WO2003044209A1 - Novel tetronic acid derivative - Google Patents

Abstract

Using the expression of a molecular chaperone GRP78 as an indication, attempts have been made to search for a substance capable of controlling the expression of GRP78. As a result, versipelostatin (also called JL68) represented by the formula (I), which is a novel tetronic acid derivative having an activity of inhibiting the GRP78 expression, is isolated from a metabolite of Streptomyces versipellis 4083-SVS6 strain. This versipelostatin can be obtained by culturing the Streptomyces versipellis 4083-SVS6 strain and collecting from the culture supernatant.

Description

 Description New tetronic acid derivative Technical field

 The present invention relates to a novel tetronic acid derivative, and particularly to a tetronic acid derivative derived from actinomycetes. Background art

 The endoplasmic reticulum is an organ where protein synthesis is performed, and is an organelle that folds the produced protein, modifies the sugar chain, and transports the protein. recent years

However, the stress in the endoplasmic reticulum is attracting attention, for example, in relation to the onset of Alzheimer's disease (AD). It is thought that endoplasmic reticulum stress is induced by disruption of physiological balance or external factors such as protein folding, sugar chain modification, and protein transport normally performed in the endoplasmic reticulum. For example, drugs such as tunicamycin ゃ brefeldin A induce ER stress. Sinicamycin inhibits glycosylation of proteins in the endoplasmic reticulum, causing unfolding protein to accumulate in the endoplasmic reticulum. Such accumulation of unfolded proteins causes ER stress. Brefeldin A inhibits protein transport between the endoplasmic reticulum and the Golgi apparatus, thereby accumulating proteins and inducing endoplasmic reticulum stress.

In response to these ER stresses, the ER has a stress response mechanism. For example, Ire, a stress sensor, is known to recognize unfolded proteins, induce the expression of molecular chaperones such as GRP78, and promote protein folding. However, such stress responders It has been suggested that if the ER stress cannot be resolved even by the structure, the cells die by apoptosis.

 Clinical findings on ER stress have also been reported. Analysis of the genetic predisposition to familial Alhiima disease has detected mutations in preselinin-1 (PS1), a membrane protein that is mainly located in the endoplasmic reticulum (ER). It has been reported that this PS1 mutation promotes the production of beta-amyloid, which causes the formation of senile plaques characteristic of Alheimer's disease. It has been reported that sensitivity is increased and apoptosis is easily induced (Guo, Q. et al., J. Neurosci. 17: 4212-4222 (1997), Guo,

Q. et al., Nat. Med. 5: 101-106 (1999)). It has been shown that cell death due to such PS1 mutations can be suppressed by treatments that can reduce endoplasmic reticulum stress, such as treatment with drugs that inhibit calcium release from the endoplasmic reticulum—antioxidants.

 On the other hand, it has been found that DNA topoisomerase II is degraded when stress is applied to human cancer cells. At this time, it is known that cancer cells show resistance to the anticancer drug VP-16 targeting DNA topoi somerase II. Furthermore, it has been found that the expression of GRP78, a molecular chaperone, is increased at this time (Yun,

J. et al., Oncol. Res., 7: 583-590, 1995). In addition, a study of the stress response in human solid tumors and the drug resistance of cancer cells when a proteasome inhibitor was used showed that the degradation of Topoi somerase II due to the stress response was suppressed and the sensitivity to anticancer drugs was improved. (Ogi so, Y. et al., Cancer Res., 60: 2429-2434, 2000)

 In addition, the response to endoplasmic reticulum stress is increased in solid tumors, and it has been suggested that this increased stress response may result in resistance to anticancer drugs. It has also been reported that inhibition of the induction of molecular chaperones such as GRP78 described above enhances sensitivity to anticancer drugs (Koomagi, R. et al., Anticancer Res.

19: 4333-6 (1999), Fernandez, PM et al., Breast Cancer Res. Treat. 59: 15-26 ( 2000), Katschinski, DM et al., J. Cancer Res. Clin. Oncol. 127: 425-32 (2001)) o GRP78 has also been found to be an excellent target for anticancer agents (Jamor a, C. Acad. Sci. 93: 7690-7694, 1996; Toiida, A. and Ts uruo, T., Anti-Cancer Drug Design, 14: 169-177, 1999; Miyake, H. et al., Cancer. Cells. J Cell. Biochem., 77: 396-408, 2000; Lee, AS., TRENDS in Biochem. Sci., 26: 504-510, 2001).

 Thus, regulating the endoplasmic reticulum stress response mechanism by molecular chaperones such as GRP78 expression is important for treating diseases such as Alzheimer's disease and improving sensitivity to anticancer drugs. Further, such a substance capable of regulating the endoplasmic reticulum stress response mechanism is useful for the development of a therapeutic drug for Alzheimer's disease or an anticancer drug. Disclosure of the invention

 Therefore, an object of the present invention is to provide a substance capable of regulating an endoplasmic reticulum stress response mechanism and a method for producing the same.

In view of the above problems, the present inventors have tried to search for a substance capable of regulating the expression of GRP78 from natural materials using the expression of GRP78, one of molecular chaperones, as an index. In this search, an expression vector holding a cassette in which a luciferase gene was linked as a repo overnight gene was introduced into cells downstream of the GRP78 promoter, and the cells were used for screening for the compound. In the screening, the cells were contacted with various crude drugs, fungi, actinomycetes and other metabolites, and further administered with tunicamycin to induce ER stress. In general, the expression of GRP78 was increased due to the induction of endoplasmic reticulum stress by the administration of tunicamycin, but the increased expression was suppressed in the group to which a metabolite derived from a certain actinomycete Streptomyces strain was added. Purification of the active substance from this metabolite using the activity that can suppress the expression of GRP78 despite the induction of endoplasmic reticulum stress as an index resulted in the isolation and identification of a novel tetronic acid derivative, versipelostatin (also known as JL68). Was successful. Also Strain producing this active substance could also be identified.

