JP3232676B2 - New peptides and antibacterial agents - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a novel peptide or a salt thereof and an antibacterial agent containing them as an active ingredient.

[0002]

2. Description of the Related Art In recent years, along with the development of protein engineering, research and development of not only conventional peptides obtained from nature but also synthesized peptides have been actively conducted.

[0003] The development of peptides having an antibacterial activity has been actively carried out conventionally, and the following reports have been made. For example, cyclic peptide derivatives (JP-A-58-21)
No. 3744), polypeptides useful as antibacterial agents against yeasts (JP-A-60-130599), novel glycopeptides (JP-A-61-251699), oligopeptides having an effect on Gram-positive bacteria ( JP-A-62-22798) and peptide antibiotics (JP-A-63-17897).

It is known that polypeptides containing a large amount of basic amino acids such as polylysine and protamine exhibit antibacterial action.

[0005]

However, many of these prior arts do not fall into the scope of basic research, suggesting that the development of peptide-based antibacterial agents is insufficient. In any case, a new antibacterial agent having a high effect on undesirable pathogens and other microorganisms is always desired, and the development of novel peptides that can be put to practical use as an antibacterial agent is important.

[0006]

Means for Solving the Problems From such a viewpoint, the present inventors have searched for useful novel peptides, and as a result, have found a peptide having a strong antibacterial action and completed the present invention. .

That is, the present invention relates to a novel peptide represented by the following amino acid sequence or a salt thereof:
It is an antibacterial agent containing them as an active ingredient.

Arg-Leu-Lys-Cys-Leu
-Leu-Leu-Cys-Arg-Leu-Lys-
Y (where Y is a carboxyl group (—COOH) of a lysine residue at the carboxyl terminus of the peptide of the present invention;
Alternatively, it represents a carbamyl group (—CONH ₂ ) in which the carboxyl group is amidated. ).

In the present invention, the amino acid is IUPAC-IUB
Abbreviations based on commission on Biological Nomenclature and conventional abbreviations in the field may be indicated, and examples thereof are as follows.

Arg: arginine, Leu: leucine,
Cys: cysteine, Lys: lysine.

That is, the present invention provides a novel peptide having a strong antibacterial activity and a salt thereof, and an antibacterial agent containing them as an active ingredient.

The peptide of the present invention comprises arginine (Ar
g) is an N-terminal, basic peptide composed of 11 amino acids, containing many basic amino acids such as arginine and lysine, and having a carboxyl terminal lysine (Lys). Also, peptides in which the carboxyl group of lysine, which is the C-terminal amino acid, exists as it is as a carboxyl group, and peptides in which the carboxyl group is amidated to exist as lysine amide have strong antibacterial activity. It is included in the invention.

In the present invention, these novel peptides having a carboxyl group at the C-terminus are referred to as T09.
8. The one in which the carboxyl group at the C-terminus is amidated is referred to as T098N.

The peptides T098 and T098N of the present invention
Can both take the form of salts, and include, for example, addition salts of organic or inorganic acids such as trifluoroacetic acid, methanesulfonic acid, hydrochloric acid, sulfuric acid, phosphoric acid and the like.

The peptide of the present invention can be easily synthesized by a solid phase method commonly used for peptide synthesis. For example, Journal of Chemical Society, Vol. 85, pp. 2149-2154 (1963)
Can be synthesized according to the method of Merrifield et al. It can also be synthesized by a commercially available peptide synthesizer or the like. In addition, the gene encoding the amino acid sequence of the peptide of the present invention can be produced in a large amount in microbial cells, plant cells, and animal cells using genetic engineering techniques. The resulting crude synthetic peptide was subjected to gel filtration, reverse phase HPL
C. Purification can be further enhanced by means used for ordinary protein / peptide purification such as ion exchange column purification.

The peptide of the present invention can be used as an antibacterial agent in various compositions depending on the purpose. That is, it can be used as an antibacterial agent alone or in combination with an appropriate liquid, solid, or the like. It can also be used in combination with other drugs. For example, it can be used as a preservative and disinfectant for contact lens cleaning solutions, ointments, soaps, shampoos, medicines, foods, feeds, pet foods, toothpastes, and the like.

