Dual-target antifungal peptides, methods of making and using the same

Abstract

This invention provides two dual-target antimicrobial peptides, their preparation methods, and applications, belonging to the field of bioengineering. Their sequences are shown as NLR-2 and NIR-3, with the N-terminus linked to the carboxyl group of 3-(1-naphthyl)acrylic acid. The two antifungal peptides of this invention exhibit good bactericidal activity against Cryptococcus neoformans under physiological conditions (1 μM), and also demonstrate good antifungal activity in corn feed, effectively reducing the toxin production in corn feed caused by Fusarium graminearum. Therefore, the antifungal peptides of this invention possess excellent bactericidal function, can target fungal membrane structures, achieving highly efficient membrane-breaking and bactericidal effects, and can be used as novel antifungal agents.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering technology, specifically involving two dual-target antifungal peptides, their preparation methods, and applications. Background Technology

[0002] Antimicrobial peptides (AMPs), as primary components of the host's own defense system, are widely distributed in animals, plants, and microorganisms and are considered one of the most promising antibiotic alternatives. Membrane-active antimicrobial peptides (AMPs) selectively disrupt the integrity of microbial membranes and are considered a new generation of antibiotics against multidrug-resistant pathogenic fungi. Accurately understanding the interactions between membrane-active drugs and biomembrane components is crucial for elucidating their molecular mechanisms and optimizing their targeting specificity, and is also a prerequisite for reducing potential side effects. Ergosterol is a key sterol component in the cell membrane of fungi (including molds and yeasts). Decreased ergosterol levels in fungal cell membranes easily lead to cell membrane damage and even death; therefore, most antifungal drugs target key enzymes involved in the ergosterol biosynthesis pathway. Dual-target drug design is considered a more effective strategy, capable of exerting a synergistic inhibitory advantage in the disease system similar to combination therapy, thereby achieving a "1 + 1 > 2" therapeutic effect. Developing novel antifungal peptides and compounds in this way holds promise for improving the efficacy of antifungal peptides, reducing toxicity, and minimizing drug resistance. Summary of the Invention

[0003] Based on the aforementioned problems in the technical field, the purpose of this invention is to provide two dual-target antifungal peptides that can self-assemble into nanoparticles in ultrapure water, exhibiting good killing effects against Cryptococcus neoformans and Fusarium graminearum in a physiological environment. Furthermore, the antifungal peptides of this invention can effectively disrupt the fungal cell membrane structure, thereby killing fungi.

[0004] The technical solution adopted in this invention is as follows: two dual-target antifungal peptides, NLR-2 and NLR-3, whose amino acid sequences are shown in SEQ ID No.1 and SEQ ID No.2, respectively, and whose N-terminus is linked to the carboxyl group of 3-(1-naphthyl)acrylic acid.

[0005] Another objective of this invention is to provide two methods for preparing dual-target antifungal peptides NLR-2 and NLR-3, as detailed below:

[0006] Step S1: Using a heptapeptide repeat sequence template (LKAIRRR) n n = 2, 3, which act as hydrophobic supports to provide hydrophobic forces;

[0007] Step S2: Select 3-(1-naphthyl)acrylic acid to link with it to construct a polypeptide;

[0008] Step S3: The peptides were prepared by solid-phase chemical synthesis and mass spectrometry identification. The nanoscale morphology, in vitro cytotoxicity, hemolytic activity and bactericidal activity of the peptides were then determined. Finally, they were named antimicrobial peptides NLR-2 and NLR-3.

[0009] Furthermore, the self-assembly methods of the two dual-target antifungal peptides NLR-2 and NLR-3 mentioned above are as follows: at a concentration of 64 μM, incubation at 37°C for 24 hours can self-assemble into nanostructures.

[0010] Another objective of this invention is to provide the application of the aforementioned dual-target antifungal peptides NLR-2 and NLR-3 in preventing corn mold caused by Fusarium graminearum.