 From the above findings, the present invention provides this novel tetronic acid derivative versipelostatin, a method for producing the same, and a microorganism that produces the compound. Specifically, the present invention provides:

A versipelostatin compound, represented by

 (2) The method for producing a compound according to (1), wherein a bacterium producing the compound according to (1) above belonging to the genus Streptomyces is cultured, and the compound is collected from the culture.

 (3) The method according to the above (2), wherein the bacterium producing the compound according to the above (1) is Streptomyces basilis strain 4083-SVS6 strain (FERM BP-8179),

(4) A microorganism belonging to the genus Streptomyces and producing the compound described in (1) above.

(5) The microorganism according to the above (4), which is a Streptomyces bacillus 4083-SVS6 strain (FERM BP-8179).

 (6) A composition comprising the compound according to (1) or a pharmacologically acceptable salt thereof.

(7) An anticancer agent comprising the compound according to (1) or a pharmacologically acceptable salt thereof. (8) The anticancer agent according to (7), which induces cell death on cancer cells in a physiological stress state.

 (9) The anticancer agent according to the above (8), wherein the physiological stress state is a hypotrophic state or a hypoxic state.

 (10) The antitumor agent according to any one of the above (7) to (9), which has an anticancer effect on solid cancer.

 The novel tetronic acid derivative vers ipelos tat in (hereinafter abbreviated as VST) of the present invention has a structure represented by the above formula (I), and has physicochemical properties shown in Examples described later. The VST compound of the present invention also includes salts, solvates and the like. VST salts include 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) Salts, nickel salts, etc.), inorganic salts (acetates, ammonium salts), organic amine salts (dibenzylamine salts, dalcosamine salts, ethylenediamine salts, getylamine salts, triethylamine salts, dicyclohexylamine salts, jetanolamine salts, tetramethyl salts) Ammonium salts) and amino acid salts (glycine salts, lysine salts, arginine salts, orditin salts, asparagine salts, etc.). In addition, VST has a hydroxyl group in the molecule as shown in the above formula (I), and can be converted into derivatives of various ethers, esters and the like at those functional groups. These derivatives are included in the present invention as long as they retain VST biological activity.

The above-mentioned VST can be obtained by culturing the producing microorganism and collecting it from the culture. As a VST-producing microorganism, the actinomycete Streptomyces' Vaciaris 4083-SVS6 strain can be preferably used. S. cerevisiae var. 4083—SVS6 strain was isolated from a soil sample collected in Miyoshi City, Hiroshima Prefecture by the present inventors. This strain has been identified according to the method of the International Streptomyces' Project (ISP) and has the following somatic characteristics: It is on the street.

 This strain does not disrupt the basal hypha. These aerial hyphae form a long main axis, and form a spiral spore chain of 10 to 50 or more at the irregularly branched tip. The spores are 0.3-0.5 m wide and 0.7-1.0 m long, non-motile, cylindrical or elliptical, with a smooth spore surface. Black moistening of the spore surface is observed on glycerin-asparagine agar, inorganic salt starch agar, yeast malt agar, oatmeal agar, and the like. Sclerotia, sporangia, and other special forms are not observed. The cell wall chemical type was type (I) having LL-diaminodimeric acid, and the GC content of DNA was 71.3 mol%.

Table 1 shows the culture characteristics of this strain when cultured on various culture media for 28 days. Although the flora color was a gray series on the colony surface, a hygroscopic mass was observed on the spore surface after 2 weeks. The color of the reverse side is a pale yellow to unsharp color such as dark brownish gray, and does not change with pH. No diffusible pigment was observed except for the production of melanin-like pigment. The color tones are based on the Container Harmony Manual (1958 edition) of Container Corporation of America, Inc. (The Color Harmony Manual). Was.

table 1

Medium The flora color of the colony surface 集 裏面 裏面 色 Γ Γ 裏面 裏面 裏面 裏面 裏面 裏面 裏面 裏面 裏面 裏面 裏面 裏面 裏面 裏面 裏面Gray series Light brown taste Gray None Raki "agar agar" Lyserin 'Asha. Gray series Light yellowish brown None Raki "n agar Inorganic salt Gray series Light brownish gray None Starch agar Chi. Shin Agar No thread Dark brown gray Light brown gray Nutritional agar No thread Light brown gray None

 From light brown

Yeast, malt agar Gray series Brown None

 From brown taste gray

Otomir's agar Gray series Dark brownish brown None The physiological properties of this strain when cultured for 28, 2 to 21 days are shown in Table _2c. Table 2 Growth temperature range 25 40 ° C

 27 37 ° C melanin-like pigment

 Tyrosine agar + peptone · yeast iron agar + tryptone · yeast · broth + hydrolysis of starch + liquefaction of gelatin + peptone conversion of skim milk + coagulation of skim milk + reduction of nitrate

Assimilation of carbon sources

 D-glucose + realabinose +

D-xylose +

D—Fructose + Sucrose +

L-rhamnose + rough-inos + i-inos

D—Mannit

Galactose + adnitol

Cellobiose + inulin

In addition, "+" indicates demand, and "one" indicates non-requirement. This strain is mesophilic and produces melanin-like pigments. This strain is a strain that belongs to the genus Streptomyces (trep tomyces) due to its spirally linked spore form and cell wall chemical type (I). Based on the above properties, a search was made for Streptomyces species listed in the “List of Approved Bacteria Names, 1980” and the list of valid names thereafter, and Streptomyces bacillus as a closely related species was searched. Elected. Table 3 shows a comparison between this strain and this closely related species.

