JP3362178B2 - Novel peptide and AIDS virus (HIV-1) protease inhibitor containing the same - Google Patents

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel peptide present in a hydrolyzate of an oyster (oyster) heat-denatured protein, and an AIDS virus (HIV-1) characterized by containing the peptide as an active ingredient. ) Protease inhibitors.

[0002]

2. Description of the Related Art AIDS virus (HIV-1) protease cleaves the precursor protein of HIV-1 to produce the virus's own enzyme and structural protein. This process is essential for the formation of viral particles and HIV-1 protease inhibitors are said to be useful in the treatment of AIDS. In HIV-1 protease, a protein consisting of 99 amino acids forms a homodimer and expresses its proteolytic activity. The enzyme has Asp-Th at the active center.
Since it has characteristics as an aspartic protease such as having an r-Gly sequence, it is said to be inhibited by pepstatin or acetylpepstatin (Ki = 35nM, pH5.0) (FEBS Letters, Vol.2 47, p. .113, 1989). In addition, HIV-1 protease specifically recognizes the substrate cleavage site having the structure of Phe-Pro and Tyr-Pro and cleaves the peptide bond on the imino group side of the proline residue, which is a mammalian enzyme. It is an endo-type peptidase having the following characteristics (Biochem. Biophys. Res. Commun., Vol. 156,
p297, 1988). Focusing on this point, Ro31-, an inhibitor with a structure that mimics the transition state of substrates including Phe-Pro
8959 (Saquinavir, IC ₅₀ <0.4 nM), KNI-272 (K _i = 0.00
55 nM) etc. are synthesized in large numbers (Science, Vol.248,
p358, 1990, History of Medicine, Vol.177, p.962, 1996, Protein Nucleic Acid Enzyme, Vol. 43, p.725, 1998, Protein Nucleic Acid Enzyme,
Vol.43, p744, 1998).

As described above, various synthetic HIV-1 protease inhibitors have been studied for the purpose of treating AIDS, and some of them have been approved as pharmaceuticals.
However, most of the inhibitors that have been developed so far are peptides synthesized by mimicking the transition state of pepstatin and substrates, and are not found in natural products. Moreover, due to the emergence of resistant viruses against them, it cannot be said that the inhibitory activity is sufficiently satisfactory, and the more potent HIV-
1 Protease inhibitors are needed.

[0004]

SUMMARY OF THE INVENTION Therefore, an object of the present invention is to provide a novel HIV-1 protease inhibitor which can be used as a research reagent or medicine for AIDS.

[0005]

[Means for Solving the Problems] The present inventors have developed a novel HI
As a result of examining various natural product materials as a target for searching V-1 protease inhibitors, oysters are rarely contaminated with infectious organisms, and AIDS infected persons with weak immunity eat oysters rawly Has been pointed out (Gastr
oenterology, Vol.92, p.796, 1987, J.Am.Diet.Asso
c., Vol.94, p.312, 1994), and it is possible that oysters produce some antiviral and antibacterial substances as a defense against contamination by such infectious organisms. I thought it might be. Accordingly, the present inventors obtained a denatured protein by heat-treating oysters and further hydrolyzing it with a protease, and found a low-molecular peptide having HIV-1 protease inhibitory activity in the hydrolyzate, We succeeded in isolating and purifying the peptide and determining its amino acid sequence, and completed the present invention.

That is, the present invention provides the peptide of (a) or (b) below: (a) A peptide comprising the amino acid sequence represented by SEQ ID NO: 1. (B) One amino acid in the amino acid sequence of SEQ ID NO: 1 has a deleted, substituted or added amino acid sequence, and has the amino acid sequence of Leu Leu Glu Tyr Ser.
And, a peptide having the AIDS virus (HIV-1) protease inhibitory activity. Deletion, substitution of the above amino acids,
The addition can be performed by a known technique (eg, individual phase synthesis method).

Specifically, such a peptide is a peptide having an amino acid sequence of SEQ ID NO: 1 in which isoleucine at the C terminus is replaced with leucine.
Another example is a peptide consisting of the amino acid sequence of SEQ ID NO: 1 with the C-terminal isoleucine deleted. The present invention also comprises an AIDS virus (HIV-
1) It is a protease inhibitor. Hereinafter, the present invention will be described in detail.

