JP2798711B2 - Polypeptide antiviral agent - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Object of the Invention] (Field of Industrial Application) The present invention relates to an antiviral agent containing a polypeptide as an active ingredient.

(Problems to be Solved by the Prior Art and the Invention) The present inventors have conducted intensive studies to find a novel substance having an affinity for lipopolysaccharide (hereinafter referred to as “LPS”). The polypeptide was extracted and isolated, and the polypeptide was successfully synthesized by the solid phase synthesis method.Furthermore, the polypeptide was found to exhibit strong affinity for LPS, and has been published internationally as WO89 / 01492. .

The present inventors have further conducted intensive studies on the pharmacological activity of the present polypeptide, and as a result, have found that the present polypeptide has an antiviral effect, thereby completing the present invention.

[Configuration of the Invention] (Means for Solving the Problems) The present invention provides the following formula (I): (Where Lys is lysine, Trp is tryptophan, Cys
Represents cysteine, Phe represents phenylalanine, Arg represents arginine, Val represents valine, Tyr represents tyrosine, Gly represents glycine, Ile represents isoleucine; X represents a hydroxyl group or an amino group. ) Or an analog thereof as an active ingredient.

The compound used in the present invention is a polypeptide consisting of 17 amino acids, and the carboxyl group of arginine, which is the C-terminal amino acid in the extracted and isolated state, is amidated, but may be converted to an acid by hydrolysis. No particular effect is exerted on the antiviral action.

The polypeptide used in the present invention is, for example, a horseshoe crab blood cell (Tachypleus tridentatus and Ta
chypleus gigas).

That is, horseshoe crab blood cells are hypotonically extracted, and the insoluble fraction obtained as a residue is subjected to acidic conditions, for example, diluted hydrochloric acid, diluted nitric acid,
By extracting under a strongly acidic condition due to the presence of a mineral dilute acid such as dilute sulfuric acid; a lower fatty acid such as acetic acid, the resulting extract is purified by a conventional method, for example, gel filtration, chromatography or other purification means. Can be isolated.

The polypeptide used in the present invention can also be produced by a solid phase synthesis method.

That is, after the N-protected arginine is bound to an amino-containing insoluble resin via a spacer having a functional group capable of binding to a carboxyl group and a carboxyl group in some cases, the following formula: (The symbols in the formula are as defined above.) The protected amino acids from position 16 to position 1 of the polypeptide shown in the formula are sequentially bonded according to a solid phase synthesis method, and then the insoluble resin and the protecting group of the amino acid are removed. And the following equation (II): (The symbols in the formula are as defined above.), The positions 3 and 16, and 7
The cysteines at the 12th and 12th positions can be bonded via the respective mercapto groups to form a disulfide bond.

The above-mentioned insoluble resin having an amino group includes a carboxyl group of N-protected arginine or a carboxyl group of a spacer which is optionally bonded to the carboxyl group via the amino group, and can be subsequently desorbed. Any object can be used.

Examples of such insoluble resins include aminomethyl resins (aminomethylated (styrene-divinylbenzene copolymer, benzhydrylamine resin, methylbenzhydrylamine resin, 4- (aminomethyl) phenoxymethyl resin, and the like). Benzhydrylamine resin, methylbenzhydrylamine resin, 4- (aminomethyl)
When a phenoxymethyl resin is used, an amide is directly obtained by cleavage, but an aminomethyl resin is preferable in terms of yield.

Examples of the spacer having a carboxyl group and a functional group capable of binding to the carboxyl group present in the above-mentioned case include, but are not particularly limited to, those capable of converting the carboxyl group of arginine into p-carboxymethylbenzyl ester.

4- in which such a spacer is bound to a protected arginine
(T-Butoxycarbonyl- G -tosyl-L-arginyloxymethyl) phenylacetic acid was obtained from JPTam et al. (Synthesis
(1979) p.955-957).

