JP2001103990A - New fom-8,108 substance and method for producing the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a substance having inhibitory activity against sphingomyelinase, useful for preventing and treating diseases such as arteriosclerosis, inflammation, diabetes, etc., and immunological function-related diseases. SOLUTION: This substance is obtained by culturing a fungus capable of producing FOM-8,108 substance in a medium, accumulating the FOM-8,108 substance in a culture solution and collecting the FOM-8,108 substance from the culture product.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a novel FOM-8108 substance having sphingomyelinase inhibitory activity and a method for producing the same.

[0002]

BACKGROUND OF THE INVENTION Interleukin, a cytokine
It is known that intracellular information transmission by stimulation of 1β or tumor necrosis factor-α is involved in diseases such as inflammation, arteriosclerosis, diabetes, and immune regulation mechanisms. Recently, it has been revealed that a hydrolysis reaction of sphingomyelin, one of the lipids as a biological membrane component, is involved in this information transmission pathway. (YA Hannun et al., Journal.
Of Biological Chemistry, 269, 31
25, 1994; Kolesnick and D.K.
W. Golde, Cell, 77, 325, 1994
Year; M. Boucher et al., Journal of Existence Medicine, 18, 2059, 199.
5 years). However, a prophylactic or therapeutic drug for the disease from a drug that specifically and strongly inhibits sphingomyelinase, a sphingomyelin hydrolase, has not yet been put to practical use.

[0003]

Sphingomyelinase is an enzyme that uses sphingomyelin, one of the lipids as a component of a biological membrane, as a substrate and decomposes it into ceramide and phosphocholine. Ceramide, a degradation product of sphingomyelin, is known to be involved in the regulation of cell differentiation, proliferation and related signaling (Kiyohide Kojima and Keiko Koizumi, Protein Nucleic Acid Enzyme, 36,
629, 1991).

The cytokine interleukin-1
β and tumor necrosis factor-α bind to receptors on target cells,
It has been shown that sphingomyelinase is involved when signals are transmitted intracellularly (KA Dress).
ler et al., Science, 252, 1715, 199.
2 years).

[0005] Interleukin-1β and tumor necrosis factor-
α is infiltration of lymphocytes in inflammatory diseases such as rheumatoid arthritis and collagen disease (SB Mixel et al., Proceeding of National Academy of Sciences of USA, 78, 2474,
1980; Tanaka et al., Journal of
Immunology, 143, 1584, 1989),
Adhesion and migration of monocytes in the onset and progression of atherosclerosis and proliferation of vascular smooth muscle cells in the intima (JS Puber
Et al., Journal of Immunology, 137, 18
93, 1986; A. Neiken et al., Journal of Clinical Investigation, 8
8, 1121, 1991; W. Raines
Et al., Science, 243, 393, 1989),
Denatured low density lipoprotein formation that causes atherosclerosis (SL Schissel et al., Journal of
Clinical Investigation, 98, 145
5, 1996), and immune diseases such as transplantation versus host disease due to activation of immune cells such as T cells (P. Scheurich).
Et al., Journal of Immunology, 138, 17
86, 1987) and the development of insulin resistance in non-insulin dependent diabetes (BM Spiegel).
Man et al., Journal of Biological Chemistry, 268, 6823, 1993).

[0006] Therefore, substances that inhibit sphingomyelinase activity can block signal transduction of interleukin-1β and tumor necrosis factor-α, and are expected to be effective in treating pathological conditions involving these cytokines. Is done. Under such circumstances, providing a substance that inhibits sphingomyelinase activity is expected to be useful for treating diseases such as inflammation, arteriosclerosis, and diabetes, and diseases related to immune function.

[0007]

The present inventors have conducted various studies on metabolites produced by microorganisms. As a result, the present inventors found that sphingomyelinase inhibitory activity was found in a culture of FOM-8108 strain newly isolated from seawater. Have been found to be produced. Next, the active substance that inhibits the sphingomyelinase was separated and purified from the culture, and as a result, a substance having a chemical structure represented by the following formula [I] was found. Since a substance having this chemical structure has not been known at all, this substance is referred to as a FOM-8108 substance. The present invention has been completed based on such findings, and has the following formula [I]

[0008]

Embedded image The FOM-8108 substance represented by

[0009] The present invention further belongs to the aspergillus oryzae,
A microorganism having an ability to produce the FOM-8108 substance according to claim 1 is cultured in a culture medium, and the FOM-8108 is contained in the culture solution.
Accumulate 108 substances and remove FOM-810 from the culture.
The present invention relates to a method for producing FOM-8108, which collects 8 substances.