The peptide of the present invention can be used as an agent for treating and preventing bacterial diseases. In this case, any administration method and dosage can be selected depending on the type and symptom of the disease, as long as the antimicrobial polypeptide of the present invention has activity.

When the peptide of the present invention is used as a pharmaceutical for an antibacterial agent, when a therapeutic agent containing an effective amount of the peptide of the present invention is clinically administered, it is administered by the oral or parenteral route. The dosage forms include tablets, dragees, pills, capsules, powders, troches, solutions, suppositories, injections, and the like, which are excipients and pharmaceutically acceptable excipients.
nt). The following can be exemplified as the excipient. Lactose, sucrose, glucose, sorbitol, mannitol, potato starch, amylopectin, other various starches, cellulose derivatives (eg, carboxymethylcellulose, hydroxyethylcellulose, etc.), gelatin, magnesium stearate, polyvinyl alcohol, calcium stearate, polyethylene glycol wax Vegetable oils such as gum arabic, talc, titanium dioxide, olive oil, peanut oil, sesame oil, paraffin oil, neutral fat base, ethanol, propylene glycol, physiological saline,
Sterile water, glycerin, colorants, seasonings, thickeners, stabilizers, isotonic agents, buffers, and the like, and other pharmaceutically acceptable excipients.

The antimicrobial agent of the present invention contains 0.001 to 85% by weight, preferably 0.005 to 60% by weight of the peptide of the present invention.
% By weight.

The dose of the therapeutic agent of the present invention mainly depends on symptoms, but it is 1 to 500 m / day of adult body weight.
g, preferably 1 to 50 mg.

Since the antibacterial agent of the present invention is a protein, it is considered that it has little toxicity at least when taken orally. Therefore, the peptide of the present invention is particularly useful in use as a drug for humans and animals and as a food or feed additive. For example, by adding the peptide of the present invention to foods such as gums and toothpaste, it is useful for sterilization in the oral cavity and prevention and treatment of caries and periodontal disease. In addition, in the culture of fish, it is possible to prevent the cultured fish from being infected by adding the peptide of the present invention to the feed used.

In other words, in other words, the peptide of the present invention can be used as a preservative or pharmaceutical preparation containing the substance as an active ingredient and as a food additive.

[0023]

The present invention will be described in more detail with reference to the following examples.

Example 1 Synthesis of Peptide The peptide of the present invention was synthesized by the solid phase method described below.

The functional groups other than the carboxyl group of the amino acid used in the peptide synthesis, that is, the amino group, the hydroxyl group, the guanidyl group, and the sulfhydryl group are those protected with a general protecting group commonly used in amino acid and peptide chemistry. Was. For example, an α-amino group is a t-butoxycarbonyl group (Boc group), an ε-amino group is a 2-chlorobenzyloxycarbonyl group, a hydroxyl group is a benzyl group, a guanidyl group is a tosyl group, and a sulfhydryl group is a p-methylbenzyl group. The protected amino acid was used.

First, a polystyrene resin in which 0.2 mmol of lysine, which is a carboxyl-terminal amino acid protected with a Boc group and a 2-chlorobenzyloxycarbonyl group, is bonded with the carboxyl group is sufficiently washed with methylene chloride. The Boc group was selectively removed using a solution of 50% trifluoroacetic acid in methylene chloride. Next, as viewed from the carboxyl terminal side of the peptide of the present invention, 3 mol equivalent of an amino acid (protected amino acid) corresponding to the next amino acid and having a functional group other than a carboxyl group protected, and dicyclohexylcarbodiimide (condensing agent) DCC) 3 molar equivalents to 4
The reaction was allowed to react for a period of time to form a peptide bond. After washing with methylene chloride, the above operation was repeated to sequentially synthesize a peptide from the carboxyl terminal. After the condensation of the amino acid corresponding to the amino group terminal was completed, all the protecting groups of the peptide synthesized with hydrogen fluoride were removed, and the synthesized peptide was cleaved from the resin.