The protected amino acid is an amino acid in which a functional group is protected with a protecting group by a known method, and various protected amino acids are commercially available. When synthesizing the polypeptide of the present invention, it is preferable to select one of the following protecting groups. First, the protecting group for the α-amino acid of the amino acid is Boc (t
-Butyloxycarbonyl) or Fmoc (9-fluorenylmethyloxycarbonyl). The protecting groups for the guanidino group of Arg include Tos (tosyl), NO ₂ (nitro), and Mtr
(4-methoxy-2,3,6-trimethylbenzenesulfonyl). Bzl is a protective group for the mercapto group of Cys.
(Benzyl), M.Bzl (4-methoxybenzyl), 4
-MeBzl (4-methylbenzyl), Acm (acetamidomethyl), Trt (trityl), Npys (3-nitropyridinesulfenyl), t-Bu (t-butyl), t-BuS (t
-Butylmercapto), but 4-MeBzl, Ac
m and Npys are preferred. The hydroxyl protecting group for Try is Bzl, Cl ₂
Bzl (2,6-dichlorobenzyl), t-Bu, or need not be protected. The protecting group for the ε-amino group of Lys is Z (benzyloxycarbonyl), ClZ (2-
Chlorobenzyloxycarbonyl), Boc and Npys. It is necessary to select an appropriate protecting group according to the peptide synthesis conditions.

Coupling of protected amino acids is accomplished by conventional condensation methods, such as DCC
(Dicyclohexylcarbodiimide) method, active ester method, mixed or symmetric acid anhydride method, carbonyldiimidazole method, DCC-HOBt (1-hydroxybenzotriazole) method, diphenylphosphoryl azide method, etc. The DCC-HOBt method and the symmetric acid anhydride method are preferred. These condensation reactions are usually performed in an organic solvent such as dichloromethane or dimethylformamide or a mixed solvent thereof. As a reagent for removing an α-amino group-protecting group, trifluoroacetic acid / dichloromethane,
HCl / dioxane, piperidine / dimethylformamide and the like are used, and are appropriately selected depending on the kind of the protecting group. The degree of the signal of the condensation reaction at each stage of the synthesis is examined by the method of E. Kaiser et al. [Anal. Biochem., 34, 595 (1970)] (ninhydrin reaction method).

As described above, a protected peptide resin having a desired amino acid sequence can be obtained.

When an aminomethyl resin is used as the insoluble resin, for example, the resin can be eliminated by treating it with ammonia in a suitable solvent. Then, by treating with hydrogen fluoride, a polypeptide represented by the above formula (II) from which all protecting groups have been eliminated can be obtained.
When a benzhydrylamine resin, a methylbenzhydrylamine resin, or a 4- (aminomethyl) phenoxymethyl resin is used as the insoluble resin, the resin and the protecting group are simultaneously eliminated by treating with hydrogen fluoride. Can be.

Then, preferably, the mercapto group of cysteine is reduced by 2-mercaptoethanol or the like to make sure that the mercapto group is in a reduced form. Can be obtained as

For the oxidation treatment at this time, a known method can be used, and usually, oxygen or ferricyanate (for example,
An oxidizing agent such as potassium ferricyanide) is used.

The polypeptide thus obtained is isolated by conventional means of the polypeptide, for example, extraction, recrystallization, various types of chromatography (gel filtration, ion exchange, distribution, adsorption, reverse phase), electrophoresis, countercurrent distribution and the like. Although it can be purified, the method by reversed phase high performance liquid chromatography is most effective.

The thus obtained polypeptide of the formula (I) is a vesicular stomatitis virus (Vesicular Stomat).
It is effective against various viruses such as itis virus, influenza virus, human immunodeficiency virus (HIV), and has an inactivating effect, and can be widely applied as an antiviral agent.

(Examples of the Invention) Hereinafter, the present invention will be described in more detail with reference to Preparation Examples and Examples, but these do not limit the scope of the present invention in any way.