[0008]

BEST MODE FOR CARRYING OUT THE INVENTION The peptide of the present invention can be isolated from, for example, a hydrolyzate of oyster protein denatured by heating. Here, the type of oyster for isolating the peptide is not particularly limited, but oysters (Crassostrea giga
s), Suminotogaki (C. ariakensis), Itagaki (Ostrea)
dense lamellosa) and the like are preferably used. It can also be isolated from a hydrolyzate of a protein of another organism having the same amino acid sequence portion as the peptide of the present invention. Such organisms include corn (Zea mays) and yeast (Sac
charomyces cerevisiae), Photobacterium leiognathi
Etc. are illustrated.

The peptide of the present invention can also be produced by a conventional peptide synthesis method in which amino acids are introduced stepwise by an organic chemical synthesis method, or a genetic engineering method. The method for isolating and purifying the peptide of the present invention from oyster will be specifically described below. Put the shelled oysters in distilled water, heat denaturate at 80-100 ° C for 5-15 minutes, and then homogenize.

Tris-hydrochloric acid buffer was added to the obtained homogenate, and 0.1 to 10% by weight, preferably 1 to 4% by weight of protease was added to the homogenate.
Stir at ~ 70 ° C for 0.5-24 hours to allow enzymatic hydrolysis reaction. The precipitate generated by the reaction is removed by filtration, and the filtrate is further filtered using an ultrafiltration membrane to recover a low molecular weight substance having a molecular weight of 10,000 or less. Examples of the ultrafiltration membrane used include YM-10 (Amicon).

The above low molecular weight substance was loaded on the column and
Elution with methanol collects a fraction having HIV-1 protease inhibitory activity. Then, this fraction was loaded on a column to collect a fraction eluted with distilled water, and the obtained fraction was further loaded on a column and eluted with a linear concentration gradient of 0 to 1 M sodium chloride. The active fraction is collected.

The active fraction is subjected to HPLC and fractionated by elution with a linear gradient of 0 to 63% methanol. The active fraction was further subjected to HPL by a linear gradient of 0-63% acetonitrile.
Fractionate with C and collect the active fraction eluted around 42% acetonitrile. The peptide of the present invention having HIV-1 protease inhibitory activity can be obtained by drying and collecting the collected active fraction under reduced pressure.

The above-mentioned protease is a microbial-derived protease such as Bacillus thermoproteolyticu.
s, Bacillus subtilis, Bacillus megaterium, Pseudom
Proteases derived from onasaeruginosa and the like can be mentioned. Specifically, thermolysin derived from Bacillus thermoproteolyticus (manufactured by Sigma), neutral protease derived from Bacillus subtilis, and the like can be preferably used.

The obtained hydrolyzate can be obtained by purification by a conventional means such as ultrafiltration membrane, gel filtration, ion exchange chromatography, high performance liquid chromatography (HPLC) using a reversed phase column, and the like. You can When the peptide of the present invention is obtained by a peptide synthesis method, a solid phase peptide synthesis method or a liquid phase peptide synthesis method may be used, and for example, Nobuo Izumiya, “Basics and Experiments of Peptide Synthesis”.
It may be performed according to the method described in (Published by Maruzen).

It is also possible to carry out by a peptide synthesis method utilizing the reverse reaction of hydrolase. In this case, for example, the method described in "Protease" edited by Eiji Ichishima (published by the Academic Society Publishing Center), etc. You can follow the procedure. In this synthetic method, thermolysin and carboxypeptidase Y are used.
Using an enzyme such as the above, both the enzyme and the protected amino acid of the raw material are generally added at a concentration higher than the value in the decomposition reaction, and if necessary, an organic solvent such as methanol or dimethylformaldehyde is added to react with the amino acid to react. Peptide bonds are formed sequentially.

When the peptide of the present invention is obtained by a genetic engineering method, it is obtained by transforming or transducing a host cell with a recombinant DNA into which a DNA containing a gene encoding the peptide is inserted. It can be carried out by a method known per se, in which production is performed using a recombinant host.