The obtained crude peptide was purified by reverse phase HPLC. That is, 1/10 amount of the crude synthetic peptide was purified by HPLC under the following conditions, the main peak was collected, and further lyophilized to obtain 15 mg of peptide T098 of the present invention having a purity of 98% or more. .

Column: YMC-PACK (R-ODS-5 S-5 ODS 4.6
× 250 mm C18) Solvent: A: 0.1% trifluoroacetic acid aqueous solution, B: 0.1%
Acetonitrile trifluoroacetate solution Gradient conditions: 5 to 56% linear gradient of solution B Flow rate: 1.5 ml / min.

In the case of synthesis of T098N, Boc
Using a polystyrene resin in which 0.2 mmol of lysine amide, which is a carboxyl-terminal amino acid protected by a carboxyl group, is bound by the carboxyl group, the synthesis procedure is exactly the same as in T098, and the purification method The same method was used. As a result, 14 mg of purity 96.
5% or more of T098N was obtained.

The synthesized T098 and T098N were subjected to Edman degradation with a gas-phase amino acid sequencer (Applied Biosystems, Inc., Model 177A) to obtain PTH.
-The amino acid was identified and analyzed with a PTH-analyzer (Applied Biosystems, Inc., model 120A), and the amino acid sequence was confirmed. The results were as follows.

Arg-Leu-Lys-Cys-Leu
-Leu-Leu-Cys-Arg-Leu-Lys.

Example 2 Measurement of Antimicrobial Activity The antimicrobial activity was measured according to Journal of Biochemistry, Vol. 211, pp. 727-734 (1983).
Performed according to the method described. That is, each bacterium is
After shaking culture in an ic medium 3 (manufactured by Difco) medium, the concentration of the bacterium was adjusted so that the absorbance at 660 nm became about 0.3, and 10 μl thereof was added to 190 μl of the above-mentioned medium to obtain a test cell body fluid. After dissolving the lyophilized product of the peptide of the present invention in purified water, adding 200 μl of the peptide sample solution prepared by sequentially diluting with purified water, culturing at 37 ° C. for 4 hours, and measuring the absorbance at 660 nm. did. The test was performed using purified water instead of the sample solution. The antibacterial activity was expressed as the concentration of the peptide of the present invention when giving 50% growth compared to the growth of the target cells. The results are shown in Table 1.

[0033]

[Table 1]

As is clear from Table 1, it was found that the peptides T098 and T098N of the present invention exhibited strong antibacterial activity against a wide range of microorganisms including gram-positive bacteria, gram-negative bacteria and fungi.

[0035]

According to the present invention, a novel peptide can be provided. The novel peptide of the present invention has antibacterial activity and has little toxicity. Therefore, various uses utilizing this property are conceivable. In particular, use as an antibacterial agent or a food additive containing the present substance as an active ingredient can be expected. In particular, since this substance is a protein and has excellent heat stability, it is promising as an antibacterial food additive for foods requiring heat treatment in the processing step.

[0036]

[Sequence list] Sequence length: 11 Sequence type: amino acid Topology: linear Sequence type: peptide Sequence: Arg Leu Lys Leu Leu Cys Arg Leu Lys

Claims (2)

(57) [Claims] 1. A novel peptide represented by the following amino acid sequence formula or a salt thereof. Arg-Leu-Lys-Cys-Leu-Leu-L
eu-Cys-Arg-Leu-Lys-Y (where Y is a carboxyl group (—COOH) of a lysine residue at the carboxyl terminal of the peptide of the present invention;
Alternatively, it represents a carbamyl group (—CONH ₂ ) in which the carboxyl group is amidated. ) 2. An antibacterial agent containing as an effective ingredient a novel peptide represented by the following amino acid sequence formula or a salt thereof. Arg-Leu-Lys-Cys-Leu-Leu-L
eu-Cys-Arg-Leu-Lys-Y (where Y is a carboxyl group (—COOH) of a lysine residue at the carboxyl terminal of the peptide of the present invention;
Alternatively, it represents a carbamyl group (—CONH ₂ ) in which the carboxyl group is amidated. )