Antifungal peptides and pharmaceutical compositions
A novel antifungal peptide with a compact amino acid sequence addresses production inefficiencies and resistance issues by offering potent antifungal activity and synergistic potential with existing drugs.
Patent Information
- Authority / Receiving Office
- JP · JP
- Patent Type
- Applications
- Current Assignee / Owner
- Filing Date
- 2024-09-20
- Publication Date
- 2026-04-02
AI Technical Summary
Conventional antifungal peptides, such as Pezadeftide and OsAFP1, suffer from low production efficiency due to their large number of amino acids, and there is a lack of diversity in mechanisms of action, leading to the emergence of drug-resistant fungal strains.
Development of an antifungal peptide with a specific amino acid sequence of 7 to 13 residues, particularly with the sequence RKCFCKK, which can be amidated at the C-terminus, and optionally combined with additional sequences at the N-terminus, exhibiting antifungal activity and improved production efficiency.
The peptide achieves high antifungal activity against Candida and budding yeasts, reduces production costs, and prevents the development of drug-resistant strains through a distinct mechanism of action, potentially enhancing effects when combined with conventional antifungal drugs.
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Abstract
Description
Technical Field
[0001] The present invention relates to an antifungal peptide and a pharmaceutical composition.
Background Art
[0002] Mycoses are roughly classified into superficial mycoses such as pediculosis and deep mycoses such as Aspergillus pneumonia.
[0003] Superficial mycoses are serious diseases that are said to affect one in five to ten people, causing itching, skin redness, inflammation, etc., and greatly affecting the lives of patients.
[0004] Conventionally, for the treatment of superficial mycoses, five types of antifungal drugs, namely imidazole-based, morpholine-based, thiocarbamate-based, allylamine-based, and benzylamine-based, have been mainly used.
[0005] Examples of imidazole-based antifungal drugs include lanoconazole and luliconazole. Examples of morpholine-based antifungal drugs include amorolfine hydrochloride. Examples of thiocarbamate-based antifungal drugs include liranaftate. Examples of benzylamine-based antifungal drugs include butenafine hydrochloride. Examples of allylamine-based antifungal drugs include terbinafine hydrochloride.
[0006] Although lanoconazole, luliconazole, amorolfine hydrochloride, liranaftate, and butenafine hydrochloride have different pathways, they all ultimately inhibit the growth of fungi bacteriostatically by suppressing the synthesis of ergosterol, which is one of the cell membrane components. In addition, terbinafine hydrochloride selectively inhibits squalene epoxidase in fungal cells, resulting in the accumulation of squalene and a decrease in ergosterol content, showing an antifungal effect.
[0007] As described above, all of the antifungal drugs used for superficial mycoses have mechanisms of action that act on cell membrane components, and their lack of diversity has led to the problem of drug-resistant strains emerging.
[0008] On the other hand, Non-Patent Document 1 describes Pezadeftide as a modified plant-derived defensin. Pezadeftide has antifungal activity by inducing hyperpolarization of the mitochondrial membrane or causing the production of reactive oxygen species, which may explain why resistant strains are less likely to emerge.
[0009] Furthermore, Non-Patent Document 2 describes OsAFP1, a defensin that exhibits antifungal activity in the innate immune system of rice. OsAFP1 potently induces apoptosis in Candida fungi at a concentration of 16 μM. This mechanism of action is not observed in conventional antifungal drugs. [Prior art documents] [Non-patent literature]
[0010] [Non-Patent Document 1] Matthew JA H et al., Defensin-lipid interactions in membrane targeting: mechanisms of action and opportunities for the development of antimicrobial and anticancer therapeutics. Biochemical Society Transactions, 50, 423-437, 2022.
[0011] [Non-Patent Document 2] A. Ochiai. et al., Rice Defensin OsAFP1 is a New Drug Candidate against Human Pathogenic Fungi. Scientific Reports, 8, 11434, 2018. [Overview of the project] [Problems that the invention aims to solve]
[0012] However, Pezadeftide, described in Non-Patent Document 1, is a polypeptide consisting of 51 amino acids linked by peptide bonds, and has the problem of low production efficiency due to its large number of amino acids. Similarly, OsAFP1, described in Non-Patent Document 2, induces apoptosis in fungi, and although its mechanism of action differs from the aforementioned imidazole-based antibacterial agents, it is a polypeptide consisting of 49 amino acids, and therefore also suffers from the problem of low production efficiency.
[0013] The present invention has been made in view of these circumstances, and aims to provide an antifungal peptide with higher production efficiency compared to conventional peptides having antifungal activity, and a pharmaceutical composition containing the same as an active ingredient. [Means for solving the problem]
[0014] As a result of diligent research to achieve the above objective, the inventors discovered that the amino acid sequence in the region near the C-terminus of OsAFP1 has high antifungal activity, and thus completed the present invention. That is, one aspect of the present invention that solves the above problem includes the following aspects.