[0011] The beneficial effects and advantages of this invention are as follows: The dual-target antifungal peptides NLR-2 and NLR-3 of this invention form nanoparticles in a physiological environment, which is more conducive to disrupting fungal cell membranes. Under physiological conditions, they possess direct bactericidal activity and have excellent killing effects against Cryptococcus neoformans and Fusarium graminearum. In summary, NLR-2 and NLR-3 are self-assembled antifungal peptides with high application value. Attached Figure Description

[0012] Figure 1 Mass spectra of NLR-2 (a) and NLR-3 (b);

[0013] Figure 2 Chromatograms of NLR-2 (a) and NLR-3 (b);

[0014] Figure 3 Transmission electron microscopy nanoscale characterization images of NLR-2 (a) and NLR-3 (b);

[0015] Figure 4 For the determination of NLR-2 and NLR-3 cytotoxicity;

[0016] Figure 5 For the determination of the hemolytic activity of NLR-2 and NLR-3;

[0017] Figure 6 NLR-2 and NLR-3 are effective against Cryptococcus neoformans. Crytococcus Neoformans 32609, Fusarium graminearum Fusarium graminearum 37687's killing activity;

[0018] Figure 7 For NLR-2 and NLR-3 pairs Crytococcus Neoformans 32609 (a) Fusarium graminearum 37687(b) Effects on extracellular membrane permeability;

[0019] Figure 8 For NLR-2 and NLR-3 pairs Crytococcus Neoformans 32609 (a) Fusarium graminearum 37687 (b) Effects of ergosterol on the cell membrane;

[0020] Figure 9 To observe NLR-2 and NLR-3 pairs using scanning electron microscopy Crytococcus Neoformans 32609 (A) Fusarium graminearum The morphological effects of 37687 (B) are shown in (a) physiological environment control group, (b) physiological environment NLR-2 peptide treatment group, (c) physiological environment NLR-3 peptide treatment group, and (d), (e) and (f) are magnified views of the local areas. Detailed Implementation

[0021] The present invention will be further described in detail below with reference to the embodiments and accompanying drawings, but the embodiments of the present invention are not limited thereto. Example 1

[0022] Design of NLR-2 and NLR-3: Selection of heptapeptide repeat sequence template (LKAIRRR)

[0023] n

[0024] The n=2,3 group serves as a hydrophobic core and promotes the self-assembly of peptide molecules by linking 3-(1-naphthyl)acrylic acid. The naphthyl group can simultaneously dock to the dual-target active regions of SE and CYP 51 on the cell membrane, inhibiting ergosterol synthesis and thus enhancing membrane disruption ability. The amino acid sequence of the peptide is shown in Table 1.

[0025] Table 1. Amino acid sequences of dual-target antifungal peptides NLR-2 and NLR-3

[0026]

[0027] Example 2

[0028] The dual-target antifungal peptides NLR-2 and NLR-3 were synthesized using a solid-phase chemical synthesis method.

[0029] 1. The preparation of antimicrobial peptides was carried out sequentially from the C-terminus to the N-terminus using a peptide synthesizer. 3 g of RINK resin (0.3 mmol / g degree of substitution) was weighed into a 150 mL reactor and soaked in 50 mL of dichloromethane (DCM). After 2 hours, the resin was washed with 3 times its volume of nitrogen-dimethylformamide (DMF), then dried. This process was repeated four times. The resin was then dried and set aside for later use. A certain amount of 20% piperidine (piperidine / DMF) was added to the reactor, and the mixture was shaken on a decolorizing shaker for 20 min to remove the Fmoc protecting groups from the resin. After deprotection, the resin was washed four times with 3 times its volume of DMF, and then dried.

[0030] 2. Take a small amount of resin and test it using the ninhydrin (Ninhydrin hydrate) method (two drops each of test A and test B, react at 100℃ for 1 min). If the resin turns colored, it indicates successful deprotection. Weigh an appropriate amount of the first amino acid at the C-terminus and an appropriate amount of 1-hydroxy-benzotriazole (HOBT) into a 50 mL centrifuge tube, add 20 mL of DMF to dissolve them, then add 3 mL of N,N-diisopropylcarbodiimide (DIC) and shake well for 1 min. After the solution becomes clear, add it to the reactor, and then place the reactor in a shaker at 30℃ for reaction.

[0031] 3. After 2 hours, cap the resin with a certain amount of acetic anhydride (acetic anhydride: DIEA: DCM = 1:1:2) for half an hour, then wash four times with 3 times the resin volume of DMF, and dry it for later use. Add a certain amount of 20% piperidine (piperidine / DMF = 1:4) to the reactor, and shake it on a decolorizing shaker for 20 minutes to remove the Fmoc protecting groups on the resin. After decolorization...

