WO2006101073A1

WO2006101073A1 - Anti-cancer agent and bacterium capable of producing novel compound prunustatin

Info

Publication number: WO2006101073A1
Application number: PCT/JP2006/305540
Authority: WO
Inventors: Toru Natsume; Kazuo Shinya
Original assignee: National Institute Of Advanced Industrial Science And Technology
Priority date: 2005-03-22
Filing date: 2006-03-20
Publication date: 2006-09-28
Also published as: JPWO2006101073A1

Abstract

[PROBLEMS] To search a substance capable of inhibiting the induction of the expression of GRP78 to find a novel anti-cancer agent. [MEANS FOR SOLVING PROBLEMS] A novel compound represented by the chemical formula 1 is now found as a substance capable of inhibiting the induction of the expression of GRP78 and named 'prunustatin'. Prunustatin has a structure similar to that of a known compound SW-163A and can be produced by Streptomyces Violaceoniger strain 4521-SVS3 or the like. A pharmaceutical composition comprising at least the compound is likely to be effective as an anti-cancer agent.

Description

Specification

Anticancer drugs and bacteria producing new compound branastatin

Technical field

[0001] The present invention relates to a novel compound "branastatin" that suppresses the expression of GRP78, a compound that suppresses the expression of GRP78 (neoantimycin analogs such as "SW163A"), an anticancer agent containing at least the compound, In addition, the present invention relates to an actinomycete strain “Streptomyces violaceoniger 4521—SVS3 strain” capable of producing the compound.

Background art

[0002] The endoplasmic reticulum is one of the organelles of eukaryotes and performs protein synthesis, folding, sugar chain modification, and the like. The state in which immature proteins accumulate in the endoplasmic reticulum is called ER stress. For example, under stress conditions such as glucose starvation, protein folding is incomplete and immature proteins accumulate in the endoplasmic reticulum, resulting in endoplasmic reticulum stress.

A molecular chaperone refers to a protein that controls folding of a synthetic protein during protein synthesis. In eukaryotes, the “HSP (Heat Shoes Protein) 70” family and the “Chaperonin (Hsp60)” family are known as representatives.

It is known that cells have a defense mechanism against endoplasmic reticulum stress. Accumulation of incompletely folded proteins in the endoplasmic reticulum induces molecular chaperone expression. Cells induce protein chaperone expression to promote protein folding and protect cells from endoplasmic reticulum stress.

When endoplasmic reticulum stress is not eliminated by induction of molecular chaperone expression, a pathway leading to apoptosis (cell death) is activated (see Non-Patent Document 1).

Endoplasmic reticulum stress is known to be induced by compounds such as tunicamycin.

[0003] GRP78 (Glucose-Regulated Protein 78—kD) is HSPA5 (Heat—Shoe k 70kD Protein 5) or i BIP (immunoglo Dulin heavv chain—Binding). Protein (also called protein), a stress-responsive molecular chaperone belonging to the HSP70 family and present in the endoplasmic reticulum.

It is known that expression of GRP78 is induced in animal cells under glucose starvation conditions or in the presence of tunicamycin (see Non-Patent Document 1, etc.).

[0004] Solid tumors are generally known to have glucose-starved regions unlike normal tissues (see Non-Patent Document 3, etc.), and the expression of GRP78 is induced. It has been reported (see Non-Patent Document 4 etc.). In addition, it has been reported that GRP78 expression suppression suppresses apoptosis induction under glucose starvation conditions (see Non-Patent Document 5).

Each of the above findings indicates that apoptosis can be induced by suppressing the induction of GRP78 expression in the state of starvation of gnolecose, that is, the starved cells (cancer cells) can be killed by suppressing the induction of GRP78 expression. Suggest a possibility.

Therefore, various attempts have been made to search for a substance that suppresses the induction of GRP78 expression as a new pile cancer agent (see Patent Document 1, Non-Patent Document 3 to Non-Patent Document 9, etc.).

