JPH0827184A - Peptide compound, its production and anti-human immunodeficiency viral agent - Google Patents

Abstract

PURPOSE:To obtain a low-toxic new peptide compound excellent in anti-human immunodeficiency viral activity, thus useful for AIDS treatment, etc., as an anti-human immunodeficiency viral agent, by culturing peptide compound- productive bacteria belonging to Streptomyces. CONSTITUTION:This new peptide compound expressed by the formula is obtained from a cultured product obtained by culturing peptide compound- producing bacteria belonging to Streptomyces [e.g. Actinomycetes SKH-2344 strain (FERM P-14239)]. This compound, which is excellent in anti-HIV activity and has low toxicity, can be taken in large quantities for a long period of time, thus being highly useful as a new AIDS medicine. Specifically, this compound is obtained by the following process: Streptomyces SKH-2344 strain is cultured in a medium at 25 deg.C for 5 days followed by centrifugation and then conducting a microbial collection, the resultant microbes are bacteriolyzed in methanol followed by making an extraction, and the extract thus obtained is concentrated to dryness, and the resultant extract is subjected to column chromatography and purified.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a peptide compound, and more particularly to an anti-HIV (anti-human immunodeficiency virus) active substance NP-06, a method for producing the same, and an anti-HIV active substance-containing anti-HIV active substance as an active ingredient. Regarding HIV agents.

[0002]

2. Description of the Related Art In recent years, acquired immunodeficiency diseases (hereinafter referred to as AID
The number of patients referred to as S) is increasing worldwide. Particularly in Asia and Africa, HIV, which is the cause of AIDS, is in a state of infection and explosion, and it is considered that AIDS patients will rapidly increase mainly in these areas in the future.

HIV is a virus of the family of retroviridae, which has RNA as a gene body. HIV infection is specific to the CD4 (cluster of differentiation antigen 4) protein present on the surface of T cells through a glycoprotein called gp-120 on the surface of the virus particles, which virus particles invaded into the blood. It begins with an adsorption process that physically binds. The virus particles adsorbed on the cell surface release the HIV gene (RNA) into the cells to unshell, and the HIV gene then undergoes DN transcription by reverse transcriptase.
It is rewritten to A (reverse transcription process). Then, the HIV genetic information rewritten in DNA is further incorporated into the DNA of the host cell by integrase, and the viral infection is established.

In infected cells, viral proteins are produced based on the genetic information of the virus. It has been clarified that the produced viral proteins include proteins called Tat and Rev that act as viral growth regulators. The viral protein produced in the host cell is converted into a viral constituent protein by cleavage with protease or modification of sugar chain by glycosidase (maturation process). Next, the synthesized viral proteins and viral genes are assembled to form new viral particles (packaging process).

The newly produced virus particles are released outside the cell (release process) and infect new cells. H
Upon infection with IV, immune cells lose their inherent immune function or cause giant cell formation, which usually leads to cell death. HIV-infected patients are severely immunodeficient due to the loss of immune cells, which causes opportunistic infections due to pathogenic bacteria such as cytomegalovirus and carinii protozoa. Therefore, at present, it is considered that drug treatment of administering a drug that inhibits intracellular proliferation of virus or cell infection brings about the effect of recovering immune function or prolonging the life of infected patients.

[0006] Recently, the life cycle of HIV in human immune cells (T cells) has been elucidated in more detail, and in particular, the compounds having the inhibitory activity of the enzyme and the receptor involved in the growth of the virus are being investigated. , Has found potential as a therapeutic agent for AIDS. By the way, currently AZT (azitothymidine), DD
Three types of nucleic acid compounds, C (dideoxycytidine) and DDI (dideoxyinosine), have been approved as therapeutic agents for AIDS. These nucleic acid drugs are HI
It exerts anti-HIV activity by acting on the reverse transcription process in the V life cycle.

However, when drug treatment is carried out using these nucleic acid compounds, there is a problem that anti-HIV activity decreases or disappears due to the emergence of drug-resistant strains, and there are strong side effects and so on, which results in long-term continuous use. The problem of inability to administer has become clear, and there is a limit to the current treatment of AIDS with only approved drugs. Under such circumstances, it is considered that future AIDS treatment will be predominantly a combination therapy using a plurality of drugs having different modes of action. Therefore, at present, a new adsorption inhibitor, deshelling inhibitor, non-nucleic acid reverse transcription inhibitor, protease inhibitor, T
The search and development of compounds that suppress HIV growth specifically are being promoted worldwide, centering on at inhibitors and glycosylation inhibitors.

