JPH05194594A - New peptide - Google Patents

Abstract

PURPOSE:To obtain a peptide having TNF inhibiting activity and expected in effect for suppressing side reactions of various kinds of TNFs. CONSTITUTION:Peptides of sequence number 1 (peptide of formula I), sequence number 2 (peptide of formula II) and sequence number 3 (peptide of formula III), etc., and physiologically permissible salt thereof, with the proviso that an amino of N end of the peptide of formula I, etc., may be modified with acetyl, tert-butoxycarbonyl or benzyloxycarbonyl and carboxyl of C end may be amide. The peptides of the sequence number 1, etc., are synthesized using reagents such as diisopropylethylamine, trifluoroacetic acid, neutralizer, methanol, etc., and adopting a solid phase method by means of an automatically synthesizing equipment of peptide. Then the elimination of a protecting group and cutting out from a resin are carried out under high temperature after using the reagent under low temperature to provide the short-chain type peptide.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a tumor necrosis factor (TNF) -related peptide, and further to a peptide having an inhibitory activity against TNF or a physiologically acceptable salt. Currently, multifaceted research on cancer treatment is being conducted. TNF is a kind of cytokine that has many actions on cells, and binds to a specific cell surface receptor to initiate the action. For example, it kills tumor cells and virus-infected cells, or amplifies the antibacterial action of granulocytes, and thus acts beneficially on the organism.

However, in some diseases, overproduction of TNF exerts harmful effects such as septic shock, loss of appetite, and weight loss, and therefore TNF is a cachecti.
It is also called n) and has the side effect of adversely affecting the living body. Therefore, there is a need for the development of TNF analogs that have higher potency and fewer side effects, or agonists that cause actions similar to TNF, that is, agonists. On the other hand, it is also important to appropriately control the excessive action of endogenously formed TNF or exogenously administered TNF, or to find a substance that antagonizes their side effects or inhibits those side effects. It is an issue. Regarding the former TNF analog, various TNF mutants have been investigated [CR Goh et al.
Protein Engng, 4 , 385 (1991)]. Regarding the latter, as a substance showing these actions, TBP-I corresponding to the extracellular domain of TNF receptor protein from human urine [I.
Olsson et al., Eur. J. Haematol., 42 , 270 (1989) and H. Engelmann, J. Bio. Chem., 264 , 11974 (1989)].
And TBP-II [H. Engelmann et al., J. Bio. Chem., 265 ,
1531 (1991)], a TNF-binding protein having a molecular weight of about 30 Kda has been isolated, and its inhibitory effect on the action of TNF has been reported. These partial proteins of TNF receptor and other protein preparations such as anti-TNF monoclonal antibody are in the stage of being investigated for their potential as therapeutic agents for endotoxin shock associated with sepsis.

[0003]

2. Description of the Related Art In order to obtain the above-mentioned proteins reported hitherto, it is first necessary to produce the target protein by gene and cell engineering, and carefully separate and purify it from the culture solution. To obtain the product, special equipment and skilled technicians are required, and it takes a long period of time and a lot of labor. Therefore, with conventional proteins, the cost is high, the produced antagonist or inhibitor against TNF is expensive, and mass production is difficult. At present, TNF antagonists or inhibitors have not reached the market.

[0004]

DISCLOSURE OF THE INVENTION The present invention has searched for a peptide which has an inhibitory activity against TNF and which can be produced inexpensively and in large quantities. For the first time, we succeeded in identifying the chain and found the short-chain peptide of the present invention.

[0005]

The present invention relates to a peptide having an inhibitory activity against TNF, or a physiologically acceptable salt thereof, that is, more specifically, the following amino acid sequence: 1) SEQ ID NO: 1 (2) SEQ ID NO: 2 (3) SEQ ID NO: 3 (4) SEQ ID NO: 4 (5) SEQ ID NO: 5 (6) SEQ ID NO: 6 (7) SEQ ID NO: 7 (8) SEQ ID NO: 8 (9) SEQ ID NO: 9 and / or (10) Peptide represented by SEQ ID NO: 10 and physiologically acceptable salt thereof

(In the formula, the N-terminal amino group of the peptide of each SEQ ID NO: may be modified with an acetyl group, a t-butoxycarbonyl group or a benzyloxycarbonyl group.
The terminal carboxyl group may be an amide group. Two Cys in the peptide of SEQ ID NO: 10 bind to each other to represent a disulfide bond in the molecule. ).