[0010] The present invention further relates to the genus Aspergillus oryzae,
The microorganism having the ability to produce FOM-8108 substance is a strain with incomplete spore-free strain FOM-8108.
(Agonomycetes Strain FOM-
8108 FERM P-17591)
8108 material. The present invention further relates to a microorganism belonging to the order of Myxomycetes and having the ability to produce FOM-8108 substances.

FOM-810 represented by the above formula [I]
Microorganisms having the ability to produce eight substances belong to the order of the spore-free bacterium. For example, the present inventors have newly isolated incomplete filamentous fungal nets from the sea sand on the coast of Enoshima, Agonomycetes
(Mycelia Sterilia) Strain F
OM-8108 strain. The present invention FOM-810
The bacteriological properties of the eight strains are as follows.

1. Morphological properties The strain grew well in a dark place on a potato dextrose agar medium, a wort agar medium, an oatmeal agar medium, a potato carrot agar medium, a V-8 juice agar medium, a Miura agar medium, etc. No sexual or asexual spores were formed in the culture for a maximum of 4 weeks. Also,
Similarly, formation of sexual spores and asexual spores was not observed under natural light and black light blue. Production of a reddish brown soluble pigment was observed on the potato dextrose agar medium. The hypha is colorless and has a partition.

2. Various properties in culture Tables 1 and 2 below show the results of macroscopic observation when cultured on various agar media at 25 ° C for 14 days.

[0014]

[Table 1]

[0015]

[Table 2] In these media, the formation of small sclerotium and sclerotium was not observed with the growth of the bacteria.

Physiological properties 1) Optimum growth conditions The optimum growth conditions of this strain are pH 4-6, growth temperature 11-
30 ° C. 2) Range of growth The growth range of this strain is pH 3-10, growth temperature 6-34.
° C. 3) distinction between aerobic and anaerobic aerobic

Based on the morphological characteristics, culture characteristics and physiological characteristics of the above-mentioned strain FOM-8108 of the present invention, the strain was compared with known strains. It became clear that there was. The strain was strain FOM-8108 (Strain FOM-8108).
-8108) has been deposited at the Institute of Biotechnology and Industrial Technology, Institute of Industrial Science and Technology, located at 1-3 1-3 Higashi, Tsukuba, Ibaraki Prefecture. The deposit date is October 4, 1999, and the deposit number is FERM P-17591.

As the FOM-8108 substance-producing bacterium used in the present invention, the above-mentioned incomplete spore-free mycelia strain
FOM-8108 strain, but a natural mutant, X
Line irradiation, ultraviolet irradiation, N-methyl-N'-nitro-N-
FOM-8108 substance belonging to the order of incomplete spores, including artificial mutant strains, cell fusion strains, and genetically engineered strains that can be obtained by mutation treatment of nitrosoguanidine, 2-aminopurine, etc., and represented by the above formula [I] All of the strains producing are usable in the present invention.

In practicing the present invention, first, a FOM-8108 substance-producing bacterium belonging to the order of incomplete spores is cultivated in a medium. The above FOM-81
As a nutrient source suitable for the production of the 08 substance, a nutrient medium containing a carbon source that can be assimilated by microorganisms, a nitrogen source that can be digested, and, if necessary, inorganic salts and vitamins is used. As the carbon source, sugars such as glucose, fructose, maltose, lactose, galactose, dextrin and starch, and vegetable oils such as soybean oil are used alone or in combination.

As the nitrogen source, peptone, yeast extract,
Meat extract, soybean flour, cottonseed flour, corn steep liquor, malt extract, casein, amino acids, urea, ammonium salts, nitrates and the like are used alone or in combination. Other phosphates and magnesium salts as needed
Salts such as calcium salt, sodium salt and potassium salt, heavy metal salts such as iron salt, manganese salt, copper salt, cobalt salt and zinc salt, vitamins, and others suitable for production of the FOM-8108 substance of the present invention are appropriately added. Is done.

In the culturing, when foaming is severe, an antifoaming agent such as a liquid paraffin, animal oil, vegetable oil, silicone or a surfactant may be added as necessary. In the above culture, the medium may be liquid or solid as long as it contains the above-mentioned nutrient source, but it is usually preferable to culture using a liquid medium. In the case of small-scale production, culturing using a flask is preferred. In order to industrially produce the target substance in large quantities, it is preferable to carry out aeration and agitation cultivation similarly to other fermentation products.

When culturing is carried out in a large tank, a relatively small amount of medium is first inoculated with the bacterium in the production process, and then the culture is transferred to a large tank in order to prevent the growth of the bacterium from delaying. Therefore, it is preferable to perform production culture there. In this case, the composition of the medium used for the preculture and the composition of the medium used for the production culture may be the same, or both may be changed if necessary.