[0032] After treatment, wash four times with DMF, then dry.

[0033] 4. Take a small amount of resin and test it using the ninhydrin (Ninhydrin hydrate) method (two drops each of test A and test B, react at 100℃ for 1 min). If the resin is colored, the deprotection is successful. Weigh an appropriate amount of the second amino acid and HOBT into a 50 mL centrifuge tube, add 25 mL of DMF to dissolve them, then add 2.5 mL of DIC and shake well for 1 min. After the solution becomes clear, add it to the reactor, and then place the reactor in a shaker at 30℃ for reaction. After 1 hour, take a small amount of resin for testing using the ninhydrin method (two drops each of test A and test B, react at 100℃ for 1 min). If the resin is colorless, the reaction is complete; if the resin is colored, the condensation is incomplete, and the reaction should continue. After the reaction is complete, the resin is washed four times with DMF, then dried. A certain amount of 20% piperidine (piperidine / DMF = 1:4) is added to the reactor, and the mixture is shaken on a decolorizing shaker for 20 minutes to remove the Fmoc protecting groups from the resin. After deprotection, the resin is washed four times with DMF, then dried to check if the protection has been removed.

[0034] 5. After the last amino acid is added, the protection is removed, and the resin is washed four times with DMF. Then, the resin is dried with methanol. The peptide is then cleaved from the resin using 95% cleavage buffer (trifluoroacetic acid: 1,2-ethylenedithiol: 3, isopropylsilane: water = 95:2:2:1) (10 mL cleavage buffer per gram of resin), and centrifuged four times with ice-cold ether (cleavage buffer: ether = 1:9). Finally, the peptide is purified by HPLC and then lyophilized to obtain a peptide of a certain purity.

[0035] 6. Add naphthylacrylic acid, and the carboxyl group of 3-(1-naphthyl)acrylic acid condenses with the amino group of the last amino acid at the N-terminus;

[0036] 7. Identification of peptides: The peptides obtained above were analyzed by electrospray ionization mass spectrometry, such as... Figure 1 The molecular weight shown in the mass spectrum is basically consistent with the theoretical molecular weight in Table 1, and the purity of the polypeptide is greater than 95%. Example 3

[0037] Transmission electron microscopy nanoscale characterization images of NLR-2 and NLR-3:

[0038] Nanomorphology Analysis: To further analyze the nanomorphology of NLR-2 and NLR-3, the peptide (2.56 mM) was diluted to 64 μM in deionized water and incubated at 37 °C for 24 hours. The samples were spread onto glass slides and, after air drying, coated with metal using a Polaron SC7640 sputtering system. The samples were observed at 5 kV using a Hitachi S-4800 SEM (Hitachi, Japan). The results are shown below. Figure 3 .

[0039] from Figure 3 It can be seen that (a) NLR-2 and (b) NLR-3 form spherical nanoparticles under physiological conditions; Example 4

[0040] In vitro cytotoxicity, hemolytic activity, and bactericidal activity assays of NLR-2 and NLR-3:

[0041] 1. Cytotoxicity assay: Cells frozen in liquid nitrogen were thawed and seeded into a medium containing 10% fetal bovine serum and 1% penicillin-dextrose antibiotics, and passaged at 37°C and 5% CO2. The cultured cells were then digested with 0.25% trypsin and adjusted to a pH of 2–4 × 10⁶ cells / mL with culture medium. 5 cells / mL. 50 µL of cell suspension was mixed with 50 µL of peptides of different concentrations in a 96-well plate and incubated at 37°C and 5% CO2 for 24 h. Then, 25 µL of MTT (5 mg / mL) was added to each well, and incubation continued for another 4 h. After incubation, the supernatant was discarded, and the crystals at the bottom of the wells were dissolved in 100 µL of DMSO. The absorbance of each well was measured at 570 nm using a microplate reader. Culture medium wells served as blank controls. Results are shown below. Figure 4 .

[0042] from Figure 4 It can be seen that even after treatment with high concentrations of NLR-2 and NLR-3, the cell survival rate of HEK293T cells is greater than 50%, indicating that NLR-2 and NLR-3 have good biocompatibility and have the potential to become antibiotic alternatives.

