JP5686981B2 - Novel compound MK844-mF10 substance, production method thereof, and use thereof - Google Patents

Description

  The present invention relates to a novel compound having an excellent antibacterial activity against a wide range of pathogenic bacteria such as drug-resistant bacteria and phytopathogenic bacteria, or an enzyme inhibitory activity against an enzyme possessed by the bacteria, a method for producing the same, and use thereof In addition, the present invention relates to a novel microorganism that is a microorganism producing the novel compound.

  Conventionally, many antibacterial agents have been used as therapeutic agents for bacterial infections. Many of the antibacterial agents known so far act by inhibiting bacterial nucleic acid synthesis, protein synthesis, peptidoglycan synthesis and the like. Since these target sites are single and mainly intended to inhibit the metabolic synthesis pathway, resistant bacteria to these antibacterial agents are likely to appear, and in recent years, in particular, they have many resistances to multiple antibiotics. The emergence of drug-resistant bacteria is a problem.

For example, Staphylococcus aureus is known as a causative bacterium for purulent diseases, pneumonia, food poisoning, etc., but methicillin-resistant Staphylococcus aureus has acquired resistance to many drugs such as the antibiotic methicillin. : MRSA) has become a major clinical problem. Currently, vancomycin, teicoplanin, arbekacin, linezolid and the like are used as typical therapeutic agents for MRSA, but it is generally considered difficult to completely eliminate MRSA. As for vancomycin, the appearance of vancomycin-resistant Staphylococcus Aureus (VRSA) has already been reported, and it is said that sufficient caution is required for its use.

  In order to overcome the problem of such drug-resistant bacteria, it is desired to develop a novel antimicrobial agent against microorganisms by a new mechanism of action different from that of conventional antibacterial agents (for example, see Non-patent Document 1).

  On the other hand, bacteria are known to have receptors that accept changes in response to the environment and information transmission mechanisms that control the expression of each gene. A typical example is a two-component system. . The two-component control system is an environmentally responsive gene expression control system composed of a sensor protein exhibiting histidine kinase activity and a regulator that is a DNA-binding protein. Bacteria are used in various ways to cope with various environmental changes. (See Non-Patent Document 2, for example).

  As a two-component control system of bacteria, there is an information transmission system involving YycF (also referred to as WalR) and YycG (also referred to as WalK) of Gram-positive bacteria, and it is known that inhibition of this will kill the bacteria. (For example, refer nonpatent literatures 3-6) and the antibacterial agent which shows antibacterial power to the Gram positive bacterium by inhibiting the said information transmission system is anticipated.

  In addition, the pathogenicity of soft rot fungi that infect crops such as Chinese cabbage and potato and cause enormous damage to agricultural production has three types of two-component control systems, PehS / PehR (see, for example, Non-Patent Document 7), PmrB / PmrA. (For example, refer nonpatent literature 8), it is known that pathogenicity is regulated by ExpS / ExpA (for example, refer nonpatent literature 9), and control of soft rot fungus by controlling these pathogenicity is carried out. Be expected.

  Although there are the above findings, satisfactory antibacterial agents and enzyme activity inhibitors have not yet been obtained, and the development of excellent antibacterial agents and the like is desired at present.

Sievert DM, et al: Staphylococcus aureus Resistant to Vancomycin-United States, 2002. MMWR July 5, 2002; 51: 565-567. Bioscience and Industry, Vol.58, No.4 Fablet, C.I. and Hoch, A.A. A. , J. et al. Bacteriol. , 180, 6375-6383, 1998 Marti, P.M. K. Li, T .; Sun, D .; , Biek, D.M. P. and Schmid, M.M. B. , J. et al. Bacteriol. , 181, 3666-3673, 1999 Lange, R.A. , Wagner, C.I. DeSaizieu, A .; , Flint, N .; Monos, J .; , Stigger, M .; , Caspers, P .; , Kamber, M .; Keck Wolfgang, Amrein, K .; E. Gene, 237, 223-234, 1999. Beier, D.D. and Frank, R.A. , J. et al. Bacteriol. , 182, 2068-2076, 2000 Eriksson, A.D. R. B. , Andersson, R .; A. Pirhonen, M .; , And Palva, E .; T. T. et al. , Mol. Plant-Microbe Interact. , 11, 743-752, 1998 Hytiainen, H .; Sjoblom, S .; Palomaki, T .; Tuikkala, A .; , And Palva, E .; T. T. et al. , Mol. Microbiol. , 50, 795-807, 2003 Flego, D.D. , Marits, R .; Eriksson, A .; R. B. , Koiv, V .; , Karlsson, M .; -B. , Hekinheim, R .; , And Palva, E .; T. T. et al. , Mol. Plant-Microbe Interact. , 13, 447-455, 2000

  An object of the present invention is to solve the above-described problems and achieve the following objects. That is, the present invention provides an excellent antibacterial activity against a wide range of pathogenic bacteria such as drug-resistant bacteria and phytopathogenic bacteria by inhibiting the two-component control system of the bacteria, or an enzyme against the enzymes possessed by the bacteria. It is an object of the present invention to provide compounds having inhibitory activity, methods for producing the same, microorganisms that produce the compounds, compound-containing compositions using the compounds, antibacterial agents, and enzyme activity inhibitors. .

