Bacterium-friendly selective antifungal peptide HRI and application thereof
By designing an antifungal peptide HRI with the amino acid sequence HRIWHIKIH, selective antifungal activity without targeting symbiotic bacteria was achieved, inhibiting MMP-9. This solves the problem of insufficient selectivity in existing antifungal drugs and improves treatment efficacy and bacterial balance.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- TECHNICAL INST OF PHYSICS & CHEMISTRY - CHINESE ACAD OF SCI
- Filing Date
- 2026-01-05
- Publication Date
- 2026-07-07
AI Technical Summary
Existing antifungal drugs lack selectivity against pathogenic fungi and symbiotic bacteria, leading to dysbiosis and drug resistance, making it difficult to effectively treat fungal infections.
We designed a microbiome-friendly selective antifungal peptide, HRI, with the amino acid sequence HRIWHIKIH. It has the property of selectively resisting pathogenic fungi but not symbiotic bacteria, and can inhibit matrix metalloproteinase MMP-9 to maintain microbiome balance.
It effectively combats fungi while maintaining the balance of the gut microbiota, reduces side effects, inhibits MMP-9 activity, lowers the risk of drug resistance, and improves the treatment effect of infections.
Smart Images
Figure CN121471309B_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of novel antibacterial materials technology. More specifically, it relates to a microbiome-friendly selective antifungal peptide, HRI, and its applications. Background Technology
[0002] Fungal infections, a long-underestimated global public health challenge, have garnered significant attention in recent years due to their high morbidity and mortality rates. The World Health Organization (WHO) lists Cryptococcus neoformans, Candida auris, Aspergillus fumigatus, and Candida albicans as "critical priority" pathogens. These pathogens typically possess multiple virulence factors, enabling them to colonize multiple sites in the body and cause corresponding diseases through immune evasion mechanisms and biofilm formation. In hospital settings, filamentous fungi (molds) and Candida are the two most common pathogens. Candida-related infections often begin with local colonization on mucosal surfaces (such as the oral cavity, pharynx, and genital tract). However, in immunocompromised individuals, patients with chronic underlying diseases, or those in intensive care units, these initially localized infections can easily develop into life-threatening invasive fungal diseases, further exacerbating the disease burden and treatment challenges.
[0003] Currently, the treatment of fungal infections mainly relies on four classes of antifungal drugs: polyenes, azoles, echinocandins, and pyrimidine analogs such as 5-fluorocytosine. Although these four classes of drugs are widely used in clinical treatment, their relatively simple antibacterial mechanisms make them highly susceptible to drug resistance in fungi.
[0004] Antimicrobial peptides (AMPs) are naturally occurring antimicrobial molecules widely found in plants, animals, humans, and microorganisms. They are characterized by abundant sources, structural diversity, broad-spectrum antimicrobial activity, and ease of production. In recent years, antimicrobial peptides have attracted widespread attention as a novel antimicrobial strategy due to their diverse antimicrobial mechanisms and low likelihood of inducing drug resistance. On the one hand, they can inhibit the synthesis of proteins and nucleic acids; on the other hand, they can induce cell membrane disturbances through different structures such as barrel-shaped, carpet-shaped, and ring-shaped structures, leading to the rupture of bacterial or fungal biofilms. However, the application of natural antimicrobial peptides is greatly limited due to drawbacks such as high hemolytic toxicity, poor protease stability, and high synthesis costs. More importantly, most natural antifungal peptides rely on nonspecific membrane targeting mechanisms, resulting in a lack of selectivity between pathogenic fungi and beneficial symbiotic bacteria. This "indiscriminate attack" can disrupt the protective microbial barrier, causing dysbiosis and creating favorable conditions for secondary colonization of pathogens, forming a vicious cycle of treatment-dysbiosis-reinfection. For example, when treating oral candidiasis, it is necessary to avoid killing the commensal bacteria *Streptococcus pyogenes*; even in the intestinal environment, any broad-spectrum antifungal agent, if it indiscriminately kills the intestinal commensal flora, may lead to more serious consequences such as *Clostridium difficile* infection. Therefore, addressing these issues through the rational design of antimicrobial peptides is currently a hot research topic. Summary of the Invention
[0005] The first objective of this invention is to design a novel antifungal peptide that is selectively effective against pathogenic fungi but not against bacteria, especially symbiotic bacteria.