[0015] [1] An antifungal peptide comprising the amino acid sequence shown in SEQ ID NO: 1, consisting of 7 to 13 amino acid residues, and possessing antifungal activity. (However, if the amino acid sequence shown in SEQ ID NO: 1 is located at the C-terminus of the antifungal peptide, the carboxyl group at the C-terminus of the amino acid sequence may be amidated.)
[0016] [2] The antifungal peptide according to [1], comprising an amino acid sequence obtained by linking, to the N-terminus of the amino acid sequence shown in SEQ ID NO: 1, one amino acid sequence selected from the group consisting of the amino acid sequences shown in SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6.
[0017] [3] The antifungal peptide according to [1], consisting of the amino acid sequence shown in SEQ ID NO: 1, the amino acid sequence shown in SEQ ID NO: 7, the amino acid sequence shown in SEQ ID NO: 8, the amino acid sequence shown in SEQ ID NO: 9, the amino acid sequence shown in SEQ ID NO: 10, the amino acid sequence shown in SEQ ID NO: 11, or the amino acid sequence shown in SEQ ID NO: 12.
[0018] [4] A pharmaceutical composition comprising the antifungal peptide according to any one of [1] to [3] and a pharmaceutically acceptable carrier.
Advantages of the Invention
[0019] According to the present invention, it is possible to provide an antifungal peptide having a smaller number of amino acids and a higher production efficiency compared to conventional peptides having antifungal activity, and a pharmaceutical composition containing the same as an active ingredient.
Brief Description of the Drawings
[0020] [Figure 1] FIG. 1 is a drawing showing the amino acid sequences of eight partial peptide fragments of OsAFP1. [Figure 2] FIG. 2 is a drawing showing the MIC measured for OsAFP1, peptide fragment R7, and a variant of peptide fragment R7, and the regions overlapping with each peptide fragment in the amino acid sequence of OsAFP1. [Figure 3] FIG. 3 is a graph showing the survival rate of Saccharomyces cerevisiae BY4742 strain in the presence of antifungal peptide 7. [Figure 4]Figure 4 is a graph showing the survival rate of Candida albicans CAI4 strain in the presence of antifungal peptide 7 measured in Experimental Example 2. [Figure 5] Figure 5 is a microscopic image after incubating fungi in the presence of antifungal peptide 7 and an apoptosis detection reagent. [Figure 6] Figure 6 is a graph showing the transition of the number of viable cells of fungi incubated in the presence of antifungal peptide 7 and an apoptosis detection reagent and the number of cells of fungi induced to apoptosis. [Figure 7] Figure 7 is an image showing the result of subjecting antifungal peptide 7 and OsAFP1 to a lipid binding assay. [Figure 8] Figure 8 is a graph showing the result of Gene Ontology analysis of a group of genes whose expression increased by the addition of antifungal peptide 7. [Figure 9] Figure 9 is a graph showing the result of Gene Ontology analysis of a group of genes whose expression decreased by the addition of antifungal peptide 7. [Figure 10] Figure 10 is a drawing showing seven genes for which the MIC of antifungal peptide 7 against fungi in which genes with large changes in gene expression by the addition of antifungal peptide 7 were knocked out was more than twice the MIC against the same wild-type fungi. [Figure 11] Figure 11 is a graph showing the ratio of the number of viable bacteria of fungi incubated in the presence of fluconazole and antifungal peptide 7.
Mode for Carrying Out the Invention
[0021] Hereinafter, preferred embodiments of the present invention will be described in detail while referring to the drawings as appropriate.
[0022] A preferred embodiment of the present invention is an antifungal peptide comprising the amino acid sequence shown in SEQ ID NO: 1, consisting of 7 to 13 amino acid residues, and possessing antifungal activity. However, if the amino acid sequence shown in SEQ ID NO: 1 is located at the C-terminus (C-terminal region) of the antifungal peptide, the carboxyl group at the C-terminus of the amino acid sequence shown in SEQ ID NO: 1, i.e., the carboxyl group of the lysine (K) residue located at the C-terminus, may be amidated. Sequence ID 1:RKCFCKK
[0023] An example of an amino acid sequence in which the carboxyl group at the C-terminus of the amino acid sequence shown in Sequence ID No. 1 is amidated is the amino acid sequence shown in Sequence ID No. 12, but is not limited to this. Sequence ID 12: HGLERKCFCKK-NH2 The "-NH2" designation indicates that the C-terminal carboxyl group is amidated.