When culturing is carried out under aeration and stirring conditions, known methods such as stirring with a propeller or other machine, rotation or shaking of a meter, pumping, and air blowing are appropriately used. Use sterilized air for ventilation. The culture temperature can be appropriately changed within a range in which the FOM-8108 substance-producing bacterium of the present invention produces the FOM-8108 substance of the present invention, but it is usually preferable to culture at 20 to 30 ° C, preferably around 27 ° C. The culture pH is usually 5
-8, preferably around 7. The culture time varies depending on the culture conditions, but is usually about 3 to 8 days.

The FOM-8 of the present invention thus obtained
108 substances are present in the culture filtrate. The target FOM-8108 substance is collected from the culture by extracting the culture filtrate with a water-immiscible organic solvent such as ethyl acetate. In addition to the above extraction method, known methods used for collecting fat-soluble substances, such as adsorption chromatography, gel filtration chromatography, thin-layer chromatography, centrifugal countercurrent distribution chromatography, high-performance liquid chromatography, etc., appropriately combined, Alternatively, by repeating, the FOM-8108 substance is separated into each component,
It can be purified.

The physicochemical properties of the FOM-8108 substance according to the present invention are as follows. (1) Property: brown needle-like (2) Molecular weight: 171 (by high-speed atom bombardment mass spectrometry) (3) Molecular formula: C ₇ H ₅ O ₃ Cl (4) Specific rotation: [α] _D ²⁵ = 0 (c) = 1.0 in methanol) (5) Ultraviolet absorption maximum (in methanol): As shown in FIG. 1, 258 nm (ε = 37100), 331 nm (ε
= 1500).

(6) Infrared absorption maximum (KBr tablet): FIG.
As shown in the figure, it has a maximum absorption at 1109, 1655, and 3438 cm ^-1 . (7) Proton nuclear magnetic resonance spectrum (in deuterated methanol): as shown in FIG. (8) ¹³ C nuclear magnetic resonance spectrum (in deuterated methanol):
As shown in FIG. (9) Solubility in solvent: chloroform, toluene,
Soluble in ethyl acetate and methanol, insoluble in hexane and water. (10) Color reaction: positive for sulfuric acid and iodine. (11) Distinguishing between acidic, neutral and basic: weakly acidic substances

As described above, as a result of examining various physicochemical properties and spectrum data of the FOM-8108 substance of the present invention, it was determined that the FOM-8108 substance of the present invention has a chemical structure represented by the following formula [I]. Was done.

[0028]

Embedded image As described above, various physicochemical properties of the FOM-8108 substance of the present invention have been described in detail, but no compound corresponding to such properties has been reported so far, and therefore, the FOM-8108 according to the present invention has not been reported. The substance was determined to be a new substance.

Next, the biological properties of the FOM-8108 substance of the present invention will be described in detail. (1) Inhibitory activity on rat brain-derived neutral sphingomyelinase The effect on rat brain-derived neutral sphingomyelinase was determined by the method of Murakami and Arima et al. (Journal of Neurochemistry, vol. 52, 611-
618, 1989).

That is, using a membrane fraction prepared from rat brain as an enzyme source, 20 mM HEPES-NaOH buffer (pH 7.4), 6.5 mM MgCl ₂ , 0.1% Triton X-100, 25 μM [N-methyl] ⁻³ H] sphingomyelin (0.006 μCi), and add 5
0 μl was used. After reaction at 37 ° C. for 30 minutes, chloroform:
200 μl of a mixture of methanol (2: 1, v / v) is added to separate [ ³ H] sphingomyelin as a raw material and [ ³ H] phosphocholine as a reaction product. The upper layer was placed in a 50 μl vial, the amount of [ ³ H] phosphocholine was quantified using a liquid scintillation counter, and the neutral sphingomyelinase activity was measured. F that inhibits this enzyme by 50%
The result of calculating the concentration of the OM-8108 substance was 0.2 μg.
/ Ml.

(2) Effect on human placenta-derived acid sphingomyelinase The effect on human placenta-derived acid sphingomyelinase was partially described by the method of Jones et al. (Biochemical Journal, 195, pp. 373-382, 1981). The modification was performed.

That is, an acid sphingomyelinase (manufactured by Sigma) derived from human placenta was used as an enzyme source for 250 m
M sodium acetate buffer (pH 5.0), 0.1% NP
-40 (manufactured by Sigma), 25 μM [N-methyl- ³ H]
Sphingomyelin (0.006 μCi) was added to bring the total volume to 50 μl. After reaction at 37 ° C. for 30 minutes, 200 μl of a chloroform: methanol (2: 1, v / v) mixed solution was added.
In addition, [ ³ H] sphingomyelin and the reaction product [ ³ H] phosphocholine are separated. The upper layer was placed in a 50 μl vial, [ ³ H] phosphocholine was quantified using a liquid scintillation counter, and the acid sphingomyelinase activity was measured. The FOM-8108 substance of the present invention did not show any inhibitory activity against acid sphingomyelinase at a concentration of 100 μg / ml.

