JP2989662B2 - Benaltin, a novel enzyme inhibitor, and its production - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to a novel enzyme inhibitor, benaltin or a salt thereof, and a method for producing the same.

More specifically, the present invention provides a novel substance having an enzyme inhibitory activity against pyroglutamyl peptidase, benaltin,
And a method for producing benaltin by culturing a microorganism belonging to the genus Streptomyces.

[Problems to be solved by conventional technology and invention]

As compounds having similar physicochemical properties of the enzyme inhibitor benaltin according to the present invention, vanoxonine [F. Kanai et al.
l. “J. Antibiotics”, 36, 656-660 (1983)] and some peptide antibiotics, and these are different from those of Benaltin in physicochemical and biological properties. Was confirmed.

Pyroglutamyl peptidase, which is an inhibitory activity of this substance, benaltin, uses in vivo a peptide hormone (bioactive peptide) having various physiological functions such as neurotensin, luteinizing hormone releasing hormone, and thyroid stimulating hormone releasing hormone as a substrate. It is known to be involved in degradation, metabolism, and regulation of the function of peptide hormones or the development of new physiological activities.

Heretofore, many enzyme inhibitors have been reported, and some of them have been put into practical use as drugs. However, no inhibitor for pyroglutamyl peptidase has been reported yet. Therefore, inhibitors with high specificity for pyroglutamyl peptidase are desired. An object of the present invention is to provide a biologically active substance benaltin having a highly specific pyroglutamyl peptidase inhibitory activity and a method for producing the same.

[Means for solving the problem]

The present inventors isolated a large number of microorganisms from soil and searched for substances produced by them, and found that certain microorganisms produce a bioactive substance that exhibits an inhibitory effect on pyroglutamyl peptidase. Was. As a result of the study, it was confirmed that this substance was successfully isolated, and that it had a structure represented by the following formula (I). Upon further detailed investigation, the bioactive substance was found to be a novel antibiotic, and was named Benaltin.

The present invention has been completed based on the above findings.

Therefore, according to the first invention, the formula (I) Or a salt thereof is provided.

The enzyme inhibitor benaltin according to the present invention has the following properties.

1) Physicochemical properties of benaltin (1) Appearance: white powder (2) Melting point: 178-180 ° C (3) Molecular formula: C ₁₇ H ₂₅ N ₅ O ₇ (4) Elemental analysis value: C ₁₇ H ₂₅ N ₅ O ₇ .HCl.1 / 2H ₂ O C 44.46%, H 6.28% N 14.54%, O 26.08% (5) Mass spectrum: FAB-MS (pos.) M / z 412 (M +
H) ⁺ (neg.) M / z 410 (M-
H) ^- (6) Specific light intensity: ▲ [α] ²⁰ _D ▼ -2.5 ° C (c1.0, H
₂ O) (7) Ultraviolet absorption spectrum: shown in FIG. 1 of the accompanying drawings.

λ _max , nm (ε) (in aqueous solution): 246 (8200), 308 (24
00) λ _max , nm (ε) (in NaOH aqueous solution): 252 (sh), 334
(3800) (8) Infrared absorption spectrum: shown in FIG. 2 of the accompanying drawings.

(9) ¹ H-NMR spectrum (400 MHz, DMSO-d ₆ ): shown in FIG. 3 of the accompanying drawings.

(10) ¹³ C-NMR spectrum (100 MHz, DMSO-d ₆ ): shown in FIG. 4 of the accompanying drawings.

δ (ppm): 173.4 (s), 170.6 (s), 168.1 (s),
157.0 (s), 149.9 (s), 146.5 (s), 118.5 (d), 11
7.5 (d), 116.4 (d), 115.9 (s), 66.3 (d), 57.8
(D), 52.4 (d), 40.8 (t), 28.9 (t), 24.5
(T), 19.6 (q) (11) Solubility: soluble in water, methanol and dimethylsulfoxide, not soluble in chloroform, ethyl acetate and n-hexane.

(12) Thin-layer chromatography (silica gel; Merck): (13) Color reaction: Positive for 10% sulfuric acid, Sakaguchi, iodine, molybdic acid reagents.

2) Enzyme inhibitory activity of benaltin Pyroglutamyl peptidase inhibitory activity
"Anal. Biochem." 53, 321 (1973).

