JPH111480A - New substances f-9775a and f-9775b - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain new compounds, comprising F-9775A and/or F-9775B, having inhibiting actions on cathepsin K and useful as a therapeutic agent for osteoporosis, etc. SOLUTION: The F-9775A is represented by formula I and/or the F-9775B is represented by formula II. The compounds are obtained by culturing a microorganism, belonging to the genus Paecilomyces and capable of producing the F-9775A and/or F-9775B, e.g. a Paecilomyces carneus SANK 15996 (FERM BP-5916) strain and isolating and purifying the F-9775A and/or F-9775B from the cultured solution. Physicochemical properties of the F-9775A are as follows: property: red needlelike crystal; melting point: >270 deg.C (decomposition); specific rotatory power [a]<25> D 0 deg. (c 0.52. methanol); circular dichroism(CD) spectrum: a plain curve (without Cotton effect); molecular formula: C21 H16 O8 ; molecular weight: 396; solubility: sparingly soluble in methanol, ethanol and dimethyl sulfoxide.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to novel compounds F-9775A and F-9775B useful as a therapeutic agent for osteoporosis and the like, a process for producing the compounds, a medicine comprising the compounds, and a bacterium producing the compounds.

[0002]

2. Description of the Related Art Osteoporosis is a disease in which bone mass is reduced.
Spinal compression fractures, femoral neck fractures,
Humeral neck fracture, forearm distal bone fracture and the like (Takuo Fujita, Osteoporosis-Treatment and Diagnosis-, 1993,
See Life Sciences). In addition, osteoporosis is one of the diseases having an extremely large social impact in an aging society, because the morbidity rate increases as the elderly age. Bone is a seemingly static tissue, but it actually repeats bone formation and bone resorption at all times, and maintains a normal state by maintaining the balance. However, when the balance is lost and bone resorption exceeds bone formation, bone mass is reduced and osteoporosis is exhibited (see Takuo Fujita, Osteoporosis-Treatment and Diagnosis-, 1993, Life Science).

[0003] Therefore, bone resorption inhibitors are useful as therapeutic or preventive agents for osteoporosis. Conventionally, as bone resorption inhibitors, estrogen preparations, calcitonin, etc. have been used, but side effects or escape phenomena are seen,
Sufficient therapeutic effects have not been obtained, and bone resorption inhibitors with different mechanisms are expected (see Trends in the development of therapeutic agents for osteoporosis, NEW CURRENT, 1996, Volume 7, pages 1-50).

[0004] Osteoclasts are responsible for bone resorption,
It comes into contact with the bone surface, decomposes the bone matrix following decalcification, and performs bone resorption. Most of the bone matrix is occupied by type I collagen, and it has been reported that cysteine protease is greatly involved in the degradation of type I collagen. In fact, it has been reported that administration of E-64, a cysteine protease inhibitor, to rabbits suppresses bone resorption (PA Hill et al .: J. Cell Biochem., 56, 118).
-130 (1994)).

[0005] Cathepsin L (see Kakegawa et al., FEBS Letters, Vol. 321, pp. 247-250 (1993)) has been known as a cysteine protease involved in the degradation of type I collagen. This enzyme inhibitor is expected to be a therapeutic agent for osteoporosis or the like by suppressing bone resorption, and inhibitors for the enzyme have been widely studied. Examples of such inhibitors include leupeptin (Nishimura et al .: Biol. Pharm. Bull., Vol. 18, p. 945-950), catestatins (Je-Tae Woo et al .: Biosci. Biotech. Bioche
m., 59, pp. 350-352 (1995);
No. 98), epoxy succinic acid derivative (Kubotani, etc .: EP-
655447), petidyl vinyl sulfone derivative (D.
Bromme et al .: Biochem. J., vol. 315, pp. 85-89 (1996)), E-64 (BJ Gour-Salin et al .: Bioorg. Chem., 22).
Vol., Pp. 227-241 (1994)), chymostatinols (see Hamano et al., JP-A-8-3188), TAK-726 (Soda, et al .: see JP-A-8-283221), YM-5740.
9 (Amuro et al .: see Japanese Patent Application Laid-Open No. 9-87265) is known.

In recent years, cathepsin K, a cysteine protease that is selectively and abundantly expressed in osteoclasts, has been cloned by Inokas (T. Inaoka).
Et al .: Biochem. Biophy. Res. Comm., 206, 89-96 (1
995). More recently, the conformation of cathepsin K has been determined by X-ray crystallography (ME McGrath et al .: Natu
re Structure Biology, 4, 105-109 (1997); B. Zh
ao et al .: Nature StructureBiology, 4, 109-111 (199
7)). From the selectivity of the expression of cathepsin K, it is considered that the specific inhibitor of the enzyme selectively acts on the suppression of bone resorption, and can be a therapeutic agent for osteoporosis or the like with few side effects. Cathepsin K inhibitor research has only just begun, and in addition to the above-mentioned cathepsin L inhibitor petidylvinylsulfone derivative and E-64,
APC3328 (JT Palmer et al., J. Med. Chem., 38)
Vol. 3193-3196 (1995)) also inhibits cathepsin K non-specifically. However, an inhibitor specific to cathepsin K is not yet known.

[0007]

SUMMARY OF THE INVENTION The present inventor has sought a highly safe and highly selective cathepsin K inhibitor for a microbial resource in search of a highly safe therapeutic agent for osteoporosis and the like. Neuss (Paecilomyces carneu
s) A novel substance having a stronger cathepsin K inhibitory activity was isolated and purified from the culture of the SANK15996 strain, thereby completing the present invention.