Preparation Example 1 A. Extraction and Purification of Polypeptide 2
0mM Tris-HCl / 50mM sodium chloride / pH8.0 buffer 150ml
And a high-speed homogenizer (Hiscotron) ; Nippon Seimitsu
After homogenizing for 3 minutes with an industrial
(00 rpm, 30 minutes, 4 ° C) to separate into a supernatant and a precipitate. Sinking
The above operation was repeated twice for the sediment fraction,
After sufficient extraction of the soluble components, the insoluble fraction (precipitate) is removed.
Obtained.

150 ml of 20 mM hydrochloric acid was added to the insoluble fraction, homogenized with a high-speed homogenizer for 3 minutes, and centrifuged to obtain a supernatant of a hydrochloric acid extract. This operation was repeated three times in total to obtain a total of about 400 ml of the extract. The supernatant fraction was concentrated to dryness by freeze-drying.

The concentrated hydrochloric acid extract was redissolved in 20 mM hydrochloric acid,
The mixture was added to a Sephadex G-50 (3.0 × 90.0 cm) column (pre-equilibrated with 20 mM hydrochloric acid), and gel filtration was performed. LPS
Factor C (a horseshoe crab blood coagulation serine protease precursor; LPS susceptibility factor named by the present inventors, Nakamura, et al., Eur.
Eluted fractions that inhibit the activation of J. Biochem., 154 , 511 (1986)) were collected and the pH of the pooled fractions was adjusted to 6.0 with aqueous sodium hydroxide.

The sample was applied to a CM-Sepharose CL-6B column which had been equilibrated with 20 mM acetate buffer (pH 6.0), and elution was performed at 0 to 0.
The measurement was performed with a gradient of 20 mM acetate buffer (pH 6.0) containing 3 M sodium chloride. Collect the fractions inhibiting factor C activation, LP
This was used as a final purified sample of the S-binding substance (the polypeptide used in the present invention). Yields ranged from about 50 g to about 30 mg of blood cells.

B. Purity Assay (1) SDS Polyacrylamide Gel Electrophoresis A 12% polyacrylamide gel electrophoresis containing 8M urea (Weber-) was used for the LPS-binding polypeptide in the absence or presence of a reducing agent (β-mercaptoethanol). Osborn), Coomassie Brilliant Blue (Coomassie Bril)
liant Blue) When stained with R-250, both had a molecular weight of about 2
000 single bands. The results are shown in FIG. First
In the figure, the left band is the band in the absence of the reducing agent, the middle band is the band in the presence of the reducing agent, and the right band is the standard protein [SDS-PAGE Ma].
rker III, Fluka AG (Switzerland)]
6.9 kDa), myoglobin I + II (14.4 kDa), myoglobin I (8.2 kDa), myoglobin II (6.2 kDa), and myoglobin III (2.5 kDa).

(2) using reverse phase high performance liquid chromatography the reversed phase high-performance liquid chromatography (column polypeptides for use in the invention Cosmosil 5C ₁₈ P, elution of the peptide 0-98% gradient system of 0.1% trifluoroacetic acid / acetonitrile ) Showed a single peak.

C. Amino acid composition value The sample was hydrolyzed with 5.7 M hydrochloric acid at 110 ° C for 24, 48, and 72 hours, and then analyzed with a Hitachi 835 automatic amino acid analyzer. For semicystine, 110 ° C after formic acid oxidation of sample
Hydrolyzed with 5.7M hydrochloric acid for 24 hours, and for tryptophan, samples were treated with 3M mercaptoethanesulfonic acid.
After hydrolysis at 110 ° C. for 24 hours, each was analyzed with an amino acid analyzer. The molecular weight obtained by SDS polyacrylamide gel electrophoresis revealed that this peptide was a simple basic polypeptide composed of 17 amino acids. Table 1 shows the results of amino acid analysis.