[0024] As will be described in detail later in the examples, the peptide containing the amino acid sequence shown in SEQ ID NO: 1 has antifungal activity. For this reason, the antifungal peptide of this embodiment, which contains the amino acid sequence shown in SEQ ID NO: 1 and has 13 or fewer amino acid residues, has higher production efficiency and can significantly reduce the cost required for peptide synthesis compared to conventional peptides that have antifungal activity. The antifungal peptide may consist only of the amino acid sequence shown in SEQ ID NO: 1, or it may be a peptide in which amino acid residues are further added to the N-terminus or C-terminus of the amino acid sequence shown in SEQ ID NO: 1. The antifungal peptide of this embodiment may be an artificially synthesized peptide.
[0025] In this specification, antifungal activity refers to the effect of inhibiting, suppressing, or killing the growth of one or more fungi. Specifically, when the MIC (Minimum Inhibitory Concentration) of a peptide is measured in the same manner as in Experimental Example 2 of the Examples described in detail later, the peptide is considered to have antifungal activity if the MIC value is 256 μM (mol / L) or less.
[0026] As will be detailed later in the examples, the antifungal peptide of this embodiment has been shown to exhibit antibacterial activity against Candida pathogens and budding yeasts. Since Candida pathogens and budding yeasts are quite distantly related species in the phylogenetic tree, even though they are both fungi, the antifungal peptide of this embodiment is expected to have antibacterial activity against a wide range of fungal species.
[0027] The antifungal peptide may be an antifungal peptide used to inhibit the growth of fungi, an antifungal peptide used to inhibit the growth of Candida fungi (antimicrobial peptide against Candida fungi), an antifungal peptide used to inhibit the growth of Candida albicans (antimicrobial peptide against Candida albicans), an antifungal peptide used to inhibit the growth of Saccharomyces fungi (antimicrobial peptide against Saccharomyces fungi), or an antifungal peptide used to inhibit the growth of Saccharomyces cerevisiae (antimicrobial peptide against Saccharomyces cerevisiae).
[0028] Furthermore, antifungal peptides can be used as antibacterial agents on their own, or they can exert antibacterial effects by being mixed with fibers, polymer materials, membranes, etc.
[0029] As will be detailed later in the examples, the antifungal peptide of this embodiment exerts high antifungal activity by inducing apoptosis in fungi. This mechanism of action is the same as that of OsAFP1's antibacterial activity. Thus, because the antifungal peptide of this embodiment has a different mechanism of action from conventional antifungal drugs such as imidazoles, it is possible to suppress the development of drug-resistant strains.
[0030] The antifungal peptide of this embodiment may be used in combination with conventional antifungal drugs such as fluconazole. By using the antifungal peptide in combination with conventional antifungal drugs that have a different mechanism of action, a higher antifungal effect can be obtained through a synergistic effect.
[0031] The antifungal peptide preferably contains an amino acid sequence in which one amino acid sequence selected from the group consisting of the amino acid sequence shown in SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6 is appended to the N-terminus of the amino acid sequence shown in SEQ ID NO: 1. In particular, the antifungal activity of the antifungal peptide can be enhanced by including any of SEQ ID NOs from 2 to 6 at its N-terminus. Sequence ID 2: HGLE Sequence ID 3: KSHGLE Sequence ID 4: HGLR Sequence ID 5: HGLL Sequence ID 6: SHGLE
[0032] In an antifungal peptide consisting of an amino acid sequence in which one amino acid sequence selected from the above group is attached to the N-terminus of the amino acid sequence shown in Sequence ID No. 1, the amino acid residue one C-terminal to the C-terminus of each amino acid sequence constituting the above group is R (arginine), which is located at the N-terminus of the amino acid sequence shown in Sequence ID No. 1.