[0008] For example, as an adsorption inhibitor, soluble CD4 (DHSmith, RAByrn, SAMar) obtained by solubilizing CD4 is used.
sters, T.Gregory, JEGroopman and DJCapon, Scienc
e, 238,1704 (1987)), dextran sulfate, heparin sulfate, and other sulfated polysaccharides (MA Skinner, R. Ting, A. Langlois,
KJWeinhold, HKLyerly, K.Javaherian and, TJMatth
ews, AIDS Res. Hum. Retorovirus, 4,187 (1988) and others);

As a non-nucleic acid reverse transcription inhibitor, TIBO
(Tetrahydro-imidazo-benzodiazepin-one
And Thion) (Pauwels, R., Andries, K., Desmyter,
J., et.al., Nature, 343: 470 (1990) and others),
HEPT (1-[(2-hydroxyethoxy) methyl]
-6- (Phenylthio) thymine) (Miyasaka, T., Tanaka,
H., Baba, M., Hayakawa, H., et.al., J.Med.Chem. (Journal of Medical Chemistry), 32, 2507 (1989)), as a protease inhibitor, Ro 31-8959 ( Roberts, NA, Ma
rtin, JA, Kinchington, D., et.al., Science, (Science) 248,358 (1990)), KNI-272 (Mimoto, T., Imai, J., Kisa
nuki, S., Enomoto, H., et.al., Chem.Pharm.Bull. (Chemical Pharma-Tikal Burtin), 40,2251 (199
2)) etc. are found.

[0010] In addition, searching for anti-HIV active substances from natural products in search of the possibility of new compounds is becoming popular all over the world, and already, for example, RP-719.
55 (Gerard, H., Catherine, D., Jean-Francois, M. and Je
Anti-HIV active substances such as an, L., J. Antibiotics, (Journal of Antibiotics) 46, 1756 (1993) have been found. However, an anti-HIV agent having excellent anti-HIV activity and low toxicity, or a lead compound which can be an anti-HIV agent has not been found yet.

[0011]

Therefore, the present inventors have investigated the secondary metabolites of microorganisms isolated from soil by a screening method (MTT assay method) which can effectively measure anti-HIV activity in vitro. As a result of the screening, a lead compound which has an excellent anti-HIV activity and can be a low-toxicity anti-HIV agent and a method for producing the same have been found, and the present invention has been completed.

Therefore, the first object of the present invention is to provide anti-HIV
An object of the present invention is to provide a lead compound which is an anti-HIV agent having excellent action and low toxicity. A second object of the present invention is to provide a method for producing a lead compound which is excellent in anti-HIV action and has low toxicity and can be an anti-HIV agent. Furthermore, the third object of the present invention is to provide anti-HIV
It is an object of the present invention to provide an anti-HIV agent containing a lead compound as an active ingredient, which has an excellent action and can be an anti-HIV agent having low toxicity.

[0013]

The above-mentioned various objects of the present invention have been achieved by a peptide compound represented by the following chemical formula 2, a method for producing the same, and an anti-HIV agent containing the same.

Embedded image

The present invention will be described in detail below. The present inventors used a screening method (MTT assay method) capable of effectively measuring anti-HIV activity in vitro for the purpose of providing an anti-HIV agent excellent in anti-HIV action. This method is for determining the infection death of CD4-positive cells caused by HIV infection and the suppression of the infection death by a drug, and anti-HIV activity and cytotoxicity can be measured simultaneously and easily. There is an excellent way.

The present inventors used this screening method to screen the secondary metabolites of microorganisms isolated from soil, and found a substance particularly excellent in anti-HIV activity, which was designated as NP-06. I named it. Anti-H above
The IV active substance NP-06 is a substance having the structure represented by Chemical Formula 2 and having the following physicochemical properties.

