JPH0656888A - Novel peptide and antigerminal agent - Google Patents

Abstract

(57) [Summary] (Modified) [Structure] A peptide represented by the following amino acid sequence formula or a salt thereof, and an antibacterial agent containing these as active ingredients. Arg-Ser-Leu-Cys-Leu-Leu-H
is-Cys-Arg-Leu- Lys-Y (Y is a peptide of the carboxyl groups of lysine residues at the carboxyl terminus (-COOH) or, indicating the carboxyl group is amidated carbamyl (-CONH _2).) [Effect] Since it has excellent antibacterial activity and is hardly toxic, it is used as an antibacterial agent.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD 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 With the development of protein engineering in recent years, research and development of not only conventional peptides obtained from nature but also synthesized peptides have been actively conducted.

Peptides having an antibacterial action have been actively developed for a long time, and the following reports have been made. For example, a cyclic peptide derivative (JP-A-58-21)
3744), a polypeptide useful as an antibacterial agent against yeasts (JP-A-60-130599), a novel glycopeptide (JP-A-61-251699), an oligopeptide having an effect on Gram-positive bacteria ( JP-A-62-22798), peptide antibiotics (JP-A-63-17897) and the like.

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

[0005]

However, many of these conventional techniques are beyond the scope of basic research, which suggests that the development of peptide antibacterial agents is insufficient. In any case, a new antibacterial agent having a high effect against undesired pathogens and other microorganisms is always desired, and the development of novel peptides that can be put into practical use as an antibacterial agent is important.

[0006]

From such a point of view, the present inventors searched for useful novel peptides, and as a result, discovered a peptide having a strong antibacterial action and completed the present invention. .

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

Arg-Ser-Leu-Cys-Leu
-Leu-His-Cys-Arg-Leu-Lys-
Y (where Y is the carboxyl group (—COOH) of the 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. ).

In the present invention, the amino acid is IUPAC-IUB.
It may be indicated by an abbreviation based on commission on Biological Nomenclature or an abbreviation commonly used in the field, and examples thereof are as follows.

Arg: arginine, Ser: serine, C
ys: cysteine, Leu: leucine, His: histidine, Lys: lysine.

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

The peptide provided by the present invention is composed of 11 amino acids with arginine (Arg) as the N-terminal, contains a large amount of basic amino acids such as arginine, lysine, and histidine, and has a carboxyl-terminal lysine (Ly).
s) is a basic peptide. In addition, a peptide in which the carboxyl group of lysine, which is the amino acid at the C-terminal, exists as a carboxyl group as it is, and a peptide in which the carboxyl group is amidated and exists as lysine amide also have strong antibacterial activity. It is included in the invention.

In the present invention, those novel peptides having a carboxyl group at the C-terminus are T09.
6. The product in which the C-terminal carboxyl group is amidated is referred to as T103.

The peptides T096 and T103 of the present invention are
Both can take the form of salts, which 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, Volume 85, pages 2149-2154 (1963).
It is possible to synthesize according to the method of Merrifield et al. It can also be synthesized by a commercially available peptide synthesizer or the like. In addition, by using a gene encoding the amino acid sequence of the peptide of the present invention, it is possible to produce a large amount in microbial cells, plant cells, and animal cells using a genetic engineering technique. The obtained crude synthetic peptide was subjected to gel filtration and reverse phase HPL.
Further purification can be achieved by means commonly used for protein / peptide purification such as C and ion exchange column purification.

The peptide of the present invention can be expected to 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 a suitable 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 a disinfectant for contact lens cleaning liquid, ointment, soap, shampoo, medicine, food, feed, pet food, toothpaste and the like.

Further, the peptide of the present invention can be used as a drug for treating and preventing bacterial diseases. In this case, any administration method and dose can be selected as long as the antibacterial polypeptide of the present invention has activity depending on the type and symptom of the disease.

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

The antibacterial 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.
It may be contained in a weight percentage.

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

Further, since the antibacterial agent according to the present invention is a protein, it is considered that the antibacterial agent has little toxicity at least when taken orally. Therefore, the present antibacterial polypeptide is particularly useful as a drug for humans and animals and a food or feed additive.
For example, by adding the present antibacterial polypeptide to foods such as gum and toothpaste, it is useful for sterilization in the oral cavity and prevention and treatment of tooth decay and periodontal disease. In addition, in the cultivation of fish, it is possible to prevent the cultured fish from infection by adding the polypeptide of the present invention to the feed used.

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

[0023]

EXAMPLES The present invention will be described in more detail below with reference to examples.

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

Functional groups other than the carboxyl group of amino acids used for peptide synthesis, that is, amino groups, hydroxyl groups, guanidyl groups, and sulfhydryl groups are protected by ordinary protecting groups commonly used in amino acid and peptide chemistry. I was there. For example, α-amino group is t-butoxycarbonyl group (Boc group), ε-amino group is 2-chlorobenzyloxycarbonyl group, hydroxyl group is benzyl group, guanidyl group is tosyl group, sulfhydryl group is p-methylbenzyl group. A protected amino acid was used.