Physiologically acceptable salts include alkali metal salts or alkaline earth metal salts, salts with physiologically acceptable amines and inorganic or organic acids such as hydrochloric acid, sulfuric acid, phosphoric acid, formic acid, acetic acid. , Methanesulfonic acid, citric acid, tartaric acid, lactic acid, oleic acid, fumaric acid, malic acid, cinnamic acid, malonic acid, glutamic acid, aspartic acid, muxic acid, benzoic acid, gluconic acid, oxalic acid, ascorbic acid, acetylglycine The salt and the like can be mentioned.

The peptide of the present invention can be easily obtained by applying a general peptide synthesis method. As the peptide synthesis method, activated ester method, mixed acid anhydride method, C-terminal activation method such as azide method, coupling method such as carbodiimide, N-carboxy anhydride (NCA) method, redox method, enzyme method or Solid phase method developed by Merrifield [RB Merri
field, Angew. Chem. Int. Ed. Engl., 24 , 799 (198
5)] etc. In addition, ligation of two cysteine residues via a disulfide bridge is preferably performed by an oxidation reaction using air or iodine.

The biological evaluation of these peptides was carried out by the following method. a) Measurement of the inhibitory effect of the synthetic peptide of the present invention on TNF activity using LM or A431 cells. b) Measurement of TNF binding inhibitory activity using LM, HL-60 or A431 cells. c) Measurement of binding ability of the synthetic peptide of the present invention to TNF in a cell-free system. Hereinafter, the present invention will be described more specifically by way of examples, but the present invention is not limited thereto.

[0010]

【Example】

Example 1 Synthesis of SEQ ID NO: 1 A solid phase method was used for peptide synthesis, and an automatic peptide synthesizer was used.
(Applied Biosystems model 430A or 431A) was used and the program was carried out by the company. If the reagent used in the synthesizer is 430A, 1.
2. trifluoroacetic acid 3. nutrizer (ethanolamine) 4. methanol 5. hydroxybenzotriazole 6. dicyclohexylcarbodiimide 7. dichloromethane 8. dimethylformamide. In the case of 431A, N-methylpyrrolidone was used instead of dimethylformamide. The amino acid used in the synthesis was Boc-Al.
a, Boc-Arg (Mts), Boc-Asn, Boc-Asp (OBzl), Boc-Cys (4
-CH ₃ OBzl), Boc-Glu (OBzl), Boc-Gln, Boc-Gly, Boc-Hi
s (Dnp), Boc-Ile, Boc-Leu, Boc-Lys (Cl-Z), Boc-Met
(O), Boc-Phe, Boc-Pro, Boc-Ser (Bzl), Boc-Trp, Boc-
Tyr (Br-Z) and Boc-Val. PAM resin was used for the synthesis of C-terminal carvone type peptide, and P-methyl BHA resin was used for the synthesis of C-terminal amide type peptide. Elimination of protecting groups,
Cleavage from the resin followed the manual of Applied Biosystems. The reagents (peptide resin 1 g scale) used in those methods are as follows. 1. General method (Boc-Met
(O)), a peptide resin synthesized without using Boc-Tyr (CHO)); thioanisole: 1,2-ethanedithiol (2: 1)
1.5 ml, trifluoroacetic acid 10 ml, trifluoromethanesulfonic acid 1 ml 2. Special method (Boc-Met (O)), peptide resin synthesized using Boc-Tyr (CHO)); 2-1 Low temperature conditions,
m-cresol 1 ml, dimethyl sulfide 3 ml, trifluoroacetic acid 5 ml, trifluoromethanesulfonic acid 1 ml 2-2
Then, high temperature conditions (the same as the general method) are performed.

