JPH1017593A - New peptide, its production and inhibitor of cell cycle comprising the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To produce a new peptide RK-F1010, having inhibiting activities against the cell cycle and useful as an inhibitor of the cell cycle and an antitumor agent, etc., by culturing a mold, belonging to the genus Aspergillus and having the ability to produce the peptide RK-F1010. SOLUTION: This new peptide RK-F1010 is represented by the formula, has excellent inhibiting activities against the cell cycle and antitumor activities and is useful as an inhibitor of the cell cycle and an antitumor agent, etc. The new peptide is obtained by inoculating a mold Aspergillus terreus 95F-1 strain (FERM P-15629) belonging to the genus Aspergillus into a culture medium, culturing the strain at 28 deg.C for 96hr by shaking culture, then extracting the cultured solution with a 70% acetone, vacuum concentrating the extract solution, regulating the resultant aqueous solution to pH7.0, extracting the regulated aqueous solution with ethyl acetate, concentrating the resultant extract solution, treating the obtained concentrate by a silica get column chromatography and high-performance liquid chromatography and separating and purifying the resultant fraction.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a novel peptide RK-F
The present invention relates to 1010, a method for producing the same, a cell cycle inhibitor containing the same as an active ingredient, an antitumor agent, and a filamentous fungus belonging to the genus Aspergillus that produces the same.

2. Description of the Related Art The cells constituting the human body maintain homeostasis, and for this purpose, the proliferation and differentiation of the cells are strictly controlled. Cells divide and proliferate by rotating the cell cycle consisting of a series of processes of M phase, G ₁ phase, S phase, and G ₂ phase. When an abnormality occurs in the control mechanism of the cell cycle, homeostasis is disrupted, and the possibility of cancer or immune disease is increased. Recently, the regulation mechanism of the cell cycle is being elucidated at the molecular level, and the possibility of substances that regulate the cell cycle, such as antitumor agents and immunosuppressants, is being shown. Conventional drugs for these uses have problems in terms of toxicity and side effects, and development of new compounds suitable for these uses has been demanded.

[0002]

SUMMARY OF THE INVENTION An object of the present invention is to provide a novel compound having a cell cycle inhibitory activity, a method for producing the same, a cell cycle inhibitor containing the compound as an active ingredient, and an antitumor agent.

Means for Solving the Problems The present inventors have conducted intensive studies to solve the above problems, and as a result, a filamentous fungus belonging to the genus Aspergillus isolated from soil collected in Naha City, Okinawa Prefecture.
The present inventors have found that the 95F-1 strain produces a substance that inhibits the cell cycle, and have completed the present invention. That is, the present application provides the following inventions. A. A peptide RK-F1010 represented by the following formula (1).

[0003]

Embedded image

B. A method for producing peptide RK-F1010, comprising culturing a filamentous fungus 95F-1 belonging to the genus Aspergillus that produces peptide RK-F1010 in a medium, and isolating and collecting peptide RK-F1010 from the culture. . C. A cell cycle inhibitor comprising the peptide RK-F1010 as an active ingredient. D. An antitumor agent containing the peptide RK-F1010 as an active ingredient. E. FIG. A filamentous fungus Aspergillus terreus 95F-1 strain belonging to the genus Aspergillus that produces peptide RK-F1010.

[0005]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail. The strain producing the novel peptide RK-F1010 of the present invention is a strain newly isolated from soil collected by the present inventors in Naha City, Okinawa Prefecture, and has the microorganism name `` Aspergillus terreus 95F-1 (Aspergillus terreus). 95F-1 ''
And deposited as Microorganism Deposit Number "FERM P-15629" on May 17, 1996 at the Institute of Biotechnology and Industrial Technology, National Institute of Advanced Industrial Science and Technology. The filamentous fungus Aspergillus terreus 95F-1 producing RK-F1010 of the present invention has the following biological properties.

[Characteristics on Medium] Czapec yeast extract agar (CYA; see Table 1) on a plate: Cultured at 25 ° C. for 7 days, growth is moderate, colony diameter is 26 to 29 mm, surface is Felt-like, radially and concentrically wrinkled, slightly raised overall. Spore formation is moderate near the center of the colony, and secretion of a colorless and transparent exudate is also observed. The color of the colony surface is
A pale reddish yellow near the center where sporulation is observed [10YR
8.5 / 6], yellowish white [5Y9 / 1] near the periphery where active aerial hyphae are formed, and pale yellow [5Y9 / 6] near the boundary between the two.
Present. The back surface of the colony is wrinkled similarly to the front surface, and the color tone is yellow-red [5YR6 / 12] near the center, and in other parts, it changes from pale reddish yellow [10YR8.5 / 6] to reddish yellow [10YR7 / 12]. Slightly secretes a yellow soluble dye into the medium.