Properties: White powder Molecular formula: C ₉₇ H ₁₃₁ O ₂₆ N ₂₃ S ₄ Molecular weight: 2163 Solubility: Easily soluble in methanol, dimethylformamide and dimethylsulfoxide, sparingly soluble in water, ethyl acetate and chloroform Acid hydrolysis product : Glycine (4), cystine (2),
Alanine (2), valine (2), phenylalanine (2), leucine (1), aspartic acid (2), serine (1), isoleucine (1), tyrosine (1) IR (KBr) cm ^-1 : 701,746, 1178,1219,1337,1390,
1407,1454,1517,1649 (C = O), 2934,2964,3009,3082 (C = C),
3306,3317,3376,3387 (OH, NH) Optical rotation: [α] ^D ₂₀ = -75 ° ( ^C 0.1%, MeO
H) In determining the structure, various NMR measurements and chemical decomposition methods were used based on the data obtained from amino acid analysis, MASS spectrum measurement, and IR measurement.

The NP-06 of the present invention is RP-71955 (Gerard, H., Cath, previously reported by Gerard et al.
erine, D., Jean-Francois, M., and Jean, L., J.Antibiotic
s, 46, 1756 (1993)), and it was revealed that they have a very similar structure in amino acid sequence. That is, NP-06 and RP-71955
The difference is that the types of amino acids are different at the two positions.

The anti-HIV active substance NP-06 of the present invention is
It is isolated from a culture obtained by culturing a microorganism SKH-2344 belonging to the genus Streptomyces, which is isolated from soil collected from Jakuchikyo, Yamaguchi Prefecture.
P-06 is produced by inoculating the nutrient source-containing medium with the SKH-2344 strain and aerobically culturing, and the NP-06 producing strain that can be used in the present invention is the above strain. However, it is not limited as long as it belongs to the genus Streptomyces and has the ability to produce NP-06.

The SKH-2344 strain is a microorganism having the following mycological properties. 1. Morphological characteristics The strain was cultured in a liquid medium containing 1% yeast extract and 1% glucose, and the bacterial cells were grown at 110 ° C. using 6N hydrochloric acid.
When hydrolyzed at 18 ° C. for 18 hours and then analyzed by thin layer chromatography, L, L-diaminopimelic acid was detected. In addition, microscopic observation of the product grown on an agar plate medium showed that the aerial mycelium has a spiral shape.
No other remarkable morphology was observed.

2. Growth condition on various media (27 ℃
Culture for 14 days, color tone is based on the standard color table issued by Japan Standards Association) (1) Yeast extract / malt extract agar medium Growth: Good aerial hyphae: a lot of aerial hyphae color tone: 5RP 6/2 Back side color tone: 5R 2/2 soluble pigment : None

(2) Oat mill agar medium Growth: good Aerial mycelium: abundant aerial mycelium color tone: 7.5R 5/2 Backside color tone: 5Y 4/2 Soluble pigment: None (3) Starch / inorganic salt agar medium Growth: good energy Mycelia: Abundant Aerial mycelial color tone: 7.5R 8/2 Back side color tone: 2.5GY 3/2 Soluble pigment: None

(4) Glycerin / asparagine agar medium Growth: Good aerial hyphae: little Airborne hyphae color tone: white Backside color tone: 2.5GY 4/2 Soluble pigment: None (5) Tyrosine agar medium growth: good aerial hyphae: rich aerial Mycelial color tone: 7.5R 7/2 Back side color tone: 2.5GY 3/2 Soluble pigment: None

(6) Sucrose / Nitrate Agar Medium Growth: Good Aerial mycelium: None Aerial mycelial color tone: -Backside color tone: 2.5GY 6/2 Soluble pigment: None (7) Glucose / asparagine agar medium Growth: Good aerial mycelium : Less aerial mycelial color tone: white Back side color tone: 2.5GY 4/2 Soluble pigment: None

(8) Nutrient agar medium Growth: Good aerial hyphae: None Aerobic hyphae color tone: -Backside color tone: 2.5Y 9/4 Soluble pigment: None (9) Bennett agar medium growth: Good aerial hyphae: Low aerial hyphae color tone : White Backside color: 2.5GY 6/2 Soluble pigment: None

3. Growth temperature range (yeast extract / malt extract agar medium, 7 days) 7 ℃ Growth: Not 10 ℃ Growth: Normal 14 ℃ Growth: Normal 17 ℃ Growth: Normal 21 ℃ Growth: Good 25 ℃ Growth: Good 29 ℃ Growth: Good 32 ℃ Growth: Good 37 ℃ Growth: Normal 40 ℃ Growth: No 45 ℃ Growth: No 50 ℃ Growth: No