[0006] A second objective of this invention is to provide the application of the above-mentioned antifungal peptide in selectively combating pathogenic fungi.
[0007] A third object of the present invention is to provide a drug comprising the above-mentioned antifungal peptide.
[0008] To achieve the above objectives, the present invention adopts the following technical solution:
[0009] In a first aspect, the present invention provides a microbiome-friendly selective antifungal peptide HRI, wherein the amino acid sequence of the antifungal peptide HRI is: HRIWHIKIH.
[0010] Furthermore, the carboxyl terminus of the antifungal peptide HRI is amidated.
[0011] The antifungal peptide HRI of this invention also has an important function: it exhibits excellent inhibitory effects on matrix metalloproteinase-9 (MMP-9). During fungal infection, overexpressed MMP-9 degrades key components such as type IV collagen in the basement membrane and extracellular matrix, making it easier for fungal hyphae to invade and penetrate tissues. The antifungal peptide HRI of this invention inhibits MMP-9 activity by binding to it, helping to maintain the integrity of the host's physical barrier and reducing infection-related tissue damage, demonstrating significant advantages over existing antifungal drugs.
[0012] Furthermore, the antifungal peptide HRI exhibits selectivity between pathogenic fungi and symbiotic bacteria, meaning it can fight only pathogenic fungi in fungal infections without fighting beneficial symbiotic bacteria. This helps maintain flora balance, reduce side effects, and improve the effectiveness of infection treatment.
[0013] Furthermore, the pathogenic fungus is a yeast.
[0014] Furthermore, the yeast fungus is Candida albicans or Cryptococcus neoformans.
[0015] Furthermore, the symbiotic bacteria is Streptococcus pyogenes.
[0016] In a second aspect, the present invention provides the use of the above-mentioned antifungal peptide HRI in at least one of the following:
[0017] 1) Application in the preparation of products that inhibit the activity of pathogenic fungi or kill pathogenic fungi;
[0018] 2) Use in the preparation of products for the prevention and / or treatment of diseases caused by pathogenic fungal infections.
[0019] Furthermore, the pathogenic fungus is a yeast.
[0020] Furthermore, the yeast fungus is Candida albicans or Cryptococcus neoformans.
[0021] Furthermore, the diseases mentioned include candidal vaginitis, oral candidiasis, fungal dermatitis, or fungal enteritis, etc.
[0022] Thirdly, drugs containing the aforementioned antifungal peptide HRI also fall within the scope of protection of this invention. In addition to the aforementioned antifungal peptide HRI, the drugs also include pharmaceutically acceptable excipients, and are formulated into various dosage forms, such as ointments, patches, gels, liniments, lotions, aerosols, sprays, drops, suppositories, enemas, vaginal tablets, effervescent tablets, and injections.
[0023] The beneficial effects of this invention are as follows:
[0024] This invention provides a microbiome-friendly selective antifungal peptide HRI, which can fight fungi while maintaining microbiome balance and is less likely to induce drug resistance. It has high biosafety and a certain inhibitory effect on matrix metalloproteinases. Attached Figure Description
[0025] The specific embodiments of the present invention will be described in further detail below with reference to the accompanying drawings.
[0026] Figure 1 The image shows a helical projection of the HRI peptide.
[0027] Figure 2 The circular dichroism chromatogram of the HRI peptide is shown.
[0028] Figure 3 The inhibitory activity of the antifungal peptide HRI against the MMP-9 enzyme was demonstrated.
[0029] .
[0030] Figure 4 The cytotoxicity of the antifungal peptide HRI is demonstrated. Detailed Implementation
[0031] To more clearly illustrate the present invention, the following description, in conjunction with preferred embodiments and accompanying drawings, further clarifies the invention. Those skilled in the art should understand that the specific description below is illustrative rather than restrictive and should not be construed as limiting the scope of protection of the present invention.
[0032] The names and sequences of the antifungal peptides used in each embodiment are as follows:
[0033] HRI: HRIWHIKIH.
[0034] The antifungal peptides mentioned above were obtained through machine learning screening and can be prepared using known synthetic methods in the prior art, such as solid-phase synthesis.