[0008]

The present invention provides (1) F-9775A represented by the following formula I:

[0009]

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(2) F-9775B represented by the following formula II:

[0011]

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(3) F-9775A belonging to the genus Pesilomyces
And / or F-9775B-producing bacteria are cultured, and F-97
(4) A method for producing F-9775A and / or F-9775B, comprising isolating and purifying 75A and / or F-9775B.
F-9775A belonging to the species Pesilomyces carneus and / or
Or a method for producing F-9775A and / or F-9775B, comprising culturing an F-9775B-producing bacterium and isolating and purifying F-9775A and / or F-9775B from the culture solution. A) A method for producing F-9775A and / or F-9775B, which comprises culturing the strain PES-Romyces carneus SANK15996, and isolating and purifying F-9775A and / or F-9775B from the culture solution.

(6) Pesilomyces carneus SANK15
996 (FERM BP-5916) strain, (7) F-9775A and / or F-
9775.

The novel compounds F-9775A and F-9775 of the present invention
B has the following physicochemical properties.

[F-9775A] 1. 1. Material properties: red needles 2. Melting point:> 270 ° C. (decomposition) Optical rotation: [a] ²⁵ _D 0 ° (c 0.52, methanol) 4. CD spectrum: plain curve (no cotton effect) Molecular formula: C ₂₁ H ₁₆ O ₈ (from high-resolution fab mass spectrum) Molecular weight: 396 (from fab mass spectrum) Ultraviolet absorption spectrum (ethanol): 208 (2
9000), 305 (4700) (sh), 364 (4700), 420 (2900) Infrared absorption spectrum (KBr): 3391, 3273,
2957,2928,2873,1712,1636,1601,1528,1515,1433,1384,
1339,1261,1232,1204,1137,1084,1041,984,967,831,80
1,722, 615,584 9. ¹ H-nuclear magnetic resonance spectrum (d, heavy methano-
): 1.85 (s), 1.99 (s), 2.33 (s), 3.51 (s), 6.03
(brs), 6.40 (brs), 6.46 (brs). ¹³ C-nuclear magnetic resonance spectrum (d, heavy methano-
): 20.3 (q), 21.0 (q), 29.4 (q), 46.0 (s), 6
1.2 (d), 88.6 (s), 108.8 (d), 116.2 (d), 121.6 (s),
128.7 (s), 132.7 (d), 133.2 (s), 136.2 (s), 141.2
(s), 147.0 (s), 150.0 (s), 157.0 (s), 176.6 (s), 17
7.1 (s), 196.8 (s), 201.3 (s) [F-9775B] 1. Material properties: red needles 2. Melting point:> 280 ° C (decomposition) Optical rotation: [a] ²⁵ _D 0 ° (c 0.52, methanol) 4. CD spectrum: plain curve (no cotton effect) Molecular formula: C ₂₁ H ₁₆ O ₈ (from high-resolution fab mass spectrum) Molecular weight: 396 (from fab mass spectrum) Ultraviolet absorption spectrum (ethanol): 227 (3
7.000), 304 (10000), 367 (4000) (sh), 418 (5000) Infrared absorption spectrum (KBr): 3288, 2980,
2926,1733,1699,1657,1598,1524,1508,1458,1433,1412,
1378,1357,1319,1357,1319,1291,1263,1209,1156,1129,
1102,1085,946,841,829,782,773,604,578. ¹ H-nuclear magnetic resonance spectrum (d, heavy methano-
): 1.87 (3H, s), 2.18 (3H, s), 2.34 (3H, s), 3.6
1 (1H, s), 6.02 (1H, s), 6.42 (1H d) ¹³ C-nuclear magnetic resonance spectrum (d, heavy methano-
): 17.7 (q), 18.8 (q), 27.5 (q), 43.6 (s), 58.8
(d), 86.2 (s), 106.7 (d), 112.2 (d), 118.4 (s), 12
5.2 (s), 130.2 (d), 130.6 (s), 135.1 (s), 141.0 (s),
144.4 (s), 148.3 (s), 154.9 (s), 173.7 (s), 175.1
(s), 194.4 (s), 199.3 (s) Solubility: poorly soluble in methanol, ethanol and dimethyl sulfoxide The novel compounds F-9775A and F-9775B of the present invention have various stereoisomers. In formulas I and II, these stereoisomers and mixtures of these isomers are all represented by a single formula. Therefore, in the present invention, all of these stereoisomers and a mixture of these stereoisomers are included, and the novel compounds F-9775A and F-9775B of the present invention are formed from solvates (eg, hydrates). When they are formed, these are all included.

For example, the novel compounds F-9775A of the present invention and
When F-9775B is left in the air or undergoes recrystallization, it may absorb moisture and adhere to adsorbed water or form hydrates. The present invention includes such a solvate.

Further, in the present invention, all compounds that are metabolized in vivo and converted into F-9775A or 9775B, so-called prodrugs, are also included in the present invention.

The novel compounds F-9775 and F-9775B of the present invention
Pairocils used in the production of
As a strain belonging to the genus omyces, for example, Paecilomyces carneus SANK 15996
(Hereinafter, abbreviated as "SANK 15996 strain"). SANK 15996 strain was isolated from soil collected in Tsukuba, Ibaraki Prefecture.

The mycological characteristics of the SANK 15996 strain are as follows.