The protease inhibitory activity of the peptide of the present invention obtained as described above can be measured by the following method. That is, a buffer solution was added to HIV-1 protease as an enzyme solution, and His-Lys-Ala-Arg-Val-Leu-p-
A solution of nitro-Phe-Glu-Ala-Nle-Ser-NH ₂ dissolved in distilled water was used as a substrate solution, the peptide sample solution of the present invention was mixed with the substrate solution, and the enzyme solution was further added to carry out an enzymatic reaction. And then release p-nitro-Phe-Glu- after stopping the reaction.
Ala-Nle-Ser-NH ₂ may be quantified by HPLC to calculate the inhibition rate.

The peptide of the present invention or a salt thereof may be used as it is, or may be formulated into a tablet, a powder, a wettable powder, an emulsion, a capsule, etc. by a commonly used solid carrier, liquid carrier, emulsifying dispersant, etc. -1 can be used as a protease inhibitor. Examples of the carrier include water, gelatin, starch, magnesium stearate, lactose, gum arabic, vegetable oil and the like.

The above-mentioned salts of the peptide of the present invention are pharmaceutically acceptable acid addition salts and salt addition salts. Examples of pharmaceutically acceptable acid addition salts include salts with inorganic acids such as hydrochloric acid, sulfuric acid, nitric acid and phosphoric acid; formic acid, acetic acid, propionic acid, glycolic acid, succinic acid, malic acid, tartaric acid, citric acid, Examples thereof include salts with organic acids such as trifluoroacetic acid, and the pharmaceutically acceptable salt addition salts include, for example, alkali metal salts such as sodium salt and potassium salt; alkaline earth metal salts such as calcium salt; Examples thereof include salts with amines such as ammonium, ethanolamine, triethylamine and dicyclohexylamine.

Examples of the use form of the AIDS virus (HIV-1) protease inhibitor containing the peptide of the present invention as an active ingredient include parenteral administration such as intravenous injection and rectal administration, or oral administration. . The content of the peptide of the present invention in the HIV-1 protease inhibitor is
Although it may be appropriately changed depending on the administration route and dosage form, for example, the peptide of the present invention is contained in an amount of 5 to 90% by weight, preferably 10 to 60% by weight based on the total weight of the preparation. Alternatively, the concentration of the peptide of the present invention in blood is 0.005 to 0.05 μM.
It is preferable to design the formulation so as to be in the range.

[0021]

EXAMPLES The present invention will be described below based on examples and test examples, but the present invention is not limited to these examples. [Example 1] (Isolation and purification of HIV-1 protease inhibitor) 100 g of Japanese oyster (Crassostrea gigas) with shells removed was placed in 300 ml of distilled water and boiled for 10 minutes for heat denaturation,
It was homogenized with a Polytron. Volume 30 ml
50 ml of Tris-HCl buffer (100 mM, p
H8.2, containing 10 mM calcium chloride), 32 mg of thermolysin (manufactured by Sigma) as a protease were further added, and the mixture was stirred at 37 ° C. for 4.5 hours. After removing the precipitate with a filter paper, the filtrate was subjected to an ultrafiltration membrane YM-10 with a molecular weight cutoff of 10,000.
(Amicon), and the low molecular weight substance passing through the membrane was recovered. This sample was loaded on a Sephadex LH-20 column (Pharmacia, 26 × 900 mm), eluted with 30% methanol, and had an inhibitory activity against HIV-1 protease (the measuring method is described in Test Example 1 below). Fractions were collected (ie 254-267 ml fractions). Then, this fraction was collected on an SP Toyopearl 650S column (Tosoh Corporation, 16 × 650 m).
m) and elute with distilled water.
(eluted to ml). This was further loaded on Super Q Toyopearl 650S column (Tosoh Corporation, 16 x 650 mm), and 0
The active fraction (eluted at 185-190 ml) eluted with a linear gradient of ~ 1 M sodium chloride was collected. This active fraction was further added to a μBondapak C18 column (Waters 3.9 ×
High performance liquid chromatography (HPLC) using 300 mm)
Fractionated by elution with a linear gradient of 0-63% methanol. The active fraction was further subjected to HPLC (0.1%) using a μBondasphere C18 column (Waters 3.9 × 150 mm).
Fractionation was performed with 0-63% acetonitrile containing trifluoroacetic acid) to obtain an active fraction (peak shape has a slight shoulder) eluted at an acetonitrile concentration of about 42%. The collected substance was dried under reduced pressure to give a final preparation.