That is, 5 mM L-pyroglutamyl acid β-naphthylamide
0.02 ml, buffer [0.2 M potassium phosphate buffer (pH 8.0)-
0.1M EDTA-0.01M DTT (5: 1: 1)] to a mixture of 0.1 ml of an aqueous solution containing liquid (benaltin) and 0.02 ml of pyroglutamyl peptidase (manufactured by Sigma) was added. At 60 ° C for 60 minutes, 0.01 ml of 16 mM NaIO ₄ was added,
The reaction was performed at 37 ° C. for 15 minutes. After the reaction, 0.1 ml of a 0.5 mg sodium citrate buffer of 2 mg / ml Fast Garnet GBC (manufactured by Sigma) was added, and the absorbance (a) at 525 nm was measured. At the same time, the absorbance (b) of the control when the same reaction was carried out without the sample was measured. At this time, the absorbances (a ') and (b') of each of the blind tests were measured. Pyroglutamyl peptidase inhibition was calculated using the formula [(1- (a
−a ′) / (bb−b ′)] × 100. 50%
The concentration of the sample showing the inhibition rate was defined as the IC ₅₀ value.

The IC ₅₀ value indicating the inhibitory activity of benaltin on pyroglutamyl peptidase was 2 μg / ml.

3) Antibacterial activity of benaltin The benaltin of the present invention did not show antibacterial activity against bacteria and fungi at a concentration of 100 μg / ml.

4) Toxicity of Benaltin Benaltin according to the present invention showed no toxicity when the mice were intravenously administered at a dose of 100 mg / kg to the mice for 2 weeks.

The second present invention relates to a method for producing an enzyme inhibitor benaltin, which comprises culturing a bacterium of the genus Streptomyces which produces the benaltin of the formula (I) and collecting the enzyme inhibitor benaltin from the culture.

An example of an enzyme-inhibiting substance, benaltin-producing bacterium used in the method of the present invention is an actinomycete isolated from the soil in Shinagawa-ku, Tokyo and having a strain number of MJ244-SF1.

 The mycological properties of the MJ244-SF1 strain are as follows.

I. Morphology MJ244-SF1 strain was obtained from a basic mycelium branched under a microscope.
It elongates straight aerial hyphae and lacks helix formation, ring branches and sporangia. The mature spore chain has a chain of 50 or more spores, and the size of the spore is about 0.6 × 0.7 to 1.2 × 1.3 microns. The spore surface is smooth.

II. Growth conditions in various media Standards shown in parentheses () in color descriptions are those of Container Corporation of America's Color Harmony Manual (Container Corporation of America).
a. Color harmony manual).

(1) Sucrose / nitrate agar medium (cultured at 27 ° C) Colorless to light yellow growth, and bright brown ash (2dc, Natura
l) Aerial mycelium is formed. No soluble dye is observed.

(2) Glucose-asparagine agar (cultured at 27 ° C) Bright ash (2ig, Slat) on yellow (2ea, Lt Wheat) growth
etan). The soluble pigment is slightly yellowish.

(3) Glycerin-asparagine agar medium (ISP-medium
Culture at 5,27 ° C) Bright gray (3fe,
Aerial mycelium of Silver Gray is formed and yellow (1 1 / 2na, Brite Y)
ellow) soluble dye.

(4) Starch / inorganic salt agar medium (ISP-medium, cultivation at 27 ° C) Aerial mycelia of bright ash (2fe, Covert Gray) grow on the growth of light yellow. The soluble pigment is slightly yellowish.

(5) Tyrosine agar medium (ISP-medium at 7,27 ° C) Light brown (3lg, Adobe Brown)
Aerial hyphae of fe Silver Gray to bright brown ash (3ig, Beige Brown) form. No soluble dye is observed.

(6) Nutrient agar medium (cultured at 27 ° C) Growth light tea (3le, Cinnamon), aerial mycelium does not grow,
No soluble dye is observed.

(7) Yeast-malt agar medium (ISP-medium culture at 2,27 ° C) Aerial mycelium of bright brown ash (3ig, Beige Brown) grows on the growth of Nibu yellow (2pe, Mustard Gold), and yellow ( 1 1 / 2p
a, Brite Yellow).