In the synthesis of the peptide of this example, Bo
Method c was adopted and 0.5 mmol Boc-Gly-PAM Resin was used.
The synthesis was completed and 2.5 g of peptide resin was obtained. The above peptide resin was suspended in dimethylformamide (60 ml), thiophenol (2.5 ml) was added, and the mixture was stirred at room temperature for 1 hr. First, the protective group Dnp for histidine was removed. The peptide resin was filtered off and washed with dichloromethane. The peptide resin was cut out by a special method according to the manual of Applied Biosystems. That is, m-cresol
(2.5 ml) and dimethyl sulfide (7.5 ml) were added to the stirred mixture while cooling in an ice bath with trifluoroacetic acid.
(12.5 ml), then trifluoromethanesulfonic acid (2.5 m
l) was added. Stirred at that temperature for 3 hours. ether
(150 ml) was added and the reaction mixture was filtered and washed 3 times with ether. Thioanisole: 1,2-ethanedithiol (2: 1) (3.8 ml) was added to the obtained mixture, and the mixture was stirred for 10 minutes.
Then trifluoroacetic acid (25 ml) was added while cooling in an ice bath. Trifluoromethanesulfonic acid (2.5 ml) was added slowly and the mixture was stirred at room temperature for 30 minutes. Chilled ether was added and the reaction mixture was filtered and washed 3 times with ether. The crude peptide obtained was trifluoroacetic acid (15 ml).
Was dissolved in, cooled, and added dropwise to the stirred ether.
The precipitate was filtered and washed 3 times with ether. It was dissolved in a 10% aqueous acetic acid solution and filtered. The crude peptide was dissolved in an aqueous acetic acid solution and developed with a 10% aqueous acetic acid solution using an open column (Bio-Gel P-2). The eluate containing the target substance was freeze-dried to obtain 1.4 g of a white powder. HPLC [ODS-80TM column (Toso
-, 21.5mmmIDx30cm), acetonitrile linear gradient / 0.1% trifluoroacetic acid], lyophilized and white powder (556mg)
Got Analytical column [ODS-80TM column (Toso-, 4.6mmID
x15 cm) Acetonitrile linear gradient / 0.1% trifluoroacetic acid] was used for HPLC to confirm that the purified product was single. A part was subjected to amino acid sequence analysis using a 477A protein sequencer manufactured by Applied Biosystems, 120APTH analyzer. After hydrolysis with 6N hydrochloric acid at 110 ° C for 20 hours, analysis was performed using an A-8700 type amino acid analyzer manufactured by Ichika Kogyo Co., Ltd., and the peptide sequence Thr Ala Ser Glu was analyzed.
Asn His Leu Arg His Ala Leu Ser AlaSer Lys Ala Arg
It was confirmed to have Lys Glu Met Gly Gln Val Glu Ile.

Example 2-10 The same reaction procedure as in Example 1 was carried out to obtain the peptides of Examples 2 to 10 shown in Table 1.

Example 11 Synthesis of SEQ ID NO: 10 Using Boc-Glu-PAM-resin in the same manner as in Example 1, 1.50 g of peptide resin was obtained. Suspend in dimethylformamide (30 ml), add thiophenol (1.5 ml),
I stirred it for a while. After filtration, the peptide resin was washed with dichloromethane. Thereafter, deprotection and cutting out from the resin were performed by a general method. Thioanisole (1.5m
l) and 1,2-ethanedithiol (0.75 ml) were added, and trifluoroacetic acid (15 ml) and trifluoromethanesulfonic acid (1.5 ml) were added with stirring under ice cooling. After 30 minutes, diethyl ether (150 ml) was added. The precipitate was filtered and washed with diethyl ether. Trifluoroacetic acid (2
0 ml) was added to dissolve the peptide, and the trifluoroacetic acid layer was added dropwise to the stirred diethyl ether. The precipitate formed was filtered and washed with diethyl ether. The precipitate was dissolved in a water-acetic acid system and purified by an open column. The powder (1.15 g) obtained by freeze-drying was dissolved in a 90% acetic acid aqueous solution, and an acetic acid solution of iodine was added. 12 hours later,
Zinc (100 mg) powder was added to decompose excess iodine, and then concentrated under reduced pressure. The residue was purified by HPLC. 45 mg of the desired product was obtained.