 Table 3 Strain Streptomyces

 VST basic spore chain helical + +

 Spore surface smooth + +

 Gray + +

 Unclear color + +

 pH sensitivity

 Diffusible pigment production

 pH sensitivity

 Melanin pigment production + +

 Starch hydrolysis + +

 Nitrate reduction

 Growth temperature 45 ° C

 Assimilation of carbon sources

 Arabinose + +

 Kiss mouth + +

 Innocent + +

 Mannit + +

 Rhamnose + +

 Rough Innocent + +

 Sucrose + +

Fractose + + As shown in Table 3, this strain is in good agreement with the characteristics of Streptomyces bacillus tr6p tomyces versipellis). Therefore, since this strain is most similar to Streptomyces 'bacillus, this strain was identified as a strain contained in Streptomyces versalis and was identified as Streptomyces' versalis. The SVS6 strain was deposited internationally as follows.

 (B) Name of international depositary organization

Name: National Institute of Advanced Industrial Science and Technology, Patent Organism Depositary Center (formerly Ministry of International Trade and Industry, National Institute of Advanced Industrial Science and Technology)

Address: 1-1-1, Higashi, Tsukuba, Ibaraki, Japan 1 Chuo No. 6 (Zip code: 3 0 5— 8 5 6 6)

 (Mouth) Deposit date (original deposit date): January 16, 2001

 (C) Accession number: F ERM B P—8 1 7 9

 In the above, Streptomyces bacillus 4083-SVS6 strain was described as the VST-producing microorganism, but the VST-producing microorganism of the present invention is not limited to this. The characteristics of the above-mentioned cells are generally easily mutated and are not constant. The nature of the cells changes naturally, or physical mutagenesis such as X-ray irradiation, scientific mutagenesis such as ethyl methyl sulfonate, or cell engineering mutations such as genetic manipulation It is a well-known fact that it can be modified artificially by means of introduction. Therefore, the VST-producing microorganism of the present invention is included in the present invention, even if it is a natural mutant or an artificial mutant of the above strain belonging to the genus Streptomyces produced by VST, as long as it can produce VST.

For cultivation of VST-producing bacteria, a conventional method for culturing actinomycetes (Shinya, K. et al., I. Antibiot. 48: 574-578 (1995)) can be used. Specifically, a nutrient medium containing a carbon source and a nitrogen source that can be assimilated by microorganisms, and optionally an inorganic salt, an organic nutrient source, and the like can be used as the medium. Examples of the above carbon sources include glucose, ara pinose, xylose, fructose, sucrose, rhamnose, and rafinoin. , Inositol, mannitol, galactose, cellobiose, melviose, molasses, starch, dextrin, cellulose, glycerin, organic acids and the like.

 Nitrogen sources include peptone, meat extract, yeast extract, dried yeast, skim milk, soy flour, corn steep liquor, cottonseed flour, casein hydrolyzate, soy protein hydrolyzate, amino acids, urea, etc. Examples include organic nitrogen sources, inorganic nitrogen compounds such as nitrates and ammonium salts, and the like.

 The above-mentioned carbon source and nitrogen source can be added to the medium alone or in combination. If necessary, the medium may contain inorganic salts such as sodium salts, potassium salts, ammonium salts, calcium salts, phosphates, sulfates, and carbonates, and cobalt and manganese.

Trace metals such as iron, magnesium, etc., trace nutrients capable of promoting the growth of VST-producing strains and VST production, growth promoting substances, precursors, etc. may be added as appropriate. Further, the pH of the medium PH is preferably around neutral, specifically pH 7.0 to 7.6. In the liquid medium, an antifoaming agent such as a vegetable oil or a surfactant may be appropriately added to suppress foaming during culturing.

 The cultivation of the VST-producing bacterium is preferably carried out by aerobic culturing such as shaking or aeration-agitation culturing as in the usual culturing of actinomyces. The culturing temperature is preferably 24 to 30 ° C, and more preferably 27. When performing large-volume liquid culture, first, the preculture using a small amount of medium is used to activate the growth of the preserved cells, and then this large amount of medium is inoculated with this preculture. It is efficient to carry out the main culture. The cultivation time can be usually 2 to 3 days for preculture and 4 to 6 days for main culture. The culture conditions such as culture temperature, aeration rate, and culture time can be appropriately adjusted and selected based on the amount of VST produced. In addition, the above culture conditions and the like can be changed according to the type of VST viable bacteria and the culture environment.

In order to collect VST from the culture, the culture is centrifuged or filtered to separate the bacterial components, and the supernatant or filtrate is collected. Recovered supernatant or Purification of VST from the filtrate can be performed using the biochemical activity of VST, for example, the GRP78 expression suppression activity shown in Example 1 or the like, or physical properties such as HPLC analysis. . As an example, purification of VST can be performed by the following operation.

