KR100232642B1 - Antifungal peptide against trichosporon beigelii - Google Patents

Abstract

The present invention relates to the use of a synthetic peptide consisting of an amphiphilic site, a flexible site and a hydrophobic site as an antifungal peptide that acts on a strain of Trichosporon beigelii, which is a pathogenic fungus, and the antifungal peptide of the present invention. Low cytotoxicity can be widely used to prevent and treat white piedra caused by tricosphorone base jelly.

Description

Antifungal Peptides Act on Trichosporon beiglii

The present invention relates to antifungal peptides that act on Trichosporon beigelii and the like.

More specifically, the present invention relates to the use of the synthetic peptide consisting of an amphiphilic site, a flexible site and a hydrophobic site as an antifungal peptide acting on the pathogenic fungus Tricosporon Bay jelly strain, etc. Peptides have low cytotoxicity and can be widely used for preventing and treating white piedra caused by tricosphorone base jelly.

Tricosporon beigelii is a pathogenic fungus that infects human hair, beards, eyebrows, armpits, pubic hair, etc., when it is softened, hair becomes viscous, and the color itself is viscous. It is known to cause a disease called white piedra, which turns into a light brownish white color and damages the hair itself (Avram, A., et al., Dermatol. Venereol., 114: 819, 1987; Kreger-van Rij, NJW, Amsterdam, Elsevier Science Publishers., 933-962, 1984). Infections represented by these tricosphorone base jelly are widely found in Europe, Asia, and South America, regardless of temperate or tropical climate, and rarely in the southern part of the United States. In addition, Tricosporon Bay Jelly has been found in spider monkeys and horses, but it is known that it is not contagious (Kaplan, W., J. Am. Vet. Med. Assoc., 184: 113, 1959). In particular, such hirsutism is found in young men (Kalter, D. C., et al., J. Am. Acad. Dermatol., 14: 982, 1986).

Fungal infections, including tricosphorone bay jelly, have a limit in developing antifungal agents that act on metabolic processes due to the characteristics of fungi, which are eukaryotic cells, unlike bacterial infections, and many new attempts have been made to develop therapeutics for them. .

Amphipathic peptide (amphipathic peptide) has a structure similar to the lipid component of the cell membrane and has the property of binding to the lipids of the cell membrane. These amphiphilic peptides are reported to destroy microorganisms by binding to the cell membrane lipids of the microorganisms and destroying the cell membranes of the microorganisms or by affecting and changing the potential of the cell membranes.

Therefore, many studies have been made on the antimicrobial activity of the amphiphilic peptides, and many studies have been attempted to develop antibiotics for the bacteria using the affinity peptides. Representative amphiphilic peptides reported to date include magainin 2 (MA 2) peptides, meellitin (ME) peptides and cecropin A peptides.

First, the marganine 2 (amino acid sequence: GIGKFLHSAKKFGKAFVGEIM NS) peptide has been known to have antifungal, anticancer and probiotic effects with antimicrobial activity without cytotoxicity (Zosloff, M., Proc. Natl. Acad. Sci. USA, 84 : 5449, 1987). The melittin (amino acid sequence: GIGAVLKVLTTGLPALISWIK RKQQ) peptide is a peptide that accounts for more than 50% of solids in the components of bee venom. It was reported to be well destroyed and high antimicrobial activity against microorganisms Gram-negative and positive bacteria (Habermann, E., Science, 177: 314, 1972; Steiner, H. et al., Nature, 292: 246, 1981; Tosteson , MT, et al., Biochemistry., 228: 337, 1987).

In addition, the secropin family of amphiphilic peptides was first found in Drosophila and subsequently in silkworm pupa and small intestine of pigs. Antifungal and anticancer activity has been reported to be insignificant (Boman, HG and Hultmark, D., Annu. Rev. Microbiol., 41: 103, 1987).

In addition to the above studies on the activity of the amphiphilic peptides and the characteristics of their amino acid sequence and protein structure, the characteristics of the sequences are closely related to the antibacterial, antifungal and anticancer activity of the peptides. You can check it. Thus, by using the amino acid sequence of the amphiphilic peptide to substitute a similar amino acid at a specific site of the sequence, or by recombining some sequences to produce a fusion peptide (hybrid peptide), or by inverting some functional sites of the peptide sequence It is possible to prepare new synthetic peptides with better antibacterial, antifungal or anticancer activity (Chan, HC, et al., FEBS Lett., 259: 103, 1989; Boman, HG, et al., FEBS Lett. 259: 103, 1989; Wade, D., et al., Int. J. Pept. Prot. Res., 40: 429, 1992).