Colony growth on marine agar reaches a diameter of 20-23 mm at 23 ° C. for 2 weeks. The colony surface becomes powdery with sporulation intensified, becomes wool-shaped at the center, becomes white at first, and becomes yellowish-pink at the center as the culture is continued. The back is olive,
It may become dark green by continuing the culture.
No odor occurs. The vegetative mycelium is slightly thin, smooth, width 2
μm, the conidiophores grow singly and stand upright from the buried hypha,
Attach 1-4 of the wheeled phialides. Phialides are 14-17 x 2.5 μm, with a cylindrical base and tapering towards an elongated neck. Conidia are 2.5 x 2.5-3 μm, subglobular, rough to acicular, long chained and divergent. These features are described in the literature (Samson, RA (1974)
Paecilomyces and some allied hyphomycetes. Studies
in Mycology 6: 9-74.), which is consistent with the characteristics of Paecilomyces carneus. Therefore, this strain was identified as Paecilomyces carneus.

This strain is Paecilomyces carneus (Duche
& Hein) Brown & Smith SANK15996 shares, 1997
April 15, 2000, deposited with the Institute of Biotechnology, Institute of Industrial Science and Technology, Ministry of International Trade and Industry, and assigned the deposit number FERM BP-5916.

The bacteria producing F-9775A and F-9775B have been described above. However, it is well known that the properties of bacteria are not constant and easily change naturally or artificially. The same applies to the SANK15996 strain of the present invention. The SANK15996 strain according to the present invention contains all the mutant strains. These mutants also include those obtained by genetic methods such as recombination, transduction, and transformation. That is, SA producing F-9775A and F-9775B
The NK15996 strain, its mutant strains and strains that cannot be clearly distinguished therefrom are all included in the SANK15996 strain of the present invention.

The novel compounds F-9775A and F-9775B of the present invention
In order to obtain these, the culture of these microorganisms is carried out in a medium for producing a general fermentation product. Such a medium contains a carbon source, a nitrogen source, and inorganic salts that can be used by microorganisms. Tap water is used for the preparation of the medium.

Generally, glucose, fructose, maltose, sucrose, mannitol, and the like are used as carbon sources.
Glycerol, dextrin, oatmeal, rye,
Corn starch, potato, soybean flour, cottonseed oil, molasses, citric acid, tartaric acid and the like can be used alone or in combination. Generally, these carbon sources are used in an amount of 1 to 10% by weight of the medium. Can be

As the nitrogen source, a substance containing a protein is generally used in the fermentation step. Suitable nitrogen sources include soybean flour, bran, peanut flour, cottonseed oil, cottonseed flour, casein hydrolyzate, pharmamin, fishmeal, peptone, meat extract, yeast extract, malt extract, sodium nitrate, ammonium nitrate, ammonium sulfate And so on. The nitrogen source is used alone or in combination with 0.2-0.6% by weight of the medium volume.

The inorganic salts to be taken into the medium are ordinary salts which give ions such as sodium, ammonium, calcium, phosphonate, sulfate, chloride, carbonate and the like. It also contains trace metals such as potassium, cobalt, manganese, and magnesium. In liquid culture, silicone oil, vegetable oil, and surfactant are used as antifoaming agents.

F-9775A and F-9775B can be obtained by aerobically culturing the strain SANK15996. As the aerobic culturing method, a liquid shaking culturing method, an aerated stirring liquid culturing method and the like are employed.
Suitably containing the aforementioned carbon source, nitrogen source, and inorganic salt, 12
The medium is heat-sterilized at 0 ° C for 20 minutes, inoculated from the slant into the culture medium, and precultured at 20 ° C for 7 days. Thereafter, the above-mentioned preculture solution is inoculated into the same culture medium, and main culture is performed at 20 ° C for 7 days. The cultivation is performed in an incubator such as a flask, a jar, or a tank.

After completion of the culture, celite is added to the culture solution, and the mixture is filtered to separate the cells into the supernatant and the supernatant. Adjust the filtrate to pH 3,
Extract with ethyl acetate. This ethyl acetate extract may further contain an adsorbent such as silica gel, activated carbon, Amberlite X
It can be purified using AD-1, -4 (manufactured by Rohm & Haas), Diaion HP-20, CHP-20, HP-50 (manufactured by Mitsubishi Kasei Co., Ltd.). The partially purified product thus obtained is further subjected to distribution column chromatography using Sephadex LH-20 (manufactured by Pharmacia) or the like, or a roving column using octadecyl or decylated silica gel as a filler. [RP-8, 18 (manufactured by Merck)] Low-pressure reverse-phase column chromatography, purified by HPLC using Segashu ODS-H, S; Pegacil ODS (manufactured by Senshu Kagaku); Novapack (manufactured by Millipore) I can do it.

[0029]

BEST MODE FOR CARRYING OUT THE INVENTION The cathepsin K inhibitory action of F-9775A or F-9775B of the present invention can be calculated by the following method.