[0007] On CYA agar plate: When cultured at 37 ° C for 7 days, the growth is good and the diameter of the colony reaches 48 to 56 mm. The surface has a fluffy to felt shape and the others are CY
A, shows the same properties as the colony at 25 ° C. Malt extract agar (MEA; see Table 1) on a plate: 25 ° C,
After 7 days of culture, the growth was moderate and the colony diameter was 19
At ~ 20 mm, the growth rate is slightly slower than CYA. The surface is felt to velvety, wrinkles radially and concentrically like CYA, but forms flat colonies with no growth thickness. Spore formation is moderate near the center of the colony. The color tone of the colony surface is pale reddish yellow [10YR8.5 / 6] near the center where sporulation is observed, and yellowish white [5Y9 / 1] near the periphery where active aerial hyphae are actively formed. The color on the back of the colony is reddish yellow [10YR7 / 12] near the center, and pale reddish yellow [10YR8.5 / 6] in other areas.

CYA agar containing 20% sucrose (C
Y20S; see Table 1) On a plate: Cultured at 25 ° C for 7 days, growth is moderate, colony diameter is 26 to 29 mm, C
It shows almost the same properties as YA, secretes a little more soluble dye, and no secretions of exudate. The color of the colony surface is light reddish yellow near the center where sporulation is observed.
[10YR8.5 / 6], the color changes from yellowish white [5Y9 / 1] to very pale yellow [5Y9 / 3] near the periphery where aerial hyphae are actively formed. The color of the back of the colony is yellowish red [5YR6 / 12] to bright reddish yellow
Presents [10YR8 / 11]. Other various agar plates: Table 2 shows the results of macroscopic colony observation when cultured at 25 ° C for 7 days. In addition, the color display quoted the system color name of the Japan Standards Association and JIS color name book (1985).

[Morphology] This strain was grown on MEA at 25 ° C and 14 ° C.
When the colony formed during the culture for 1 day was observed with an optical microscope, the tip of the conidium stalk extending from the basal hypha swelled in a sphere, and the confluent, short-cylindrical conidial head characteristic of Aspergillus sp. Are observed in large numbers. Most of the conidial heads formed on the 7th day of culture are immature radial, and the color tone is white to extremely faint brown. On the 14th day of culture, light yellowish brown and short cylindrical conidial heads develop and become dominant. Become. However, places where immature radial conidial heads remain dominant are found near the center of the colony and in irregular sector-like portions. The conidiophore is slightly bent, colorless and thick-walled, the surface is smooth, 200-700 μm in length, and 4.6 in diameter.
88.0 μm, wall thickness 0.4-1.0 μm. The diameter of the tip of the conidium swelling in a spherical shape is 14 to 2
Metre was formed on the surface of 6 μm, 2/3 or more excluding the base. The size of the metre is 4.0-7.0 × 1.8-3.0 μm.
The phialide is in the form of a flask and the size is 5.8 to 8.0 x 1.6.
22.0 μm. Conidia are spherical to subspherical in shape, the surface is smooth, and the diameter is 2.0 to 2.6 μm. In some basal hyphae, aleurioconidia, which is subglobular to drop-shaped, are single-sided. The size of the aleurio-type conidia is 5.0-9.0 × 3.4-5.0 μm. Although the culture was extended to 3 weeks, formation of sexual reproductive organs such as ascocarps was not observed.

[0010]

[Table 1] ────────────────────────────────── CYA * 1 MEA * 2 CY20S * 3 Hydrogen phosphate Dipotassium (K ₂ HPO ₄ ) O.1%-O.1% Yeast extract O.5%-O.1% Sucrose 3.0%-20.0% Malt extract (Malt extract)-2.0 % - peptone (peptone) - O.1% - glucose (glucose) - 2.0% - sodium nitrate _{(NaNO 3) O.3% - O.3} % potassium chloride (KCl) O.05% - O.05% sulfuric acid magnesium heptahydrate _{O.05% - O.05% (MgSO 4} · 7H 2 O) iron (II) sulfate heptahydrate _{O.001% - O.001% (FeSO 4} · 7H 2 O) zinc sulfate heptahydrate _{O.001% - O.001% (ZnSO 4} · 7H 2 O) copper (II) sulfate pentahydrate _{O.0005% - O.0005% (CuSO 4} · 5H 2 O) agar (agar ) 1.5% 2.0% 1.5% pH 6.0 6.0 6.0 * 1: Zapec / Yeast extract agar * 2: Malt extract agar * 3: 20% sucrose-containing Zapec / yeast extract agar

[0011]