Example 12 1. Experiment for measuring binding ability of peptide to ¹²⁵ I-TNF by ELISA ball The peptide was immobilized on Sumitomo-aminoball using glutaraldehyde. After washing with phosphate buffer (PBS),
% Bovine serum albumin (BSA) inhibited TNF binding to balls. ¹²⁵ I-TNF (Amersham; specific activit
(y: 15-30 TBq / mmol) was added, and carbon dioxide culture was performed at 37 ° C. for 18 hours. After washing with PBS, the radioactivity was measured with a gamma counter. On the other hand, non-specific binding was measured in the coexistence of 200-fold unlabeled TNF. Specific binding was calculated from the difference between the two measured values.

Specific binding-cpm ratio [Example number of each synthetic peptide, concentration (4 mM)] No addition Example 2 Example 7 Example 11 Specific binding-cpm ratio 1 4 1.5 2

No addition Example 2 Example 9 Example 10 Specific binding-cpm ratio 1 3.5 2.3 2

2. Experiment for measuring the binding ability of the peptide to ¹²⁵ I-TNF using a plate The peptide was immobilized on a Nunc Maxi plate. After washing with phosphate buffer solution (PBS), binding of TNF to balls was inhibited by 3% BSA. ¹²⁵ I-TNF (Amersham; specific activit
(y: 15-30 TBq / mmol) was added, and carbon dioxide culture was performed at 37 ° C. for 18 hours. After washing with PBS, the radioactivity was measured with a gamma counter. On the other hand, non-specific binding was measured in the coexistence of 200-fold unlabeled TNF. Specific binding was calculated from the difference between the two measured values.

Specific binding-cpm ratio-Synthetic peptide concentration (mM) no addition Example 2 Example 3 Example 5 Example 8 (4mM) (4mM) (0.5mM) (1mM) Specific binding-cpm ratio 1 3 6 2.2 4

Example 13 Cytotoxic activity test Mouse LM cells contained D containing 5% bovine serum and 0.2% glucose.
It was cultured in ulbecco's Modified Eagle Medium (DMEM). 9
A solution of peptide and TNF was added to a 6-well microplate, precultured at room temperature for 1 hour, LM cells per 0.8-1x10 ⁴ / well were added, and stained with crystal purple after 3 days. To determine the cytotoxic activity, extract the dye absorbed in living cells and use a microplate reader to measure 550 nm.
It was measured at.

Effect of synthetic peptide on cytotoxic activity of TNF on LM cells Absorbance TNF-free 100 100 100 TNF (0.51 ng / ml) 22 52 66 TNF (0.51 ng / ml) + Example 1 (1 mM / ml) -78-TNF (0.51ng / ml) + Example 2 (4mM / ml) 86--TNF (0.51ng / ml) + Example 4 (4mM / ml) 94--TNF (0.51ng / ml) + Implementation Example 6 (2 mM / ml)--88 In the presence of the synthetic peptides of Examples 1, 2, 4 and 6, TNF
It shows that the cytotoxicity of is suppressed.

Example 14 Measurement of Binding Inhibitory Activity The human epithelial cancer cell line A-431 was treated with 10% fetal bovine serum (fetal bov).
It was cultured in DMEM containing ine serum, FBS) and 4.5 g / ml glucose. The cells were cultured in a 12-well plate at 1 × 10 ⁵ / well, and after 3 days, they were used in a semiconfluent state. Upon use, the supernatant was removed by suction and then washed twice with DMEM containing 10% FBS. Peptide and ¹²⁵ I-TNF
After pretreatment with CO2 incubator at 37 ° C for 18 hours, the reaction solution was added to the cells and kept in ice.
Sensitized for 90 minutes. After completion of the reaction, the supernatant was removed by suction and washed, and then a 1N sodium hydroxide aqueous solution was added to lyse the cells. This lysate was collected and the radioactivity was measured with a gamma-counter.