(8) Automill agar medium (ISP-medium, cultivation at 3,27 ° C) Aerial mycelia of bright brown ash (3ig, Brige Brown) grow on the growth of light yellow, and dissolution of light yellow (1ga, Lt Lemon Yellow) Produces sex pigments.

(9) Glycerin / nitrate agar medium (cultured at 27 ° C.) The growth is light yellow tea (2ic, Honey Gold), and aerial hypha does not grow. The soluble pigment is slightly yellowish.

(10) Starch agar medium (cultured at 27 ° C) Aerial mycelia of bright brown ash (3ge, Beige) grow on the growth of light yellow. The soluble pigment is slightly yellowish.

(11) Lime malate agar medium (cultured at 27 ° C) White aerial hyphae grow slightly on light yellow growth, and no soluble pigment is observed.

(12) Cellulose (cultured at 27 ° C) After cultivation, observation was made for 3 weeks, but no growth was observed.

(13) Gelatin stab culture In a simple gelatin medium (cultured at 20 ° C), the growth is light yellow,
Aerial mycelium does not set. The soluble pigment has a yellow-brown tint.

In a glucose / peptone / gelatin medium (cultured at 27 ° C), the growth is initially light yellow to light yellow (5na, Brite
Yellow), and from 16 days after cultivation, the growth is pale yellow, aerial mycelia do not form, and a yellow (1pa, Lemon Yellow) soluble pigment is produced.

(14) Skim milk (cultured at 37 ° C) Light yellow tea (2ne, mustard gold) develops, but aerial mycelia do not form. The soluble pigment has a yellow tint.

III. Physiological properties (1) Growth temperature range East starch agar medium (soluble starch 1.0
%, Yeast extract 0.2%, string agar 3.0%, pH 7.0) at 14 ° C, 20 ° C, 27 ° C, 30 ° C, 37 ° C, and 50 ° C. Grew at the same temperature,
The optimum temperature is around 27-30 ° C.

(2) Liquefaction of gelatin (15% simple gelatin medium, culture at 20 ° C; glucose / peptone gelatin medium, 27 ° C culture) 21 days after culture in any of 15% simple gelatin medium and glucose / peptone gelatin medium No liquefaction was observed.

(3) Starch hydrolysis (starch inorganic salt agar medium and starch agar medium, both cultivated at 27 ° C.) In any medium, water degradability was observed on day 4 after culturing, and the action was stronger.

(4) Coagulation of defatted milk, peptone formation (defatted milk, cultivation at 37 ° C) Coagulation state appears around 10 days after culturing, and after 2 days,
Immediately, peptone conversion begins. Peptone formation progresses very slowly, and is not completed even after 30 days of culture.

(5) Melanin-like pigment formation (trypton yeast,
Broth, ISP-medium 1; peptone yeast iron agar, I
SP-medium 6; tyrosine agar, ISP-medium 7; all 27
C) The culture was positive on any of tryptone yeast broth, peptone yeast iron agar and tyrosine agar.

(6) Utilization of carbon source (Pridham Gottlieb agar medium, ISP-medium 9; cultured at 27 ° C.) L-arabinose, D-xylose, fructose, sucrose, inositol, rhamnose, raffinose, D -Does not use mannitol or lactose.

(7) Dissolution of malate lime (maleate lime agar medium, 27
Dissolve the lime malate around the growth from around 10 days after the culture. Its effect is weak to moderate.

(8) Nitrate reduction reaction (peptone water containing 0.1% potassium nitrate, ISP-medium 8; cultured at 27 ° C) is positive.

(9) Decomposition of cellulose (synthetic solution containing filter paper pieces, cultivation at 27 ° C.) No growth was observed after 3 weeks of observation.

To summarize the above properties, the MJ244-SF1 strain elongates a straight aerial hyphae, and does not show helix formation, ring-shaped branches and spores. The spore surface is smooth. In various media, light gray to light brown ash aerial mycelia develop on light yellow to yellow development, producing a yellow soluble pigment.
The production of melanin-like pigment is positive in any medium. The starch hydrolyzability is stronger and the proteolytic power is weaker.