[0005] Here, a compound related to the present invention, a neoantimycin analog, will be described. Neoantimycin analog is a compound represented by the following general formula:

[Chemical 5]

[0006] SW163A is a compound of R force S methyl group, R is antisobutyl group, SW163B Is a compound having an R-catyl group and an R-force S-isopropyl group. Neoantimycin is

1 2

, R force S is an isopropyl group, R is an antibutyl group compound.

1 2

SW-163A and SW-163B are substances identified from metabolites of the actinomycete “Streptomyces” genus, and although there are reports as immunosuppressants (Non-patent Document 10), they have specific actions. · The use is not reported. Neoantimycin is known for its chemical structure (see, for example, Non-Patent Document 11), but no specific action has been reported. The chemical structure of SW-163A is shown in [Chemical 4] below.

[Chemical 4]

Patent Document 1: PCT / JP02 / 12237

Non-patent document 1: Biochemical Dictionary 3rd edition (Tokyo Kagaku Doujin): p422 (Gnore course regulatory protein), p607 (GRP94).

Non-Patent Document 2: Special Issue on Experimental Medicine, “Maturity / Developing Apoptosis Research”, Yodosha, 2004: p66-72 (apoptosis caused by endoplasmic reticulum stress)

Non-patent literature 3: om omida A et al, Drug resistance mediated by cellular stress response to the microenvironment of solid tumors Anticancer Drug Des (2): 169-77 (1999) Non-patent literature 4: Jamora C, et al, 'Inhibition of tumor progression by suppression ofstr ess protein GRP78 / Bip induction in fibrosarcoma B / C10ME "Proc Natl Acad SciUS A. (15): 7690-4 (1996)

Non-Patent Document 5: Miyake H., et al 'Stress protein GRP78 prevents apoptosis induced b ycalcium ionophore, ionomycin, in human prostate cancer cells "J Cell Biochem. (3): 396-408 (2000)

Patent Document 6: Hae-Ryong Park, et al "Versipelostatin, a novel GRP78 / Bip molecula rchaperone down-regulator of microbial origin etrahedron Letters (43): 6941-6945 (2002)

Non-Patent Document 7: Park HR, et al "Effect on tumor cells of bloc ing survival response to glucose deprivation" J Natl Cancer Inst. (17): 1266- 7 (2004)

Non-Patent Document 8: Park HR., Et al Effect on tumor cells of blocking survival response to glucose deprivation "J Natl Cancer Inst (17): 1300-10 (2004)

Non-Patent Document 9: Lee AS., Et ai The glucose-regulated protein: stress induction and clinical application Trends Biochem Sci. (8): 504_10 (2001)

Non-Patent Document i¾ 0: Takahashi K., et al "Sw-163A and B, novel immunosuppressantspro duced by Streptomyces sp." J Antibiot (11): 867_73 (2001)

Patent Document 11: Takeda Y., et al "Nuclear magnetic resonance and biosynthetic studies and structures elucidation of isoneoantimycin, a minormetabolite r elated to neoantimycin." J Nat Prod (8): 978- 81 (1998)

Disclosure of the invention

Problems to be solved by the invention

[0008] As described above, a substance that suppresses induction of GRP78 expression in cancer cells may be effective as an anticancer agent.

Accordingly, the main object of the present invention is to search for a substance that suppresses the induction of GRP78 expression and to find a novel anticancer agent.

Means for solving the problem

As a result of the above search, the present inventor has found a novel compound represented by the following general formula [I 匕 1] as a substance that suppresses the induction of GRP78 expression, and has demonstrated that Brunastatin (Pm nustatin) Named. Of the chemical structures of pranastatin, R

1 2 Antisobutyl group compound was named pranastatin A.

[0011] Accordingly, in the present invention, a novel compound, blanastatin, represented by the following general formula [I 匕 1] Or a salt thereof, and a novel compound, blanastatin A represented by the following chemical formula [Dig 2], or a salt thereof. In the general formula [Chemical Formula 1], the substituents R and R are

1 2

Any of the following amino acids Represents the side chain structure. In the above structure, each of the substituents R and R is a methyl group

1 2

In particular, the case of any one of the following: an isopropyl group (palin side chain structure) or an antibutyl group (leucine side chain structure) is particularly preferred.