4. Utilization of sugar D-glucose Growth: Good D-Fructose Growth: Good L-Rhamnose Growth: Good Raffinose Growth: Normal D-Xylose Growth: Normal L-Arabinose Development: Good Sucrose Growth: Good Inositol Development: Good D-mannitol : Good

From the above properties, it is clear that the SKH-2344 strain is a microorganism belonging to the genus Streptomyces. This strain was confirmed by the FERM P-
Deposited as 14239. The method for culturing the above-mentioned microorganism is basically similar to the method for culturing a general microorganism,
Usually, it is preferable to carry out the shaking culture method or aeration stirring culture method using a liquid medium. The medium used for culturing is
Any medium containing a nutrient source that can be used by microorganisms belonging to the genus Streptomyces may be used, and any medium such as various synthetic media, semi-synthetic media and natural media can be used.

The carbon source of the medium composition is glucose,
Sucrose, fructose, glycerin, dextrin, molasses, corn steep liquor, organic acids and the like can be used alone or in combination. As the nitrogen source, organic nitrogen sources such as peptone, meat extract, yeast extract, soybean powder, casein, urea and amino acid, and inorganic nitrogen sources such as ammonium sulfate and sodium nitrate can be used alone or in combination. If necessary, sodium salts, potassium salts, magnesium salts, phosphates, other heavy metals and vitamins can also be used. When foaming is remarkable during the culture, a known antifoaming agent such as adecanol or silicone oil can be appropriately added to the medium.

It is desirable that the pH of the medium is around neutral, at which the microorganisms grow well. The temperature of the medium is preferably 10 to 37 ° C., at which the microorganisms grow well, and particularly preferably 25 to 30 ° C. Further, the culture time is generally about 3 to 6 days. These culture conditions, depending on the conditions such as the type and characteristics of the microorganism used,
It can be changed appropriately. By the above culture, NP-
06 is accumulated in the cells and in the medium.

NP-06 produced by the above culture
Was isolated after the accumulation of NP-06 in the culture (bacteria and medium), preferably when the accumulation was maximized, according to a general method for collecting the fermentation product, for example salting out. Law,
Various methods such as extraction method, various gel chromatography, ion exchange chromatography, adsorption chromatography and the like are used alone or in combination. In addition, NP-06
Can be detected by bioassay method, HPLC method, TLC method and the like.

That is, NP-0 produced by the above culture
Since 6 exists both in the bacterial cells and in the medium, after the culture filtrate and the bacterial cell solids are separated by a means such as filtration and centrifugation, a solvent such as methanol or acetone is used for the bacterial cell solids. Then, NP-06 is extracted, and if necessary, the organic solvent is further distilled off under reduced pressure with an evaporator or the like to obtain an extract concentrate.

From the extract and the culture filtrate, Amberlite IRA-402, Amberlite IRA-904,
Amberlite IRA-958, Amberlite IRA
By adsorbing and eluting with -68, Amberlite IRA-93 or Amberlite IRA-35 (trade name of anion exchange resin manufactured by Organo Corporation), the target substance can be efficiently recovered. Further, the target substance may be extracted and recovered with n-butanol, ethyl acetate or the like.

Then, the extract containing the target substance was concentrated, and then the product was diluted with Diaion HP-20 column and Sephadex L.
It is purified by chromatography using an H-20 column, silica gel column, reverse phase column and the like. An extremely excellent anti-HIV containing NP-06 as an active ingredient is obtained by adding crystalline cellulose or the like to the thus-obtained NP-06, and forming a tablet as required by a known method, for example.
The agent can be obtained.

[0034]

The peptide compound of the present invention, NP-0.
Since 6 has an excellent anti-HIV effect and low toxicity, it can be taken in a large amount in a long period of time and is extremely promising as a new AIDS therapeutic drug.

[0035]

EXAMPLES The present invention will be described in more detail with reference to the following Examples, but it should not be construed that the invention is limited thereto.
Incidentally,% means% by weight unless otherwise specified.

Example 1 Using a 30 liter jar fermenter, actinomycete SKH-2344 strain was treated with 20 liter of medium (glucose 20 g, starch 10 g, malt extract 1 g, dry yeast 4 g, soybean powder 25 g, salt 2).
g, dipotassium hydrogen phosphate 54 mg, water 1 liter, p
After culturing for 5 days at 25 ° C. by H7.3), the cells were separated into the culture supernatant by centrifugation.