D. Determination of amino acid sequence and identification of C-terminal argininamide The amino acid sequence was determined using about 23 μg of intact standard,
From the amino terminus, up to 15 residues (excluding half cystine) could be identified using a Beckman 890D sequencer. In addition, a reductively-alkylated sample (the polypeptide used in the present invention was obtained from MA Hermodson, et al., Biochemistr.
Using about 36 μg of S-pyridylethylated by the method of y, 12 , 3146 (1973), up to 16 residues (including half cystine) could be identified. The remaining 17th amino acid residue (C-terminal residue) was estimated to be arginine from the amino acid analysis. However, C-terminal arginine was not detected at all even when intact samples or pyridylethylated samples were digested with carboxypeptidase (hereinafter referred to as “CPase”) Y and B. Therefore, once sample 30
After hydrolysis under mild conditions at 110 ° C for 10 hours with mM hydrochloric acid,
It was treated with CPaseB again. As a result, about 0.5 mol of arginine was released per mol of the polypeptide of the present invention,
It was determined that the carboxyl group of the C-terminal arginine had a strong possibility of being amidated. When the theoretical molecular weight (calculated value MW = 2264) of the amide compound completely coincided with the actually measured value by mass spectrometry, it was confirmed that the carboxyl group of the C-terminal arginine was amidated.

E. Identification of Disulfide (S—S) Bonds Four semicystines were identified in the polypeptides used in the present invention, but all of them are disulfide bonded, indicating the presence of a reducing agent (dithiothreitol). Comparative experiments on S-pyridylethylation under or in the absence of clarified. Therefore, in order to identify the position of the disulfide bond, the intact sample was digested with trypsin under conditions that do not cause disulfide bond exchange reaction (acidic conditions, pH 6.5), and the digest was subjected to the reverse phase high-performance liquid described above. Separation was carried out by chromatography (Cosmosil5C ₁₈ P column, elution of peptide using 0.1% trifluoroacetic acid / acetonitrile system). When the amino acid composition of the obtained peptide was examined, it was found that the third and the 16th, and the 7th and the 12th from the amino terminal were disulfide bonded.

F. LPS Binding Activity of Polypeptide The polypeptide used in the present invention is 0.1 μg / ml LPS (E.
E. coli 0111: Activation of factor C (expressed as "factor") by 0.05 μM (0.12 μg / ml).
%, Completely inhibited by 1 μM (2.3 μg / ml). Further, in the double diffusion test using 1% agarose gel, it was observed that the polypeptide used in the present invention formed a polymer complex with LPS and formed a sedimentation line.

Test results on the inhibitory effect of the polypeptide used in the present invention on the activation of factor C by LPS are shown in FIGS. 2 and 3. 2 and 3 show the results in the absence and presence (1M) of chloride and sodium, respectively. As a control, polylysine, a high-molecular-weight basic substance having a property of electrically binding to LPS, first revealed by the present inventors, was used. In FIGS. 2 and 3,
(○) and (●) indicate the results of the polypeptide and polylysine used in the present invention, respectively.

From these results, the polypeptide used in the present invention is:
It turns out that it has a strong affinity which is not affected by salt concentration, rather than a mere electrical coupling with LPS.

G. Measurement of Absorbance FIG. 4 shows the ultraviolet absorption spectrum of the aqueous solution of 99.2 μg / ml of the polypeptide used in the present invention. It has an absorption peak at 276 nm. Since the absorbance at 280 nm is 0.3842, 1
The absorbance at 280 nm of the% aqueous solution is calculated to be 38.7.