[Chemical 1]

In addition, as a result of the search, the compound represented by the general formula [Chemical Formula 5] “Thin analog”. ) Has also been found to have an action of suppressing the expression of GRP78. Therefore, in the present invention, the neoantimycin analog represented by the following general formula [Chemical Formula 3] or a salt thereof, which suppresses the expression of GRP78, and the following chemical formula [i 匕 4] which suppresses the expression of GRP78. SW-163A represented by the formula: In the following general formula [Chemical Formula 3], the substituents R and R are glycine, alanine, valine, leucine, isoform, respectively.

1 2

Represents the side chain structure of any of the amino acids of leucine, serine, threonine, cysteine, methionine, asparagine, gnoretamine, proline, phenylalanine, tyrosine, tryptophan, aspartate, gnoletamic acid, lysine, arginine, and histidine. Among the above structures, the substituents R and R forces are methyl group (side chain structure of alanine) and isopropyl group (palin), respectively.

1 2

In particular, the case of either an antisobutyl group (side chain structure of leucine) is particularly preferred.

[Chemical 3]

[Chemical 4]

[0013] As described above, a substance that suppresses the induction of GRP78 expression in cancer cells may be effective as an anticancer agent. Therefore, a pharmaceutical composition containing at least one of the above-described compounds may be effective as an anticancer agent.

[0014] It should be noted that each of the above-mentioned compounds is, for example, Streptomyces violaceoniger 4521— SVS3 strain (Tsukuba Rinhito, Ibaraki Pref. It can be produced as a metabolite such as the deposit at the research institute and the Patent Biological Depositary, and the deposit number FERM BP-10548 (deposited March 3, 2006).

The invention's effect

[0015] Since the compound according to the present invention is presumed to suppress the induction of GRP78 expression and induce apoptosis (cell death) of cancer cells, it may be effective as a pile cancer agent.

Brief Description of Drawings

[0016] FIG. 1 is a graph showing that branastatin has an inhibitory effect on GRP78 expression induction.

FIG. 2 is a graph showing that SW_163A also has an inhibitory effect on GRP78 expression induction. (Inhibition of GRP78 promoter-regulated luciferase production in HT1080 G-L cells by Prunus tation A and SX-163A (black circle: Prunustation A, black triangle: SW-163A)

[Fig. 3] A graph showing that branastatin has almost no effect on the induction of HSP70 expression.

[Fig.4] Branastatin activity Induction of GRP78 expression only under glucose starvation conditions Western plot photograph showing that it suppresses.

FIG. 5 is a graph showing that branastatin force S induces apoptosis (cell death).

[Fig. 6] Electron micrograph (low magnification) of Streptomyces violaceoniger 4521-3 ¥ 33.

[FIG. 7] Electron micrograph (high magnification) of Streptomyces violaceoniger 4521—SVS3 strain.

BEST MODE FOR CARRYING OUT THE INVENTION

<About the compound according to the present invention>:

As an example of the blanastatin according to the present invention, the physical properties of blanastatin A are shown in Table 1.

[0018] Each data is data measured for branastatin A separated and purified by the following procedure. First, the cells were centrifuged from the culture solution (2 L) of Streptomyces violaceoniger 4521-SVS3 strain, and the cultured cells were extracted with acetone. Next, the acetone extract was concentrated, extracted twice with ethyl acetate, dehydrated with Na 2 SO, and then under reduced pressure.

twenty four

Concentrated to dryness. Next, the crude extract was subjected to silica gel column chromatography and developed with n-hexane-ethyl acetate (2: 1), and the activated fraction was concentrated. Next, it was subjected to Sephadex LH-20 column chromatography (inner diameter: 20 mm, length: 500 mm), and gel filtration was performed with black mouth form-methanol (1: 1), and the active fractions were collected and concentrated. Furthermore, 2.5 mg of pranastatin A from 2 L of culture fluid was isolated by reverse-phase HPLC (PEGASIL ODS, inner diameter 20 mm, length 250 mm) using 80% methanol as a developing solvent.