The entire translation region of human cathepsin K cDNA has already been reported (T. Inaoka et al: Biochem. Biophys. Res. Comm.
um., 206, based on the nucleotide sequence of human cathepsin K of 89-96 ^{(1995)), Quick-Screen TM Human cDNA} Library Pa
nel (Clontech Laboratories: CLONTECH Lab
oratories, Inc.) (λgt1
1) Using the polymerase chain reaction (Polymerase Ch
ain Reaction: Hereinafter referred to as “PCR”. ) Method. Next, the protein encoded by this cDNA contains a signal peptide region. The signal peptide region is removed from this cDNA, and a fragment corresponding to the region corresponding to the pro-mature is amplified by PCR. An expression plasmid inserted into pTrcHisB (Stratagen Ltd .: STRATAGEN Ltd.) was prepared. Escherichia coli M15 [pREP4] strain (Nal ^S ,
^{Str S, rif S, lac -} , ara -, gal -, mtl -, F -, recA +, uvr
⁺ ) (QIAGEN GmbH, Qiagen) was transformed to obtain a transformed strain. Human cathepsin K expressed from this transformant was purified using Xpress ^™ System protein purification.
After purification using (Invitrogen: INVITOROGEN BV), a refolding operation was performed. Further, the pH was lowered to 4.0, and auto-digestion (auto-processing) was performed to obtain activated human cathepsin K. To the obtained activated human cathepsin K, 40 μM of Z-phenylalanine-arginine-aminomethylcoumarin (Z-Ph
e-Arg-AMC (Peptide Research Institute)) and the test sample
The reaction was carried out at 150C for 15 minutes. Stop solution (100 mM monochloroacetic acid), 30 mM sodium acetate (s
odium acetate), 70mM acetic acid, (pH4.3))
Fluorometer (Labsystems Fluols from Labsystems)
oskan II), the fluorescence of the released aminomethylcoumarin was measured at an excitation wavelength of 355 nm and a fluorescence wavelength of 460 nm. As a control, the solvent used for dissolving the test sample was added instead of the test sample, and the enzyme activity at this time was determined as 100%, and the remaining enzyme activity was determined. 6 for each test sample
Serial dilution was performed, and the IC ₅₀ value was calculated according to the following equation.

Cathepsin K inhibitory action = [1- (A / B)] × 100 (%) A: Amount of aminoethylcoumarin produced in the presence of inhibitor B: Amount of aminoethylcoumarin produced in the absence of inhibitor The cathepsin L inhibitory effect of F-9775A or F-9775B of the present invention can be calculated by the following method.

The substrate cathepsin L enzyme prepared by the method of Hamano et al. (See JP-A-8-3188) is reacted with the substrate Z-phenylalanine-arginine-aminomethylcoumarin and a test sample, and the aminoethyl produced by the enzyme is reacted. Coumarin was quantified by fluorescence. As a control, a solution obtained by adding only a solvent in which a test sample was dissolved was used, and the remaining enzyme activity was determined by setting the enzyme activity at this time to 100%. For each test sample, the IC ₅₀ value was calculated according to the following formula.

Cathepsin L inhibitory action = [1- (A / B)] × 100 (%) A: Amount of aminoethylcoumarin produced in the presence of inhibitor B: Amount of aminoethylcoumarin produced in the absence of inhibitor F -9775 and F-9775B are novel compounds not described in the literature,
It has a cathepsin K inhibitory action and is useful as an agent for preventing and treating bone metabolic diseases such as osteoporosis.

When the F-9775A or F-9775B of the present invention is used as a medicament, it is mixed with itself or a pharmaceutically acceptable carrier or excipient according to a conventional method to obtain a powder, granule, tablet, capsule or the like. It can be safely administered orally or parenterally in the form of injections and the like. These formulations contain excipients, lubricants,
It is manufactured by a known method using additives such as a binder, a disintegrant, a stabilizer, a flavoring agent, and a diluent. Examples of the excipient include sugar derivatives such as lactose, sucrose, glucose, mannitol, and sorbite; starch derivatives such as corn starch, potato starch, α-starch, dextrin, carboxymethyl starch; and crystalline cellulose; Low-substituted hydroxypropyl cellulose, hydroxymethyl cellulose,
Cellulose derivatives such as carboxymethylcellulose, carboxymethylcellulose calcium, internally cross-linked carboxymethylcellulose; organic excipients such as gum arabic, dextran, pullulan; and light anhydrous silicic acid;
Inorganic excipients such as silicate derivatives such as synthetic aluminum silicate and magnesium aluminate metasilicate; phosphates such as calcium phosphate; and sulfates such as calcium sulfate. As the lubricant, for example, stearic acid,
Metal stearate such as calcium stearate, magnesium stearate; talc; colloidal silica;
Bea gum; waxes such as gay wax; boric acid; adipic acid; sulfates such as sodium sulfate; glycol; fumaric acid; sodium benzoate; DL-leucine; sodium fatty acid; sodium lauryl sulfate, magnesium lauryl sulfate and the like. Lauryl sulfate; and the above starch derivatives. Examples of the binder include polyvinylpyrrolidone, macrogol and substances similar to the above-mentioned excipients. Examples of the disintegrant include substances similar to the above-mentioned excipients, chemically modified starch such as croscarmellose sodium, carboxymethyl starch sodium, cross-linked polyvinyl pyrrolidone, and celluloses. Can be done. Examples of the stabilizer include paraoxybenzoic acid esters such as methylparaben and propylparaben; chlorobutanol, benzyl alcohol-
Alcohols such as phenol and phenethyl alcohol; benzalkonium chloride; phenols such as phenol and cresol; thimerosal; dehydroacetic acid; and sorbic acid. Examples of the flavoring agent include, for example, commonly used sweeteners, sour agents, flavors and the like.

The dosage of the F-9775A and F-9775B of the present invention varies depending on the target disease, the administration route, the number of administrations, and the like.
It is desirable to administer g in one to several divided doses according to the symptoms. In the case of intravenous administration, 0.01 to 100 mg per day
Is preferably administered once or several times according to the symptoms.

[0036]

The present invention will be described in more detail with reference to the following examples, test examples and preparation examples, but the present invention is not limited to these examples.