In order to solve the above-mentioned problems, the present inventors have paid attention to a two-component control system that is a main information transmission mechanism of bacterial cells, and as a result of extensive studies, as a new microorganism, a strain belonging to the genus Streptomyces ( Streptomyces ) It was found that this strain produced a compound having a novel structural skeleton and having antibacterial activity or enzyme inhibitory activity. The inventors of the present invention have confirmed that this is a novel compound by analyzing the chemical structure of the compound, and have completed the present invention. In addition, the present inventors named this novel compound MK844-mF10 substance.

The present invention is based on the above findings by the present inventors, and means for solving the above problems are as follows. That is,
<1> A compound represented by the following structural formula (A).
<2> A method for producing a compound represented by the following structural formula (A),
A culture step for culturing a microorganism belonging to the genus Streptomyces and having the ability to produce a compound represented by the following structural formula (A):
A collecting step of collecting a compound represented by the following structural formula (A) from the culture obtained in the culturing step;
It is a manufacturing method of the compound characterized by including.
<3> A microorganism belonging to the genus Streptomyces and having the ability to produce a compound represented by the following structural formula (A) is a Streptomyces sp. MK844-mF10 having a deposit number of NITE P-903. It is a manufacturing method of the compound as described in said <2> which is a strain | stump | stock.
<4> A microorganism which belongs to the genus Streptomyces and has the ability to produce a compound represented by the following structural formula (A).
<5> The microorganism according to <4>, which is a Streptomyces sp. MK844-mF10 strain having an accession number of NITE P-903.
<6> A compound-containing composition comprising a compound represented by the following structural formula (A).
<7> An antibacterial agent comprising the compound-containing composition according to <6>.
<8> An enzyme activity inhibitor comprising the compound-containing composition according to <6>.

  According to the present invention, the above-mentioned problems can be solved and the above-mentioned object can be achieved. By inhibiting the bacterial two-component control system, a wide variety of pathogenic bacteria such as drug-resistant bacteria and plant pathogenic bacteria can be obtained. On the other hand, a compound having an excellent antibacterial activity or an enzyme inhibitory activity against the enzyme of the bacterium, a method for producing the same, a microorganism that is a bacterium producing the compound, and a compound-containing composition using the compound Products, antibacterial agents, and enzyme activity inhibitors.

FIG. 1 is an infrared spectrum chart of the compound represented by the structural formula (A) (MK844-mF10 substance) measured by the KBr tablet method. Vertical axis: transmittance (%), horizontal axis: wave number (cm ⁻¹ ). FIG. 2 is a chart of ultraviolet absorption spectra of the compound represented by the structural formula (MK844-mF10 substance) in methanol, in methanol containing 0.005M hydrochloric acid, and in methanol containing 0.005M sodium hydroxide. Vertical axis: absorbance (Abs), horizontal axis: wavelength (nm). FIG. 3 is a chart of a proton nuclear magnetic resonance spectrum at 600 MHz measured in deuterated methanol of the compound represented by the structural formula (MK844-mF10 substance). Horizontal axis: ppm unit. FIG. 4 is a chart of a carbon-13 nuclear magnetic resonance spectrum at 150 MHz measured in deuterated methanol of the compound represented by the structural formula (A) (MK844-mF10 substance). Horizontal axis: ppm unit.

(New compound)
The compound of the present invention is a novel compound isolated by the present inventors (hereinafter sometimes referred to as “MK844-mF10 substance”).

-Physicochemical properties-
The physicochemical properties of the compound represented by the structural formula (A) are as follows.
(1) The appearance is yellow powder.
(2) The molecular formula is represented by C ₃₈ H ₃₈ O ₁₃ .
(3) The experimental value by high resolution mass spectrometry (HRESIMS: negative ion mode) is m / z 701.2243 (M−H) ⁻ , and the calculated value is m / z 701.2229 (C ₃₈ H ₃₇ O ₁₃ ).
(4) The specific rotation is [α] _D ²³ = + 102.1 ° (c 0.15, MeOH).
(5) The infrared absorption spectrum is as shown in FIG.
ν _max (KBr) cm ⁻¹ : 3,700 to 3,200, 2,929, 1,710, 1,675, 1,631, 1,592, 1,274, 1,124, 1,078
(6) The ultraviolet absorption spectrum is as shown in FIG.
λ _max nm (ε):
In methanol: 275 (43,000), 292 (42,000), 305 (3,500), 406 (5,700)
In 0.005M hydrochloric acid-containing methanol: 275 (42,700), 292 (41,800), 305 (3,500), 406 (5,700)
In methanol containing 0.005M sodium hydroxide: 277 (36,000), 295 (38,700), 308 (3,400), 509 (4,800)
(7) As a proton nuclear magnetic resonance spectrum, a proton NMR spectrum measured in deuterated methanol at 600 MHz is as shown in FIG.
(8) As a carbon 13 nuclear magnetic resonance spectrum, a carbon 13 NMR spectrum measured in deuterated methanol at 150 MHz is as shown in FIG.

  Whether or not the compound has a structure represented by the structural formula (A) can be confirmed by various analysis methods selected as appropriate, for example, the mass spectrometry, the infrared absorption spectrum, the ultraviolet absorption spectrum. The proton nuclear magnetic resonance spectrum, the carbon 13 nuclear magnetic resonance spectrum, and the like can be analyzed.

  The MK844-mF10 substance may be obtained from a microorganism that produces the MK844-mF10 substance, or may be obtained by chemical synthesis. It is preferably obtained by a method.