[table 1]

Prunustatin A

Appearance Pale yellow powder

MP 103 to 106 ° C

[afo +24.7. (C 0.32, CHCI ₃ )

Molecular formula C3 H 0N2O12

HRFAB-MS (mlz)

Found 669.2668 (M + H) ⁺

Calcd 669.2660

UV ^? ^H nm (e) 340 (6,500)

IR v 瞧 (KBr) cm " ¹ 3400, 1750, 1720,

1640, 1250 In Table 1,

(1) “Appearance” indicates the property of the substance, and “Pale yellow powder” indicates a blue-yellow powder.

(2) “MP” indicates melting point.

(3) [H] indicates specific rotation. “27” indicates the measurement temperature, and “D” indicates that the measurement was performed using the sodium D line. “C0.32” is the solution concentration and “CHC1” is measured in the mouthpiece form.

Three

It shows that.

(4) “Molecular formula” indicates a molecular formula.

(5) “HR_FAB MS (High-Resolution Fast Atom Bombardment Mass Spectra)” indicates the exact mass (m / z) of blanastatin A measured with a high-resolution FAB mass spectrometer. “Found” indicates the measured value ([M + H] ⁺ ), and “calcd” indicates the calculated value.

(6) “UV” is the ultraviolet absorption spectrum, “nm (ε)” is the maximum absorption, and “MeOH” is the max.

It shows that it measured in methanol. (7) "IR" is the infrared absorption spectrum, "υ cm" J is the maximum absorption, and "KBr" is the bromide power

max

It shows that it was measured by the lithium tablet method.

The ¹³ C-nuclear magnetic resonance spectrum and ¹ H-nuclear magnetic resonance spectrum of pranastatin A are shown in Table 2. In Table 2, “CDC1” indicates that measurement was performed using a heavy chloroform solution.

Three

The

[Table 2]

Ή and C NMR were observed at 500 and 125 MHz, respectively.

[0021] Neoantimycin analogs (SW-163A, SW-163B, neoantimycin

) Is known (see Non-Patent Documents 10 and 11).

[0022] In addition, each compound according to the present invention includes salts, solvates and the like thereof. As salt For example, alkali metal salts (sodium salt, potassium salt, lithium salt, etc.) alkaline earth metal salts (calcium salt, magnesium salt, etc.), metal salts (aluminum salt, iron salt, zinc salt, copper salt, nickel salt, etc.) ), Inorganic salt (acetate, ammonium salt, etc.), organic amine salt (dibenzilamine salt, darcosamine salt, ethylenediamine salt, jetylamine salt, triethylamine salt, dicyclohexylamine salt, diethanolamine salt, tetramethylammonium salt, etc.) Amino acid salts (such as glycine salt, lysine salt, arginine salt, ornithine salt, and asparagine salt) can be applied.

<About the pile cancer agent according to the present invention>:

The pile cancer agent according to the present invention should contain at least the strength of branastatin (including their salts) or neoantimycin analog (including their salts) according to the present invention. It is not limited narrowly by the dosage form. Moreover, when a composition other than the compound according to the present invention is included, it is also included in the pile cancer agent according to the present invention.

That is, the anticancer agent according to the present invention may be formulated into tablets, capsules, granules, powders, syrups, injections, and the like. In addition, in the components of the anticancer agent according to the present invention, an excipient, a binder, a disintegrant, a lubricant, a flavoring agent, a solubilizing agent, a suspending agent, a coating agent, and the like may be appropriately added. .

<Concerning Production Microorganism and Production Method of Compound of the Present Invention (Example)>:

Planastatin and neoantimycin analogs can be produced using, for example, Streptomyces violaceoniger 4521-SVS3 strain of actinomycetes. This strain was deposited (Tsukuba Rinto, Ibaraki Pref. 1-1-1) Deposited at the National Institute of Advanced Industrial Science and Technology, the Patent Biological Depositary, located in the 6th central area, accession number FERM BP- 10548 March 3, 2018). The identification of this strain will be described later.