[0033] For example, the amino acid sequence shown in Sequence ID No. 2, when concatenated to the N-terminus of the amino acid sequence shown in Sequence ID No. 1, is the amino acid sequence shown in Sequence ID No. 7 below. Sequence ID 7: HGLERKCFCKK
[0034] Furthermore, the amino acid sequence obtained by concatenating the amino acid sequence shown in SEQ ID NO: 3 to the N-terminus of the amino acid sequence shown in SEQ ID NO: 1 is the amino acid sequence shown in SEQ ID NO: 9 below. Sequence ID 9: KSHGLERKCFCKK
[0035] Furthermore, the amino acid sequence obtained by concatenating the amino acid sequence shown in SEQ ID NO: 4 to the N-terminus of the amino acid sequence shown in SEQ ID NO: 1 is the amino acid sequence shown in SEQ ID NO: 10 below. Sequence ID 10: HGLRRKCFCKK
[0036] Furthermore, the amino acid sequence obtained by concatenating the amino acid sequence shown in SEQ ID NO: 5 to the N-terminus of the amino acid sequence shown in SEQ ID NO: 1 is the amino acid sequence shown in SEQ ID NO: 11. Sequence ID 11: HGLLRKCFCKK
[0037] Furthermore, the amino acid sequence obtained by concatenating the amino acid sequence shown in SEQ ID NO: 6 to the N-terminus of the amino acid sequence shown in SEQ ID NO: 1 is the amino acid sequence shown in SEQ ID NO: 13 below. Sequence ID 13: SHGLERKCFCKK
[0038] Peptides having the amino acid sequence shown in Sequence ID No. 8, which is obtained by adding VC to the C-terminus of the amino acid sequence shown in Sequence ID No. 1, also exhibit excellent antifungal activity. Sequence ID 8:RKCFCKKVC
[0039] Furthermore, examples of peptides according to this embodiment include peptides consisting of the amino acid sequence shown in SEQ ID NO: 21 and peptides consisting of the amino acid sequence shown in SEQ ID NO: 22. Sequence ID 21: GLERKCFCKK Sequence ID 22: LERKCFCKK
[0040] For each of the above amino acid sequences, as long as the total number of amino acids is within the range of 13 or less and the antifungal activity described above is observed, it is possible to add further amino acid residues to the N-terminus or C-terminus, or to amidate the carboxyl group at the C-terminus.
[0041] As will be shown in the examples later, antifungal activity can be improved by amidating the C-terminal carboxyl group. This is likely because amidation confers resistance to fungal degrading enzymes such as proteases, but it is not limited to any specific theory.
[0042] [Pharmaceutical composition] Another preferred embodiment of the present invention comprises an antifungal peptide as detailed in the above embodiment and a pharmaceutically acceptable carrier. The pharmaceutical composition of this embodiment contains the above-mentioned antifungal peptide as the active ingredient for antifungal activity.
[0043] A "pharmaceutically acceptable carrier" means a carrier that does not inhibit the physiological activity of the active ingredient and does not exhibit substantial toxicity to the target of administration. "Substantial toxicity" means that the ingredient does not exhibit toxicity to the target of administration at the doses normally used. In the pharmaceutical composition of this embodiment, the pharmaceutically acceptable carrier is a carrier that does not inhibit the antifungal activity of the antifungal peptide according to the above embodiment and does not exhibit substantial toxicity to the target of administration. A pharmaceutically acceptable carrier includes all known pharmaceutically acceptable ingredients that are typically considered inactive ingredients. A pharmaceutically acceptable carrier is not particularly limited, but examples include solvents, diluents, bases, vehicles, excipients, flow promoters, binders, granulators, dispersants, suspending agents, wetting agents, lubricants, disintegrants, solubilizers, stabilizers, emulsifiers, fillers, etc. A pharmaceutically acceptable carrier may be used alone or in combination of two or more types.
[0044] The pharmaceutical composition may contain other components in addition to the above components. The other components are not particularly limited, and any components commonly used in the pharmaceutical field may be used without particular restriction. Examples of other components include pharmaceutical additives other than those mentioned above. Examples of pharmaceutical additives include, but are not limited to, preservatives (e.g., antioxidants), chelating agents, flavoring and odor-masking agents, sweeteners, thickeners, buffering agents, and coloring agents. The pharmaceutical composition may contain active ingredients other than the antifungal peptide according to the above embodiment. Examples of active ingredients include, but are not limited to, other antifungal agents, antibiotics, anti-inflammatory agents, antipyretics, and analgesics.
[0045] The dosage form of the pharmaceutical composition is not particularly limited and can be any dosage form commonly used for pharmaceutical preparations. The pharmaceutical composition of this embodiment may be an oral preparation or a parenteral preparation, but a parenteral preparation is preferred. Examples of oral preparations include tablets, coated tablets, pills, powders, granules, capsules, syrups, fine granules, liquids, drops, emulsions, etc. Examples of parenteral preparations include injections, suppositories, nasal drops, enteral preparations, ointments, creams, topical solutions, inhalants, etc. Pharmaceutical compositions of these dosage forms can be formulated according to standard methods (for example, methods described in the Japanese Pharmacopoeia).
[0046] The pharmaceutical composition can administer a therapeutically effective amount of the antifungal peptide according to the above embodiment. "Therapeutic effective amount" means the amount of drug effective for treating or preventing the target disease. For example, the therapeutically effective amount of the antifungal peptide may be an amount effective in inhibiting or killing the growth of fungi. The therapeutically effective amount may be appropriately determined based on the patient's symptoms, weight, age, and sex, as well as the dosage form and method of administration of the pharmaceutical composition.