Preparation Example 2 A. Introduction of Arginine into Aminomethyl Resin (1) Synthesis of 4- (bromomethyl) phenylacetic acid phenacyl ester 3.98 g (20 mmol) of α-bromoacetophenone and 3.49 g of potassium fluoride in 75 ml of acetonitrile at room temperature (60mm
ol) were suspended, and while stirring, 4.58 g (20 mmol) of 4- (bromomethyl) phenylacetic acid was divided into 6 equal portions and added at 30 minute intervals,
Stirring was continued for 2 hours. After the completion of the reaction, insolubles are filtered off,
The solvent was distilled off from the filtrate, and the residue was redissolved in ethyl acetate.
The extract was washed twice with a saturated aqueous solution of sodium hydrogencarbonate and once with distilled water, citric acid and distilled water, and dried over sodium sulfate. Ethyl acetate was distilled off and crystallized from petroleum ether,
5.5 g of the desired product (mp 84-85 ° C.) were obtained. This was recrystallized to obtain 5.2 g of crystals having a melting point of 85 to 86 ° C (yield: 75%).

(2) 4- (t-butoxycarbonyl- G -tosyl-L
Synthesis of -arginyloxymethyl) phenylacetic acid 4.71 g (11 mmol) of t-butoxycarbonyl- G -tosyl-L-arginine, 3.47 g (10 mmol) of 4- (bromomethyl) phenylacetic acid phenacyl ester, 1.28 g of potassium fluoride ( 22 mmol), 0.8 ml (44 mmol) of water, 50 ml of acetonitrile and 10 ml of dimethylformamide at room temperature
Stir vigorously for 24 hours. Natural filtration of the insolubles generated,
The filtrate was concentrated by evaporation to 15-20 ml. After addition of 80 ml of ethyl acetate, twice with a saturated aqueous sodium hydrogen carbonate solution, once with distilled water, twice with a saturated aqueous citric acid solution,
Washed once with distilled water and dried over sodium sulfate. The solvent was distilled off, and the residue was treated with petroleum ether to obtain semi-solid 4- (t-butoxycarbonyl- G -tosyl-L-arginyloxymethyl) phenylacetic acid phenacyl ester.

This was dissolved in 105 ml of acetic acid, 19 ml of water and 13.1 g of zinc were added, and the mixture was vigorously stirred at room temperature for 5.5 hours. The zinc was filtered off using Hyflo Super Cell and ethyl acetate, 400 ml of ethyl acetate and 300 ml of water were added to the filtrate, the ethyl acetate layer was separated, washed with water 10 times, dried over sodium sulfate, and the solvent was distilled off. The residue was triturated in petroleum ether to give 4.77 g of 4- (t-butoxycarbonyl- G -tosyl-L-arginyloxymethyl) phenylacetic acid. This substance was obtained as one spot of almost pure product by thin-layer chromatography.

(3) 4- (t-butoxycarbonyl- G -tosyl-L
Synthesis of (arginyloxymethyl) phenylacetamidomethyl resin 4- (t-butoxycarbonyl- G -tosyl-L-arginyloxymethyl) phenylacetic acid 577 mg (1.0 mmol)
l), 2.00 g of aminomethyl resin (sold by Peptide Research Laboratories, 1% cross-linked) and 206 mg (1.0 mmol) of DCC are coupled in dichloromethane by a conventional method.
A coupling of 0.284 mmol was confirmed.

B. Introduction of Cysteine at position 16 1.0 g (0.284 mmol Arg (Tos) / g resin) of 4- (t-butoxycarbonyl- G -tosyl-L-arginyloxymethyl) phenylacetamidomethyl resin was added four times with 25 ml of dichloromethane. Washed for 1 minute each and filtered. 30 to this resin
25% trifluoroacetic acid solution (solvent: dichloromethane)
Was added and stirred for 30 minutes to remove the Boc group. The obtained resin was sequentially treated with each of the following solvents (25 ml), and filtered after each treatment.

Dichloromethane (1 time, 1 minute) dioxane (1 time, 1 minute) dichloromethane (1 time, 1 minute) dioxane (1 time, 1 minute) dichloromethane (2 times, 2 minutes each) 10% triethylamine (dichloromethane solution) (1
Times, 2 minutes), (1 time, 5 minutes) dichloromethane (4 times, 1 minute each) Then, the resin was added to 25 ml of dichloromethane and 3.5 equivalents of the protected amino acid, ie, Boc-
The mixture was stirred with 310 mg (0.994 mmol) of Cys (4-MeBzl) for 1 minute. DCC 205mg (0.994mmol) dichloromethane solution 25m
l was added and stirred for 2 hours. The obtained resin was sequentially treated with each of the following solvents (25 ml), and filtered after each treatment.