Inhibitory effect of synthetic peptide on binding between TNF and A-431 cells cpm (%) ¹²⁵ I-TNF (0.106nM) 100 ¹²⁵ I-TNF (0.106nM) + Example 1 (4mM) 57 ¹²⁵ I- TNF (0.106 nM) + Example 2 (4 mM) 25 The synthetic peptide of Example 2 more strongly inhibited the binding between TNF and A-431 cells than the synthetic peptide of Example 1.

[Sequence list]

SEQ ID NO: 1 Sequence length: 25 Sequence type: Amino acid Topology: Linear Sequence type: Peptide Sequence Thr Ala Ser Glu Asn His Leu Arg His Ala Leu Ser Ala Ser Lys 1 5 10 15 Ala Arg Lys Glu Met Gly Gln Val Glu Ile 20 25

SEQ ID NO: 2 Sequence length: 20 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Leu Pro Ala Gln Val Ala Phe Thr Pro Tyr Ala Pro Glu Pro Gly 1 5 10 15 Ser Thr Ala Arg Leu 20

SEQ ID NO: 3 Sequence length: 20 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Leu Val Pro His Leu Gly Asp Arg Glu Lys Arg Asp Ser Val Ala 1 5 10 15 Pro Gln Gly Lys Tyr 20

SEQ ID NO: 4 Sequence length: 21 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Ser Leu Ala Leu Asn Gly Thr Val His Leu Ser Ala Gln Lys Asn 1 5 10 15 Thr Val Ala Thr Ala His 20

SEQ ID NO: 5 Sequence length: 11 Sequence type: Amino acid Topology: Linear Sequence type: Peptide

SEQ ID NO: 6 Sequence length: 17 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Ser Lys Ala Arg Lys Glu Met Gly Glu Val Glu Ile Ser Ser Ala 1 5 10 15 Thr Val

SEQ ID NO: 7 Sequence length: 14 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Ser Leu Ala Leu Asn Gly Thr Val His Leu Ser Ala Gln Glu 1 5 10

SEQ ID NO: 8 Sequence length: 25 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Thr Val Asp Arg Thr Val Ala Gly Ala Arg Lys Asn Gln Tyr Arg 1 5 10 15 His Tyr Trp Ser Glu Asn Leu Phe Gln Gly 20 25

SEQ ID NO: 9 Sequence length: 21 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Gln Glu Lys Gln Asn Thr Val Ala Thr Ala His Ala Gly Phe Phe 1 5 10 15 Leu Arg Glu Asn Glu Gly 20

SEQ ID NO: 10 Sequence length: 14 Sequence type: Amino acid Topology: Sequence type: Peptide Sequence Ser Leu Cys Leu Asn Gly Thr Val His Leu Ser Cys Gln Glu 1 5 10

SEQ ID NO: 11 Sequence length: 20 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Leu Pro Ala Gln Val Ala Phe Thr Pro Tyr Ala Pro Glu Pro Gly 1 5 10 15 Ser Thr Ala Arg LeuNH ₂ 20

─────────────────────────────────────────────────── ───

[Procedure amendment]

[Submission date] December 11, 1992

[Procedure Amendment 1]

[Document name to be amended] Statement

[Correction target item name] 0010

[Correction method] Change

[Correction content]

[0010]