The cells were stirred overnight in 10 liters of methanol to lyse them, and the residue was removed by filtration. After the methanol solution was concentrated to dryness under reduced pressure, the obtained solid content was dissolved in water-saturated butanol and washed with an equal amount of water,
The active ingredient was recovered in the butanol layer. On the other hand, the dried solid matter obtained by concentrating the culture supernatant under reduced pressure was immersed in 1 liter of methanol overnight, and then the insoluble matter was removed by filtration. Then, the methanol solution was concentrated to dryness under reduced pressure, the obtained solid content was dissolved in water-saturated butanol, and washed with an equal amount of water to recover the active ingredient in the butanol layer.

Two butanol solutions containing the active ingredient were combined, the butanol was distilled off, and the obtained active ingredient was dissolved in a small amount of methanol and subjected to silica gel column chromatography (column diameter 4 cm × length 25 cm). .
As a developing solvent, ethyl acetate / methanol / water (10:
The mixed solvent of 5: 0.25) was used. About 2 column volume
The active ingredient was eluted when a double volume of developing solvent was passed.
The active fractions were collected, concentrated under reduced pressure, and then subjected to high performance liquid chromatography (column; YMC ODS-AQ column diameter 2 cm ×).
30 cm in length). As a transfer solvent, 0.5%
A 38% aqueous solution of acetonitrile containing trifluoroacetic acid was used. The active fractions were collected and the solvent was distilled off to obtain a white powder. NP-0 obtained from 20 liters of culture
6 was about 100 mg.

Next, the obtained white powder NP-06 was subjected to the following analysis and various activity tests. 1. Amino acid analysis of NP-06 The constituent amino acid analysis of NP-06 was performed by the hydrochloric acid hydrolysis method as follows. The constituent amino acids of NP-06 were analyzed by the hydrochloric acid hydrolysis method. In a test tube for amino acid decomposition containing 0.2 mg of a dried powder of NP-06 under reduced pressure, 6
200 μl of N hydrochloric acid was added and the tube was sealed under reduced pressure. After hydrolysis at 110 ° C. for 24 hours and 72 hours respectively,
The sample was vacuum dried over NaOH.

After dissolving the obtained solid content in 50 μl of water, amino acid analysis was performed using an amino acid analyzer.
The amino acid analysis results (actually measured values) are shown in Table 1.

[Table 1] Since tryptophan decomposes during the acid hydrolysis reaction, it is not detected by the above method, and asparagine is decomposed into aspartic acid.

2. C-terminal amino acid analysis of NP-06 The C-terminal amino acid analysis of NP-06 was performed by the hydrazinolysis method. First, 100 mg of anhydrous hydrazine was added to a test tube for decomposing amino acid containing 2 mg of vacuum dried powder of NP-06.
μl was added and the tube was sealed under reduced pressure. After carrying out a decomposition reaction at 100 ° C. for 18 hours, the solution was passed through a 2 ml DEAE resin column (trade name of a column manufactured by Tosoh Corporation) to remove a by-product hydrazide and purify the C-terminal amino acid. .

The obtained C-terminal amino acid is TLC [TL
C plate: silica gel 60F ₂₄₅ manufactured by Merk, developing solvent:
(1) Chloroform / methanol / 28% ammonia water (2: 2: 1) (2) n-butanol / acetic acid / water (3:
1: 1), detection: ninhydrin reagent], and as a result, it was found to be tryptophan.

3. NMR structure analysis of NP-06 Equipment used: JNM EX-400 (trade name of JEOL) Measurement conditions: 1) Measurement solvent: DMSO-d6 (internal standard T
MS), sample concentration: 100 mM. Measurement temperature: 25 ° C. 2) Measurement _{solvent: CD 3 OH-H 2 O} (50% -50%)
(Internal standard TMS), sample concentration: 10 mM. Measurement temperature:
40 ° C. Measurement data: HH-COSY spectrum (DQF-CO
SY spectrum) HOHAHA spectrum NOESY spectrum

(1) HH-COSY, HOHAHA spectrum analysis results HH-COSY, HOHAHA spectrum analysis revealed that among 21 amino acids, leucine (Leu), isoleucine (ILe), valine (Val) × 2, alanine ( 11 amino acids of Ala) × 2, glycine (Gly) × 4, and serine (Ser) were confirmed. The remaining 10
Amino acid residues represent the same AMX spin series, H
No distinction was possible with the H-COZY and HOHAHA spectra.