[Table 2] 培 地 Medium Growth Colony color Spore soluble pigment (colony Diameter) Secretion of formation Colony properties Front and back valley Medium Yellowish white Light reddish yellow Medium Slightly shrouded (14-15mm) (5Y9 / 1), (10YR8.5 / 6) Secretory Dextrose cold felt-like or dull reddish yellow red agar medium et al velvet-like yellow (10YR7 / 7) (5YR6 / 12) slightly meal cold moderate white of white redness of moderate auto yellowish (26-27mm) (5Y9 / 1 ), (10YR7 / 12) Secreted natural medium Felt-like or pale reddish yellow Yellow-red is almost velvety (10YR8.5 / 6) (5YR6 / 12) Almost diffused Not dull reddish yellow (10YR7) / 7) Sabouraud Medium yellowish white Light reddish yellow None Slight agar medium (28-34mm) (5Y9 / 1), (10YR8.5 / 6) Secreted felt-like YpSs agar Medium yellowish white pale reddish Yellowish medium slightly Medium (23-28mm) (5Y9 / 1), (10YR8.5 / 6) Secretion Fluffy to pale reddish yellowish yellow velvet (10YR8.5 / 6) (5YR6 / 12)

[Physiological properties] Growth pH range and optimum pH: Grow in a YpSs liquid medium in the range of pH 2 to 10, and the optimum pH is 5 to 7. Growth temperature range and optimum temperature: Growing in the range of 16-42 ° C in Sabouraud liquid medium, the optimum growth temperature is 36.
３９39 ° C. Aerobic, anaerobic distinction: aerobic

From the above morphological characteristics and culture characteristics,
This strain is an incomplete phylum, Aspergillus
s) is a strain of the genus
Keisuke Tsubaki ed., "A fungus picture book" Kodansha (1978), Raper (K.
B. Raper) and DI Fennell, The Genus Aspergillus, The Williams and Wilkins Company (The
Williams & Wilkins Company (1965) and MAKlich, Pitt (JIPitt), Laboratory Guide to Common Aspergillus Specialties and There Tereomorphs (A Laboratory)
guide to common Aspergillus species and their tel
eomorphs)] Commonwealth Scientific and Industrial Research Organization, Division of Food Processing (Commonwealth Scientific and Industrial Research)
Organization, Division of Food Processing) (1988
Year)).
As a result, this strain became Aspergillus terreus ( Asperg
illus terreus Thom), and the strain was called "Aspergillus terreus 95F-1".
(Aspergillus terreus 95F-1). "

The method of culturing the above filamentous fungus strain 95F-1 and collecting RK-F1010 (hereinafter sometimes referred to as “the compound of the present invention”) of the structural formula represented by the formula (1) from the culture is as follows. Specifically, it is described in the production examples described later, but it can be carried out generally according to a conventional method. That is, it can be carried out by appropriately using means generally used for collecting microbial metabolites. For example, various ion-exchange resins, non-ionic adsorption resins, gel filtration chromatography, activated carbon, alumina, silica gel and other adsorbents and high-performance liquid chromatography, or crystallization, concentration under reduced pressure, freeze drying and other means. Each of them can be used alone or in combination as appropriate, or repeatedly.

[0015]

EXAMPLES The present invention will be specifically described below with reference to examples and the like. [Production Example] Glucose 2.5%, soluble starch 1%, meat extract 0.5%, yeast 0.3%, polypeptone 0.5%, salt 0.2
%, Potassium monophosphate 0.05%, magnesium sulfate 0.
A 95F-1 strain of the present invention was inoculated into a medium (pH 5.8) having a composition of 05% and soybean flour 2%, followed by shaking culture at 28 ° C for 96 hours. 7 L of the culture solution was extracted using 10 L of 70% acetone. The aqueous solution obtained after concentration under reduced pressure was adjusted to pH 7.0, and extraction was repeated twice with 3.5 L of ethyl acetate. After extraction, all the ethyl acetates were combined and concentrated under reduced pressure to obtain 18.5 g of a brown syrup. Dissolve 18.5 g of this syrup in 10 ml of chloroform, infiltrate a silica gel column (diameter 6 cm, length 90 cm) prepared with chloroform, first flow 1 L of chloroform, and then gradually change the mixing ratio of the chloroform-methanol solution. (99: 1, 49:
1, 19: 1, 9: 1) 1 L each, and finally 1 L methanol. The activity eluted in a fraction of a chloroform-methanol solution (19: 1). This was concentrated under reduced pressure to obtain 7.87 g of a brown oily substance. Further, this brown oily substance was dissolved in ethanol, applied to a column of Sephadex LH-20 (manufactured by Pharmacia) (diameter 4.5 cm, length 120 cm) prepared with the same solution, and eluted with methanol. The active fraction was collected and concentrated under reduced pressure to obtain 4.06 g of a yellow oily substance. Finally, fractionation was performed by high performance liquid chromatography using an ODS column (diameter 2 cm, length 25 cm; Senshu Pack, manufactured by Senshu Science). The solvent used was acetonyl-water (3: 1) at a flow rate of 9.0 ml / min, and fractions of 13 to 15 minutes were separated to isolate 53.3 mg of RK-F1010 as a pale yellow powder.