[0043] 2. Hemolytic Activity Assay: 1 mL of fresh blood from healthy individuals was collected and centrifuged (4℃, 1000×g, 5 min). The blood was washed three times with HEPES inner membrane buffer (pH=7.4), the supernatant was discarded, and the precipitated blood cells were collected and resuspended in HEPES inner membrane buffer to 10 mL. An equal volume of the diluted red blood cell suspension was then placed in a 96-well plate and mixed with peptide solutions of different concentrations. After incubation at 37℃ for 1 h, the plate was centrifuged (1000×g, 10 min), and the supernatant was transferred to a new 96-well plate. Wells treated with 0.1% Triton X-100 served as positive controls, and wells without peptide treatment served as negative controls. The absorbance was measured using a microplate reader at a wavelength of 570 nm. A peptide concentration that caused 50% hemolytic activity was defined as cytotoxic. The test results are attached. Figure 5 .

[0044] pass Figure 5 It can be seen that NLR-2 and NLR-3 (128 μM) did not cause hemolysis at high concentrations, which indicates that NLR-2 and NLR-3 have good biocompatibility.

[0045] 3. Bactericidal activity assay: The minimum inhibitory concentration (MIC) of the antimicrobial peptide was determined using the microdilution method. Different concentrations of peptide were added to BSA solution in 96-well plates, followed by the addition of an equal volume of solution with a final concentration of 1×10⁻⁶. 5 Cryptococcus neoformans CFU / mL Crytococcus Neoformans 32609 / Fusarium graminearum Fusarium graminearum The final peptide concentrations in the 37687, 96-well plates ranged from 0.5 to 64 μM. After incubation at 30°C for 72 hours, 50 μL was aspirated and serially diluted in PBS. The minimum bactericidal concentration (MCC) was determined by plate counting to kill 99.99% of the bacteria. Results are shown below. Figure 6 .

[0046] Depend on Figure 6 It can be seen that, under physiological conditions, antifungal peptides possess bactericidal capabilities; 1 μM of NLR-2 and NLR-3 can kill Cryptococcus neoformans. 32 μM of NLR-2 and 16 μM of NLR-3 can kill Fusarium graminearum. Example 5

[0047] Mechanism of action determination: Prepare Fusarium graminearum spore suspension / Cryptococcus neoformans suspension, and dilute the culture to 1 × 10⁻⁶ using HEPES buffer. 5CFU / mL. This was then mixed with an N-phenyl-1-naphthylamine (NPN, Sigma-Aldrich) fluorescent probe and incubated in the dark at 30°C for 30 min. Different amounts of the peptide (50 μL) were then added to 96-well plates. Fluorescence intensity was measured using an F-4500 fluorescence spectrophotometer at an excitation wavelength of 350 nm and an emission wavelength of 420 nm. Results are shown below. Figure 7 .

[0048] from Figure 7 It can be seen that NLR-2 and NLR-3 can significantly increase fluorescence intensity, which indicates that NLR-2 and NLR-3 can increase the permeability of the fungal outer membrane. Example 6

[0049] A suspension of Fusarium graminearum spores was inoculated into 20 mL of PDB medium to achieve a spore concentration of 1 × 10⁻⁶. 6 CFU / mL was collected and incubated in a constant temperature shaking incubator at 28℃ and 150 r / min for 12 h. A certain concentration of peptide was then added to PDB medium containing *Fusarium graminearum* cultured for 12 h, resulting in peptide concentrations of 1×MIC and 2×MIC for the experimental groups, and 1×MIC for the control group containing sodium benzoate. Each concentration was repeated in triplicate. The culture was continued in a constant temperature shaking incubator at 28℃ and 150 r / min under natural light. The culture was stopped after 3 days, and the cultures were filtered.

[0050] Single colonies were picked and cultured in PDB medium until the logarithmic growth phase. The culture was then transferred to PBS solution for washing the cells three times, resulting in a final dilution of 1 × 10⁻⁶. 6 CFU / mL. Add 1×MIC and 2×MIC peptides to the bacterial cells and incubate for 2 hours. The control group is amphotericin B at a concentration of MIC. Collect the bacterial cells by centrifugation.