[0047] The pharmaceutical composition may be administered as a single dose or as a repeated dose. In the case of repeated administration, the administration interval should be appropriately determined based on the patient's symptoms, weight, age, and sex, as well as the dosage form and method of administration of the pharmaceutical composition. For example, the administration interval may be every few hours, two to three times a day, once a day, once every two to three days, once a week, once a month, once every few months, etc.
[0048] The pharmaceutical composition and the antifungal peptide described above may be a pharmaceutical composition or antifungal peptide for use in the treatment or prevention of superficial mycoses, or a pharmaceutical composition or antifungal peptide for use in the treatment or prevention of deep mycoses, but it is preferable that they be a pharmaceutical composition or antifungal peptide for use in the treatment or prevention of superficial mycoses.
[0049] Furthermore, the pharmaceutical composition and the antifungal peptide described above may be a pharmaceutical composition or antifungal peptide used for the treatment or prevention of superficial mycoses caused by fungi of the genus Candida, a pharmaceutical composition or antifungal peptide used for the treatment or prevention of superficial mycoses (such as candidiasis) caused by Candida albicans, or a pharmaceutical composition or antifungal peptide used for the treatment or prevention of superficial mycoses caused by Ascomycetes.
[0050] Examples of superficial fungal infections that can be treated with the pharmaceutical composition of this embodiment include, but are not limited to, dermatophytosis such as athlete's foot and candidiasis.
[0051] The pharmaceutical composition of this embodiment can be prepared as follows, as an example of a topical ointment.
[0052] First, melt 5g of beeswax, which is used as a thickener and moisturizer, in a double boiler, then add 50g of white petrolatum, which is used as a base and moisturizer, and mix until uniform.
[0053] Next, 5 mL of glycerin (as a thickening and moisturizing agent), 10 mL of purified water, and 5 mL of ethanol (as a thickening and penetration enhancer) are mixed in a separate container and gradually added to the beeswax and petrolatum mixture. The mixture is then stirred until it has completely cooled. The resulting ointment is then placed in a sterile container and sealed, for example.
[0054] Although preferred embodiments have been described in detail above, the present invention is not limited to these embodiments, and various modifications are possible within the scope of the invention as described in the claims. [Examples]
[0055] The present invention will be described below with reference to examples, but the present invention is not limited to the following examples. In these examples, OsAFP1 was synthesized using a strain of E. coli transformed by introducing a plasmid, while the other peptides were chemically synthesized. The purity of the peptides was 95% or higher.
[0056] [Experimental Example 1] In this experiment, because OsAFP1 (see SEQ ID NO: 14 for its amino acid sequence) has a large number of amino acid residues, eight fragments that are parts of OsAFP1 but in different regions were prepared to facilitate synthesis, and the antifungal activity of each peptide fragment was measured. Sequence ID 14: RHCLSQSHRFKGMCVSSNNCANVCRTESFPDGECKSHGLERKCFCKKVC
[0057] Figure 1 shows the amino acid sequences of the eight partial peptide fragments R1-R8 of OsAFP1. Table 1 below shows the antifungal activity (IC) of each peptide fragment R1-R8. 50 ) is shown. IC as referred to here 50 This refers to the turbidity of the bacterial cells (OD) in the presence of peptide fragments. 650 ) is the bacterial turbidity (OD) in the absence of peptide fragments. 650 This is the peptide fragment concentration that is 50% or less of the total peptide fragment concentration.
[0058] [Table 1]
[0059] As shown in Table 1, high antifungal activity was observed in peptide fragment R1 located at the N-terminus of OsAFP1 and peptide fragment R7 located near the C-terminus. Furthermore, separate examination of interactions with the cell membrane revealed that peptide fragment R1 has a different mechanism of action than OsAFP1, while peptide fragment R7 retains a similar mechanism of action to OsAFP1.
[0060] [Experimental Example 2] In this experiment, the antifungal activity (MIC) of peptide fragment R7 and its variants, which showed similar activity to OsAFP1 in Experimental Example 1, was measured as follows. One peptide fragment or fluconazole was used as a positive control group in each system, and Candida albicans strain CAI4 was used as the fungus. In addition, half-concentration PDB liquid medium containing uridine at a concentration of 25 μg / mL was used as the culture medium for the fungus.
[0061] First, 1 mg of each peptide was dissolved in pure water, and the concentration was adjusted to 10, 20, 40, 80, 160, 320, 640, or 1280 μM.