<Application>
The MK844-mF10 substance has excellent antibacterial activity and excellent enzyme inhibitory activity, as shown in Test Examples 1 and 2 described later. Therefore, the MK844-mF10 substance can be suitably used, for example, as an active ingredient such as a compound-containing composition, antibacterial agent, enzyme activity inhibitor, etc. of the present invention described later.

(Method for producing compound)
The production method of the compound of the present invention, that is, the MK844-mF10 substance includes at least a culture step and a collection step, and further includes other steps as necessary.

<Culture process>
The culturing step is a step of culturing a microorganism belonging to the genus Streptomyces and having an ability to produce an MK844-mF10 substance.

The microorganism is not particularly limited as long as it belongs to the genus Streptomyces and has the ability to produce an MK844-mF10 substance, and can be appropriately selected according to the purpose. Streptomyces sp. MK844-mF10 strain (NITE P-903, details are described in the item of microorganisms of the present invention described later) and the like. In addition, other strains capable of producing the MK844-mF10 substance can be isolated from the natural world by conventional methods. In addition, the production ability of the MK844-mF10 substance, including the Streptomyces sp. MK844-mF10 strain, is subjected to irradiation and other mutation treatments, thereby increasing the productivity of the MK844-mF10 substance. It is also possible. Furthermore, production of MK844-mF10 substance by genetic engineering techniques is also possible.

In the culture, a producing bacterium producing MK844-mF10 substance (hereinafter sometimes simply referred to as “MK844-mF10 substance producing bacterium”) in a nutrient medium (hereinafter sometimes simply referred to as “medium”). It is done by inoculating and culturing at a good temperature for the production of the MK844-mF10 substance.
There is no restriction | limiting in particular as said nutrient medium, According to the objective, it can select suitably, For example, the well-known thing utilized conventionally for culture | cultivation of actinomycetes can be used.
The nutrient source added to the nutrient medium is not particularly limited and can be appropriately selected according to the purpose. For example, as a nitrogen source, commercially available soybean flour, peptone, yeast extract, meat extract, corn Steep liquor, ammonium sulfate, etc. can be used, and carbohydrates such as tomato paste, glycerin, starch, glucose, galactose, dextrin, bacto-soyton, fat, etc. can be used as the carbon source. Furthermore, inorganic salts such as sodium chloride and calcium carbonate can be added to the medium for use, and in addition, a trace amount of metal salt can be added to the medium for use.
These materials may be any material that can be used by the MK844-mF10 substance-producing bacteria and can be used for the production of the MK844-mF10 substance, and all known culture materials can be used.

  The seed medium for production of the MK844-mF10 substance (hereinafter sometimes referred to as “production medium”) is not particularly limited and may be appropriately selected depending on the intended purpose. The growth obtained from the slope culture of the MK844-mF10 substance-producing bacteria can be used.

The culture method is not particularly limited and may be appropriately selected depending on the intended purpose. A culture method under aerobic conditions is preferred.
The temperature of the culture is not particularly limited as long as the growth of the MK844-mF10 substance-producing bacteria is not substantially inhibited and the MK844-mF10 substance can be produced, depending on the producing bacteria used. Although it can select suitably, 25 to 35 degreeC is preferable.
There is no restriction | limiting in particular as the period of the said culture | cultivation, According to accumulation | storage of MK844-mF10 substance, it can select suitably. Usually, the accumulation of the MK844-mF10 substance is highest in 3 to 10 days of culture.

<Collection process>
The collection step is a step of collecting MK844-mF10 substance from the culture obtained in the culture step.
Since the MK844-mF10 substance has the physicochemical properties described above, it can be collected from the culture according to the properties.

The collection method is not particularly limited, and a method used for collecting a metabolite produced by a microorganism can be appropriately selected. For example, a method of extraction using a solvent that is not mixed with water, a method of using a difference in adsorption affinity for various adsorbents, a method of gel filtration, chromatography using countercurrent distribution, or the like may be used alone or in combination.
From the separated cells, the MK844-mF10 substance is extracted from the cells by an extraction method using an appropriate organic solvent or an elution method by disrupting the cells, and is isolated and purified in the same manner as described above. Can be collected.

  As described above, the production method can be carried out, whereby the MK844-mF10 substance can be suitably obtained.

(Microorganism)
The microorganism of the present invention belongs to the genus Streptomyces and is characterized by having the ability to produce the above-described compound of the present invention, that is, the MK844-mF10 substance. The microorganism has an ability to produce an MK844-mF10 substance, and therefore, there is no particular limitation as long as it is a microorganism that can be used as an MK844-mF10 substance producing microorganism in the above-described method for producing a compound of the present invention. Can be appropriately selected according to the purpose.

  Among these microorganisms, in particular, in the Center for Microbial Chemistry, Foundation for Microbial Chemistry, a microorganism with a strain number of MK844-mF10 strain, which is an actinomycete isolated from soil in Hokuto City, Yamanashi Prefecture, is used. It is preferable. The mycological properties of the MK844-mF10 strain are as follows.

1. Form MK844-mF10 strain extends straight aerial hyphae from branched basic hyphae. Mature spore chains link more than 10 cylindrical spores. The size of the spore is about 0.5 μm to 0.7 μm × 0.6 μm to 1.1 μm, and the surface of the spore is smooth or spiny. No roticular branches, mycelial bundles, spore capsules, or motile spores are observed.