[0035] Example 1: Design of the antifungal peptide HRI
[0036] The helical wheel projection of the HRI peptide was generated from the HeliQuest website (http: / / heliquest.ipmc.cnrs.fr). For example... Figure 1 As shown, light blue represents histidine (H), dark blue represents arginine (R) and lysine (K), and yellow represents other hydrophobic amino acids; the arrows indicate the direction of the hydrophobic torque, and their lengths indicate the magnitude of the hydrophobic torque. HRI is composed of 1 arginine, 3 isoleucines, 3 histidines, 1 lysine, and 1 tryptophan.
[0037] Example 2: Circular dichroism spectroscopy of the antifungal peptide HRI
[0038] The peptides were diluted to appropriate concentrations with ultrapure water, 25 mM sodium dodecyl sulfate (SDS), and 50% trifluoroacetic acid (TFE), respectively, and allowed to stabilize overnight at room temperature. The peptides were then added to a quartz cuvette with a 1 cm path length, and the CD values in the range of 190–250 nm were measured using a circular dichroism spectrometer (JASCO-J815).
[0039] like Figure 2 As shown, the peptide exhibits a polyproline type II (PPII) conformation in water, with a large negative peak at 197 nm and a small positive peak at 220 nm. Under biofilm simulation conditions (SDS and TFE), it can transform into an α-helix conformation, exhibiting a negative characteristic peak at 207 nm and a weak negative characteristic peak around 220 nm. The antifungal peptide can disrupt membrane integrity through the α-helix conformation, ultimately leading to fungal cell lysis and death.
[0040] Example 3: Antifungal performance test of antifungal peptide HRI
[0041] The antifungal activity of the antifungal peptide HRI against fungi (Candida albicans ATCC10231, Candida albicans SC5314, and Cryptococcus neoformans var. grubii ATCC208821) was determined by microbroth method.
[0042] First, culture the fungus in the logarithmic growth phase. Single colonies were picked and placed in the appropriate culture medium (C. albicans on Sabouraud dextrose agar, C. neoformans on YM medium), and incubated at 30°C with shaking at 160 rpm for 24 h until the logarithmic growth phase. The fungal suspension was then diluted to 10⁻¹⁰ with the appropriate liquid medium. 3 CFU / mL was prepared for use. Sample preparation utilized a serial dilution method. First, 200 μL of the antifungal peptide HRI solution was added to the first column of wells in a 96-well plate, and 100 μL of physiological saline was added to the remaining wells. 100 μL was then added from each subsequent column of wells until the tenth column was reached; after mixing, 100 μL was discarded. 100 μL of the solution was added to each well containing different concentrations of the antifungal peptide HRI. 3 CFU / mL bacterial suspension. A negative control was prepared using a mixture of 100 μL physiological saline and 100 μL liquid culture medium. 3 A CFU / mL bacterial suspension was used as a positive control. The treated 96-well plates were incubated at 30°C for 24 hours, and the OD620 value was measured using a microplate reader. The lowest antifungal peptide HRI concentration at which the antibacterial rate was greater than 90% or the wells showed a visible change from turbid to clear was recorded as the minimum inhibitory concentration (MIC).
[0043] The minimum fungicidal concentration (MFC) of the antifungal peptide HRI was determined using a spot plate test. 3.5 μL of bacterial culture was taken from each well and added to the appropriate agar medium. After the medium was allowed to dry, it was inverted and incubated at 30°C for 24 hours. Colony growth was observed, and the concentration at which no colonies grew was defined as the MFC.
[0044] The antifungal peptide HRI showed excellent antifungal activity against fungi, with a minimum MIC of 2 μg / mL. See Table 1.
[0045] Table 1: Antifungal activity of the antifungal peptide HRI (μg / mL)
[0046]
[0047] Example 4: Antibacterial performance test of antifungal peptide HRI
[0048] The antifungal peptide HRI was determined using the microbroth method against Gram-positive bacteria (Staphylococcus aureus). S. aureus ATCC6538), Gram-negative bacteria (Pseudomonas aeruginosa) P. aeruginosaThe antibacterial activity of PAO1 and symbiotic bacteria (Streptococcus pyogenes BNCC357872).