Embodiment 1 [Culture] Medium A shown in Table 1 below was heat-sterilized at 120 ° C. for 20 minutes. One loopful of Paecilomyces carneus SANK15996 strain was inoculated from a slant into a 500 ml Erlenmeyer flask containing 100 ml of the same medium after sterilization, and cultured at 20 ° C. for 7 days.
The same culture medium A was dispensed in 15 L portions into two 30 L jar incubators, and the pre-cultured solution was cultured under aeration and stirring at 20 ° C. for 7 days.

[0038]

Table 1 Medium A Medium composition Glycerin 50 g Malt extract (Difco) 5 g Potato 50 g East extract (Difco) 5 g Nissan deform CB-442 10% acetone solution (Nippon Oil & Fat) 0.5 ml Tap water 1000 ml [Isolation]・ Purification] Celite (1.5 kg) in the culture end solution (27 L)
Was added, and the cells were separated by filtration. The filtrate (28 L) was adjusted to pH 3.0 and extracted twice with ethyl acetate (20 L, 30 L). After the extract was washed with tap water (25 L) and saturated saline (27 L) in that order, the extract was dehydrated with anhydrous Na ₂ SO ₄ (1.0 kg). Ethyl acetate was distilled off under reduced pressure, and 3.0 g of an oily substance (9.3 g) obtained was partitioned between hexane and 90% aqueous methanol. From the active 90% aqueous methanol layer, ethyl acetate was distilled off under reduced pressure to obtain an active fraction (8.4 g). Of which, 3.0 g
With HPLC column [Nova pack (Millipore) 40 fx 21
0 mm] and the solvent (50% aqueous methanol + 0.3
% Trifluoroacetic acid) at a flow rate of 50 ml / min, and the fractions having a retention time of 7-11 minutes were collected to obtain a crude active fraction (391.4 mg). 330 mg of this crude active fraction was again added to an HPLC column [Novapack (Millipore) 40 f
x 210 mm] and the solvent (40% aqueous methanol +
Chromatography was performed using 0.2% trifluoroacetic acid) at a flow rate of 50 ml / min, and the retention time was 17.5 to 18.5 minutes (3.
The fractions of 4.3 mg) and 31-37 minutes (101.4 mg) were collected. The former was recrystallized from 10% aqueous ethanol (20 ml) to give an F-9775B fraction (11.1 mg). The latter was also recrystallized from 10% aqueous ethanol (20 ml) to obtain the F-9775A fraction.
(79.8 mg).

Test Example 1 [Cloning of cDNA of human cathepsin K] In order to clone the entire translation region of human cathepsin K by the PCR method, it was reported previously (T. Inaoka et al: Biochem. Biophys.
Cathepsin K of Res. Commum., 206, 89-96 (1995)).
And the primers shown in SEQ ID NO: 1 and SEQ ID NO: 2 based on the base sequence of Oligo 1000 DNA Synthesizer (Oligo
1000 DNA synthesizer: BECKMAN Co.,
Ltd.)).

Sense primer: 5'-GCAGGATGTGGGGGC
TCAAG-3 '(SEQ ID NO: 1) Antisense primer: 5'-GGAGTCACATCTTGGGGAAG-
3 ′ (SEQ ID NO: 2) The template for the PCR method is Quick-Screen ^™ Human cDNA Li
brary Panel (Clontech: CLONTECH Laborator
ies, Inc.). Quick-Screen ^TM HumancDNA Lib
1 human lung cDNA (λgt11) contained in the rary Panel
μl and 1 μl of each of the above primers 20 μM,
According to the protocol of Gibco BRL, PCR was performed with a reaction volume of 100 μl. The PCR conditions were 94 ° C, 2 minutes, 94 ° C, 1 minute, 55 ° C, 2 minutes.
The reaction was repeated 25 times at 72 ° C. for 3 minutes for 25 minutes, and finally kept at 72 ° C. for 7 minutes. When 10 µl of the PCR reaction solution was confirmed by 1% agarose gel electrophoresis, it was found in the literature (T. Inaoka et al: Biochem. Biophys. Res. Commu.
m., 206, 89-96 (1995)).
A band corresponding to 99 bp) was confirmed.
Cathepsin K cDNA fragment amplified by the
Using a Cloning Kit (Invitrogen BV), use the pCR ^™ attached to the kit.
It was cloned directly using the II plasmid. That is, P
2 μl of CR reaction solution, 2 μl of pCR ^™ II plasmid solution,
4 μl of sterile distilled water, 1 μl of a ligation buffer included in the kit, and 1 μl of a ligase included in the kit were mixed and reacted at 16 ° C. overnight. Using 1 μl of the reaction solution,
A Escherichia coli attached to Cloning Kit
INV αF competent cells were transformed. A plasmid was extracted from the obtained transformant, and the restriction enzyme EcoRI was used.
To confirm that a plasmid into which human cathepsin K cDNA was inserted was obtained and named pCK1. Human cathepsin KcD inserted into pCK1 by methods well known in the art
The nucleotide sequence of NA was determined. As shown in (SEQ ID NO: 3), a previously reported sequence (T. Inaoka et al: Biochem. Biophy
s. Res. Commum., 206, 89-96 (1995)), but the amino acid (phenylalanine) encoded by the DNA at this position was unchanged. Therefore, this human cathepsin Kc
An expression system in Escherichia coli was constructed using the DNA.

Test Example 2 [Construction of expression vector for human cathepsin K in Escherichia coli]
In order to insert only the pro-mature region from the human cathepsin K cDNA obtained in Test Example 1 into an expression vector, PCR was performed using the sense primer shown in SEQ ID NO: 4 and the antisense primer shown in SEQ ID NO: 2 described above. Only the pro-mature region was amplified by the method.