Practically, synthetic peptides mag A and mag G, etc., having anticancer effects, have been produced by applying the amphiphilic peptides, and their efficacy has been reported (Ohsaki, et al., Cancer Res., 52: 3534-3538, 1992). ). In addition, synthetic peptides have been developed that exhibit antifungal activity with an affinity region of marganine 2 and melittin peptides and an amino acid sequence of a flexible and hydrophobic region, which are particularly specific to the genus Aspergillus. It has been known to function and has been patented as an antifungal peptide (Korean Patent Application No. 96-27996). In addition, the inventors of the present invention have prepared fusion peptides having an amino acid sequence recombining the respective sites, such as conventional secropin A, maganin 2 or melittin peptide, and confirmed the excellent anti-cancer effect and have applied for a patent (Korea Patent Application No. 96-52673).

Against this background, the present inventors have studied various synthetic peptides having amphiphilicity to develop new antifungal agents that can be used in the human body. Thus, the inventors of SEQ ID NO: 1 and SEQ ID NO: 2 have already been reported to have anticancer effects as recombinant melittin peptides. The synthetic peptide (Korean Patent Application No. 96-52673) was found to be effective in the pathogenic fungus Tricosporon beigelii and the like, and their antifungal activity and cytotoxicity were confirmed to find new antifungal peptides. The present invention has been completed by providing a use.

It is an object of the present invention to provide an antifungal peptide that can be used in the human body.

Specifically, the present invention provides an amphiphilic peptide having an amino acid sequence of SEQ ID NO: 1 and SEQ ID NO: 2 and having an anticancer effect as an antifungal peptide. The present invention can include all peptides in which some sequences are added to the synthetic peptide having the amino acid sequence or substituted with similar amino acids as antifungal peptides, and antifungal in which the amino group at its amino terminus or the carboxyl group at the carboxyl terminus is substituted with another functional group. Sex peptides.

It is an object of the present invention to provide an antifungal peptide that specifically acts on Trichosporon beigelii strains.

In addition, the present invention provides a use of the pharmaceutical composition comprising the antifungal peptide as an active ingredient for the prevention and treatment of infection caused by the pathogenic fungus Tricosporon beigelii. Specifically, the antifungal peptide of the present invention may be used as a therapeutic agent for hirsutism, and may be used to prevent the infection by adding to detergents such as shampoos.

Figure 1 shows that the synthetic peptide of the present invention by inhibiting the growth of tricosporon Bay jelly cells by MTT method.

PC: positive control; NC: negative control, ME: melittin; CA: cecropin; S: synthetic peptide of SEQ ID NO: 2.

Figure 2 is a photograph showing the number of viable cells by treating the synthetic peptides of the present invention to tricosporon Bay jelly cells and then plated in YM agar medium.

A: positive control; B: cecropin; C: melittin; D: Synthetic peptide of SEQ ID NO: 2.

In the present invention, in order to develop an amphiphilic peptide having no cytotoxicity and exhibiting excellent antifungal activity, various synthetic peptides composed of amphiphilic sites, flexible sites and hydrophobic sites are investigated.

Specifically, in the present invention, the amphiphilic region (SECROPIN SEQ ID NO: 1-8) of the cecropin is located at the amino terminal, and the flexible portion and the hydrophobic portion of the melittin (SEQ ID NO: 1-12) is located at the rear thereof. A synthetic peptide consisting of 20 amino acids (see SEQ ID NO: 1; see Korean Patent Application No. 96-52673) and threonine of amino acids 18 and 19 located at its carboxy terminus with lysine. Examination of the synthetic peptide (see SEQ ID NO: 2) confirms excellent antifungal activity. In this case, the present invention uses an antifungal peptide wherein the carboxy terminus of the peptide is aminated.

The present invention may include all synthetic peptides added with some amino acid sequences or substituted with similar amino acids as the antifungal peptides to the amphiphilic synthetic peptides, and the antifungals having an amino group or a carboxyl group at the amino terminal thereof substituted with other functional groups. Sex peptides.

In addition, the present invention confirms that the antifungal peptides exhibit excellent antifungal activity specifically to Trichosporon beigelii, which are pathogenic fungi.

The peptide of the present invention is synthesized by the Merrifield solid phase method using a Fmoc amino group protective container (Merrifield, RBJ Am. Chem. Soc. 85: 2149, 1963). Peptide synthesized above was separated and purified by HPLC column using reversed-phase C ₁₈ column, and its molecular weight was measured by Matrix-Assisted Laser Desorption Ionization (MALDI) mass spectrometry to confirm whether the same peptide as designed was synthesized (Hill , et al., Rapid Commin, Mass Spectrometry, 5: 395, 1991).