The 2,6-diaminopimelic acid contained in the cell wall of the bacterial cell of this strain is of the LL-type, and considering the above properties, the MJ244-SF1 strain is Streptomyces (Streptomyces).
It clearly belongs to the genus yces). From these properties, a search for known strains closely related to the MJ244-SF1 strain reveals the following four species. That is, Streptomyces cinereoruber (Internat
ional Journal of Systematic Bacteriology, 18, 18
P. 1968), Streptomyces xanthopheus (St.
reptomyces xanthophaeus; International Journal of S
ystematic Bacteriology, 18, 180, 1968), Streptomyces flavotricin
i; International Journal of Systematic Bacteriolog
y, Vol. 18, p. 114, 1968 and Vol. 30, p. 382, 1980) and Striptomyces zao
myceticus; International Journal of Systematic Bact
eriology, vol. 22, p. 374, 1972). Currently these species
The MJ244-SF1 strain is under comparative study.

MJ244-SF1 strain was transformed into Streptomyces sp.
omyces sp.) MJ244-SF1. The MJ244-SF1 strain was applied for deposit with the Research Institute of Microbial Industry and Technology,
On October 12, 2000, it was accepted as a micro-organism research bacterium No. 11769 (FERM P-11769).

The MJ244-SF1 strain is variable in its properties, as seen in other actinomycetes, for example, a mutant strain of the MJ244-SF1 strain or derived therefrom (naturally occurring or inducible),
Any zygote or gene recombinant that produces the new enzyme inhibitor benaltin can be used in the present invention.

In the method of the present invention, the above-mentioned benaltin-producing bacteria are cultured in a medium containing nutrients that can be used by ordinary microorganisms.
As a nutrient source, known nutrients conventionally used for cultivation of actinomycetes can be used. For example, as a carbon source, glucose, galactose, dextrin, starch, molasses,
Animal and vegetable oils can be used. Nitrogen sources include soy flour, wheat germ, corn steep liquor, cottonseed meal,
Meat extract, peptone, yeast extract, ammonium sulfate,
Sodium nitrate, urea and the like may be used. In addition, it is effective to add, if necessary, sodium, potassium, calcium, magnesium, cobalt, hydrochloric acid, phosphoric acid, sulfuric acid and other inorganic salts capable of generating ions. In addition, organic and inorganic substances that promote the growth of bacteria and promote the production of benaltin can be appropriately added.

As a culture method, a culture method under aerobic conditions, particularly a submerged culture method is most suitable. A suitable temperature for culturing is 15-37 ° C, but in most cases, culturing is performed at around 26-30 ° C. The production of benaltin varies depending on the culture medium and the culture conditions, but the maximum amount of the shaken culture and the tank culture usually reaches the maximum within 2 to 7 days. When the accumulated amount of benaltin during the cultivation reaches the maximum, the culture is stopped, and the target substance benaltin is isolated and purified from the culture solution.

When collecting from a culture of benaltin obtained by the present invention, a usual separation means utilizing its properties,
For example, it can be purified by filtration, adsorption or distribution column chromatography, gel filtration, dialysis, precipitation, etc., alone or in an appropriate combination. For example, benaltin is adsorbed on an adsorbent such as activated carbon or Diaion HP-20 (manufactured by Mitsubishi Kasei) and eluted with a mixture of an organic solvent and water. For example, benaltin is eluted from the adsorbent with 50% methanol, 50% acetone or the like.

To further purify benaltin, an adsorbent such as silica gel (Wakogel C-200, manufactured by Wako Pure Chemical Industries), DEAE-Sephadex (manufactured by Pharmacia), Sephadex G-10 (manufactured by Pharmacia), or the like is used. Chromatography or countercurrent distribution may be performed.

Following the cultivation process of Benaltin and the purifying process, the measurement of the anti-pyroglutamyl peptidase activity described above,
And developing solvent by silica gel thin layer chromatography
_{BuOH-AcOH-H 2 O (} 4: 1: 2) and the Rf of Benaruchin when
Performed based on a value of 0.34.

Examples of the present invention will be described below, but these are merely examples and do not limit the present invention. Of course, many alternatives or modifications not illustrated herein may be used.

Example 1 As a seed medium and a production medium, galactose 2.0%, dextrin 2.0%, bactosoyton (manufactured by Difco) 1.0
%, Corn steep liquor (Iwaki) 0.5
%, Ammonium sulfate 0.2%, and calcium carbonate 0.2%. The pH before sterilization was adjusted to 7.4 before use.