Sense primer: 5'-GGATCCTCTGTACCC
TGAGGAGATAC-3 '(SEQ ID NO: 4) A recognition sequence for the restriction enzyme BamHI was added to the sense primer. The pCK1 obtained in Test Example 1 was used as a PCR template, and the PCR reaction conditions were the same as those described in Test Example 1. After confirming the amplified fragment by 1% agarose gel electrophoresis, a transformant was obtained in the same manner as in Test Example 1, using the TA cloning kit shown in Test Example 1. A plasmid was extracted from the obtained transformant,
After treatment with EcoRI, it was confirmed that a plasmid into which cDNA of the human cathepsin K pro-mature region had been inserted was obtained and named pCKpro-mat1. Next, pCKpro-mat1 is
After treatment with oRI and BamHI, 1% agarose gel electrophoresis was performed, and Ba containing the pro-mature region of human cathepsin K was analyzed.
The mHI-EcoRI fragment was extracted using a centrifuge tube with a DNA recovery filter (Takara Shuzo Co., Ltd.). BamHI-EcoRI fragment D containing the pro-mature region of the extracted human cathepsin K
NA and pTrcHisB (STRATAG)
EN Ltd.,) treated with EcoRI and BamHI and DNA
Ligation kit version 1 (Takara Shuzo Co., Ltd.) was used and ligated at 16 ° C for 30 minutes.
109 strains were transformed. A plasmid was extracted from the obtained transformant, treated with restriction enzymes EcoRI and BamHI,
The plasmid in which the cDNA of the human cathepsin K pro-mature region was inserted between EcoRI and BamHI of the expression vector pTrcHisB was confirmed and named pTrcpmCK4.

Test Example 3 [Expression of human cathepsin K in Escherichia coli and purification of denatured human cathepsin K] Escherichia coli M15 [pREP4] strain (Nal ^S , Str ^S ,
^{rif S, lac -, ara -} , gal -, mtl -, F -, recA +, uv
r ⁺ ) (QIAGEN GmbH, Qiagen) was transformed with the expression plasmid pTrcpmCK4 prepared in Test Example 2. The transformant was used to prepare a 2 × YT medium (2 × YT: 16 g Bacto tryptone (Difco: Difco)) containing 10 μg / ml ampicillin and 50 μg / ml kanamycin, 10 g yeast extract (Difco) (5 g sodium chloride / 1 L) was inoculated with one platinum loop and inoculated at 37 ° C. overnight, and 1 ml was inoculated into 100 ml of the same medium. After culturing at 37 ° C. for two and a half hours, isopropyl β-D (−)-thiogalactopyranoside (isopropylβ-D (−)) was adjusted to a final concentration of 1 mM.
-thiogalactopyranoside (IPTG)), and the cells were further cultured for 5 hours. After completion of the culture, the cells are centrifuged (8,00
(0 rpm, 5 minutes). 10ml Native binding buffer
: The cells were suspended in 10 ml of 20 mM sodium phosphate buffer (pH 7.8), 500 mM sodium chloride) and disrupted by ultrasonication. After centrifugation (10,000 rpm, 10 minutes), the precipitate was washed with 10 ml of Denaturing binding buffer I.
I: Suspended in 20 mM sodium phosphate buffer (pH 7.8), 500 mM sodium chloride, 8 M urea). Centrifugation (10,000
After 10 minutes (rpm, 10 minutes), the supernatant was recovered and used as a denatured human cathepsin K solution. Next, this modified human cathepsin K
From the solution, denatured human cathepsin K was purified using Xpress ^™ System protein purification (Invitrogen: INVITOROGEN BV). That is, ProBon attached to the kit
d ^TM column
After equilibration with buffer I, the denatured human cathepsin K
The solution was applied to a ProBond ^™ column. 14 ml of denaturalizing binding buffer I, 14 ml of denaturalizing binding buffer II (20
mM sodium phosphate buffer (pH 6.0), 500 mM sodium chloride, 8 M urea, 14 ml of denaturalizing binding buffer III (20 mM sodium phosphate buffer (pH 5.3), 500 mM sodium chloride, 8 M urea ), And then wash with 10 ml of denaturalizing / elution buffer (20 ml).
Elution was performed with mM sodium phosphate buffer (pH 4.0), 500 mM sodium chloride, and 8 M urea. 10 mg of the denatured human cathepsin K was contained in 10 ml of the eluted fraction, which was used as a purified denatured human cathepsin K solution (1 mg / ml).

Test Example 4 [Activation of human cathepsin K] Dithiothreitol (DT) was added to the purified and denatured human cathepsin K solution obtained in Test Example 3 so that the final concentrations were 10 mM and 1 mM, respectively.
T)), disodium ethylenediaminetetraacetate (ethylene
diamine tetra acetic acid, disodium salt:
TA ”. ) Was added and allowed to stand at room temperature for 2 hours. Add 1 ml of this solution at 4 ° C to 100 ml
Buffer (refolding buffer: 50 mM potassium phosphate buffer
(pH10.7), 2.5mM EDTA, 1mM reduced glutathione (reduc
ed formed glutathione), 0.1mM oxidized glutathione (o
xidized formed glutathione)) at 1.5 ml / hour and stirred at 4 ° C overnight. After the completion of this operation, insolubles were removed by centrifugation (10,000 rpm, 15 minutes), and the supernatant was recovered. At this time, the protein concentration of the supernatant was 5 μg / ml.
Had become. The collected supernatant (about 100 ml) is adjusted to a final concentration of 10 μM by using pepstatin A.
Was added, and pH 4 was adjusted with 2M sodium acetate buffer (pH 3.0).
After adjusting to 0, the solution was allowed to stand at room temperature for 2 hours to obtain an activated human cathepsin K solution (about 110 ml).