(2) Results of NOESY spectrum analysis The 10 amino residues which could not be identified by the HOHAHA spectrum were identified. Phenylalanine (P
he) × 2, tyrosine (Tyr), tryptophan (T
Each amino acid residue of rp) was identified by the NOE correlation peak between the phenyl group and β-proton. Regarding the remaining 6 amino acid residues, cysteine (Cys) x 4 and asparagine (As
n), aspartic acid (Asp) and the chemical shift value of Hβ.

Table 2 shows the chemical shift values (ppm) of the identified amino acids.

[Table 2]

(3) Amino acid sequence of NP-06 The sequence of 21 amino acid residues identified by NMR was
It was determined by reading the NOE intensity between Hα _{i and} HN _{i + 1} . For the C-terminal amino acid, only the correlation peak between HN of Trp (21) and Hα of Phe (20) was observed, so Trp (21) was determined to be the C-terminal amino acid. This result is consistent with the result of C-terminal amino acid analysis by the hydrazine decomposition method. Figure 1 shows the NOE strength analysis results.
The amino acid sequence of NP-06 is shown in FIG.

(4) Primary structural analysis of NP-06 The important point in the primary structural analysis of NP-06 is:
It is the combination of the four Cys forming the SS bond, and the state of the N-terminal amino group of Cys (1). So, these two points are detailed in the Longe Range NO
The E correlation peak was examined. Cys (1) HN and Asp
From the correlation peak of (9) with Hβ, it was confirmed that the N-terminal of Cys (1) and the β-position carboxyl group of Asp (9) were amide-bonded. Cys (1) Hβ and Gly
From the correlation peak of (14) with Hα, it was confirmed that Cys (1) and Cys (13) were SS-bonded. C
From the correlation peak of Hβ of ys (7) and Hβ of Cys (19), it was confirmed that Cys (7) and Cys (19) were SS-bonded. The analysis result is shown in FIG.

Based on the above results, the primary structure of NP-06 was determined to be Chemical formula 3.

Embedded image This structure is based on the recently reported RP-719, as described above.
Very similar to 55, but with Val (4), ILe
The position of (17) is different.

4. Anti-HIV activity of NP-06 The anti-HIV activity of NP-06 was measured by the following test method using MT-4 cells. This method determines the infection death of CD4 positive cells caused by HIV infection and its suppression by drugs, and is an excellent method that can simultaneously and simply measure anti-HIV activity and cytotoxicity. Is.

(Test Method) 1 × 10 ⁴ HIV-1
(HTLV-3B) -infected MT-4 cells and uninfected MT-4 cells with various concentrations of NP-06,
Added to each well of a 6-well microplate. CO _{2 at} 37 ° C
After culturing for 5 days in an incubator, 3- (4,5-dimet
hyl-2-thiazolyl) -2,5-diphenyl-2H tetrazolium bromi
de (MTT) was added, and the culture was continued for another 2 hours. During this period, MTT taken up by living cells was reduced by an enzyme contained in mitochondria in the cells to produce a blue-violet water-insoluble pigment (formazan). A 2-propyl alcohol acidic hydrochloric acid solution containing 5% Triton X-100 was added to solubilize the produced dye, and then the specific absorbance at 595 nm and the nonspecific absorbance at 655 nm were measured using a microplate reader (BIO-RAD model). 3550)
The difference between the two absorbances was determined by measuring with.

Computer processing (BIO-RAD Microplate Manager for Macintosh) using the obtained numerical values
) Is carried out and 50% cell death inhibitory concentration (EC ₅₀ ) and 50%
The cytotoxic concentration (CC ₅₀ ) was calculated and the therapeutic index (CC ₅₀ / E
_C50 ) was calculated. Table 3 shows the results of anti-HIV assay for NP-06 and dextran sulfate sodium (molecular weight 5000) (comparative drug), which has already been reported to show an anti-HIV effect.

[Table 3]

5. NP-06 giant cell formation inhibitory effect It is known that when HIV infects T cells, the infected cells fuse with each other to form giant cells. So NP
The inhibitory effect of −06 on giant cell formation was examined. (Test method) 5 × 10 ⁴ HIV-1 non-infected MOLT
Cells and HIV-1 persistently infected MOLT-4 cells with various concentrations of NP-06 were added to each well of a 96-well microplate and cultured for 24 hours in a CO ₂ incubator at 37 ° C. Observe or take a picture to observe the degree of multinucleated giant cell shape inhibitory action, anti-HIV
-1 activity (HIV-1 adsorption inhibitory activity) was evaluated.