Dichloromethane (1 time, 1 minute) Isopropanol (1 time, 1 minute) Dichloromethane (1 time, 1 minute) Isopropanol (1 time, 1 minute) Dichloromethane (3 times, 1 minute each) C. In the same manner as in the introduction B, the resin obtained above was added to the above-mentioned formula (II)
The protected amino acids corresponding to each of the constituent amino acids from position 15 to position 1 of the polypeptide shown by were coupled in order. Table 2 shows the protected amino acids used in each reaction step. All protected amino acids used were 3.5% of total arginine
Is equivalent.

The coupling of Boc-Arg (Tos) was carried out by the DCC-HOBt method using HOBt in a molar amount of 2 times the amount of DCC.

After the introduction of the 1st amino acid, the resin peptide is collected on a glass filter using dichloromethane, and dried under reduced pressure.
1.781 g of dry resin peptide was obtained.

D. Elimination of resin The resin is eliminated by treating the dried resin peptide obtained in C with ammonia (anhydrous) in methanol and dimethylformamide, the following formula: 0.765 g (0.196 mmol) of the protected polypeptide represented by was obtained.

Molecular weight: 3904 e. Elimination of the protecting group The protected polypeptide obtained in D is treated with hydrogen fluoride in anisole in the presence of ethanedithiol and then treated with hydrochloric acid to give the following formula: 470 mg (0.186 mmol) of the polypeptide hydrochloride represented by was obtained.

Molecular weight: 2523 F. Cyclization of polypeptide 30 mg of the polypeptide hydrochloride obtained in E was added to 0.1 M Tris-HCl containing 200-fold molar excess of 2-mercaptoethanol.
(PH 8.5) overnight at room temperature. Then 1
The mixture was treated with a Sephadex G-10 column equilibrated with acetic acid to obtain a polypeptide-containing fraction. Dilute 10-fold with water (0.1 mg / ml), adjust the pH to 8.5 with 0.5 M NaOH, and add
After standing for a time, it was freeze-dried. Then, it was treated with a Sephadex G-10 column equilibrated with 1% acetic acid to obtain 8.5 mg of an oxidized polypeptide.

This was subjected to reversed-phase high-performance liquid chromatography (the column was TS
K-gel ODS-120T (0.46 × 25cm), elution of peptide
0.01M formic acid-triethylamine (pH 4.5) (A) -A20%
Using acetonitrile (B) containing)
The peak coincides with the peak of the natural polypeptide obtained in Preparation Example 1, and the peak of the mixture of the two is doubled.
Both were found to be identical.

Example 1 Antiviral activity against vesicular stomatitis virus and influenza virus 1. Materials and methods (1) Virus and cells used Vesicular Stomatitis Viru
s) (New Jersey strain) (hereinafter referred to as “VSV”) was grown on Vero cells derived from monkey kidney. Influenza virus (A / Yamagata / 120/86, H1N1) is derived from canine kidney Madin
After growing in Darby caninekidney cells, the cells were washed twice by ultracentrifugation (24,000 rpm, 90 minutes) to remove trypsin (10 μg / ml) in the culture solution.

(2) Reaction between polypeptide and virus The polypeptide (hereinafter, referred to as “LBP”) (20 μl) obtained in Preparation Example 1 at a constant concentration diluted with phosphate buffered saline (hereinafter, referred to as “PBS”) was used. Virus solution (20μl)
Was mixed and reacted at 37 ° C. in a 5% CO ₂ incubator for 60 minutes or 120 minutes, and then the virus was quantified. PBS was used instead of LBP as a control. In the reaction between LBP and trypsin, the final concentrations were adjusted to 125 μg / ml and 10 μg / ml, respectively.