2. Regarding the description of the color of growth in various media The standard shown in [] was the color harmony manual of Container Corporation of America (color harmony manual of Container Corporation of America).
(1) Yeast / malt agar medium (ISP-medium 2, 27 ° C. culture)
On the growth of ash red tea [5 lg, Cocoa Brown to 6 1/2 ni, Rose Brown], aerial mycelia of tea white [3 dc, Natural] were grown and tea [5 pi, Copper Brown] Produces soluble pigment.
(2) Oatmeal agar medium (ISP-medium 3, 27 ° C. culture)
On the development of tea [5 pi, Copper Brown] to dark tea [5 nl, Chocolate], an aerial mycelium of gray [13 cb, Pearl Gray] grows. Produces a soluble pigment of light tea.
(3) Starch / inorganic salt agar medium (ISP-medium 4, 27 ° C. culture)
On the development of tea [5 pi, Copper Brown] to dark tea [6 pl, Deep Brown Mahogany], an aerial mycelium of gray [13 ba, Alabaster Tint] grows. Produces a soluble pigment of light tea.
(4) Glycerin / asparagine agar medium (ISP-medium 5, 27 ° C. culture)
Growth is ash red tea [5 ni, Cocoa Brown], aerial mycelia do not grow, and soluble pigments take on tea.
(5) Tyrosine agar medium (ISP-medium 7, 27 ° C. culture)
Growth is bright tea [4 ng, Lt Brown to 4 ne, Luggage tan], aerial hyphae does not grow, and soluble pigments take on tea.
(6) Sucrose / Nitrate agar medium (27 ° C culture)
On the growth of light yellow [2 db, Ivory], aerial mycelia of tea white [3 cb, Sand] are formed slightly and the soluble pigment is light pink.

3. Physiological properties (1) Growth temperature range Yeast starch agar medium (soluble starch 1.0 mass%, yeast extract 0.2 mass%, string agar 2.6 mass%, pH 7.0), 10 ° C., 20 As a result of testing at each temperature of 24 ° C., 24 ° C., 27 ° C., 30 ° C., 37 ° C., and 50 ° C., growth at 10 ° C., 37 ° C., and 50 ° C. was not observed, and in the range of 20 ° C. to 30 ° C. Growing up. The optimum temperature for growth is 24 ° C to 27 ° C.
(2) Starch hydrolysis (starch / inorganic salt agar medium, ISP-medium 4, 27 ° C. culture)
Starch water disintegration is not observed.
(3) Production of melanin-like pigment (tripton yeast pros, ISP-medium 1; peptone yeast iron agar medium, ISP-medium 6; tyrosine agar medium, ISP-medium 7; all at 27 ° C.)
Neither medium is observed.
(4) Availability of carbon source (inorganic salt agar medium, medium obtained by removing starch from ISP-medium 4; culture at 27 ° C.)
It grows using D-glucose, L-arabinose, D-xylose, and rhamnose, and does not use D-fructose, sucrose, inositol, raffinose, and D-mannitol.

4). Cell component 2,6-diaminopimelic acid in the cell wall is LL-type.

5. 16S rRNA gene analysis The partial base sequence (1,481 bp) of the 16S rRNA gene was determined and compared with the data of known strains registered in the DNA database. As a result, the base sequence of MK844-mF10 strain showed high homology with the 16S rRNA gene base sequence of Streptomyces genus Streptomyces as shown below. That is, Streptomyces Yochonenshisu (Streptomyces yeochonensis: NBRC 100782 ^T and 98% homologous), Streptomyces Paushisuporeusu (S.paucisporeus (NBRC 1413 ^T and 98% homologous), Streptomyces Hopei ene cis (S.hebeiensis: YIM 001 ^T and 97% Homology), etc. In addition, the value in the parenthesis indicates the homology value of the base sequence.

Summarizing the above properties, the MK844-mF10 strain elongates straight aerial hyphae from the well-branched basic mycelium and links cylindrical spores. In various media, brown and white aerial mycelia are grown on the growth of ash red tea to dark tea. Produces soluble pigments of light pink to brown. The optimum temperature for growth is 24 ° C to 27 ° C. The starch hydrolyzability and the production of melanin-like pigments are negative.
2,6-Diaminopimelic acid in the cell wall of MK844-mF10 strain is LL-type.
When the partial base sequence of 16S rRNA gene of MK844-mF10 strain was analyzed and compared with the data of known strains, it showed high homology with Streptomyces genus actinomycetes.

From the above results, the MK844-mF10 strain is considered to belong to the genus Streptomyces . Therefore, the MK844-mF10 strain was designated as Streptomyces sp. MK844-mF10.
The MK844-mF10 strain was applied for deposit at the Patent Microorganism Deposit Center of the National Institute of Technology and Evaluation, and was commissioned as NITE P-903 on February 23, 2010.

  As seen in other bacteria, the MK844-mF10 strain is susceptible to changes in properties, but for example, mutant strains (naturally occurring or inducible) derived from the MK844-mF10 strain, Even if it is a gene recombinant, etc., those having the ability to produce the MK844-mF10 substance are included in the microorganism of the present invention.

(Compound-containing composition)
The compound-containing composition of the present invention contains at least the compound of the present invention, that is, the MK844-mF10 substance, and further contains other components as necessary.

  There is no restriction | limiting in particular as content of MK844-mF10 substance in the said compound containing composition, According to the objective, it can select suitably. The compound-containing composition may be the MK844-mF10 substance itself.