[0049] First, culture the bacteria in the logarithmic growth phase. Pick a single colony and place it in the appropriate medium (TSB for Staphylococcus aureus, LB for Pseudomonas aeruginosa, and BHI for Streptococcus pyogenes, microaerophilic culture), and incubate at 37°C with shaking for 18-24 hours until the logarithmic growth phase. Dilute the bacterial suspension to 10⁻⁶ using cationic-adjusted Mueller Hinton II medium. 5 CFU / mL was prepared for use. Sample preparation utilized a serial dilution method. First, 200 μL of the antifungal peptide HRI solution was added to the first column of wells in a 96-well plate, and 100 μL of physiological saline was added to the remaining wells. 100 μL was then added from each subsequent column until the tenth column was reached; after mixing, 100 μL was discarded. 100 μL of the solution was added to each well containing different concentrations of the antifungal peptide HRI. 5 CFU / mL bacterial suspension. A negative control was prepared using a mixture of 100 μL physiological saline and 100 μL cationic-adjusted MH medium, and a negative control was prepared using a mixture of 100 μL physiological saline and 100 μL 10... 5 A CFU / mL bacterial suspension was used as a positive control. The prepared 96-well plates were incubated at 37°C for 24 hours, and the OD620 value was measured using a microplate reader. For Streptococcus suis, the 96-well plates containing the sample and bacterial suspension were placed in an anaerobic chamber with a microaerophilic gas-generating bag to ensure a microaerophilic environment, and then incubated at 37°C for 24 hours. The lowest antifungal peptide concentration (HRI) at which the antibacterial rate was greater than 90% or the wells visibly changed from turbid to clear was recorded as the minimum inhibitory concentration (MIC).
[0050] The minimum bacterial concentration (MBC) of the antifungal peptide HRI was determined using a spot plate test. 3.5 μL of bacterial culture was taken from each well and added to the appropriate agar medium. After the medium was allowed to dry, it was inverted and incubated at 37°C for 24 hours. Colony growth was observed, and the concentration at which no colonies grew was defined as the MBC.
[0051] The antifungal peptide HRI was determined to have no significant antibacterial activity against either Gram-positive Staphylococcus aureus or Gram-negative Pseudomonas aeruginosa, but both its MIC and MBC were very high (see Table 2).
[0052] More importantly, the antifungal peptide HRI of the present invention exhibits excellent selective antimicrobial activity between pathogenic Candida albicans and symbiotic Streptococcus pyogenes, with a selective index (SI), i.e., MIC (symbiotic bacteria) / MIC (pathogenic fungi), of 1024, as shown in Table 3.
[0053] Table 2: Antibacterial activity of antifungal peptide HRI (μg / mL)
[0054]
[0055] Table 3: Selectivity index of antifungal peptide HRIs for target fungi and symbiotic bacteria
[0056]
[0057] Example 5: Inhibitory effect of antifungal peptide HRI on MMP-9
[0058] First, activate the MMP-9 protein. Specifically, dissolve the MMP-9 protein in 0.2 mL of assay buffer to obtain a stock solution with a concentration of 100 ng / μL. Then, dilute the stock solution to 10 ng / μL with assay buffer for later use. Dilute 1 M APMA 500-fold with assay buffer to obtain a 2 mM APMA working solution. Prepare the enzyme reaction solution at 25 μL per well and 5 ng of enzyme per well, and incubate at 37°C for 2 h to activate MMP-9.
[0059] The antifungal peptide HRI sample was prepared into a working solution of 256 μg / mL using ultrapure water. 25 μL of activated enzyme reaction solution and 25 μL of sample solutions of different concentrations were added to each well of a 96-well plate. An equal volume of Assay Buffer was used as a negative control, and a mixture of activated enzyme reaction solution and Assay Buffer was used as a positive control. The reaction was carried out at 37℃ for 10 min. After incubation, MMP Green Substrate was diluted 1:100 with Assay Buffer to obtain the substrate working solution. 50 μL of the substrate working solution was added to each well, and the reaction was continued at room temperature in the dark for 30 min. The fluorescence intensity at Ex / Em = 490 / 525 nm was detected using a microplate reader. The inhibitory effect of MMP-9 was calculated using the following formula:
[0060]
[0061] OD525 (sample) indicates a well containing MMP-9 protease and the sample;
[0062] OD525 (positive) indicates that the well contains MMP-9 protease and detection buffer;
[0063] OD525 (negative) indicates that the well contains only the detection buffer.