[0062] 20 μL of the peptide solution thus obtained, and 80 μL of fungal culture medium that had been pre-cultured twice (final turbidity OD) 650 Each well of a 96-well microculture plate was added to a solution of 0.01 (0.01). This allowed for final concentrations of 0, 2, 4, 8, 16, 32, 64, 128, or 256 μM. Alternatively, fluconazole was mixed with the fungal culture medium using the same procedure instead of the peptide solution to create a baseline positive control group.
[0063] Subsequently, 96-well microculture plates were incubated at 30°C for 16 hours, and the number of viable fungi (CFU: colony forming unit) in each well was measured. The growth inhibition rate (%) of the fungi was calculated, with the group with a peptide concentration of 0 μM set as 100%. More specifically, the growth inhibition rate was calculated by dividing the number of viable fungi in the presence of each peptide by the number of viable fungi in the group with a peptide concentration of 0 μM, multiplying by 100 to obtain the survival rate [%], and then subtracting this from 100%. The peptide concentration at which the growth inhibition rate was 95% or higher (in other words, the growth of the test fungi was suppressed to 5% or less) was defined as the MIC.
[0064] Figure 2 shows the measured MICs for OsAFP1, peptide fragment R7, and a modified version of peptide fragment R7, as well as the regions in the amino acid sequence of OsAFP1 that overlap with each peptide fragment.
[0065] As shown in Figure 2, high antifungal activity was confirmed for peptides consisting of the amino acid sequences of SEQ ID NOs: 1, 7, 8, 9, 10, 11, 12, and 16, which have the amino acid sequence shown in SEQ ID NO: 1. Therefore, it was suggested that the amino acid sequence shown in Sequence ID No. 1 is essential for exhibiting high antifungal activity.
[0066] Furthermore, particularly high antifungal activity was confirmed for peptides consisting of the amino acid sequences shown in SEQ ID NOs: 7, 9, 10, 11, and 12, which are created by concatenating the amino acid sequences shown in SEQ ID NOs: 2, 3, 4, or 5 to the N-terminus of the amino acid sequence in SEQ ID NO: 1. Therefore, the amino acid sequences shown in SEQ ID NOs: 2, 3, 4, or 5 were suggested to potentially enhance the antifungal activity of peptides having the amino acid sequence shown in SEQ ID NO: 1.
[0067] Furthermore, the peptide consisting of the amino acid sequence shown in SEQ ID NO: 12, in which the amino acid sequence shown in SEQ ID NO: 2 is attached to the N-terminus of the amino acid sequence in SEQ ID NO: 1 and the carboxyl group at the C-terminus is amidated, showed higher antifungal activity compared to the peptide consisting of the amino acid sequence shown in SEQ ID NO: 7, which is not amidated. These results suggest that amidation of the C-terminus of the peptide may improve its antifungal activity.
[0068] [Experimental Example 3] In this experiment, the survival rate of fungi was measured in the same manner as in Experimental Example 2, except that Saccharomyces cerevisiae strain BY4742 was used as the test fungus for the peptide consisting of the amino acid sequence shown in Sequence ID No. 7 (hereinafter also referred to as "antifungal peptide 7").
[0069] Figure 3 is a graph showing the survival rate of Saccharomyces cerevisiae strain BY4742 in the presence of antifungal peptide 7. Figure 4 is a graph showing the survival rate of Candida albicans strain CAI4 in the presence of antifungal peptide 7, as measured in Experimental Example 2. In Figures 3 and 4, "Control" represents the group without antifungal peptide 7, and the figures show the percentage of viable cells in each group with antifungal peptide 7 added, compared to the viable cell count in the Control group (100%).
[0070] As shown in Figure 3, antifungal peptide 7 suppressed the survival rate of Saccharomyces cerevisiae strain BY4742 to 5% or less at concentrations of 16 μM or higher.
[0071] Furthermore, as shown in Figures 2 and 4, antifungal peptide 7 was able to suppress the survival rate of Candida albicans CAI4 strain to 5% or less at concentrations of 32 μM or higher.
[0072] These results suggest that antifungal peptide 7 can inhibit the growth of both Candida fungi and budding yeast, thus demonstrating antifungal activity against a wide variety of fungi.
[0073] [Experimental Example 4] This experiment investigated the apoptosis-inducing effect of antifungal peptide 7. Half-concentration PDB liquid medium containing uridine at a concentration of 25 μg / mL was used as the culture medium for the fungi.
[0074] In detail, first, 1 mg of antifungal peptide 7 was dissolved in pure water and diluted to a concentration of 640 μM.
[0075] 20 μL of the antifungal peptide 7 solution obtained in this way, and 80 μL of culture medium of the fungus (Candida albicans strain CAI4) that had been pre-cultured twice (final turbidity OD) 650 (=0.01) was added to a 1.5 mL microcentrifuge tube and thoroughly mixed. The final concentration of antifungal peptide 7 at this time was 128 μM, which is four times the MIC.