There is no restriction | limiting in particular as said other component, For example, it can select suitably from the pharmacologically acceptable support | carrier according to the objective, For example, ethanol, water, starch, etc. are mentioned. There is no restriction | limiting in particular as content of the said other component in the said compound containing composition, According to the objective, it can select suitably within the range which does not impair the effect of MK844-mF10 substance.
In addition, the said compound containing composition may be used individually by 1 type, and may be used in combination with the pharmaceutical which uses another component as an active ingredient. Moreover, you may use the said compound containing composition in the state mix | blended in the pharmaceutical which uses another component as an active ingredient.

<Dosage form, administration>
-Dosage form-
There is no restriction | limiting in particular as a dosage form of the said compound containing composition, According to the objective, it can select suitably, For example, a powder form, a capsule form, a tablet form, a liquid form etc. are mentioned. The compound-containing composition of these dosage forms can be produced according to a conventional method.

The dosage form for use as the agricultural and horticultural fungicide is not particularly limited and may be appropriately selected depending on the intended purpose. Usually, it is a solid carrier, liquid carrier, surfactant, and other formulation aids. It can be prepared in an appropriate form such as emulsion, wettable powder, liquid, flowable (sol), powder, granule, fine granule, tablet, etc. by conventional formulation as a mixture with the agent.
Various surfactants (or emulsifiers) are used for the purpose of emulsions, wettable powders, liquids, flowable (sol) agents, powders, granules, fine granules, tablets and the like. There is no restriction | limiting in particular as such surfactant, According to the objective, it can select suitably, For example, nonionic type (polyalkyl ether, polyoxyethylene alkyl ester, polyoxyethylene sorbitan alkyl ester etc.), Examples include anion type (alkyl succinic acid alkyl sulfate, aryl sulfonate, etc.), cation type (alkyl amines, polyoxyalkyl amines, etc.), amphoteric type (sulfate ester salts, etc.), and the like. These may be used alone or in combination of two or more. Besides these, various adjuvants such as polyvinyl alcohol, carboxymethyl cellulose, gum arabic, polyvinyl acetate, sodium alginate, gelatin, gum tragacanth and the like can be used.

-Administration-
There is no restriction | limiting in particular as an administration method of the said compound containing composition, For example, it can select suitably according to the dosage form of the said compound containing composition, etc., It can administer orally or parenterally.
The dosage of the compound-containing composition is not particularly limited, taking into consideration various factors such as the age, weight, constitution, symptom of the administration subject, and the presence or absence of administration of a pharmaceutical comprising other ingredients as active ingredients. It can be selected appropriately.
There is no restriction | limiting in particular as an administration time of the said compound containing composition, According to the objective, it can select suitably.
The animal species to be administered with the compound-containing composition is not particularly limited and may be appropriately selected depending on the intended purpose. For example, humans, monkeys, pigs, cows, sheep, goats, dogs, cats, mice , Rats, birds and the like.
Moreover, there is no restriction | limiting in particular as an administration method in the case of using as said agricultural and horticultural fungicide, dosage amount, administration time, and administration object, According to the objective, it can select suitably.

<Application>
Since the compound-containing composition contains the MK844-mF10 substance, it has at least one of an antibacterial action and an enzyme activity inhibitory action.

(Antimicrobial agent, enzyme activity inhibitor)
<Antimicrobial agent>
The antibacterial agent of this invention contains the said compound containing composition of this invention at least, and contains another component suitably as needed.

  There is no restriction | limiting in particular as content of the said compound containing composition in the said antibacterial agent, According to the objective, it can select suitably. The antibacterial agent may be the compound-containing composition itself.

There is no restriction | limiting in particular as said other component, For example, it can select suitably from the pharmacologically acceptable support | carrier according to the objective, For example, ethanol, water, starch, etc. are mentioned. There is no restriction | limiting in particular as content of the said other component in the said antibacterial agent, It can select suitably according to the objective within the range which does not impair the effect of MK844-mF10 substance.
In addition, the said antibacterial agent may be used individually by 1 type, and may be used in combination with the pharmaceutical which uses another component as an active ingredient. Moreover, you may use the said antibacterial agent in the state mix | blended in the pharmaceutical which uses another component as an active ingredient.

Since the antibacterial agent contains MK844-mF10 substance, the antibacterial agent has excellent antibacterial activity against drug-resistant bacteria as shown in Test Example 1 described later.
Therefore, the antibacterial agent can be suitably used for the prevention or treatment of infectious diseases caused by drug-resistant bacteria. The antibacterial agent can also be suitably used as an agricultural and horticultural fungicide.

<Enzyme activity inhibitor>
The enzyme activity inhibitor of the present invention includes at least the compound-containing composition of the present invention, and optionally includes other components as necessary.
The enzyme activity inhibitor can suitably inhibit histidine kinase activity.

  There is no restriction | limiting in particular as content of the said compound containing composition in the said enzyme activity inhibitor, According to the objective, it can select suitably. The enzyme activity inhibitor may be the compound-containing composition itself.

There is no restriction | limiting in particular as said other component, For example, it can select suitably from the pharmacologically acceptable support | carrier according to the objective, For example, ethanol, water, starch, etc. are mentioned. There is no restriction | limiting in particular as content of the said other component in the said enzyme activity inhibitor, According to the objective, it can select suitably within the range which does not impair the effect of MK844-mF10 substance.
In addition, the said enzyme activity inhibitor may be used individually by 1 type, and may be used in combination with the pharmaceutical which uses another component as an active ingredient. Moreover, you may use the said enzyme activity inhibitor in the state mix | blended in the pharmaceutical which uses another component as an active ingredient.