[0064] Tests showed that, at the same sample concentration, the antifungal peptide HRI exhibited superior inhibitory effects on MMP-9 compared to the clinically commonly used MMP inhibitor doxycycline (DOX) (see reference: Payne JB, et al. Curr Oral Health Rep. 2015;2(1):20-29.). Figure 3 .
[0065] Example 6 Cytotoxicity of the antifungal peptide HRI
[0066] L929 cells frozen in liquid nitrogen were rapidly thawed and revived in a 37°C water bath. Then, an appropriate amount of DMEM medium containing 10% FBS and 1% P / S was added, and the cells were cultured for 2-3 passages in a 37°C, 5% CO2 incubator. After the cells adhered to the culture flask and reached 80% confluence, they were digested with 500 μL of trypsin, resuspended in 2 mL of DMEM medium, and counted.
[0067] Except for the negative control group, cells were seeded at a density of 8000 cells per well in 96-well plates and incubated for 24 h to allow cell adhesion and growth. DMEM was used to prepare peptide solutions. The old culture medium in the 96-well plates was then discarded and replaced with 100 μL of DMEM containing different concentrations of the samples. 100 μL of pure DMEM was added to the positive control group, and 100 μL of PBS buffer was added to the negative control group. After culturing for another 24 h, the old medium was aspirated, and 100 μL of DMEM containing 10% CCK-8 was added to each well. The plates were incubated at 37°C for 30 min–1 h. The absorbance at 450 nm was measured using a microplate reader, and cell viability was calculated using the following formula:
[0068]
[0069] OD450 (peptide) indicates a well containing L929 cells, a peptide, and culture medium;
[0070] OD450 (positive) indicates that the well contains L929 cells and culture medium;
[0071] OD450 (negative) indicates that the well contains only culture medium.
[0072] The results are as follows Figure 4As shown in Table 4, the IC50 value of the peptide HRI is 1.170 mg / mL, and its selectivity index (Selectivity = IC50 / MIC90) is 585, indicating that its toxicity to mammalian cells is also very low.
[0073] Table 4: Selectivity index of peptides (IC50 / MIC)
[0074]
[0075] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation of the present invention. For those skilled in the art, other variations or modifications can be made based on the above description. It is impossible to exhaustively list all the implementation methods here. All obvious variations or modifications derived from the technical solutions of the present invention are still within the protection scope of the present invention.
Claims
1. A microbiome-friendly selective antifungal peptide HRI, characterized in that, The amino acid sequence of the antifungal peptide HRI is: HRIWHIKIH.
2. The antifungal peptide HRI according to claim 1, characterized in that, The antifungal peptide HRI is amidated at its carboxyl terminus.
3. The antifungal peptide HRI according to claim 1, characterized in that, The antifungal peptide HRI has a matrix metalloproteinase inhibitory effect.
4. The antifungal peptide HRI according to claim 1, characterized in that, The antifungal peptide HRI is selective between pathogenic fungi and symbiotic bacteria.
5. The antifungal peptide HRI according to claim 4, characterized in that, The pathogenic fungus is a yeast; The yeast fungus is Candida albicans or Cryptococcus neoformans.
6. The antifungal peptide HRI according to claim 4, characterized in that, The symbiotic bacteria are Streptococcus pyogenes.
7. The use of the microbiome-friendly selective antifungal peptide HRI as described in any one of claims 1-6 in at least one of the following: 1) Application in the preparation of products that inhibit the activity of pathogenic fungi or kill pathogenic fungi; 2) Use in the preparation of products for the prevention and / or treatment of diseases caused by pathogenic fungal infections; The pathogenic fungus is a yeast; The yeast fungus is Candida albicans or Cryptococcus neoformans.
8. The application according to claim 7, characterized in that, The diseases mentioned are candidal vaginitis, oral candidiasis, fungal dermatitis, or fungal enteritis.
9. A drug for antifungal use, characterized in that, The drug comprises the microbiome-friendly selective antifungal peptide HRI as described in any one of claims 1-6.
10. The medicament according to claim 9, characterized in that, The dosage forms of drugs include ointments, patches, gels, liniments, lotions, aerosols, sprays, drops, suppositories, enemas, vaginal tablets, effervescent tablets, or injections.