[0026] The strain can be cultured by the same method as that for ordinary actinomycetes (Strebtomyces). For example, the culture is performed at a culture temperature of 27 ° C (24-30 ° C) and aerobic conditions (shaking culture, aeration and agitation culture, etc.).

When performing large-scale liquid culture, for example, the main culture may be performed after first performing the culture for 2 to 3 days. Since the growth of the strain can be activated by pre-culturing with a small amount of medium, the efficiency of the culture should be improved by ingesting the medium into a large amount of medium after pre-culture. Can do.

The culture conditions such as culture temperature, aeration volume, and culture time can be changed as appropriate.

[0027] The separation and purification of the blanastatin or neoantimycin analog according to the present invention can be performed by a known method.

For example, after first centrifuging or filtering the culture solution, the bacterial cell components are separated, and the supernatant or filtrate is recovered.

Next, pranastatin is purified. Brunastatin can be purified by, for example, adsorption column chromatography using a carrier such as silica gel, gel filtration column chromatography using a gel filtration resin, ion exchange chromatography using anion exchange or cation exchange resin, HPLC. It can be performed by a method utilizing suppression of the expression activity of GRP78.

[0028] In addition, SW-163A can also be obtained, for example, by reducing pranastatin A using NaBH4 (sodium borohydride).

Example 1

[0029] Example 1 is an experiment showing that branastatin according to the present invention has an inhibitory effect on GRP78 expression induction.

[0030] The outline of the experiment is as follows.

“Luciferase” is an enzyme that catalyzes the oxidation reaction of the luminescent substance noreciferin. Luciferin is oxidized and colored by the action of luciferase.

Therefore, first, a GRP78 promoter gene and a luciferase gene (reporter gene) are inserted into a vector. The luciferase gene is inserted downstream of the promoter. Next, the vector is introduced into cancer cells.

Thus, when a substance that promotes the expression of GRP78 is supplied to cancer cells, the candidate substance acts on the promoter site and expresses luciferase. Since the expressed luciferase oxidizes luciferin and develops color, the induction of GRP78 expression can be detected by detecting the color intensity.

In this experiment, HT1080G-L cells were used as cancer cells.

[0031] The results are shown in FIG. In the figure, the vertical axis “Relative luciferase activity” is Lucifera Ze activity (detected color intensity). “Control” is the color intensity in the case of no additive, “2—

“DG” is the color intensity when 1-onM of 2-deoxyglucose is added to the cancer cells, and “PA” is the color intensity when blanastatin A is added to the cancer cells. Represent each.

Deoxygnolecose is a gnorecose metabolism inhibitor and is found in cancer cells.

, A compound that induces endoplasmic reticulum stress.

[0032] As shown in the second bar from the left in Fig. 1, when doxyglucose was supplied to cancer cells, the expression of GRP78 was induced. That is, endoplasmic reticulum stress in cancer cells is G

It was confirmed to induce the expression of RP78.

[0033] On the other hand, as shown by the bars from the third to the right in the left of Fig. 1, when dexignolecose and branastatin A are supplied to cancer cells, the induction of GRP78 expression is suppressed. It was. In addition, the greater the supply amount of branastatin A, the greater the suppression of GRP78 expression induction.

[0034] The above results indicate that the pranastatin A-force cancer cell according to the present invention has an action of suppressing the expression induction of GRP78.

Example 2

[0035] Example 2 is an experiment showing that the neoantimycin analog according to the present invention also has an inhibitory effect on GRP78 expression induction. The outline of the experiment is almost the same as in Example 1.

[0036] The results are shown in FIG. In the figure, the vertical axis “Relative luciferase activity” indicates luciferase activity (detected color intensity), and the horizontal axis “Concentration (nM)” indicates the concentration of pranastatin A or SW-163A. In the figure, black circles indicate the luciferase activity of pranastatin A, and black triangles indicate the luciferase activity of SW163A.

[0037] As shown in FIG. 2, when SW-163A was supplied to cancer cells, the color intensity decreased as with pranastatin A. In both compounds, the color intensity decreased with increasing concentration. The IC value (concentration at which color intensity is inhibited by 50%) of branastatin A is 1

50

9nM, SW— The IC value of 163A was 12.9 nM.