Since the enzyme activity inhibitor contains MK844-mF10 substance, as shown in Test Example 2 described later, a wide range of gram-positive bacteria including drug-resistant bacteria, phytopathogenic bacteria, and the like, and enzymes possessed by gram-negative bacteria. It has excellent enzyme inhibitory activity.
Therefore, the enzyme activity inhibitor can suppress the pathogenicity of a wide range of gram-positive bacteria and gram-negative bacteria including drug-resistant bacteria, and is suitably used for the prevention or treatment of infectious diseases caused by the bacteria. Is possible. Moreover, it can utilize suitably also as an active ingredient of the agricultural and horticultural fungicide.

<Dosage form, administration>
-Dosage form-
There is no restriction | limiting in particular as a dosage form of the said antibacterial agent and the said enzyme activity inhibitor, According to the objective, it can select suitably, For example, a powder form, a capsule form, a tablet form, a liquid form etc. are mentioned. These antibacterial agents and enzyme activity inhibitors in these dosage forms can be produced according to conventional methods.

The dosage form for use as the agricultural and horticultural fungicide is not particularly limited and may be appropriately selected depending on the intended purpose. Usually, it is a solid carrier, liquid carrier, surfactant, and other formulation aids. It can be prepared in an appropriate form such as emulsion, wettable powder, liquid, flowable (sol), powder, granule, fine granule, tablet, etc. by conventional formulation as a mixture with the agent.
Various surfactants (or emulsifiers) are used for the purpose of emulsions, wettable powders, liquids, flowable (sol) agents, powders, granules, fine granules, tablets and the like. There is no restriction | limiting in particular as such surfactant, According to the objective, it can select suitably, For example, nonionic type (polyalkyl ether, polyoxyethylene alkyl ester, polyoxyethylene sorbitan alkyl ester etc.), Examples include anion type (alkyl succinic acid alkyl sulfate, aryl sulfonate, etc.), cation type (alkyl amines, polyoxyalkyl amines, etc.), amphoteric type (sulfate ester salts, etc.), and the like. These may be used alone or in combination of two or more. In addition to these, various adjuvants such as polyvinyl alcohol, carboxymethyl cellulose, gum arabic, polyvinyl acetate, sodium alginate, gelatin, and tragacanth gum can be used.

-Administration-
The administration method of the antibacterial agent and the enzyme activity inhibitor is not particularly limited, and can be appropriately selected according to the dosage form of the antibacterial agent and the enzyme activity inhibitor, for example, orally or parenterally. Can be administered.
The dosage of the antibacterial agent and the enzyme activity inhibitor is not particularly limited, and includes various factors such as the age, weight, constitution, symptom of the administration target individual, and the presence / absence of administration of a drug containing other ingredients as active ingredients. Can be selected as appropriate.
There is no restriction | limiting in particular as an administration time of the said antibacterial agent and the said enzyme activity inhibitor, According to the objective, it can select suitably.
The animal species to be administered with the antibacterial agent and the enzyme activity inhibitor is not particularly limited and may be appropriately selected depending on the intended purpose. For example, humans, monkeys, pigs, cows, sheep, goats, dogs , Cats, mice, rats, birds and the like.
Moreover, there is no restriction | limiting in particular as an administration method in the case of using as said agricultural and horticultural fungicide, dosage amount, administration time, and administration object, According to the objective, it can select suitably.

  EXAMPLES The present invention will be specifically described below with reference to examples, comparative examples, and test examples, but the present invention is not limited to these examples and test examples. In the following examples, comparative examples, and test examples, “%” represents “% by mass” unless otherwise specified.

(Example 1: Production of compound)
-Culture process-
Streptomyces sp. MK844-mF10 strain (deposited as NITE P-903) cultured on an agar slant medium, galactose 2%, dextrin 2%, glycerin 1%, Bacto Soyton (manufactured by Difco) 1%, Dispense 110 mL of a liquid medium (adjusted to pH 7.0) containing 0.5% corn steep liquor, 0.2% ammonium sulfate and 0.2% calcium carbonate into an Erlenmeyer flask (500 mL volume). Inoculated into medium sterilized at 120 ° C. for 20 minutes. Thereafter, it was subjected to rotary shaking culture at 30 ° C. for 2 days to obtain a seed culture solution.

  A liquid medium (adjusted to pH 7.0) containing 15 g of pressed wheat (Musso) and 25 g of water was dispensed into an Erlenmeyer flask (500 mL volume) and sterilized at 120 ° C. for 20 minutes by a conventional method to obtain a production medium. This production medium was inoculated with 7 mL / flask of the above-described seed culture medium, and statically cultured at 30 ° C. for 14 days in the dark.

-Sampling process-
Ethanol extract was obtained by adding 2.2 liters of ethanol to 1,160 g (29 flasks) of the culture thus obtained, followed by extraction at room temperature for 24 hours and centrifugation. Subsequently, 10 liters of water was added to the ethanol extract and stirred well, and this was passed through a Diaion CHP-20P (300 mL, manufactured by Mitsubishi Chemical Corporation) column to adsorb the MK844-mF10 substance. The column was washed with 1.6 liters of 50% methanol water and then eluted with 1.5 liters of acetone. The acetone eluate containing this MK844-mF10 substance was concentrated to dryness, 70 mL of hexane and 50 mL of methanol were added and dissolved, and the mixture was sufficiently stirred. When this hexane-methanol mixture was allowed to stand, it separated into two layers, and the MK844-mF10 substance was contained in the lower layer. This was collected and concentrated to dryness under reduced pressure to obtain 2.6 g of a crude extract containing MK844-mF10 substance.