Dispense 110 ml of the above medium into a 500 ml Erlenmeyer flask 12
Sterilize at 0 ° C for 20 minutes, and add 1 to 1 of the slope culture of Streptomyces sp. Strain MJ244-SF1 (FERM P-11769).
Two loops of platinum loop were inoculated and cultured at 27 ° C. at 180 rpm for 2 days. 2 ml of this seed culture was transferred to a 500 ml Erlenmeyer flask in which 110 ml of the medium was dispensed and sterilized, and cultured for 2 days under the same conditions as described above. After completion of the culture, diatomaceous earth was added to the culture solution, followed by filtration to obtain a culture filtrate.

Example 2 2 L of the culture filtrate obtained in Example 1 was applied to a column of 250 ml of activated carbon, washed with water, and eluted with 50% aqueous acetone (750 ml). The eluate containing the active fraction was concentrated under reduced pressure and freeze-dried to obtain about 3.2 g of active coarse powder. 1 g of the powder was eluted in a small amount of water, and 200 ml of Diaion HP-20 (manufactured by Mitsubishi Kasei) was eluted.
After washing with water, the column was eluted with 50% acetone water. The eluate was concentrated and freeze-dried to obtain 573 mg of a gray active powder.

This powder was dissolved in a small amount of water and filled with water. Sephadex G-10 (Pharmacia Fine Chemical Co., Ltd.)
The residue was applied to a 700 ml column, subjected to chromatography using water as a developing solution, and the active fraction was concentrated and lyophilized to obtain 117 mg of an active white powder.

Further, this white powder was subjected to Centrifugal Partition Chromatography-LLN (Centrifugal P
Purification was performed by centrifugal liquid-liquid chromatography using a solvent system of butanol-acetic acid-water (750: 50: 750) using artition Chromatography-LLN (model NMF, manufactured by Miki Engineering Co., Ltd.). The upper layer of the solvent system was used as the stationary phase solution, the lower layer was used as the mobile phase solution, and developed at 20 ° C., 1,000 rpm, 4 ml / min.The obtained active fraction was concentrated and lyophilized to give 66 mg of white powder of benaltin. Obtained.

Example 3 20 mg of benaltin white powder was dissolved in 5 ml of 0.1N HCl, concentrated and freeze-dried to obtain 22 mg of benaltin hydrochloride white powder.

〔The invention's effect〕

Benaltin, a novel substance of the present invention, is degraded and metabolized using a peptide hormone (bioactive peptide) having various physiological functions in vivo as a substrate, and pyroglutin is involved in the regulation of the function of the peptide hormone or the expression of a new physiological activity. Since it has an inhibitory activity on tamyl peptidase, it is expected to be useful as a drug having a new physiological action. Benaltin or an acid addition salt thereof, for example, hydrochloride,
Sulfates and the like can be expected to be used as immunomodulators and as effective medicaments for treating various pituitary hormone secretion disorders.

[Brief description of the drawings]

FIG. 1 shows an ultraviolet absorption spectrum of benaltin in an aqueous solution. FIG. 2 shows an infrared absorption spectrum of benaltin in a potassium bromide tablet. FIG. 3 shows that 40% of benaltin in heavy dimethyl sulfoxide.
¹ shows a ¹ H-NMR spectrum at 0 MHz. FIG. 4 shows that 10% of benaltin in deuterated dimethyl sulfoxide.
1 shows a ¹³ C-NMR spectrum at 0 MHz.

Continuation of the front page (51) Int.Cl. ⁶ Identification symbol FI (C12P 21/02 C12R 1: 465) (72) Inventor Masahiro Hachi 3-25-10 Hiyoshihoncho, Kohoku-ku, Yokohama-shi, Kanagawa-ken (58) Field (Int.Cl. ⁶ , DB name) C07K 5/06 C12P 21/00-21/02 C12N 9/99 CA (STN) REGISTRY (STN) BIOSIS (DIALOG) WPI (DIALOG)

Claims (2)

(57) [Claims] (1) The following formula (I) An enzyme inhibitor benaltin represented by or a salt thereof. 2. A method for producing the enzyme inhibitor benaltin, which comprises culturing a benartin-producing bacterium belonging to the genus Streptomyces and collecting the enzyme inhibitor benaltin according to claim 1 from the culture.