number n of its dopa and basic amino acids ranges from 1 to 50.

2. The dopa antifungal polypeptide according to claim 1, characterized in that, The molecular sequence, wherein the dopa and basic amino acids are arranged in an alternating or block arrangement, specifically: Dopa-A, A-Dopa, Dopa-Dopa-AA, AA-Dopa-Dopa, Dopa-Dopa-A, or Dopa-AA; the ratio of dopa to basic amino acids is 4 to 1: 1 to 4.

3. The dopa antifungal polypeptide according to claim 1, characterized in that, The molecular series, in which any other non-functional amino acid B is inserted into the sequence, has the molecular sequence Dopa-AB, Dopa-BA, or B-Dopa-A; wherein B is alanine, glycine, phenylalanine, leucine, or valine.

4. The dopa-antifungal polypeptide according to claim 1, characterized in that, The molecular sequence (Dopa-A)n is modified at its N-terminus or C-terminus to obtain dopa antifungal polypeptide derivatives with structures XY-(Dopa-A)n or (Dopa-A)nYX, respectively. The modifying group X is an aliphatic chain, a methoxycarbonyl-phenylalanine-phenylalanine Fmoc-FF assembly group, a naphthalenedicarboximide-phenylalanine-phenylalanine Nap-FF group, or a fluorescein isothiocyanate group. The modifying group X can promote assembly. Y represents the linking bond between the modifying group X and the N-terminus or C-terminus, including amide bonds and disulfide bonds.

5. The use of the dopa antifungal polypeptide according to claim 1, characterized in that, The aforementioned dopa antifungal polypeptide has antibacterial activity against dermatophytes, Candida spp., Cryptococcus spp., Sporotyphi spp., and Ascomycetes spp.

6. The application of the dopa antifungal polypeptide according to claim 1, characterized in that, The aforementioned dopa antifungal polypeptide has applications in the preparation of topical formulations, antifungal coatings, and daily disinfection and protection against fungi.

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