First, a polystyrene resin in which 0.2 mmol of lysine, which is a carboxyl-terminal amino acid protected by Boc group and 2-chlorobenzyloxycarbonyl group, is bound to the carboxyl group is thoroughly washed with methylene chloride, and then 50 The Boc group was selectively removed using a solution of trifluoroacetic acid in methylene chloride. Next, when viewed from the carboxyl terminal side of the peptide of the present invention, 3 mol equivalents of an amino acid (protected amino acid) corresponding to the following amino acid and having a functional group other than the carboxyl group protected, and dicyclohexylcarbodiimide (a condensing agent) DCC) 3 molar equivalents to 4
Coupling was carried out by reacting for a time to form a peptide bond. After washing with methylene chloride, the above procedure was repeated to successively synthesize peptides from the carboxyl terminus. After the condensation of the amino acid corresponding to the amino group end was completed, all the protecting groups of the peptide synthesized with hydrogen fluoride were removed, and the synthesized peptide was released from the resin.

The resulting crude synthetic peptide was purified by reverse phase HPLC. That is, 1/10 amount of the crude synthetic peptide was purified by HPLC under the conditions shown below, the main peak was separated, and further freeze-dried to obtain 16 mg of peptide T096 of the present invention having a purity of 96% 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%
Trifluoroacetic acid acetonitrile solution Gradient conditions: Linear gradient of 5-56% of solution B Flow rate: 1.5 ml / min.

In the case of synthesis of T103, 0.2 mmol of lysine amide, which is a carboxyl-terminal amino acid protected by Boc group, is bound to the carboxyl group by using a polystyrene resin. The same method was used for the purification, and the same purification method was used. As a result, 13 mg of T103 having a purity of 97% or more was obtained.

The synthesized T096 and T103 were subjected to Edman degradation using a gas phase amino acid sequencer (Model 177A of Applied Biosystems) to obtain PTH-
The amino acids were identified and analyzed with a PTH-analyzer (Type 120A, Applied Biosystems, Inc.), and the results of confirming the amino acid sequences were as follows.

Arg-Ser-Leu-Cys-Leu
-Leu-His-Cys-Arg-Leu-Lys.

Example 2 Measurement of Antibacterial Activity The antibacterial activity was measured by Journal of Biochemistry, Vol. 211, pp. 727-734 (1983).
The procedure was as described. That is, each bacterium is called Antibiot
After shaking culture in ic medium 3 (manufactured by Difco), the concentration of the bacteria was adjusted so that the absorbance at 660 nm was about 0.3, and 10 μl thereof was added to 190 μl of the above medium to prepare a test bacterial cell fluid. After dissolving the freeze-dried product of the peptide of the present invention in purified water, 200 μl of a peptide sample solution prepared by sequentially diluting with purified water was added, and the mixture was incubated at 37 ° C. for 4 hours, and the absorbance at 660 nm was measured. did. The test was performed using purified water instead of the sample solution. The antibacterial activity is shown by the concentration of the peptide of the present invention when the growth rate of 50% is obtained as compared with the bacterial growth of the subject. The results are shown in Table 1.

[0033]

[Table 1]

As is clear from Table 1, both peptides T096 and T103 of the present invention were found to exhibit strong antibacterial activity against a wide range of microorganisms including Gram-positive bacteria, Gram-negative bacteria and fungi.

[0035]

Industrial Applicability According to the present invention, a novel peptide can be provided. The novel peptides of the present invention have broad antibacterial activity and consist only of naturally occurring amino acids. Therefore,
Although various uses of this property can be considered, the use as an antibacterial agent or a food additive containing this substance as an active ingredient is particularly expected. In particular, since this substance is a protein and has excellent heat stability, it is promising as an antibacterial food additive for foods that require heat treatment in the processing step.

[0036]

[Sequence Listing] Sequence Length: 11 Sequence Type: Amino Acid Topology: Linear Sequence Type: Peptide Sequence: Arg Ser Leu Cys Leu Leu His Cys Arg Leu Lys

Continuation of front page (51) Int.Cl. ⁵ Identification number Office reference number FI technical display area C07K 99:00

Claims (2)

[Claims] 1. A novel peptide represented by the following amino acid sequence formula or a salt thereof. Arg-Ser-Leu-Cys-Leu-Leu-H
is-Cys-Arg-Leu-Lys-Y (where Y is the carboxyl group (-COOH) of the lysine residue at the carboxyl terminal of the peptide of the present invention or a carbamyl group (-) in which the carboxyl group is amidated. CONH ₂ ).) 2. An antibacterial agent containing the novel peptide according to claim 1 or a salt thereof as an active ingredient.