The crude extract was dissolved in methanol and placed on a Sephadex LH-20 (inner diameter 36 mm × 480 mm, Pharmacia Biotech) column for chromatography. After developing 550 mL of solvent, fractions of 1 fraction were fractionated in 3 g portions. The active fractions were eluted in fractions 3 to 21, which were collected and concentrated to dryness under reduced pressure to obtain 1.4 g of MK844-mF10 substance. It was.
The MK844-mF10 substance was dissolved in a small amount of a chloroform-methanol mixed solvent and applied to a silica gel 60 (inner diameter 36 mm × 190 mm, Merck) column for chromatography. The column was washed with 300 mL of a mixed solvent of chloroform: methanol = 50: 1 (volume ratio), then 980 mL of a mixed solvent of chloroform: methanol = 9: 1 (volume ratio), and then chloroform: methanol: water = 4: 1. : Elution with 360 mL of a mixed solvent of 0.2 (volume ratio). When fraction of each fraction was fractionated by 18 g, the active fraction was eluted from fractions 30 to 70, which were collected and concentrated to dryness under reduced pressure to obtain 146 mg of pure MK844-mF10 substance.

When the physicochemical properties of the obtained MK844-mF10 substance were measured, it was as follows. From these facts, the MK844-mF10 substance was a novel compound having a structure represented by the following structural formula (A). It was confirmed that there was.
(1) The appearance is yellow powder.
(2) The molecular formula is represented by C ₃₈ H ₃₈ O ₁₃ .
(3) The experimental value by high resolution mass spectrometry (HRESIMS: negative ion mode) is m / z 701.2243 (M−H) ⁻ , and the calculated value is m / z 701.2229 (C ₃₈ H ₃₇ O ₁₃ ).
(4) The specific rotation is [α] _D ²³ = + 102.1 ° (c 0.15, MeOH).
(5) The infrared absorption spectrum is as shown in FIG.
ν _max (KBr) cm ⁻¹ : 3,700 to 3,200, 2,929, 1,710, 1,675, 1,631, 1,592, 1,274, 1,124, 1,078
(6) The ultraviolet absorption spectrum is as shown in FIG.
λ _max nm (ε):
In methanol: 275 (43,000), 292 (42,000), 305 (3,500), 406 (5,700)
In 0.005M hydrochloric acid-containing methanol: 275 (42,700), 292 (41,800), 305 (3,500), 406 (5,700)
In methanol containing 0.005M sodium hydroxide: 277 (36,000), 295 (38,700), 308 (3,400), 509 (4,800)
(7) As a proton nuclear magnetic resonance spectrum, a proton NMR spectrum measured in deuterated methanol at 600 MHz is as shown in FIG.
(8) As a carbon 13 nuclear magnetic resonance spectrum, a carbon 13 NMR spectrum measured in deuterated methanol at 150 MHz is as shown in FIG.
Moreover, about the MK844-mF10 substance obtained in Example 1, antibacterial activity was confirmed by the following test example 1, and enzyme inhibitory activity was confirmed by the following test example 2.

(Test Example 1: Antibacterial activity)
Methicillin-Sensitive Staphylococcus aureus (MSSA), Multi-Drug Resistant Staphylococcus aureus (MDRSA), Methicillin-resistant Staphylococcus aureus (MDRSA) The antibacterial spectrum of the MK844-mF10 substance against Aureus: VISA) was measured by a multiple dilution method on Mullah Hinton agar medium (manufactured by Difco) based on the standard method of the Japanese Society of Chemotherapy. The measurement results of the minimum inhibitory concentration (MIC) are shown in Table 1.

  From the results in Table 1, the MK844-mF10 substance is found in methicillin-sensitive Staphylococcus aureus (MSSA), multi-drug resistant Staphylococcus aureus (MDRSA), methicillin-resistant Staphylococcus aureus (MRSA), and vancomycin-insensitive Staphylococcus aureus (VISA). On the other hand, it was found to have antibacterial activity.

(Test Example 2: Enzyme inhibitory activity)
-(1) YycG histidine kinase activity inhibition test-
The enzyme inhibitory activity of the MK844-mF10 substance against YycG of Bacillus subtilis 168 strain ( B. subtilis 168, sold by Tokyo University of Agriculture) was examined.
Measurement of histidine kinase activity was performed as described in Biosci. Biotechnol. Biochem. 64, 919-923, 2000.
A chromosomal DNA of Bacillus subtilis 168 strain comprising a region containing only the kinase active domain of YycG (including amino acids 207 to 611 from the N-terminal) (hereinafter sometimes referred to as “ycGtru”) by PCR. And cloned into the expression vector pET-21a (+) (Novagen). A protein (hereinafter referred to as “YycG-207-611”) expressing the histidine kinase activity domain of YycG from the culture solution of the strain obtained by transforming the plasmid pET-yycGtru thus obtained into E. coli. ) Was purified.
The reaction solution for measuring histidine kinase activity was composed of 0.5 μM YycG-207-611, 50 mM Tris-HCl (pH 8.5), 100 mM potassium chloride, 100 mM ammonium chloride, and 5 mM magnesium chloride.
A predetermined concentration of MK844-mF10 substance was added to the reaction solution and incubated at 30 ° C. for 5 minutes. Then, add 2.5 μM ATP-10 μCi [γ- ³² P] ATP mixed solution to 10 μL, start the reaction, incubate at 30 ° C. for 10 minutes, terminate the reaction, and perform SDS-polyacrylamide electrophoresis Was used to determine the 50% inhibitory concentration (IC ₅₀ ) of Bacillus subtilis against YycG.
As a control, the MK844-mF10 substance was not added to the reaction solution, but 2.5 μM ATP-10 μCi [γ- ³² P] ATP mixed solution was added to make 10 μL, and the mixture was incubated at 30 ° C. for 10 minutes. It was. The results are shown in Table 2.