EXAMPLES Example 1 Synthesis of SEQ ID NO: 1 A solid phase method was used for peptide synthesis, and an automatic peptide synthesizer was used.
(Applied Biosystems model 430A or 431A) was used and the program was carried out by the company. If the reagent used in the synthesizer is 430A, 1.
2. trifluoroacetic acid 3. nutrizer (ethanolamine) 4. methanol 5. hydroxybenzotriazole 6. dicyclohexylcarbodiimide 7. dichloromethane 8. dimethylformamide. In the case of 431A, N-methylpyrrolidone was used instead of dimethylformamide. The amino acid used in the synthesis was Boc-Al.
a, Boc-Arg (Mts), Boc-Asn, Boc-Asp (OBzl), Boc-Cys (4
-CH ₃ OBzl), Boc-Glu (OBzl), Boc-Gln, Boc-Gly, Boc-Hi
s (Dnp), Boc-Ile, Boc-Leu, Boc-Lys (Cl-Z), Boc-Met
(O), Boc-Phe, Boc-Pro, Boc-Ser (Bzl), Boc-Thr (Bz
l), Boc-Trp (CHO) , Boc-Tyr (Br-Z) and Boc-Val. C
PAM resin was used for peptide synthesis of the terminal carvone type.
P-methyl BHA resin was used for terminal amide type peptide synthesis. Applied for removal of protecting groups and cutting out from resin
The Biosystems manual was followed. The reagents (peptide resin 1 g scale) used in those methods are as follows. 1. In the case of general method (Boc-Met (O)), peptide resin synthesized without using Boc-Tyr (CHO)); thioanisole: 1,2
-Ethandithiol (2: 1) 1.5 ml, trifluoroacetic acid 10 m
l, trifluoromethanesulfonic acid 1 ml 2. Special method (Boc-
Met (O)), peptide resin synthesized using Boc-Tyr (CHO)); 2-1 low temperature conditions, m-cresol 1 ml, dimethyl sulfide 3 ml, trifluoroacetic acid 5 ml, trifluoromethanesulfonic acid 1 ml 2 -2 Then, high temperature conditions (same as general method) are performed.

[Procedure Amendment 2]

[Document name to be amended] Statement

[Correction target item name] 0020

[Correction method] Change

[Correction content]

Effect of synthetic peptide on cytotoxic activity of TNF on LM cells Absorbance TNF-free 100 100 100 TNF (0.51ng / ml) 22 52 66 TNF (0.51ng / ml) + Example 1 (1mM) -78 -TNF (0.51ng / ml) + Example 2 (4mM) 86--TNF (0.51ng / ml) + Example 4 (4mM) 94--TNF (0.51ng / ml) + Example 6 (2mM) - In the presence of the synthetic peptides of Examples 1, 2, 4 and 6, TNF
It shows that the cytotoxicity of is suppressed.

[Procedure 3]

[Document name to be amended] Statement

[Correction target item name] 0026

[Correction method] Change

[Correction content]

SEQ ID NO: 4 Sequence length: 21 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Ser Leu Ala Leu Asn Gly Thr Val His Leu Ser Ala Gln Glu Lys 1 5 10 15 Gln Asn Thr Val Ala Thr Ala His 20

[Submission date] December 11, 1992

[Procedure amendment 4]

[Document name to be amended] Statement

[Correction target item name] 0028

[Correction method] Change

[Correction content]

SEQ ID NO: 6 Sequence length: 17 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Ser Lys Ala Arg Lys Glu Met Gly Gln Val Glu Ile Ser Ser Ala 1 5 10 15 Thr Val

[Submission date] December 11, 1992

[Procedure Amendment 5]

[Document name to be amended] Statement

[Name of item to be corrected] 0030

[Correction method] Change

[Correction content]

SEQ ID NO: 8 Sequence length: 25 Sequence type: Amino acid Topology: Linear Sequence type: Peptide Sequence Thr Val Asp Arg Asp Thr Val Ala Gly Ala Arg Lys Asn Gln Tyr 1 5 10 15 Arg His Tyr Trp Ser Glu Asn Leu Phe Gln Gly 20 25

Claims (2)

[Claims] (1) SEQ ID NO: 1 (2) SEQ ID NO: 2 (3) SEQ ID NO: 3 (4) SEQ ID NO: 4 (5) SEQ ID NO: 5 (6) SEQ ID NO: 6 (7) SEQ ID NO: 7 (8) SEQ ID NO: 8 (9) The peptide represented by SEQ ID NO: 9 and / or (10) SEQ ID NO: 10 and a physiologically acceptable salt thereof (wherein the N-terminal amino group of the peptide of each SEQ ID NO: is an acetyl group) , The t-butoxycarbonyl group or the benzyloxycarbonyl group may be modified, and the C-terminal carboxyl group may be an amide group. Represents a disulfide bond). 2. The peptide according to claim 1 having TNF inhibitory activity or a physiologically acceptable salt thereof.