In order to investigate the antifungal effect of the synthetic peptides prepared in the present invention, growth minimum inhibitory concentration (MIC), growth inhibition of the cells, cytotoxicity, and the like are measured.

In order to investigate the antifungal activity of the synthetic peptides of the present invention, the minimum growth concentration, the minimum concentration of the peptide that the cells cannot grow, and the growth inhibitory ability of the cells by the peptides were measured.

It is cultured in YM medium using a pathogenic fungus Trichosporon beigelii strain and the like, and inoculated onto micro titrate plate with an appropriate cell number, followed by addition of the synthetic peptide of the present invention. MTT (3- [4,5 [Dimethylthiazol-2yl] -2,5-diphenyl tetrazolium bromide) was added to measure the growth inhibitory concentration to the extent that color was developed (see Table 1 and FIG. 1). In addition, the cells were cultured by treating the synthetic peptide under the same conditions, and the growth inhibitory concentration was measured in the non-peptide group and the peptide-treated group by the MTT method to measure the growth inhibitory ability at the ratio thereof (see Table 2 and FIG. 2).

As a result, it was observed that the synthetic peptide of the present invention inhibited the growth of the cells, and in particular, the synthetic peptide of SEQ ID NO: 2 compared to 100% of the conventional melittin compared with the peptide showing the inhibition ability of 93.4% at the concentration of 3 µg / ml. It can be seen that the inhibitory ability.

In order to confirm whether the synthetic peptide of the present invention is cytotoxic, hemolysis capacity is examined. Human red blood cells are diluted with a phosphate-sodium chloride buffer solution and the like, and the synthetic peptide is mixed and reacted, followed by centrifugation, and the hemoglobin contained in the supernatant is measured by absorbance at 414 nm. The erythrocyte destructive capacity, which can determine the degree of cell destruction, is calculated by comparing the absorbance of 1% Triton-X100 with 100% cell destructive capacity and comparing it with the measured value (see Table 3). As a result, it was confirmed that the synthetic peptide of SEQ ID NO: 2 of the present invention had almost no cytotoxicity even at a concentration of 50 µg / ml.

As a result, it can be seen that the peptide of the present invention exhibits strong antifungal activity without showing cytotoxicity. The synthetic peptides of the present invention can be prepared and used in various forms of suspending agents in pharmaceutical aspects.

The invention is illustrated in more detail by the following examples.

The following examples illustrate the invention and are not intended to limit the scope of the invention.

Example 1 Peptide Design, Synthesis, Separation and Purification

The synthetic peptide of the present invention was synthesized by the Merrifield solid phase method using a Fmoc amino group protecting vessel in the reactor developed by the inventors (Merrifield, RB, J. Am. Chem. Soc., 85: 2149, 1963) .

The present invention is a sequence consisting of 20 amino acids by placing SEQ ID NO: 1-8, the affinity region of the existing secroffin, at the amino terminus of the synthetic peptide, and SEQ ID NO: 1-12, the flexibility and hydrophobic region of melittin, at the carboxy terminus. Synthetic peptides having an amino acid sequence of 1 were prepared. In addition, a peptide having the amino acid sequence of SEQ ID NO: 2 was designed by designing a peptide in which threonine Nos. 18 and 19 located at the carboxy terminus of the peptide thus designed was substituted with lysine. At this time, the carboxy terminus of these peptides was aminated (amidation).

Separation of each synthetic peptide by separation HPLC and analysis of the purity by analytical HPLC revealed that the molecular weight obtained by using MALDI mass spectrometry was 95% or higher and matched with the calculated molecular weight. It was confirmed that the peptide was synthesized.

Example 2 Growth Inhibition Concentration of Peptides

In order to investigate the antifungal activity of the synthetic peptide of the present invention, the growth inhibitory concentration, which is the minimum peptide concentration at which the cells are not split, was measured.

Tree course isophorone bay jelly (Trichosporon beigelii), a YM medium incubated at (0.3% yeast extract, 0.3% malteu extract, 0.5% peptone, 1% dextrose), and then by measuring the number of cells of 5 × 10 ⁴ / 100㎕ Cell concentrations were diluted in YM medium and inoculated in micro titrate plates. Thus, the synthetic peptides were added to each of 10, 5, 2.5 and 1.25 µg / ml, and incubated at 28 ° C. for 30 hours, followed by MTT (3- [4,5-dimethylthiazol-2-yl] -2,5-diphenyltetrazolium bromide 10μl (5mg / ml) and 10μl of 10X Sodium Phosphate-Sodium Chloride Buffer (PBS, pH 7.0) were added for 12 hours at 37 ℃ and the color developed at 570nm using a microplate reader. Absorbance was measured. As a result, the growth minimum inhibition concentration (MIC) was measured and shown in Table 1.