 The culture supernatant or filtrate is extracted with a non-hydrophilic organic solvent such as ethyl acetate, and the extract is concentrated under reduced pressure or dehydrated using a dehydrating agent to obtain crude purified VST. The crude substance is further purified to a pure product by a known method usually used for purifying fat-soluble substances, for example, adsorption column chromatography using a carrier such as silica gel, and HPLC using Senshu Pak (Senshu Ikagaku). be able to.

 However, the above purification method is an example, and the present invention is not limited to this method. Therefore, other purification means usually used by those skilled in the art, for example, adsorption column chromatography using another adsorption carrier, gel filtration using a resin for gel filtration, column chromatography, anion exchange or It is also possible to use ion exchange chromatography using a cation exchange resin. These purification means may be used alone or in appropriate combination to separate and purify VST.

The VST purified above may be used as a pharmaceutical composition, or may be used as an intermediate for producing a further compound. The dosage form of the pharmaceutical composition containing VST is not particularly limited, and can be formulated into, for example, tablets, capsules, granules, powders, syrups, and injections. In these formulations, excipients, binders, disintegrants, lubricants, flavoring agents, solubilizing agents, suspending agents, coating agents, and other auxiliary agents are added as long as they do not inhibit VST activity. Such a composition can be an effective composition for treating cancer, Alzheimer's disease, etc., but is preferably used as an anticancer agent. The anticancer agent according to the present invention is preferably an anticancer agent that induces cell death of cancer cells under physiological stress. Here, examples of the physiological stress state include a hypotrophic state and a hypoxic state, but are not limited thereto. Examples of the undernutrition state include a glucose-starved state, a low mineral, a low lipid, and a low vitamin, and a glucose-starved state is preferable.

 In addition, the anticancer agent of the present invention directly exhibits an anticancer effect on cancer cells. For example, the anticancer agent can exert a stronger anticancer effect when used in combination with other chemotherapeutic agents or radiation therapy. . Further, the anticancer agent of the present invention is an effective anticancer agent for solid tumors. BRIEF DESCRIPTION OF THE FIGURES

 FIG. 1 is a diagram showing the ultraviolet absorption spectrum of VST measured in methanol. FIG. 2 is a diagram showing the ultraviolet absorption spectrum of VST measured in methanol NaOH.

 FIG. 3 is a diagram showing an ultraviolet absorption spectrum of VST measured in methanol-HC1.

FIG. 4 is a diagram showing an infrared absorption spectrum of VST measured by a bromide lithium tablet method. In FIG. 4, wavelength number 1 is 3454 cm—2 is 2934 cm— ¹ and 3 is 2360 cm ” ¹ , 4 is 1758 cm” ¹ and 5 is 171 1 cm— ¹ and 6 is 1623 cm ” ¹ and 7 are 1575 cm - 8 1457 cm one ^1, 9 1381 cm one ^1, 10 1308 cm ^"1, 1 1 the 1208 cm- ^1, 12 is 1062 cm- 13 is 9 89 cm- 1, 14 is 934 cm" ^1, 15 indicates 867 ci " ¹ , 16 indicates 830 cm" ¹ and 17 indicates 730 cm- ¹ signal.

 FIG. 5 is a diagram showing a VST nuclear magnetic resonance spectrum.

FIG. 6 is a diagram showing a ¹³ C-nuclear magnetic resonance spectrum of VST.

FIG. 7 is a graph showing the results of measuring the inhibitory activity of VST on GRP78 expression. In the figure, the black symbol indicates the group in which GRP78 expression was induced by adding intracellular endoplasmic reticulum stress by adding VST at different concentrations and then adding tunicamycin. Open symbols indicate the group to which only VST at a different concentration was added and no tunicamycin was added. FIG. 8 shows the results of direct cytotoxicity measurements based on the cell viability when various concentrations of VST were added to various cells. Each symbol corresponds to the cell shown on the right side of the figure.

 FIG. 9 is a photograph showing the activity of VST to suppress the expression of endogenous GRP78 induced by endoplasmic reticulum stress in HeLa78C6 cells. A shows the dose dependence, B shows the evening course, and C shows the examination for each stress condition.

 FIG. 10 is a photograph showing whether VST inhibits GRP78 expression induced by various endoplasmic reticulum stresses in cancer cells with enhanced endoplasmic reticulum stress response HT-29 and # 080 cells. A shows HT-29 cells and B shows HT1080 cells.

 FIG. 11 shows the ability of VST to inhibit colony formation under various ER stress conditions in HT-29 cells.

 FIG. 12 is a diagram showing induction of apoptosis in HT1080 cells by VST under glucose-starved conditions.

 FIG. 13 is a graph showing the time course of apoptosis induction by VST in HT1080 cells under glucose-starved conditions.

FIG. 14 is a diagram showing the combined effect of the antitumor agents cisplatin and VST effective against solid cancer.

FIG. 15 shows the antitumor effect of VST on HT-29 cancer cells in nude mice at the animal level.