50

[0038] The above results indicate that SW-163A also has an action of suppressing the induction of GRP78 expression. Example 3

[0039] Example 3 is an experiment in which it was investigated whether the blanastatin according to the present invention has an inhibitory effect on the induction of HSP70 expression.

[0040] "HSP70" is a molecular chaperone belonging to the same family as GRP78, and it is known that expression is induced when heat shock (heat shock) is applied.

This experiment was conducted to examine whether pranastatin force GRP78 alone has the effect of suppressing the induction of the expression of other molecular chaperones. The outline of the experiment is almost the same as in Example 1.

[0041] The results are shown in FIG. In the figure, the vertical axis “Relative luciferase activity” indicates luciferase activity (detected color intensity). “None” is the color intensity when no heat shock is applied, “Heat shock” is the color intensity when heat shock is applied to the cancer cells, and “PA” is the addition of blanastatin A to the cancer cells In this case, the color intensity is shown respectively.

[0042] As shown in the second bar from the left in Fig. 2, when heat shock is applied to cancer cells,

HSP70 expression was induced.

[0043] On the other hand, as shown in the rightmost bar from the left in Fig. 2, when both heat shock and brustatatin A supplementation were applied to cancer cells, the induction of HSP70 expression was almost It wasn't suppressed.

[0044] The above results have little effect on the induction of the expression of branastatin HSP70 according to the present invention, that is, the effect of the brustatin according to the present invention specifically on the suppression of GRP78 expression induction. Indicates.

Example 4

[0045] Example 4 is an experiment showing that pranastatin activity according to the present invention has an action to suppress GRP78 expression only under glucose starvation.

[0046] As described above, it is known that GRP78 is induced to express under the conditions of glucose starvation or in the presence of tsuyu forcemycin or the like. Therefore, in this experiment, it was investigated whether GRP78 expression induction was suppressed even in the presence of branastatin and punicamycin. [0047] The outline of the experiment is as follows.

First, HeLa cells (cancer cells) were cultured in DMEM medium. Next, pranastatin A was added to each medium at various concentrations, and 30 minutes later, 2-deoxyglucose or tunicamycin was added at each concentration, followed by incubation for 18 hours.

Next, after the cultured cells were collected, a Western plot was performed using an anti-GRP78 antibody, and the expressed GRP78 was detected.

The results are shown in FIG. In the figure, the top photo is a Western blot photo when 2_deoxygnosole is added, and the bottom photo is a Western blot photo when Tsuyu-mycin is added. “2_DG” represents 2_deoxyglucose, “Prunusutatin A” represents pranastatin A, “TM” and “tsuyu-mycin”, and each numerical value represents an added amount. “GRP78” indicates the position of the GRP78 band, and “actin” indicates the position of the actin band. The actin band is a control indicating that the number of collected cells is almost the same in each experimental condition.

[0049] As shown in the upper Western blot photograph in Fig. 4, the GRP78 band was detected in the second and third lanes from the left. This indicates that the addition of 2-deoxyglucose induced the expression of GRP78.

On the other hand, the left force, the fourth force, and the rightmost lane, the band of GRP78 was attenuated. In particular, in the lanes from the left to the fourth force, the band almost disappeared. This indicates that the induction of GRP78 expression was suppressed by brustatatin A supplementation.

[0050] As shown in the lower Western photograph of FIG. 4, the GRP78 band was detected in the second and third lanes from the left. This indicates that GRP78 expression was induced by the addition of tunicamycin.

The same bands as those in the second and third lanes from the left were detected in each lane from the left to the seventh right. This indicates that branastatin A does not suppress the induction of GRP78 expression when GRP78 expression is induced by the addition of tsuyu forcemycin.

[0051] Therefore, the above results strongly suggest that the induction of the expression of GRP78 is suppressed only under the condition of branastatin force glucose starvation. Example 5

[0052] Example 5 is an experiment showing that the blanastatin according to the present invention induces apoptosis (cell death) as well as suppressing GRP78 expression induction.

[0053] The outline of the experiment is as follows.