The structure of the inhibitory active substance was analyzed by Protein Sequencer G1000A manufactured by Hewlett-Packard Co., and it was presumed to be a mixture of two kinds of peptides having the amino acid sequences represented by SEQ ID NOS: 1 and 2, respectively.

[Example 2] (Chemical synthesis of peptide) First, a peptide having the amino acid sequence (Leu-Leu-Glu-Tyr-Ser-Ile) represented by SEQ ID NO: 1 estimated in Example 1 (hereinafter, referred to as Peptide 1) was synthesized by the following procedure. Similarly, the amino acid sequence (Leu-Leu-Glu-Tyr-Se) represented by SEQ ID NO: 2 estimated in Example 1 was used.
r-Leu) -containing peptide (hereinafter referred to as peptide 2), and the amino acid sequence represented by SEQ ID NO: 3 (Le
A peptide having u-Leu-Glu-Tyr-Ser) (hereinafter referred to as peptide 3) was also synthesized according to this.

First, 0.5 mmol of Boc-Ile was applied to a peptide synthesizer (430A type) manufactured by Applied Biosystems.
-O-CH ₂ -PAM resin and Boc-Leu cartridge two each 2 mmol, and Boc-Glu (OBzl), Boc -Tyr (Br-Z), Boc-Ser (B
zl) cartridge is loaded, and by the anhydrous symmetry method by DCC
Leu-Leu-Glu (OBzl) -Tyr (Br-Z) -Ser (Bzl) -Ile-O-CH ₂ -PAM
Was synthesized. Boc is a t-butyloxycarbonyl group, Obzl is a benzyloxy group, Br-Z is a bromobenzyloxycarbonyl group, and Bzl is a benzyl group. All amino acids used here are in the L-form. Next, the above synthetic peptide resin was introduced into a hydrogen fluoride apparatus manufactured by Peptide Institute, and after adding 1.5 ml of anisole, 10 ml of hydrogen fluoride was introduced. After reacting at -2 ° C for 1 hour, hydrogen fluoride was removed under reduced pressure, the peptide was washed with anhydrous ether and chloroform alternately three times, and the peptide was dissolved in 60 ml of 2N acetic acid and freeze-dried. Then, the peptide was purified by HPLC under the following conditions. HPLC conditions: Column: Merck LiChrospher RP-18 (e) (10 x 250
mm) Eluent: linear gradient of 3.5-67% acetonitrile containing 0.1% trifluoroacetic acid Flow rate: 6 ml / min

When the peptides purified above were dried under reduced pressure, about 25 mg of white powder was obtained for each of the peptides. Furthermore, after the synthesized peptide was hydrolyzed with 4N methanesulfonic acid at 110 ° C for 24 hours, Hitachi L-850
The structure was confirmed by performing amino acid analysis with a 0A amino acid analyzer.

The retention time of peptide 1 by HPLC under the following conditions was 13.3 minutes, which was consistent with the retention time of the HPLC peak of the substance purified from the enzymatic hydrolyzate of oyster in Example 1 above. Also, for Peptide 2 under the following HPLC conditions
The retention time by HPLC was 13.5 minutes, which was in agreement with the retention time of the slight shoulder portion of the HPLC peak of the substance purified from the enzymatic hydrolyzate of oyster in Example 1. HPLC conditions: Column: μBondasphere C18 (Waters 3.9 x 15
0 mm) Eluent: linear gradient of 0 to 63% acetonitrile containing 0.1% trifluoroacetic acid (20 minutes) Flow rate: 1 ml / min

[Test Example 1] (Measurement of HIV-1 Protease Inhibitory Activity) (1) Method for Testing Inhibitory Activity HIV-1 protease inhibitory activity in the purification process of Example 1 was measured by the following method, and Example 2 The HIV-1 protease inhibitory activity of each peptide synthesized by was measured by the following method.