FIG. 16 is a photograph showing suppression of XBP1 activation which is deeply involved in the induction of GRP78 by VST. BEST MODE FOR CARRYING OUT THE INVENTION ''

 [Example 1] Screening

Screening of substances capable of regulating GRP78 expression in nature was performed. In detail, the test samples were prepared with the respective metabolites of crude drugs, fungi, and actinomycetes. . The screening system was prepared as follows. Insert the GRP78 promoter region (-132 to +7) containing three ERSEs into the Kpnl-Bglll site of the multicloning site of the pGL3-basic vector (Promega), and reporter downstream of the GRP78 promoter. A vector was constructed in which the luciferase gene was ligated as a gene. Further, a pgk-neo cassette (l, 867 bp) (McBurney et al., Nuclear Acid Res. 19: 5755-5761 (1991)) was used as a selection marker in this vector. Introduced into the Sai site of Gcite. The vector constructed here (hereinafter referred to as "GRP luciferase vector 1") was introduced into HeLa cells. Transfection into cells was carried out by the lipofectin method using Transastj (Promega). Cells holding the above GRP luciferase vector (these cells are referred to as “HeLa78C6 cells”). ) Was selected using a DMEM medium containing G418 (400 g / ml). The selected cells were cultured in a DMEM medium containing 10¾FCS and 400 ig / ml G418 to prepare a HeLa78C6 cell suspension (1.5 × 10 ⁵ cells, 7 ml).

To 96 Each of Uwerupureto Ueru dispensed the cell suspension (100 1) min and 6 h at _{37 ° C, 5% C0 2} . Thereafter, the test sample (equivalent to 11) was added, and incubation was carried out at 37 for 30 minutes. Thereafter, a tunica mycin solution was further added to each well to a final concentration of 2 / gZml. After the addition of tunicamycin, incubation was carried out at 37 ° C for 18 hours. Thereafter, the luciferase activity in the cells was measured using a luciferase assay kit (Luciferase Assay System: Proiega, cat * E1501) according to the attached protocol.

 The induction of endoplasmic reticulum stress by the addition of tunicamycin induced expression from the GRP78 promoter and increased luciferase activity. Was suppressed.

 [Example 2] Suppression of GRP78 expression by culture supernatant of actinomycetes 4083-SVS6 strain

Pre-culture of the actinomycetes 4083-SVS6 strain, which showed suppression of GRP78 expression in Example 1, was performed. I got it. A medium for preculture (containing 10 g starch, 10 g polypeptone, 10 g molasses and 10 g meat extract, adjusted to pH 2 per liter) was prepared and sterilized. The sterilized medium (15 mL) was dispensed into a 50-ml test tube, and this was inoculated aseptically with actinomycetes. After inoculation, the cells were cultured at 27 ° C. for 3 days while shaking the test tube (200 rpm). Next, main culture was performed. That is, the medium for main culture (1 liter Starch 25g Soybean meal 15 g, dry yeast 2g and CAC0 ₃ 4g containing, pH 7. 0) was prepared. The medium (100 ml) of the main culture was dispensed into a 500 ml Erlenmeyer flask with a bump, and the preculture (2 ml) was aseptically added thereto. After the addition, the cells were cultured at 27 ° C. for 5 days while shaking (200 rpm).

The main culture solution was separated into a culture supernatant and bacterial cells by centrifugation. Using the culture supernatant and the cell product obtained by disrupting the cells, the GRP78 expression inhibitory activity was measured for each fraction by repo overnight analysis in the same manner as in Example 1. Each Ueru of 96 © E Le plate dispensed the cell suspension (I OO L) min, at 37, and 6 h at 5% C0 _2. Thereafter, a solution (10 1) containing the culture supernatant (1 ^ 1) or the cell product (1 U) was added, and incubation was carried out at 37 for 30 minutes. Thereafter, a twemycin solution was further added to a final concentration of 2 gZml. After the addition of tunicamycin, incubation was carried out at 37 for 18 hours. Thereafter, the luciferase activity in the cells was measured using a luciferase measurement kit. As a result of the measurement, suppression of luciferase activity was observed in the group to which the actinomycetes culture supernatant was added.

 [Example 3] Purification of active substance from actinomycete 4083-SVS6 strain culture supernatant

The actinomycetes 4083-SVS6 strain was cultured in the same manner as in Example 1 except that the scale was increased to prepare a main culture solution (2 liters). 'The culture was centrifuged (10,000 n) in, 10 minutes, and the culture supernatant was collected to remove the cells. The collected culture supernatant was extracted with ethanol acetate and then dehydrated with sodium sulfate. The weight of the crude product after dehydration was 2.57 g. The concentrated acetic acid-ethanol extract after dehydration is concentrated and dried, and then dissolved in a solution of chloroform: methanol (20: 1). This solution is supplied to a silica gel column (column capacity: 3.5ΦΧ30 cm), and the same solvent Chromatography was performed. The eluted sample was measured by the reporter analysis described in Example 1 above, and an active eluted fraction (250 mg) was separated. Subsequently, the fractions having the above-mentioned activities were collected, concentrated to dryness, dissolved in methanol, and applied to an HPLC column (Senshu Pak: PEGASIL ODS C18, 20 × 250 marauder, manufactured by Senshu Ichigaku) at 1 ml / min. Chromatography using 80% MeOH at a flow rate of. The eluate was monitored by ultraviolet and visible light (254 nm), and a peak having a retention time of 28 minutes was collected. The collected eluate was dried to obtain 35.7 mg of a pure VST (also known as JL68) compound.

 The physicochemical properties of VST are shown below.