TABLE 1

Peptide Minimum Growth Inhibition Concentration for Tricosphoron Bay Jelly (MIC, μg / ml) Secropin ND Melittin 5 Synthetic Peptides of SEQ ID NO: 1 5 Synthetic Peptides of SEQ ID NO: 2 2.5

As a result of the measurement, the synthetic peptide of SEQ ID NO: 2 of the present invention compared to the conventional melittin (ME) showed a 2-fold lower growth inhibitory concentration, so that the antifungal activity was increased than the existing amphiphilic peptide.

Example 3 Measurement of Growth Inhibition of Cells

In order to measure the antifungal activity of the synthetic peptide of the present invention, the growth inhibitory ability of the mycelia by the peptide was measured in the same manner as in Example 2.

Cells were cultured by treating the synthetic peptides under the same conditions as in Example 2, and the growth inhibition was measured by the MTT method in the non-peptide group and the peptide-treated group. As a result, it was observed that the synthetic peptide of the present invention inhibited the growth of the cells, and in particular, the synthetic peptide of SEQ ID NO: 2 compared with the conventional melittin showing a 93.4% inhibitory ability at a peptide concentration of 3 µg / ml. It could be seen that. Table 2 shows the results.

TABLE 2

Peptide Growth Inhibitory Capacity for Tricosphoron Bay Jelly (%) Secropin 0 Melittin 93.4 Synthetic Peptides of SEQ ID NO: 1 58.6 Synthetic Peptides of SEQ ID NO: 2 100

Example 4 Cytotoxicity of Peptides

In order to confirm whether the synthetic peptide of the present invention is cytotoxic, erythrocyte destructive ability was examined.

Dilute human erythrocytes to 4% with 0.5 ml of sodium phosphate-sodium chloride buffer (PBS, pH 7.4), mix 0.5 ml of 400 ㎍ / ml synthetic peptide solution for 1 hour, and react at 37 ° C for 1 hour. The amount of hemoglobin contained in the supernatant by centrifugation at xg was investigated by measuring the absorbance at 414 nm. In order to investigate the degree of cell destruction, absorbance was measured by adding 1% Triton-X100, and the cell destruction capacity of 1% Triton-X100 was determined as 100%, which was calculated by comparing the hemolysis capacity of each synthetic peptide. . The results are shown in Table 3.

TABLE 3

Peptide Human erythrocyte destructive capacity (%) (50 µg / ml) Secropin A 0 Melittin 100 Synthetic Peptides of SEQ ID NO: 1 5.4 Synthetic Peptides of SEQ ID NO: 2 0.4

Synthetic peptide of SEQ ID NO: 2 of the present invention was found to show little cytotoxicity, such as cecropin, synthetic peptide of SEQ ID NO: 1). On the other hand, melittin (melittin), a bee venom used as a control, was very high at 100% cytotoxicity.

Since the antifungal peptides of SEQ ID NO: 1 and SEQ ID NO: 2 of the present invention are already known to have anti-cancer effects, they are expected to be greatly used as peptides having anti-fungal and anti-cancer effects. In particular, the antifungal peptide of SEQ ID NO: 2 has high antifungal activity and little cytotoxicity compared to conventional melittin (ME) and can be usefully used as an antifungal agent safe for human body. Specifically, it can be used as a therapeutic agent for alopecia caused by Tricosporon beigelii, and can be widely used in addition to a shampoo detergent as the preventive agent.

(Sequence list)

SEQ ID NO: 1

Sequence length: 20

Type of sequence: amino acid

Number of chains: 1 chain

Form: Straight

Type of sequence: Peptide

[SEQ ID NO 1]

(Sequence list)

SEQ ID NO: 2

Sequence length: 20

Type of sequence: amino acid

Number of chains: 1 chain

Form: Straight

Type of sequence: Peptide

[SEQ ID NO 2]

Claims (5)

An antifungal peptide having an affinity for use in a human body, characterized by having the amino acid sequence of SEQ ID NO: 1. An antifungal peptide having an affinity for use in a human body, characterized by having an amino acid sequence of SEQ ID NO: 2. A therapeutic and prophylactic agent for the infection caused by a Trichosporon beigelii strain comprising the antifungal peptide of claim 1 or 2 as an active ingredient. 4. The therapeutic and prophylactic agent for infection caused by Trichosporon beigelii strains according to claim 3, wherein the infection is white piedra. 4. The therapeutic and prophylactic agent for infections caused by Trichosporon beigelii strains according to claim 3, which is used in addition to detergents.

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