Test Example 5 [Measurement of Human Cathepsin K Inhibitory Activity] The final concentration of the active human cathepsin K solution obtained in Test Example 4 was adjusted to 2 mM.
DTT was added, and a 96-well plate (Nunc: Nunc:
75 μl was added to each well of the FluoroNunc ^™ Plates). Here, 25 μl of 40 μM Z-phenylalanine-arginine-aminomethylcoumarin (Z-Phe-Arg-AMC (Peptide Research Institute)) dissolved in 0.2% dimethyl sulfoxide and 1 μl of a test sample dissolved in 100% methanol Was added and reacted at 30 ° C. for 15 minutes. 100μ
l of stop solution (100 mM monochloroacetate)
tic acid), 30mM sodium acetate (sodiumacetate), 70m
M acetic acid (pH 4.3)) was added to terminate the reaction. After the reaction is completed, use a fluorescence meter (Lab System: Lab
ystems Labsystems FluoloskanII), excitation wavelength 3
The fluorescence of the released aminomethylcoumarin was measured at a wavelength of 55 nm and a wavelength of 460 nm. As a control, 1 μl of the solvent used for dissolving the test sample (100% methanol) was added instead of the test sample, and the enzyme activity at this time was taken as 100%, and the residual enzyme activity when the test sample was added was determined. Was. Performed serially diluted in 100% methanol for a test specimen was calculated and IC ₅₀ values make inhibition curves from percent inhibition calculated according to the following formula.

Cathepsin K inhibitory action = [1- (A / B)] × 100 (%) A: Amount of aminoethylcoumarin produced in the presence of inhibitor B: Amount of aminoethylcoumarin produced in the absence of inhibitor F The 50% inhibitory concentrations (IC ₅₀ ) of -9775A and F-9775B for cathepsin K were 28.8 mM and 14.3 mM, respectively.

Test Example 6 [Measurement of human cathepsin L inhibitory activity] Rat cathepsin L enzyme prepared by the method of Hamano et al. (Described in JP-A-8-3188) was added to a substrate, Z-phenylalanine-arginine-aminomethylcoumarin and a test sample according to the same method. After the reaction, the aminoethyl coumarin produced by the enzyme was quantified by fluorescence. As a control, a solvent (100% methanol only) in which the test sample was dissolved was added, and the activity at this time was defined as 100% to determine the residual activity. The test sample was serially diluted with 100% methanol, and an inhibition curve was created from the inhibition rate determined according to the following formula to calculate an IC ₅₀ value.

Cathepsin L inhibitory action = [1- (A / B)] × 100 (%) A: Amount of aminoethylcoumarin produced in the presence of inhibitor B: Amount of aminoethylcoumarin produced in the absence of inhibitor F The 50% inhibitory concentrations (IC ₅₀ ) of -9775A and F-9775B for cathepsin L were 98.9 mM and 61.3 mM, respectively.

[0049]

As described above, F-9775A and F-9775B
Is useful as a selective cathepsin K inhibitor as a therapeutic or prophylactic for osteoporosis and the like.

[Brief description of the drawings]

FIG. 1 is a construction diagram of an expression plasmid for human cathepsin K.

[Sequence list]