(3) Quantification of virus Quantification of virus after reacting LBP with virus
Mix the mixture into a small amount of essential medium
(Hereafter referred to as “MEM”) after 10-step dilution, 50% tis
sue culture infective dose (hereinafter “TCID ₅₀ ”) or plaque-forming unit
(Hereinafter referred to as “PFU”).

First, in the TCID ₅₀ method, 0.1 m
Each l was inoculated into 4 wells of a monolayer cell previously cultured in a 96-well microplate, cultured for 2 to 3 days, and then examined under a microscope for the presence of cytopathic effect (CPE).
Calculated by the method of h. In the PFU method, 0.1 ml of a 10-step diluted virus solution was inoculated to cells cultured in a monolayer of a plastic Petri dish having a diameter of 60 mm, and allowed to stand at 37 ° C. for 60 minutes in a CO ₂ incubator to adsorb the virus to the cells. Contains 0.6% agar
After overlaying the cells with 5 ml of MEM medium, the cells were cultured at 37 ° C. in a CO ₂ incubator. The number of plaques was calculated after 1 to 3 days of cultivation depending on the type of virus. The measurement of erythrocyte pseudomagglutinin (Hemagglutinin) (hereinafter referred to as “HA”) for influenza virus quantification was performed by diluting the virus-LBP reaction solution in two steps Then, 50 μl of the mixture and 50 μl of 0.5% chicken erythrocytes were placed in a 96-well U-shaped microplate, mixed, and then mixed at 4 ° C. for 60 minutes. Then, the virus number was determined by aggregation at the highest dilution factor.

2. Results (1) Inactivation of high concentration VSV 15 μl of VSV of about 2 × 10 ⁶ PFU / 0.1 ml and LBP (0 to 250
15 μl was incubated at 37 ° C. for 60 minutes, serially diluted from 10 ⁻² to 10 ⁻⁸ , 0.1 ml of the resulting solution was inoculated into Vero cells, and
FIG. 5 shows the measurement results of D ₅₀ and PFU.

FIG. 5 shows that LBP inactivates VSV in a concentration-dependent manner.

(2) Table 3 shows the results of examining the effects of the antiviral agent of the present invention on VSV and influenza virus.

Table 3 shows that LBP inactivates VSV and influenza virus.

Example 2 Antiviral activity against human immunodeficiency virus (HIV) 1. Materials and methods (1) Virus and cells used LAV strain (Osaka Prefectural Institute of Public Health) was used as an HIV strain subcultured with a T cell strain. As a stock solution, a virus produced in a culture solution by TALL-1 / LAV or MOLT-4 / LAV cells, which were specially infected with the LAV strain, was used.

The established T cell line is adult T cell leukemia (adul
HTLV, a virus that causes T T cell leukemia (ATL)
In addition to MT-4 cells persistently infected with -1, TALL-1 and MOLT-4 cells, which are cell lines derived from T-cell leukemia cells, and TALL-1 / HIV, each of which is an HIV persistently infected cell , MOLT-4
/ HIV and MOLT-4 / HTLV-III were used. Those cells are 1
In the presence of 5% carbon dioxide using RPMI-1640 medium (Flow, UK) containing 0% fetal calf serum (FCS), penicillin (100 μ / ml) and streptomycin (100 μg / ml) 37
Incubated at ℃.

(2) Inhibitory effect on HIV proliferation The inhibitory effect of the present peptide on HIV growth is shown in (a) below.
Based on the principle, execution was determined according to the method (b).

(A) Principle HTLV-1 (AT
LV) is a persistently infected T cell line, MT-4 cells,
It is known that HIV infection causes rapid proliferation of HIV, and MT-4 cells die in 5 to 6 days due to cytotoxicity.
Therefore, the anti-HIV effect of the drug can be determined using the cytotoxicity of MT-4 cells as a marker.