[0051] The bacterial cells were washed three times with PBS solution, centrifuged to collect the cells, and 0.1 g of wet cells were taken. 3 mL of 25% KOH-ethanol solution was added, vortexed for 2 min, and incubated at 85℃ for 4 hours. After cooling to room temperature, 1 mL of deionized water and 3 mL of n-heptane were added for extraction, vortexed for 3 min, and allowed to stand at room temperature for 1 hour to separate the layers. 1.5 mL of the upper n-heptane layer was taken, centrifuged at 12000 rpm for 10 min, and then 1 mL of the supernatant was transferred to an EP tube for liquid chromatography analysis. The results are shown in the figure. Figure 8 .

[0052] from Figure 8 As can be seen, 2 μM NLR-2 and NLR-3 significantly reduced Cryptococcus neoformans.

[0053] Crytococcus Neoformans

[0054] The ergosterol content of 32609, from Figure 8 b shows that 32 μM NLR-2 and 16 μM NLR-3 significantly reduced Fusarium graminearum.

[0055] Fusarium graminearum

[0056] The ergosterol content of 37687 indicates that NLR-2 and NLR-3 effectively inhibit... Crytococcus Neoformans 32609、 Fusarium graminearum Synthesis of 37687 ergosterol. Example 7

[0057] Fusarium graminearum spore suspension was inoculated into PDB medium to achieve a spore concentration of 1×10⁻⁶. 6 CFU / mL, 28℃, 150 r / min, culture for 72 h. Mycelia were washed with HEPES solution, and equal masses of mycelia were resuspended in NLR-2 (32 μM) and NLR-3 (16 μM) and incubated for 2 h. Mycelia were then collected. Single colonies of *Cryptococcus neoformans* were picked and cultured overnight in potato dextrose broth, then transferred to fresh potato dextrose broth and grown to mid-log phase. The fungal solution was then centrifuged and resuspended in phosphate buffer to a final concentration of OD200. 600nm =0.4, add the prepared antimicrobial peptide to a final concentration of 2 μM, and incubate at room temperature for 1 hour.

[0058] The aforementioned hyphae / cells were fixed overnight in 2.5% glutaraldehyde at 4°C. After sequential dehydration (50%, 70%, 90%, and 100%), 600 μL of tert-butanol-ethanol (1:1, v / v) and total tert-butanol were added and mixed for 15 min. The mixture was then lyophilized and coated. Finally, the samples were observed using a Hitachi S-4800 scanning electron microscope.

[0059] from Figure 9 As can be seen, in the control group, *Cryptococcus neoformans* and *Fusarium graminearum* maintained their intact membrane structures with smooth and intact surfaces. In the peptide-treated group, the antifungal peptides attached to the periphery of *Cryptococcus neoformans* and *Fusarium graminearum*, increasing outer membrane permeability and penetrating the outer membrane to reach the plasma membrane. This caused depolarization of the plasma membrane, thereby disrupting its integrity, leading to leakage of fungal contents, cell shrinkage, and ultimately, fungal death.

Claims

1. Two dual-target antifungal peptides NLR-2, NLR-3, characterized in that, Its amino acid sequences are shown in SEQ ID No.1 and SEQ ID No.2, respectively. Its N-terminus is linked to the carboxyl group of 3-(1-naphthyl)acrylic acid, targeting the two key enzymes SE / CYP51 of ergosterol on the fungal membrane.

2. The method for preparing the two dual-target antifungal peptides NLR-2 and NLR-3 according to claim 1, characterized in that, The steps are as follows: Step S1: Using a heptapeptide repeat sequence template (LKAIRRR) n n = 2, 3, which act as hydrophobic supports to provide hydrophobic forces; Step S2: Select 3-(1-naphthyl)acrylic acid to link with it to construct a polypeptide; Step S3: The peptides were prepared by solid-phase chemical synthesis and mass spectrometry identification. The nanomorphic characteristics, in vitro cytotoxicity, hemolytic activity, bactericidal activity, membrane fluidity, and ergosterol content on fungal membranes of the peptides were then determined. Finally, they were named antifungal peptides NLR-2 and NLR-3.

3. The self-assembly method of two dual-target antifungal peptides NLR-2, NLR-3 according to claim 1, characterized in that: At a concentration of 64 μM, incubation at 37°C for 24 hours allows it to self-assemble into nanostructures.

4. The use of two dual-target antifungal peptides NLR-2, NLR-3 according to claim 1 in the preparation of antifungal drugs, characterized by: Under physiological conditions, it has a killing effect on Cryptococcus neoformans and Fusarium graminearum.

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