“Propidium iodide” is one of the dyes that stain nucleic acids. It is a fluorescent dye that penetrates into dead cells and interacts with the DNA double helix in dead cells. Therefore, after supplying pranastatin A to cancer cells, we examined whether apoptosis occurred by staining dead cells with propidium iodide and counting the number of dead cells.

[0054] The specific procedure is as follows.

First, HT1080 cells (cancer cells) were treated with pranastatin A and tunicamycin or 2-deoxyglucose. Next, the cells were stained with propidium iodide. Next, the number of dead cells stained with propidium iodide was counted using an epifluorescence microscope.

[0055] The results are shown in FIG. In the figure, the vertical axis represents the cell death rate (%). When “control” is non-additive force, “TM” is when cinnamycin is added, “2-DG” is when 2-deoxyglucose is added, “PA” is pranastatin A Indicates that if there is an error.

[0056] In FIG. 5, the cell death rate markedly increased in the bar from the 12th to the rightmost from the left, compared to the bar from the 8th force ^ -th from the left.

[0057] This result strongly suggests that pranastatin induces apoptosis by suppressing the induction of GRP78 expression. That is, branastatin strongly suggests that it may be effective as an anticancer agent.

Example 6

[0058] Streptomyces violaceoniger 4521—Example of identification of SVS3 strain

It is described in 6.

[0059] This strain is an actinomycete isolated from soil collected in Kume-cho, Okiami Prefecture. Its morphological, cultural and physiological properties are as follows.

[0060] (1) Morphological properties:

The electron micrographs of this strain are shown in Figs. The aerial hyphae formed a long main axis, which was randomly branched. At the tip of the branch, a compact 3-6 rotation helical spore chain consisting of 10 or more was formed. No fragmentation of the basic mycelium was observed.

The spores were non-motile and were covered with a sheath at the beginning of the culture, and the morphology of the spores could not be discerned. However, after 3 weeks from the start of the culture, the spores protruded from the sheath and elliptical spores were observed. No sclerotia, sporangia, or other special forms were observed.

The cell wall chemical type was type (I) and contained LL-diaminopimelic acid. The GC content of DNA is 73.2. /. Met.

(2) Culture characteristics:

Table 3 shows the culture properties. The flora color of the village surface is gray, but it became damp after long-term culture. The color of the reverse side was a light yellowish color S on inorganic salt starch agar, oatmeal agar, and tyrosin agar, and red to purple on other agars and did not change with pH. No formation of diffusible dye was observed

[Table 3]

Streptomyces violaceoniger 4521-SVS3 strain culture properties

Medium Microflora color of the village surface Back color of the village Diffusible pigment Sukuguchi · Gray series Red purple None

Nitrate agar Gunolecos · Gray series 喑 Reddish gray None

Asparagine Agar GlycerinGray series Red purple None

Asparagine Agar Inorganic salt Gray series Light yellow None

Starch agar Tyrosine agar Gray series Yellowish brown None Nutrient agar No aerial mycelia Light yellow None

1―Su 卜 ·: ^; i large gray series purple gray to dark reddish purple none oatmeal · agar gray series light yellow none

(3) Physiological properties:

Physiological properties are shown in Table 4. This strain is mesophilic, and melanin-like pigment production was not observed in tyrosine agar, peptone 'yeast' iron agar, or tryptone 'east' broth.

[Table 4] Physiological properties of Streptomyces violaceoniger 4521-SVS3 strain

Growth temperature range 17 1 40. C

Optimal temperature 20-37. C

Melanin-like pigment

Tyrosine agar

Peptone 'i-St Iron Agar

Trypton East Bros

Starch hydrolysis

Gelatin liquefaction

Peptone conversion of skim milk powder

Coagulation of skim milk powder

Reduction of nitrate

Assimilation of carbon sources

D-Guzo Records +

L-arabinose

D-X

D-fructose +

Sucrose +

Leramnos +

Rough Inos

i-Innocent +

D-Manit +

Galactose +

Addonitol

Se / Lebiose +

Innurin

Melbiose + This strain has a spore chain form in which spores are long and spirally linked, and the cell wall chemical type is (I) type. Estimated to be a seed.