 (1) Property of substance: white powdery substance

 (2) Melting point: 17 to 175 ° C

(3) molecular formula: C ₆₁ H ₉ 0 ₁₇ , (3) molecular weight measured by FAB marspectrum method: 1098 (M)-, 1121 (M + Na) +

 (4) General formula (I):

(5) High resolution Accurate mass (m / z) measured by FAB mass spectrum method: Actual Measured 1121.6398 [M + Na] +, Calculated 1121.6389

(6) The maximum absorption ( _AMe0 ) of the ultraviolet absorption spectrum (Figure 1) measured in methanol

H nm (ε)): 248 (8,000), 267 (7,700), maximum absorption of ultraviolet absorption spectrum (Figure 2) measured in methanol-NaOH U _MeQH - _MH nm (ε)): 243 (11,400), 269 (8,900), maximum absorption U _Me0H _ _Hcl nm (ε) in the ultraviolet absorption spectrum (Figure 3) measured in methanol-HC1: 263 (8, 500)

 (7) Optical rotation measured in methanol: [H] D: — 52 ° (c 0.8, MeOH)

(8) Maximum absorption (IR (KBr) Amax cm— ¹ ) of the infrared absorption spectrum (Figure 4) measured by the potassium bromide tablet method: 3446, 2934, 1758, 1711, 1261, 1075

(9) 口 -nuclear magnetic resonance spectrum (Fig. 5) and ^13C- nuclear magnetic resonance spectrum (Fig. 6) measured at 25 ° C in a 1: 1 solution of heavy liquid form: deuterated methanol = 1: shown in Table 4

Table 4 χ

ΙΝΟ. Ι Ο.

 1 166.35 32 12.39 0.913

2 103.22 33 16.26 0.92

3 205.1 34 22.8 1.63

4 58.58 35 21.84 1.92,1.88

5 23.44 2.4 36 14.56 0.88

6 49.79 2.46 37 64.72 3.51,3.40

7 210 38 17.07 0.91

8 50.09 2.98,2.40 39 22.25 0.885

9 71.28 3.78 40 20.79

 10 47.18 2.29 41 21.41 1.67

11 120.75 5.84 42 19.37 1.03

12 134.16 ι100.3 4.77

13 60.01 3.07 237.26 2.11,1.65

14 139.27 3 67.6 4.638

15 135.94 5.1 4 '80.54 3.21

16 37.9 2.28 5 68.09 3.76

17 32.23 1.59, 0.59 6 '17.69 1.17

18 35.4 1.94 1 "99.3 4.92

19 91.08 3.2 2 "34.71 2.19,1.53

20 25.19 1.61 3 "73.09 3.46

21 32.23 1.49,1.10 4 "81.13 3.27

22 19.95 1.58,1.33 5 "68.1 3.6

23 34.56 1.53,1.26 6 "17.97 1.24

24 41.41 3 "-OCH3 57.25 3.36

25 126.63 5.26 1 "'98.35 5.02

26 135.31 2 '"38.06 2.10,1.61

27 31.13 2.37 3 '"68.18 4.044

28 36.85 2.21,1.75 4 '"72.97 3.22

29 87.31 5 '"69.15 3.64

30 201.81 6 '"18.03 1.22

31 23.44 2.52, 2.12

(10) HPLC analysis: Column Sens Pak (PEGASIL ODS C18, 4.6 x 250 mm, manufactured by Sensius Science), solvent 80% methanol, flow rate 1 ml / min, detection 254 nm, retention time 14 minutes

[Example 4] Quantification of VST activity Using the pure VST purified in Example 3, the activity of suppressing the expression of GRP78 promoter was quantified. This quantification was also measured by the same reporter analysis using the luciferase gene as in Example 1. Specifically, HeLa78C6 cells (5 × 10 ⁵ cels / ml, 1001) were dispensed into a 96-well plate as in Example 1. On the other hand, a VST solution (100, 50, 25, 12.5, 6.25, 3.125, 1.5625 g / ml) diluted step-by-step with a medium was prepared. Diluted to 10% with PBS (-). The solution (101) diluted with PBS was added to a well (100 cell suspension / well). After the addition, at 37, it was carried out for 30 minutes incubation at 5% C0 _2. Thereafter, a certain amount of tunicamycin solution was further added to some of the wells to a final concentration of 2 ^ gZml. After adding tunicamycin, the cells were incubated at 37 ° C for 18 hours, and the luciferase activity in the final cells was measured using a luciferase assay kit (Promega) (Fig. 7). The numerical value on the horizontal axis in FIG. 7 indicates the final concentration of VST.

 VST alone had no effect on luciferase expression from the GRP78 promoter and was comparable to the relative luciferase activity in cells without sample addition, but responded to endoplasmic reticulum stress due to the addition of tanikimycin. Luciferase expression from GRP78 was increased. However, the increase in expression due to the addition of tunicamycin was suppressed as the concentration of VST increased. These results indicate that VST suppresses the up-regulation of GRP78 expression in response to tunicamycin-induced ER stress.

 [Example 5] VST activity measurement in various cells

 In this example, the above reporter analysis and the survival rate when VST was added were measured using various other cultured cells including HeLa cells. Specifically, in addition to HeLa cells (human ovarian cancer cells), MCF-7 (human breast cancer cells), PC12NH (rat pheochromocytoma), MDA-MB-231 (human breast cancer cells), TIG-3 ( Human normal fibroblasts), HeLa786C cannon (human ovarian cancer cells), Saos_2 (human cancer cells lacking telomerase and p53), and SUSM-1 (human cancer cells lacking telomerase) were used.

The direct growth inhibitory activity of VST on these cells was measured using the MTT method. Up The cell suspension (5 × 10 ⁴ / ml) of each of the above cells was dispensed into each well of a 96-well plate for 100 ^ 1 and cultured at 37 ° C. for 6 hours. After the culture, a VST solution serially diluted in the same manner as in Example 4 was added, and the culture was further performed for 24 hours. After culturing for 24 hours, a 0.05 g / ml MTT solution (101) was added to each well, and incubation was carried out at 37 for 4 hours. Thereafter, the medium was removed, and DMSO (IOOD was added), and the absorbance was measured at 530 nm (FIG. 8). The results of determining the IC50 from these measured values are shown in Table 5.