Measurement of HIV-1 Protease Inhibitory Activity in Purification Process HIV-1 protease (produced in Escherichia coli by genetic recombination) purchased from Bachem Co. was used as a buffer solution (100 mM).
A sodium acetate buffer solution, pH 4.9, 200 mM sodium chloride, 5 mM dithiothreitol, and 10% (V / V) glycerol were included in the solution) to prepare a 0.020 mg / m enzyme solution. Also, 1 mg
/ ml His-Lys-Ala-Arg-Val-Leu-p-nitro-Phe-Glu-Ala-N
le-Ser-NH2 (manufactured by Bachem) was dissolved in distilled water to obtain a substrate solution. Put 100 μl of the above buffer solution, 10 μl of substrate solution, and 10 μl of sample solution into a 0.5 ml plastic tube and incubate at 37 ℃.
After incubating for 5 minutes at 25 ° C., 25 μl of the above enzyme solution was added and mixed well, and the reaction was carried out at 37 ° C. for 15 minutes. The reaction was then stopped by adding 15 μl of 10% trifluoroacetic acid. P-nitro-Ph released by enzymatic reaction after the reaction is stopped
e-Glu-Ala-Nle-Ser-NH2 was quantified by HPLC under the following conditions.

HPLC conditions: Column: μBondasphere 5 μC8 300Å (Waters Inc.
3.9 x 150 mm) Eluent: linear gradient of 0 to 63% acetonitrile containing 0.1% trifluoroacetic acid (20 minutes) Flow rate: 1 ml / min Detection: Absorption at 300 nm is measured. The inhibition rate was calculated by the following formula.

[0030]

[Formula 1] Inhibition rate = (A-B) / A x 100 (%) [In the formula, A: p-nitro-Phe-Glu- in the case of containing no inhibitor.
Ala-Nle-Ser-NH2 peak area, B: p-nitro-Phe-Glu-Ala-Nle-Ser-NH2 peak area when inhibitor is added]

Similarly to the measurement of HIV-1 protease inhibitory activity of the synthetic peptide, HIV-1 protease (produced in Escherichia coli by genetic recombination) purchased from Bachem Co. was used as a buffer (100 mM sodium acetate buffer, pH 4.9, It was dissolved in 200 mM sodium chloride, 5 mM dithiothreitol, and 10% (V / V) glycerol) to prepare a 0.020 mg / m enzyme solution. In addition, 1 mg / ml His-Lys-Ala-Arg-Val-Leu-p-nitro-
Phe-Glu-Ala-Nle-Ser-NH ₂ (manufactured by Bachem) was dissolved in distilled water to obtain a substrate solution. In a 0.5 ml capacity plastic tube, 100 μl of the above buffer solution, 10 μl of sample solution, 25 enzyme solution
After adding μl and incubating at 37 ° C. for 5 minutes, 10 μl of the substrate solution was added and mixed well, and the reaction was carried out at 37 ° C. for 20 minutes. The reaction was then stopped by adding 15 μl of 10% trifluoroacetic acid. Similar to the following, p-nitro-Phe-Glu-Ala-Nle-Ser-NH ₂ released by enzymatic reaction after stopping the reaction
Was quantified by HPLC and the inhibition rate was calculated.

(2) HIV-1 Protease Inhibitory Activity of Purified Inhibitor and Synthetic Peptide The HIV-1 protease inhibitory activity of the inhibitor finally purified by the μBondasphere C18 column in Example 1 was determined according to the above (1). As a result of the measurement, the inhibitor was 0.034 μM
It showed an HIV-1 protease inhibitory activity of 79% at this concentration.

Further, the peptides 1 to 3 synthesized in Example 2,
And comparative peptide 1: Leu synthesized according to Example 2
-Leu-Glu-Tyr (SEQ ID NO: 4) and comparative peptide 2: Leu
The HIV-1 protease inhibitory activity of -Glu-Tyr-Ser (SEQ ID NO: 5) was measured according to the above (1), and the peptide concentration at which the inhibitory rate was 50% was further represented by an IC ₅₀ value. Also the peptide
Regarding 1 and 2, according to the measurement method of (1) above, the change in the reaction rate of HIV-1 protease when the substrate concentration to be added is changed in 5 steps from 0.65 to 5.3 mg / ml is measured. An experiment was conducted. The value of the dissociation constant K _i of the peptide-enzyme complex was determined by expressing the obtained results in a Lineweaver-Burk plot. The obtained IC ₅₀ value and K _i value are shown in Table 1.