The results are shown in Table 4, where N
When the P-06 concentration was 10 μg / ml or more, giant cell inhibitory effect was confirmed.

[Table 4]

Example 2. As in Example 1, using a 30-liter jar fermenter, 20-liter medium (glucose 20 g, starch 10 g, malt extract 1) was used.
g, dry yeast 4 g, soybean flour 25 g, salt 2 g, dipotassium hydrogen phosphate 54 mg, water 1 liter, pH 7.3), and then culturing the actinomycete SKH-2344 strain for 5 days,
The cells and the culture supernatant were separated by centrifugation. 1 fungus
After stirring overnight in 0 liter of methanol, solids were removed by filtration. Then, the solution was concentrated under reduced pressure to distill off methanol to obtain an aqueous solution.

After adjusting the pH to 7.0 together with the culture supernatant, the anion exchange resin (Amberlite IRA-9) was used.
The solution was passed through 3 (trade name of Organo Co., Ltd.) hydrochloride type, column diameter 8 cm × 50 cm) to adsorb the active ingredient.
0.5M monopotassium phosphate solution (pH 4.5), water, 50%
After passing 3 times the amount of methanol through each column in order, the active ingredient was eluted with a 50% methanol solution containing 0.1 M acetic acid. The active fraction was dried to dryness under reduced pressure to give a crude product 32
0 mg was obtained (purity about 80%). The above results demonstrate the effectiveness of the present invention.

[Brief description of drawings]

FIG. 1 is a diagram showing the results of NOE intensity analysis of amino acids in NP-06.

FIG. 2 is a diagram showing an amino acid sequence of NP-06.

FIG. 3 is a diagram showing a result of Long range NOE correlation analysis in NP-06.

FIG. 4 is an actual measurement diagram showing a ¹ H-NMR spectrum of NP-06.

FIG. 5 is an actual measurement diagram showing a ¹³ C-NMR spectrum of NP-06.

FIG. 6 is an actual measurement diagram showing an HH-COSY spectrum of NP-06.

FIG. 7 is an actual measurement diagram showing a HOHAHA spectrum of NP-06.

FIG. 8 is an actual measurement diagram showing a NOESY spectrum of NP-06.

FIG. 9 is an actual measurement diagram showing an IR spectrum of NP-06.

FIG. 10 is an actual measurement diagram showing a MASS (FAB ⁺ ) spectrum of NP-06.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification number Internal reference number FI technical display location C12R 1: 465) (72) Inventor Kumiko Ota 2-8-1, Iida-cho, Iwakuni-shi, Yamaguchi Japan Papermaking Co., Ltd. Iwakuni Research Institute (72) Inventor Kazuhiro Watanabe 2-8-1, Iida-cho, Iwakuni-shi, Yamaguchi Japan Iwakuni Research Institute (72) Inventor Makoto Machida 2-chome, Iida-cho, Iwakuni-shi, Yamaguchi Prefecture No. 1 Inside the Iwakuni Technical Research Institute of Japan Paper Manufacturing Co., Ltd. (72) Kunimitsu Murakami 2-8-1 Iida-cho, Iwakuni City, Yamaguchi Prefecture Inside the Iwakuni Technical Research Center of Japan Paper Manufacturing Co. 5-21-1 Oji Inside Central Research Institute, Nippon Paper Industries Co., Ltd.

Claims (5)

[Claims] 1. A peptide compound represented by the following chemical formula 1. Embedded image 2. A method for producing a peptide compound, which comprises culturing a peptide compound-producing bacterium belonging to the genus Streptomyces, and collecting the peptide compound according to claim 1 from the culture. 3. The method for producing a peptide compound according to claim 2, wherein the peptide compound-producing bacterium belonging to the genus Streptomyces is actinomycete SKH-2344 strain. 4. The peptide compound according to claim 2 or claim 3, wherein the peptide compound according to claim 1 is collected and purified from the culture of the peptide compound-producing bacterium by using an anion exchange resin. Manufacturing method. 5. An anti-human immunodeficiency virus agent, which comprises the peptide compound according to claim 1 as an active ingredient.