 At VST (final concentration gZml), no decrease in viability was observed, and at a high concentration of about 10 ig / ml, a slight decrease in viability was observed in a few cell lines. At a higher concentration of 100 ml, the viability of many cell lines was reduced, but the reduction was up to 50%. Some cell lines still showed no change in viability even at a high concentration of 100 / igZml. The results showed that VST was low in cytotoxicity, and there was no direct cytotoxicity to any of the cells, especially at concentrations of 1 g / ml or less.

 Table 5

[実施例 6 ] HeLa78C6細胞株に対する VSTの生物活性

[Example 6] Biological activity of VST against HeLa78C6 cell line

 HeLa78C6 cells were cultured in DMEM medium, VST at each concentration was added 30 minutes before the addition of tunicamycin, and 2 g / ml of tunicamycin was further added, followed by treatment for 18 hours. The cells were collected and GRP78 induction was detected by Western blotting using an anti-GRP78 antibody (Stressgene). As a result, it was confirmed that VST inhibited not only the repo overnight but also the induction of the endogenous molecular chaperone GRP78 at the protein level in a concentration-dependent manner (FIG. 9A).

 Further, HeLa78C6 was cultured by the above-mentioned culture method, pretreated with 50 M VST for 30 minutes, and then collected after each addition of tunicamycin, and the protein level of GRP78 was detected by Western blotting. As a result, it was revealed that GRP78 was induced 12 to 18 hours after treatment with 2 ^ g / ml of tunicamycin, and that the induction was inhibited by VST (Fig. 9B).

 Furthermore, the GRP78 level 18 hours after the treatment with the ER stress inducer described above was detected in the same manner as in FIG. 9A. As a result, VST inhibited the induction of GRP78 against any endoplasmic reticulum stress, but its effect was more selective for 2-deoxyglucose (2-DG), which is close to physiological endoplasmic reticulum stress. (Fig. 9C).

 [Example 7] Effect of VST on GRP78 and GRP94 levels in HT-29 and HT1080 cells under various stress conditions

 As a result of Example 6, it was revealed that VST inhibits the induction of endogenous molecular chaperone GRP78 induced by endoplasmic reticulum stress.Therefore, the next cancer cells are those with enhanced endoplasmic reticulum stress response HT-29 cells and ΗΠ080 cells were used to examine the inhibition of GRP78 induction, apoptosis induction, and the effect of combination with other antitumor agents.

 Specifically, liver cancer cell HT-29 cells and HT1080 cells were cultured in RMI 1640 medium, and pretreated with 3% and VST for 30 minutes. Next, GRP78 levels 18 hours after each endoplasmic reticulum stress treatment were detected by Western blotting.

(Figure 10). As a result, VST showed that cancer cells HT-29 and And HT1080 cells also inhibited the induction of GRP78 by 2-DG, a physiological endoplasmic reticulum stress, even at a low concentration of 3M. In contrast, VST did not show a GRP78-induced inhibitory effect on tunicamycin treatment, which is a chemical endoplasmic reticulum stress, even at a concentration of 30 M. Although data are not shown, it was confirmed that the transcriptional activity of HSP70, a heat shock protein, was not affected, demonstrating that VST acts specifically on the ER stress response. Based on these results, VST showed a highly specific inhibitory effect on the endoplasmic reticulum stress response in a glucose starvation test, which is a model that reflects the actual state of solid tumors in the body. This suggests that it is possible to overcome the resistance mechanism of solid tumors where the effects of antineoplastic agents are difficult to appear. In addition, it was revealed that VST does not exert an effect on all endoplasmic reticulum stress, but this result indicates that the endoplasmic reticulum stress response pathway is not expressed through a single mechanism. However, this phenomenon was first clarified in the world by using VST.

 [Example 8] Induction of apoptosis under stress conditions

 As described above, VST was shown to specifically inhibit the expression of GRP78 by glucose starvation, a physiological endoplasmic reticulum stress. We examined whether cell growth suppression or apoptosis was induced.

The HT-29 cells were pretreated with VST at each concentration for 30 minutes, and were treated with tunicamycin, calcium ionophore A23187, glucose starvation and 2-DG stress treatment for 18 hours. Thereafter, the cells were cultured on a plate for one week, fixed with formalin, and colonies were stained and counted by crystal violet staining. As a result, VST showed a strong cell growth inhibitory effect under glucose starvation and 2-DG treatment in proportion to the GRP78 induction inhibitory activity (Fig. Lie and D). In contrast, treatment with tunicamycin did not show a cell growth inhibitory effect as well as GRP78 induction inhibition (FIGS. 11A and B). In addition, HT1080 cells were pretreated with VST for 30 minutes, the cells were fixed, the nuclei were stained with propidium iodide, and FACS analysis was performed. As a result, as shown in Figure 12A, VST Did not show any effect in the presence of glucose, as did the control, but showed a strong sub-G1 induction after 48 hours in the dark-starch state, suggesting that HT1080 cells induce apoptosis in the glucose-starved state Was done. In contrast, the number of apoptotic cells in HT1080 cells was extremely limited only by Darcos-starved treatment. In addition, as shown in FIG. 12B, it was suggested that VST induced apoptosis in ΗΠ080 cells under glucose-starved condition in a time-dependent manner.