Therefore, a strain of the genus Streptomyces that matches the above properties was searched using the “bacterial name approval list, 1980” and subsequent effective name lists.

As a result, we found Streptomyces violaseiniger as a related species. A comparison of this strain with Streptomyces violaceoniger revealed that the assimilation of arabinose and raffinose carbon sources differed, but the other properties were in good agreement (see Table 5). ) ₀

Therefore, the inventor classified this strain as Streptomyces violaceoniger.

Plexus face

-Identify the strain and the chain color and identify it as Streptomyces violaceoniger 4521—SVS3.

Form

Named. State

[Table 5] Gray screw

Vivid purple

Comparison of Streptomyces violaceoniger 4521-SVS3 with related species

― 4521-SVS3 Streptomyces violaceoniger +

+ +

Light color + +

Color + +

pH sensitivity

Diffusible pigment production

pH sensitivity

Melanin-like pigment production

Starch hydrolysis

Reduced salt

Growth temperature 45T

Assimilation of carbon sources

Arabinos +

Kisushi mouth + +

Inositol + +

Man knit + +

Rhamnose + +

Rough Inose +

Sucrose + +

Fractor Tooth + +

Example 7

[0064] Example 7 shows an example of a method for culturing Streptomyces violaceoniger 4521-SVS3 strain.

[0065] First, 15 ml of the preculture medium having the composition shown in “Table 6” was dispensed into a 50-ml large test tube, and after sterilization, the strain was inoculated into a platinum loop from the culture slant, and then 27 ° C, 3 The seeds are those cultured for a day.

[Table 6] Pre-culture medium (pH 7.2)

S t a r c h 1. 0%

P o 1 y p e p t o n 1. 0%

Mo l a s s e s 1. 0%

Next, dispense 100 ml of production medium with the composition shown in “Table 7” into a 500 ml Erlenmeyer flask with a bump and sterilize it. Next, add 2 ml of each seed to each flask and incubate on a rotary shaker at 27 ° C for 5 days.

[Table 7]

[0067] For example, since the strain can be cultured in a large amount by culturing the strain according to the procedure described above, it is possible to obtain a culture solution containing the compound according to the present invention (branastatin, SW163, etc.) with high efficiency.

Industrial applicability

[0068] The blanastatin according to the present invention has applicability as an anticancer agent.

[0069] Since pranastatin suppresses the induction of GRP78 expression only in a glucose-starved state, it is assumed that pranastatin has little effect on the reaction system of cells other than cancer cells. Therefore

The pile cancer agent according to the present invention is likely to have higher safety with fewer side effects than conventional ones.

Claims

The scope of the claims

Brunastatin or a salt thereof represented by the following general formula [ii 匕].

(Wherein the substituents R and R are glycine, alanine, valine, leucine,

1 2

It represents the side chain structure of any one of amino acids such as amino acids, serine, threonine, cysteine, methionine, asparagine, glutamine, proline, phenylalanine, tyrosine, tryptophan, aspartic acid, glutamic acid, lysine, arginine, and histidine. )

Brunastatin A represented by the following chemical formula [Ihi 2] or a salt thereof.

[Chemical 2]

An anticancer agent comprising the blanastatin or salt thereof according to claim 1 as an active ingredient. A compound comprising a compound represented by the following general formula [Dig 3] or a salt thereof as an active ingredient: Narcotic.

[Chemical 3]

(Wherein the substituents R and R are glycine, alanine, valine, leucine,

1 2

An anticancer agent containing SW163A represented by the following chemical formula [I 匕 4] or a salt thereof as an active ingredient.

[Chemical 4]

[6] Streptomyces violaceniga capable of producing the blanastatin according to claim 1. 1 4521—SVS3 strain (Tsukuba Sakai Higashi 1-1-1, Ibaraki Prefecture) Deposited at the National Institute of Advanced Industrial Science and Technology, Patent Biological Depositary, located in Central 6th, Accession No. FERM BP—105 48 Deposit Date March 3).