[0034]

[Table 1]

As shown in Table 1, Peptide 1 and Peptide 2 showed almost the same HIV-1 protease inhibitory activity. Also, weak HIV-1 protease inhibitory activity was observed in peptide 3 in which the C-terminal amino acid Ile or Leu of these peptides was deleted. For comparative peptides 1 and 2, only very weak activity was observed for both.

Then, the peptide for comparison Leu-Leu-Glu was synthesized by the method according to Example 2, and commercially available Leu-Leu (manufactured by Sigma), Leu-Glu (manufactured by Bachem), and Leu. -Leu-Tyr (manufactured by Sigma), Leu-Leu-Val-Tyr (manufactured by Bachem), including H
The IV-1 protease inhibitory activity was measured according to the above (1). The results are shown in Table 2, but almost no activity was observed for any of the peptides.

[0037]

[Table 2]

Test Example 2 Effect of Synthetic Peptide on Other Proteases and Peptides Peptide 1 synthesized in Example 2 (Leu-Leu-Glu-Tyr-Se)
r-Ile: SEQ ID NO: 1), peptide 2 (Leu-Leu-Glu-Tyr-Ser
-Leu: SEQ ID NO: 2), peptide 3 (Leu-Leu-Glu-Tyr-Se
Regarding the enzyme specificity of r: SEQ ID NO: 3), the enzyme inhibition rate was examined using a typical protease shown in Table 3. The results are shown in Table 3.

[0039]

[Table 3]

Peptide 1 slightly (31%) inhibited angiotensin I converting enzyme, presumably because the peptide serves as a substrate for angiotensin I converting enzyme. Peptin and renin, which are aspartic proteases having aspartic acid at the active center like HIV-1 protease, are not inhibited by peptides 1, 2 and 3, and peptides 1, 2 and 3 are both HIV-1 proteases. It can be seen that it will be blocked.

[0041]

INDUSTRIAL APPLICABILITY The present invention provides a novel peptide having HIV-1 protease inhibitory activity. The peptide of the present invention can be used as a research reagent for studying the properties of HIV-1 protease and AIDS virus, or as a lead compound for designing a new compound. Moreover, if its function is clinically sufficiently evaluated, it can be used for the treatment of AIDS.

[0042]

[Sequence list]                                 SEQUENCE LISTING    <110> Director-General of Agency of Industrial Science and Technology <120> Novel peptides and HIV-1 virus protease inhibitor containing the          same <130> 11900228    <160> 5    <210> 1 <211> 6 <212> PRT <213> Crassostrea gigas    <400> 1 Leu Leu Glu Tyr Ser Ile  1 5    <210> 2 <211> 6 <212> PRT <213> Crassostrea gigas    <400> 2 Leu Leu Glu Tyr Ser Leu  1 5    <210> 3 <211> 5 <212> PRT <213> Artificial Sequence <400> 3 Leu Leu Glu Tyr Ser  1 5

─────────────────────────────────────────────────── ─── Continuation of front page (58) Fields surveyed (Int.Cl. ⁷ , DB name) C07K 14/435 ZNA A61K 31/00 A61K 37/64 C12N 9/99 C12N 15/00 A

Claims (4)

(57) [Claims] 1. A peptide of the following (a) or (b); (a) A peptide comprising the amino acid sequence represented by SEQ ID NO: 1. (B) One amino acid in the amino acid sequence of SEQ ID NO: 1 has a deleted, substituted or added amino acid sequence, and has the amino acid sequence of Leu Leu Glu Tyr Ser.
A peptide having an AIDS virus (HIV-1) protease inhibitory activity. 2. In the amino acid sequence of SEQ ID NO: 1, C
The peptide according to claim 1, comprising an amino acid sequence in which isoleucine at the terminal is replaced with leucine. 3. In the amino acid sequence of SEQ ID NO: 1, C
Consisting of an amino acid sequence with the terminal isoleucine deleted,
The peptide according to claim 1. 4. An AIDS virus (HIV-1) protease inhibitor comprising the peptide according to claim 1 as an active ingredient.