[0076] Next, the microcentrifuge tubes were incubated at 30°C, and 150 μL of the bacterial suspension was withdrawn at 0, 0.5, 1, 2, and 4 hours. 0.3 μL of CaspACE® FITC-VAD-FMK solution (apoptosis detection reagent, Promega) was added, and the mixture was incubated at 30°C for 30 minutes.
[0077] The samples were then washed twice with PBS to remove excess FITC-VAD-FMK solution, and fluorescence observation was performed using a fluorescence microscope equipped with a FITC filter (Ex=494nm, Em=518nm).
[0078] For over 100 fungal cells at each incubation time, total cell counting was performed under bright-field imaging and FITC fluorescence cell counting under dark-field imaging to calculate the percentage of fungal cells in which apoptosis was induced. However, only cells that emitted fluorescence as a whole were counted as cells in which apoptosis was induced. In addition, the number of viable fungal cells (CFU) at each incubation time was measured simultaneously with fluorescence observation, and the viability of the fungi was calculated by setting the number of viable cells in the control group (not treated with antifungal peptide 7) as 100%.
[0079] Figure 5 shows microscopic images of fungi after incubation in the presence of antifungal peptide 7 and an apoptosis detection reagent. Figure 5 shows bright-field and dark-field fluorescence microscopic images, with the bright areas in the dark-field image indicating areas that emit green fluorescence. Table 2 below shows the total number of fungal cells and the number of apoptotic fungal cells at different incubation times.
[0080] [Table 2]
[0081] Figure 6 is a graph showing the changes in the number of viable fungal cells and the number of apoptotic fungal cells incubated in the presence of antifungal peptide 7 and an apoptosis detection reagent.
[0082] As shown in Figure 6, it can be observed that as the incubation time increases, the number of viable fungal cells (CFU) decreases, while the proportion of cells that have undergone apoptosis (see dashed line) increases.
[0083] These results suggest that antifungal peptide 7 maintains the mechanism of action of antifungal activity through apoptosis induction of OsAFP1 before fragmentation, as there is a negative correlation between the number of viable fungal cells and the proportion of apoptotically induced cells.
[0084] [Experimental Example 5] In this experiment, a peptide-lipid binding assay was performed using PIPstrips to identify the target molecule of antifungal peptide 7 on the cell membrane. OsAFP1 before fragmentation was also subjected to the peptide-lipid binding assay.
[0085] Figure 7 shows images of the results of a lipid binding assay using antifungal peptide 7 and OsAFP1. As shown in Figure 7, antifungal peptide 7 was most strongly bound to PI(3)P (phosphatidylinositol-3-monophosphate). Similarly, OsAFP1 was also most strongly bound to PI(3)P, suggesting that antifungal peptide 7 retains the ability of OsAFP1 to bind to target molecules on the cell membrane before fragmentation.
[0086] [Experimental Example 6] In this experiment, transcriptome analysis using a microarray of Saccharomyces cerevisiae strain BY4742 was performed to confirm the effects of antifungal peptide 7 on the expression levels of various genes. The addition of antifungal peptide 7 resulted in increased expression of 64 genes (Fold change < 2) and decreased expression of 34 genes (Fold change > 0.5).
[0087] Figure 8 is a graph showing the results of gene ontology analysis of the gene group whose expression increased upon addition of antifungal peptide 7. Figure 9 is a graph showing the results of gene ontology analysis of the gene group whose expression decreased upon addition of antifungal peptide 7.
[0088] As shown in Figure 8, the genes whose expression increased upon the addition of antifungal peptide 7 included genes involved in cell wall organization and biosynthesis, ion transport, and oxidoreductase activity and transmembrane transport.
[0089] As shown in Figure 9, the genes whose expression was reduced by the addition of antifungal peptide 7 included genes involved in ion transport and intracellular amino acid metabolism, as well as genes involved in transmembrane transporter activity.
[0090] [Experimental Example 7] In this experiment, we measured the MIC (microactivity coefficient) of antifungal peptide 7 against a commercially available knockout strain of Saccharomyces cerevisiae BY4742, in which genes showing significant changes in gene expression upon addition of antifungal peptide 7 were knocked out.
[0091] Figure 10 shows seven genes in which the MIC of antifungal peptide 7 was more than double that of wild-type fungi of the same species when the genes in which antifungal peptide 7 was knocked out were found to have undergone significant changes in gene expression upon addition of antifungal peptide 7.