SEQ ID NO: 1 Sequence length: 20 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid (synthetic DNA) Hypothetical: No Antisense: No sequence GCAGGATGTG GGGGCTCAAG 20 SEQ ID NO: 2 Sequence length: 20 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acids (synthetic DNA) Hypothetical: No Antisense: No sequence GGAGTCACAT CTTGGGGAAG 20 SEQ ID NO: 3 Sequence length: 990 Sequence type: Nucleic acid Number of strands: Double-stranded Topology: Linear Sequence type: cDNA to mRNA high-specific: No Antisense: No Origin Organism name: Homo Sapiens Tissue: Lung Sequence features Symbol indicating features: CDS position: 1..987 Symbol indicating features: mat_peptide Position: 343..987 Symbol indicating features: sig_peptide Position: 1..45 Sequence ATG TGG GGG CTC AAG GTT CTG CTG CTA CCT GTG GTG AGC TTT GCT CTG 48 Met Trp Gly Leu Lys Val Leu Leu Leu Pro Val Val Ser Phe Ala Leu -114 -110 -105 -100 TAC CCT GAG GAG ATA CTG GAC ACC CAC TGG GAG CTA TGG AAG AAG ACC 96 Tyr Pro Glu Glu Ile Leu Asp Thr His Trp Glu Leu Trp Lys Lys Thr -95 -90 -85 CAC AGG AAG CAA TAT AAC AAC AAG GTG GAT GAA ATC TCT CGG CGT TTA 144 His Arg Lys Gln Tyr Asn Asn Lys Val Asp Glu Ile Ser Arg Arg Leu- 80 -75 -70 ATT TGG GAA AAA AAC CTG AAG TAT ATT TCC ATC CAT AAC CTT GAG GCT 192 Ile Trp Glu Lys Asn Leu Lys Tyr Ile Ser Ile His Asn Leu Glu Ala -65 -60 -55 TCT CTT GGT GTC CAT ACA TAT GAA CTG GCT ATG AAC CAC CTG GGG GAC 240 Ser Leu Gly Val His Thr Tyr Glu Leu Ala Met Asn His Leu Gly Asp -50 -45 -40 -35 ATG ACC AGT GAA GAG GTG GTT CAG AAG ATG ACT GGA CTC AAA GTA CCC 288 Met Thr Ser Glu Glu Val Val Gln Lys Met Thr Gly Leu Lys Val Pro -30 -25 -20 CTG TCT CAT TCC CGC AGT AAT GAC ACC CTT TAT ATC CCA GAA TGG GAA 336 Leu Ser His Ser Arg Ser Asn Asp Thr Leu Tyr Ile Pro Glu Trp Glu -15 -10 -5 GGT AGA GCC CCA GAC TCT GTC GAC TAT CGA AAG AAA GGA TAT GTT ACT 384 Gly Arg Ala Pro Asp Ser Val Asp Tyr Arg Lys Lys Gly Tyr Val Thr 1 5 10 CCT GTC AAA AAT CAG GGT CAG TGT GGT TCC TGT TGG GCT TTT AGC TCT 432 Pro Val Lys Asn Gln Gly Gln Cys Gly Ser Cys Trp Ala Phe Ser Ser 15 20 25 30 GTG GGT GCC CTG GAG GGC CAA CTC AAG AAG AAA ACT GGC AAA CTC TTA 480 Val Gly Ala Leu Glu Gly Gln Leu Lys Lys Lys Thr Gly Lys Leu Leu 35 40 45 AAT CTG AGT CCC CAG AAC CTA GTG GAT TGT GTG TCT GAG AAT GAT GGC 528 Asn Leu Ser Pro Gln Asn Leu Val Asp Cys Val Ser Glu Asn Asp Gly 50 55 60 TGT GGA GGG GGC TAC ATG ACC AAT GCC TTC CAA TAT GTG CAG AAG AAC 576 Cys Gly Gly Gly Tyr Met Thr Asn Ala Phe Gln Tyr Val Gln Lys Asn 65 70 75 CGG GGT ATT GAC TCT GAA GAT GCC TAC CCA TAT GTG GGA CAG GAA GAG 624 Arg Gly Ile Asp Ser Glu Asp Ala Tyr Pro Tyr Val Gly Gln Glu Glu 80 85 90 AGT TGT ATG TAC AAC CCA ACA GGC AAG GCA GCT AAA TGC AGA GGG TAC 672 Ser Cys Met Tyr Asn Pro Thr Gly Lys Ala Ala Lys Cys Arg Gly Tyr 95 100 105 110 AGA GAG ATC CCC GAG GGG AAT GAG AAA GCC CTG A AG AGG GCA GTG GCC 720 Arg Glu Ile Pro Glu Gly Asn Glu Lys Ala Leu Lys Arg Ala Val Ala 115 120 125 CGA GTG GGA CCT GTC TCT GTG GCC ATT GAT GCA AGC CTG ACC TCC TTT 768 Arg Val Gly Pro Val Ser Val Ala Ile Asp Ala Ser Leu Thr Ser Phe 130 135 140 CAG TTT TAC AGC AAA GGT GTG TAT TAT GAT GAA AGC TGC AAT AGC GAT 816 Gln Phe Tyr Ser Lys Gly Val Tyr Tyr Asp Glu Ser Cys Asn Ser Asp 145 150 155 AAT CTG AAC CAT GCG GTT TTG GCA GTG GGA TAT GGA ATC CAG AAG GGA 864 Asn Leu Asn His Ala Val Leu Ala Val Gly Tyr Gly Ile Gln Lys Gly 160 165 170 AAC AAG CAC TGG ATA ATT AAA AAC AGC TGG GGA GAA AAC TGG GGA AAC 912 Asn Lys His Trp Ile Ile Lys Asn Ser Trp Gly Glu Asn Trp Gly Asn 175 180 185 190 AAA GGA TAT ATC CTC ATG GCT CGA AAT AAG AAC AAC GCC TGT GGC ATT 960 Lys Gly Tyr Ile Leu Met Ala Arg Asn Lys Asn Asn Ala Cys Gly Ile 195 200 205 GCC AAC CTG GCC AGC TTC CCC AAG ATG TGA 990 Ala Asn Leu Ala Ser Phe Pro Lys Met 210 215 SEQ ID NO: 4 Sequence length: 26 Sequence type: Nucleic acid Number of strands: Single strand Topology: linear sequence type: other nucleic acid (synthetic DNA) hypothemycin Pharmaceutical: No Antisense: No sequence GGATCCTCTG TACCCTGAGG AGATAC 2
6

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁶ Identification symbol FI // (C12N 1/14 C12R 1:79) (C12P 17/06 C12R 1:79) (72) Inventor Yuichi Ishikawa Shinagawa, Tokyo 1-58, Hiromachi, Ward Sankyo Co., Ltd.

Claims (7)

[Claims] (1) F-9775A represented by the following formula I: 2. F-9775B represented by the following formula II: 3. F-9775A belonging to the genus Pesilomyces and / or
Alternatively, a method for producing F-9775A and / or F-9775B, comprising culturing an F-9775B-producing bacterium and isolating and purifying F-9775A and / or F-9775B from the culture solution. 4. An F-protein belonging to the genus Pesilomyces carneus
A method for producing F-9775A and / or F-9775B, comprising culturing 9775A and / or F-9775B-producing bacteria, and isolating and purifying F-9775A and / or F-9775B from the culture solution. . 5. A method for culturing a strain of Pesilomyces carneus SANK15996, and isolating and purifying F-9775A and / or F-9775B from the culture broth, wherein F-9775A and / or F-9775B is isolated. Manufacturing method. 6. Pesilomyces carneus SANK15996
(FERM BP-5916) strain. 7. A medicament comprising F-9775A and / or F-9775B.