[0033]

EXAMPLES The present invention will be described in more detail with reference to examples, but the scope of the present invention is not limited to the examples. 2.0% glucose, yeast extract (Oriental Yeast Co., Ltd.) in a 500 ml Erlenmeyer flask
0.2%, magnesium sulfate heptahydrate 0.05%, polypeptone (manufactured by Nippon Pharmaceutical Co., Ltd.) 0.5%, dipotassium phosphate 0.1%, agar 0.1% were dissolved in 50% natural seawater. Liquid medium (pH 6.0) was dispensed in 100 ml portions, and 12
Sterilize by autoclaving at 1 ° C. for 15 minutes, add potato dextrose agar (Difco) 1.95%, agar 0.
Strain FOM-8108 strain (FERM) cultured at 27 ° C. on an agar slant medium containing 75% and 50% natural seawater
P-17591) was inoculated with one loopful, and cultured at 27 ° C. for 4 days using a rotary shaker to obtain a seed culture solution.

Next, 100 ml of a liquid medium in which 2.4% of potato dextrose broth (manufactured by Difco) and 1% of tertiary magnesium phosphate were dissolved in 50% natural seawater were dispensed into 60 500 ml Erlenmeyer flasks. The solution was sterilized by high-pressure steam at 121 ° C. for 15 minutes, inoculated with 1 ml of a seed culture solution, and cultured at 27 ° C. for 6 days using a rotary shaker.

The culture solution is centrifuged, and the pH of the obtained supernatant is determined.
Was adjusted to 9.0, and 5 liters of ethyl acetate was added for extraction. After anhydrous sodium sulfate was added to the obtained ethyl acetate layer for dehydration, the ethyl acetate layer was concentrated under reduced pressure to 1.4.
8 g of crude substance I were obtained. This was dissolved in a small amount of hexane, and then a silica gel column (75
g, Merck Art. 1.07734), and after washing the column with hexane-ethyl acetate (100: 10), the active substance was eluted with hexane-ethyl acetate (100: 20). I got Further, this is dissolved in a small amount of methanol, and Sephadex L pre-filled with methanol is used.
Loaded on top of H-20 column (1 × 107 cm) and eluted with methanol. The active fraction was collected and concentrated under reduced pressure to obtain 75 mg of FOM-8108 substance.

[0036]

As described above, a novel FOM according to the present invention is obtained.
The -8108 substance is expected to be useful for the prevention and treatment of diseases such as arteriosclerosis, inflammation, and diabetes, and diseases related to immune function, because it shows an inhibitory activity on sphingomyelinase.

[Brief description of the drawings]

FIG. 1 shows an ultraviolet absorption spectrum (in methanol) of FOM-8108 substance of the present invention.

FIG. 2 shows an infrared absorption spectrum (KBr method) of the FOM-8108 substance of the present invention.

FIG. 3 shows a proton nuclear magnetic resonance spectrum of the FOM-8108 substance of the present invention.

FIG. 4 shows a ¹³ C nuclear magnetic resonance spectrum of the FOM-8108 substance of the present invention.

Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat II (reference) A61P 43/00 111 A61P 43/00 111 C07C 50/28 C07C 50/28 C12N 1/14 C12N 1/14 A (72) Inventor Masayoshi Arai 5-9-1, Shirogane, Minato-ku, Tokyo Inside the Kitasato Institute (72) Inventor Rokuro 5-9-1, Shirogane 5-chome, Minato-ku, Tokyo F-term in the Kitasato Institute (reference) 4B064 AD92 BA02 BE05 BE07 BE09 BE14 BG10 BH06 CA05 DA01 4B065 AA57X AC14 BD15 BD16 CA09 CA44 4C206 AA02 AA03 CB27 KA19 MA01 MA04 NA14 ZA45 ZB02 ZB11 ZC20 ZC35 4H006 AA01 AB20 AB22 AB23 AB27

Claims (4)

[Claims] [Claim 1] The following formula [I] A novel FOM-8108 substance which is a compound represented by the formula: 2. A microorganism belonging to the order of incomplete mycelia and having the ability to produce the FOM-8108 substance according to claim 1 is cultured in a medium, and the FOM-8108 substance is accumulated in a culture solution. A method for producing a novel FOM-8108 substance, comprising collecting a FOM-8108 substance from a culture. 3. FOM-81 belonging to the order of Aspergillus incomplete mycelia.
Microorganisms having the ability to produce C.08 substance are described in Spore-free Aspergillus strain FOM-8108 (Agonomiy).
cetes strain FOM-8108, FER
MP-17591). 4. FOM-81 belonging to the order of Asporophora incomplete mycelia.
A microorganism capable of producing 08 substances.