[0092] As shown in Figure 10, the study included genes involved in proteins that function near or on the cell membrane, such as YDR034W-B, ZRT1, and CTR1, as well as signaling genes such as YPK2. This result suggests that stress near the cell membrane caused by antifungal peptide 7 is involved in the survival or death of fungi.
[0093] From the above, it was suggested that in fungi exposed to antifungal peptide 7, the accumulation of antifungal peptide 7 on the cell membrane causes stress, which promotes zinc ion uptake by ZRT1 and copper ion uptake by CTR1. Furthermore, oxidative stress due to ROS production leads to protein modification of YPK2 by YKL107W, which in turn activates YPK2 signaling, and lipid transport by RTA1 is promoted through two pathways: the oxidative stress response of FRM2 and the subsequent modification of YPK2. Therefore, it is possible that apoptosis was induced by the activation of metal ion uptake by each ion transporter triggered by the accumulation of antifungal peptide 7, and by the activation of signaling due to ROS production.
[0094] [Experimental Example 8] In this experiment, we confirmed the synergistic effect between the conventional antifungal drug fluconazole and antifungal peptide 7.
[0095] In detail, first, 1 mg of antifungal peptide 7 was dissolved in pure water and diluted to concentrations of 80, 160, and 320 μM. Additionally, 10 mg of fluconazole was dissolved in pure water and diluted to concentrations of 50, 100, 200, 400, 800, and 1600 μM.
[0096] Next, 80 μL of culture medium of a fungus (Candida albicans CAI4 strain) that had been pre-cultured twice was placed in each well of a 96-well microculture plate (final turbidity OD). 650 (=0.01), 10 μL each of antifungal peptide 7 solution (final concentrations 8, 16, 32 μM) and fluconazole solution (final concentrations 0, 5, 10, 20, 40, 80, 160 μM) were added. Half-concentration PDB liquid medium containing uridine at a concentration of 25 μg / mL was used as the culture medium for the fungi. A bacterial suspension without antifungal peptide 7 and fluconazole was used as the control group.
[0097] Subsequently, 96-well microculture plates were incubated at 30°C for 16 hours, and the number of viable fungi (CFU) in each well was measured. The percentage of viable fungal cells was then calculated, with the control group set to 100%.
[0098] Figure 11 is a graph showing the percentage of viable fungi incubated in the presence of fluconazole and antifungal peptide 7. In Figure 11, "control" is the control group that did not receive fluconazole or antifungal peptide.
[0099] As shown in Figure 11, it can be seen that using antifungal peptide 7 in combination with fluconazole significantly enhances the antifungal effect compared to using fluconazole alone or antifungal peptide 7 alone (see Figure 4). Furthermore, the observed synergistic effect suggests that the antifungal activity of antifungal peptide 7 is due to a different mechanism of action than that of conventional antifungal drugs. Therefore, it is possible that using conventional antifungal drugs in combination with antifungal peptide 7 may suppress the development of drug-resistant strains. [Industrial applicability]
[0100] According to the present invention, an antifungal peptide can be provided that contains the amino acid sequence shown in SEQ ID NO: 1 and consists of 13 or fewer amino acid residues. Therefore, production efficiency can be significantly improved compared to conventional antifungal peptides. Consequently, it is suitable for industrial use. [Explanation of Symbols]
[0101] R1~R8...peptide fragments
Claims
1. Containing the amino acid sequence shown in Sequence ID No. 1, An antifungal peptide consisting of 7 to 13 amino acid residues and possessing antifungal activity. (However, if the amino acid sequence shown in SEQ ID NO: 1 is located at the C-terminus of the antifungal peptide, the carboxyl group at the C-terminus of that amino acid sequence may be amidated.)
2. At the N-terminus of the amino acid sequence shown in Sequence ID No. 1, The antifungal peptide according to claim 1, comprising an amino acid sequence obtained by concatenating one amino acid sequence selected from the group consisting of the amino acid sequence shown in SEQ ID NO: 2, the amino acid sequence shown in SEQ ID NO: 3, the amino acid sequence shown in SEQ ID NO: 4, the amino acid sequence shown in SEQ ID NO: 5, and the amino acid sequence shown in SEQ ID NO:
6.
3. The antifungal peptide according to claim 1, comprising the amino acid sequence shown in SEQ ID NO: 1, the amino acid sequence shown in SEQ ID NO: 7, the amino acid sequence shown in SEQ ID NO: 8, the amino acid sequence shown in SEQ ID NO: 9, the amino acid sequence shown in SEQ ID NO: 10, the amino acid sequence shown in SEQ ID NO: 11, or the amino acid sequence shown in SEQ ID NO:
12.
4. A pharmaceutical composition comprising an antifungal peptide according to any one of claims 1 to 3 and a pharmaceutically acceptable carrier.