The physical properties of the purified sample are shown below. [Physical and chemical properties of RK-F1010] Property Light yellow powder Melting point 92-95 ° C [α] _D ²⁵ -135.2 ゜ (c 0.10, CHCl ₃ ) Molecular formula C ₂₈ H ₄₀ N ₄ O ₃ Mass spectrometry m / z 480 (M) elemental analysis Found (%) C: 63.09 H: 8.58 N: 9.38 theoretical value (%) C: 62.92 H: 8.61 N: 10.49 ( calculated as _{C 28 H 40 N 4 O 3} · 3H 2 0) UVλ _{The max} ^MeOH nm (ε) spectrum is shown in FIG. FIG. 2 shows 230 (87000), 285 (6700) IRν _max ^KBr cm ⁻¹ spectra. 3312, 2966, 2932, 1627, 1494, 1460, shows 1093, 744 the result of the nuclear magnetic resonance spectrum DMSO-d ₆ ¹ H-NMR as measured in Table 3 and Figure 3. The results of ¹³ C-NMR measured in DMSO-d ₆ are shown in Table 3 and FIG.

[0017]

Table 3 ¹ H-NMR, ¹³ C-NMR (DMSO-d ₆ ) of RK-F1010

[0018]

Embedded image

The activity of the compound of the present invention was measured according to the following method. [Test Example 1] Cell cycle inhibitory effect Mouse breast cancer cells tsFT210 cells in which p34 ^cdc kinase, a cell cycle regulatory protein, was mutated to temperature sensitivity were used.
The tsFT210 cells are usually cultured at 32 ° C. in an RPMI1640 medium containing 5% calf serum in an incubator saturated with 5% carbon dioxide and water vapor. When tsFT210 cells are cultured at 39 ° C, the cell cycle is G2
Phase, and when it is shifted down to 32 ° C., cell division starts again and shifts to G1 phase. At the same time as the downshift
RK-F1010 was added, and the progress of the cell cycle (G2 → M → G1) was analyzed by flow cytometry and microscopic observation. Table 4 shows the results.

[0020]

[Table 4] Cell cycle inhibitory activity of RK-F1010

Test Example 2 Cell Growth Inhibitory Effect of RK-F1010 Human leukemia cells K-562 and HL-60 were cultured in RPMI1640 medium (containing 10% fetal calf serum). To this was added a series of diluted series of RK-F1010, and after culturing for 17 hours, MTT reagent was added to measure the growth. Table 5 shows the results.

[0022]

[Table 5] Cell growth inhibitory effect (minimum growth inhibitory concentration)

[0023]

Industrial Applicability The novel peptide RK-F1010 of the present invention has excellent cell cycle inhibitory activity and antitumor activity.

[Brief description of the drawings]

FIG. 1 shows an ultraviolet absorption spectrum (in methanol) of the compound of the present invention.

FIG. 2 Infrared absorption spectrum (in KBr) of the compound of the present invention
Is shown.

FIG. 3 shows the ¹ H-NMR spectrum (in dimethyl sulfoxide-d6) of the compound of the present invention.

FIG. 4 shows a ¹³ C-NMR spectrum (in dimethyl sulfoxide-d6) of the compound of the present invention.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁶ Identification code Agency reference number FI Technical display location // (C12N 1/16 C12R 1:66) (C12P 21/02 C12R 1:66) (72) Inventor Kimie Kohinata 2-1 Hirosawa, Wako-shi, Saitama Prefecture RIKEN (72) Inventor Yoshifumi Arai Taisho Pharmaceutical Co., Ltd. 3-24-1 Takada, Toshima-ku, Tokyo

Claims (5)

[Claims] 1. A peptide RK-F represented by the following formula (1):
1010. Embedded image 2. A peptide RK comprising culturing a filamentous fungus 95F-1 belonging to the genus Aspergillus that produces the peptide RK-F1010 in a medium, and separating and collecting the peptide RK-F1010 from the culture. -Manufacturing method of F1010. 3. A cell cycle inhibitor comprising the peptide RK-F1010 as an active ingredient. 4. An antitumor agent comprising the peptide RK-F1010 as an active ingredient. 5. A filamentous fungus belonging to the genus Aspergillus producing the peptide RK-F1010, Aspergillus terreus 95F-1.
stock.