 From the above results, it was clarified that VST specifically induced cell death in glucose-starved cancer cells by inhibiting the induction of GRP78.

 [Example 9] Combined activity of VST and anticancer agent

 Enhanced ER stress response promotes survival under malnutrition and has acquired resistance to antitumor agents. Therefore, we decided to study its effects on known antitumor agents. However, in experiments at the cellular level, VST alone showed strong apoptosis induction under glucose-starved conditions, so that the effect of combination with other drugs could not be sufficiently examined. Therefore, the activity was maintained even under low glucose against cancer cells. Therefore, the combination effect was examined using cisbratin, which is widely used for solid cancer treatment. Specifically, HT-29 cells were cultured by the above method, pretreated with VST for 30 minutes, and then cultured for 18 hours. Thereafter, the cells were treated with each concentration of cisbratin for 4 hours, and one week later, the ability to form colonies was examined.

 As a result, both VST and cisplatin showed enhanced activity under glucose-starved conditions, but it was found that the addition of low concentrations of VST (300 nM and 500 nM) further enhanced the effect of cisplatin. (Fig. 13).

 Cisplatin is widely used for the treatment of solid cancer, but is known to have strong side effects. Therefore, if the dose can be reduced by VST, it is expected to greatly contribute to the patient's quality of life (Q0L).

 [Example 10] Animal test

The antitumor effect of VST at the animal level was examined. Specifically, HT-29 cells The transplanted into nude mice were增殖the tumor up to 100 Yuzuru ^3. After that, each concentration of VST was administered to the tail vein, followed by follow-up, and the tumor was excised at each lapsed day and the size was compared. As a result, as shown in FIG. 14, VST did not show as strong an effect as cisplatin, but showed an antitumor effect. The effect of concomitant use with cDDP (cis bratin) was examined, but the effect of cisplatin was sufficient, and no concomitant effect was observed. Since no strong effects were observed in in vivo in animal tests, the apoptosis-inducing effect of VST on HT-29 cells was re-examined.As a result, VST showed a growth-suppressing effect in a colony formation test, but did not exhibit an apoptosis-inducing effect It became clear.

 [Example 11] Study on mechanism elucidation

 To elucidate the mechanism of VST activity expression, effects on several factors acting on the endoplasmic reticulum stress response pathway were examined. Among them, we investigated the Irela-XBPl pathway, which is an endoplasmic reticulum stress response pathway widely stored from yeast to mammalian cells. At the mRNA level, XBP1 is spliced from the precursor (XBP1 (U)) by endoplasmic reticulum stress and activated by Irela, and becomes an activated form (XBP1 (S)). Binds and induces GRP78 transcription.

A plasmid in which a Flag tag was linked to the cloned XBP1 was introduced into # 080, and pretreated with VST for 30 minutes. Six hours after each stress load, MG132 which is a proteasome inhibitor was added, and a full-length spliced XBPl was detected using an anti-Flag antibody using a sample prepared from cells cultured for further 6 hours. Fig. 15 shows the results of 2-DG, and the right side shows the formation of spliced forms of XBP1 by treatment with tunicamycin. The spliced form of SBP1 is prone to degradation by the proteasome, making its detection difficult. To solve this problem, MG132, a proteasome inhibitor, was added. Comparing the two lanes on the right in both Figures 15A and B, it was observed that the addition of VST inhibited the formation of spliced XBP1 in 2-DG treatment. On the other hand, the splicing of XBP1 by tsuyumycin was corrected. Was not inhibited by VST. This suggests that at the activation level of ΧΒΠ, the mechanisms of ER stress response differ between tunicamycin and glucose starvation. The new findings obtained from this result were first discovered by the discovery of VST. No substance other than VST has been reported to inhibit splicing of XBP1. Industrial applicability

 A novel substance (VST) has been identified from actinomycetes that can reduce GRP78 expression. In addition, a microorganism producing this novel compound, Streptomyces basilis, was isolated and identified. This VST suppressed the increase in GRP78 expression induced by the endoplasmic reticulum stress inducer, indicating that VST itself can inhibit GRP78 expression caused by stress response. Based on these results, we will use VST to develop a drug that suppresses the stress response mechanism of cancer cells during cancer treatment by chemotherapy or radiation therapy, and that gives cancer cells high sensitivity to these chemotherapeutic agents or radiation therapy. Becomes effective.

Claims

The scope of the claims

Versipelostatin dyad compound shown by.  2. The method for producing a compound according to claim 1, wherein the bacterium producing the compound according to claim 1 belonging to the genus Streptomyces is cultured, and the compound is collected from the culture.  3. The production method according to claim 2, wherein the strain producing the compound according to claim 1 is Streptomyces basilis 4083-SVS6 strain (FERM BP-8179). 4. A microorganism which belongs to the genus Streptomyces and produces the compound according to claim 1. 5. The microorganism according to claim 4, which is Streptomyces basilis 4083-SVS6 strain (FERM BP-8179).  6. A composition comprising the compound of claim 1 or a pharmacologically acceptable salt thereof. 7. An anticancer agent comprising the compound according to claim 1 or a pharmacologically acceptable salt thereof. 8. The anticancer agent according to claim 7, wherein the agent induces cell death of a cancer cell in a physiological stress state. 9. The physiological stress state is a hypotrophic state or a hypoxic state. Anticancer drugs.  10. The anticancer agent according to any one of claims 7 to 9, which has an anticancer effect on solid cancer.