-(2) VicK histidine kinase activity inhibition test-
The enzyme inhibitory activity of the MK844-mF10 substance against VicK of Streptococcus mutans (obtained from ATCC (American type culture collection)) was examined.
Measurement of histidine kinase activity was performed as described in Biosci. Biotechnol. Biochem. 64, 919-923, 2000, which was modified.
A region containing only the kinase activity domain of VicK (including the 31st to 450th amino acids from the N-terminal) (hereinafter sometimes referred to as “SMvicK31-450”) is subjected to PCR by cariogenic bacteria. It was prepared from chromosomal DNA and cloned into the expression vector pET22b (+) (Novagen). A protein expressing only the histidine kinase active domain of VicK (hereinafter referred to as “VicK-31-450”) from the culture solution of the strain obtained by transforming the plasmid pET-SMvicK31-450 thus obtained into E. coli. Is purified).
The reaction solution for measuring histidine kinase activity was composed of 0.5 μM VicK-31-450, 50 mM Tris-HCl (pH 7.5), 50 mM potassium chloride, 10 mM magnesium chloride.
1 μL of MK844-mF10 substance was added to 7 μL of this reaction solution, and incubated at 25 ° C. for 5 minutes. Thereafter, 2 μL of 12.5 μM ATP containing [ ³² P] ATP is added (final concentration 2.5 μM) to start the reaction. After incubation at 25 ° C. for 20 minutes, the reaction is terminated, and SDS-polyacrylamide gel electrophoresis is performed. Thus, a 50% inhibitory concentration (IC ₅₀ ) of caries bacteria against VicK was determined.
In addition, as a control, the MK844-mF10 substance was not added to the reaction solution, but 2 μL of 12.5 μM ATP containing [ ³² P] ATP was added and incubated at 25 ° C. for 20 minutes. The results are shown in Table 2.

-(3) PehS histidine kinase activity inhibition test-
Soft rot MAFF301393 strain for PehS of (Erwinia carotovora subsp. Carotovora MAFF301393, MAFF microbial genetic resources (MAFF) from available), it was examined enzyme inhibitory activity of the MK844-mF10 material.
Measurement of histidine kinase activity was performed as described in Biosci. Biotechnol. Biochem. 64, 919-923, 2000.
A region containing only the kinase active domain of PehS (including amino acids 209 to 484 from the N-terminus) (hereinafter sometimes referred to as “pehScM2-2”) is obtained by PCR using the soft rot MAFF301393 strain. It was prepared from chromosomal DNA and cloned into the expression vector pET-21a (+) (Novagen). From a culture solution of a strain obtained by transforming the plasmid pET-pehScM2-2 thus obtained into E. coli, a protein expressing only the PehS histidine kinase active domain (hereinafter referred to as “PehS-209-484”). Was purified).
The reaction solution for measuring histidine kinase activity was composed of 4 μM PehS-209-484, 50 mM Tris-HCl (pH 8.5), 100 mM potassium chloride, 100 mM ammonium chloride, 5 mM magnesium chloride.
A predetermined concentration of MK844-mF10 substance was added to the reaction solution and incubated at 30 ° C. for 5 minutes. Then, add 2.5 μM ATP-10 μCi [γ- ³² P] ATP mixed solution to make 10 μL, start the reaction, incubate at 30 ° C. for 20 minutes, terminate the reaction, and perform SDS-polyacrylamide electrophoresis Was used to determine the 50% inhibitory concentration (IC ₅₀ ) of soft rot fungus against PehS.
As a control, the MK844-mF10 substance was not added to the reaction solution, but a 2.5 μM ATP-10 μCi [γ- ³² P] ATP mixed solution was added to make 10 μL and incubated at 30 ° C. for 20 minutes. It was. The results are shown in Table 2.

  From the results of Table 2, it was found that the MK844-mF10 substance has an inhibitory activity against histidine kinase possessed by Gram-positive bacteria and Gram-negative bacteria. It was found that the MK844-mF10 substance has particularly strong inhibitory activity against YycG.

  Since the novel compound (MK844-mF10 substance) of the present invention has excellent antibacterial activity and excellent enzyme inhibitory activity, it can be suitably used as a new antibacterial agent and enzyme activity inhibitor.

  NITE P-903

Claims (6)

A compound represented by the following structural formula (A):
A method for producing a compound represented by the following structural formula (A),
A culture step for culturing a microorganism belonging to the genus Streptomyces and having the ability to produce a compound represented by the following structural formula (A):
A collecting step of collecting a compound represented by the following structural formula (A) from the culture obtained in the culturing step;
A process for producing a compound, comprising:
A microorganism belonging to the genus Streptomyces and having the ability to produce a compound represented by the following structural formula (A) is Streptomyces sp. MK844-mF10 strain having an accession number of NITE P-903 A method for producing the compound according to claim 2.
A compound-containing composition comprising a compound represented by the following structural formula (A).
An antibacterial agent comprising the compound-containing composition according to claim 4. A histidine kinase activity inhibitor comprising the compound-containing composition according to claim 4.