(B) Method: MT-4 cells were infected with HIV (LAV strain) at 0.001 TCID ₅₀ / cell at 37 ° C. for 1 hour, washed, and washed with various concentrations of drugs (this was obtained in Preparation Example 2). RPMI of the polypeptide of the invention
RPMI-16 containing aseptically dissolved -1640 culture solution)
1 × 10 ⁵ cell / ml in 40 medium (containing 10% fetal calf serum)
At a concentration of 1. This cell suspension was placed in a 24-well culture plate at 1 ml / well and cultured at 37 ° C. for 5 days in the presence of 5% CO ₂ . HIV-uninfected MT-4 cells were similarly cultured with the drug. After the culture, the cytotoxic effect (CPE) due to virus growth was observed, and the number of surviving MT-4 cells was counted by trypan blue staining. HI of sample
The V growth inhibitory action was evaluated using the cytotoxicity inhibition rate (%) calculated from the following equation as an index.

(3) Giant cell formation inhibitory effect The giant cell formation inhibitory effect of the present peptide was carried out based on the following (A) principle according to the method (B), and its anti-HIV effect was determined.

(B) Principle When MOLT-4 cells are mixed with MOLT-4 / HIV cells that are persistently infected with HIV, giant cells are formed in one to two days. This phenomenon is due to the CD4 receptor on MOLT-4 cell surface and MOLT-4 / H
It is thought that this occurs when gp120, an HIV envelope protein expressed on the surface of IV cells, binds. In this experiment, we can see the effect of drugs on the binding of HIV and CD4 molecules (the adsorption of HIV to lymphocytes).

(B) Method MOLT-4 cells and MOLT-4 / HTLV persistently infected with HIV
-III cells were cultured in RPMI-1640 medium (10 fetal calf serum containing various concentrations of drugs (the polypeptide of the present invention obtained in Preparation Example 2 was aseptically dissolved in RPMI-1640 culture medium)).
%) In a 1: 1 ratio (cell concentration is 5 × 10 ⁵ ce).
ll / ml), 1 ml per well was placed in a 24-well culture plate and cultured for 24 hours. After culture, the presence or absence of giant cell formation was observed by microscopy.

2. Results (1) HIV proliferation inhibitory effect of the present compound Table 4 shows the cytotoxicity inhibitory rate at each concentration of the present compound.

As can be seen from Table 4, the compound was 7.5 μg / ml to 15 μg / m
It showed a cell injury suppression rate of 80% or more at a concentration of 1 and strongly suppressed HIV proliferation.

(2) Giant cell formation inhibitory effect of the present compound The present compound inhibits giant cell formation by 100% at a concentration of 7.5 μg / ml or more.
It is suggested that it has an effect of suppressing and suppressing the adsorption (binding) of HIV to lymphocytes.

[Effects of the Invention] According to the present invention, a novel polypeptide-based antiviral agent can be provided.

[Brief description of the drawings]

FIG. 1 is a view showing the results of electrophoresis of SDS polyacrylamide gel of LBP. FIG. 2 and FIG. 3 are diagrams showing the test results on the inhibitory effect of LBP on the activation of factor C by LPS. FIG. 4 is an ultraviolet absorption spectrum of LBP. FIG. 5 is a diagram showing inactivation of VSV by LBP.

Continued on the front page (58) Fields surveyed (Int. Cl. ⁶ , DB name) A61K 38/00, 38/10 C07K 7/08 CA (STN) REGISTRY (STN) WPIDS (STN)

Claims (1)

(57) [Claims] 1. The following formula: (Where Lys is lysine, Trp is tryptophan, Cys
Represents cysteine, Phe represents phenylalanine, Arg represents arginine, Val represents valine, Tyr represents tyrosine, Gly represents glycine, Ile represents isoleucine; X represents a hydroxyl group or an amino group. ) An antiviral agent comprising a polypeptide represented by the formula (1) as an active ingredient.